DOI: 10.1002/pd.4617


Poster Abstracts of the ISPD 19th International Conference on Prenatal Diagnosis and Therapy, Washington, DC, USA, 12–15 July 2015 P1-1 Identifying Robertsonian translocations by microarraybased DNA analysis Stephanie Huang, Kara Juneau, Patrick Bogard, Kelli Davies, Eric Wang, Craig Struble, Arnold Oliphant, Jacob Zahn Ariosa Diagnostics Inc, San Jose, California, United States OBJECTIVES: A Robertsonian translocation is a whole arm exchange between two acrocentric chromosomes (human chromosomes 13, 14, 15, 21, and 22) resulting in a fused chromosome with 2 long arms. Individuals with Robertsonian translocations have a loss of 2 of the 10 p arms of the acrocentric chromosomes. This is the most common chromosomal translocation, occurring at a frequency of ~1 in 1000. Robertsonian translocation carriers have a higher risk of infertility, miscarriages, and aneuploid offspring. Female carriers of the 14:21 translocation have an estimated risk of fetal trisomy 21 of 15% in any given pregnancy. METHODS: We have developed Digital Analysis of Selected Regions (DANSRTM) assays targeting sequences that are common to the p arms of the five acrocentric chromosomes. DANSR products were hybridized onto a custom manufactured DNA microarray for DNA analysis. The Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTETM) algorithm allows one to account for both the fetal and maternal fractions in the cell free DNA sample to determine Robertsonian risk. The expectation in a Robertsonian sample is that, in the maternal fraction of the cfDNA, the DANSR assays on the five acrocentric p arms should have a concentration that is 20% less than the controls. RESULTS: 24 samples (15 Robertsonian, 9 control) were evaluated for Robertsonian translocation. FORTE analysis successfully identified the Robertsonian translocations in all 15 Robertsonian samples. CONCLUSIONS: As Robertsonian translocations are the most common chromosomal translocation found in humans and can have significant reproductive consequences, a screen for Robertsonian translocations would provide women valuable information regarding their risk for fetal trisomy. With the FORTE algorithm this analysis can be accurately carried out on cfDNA from pregnant women. P1-2 The characteristic of the small supernumerary marker chromosomes of r(4)4p13q13.3 by Affymertrix SNP Array and FISH analysis Zhiying Gao Chinese PLA General Hospital, Haidian District, Beijing, China OBJECTIVES: The aim of our study was to characterize Small supernumerary marker chromosomes (sSMCs) precisely by using Affymetrix SNP Array and FISH analysis. METHODS: Prenatal Diagnosis 2015, 35, 27–109

Chromosome karyotype was detected by G-binding analysis in amniotic fluid and umbilical cord blood. AffymetrixCytoScan 750 K Array was used to identify the size and origin of the sSMCs; FISH confirmation by using probe WSH/D4Z1 was performed in interphase cells of fetus cord blood. RESULTS: Chromosome analysis showed a karyotype of 47,XX,+mar[14] /46,XX[16]; Affymetrix Array analysis showed a result of arr 4p13q13.3 (41,593,201- 72,130,692)X2-3, revealing a trisomy mosaicism of Chromosomal fragement from 4p13-4q13.3 in length of 30.537 Mb. The fragment included the centromere with the features of trisomy mosaicism showed in the 28% interphase cells of fetus cord blood was confirmed by the FISH using the WSH/D4Z1 probe. CONCLUSIONS: This study demonstrated that the combination using chromosome GTG banding analysis, SNP Array and FISH analysis is useful and precise detection for the comprehensive of small supernumerary marker chromosomes. P1-3 CHARGE syndrome due to subchromosomal deletions: A case series illustrating the importance of the use of chromosomal microarray analysis in prenatal diagnosis Emily Hardisty, Diane Vargo, Neeta Vora, R. Matthew Coward University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States OBJECTIVES: Prenatal diagnosis of CHARGE syndrome is rare, with only a few cases published in the literature. CHD7 is the only gene that has been identified in which mutations are known to cause CHARGE syndrome; with point mutations identified in 65-70% of CHARGE syndrome patients. Chromosomal microarray analysis (CMA) can provide prenatal genetic diagnosis in the setting of prenatal ultrasound findings of structural anomalies. METHODS: We present two cases of CHARGE syndrome resulting from a subchromosomal deletion and demonstrate the clinical utility of CMA in the prenatal setting; case 1 was a missed diagnosis and occurred before the published recommendation to perform CMA prenatally on all invasively obtained samples (American College of Obstetricians and Gynecologists [ACOG] Committee Opinion 581; Wapner NEJM 2013); case 2 occurred after this recommendation was published. RESULTS: Case 1 is a 31-year-old G3P1011 who was seen following isolated prenatal diagnosis of a conotruncal heart defect by ultrasound and fetal echocardiogram. Amniocentesis was performed and karyotype was normal (46,XY) with normal FISH for 22q11 deletion syndrome. Following delivery, the neonate was confirmed to have interrupted aortic arch and truncus arteriosis, with additional findings of a cleft soft palate, bilateral profound hearing loss, and dysplastic right kidney. CMA was performed © 2015 John Wiley & Sons, Ltd.


and a 1.179 Mb deletion of 8q12.1q12.2 was identified consistent with the clinical diagnosis of CHARGE syndrome. Case 2 is a 27-year-old primagravida with prenatal ultrasound findings of a complex cardiac defect, Dandy-Walker malformation, and shortened femur measurements. Amniocentesis was performed and routine karyotype detected an apparently balanced translocation involving the proximal arms of chromosome 8 and 10 [46,XY,t(8;10)(q12;q21.2)]. SNP microarray identified a 9.3 Mb interstitial deletion of 8q12.1-8q13.2 involving 29 genes including CHD7. CONCLUSIONS: The ACOG Committee Opinion 581 recommends CMA in patients undergoing invasive prenatal testing in the setting of one or more structural abnormalities. These two cases, specific to CHARGE syndrome, illustrate the utility of prenatal CMA in making a critical prenatal diagnosis especially in cases where fetal abnormalities are present.

P1-4 Copy number variations analysis by high-resolution SNP array in fetus with congenital cardiac defects Linhuan Huang Fetal Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China OBJECTIVES: Congenital heart disease (CHD) is the most frequent birth defect, genomic copy number variations (CNVs) are important in the etiology of CHD. This study aimed to evaluate whether the application of high-resolution SNP microarrays (SNP-array) is a valuable tool for the detection of CNVs in the clinically molecular prenatal diagnosis and genetic counseling of patients with CHD, combined with the clinical and cytogenetic data (ultrasound diagnosis and G-banded karyotyping) at a single Chinese institution. METHODS: We carried out two independent strategies, conventional G-banded karyotyping to detect chromosome aberration and high-resolution SNP arrays to detect CNVs. A total of 222 patients at 12 weeks ~ 37 weeks of gestation were identified CHD with prenatal ultrasound diagnosis, and 50 of them were further delivered for whole genome SNP-array after a G-banded karyotyping analysis on 219 cases. RESULTS: 48 of 219 fetuses (21.9%) were detected abnormality by G-banded karyotyping analysis, with 43 aneuploidy (19.6%) and 5 structural abnormalities (2.3%). However, high-resolution SNP-array yielded abnormal results in 21 of 50 fetuses (42%) with 24 CNVs, including 9 pathogenic CNVs (22q11.2 deletion syndrome, 17p11.2 deletion syndrome, 5p deletion syndrome, 11q deletion syndrome and 13q deletion syndrome), 10 variants of uncertain clinical significance (deletions distributed at 14q, 4p, Xp and 16p, 235 kb ~ 33.3 Mb; duplication distributed at 1p, 2p, 3q and 4q, 570 kb ~ 48 Mb). In 43 cases with normal chromosomal karyotype, 13 cases (30.2%) had abnormal CNVs. 39 cases with isolate CHD all had normal chromosomal karyotype, but 30.8% (12/39) had abnormal CNVs. Abnormal chromosomal karyotypes were detected in 21.1% (4/21) cases with complex CHD, and 47.6% (10/21) had abnormal CNVs. CONCLUSIONS: Our study illustrates that Prenatal Diagnosis 2015, 35, 27–109

high-resolution SNP-array is a valuable tool to identify CNVs in prenatal diagnosis of CHD. Compared with conventional G-banded karyotyping, the SNP array leads to high diagnostic rate and uncover additional disease association CNVs including mosaic, and should become an integral aspect in clinically molecular diagnosis and genetic counseling. Submicroscopic deletions and duplications involving OMIM genes identified in the present study will advance the molecular understanding of etiology in CHD. P1-5 Comparative genomic hybridization in prenatal diagnosis M. Kurtser, V. Gnetetskaya, O. Malmberg, E. Kuznetsova, L. Bessonova, Ekaterina Kuznetsova Genetics Center of Mother and Child Groups of Companies, Moscow, Russian Federation OBJECTIVES: Prenatal diagnosis of chromosomal abnormalities METHODS: Classical cytogenetyc (G-banding) analysis. Fluorescence in situ hybridization (FISH) (Abbott GmbH & Co. KG). Array-comparative genomic hybridization (aCGH) (PerkinElmer) RESULTS: Molecular genetic testing by arraycomparative genomic hybridization (aCGH) significantly increases the effectiveness of the prenatal diagnosis.150 pregnancies with malformations or ultrasound markeres were analyzed in 2014. All patients underwent invasive prenatal testing by chorionic villus sampling or amniocentesis. Chromosomal abnormalities were identified in 18 cases (12%): 8 - trisomy 21, 3 - trisomy 18, 3 monosomy X, 2 - trisomy 13, 1 - mosaic trisomy 9, 1 - polyploidy. Structural chromosome imbalances by aCGH which size ranged from 0,26 to 17,7 Mb were detected in 14 cases with normal karyotype and described in OMIM database. Of the 14 alterations, nine were considered pathogenic and correlated with neurodevelopmental delay, congenital malformations and metabolic impairments. FISH - analysis with specific probes have been used in 7 cases for confirming dup 16p13.3p13.2; del 16p13.3/dup 16q23.3q24.3; del 1p36.32; del 22q11.31; del 22q13.3; del 2q37.3/ dup17p13.1p13.1 and del 4p16.3p16.1. Deletion in DMD gene (Xp21.1, 45-th - 55-th exons) was found in one cases. The deletion and Duchenne muscular dystrophy (OMIM: 310200) was confirmed by MLPA. Chromosomal microdeletions and microduplication were revealed in 3 fetus. FISH technology with subtelomeric DNA probes was applied in 2 families for confirming of the result. Balanced chromosomal rearrangements were found in one of the parents. CONCLUSIONS: Ultrasound examination is an effective method of formation of indications for invasive prenatal diagnosis. In the group of fetuses with malformations and ultrasound markeres chromosomal imbalance was detected in 17.8 %.The number of undifferentiated complexes of fetal malformations can be significantly reduced with using of molecular genetic analysis by aCGH. P1-6 Williams-Beuren syndrome with different sizes and loci of microdeletions on chromosome 7q11.23 diagnosed by chromosomal microarray analysis Lin Li, Qun Fang © 2015 John Wiley & Sons, Ltd.

Prenatal Diagnosis 2015, 35, 27–109

F, 3 M

F, 1 Y

M, 4.5 Y

M, 5 Y

F, 7 Y

F, 32 Y








arr7q11.23 (74,242,166-74,384,749)×1

arr7q11.21q11.23 (64,721,473-74,610,673)×1

arr7q11.23 (72,414,624-72,458,643)×1 (72,621,722-72,710,124)×1

arr7q11.23 (72,611,954-74,143,060)×1

arr7q11.22q11.23 (70,463,755-74,629,034)×1

arr7q11.23 (72,643,631-74,142,190)×1

arr7q11.2 (72,718,277-74,142,190)×1

arr7q11.2 (72,701,018-74,141,746)×1

arr7q11.23 (72,404,175-72,481,934)×1 (72,621,798-72,718,124)×1


145 kb




44 kb, 96 kb 9.88 Mb







ELN gene deletion

1.53 Mb

4.16 Mb

1.50 Mb

1.42 Mb

1.44 Mb

78 kb, 96 kb



11q14.3 Microduplication —




15q11.2 microduplication


Other findings

Maternal FISH: 7q11.2 deletion

Parental FISH: normal

Maternal CMA: 15q11.2 microduplication

Maternal FISH: 7q11.2 deletion

Results of parental tests

Clinical features

Phenotypically normal, her baby had epilepsy, spasm, muscle tremor and died at 4 months.

Mental retardation, feeding difficulties, impaired social interaction, upper eye lid edema.

AS, VSD, GDD, cleft palate, patellar dislocation.


SVAS, GDD, mitral valve insufficiency, urinary fistula, distinctive facial features.


SVAS, PFO, PS, distinctive facial features.

Right pelvic ectopic kidney.

Duodenal atresia, single umbilical artery.

Abbreviations: M, male; F, female; GW, gestational weeks; W, weeks; M, months; Y, years; SVAS, supravalvular aortic stenosis; PFO, patent foramen ovale; PS, pulmonary stenosis; GDD, global developmental delay; AS, aortic stenosis; VSD, ventricular septal defect.

M, 3 W



F, 27 GW


Sex, Age

M, 13




CMA results

P1–6 Table. Table 1 Clinical features and CMA results for 9 cases


© 2015 John Wiley & Sons, Ltd.


Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: Williams-Beuren syndrome (WBS) is a genetic disease caused by a common deletion on chromosome 7q11.23, which ranges from 1.55 Mb to 1.84 Mb and primarily contains the elastin (ELN) gene. Typical symptoms include supravalvular aortic stenosis (SVAS), distinct facial features, intellectual disabilities and so on. This study analyze 9 patients diagnosed with WBS by chromosomal microarray analysis (CMA), which combines single nucleotide polymorphism (SNP) analysis with microarray comparative genomic hybridization (array-CGH). METHODS: CMA was performed on 9 patients with abnormalities in prenatal screening and clinical features, such as supravalvular aortic stenosis, pulmonary stenosis, global developmental delay, ectopic kidney and characteristic facial features. Karyotyping analysis using conventional G-banded techniques (500-band resolution) was performed on 5 patients. FISH was performed following the standard technique using ELN probes on 3 pairs of parents of these patients and CMA on 1 pair. RESULTS: All 9 patients had different sizes and loci of chromosome microdeletions in the 7q11.23 region, ranging from 44 kb to 9.88 Mb. ELN gene deletions were found by CMA in 6 patients, and among them, 4 patients consisted of typical WBS deletions (1.40 Mb to 1.55 Mb), and the other 2 patients consisted of larger atypical deletions (4.16 Mb and 9.88 Mb). The ELN gene was not contained in the deletions of the remaining 3 patients in which 2 children carried 2 separated deletions and 1 adult carried a small atypical deletion (145 kb). Two patients showed additional microduplication, one was combined with 11q14.3 microduplication and another one was with maternally inherited 15q11.2 microduplication. No abnormal karyotype was found in the 5 patients who underwent G-banded karyotype analysis. The results for the 3 pairs of parents who underwent FISH showed that 2 mothers had a 7q11.23 microdeletion. In 1 pair who underwent CMA, the mother showed a 15q11.2 microduplication, the same as her son. We did not find any paternal abnormalities in the FISH or CMA analyses. CONCLUSIONS: CMA may be the most effective way to detect WBS, and the phenotypic features of WBS varied in fetuses, children and adults, influenced by the genes, deletion size and loci and many other factors.

P1-7 Prenatal genetic diagnosis in a fetus with atypical neurofibromatosis type 1 microdeletion Shaobin Lin, Jianzhu Wu, Yanmin Luo, Yi Zhou, Qun Fang, Zhiqiang Zhang Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: To analyze the relationship between a typical neurofibromatosis type 1 (NF1) microdeletion and fetal phenotype. METHODS: Fetal blood sampling was implemented on a woman with a fetus of talipes equinovarus. Both G-banded karyotyping and single nucleotide poly-morphism Prenatal Diagnosis 2015, 35, 27–109

array (SNP array) were performed on the fetal blood simultaneously, and then fluorescence in situ hybridization (FISH) was used to confirmed SNP array result. Furthermore, FISH was also carried out on the parental peripheral blood specimens to ascertain a de novo or inherited variation. RESULTS: Fetal karyotype was 46,XY by G-banding analysis on fetal blood sample. However, a 3.132 Mb microdeletion was detected at chromosome 17q11.2 by SNP array, overlapping the atypical deletion region of NF1 microdeletion syndrome. It was confirmed as a de novo microdeletion by analyzing both fetal and parental specimens with FISH. Conclusions: Talipes equinovarus might be an abnormal sonographic feature of fetus with atypical NF1 microdeletion, which could be accurately diagnosed by SNP array in fetus. P1–7 Image.

P1-8 Diagnosis of cryptic reciprocal translocation carrier: Inspiration from the abnormal SNP array results in a fetus Shaobin Lin, Yi Zhou, Yanmin Luo, Zhiqiang Zhang, Jianzhu Wu, Qun Fang Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: To investigate the clinical value of single nucleotide polymorphism array (SNP array) in the detection of cryptic reciprocal translocation carrier. METHODS: SNP array was performed on fetal blood from a fetus with abnormal sonographic features and normal karyotype. And then fluorescence in situ hybridization (FISH) was used to confirm microdeletion/microduplication. G-banded karyotyping and FISH were also carried out on parental peripheral blood specimens to figure out the origin of microdeletion/ microduplication. In addition, the relationship between abnormal sonographic features and microdeletion/microduplication was analyzed. RESULTS: SNP array detected a 4.4 Mb deletion at 1q44 and a 10.4 Mb duplication at 17q24.3q25.3 in this fetus. Based on SNP array and FISH results from both fetus and parents, the father was diagnosed as a cryptic t(1;17)(q44;q24.3) carrier and the fetus inherited a der(1)t(1;17) (q44;q24.3) from the father. Both 1q44 deletion and 17q24.3q25.3 duplication contributed to abnormal sonographic features presented in this fetus. However, whether 1q44 deletion played a major role in fetal phenotypes was needed to be further studied. CONCLUSIONS: For couples with histories of abnormal pregnancy, it was essential to perform SNP array analysis on abnormal embryo or fetus. Microdeletion/ microduplication detected in embryo or fetus would indicate that cryptic reciprocal translocation might be presented in a parent. © 2015 John Wiley & Sons, Ltd.


P1-9 Diagnostic benefit of microarray over multiple MLPA testing in prenatal cytogenetic diagnostics Merryn Macville, Servi Stevens, Sander Stegmann, John Engelen, Audrey Coumans, Christine Willekes, Ingrid Witters, Salwan Al-Nasiry, Mijntje Pieters, Nienke Muntjewerff, Suzanna Frints, Yvonne Arens, Christine de Die-Smulders Maastricht University Medical Center, Maastricht, Netherlands OBJECTIVES: Analysis of copy number variants (CNVs) by microarrays has become the standard for invasive prenatal cytogenetic testing in cases with ultrasound abnormalities. Before the introduction of microarray testing, we used karyotyping plus targeted MLPA kits for subtelomeres (P036/P070) and microdeletions (P245). Changing to genome-wide microarray testing was instigated by pioneer laboratories reporting an increase of genetically diagnosed ultrasound abnormalities of microarray testing over conventional karyotyping. In retrospect, CNVanalyses were evaluated to determine the additional diagnostic yield of microarrays over karyotyping plus multiple targeted MLPA testing. METHODS: Microarray CNV-analyses (CytoscanHD, Affymetrix, USA) were evaluated for fetuses with ultrasound abnormalities ranging from enhanced nuchal translucency (>3.5 mm) to multiple organ malformations, and testing negative for aneuploidy by a quantitative PCR region-specific assay. Aberrant microarray outcome was classified as pathogenic, benign or of ‘unknown clinical significance’ (UCS) according to ISCA guidelines. Those pathogenic CNV-regions that are not targeted in either one of the MLPA kits were considered as the additional diagnostic yield of microarray testing. RESULTS: Out of 300 CNVanalyses, 52 had an aberrant genotype (17.3%). Of these, 10 were benign (3.3%), 25 UCS (8.3%) and 17 pathogenic (5.7%). The MLPA kits would have detected 14 pathogenic cases (4.7%). The additional diagnostic yield is 1%. The pathogenic CNVs that would not have been detected by MLPA included a GATA4 deletion on 8p23.2 causing Fallot’s tetralogy, a duplication 11p15.4 causing Beckwith-Wiedemann syndrome, and a 16p11.2 microdeletion in a case of micrognathia. All 52 aberrant genotypes were communicated with the pregnant couple by the clinicians, of which 35 (11.7%) were needed to counsel a non-pathogenic finding. CONCLUSIONS: In this relatively small cohort, microarray CNV-analyses detected 1% more pathogenic genotypes than multiple targeted MLPA testing. In return, in 11.7% a counselling was needed to explain a non-pathogenic finding. Changes in laboratory diagnostics are made in the expectation of better care and service to the patient or to simplify laboratory processes and reduce cost. Laboratories that need to make a choice between microarray and multiple targeted MLPA testing should be aware of the marginal diagnostic benefit, higher cost and higher counseling burden when choosing microarray testing. P1-10 Withdrawn. P1-11 Belgian MicroArray Prenatal (BEMAPRE) database Joke Muys1, Katrien Janssens1, Olivier Vanakker2, Catheline Vilain3, Guillaume Smits3, Claude Bandelier4, Saskia Bulk5, JH Prenatal Diagnosis 2015, 35, 27–109

Caberg5, Anne De Leener3, Marjan De Rademaeker6, Thomy de Ravel8, Julie Desir9, Anne Destree9, Annelies Dheedene2, Stephanie Gaillez6, Bernard Grisart9, AC Hellin6, Sandra Janssens3, Kathelijn Keymolen6, Bjorn Menten2, Bruno Pichon3, Marie Ravoet4, Nicole Revencu4, Sonia Rombout8, Catherine Staessen6, Ann Van Den Bogaert6, Kris Van Den Bogaert7, Joris Vermeesch8, Yves Sznajer5, Bettina Blaumeiser9, Yves Jacquemyn10, Koenraad Devriendt7 1

University Hospital Antwerp, Edegem, Belgium Center for Medical Genetics, Universiteit Gent, Ghent, Belgium 3 Center for Medical Genetics, Université Libre de Bruxelles, Brussels, Belgium 4 Center for Medical Genetics, Université Catholique de Louvain, Louvain, Belgium 5 Center for Medical Genetics, Université de Liège, Liège, Belgium 6 Center for Medical Genetics, Vrije Universiteit Brussel, Brussels, Belgium 7 Center for Medical Genetics, Katholieke Universiteit Leuven, Leuven, Belgium 8 Center for Medical Genetics, IPG, Charleroi Gosselies, Charleroi Gosselies, Belgium 9 Center for Medical Genetics, Universiteit Antwerpen, Antwerp, Belgium 10 University Hospital Antwerp, Edegem, Belgium 2

OBJECTIVES: In Belgium, approximately 6% of the pregnant population undergoes an invasive procedure. As of 2013, samples for invasive prenatal diagnosis are analysed by Chromosomal Microarray Analysis (CMA). Despite the existence of publicly available copy number variant (CNV) databases, interpretation of prenatal CNVs remains difficult given the often limited phenotypic information. Concomitantly with the start of CMA in the prenatal setting, a Belgian national Ad Hoc Committee was established to cope with ambiguous situations and to reach consensus based on literature and previous experiences with similar variants. Moreover, Belgian genetic centers agreed on the establishment of a Belgian MicroArray PREnatal (BEMAPRE) database. METHODS: The framework of a national database was developed in consultation with the Belgian centers for Medical Genetics. All prenatal cases in which a pathogenic CNV or a unclassified variant (UV ) > 400 kb was detected, will be imported. Next, phenotypic data will be added from immediately after delivery. Moreover, children will be reassessed at the age of 2 to 3 years. Based on genotypic and phenotypic data from hundreds of prenatal cases, we will be able to perform meta-analysis. RESULTS: Our reporting policy is largely determined by the classification of the CNVs in 3 categories, i.e. pathogenic, benign and UV. Benign CNVs and non-actionable incidental findings are reported normal after consulting the Ad Hoc Committee. If UVs have intragenic deletions/duplications in a known gene; are mentioned in literature and/or databases; consist of deletions/duplications covering more than 18 genes or comprise an X-linked gene in a XY fetus, the likeliness of pathogenicity is evaluated. In case of strong arguments for pathogenicity, parents are tested. They are reported if parental phenotype is potentially divergent or if de novo. Known pathogenic variants, risk factors with high penetrance or ultrasound anomalies and actionable incidental © 2015 John Wiley & Sons, Ltd.


findings are reported to the parents. Since the implementation of CMA in Belgium, 7875 arrays were performed. 252 (3.20 %) were reported as having a pathogenic CNV. 8.66% (682 samples) of all arrays revealed monosomy/trisomy occurrence. Data on the relationship between indication for invasive testing, prenatal phenotype and genotype will be provided. CONCLUSIONS: The development of the BEMAPRE database which includes CNV data on all prenatal invasive tests performed in Belgium is of scientific, clinical and societal importance. It will allow easy communication about ambiguous cases among the Belgian genetic centers, but at the same time, will be made available to other scientists with questions about difficult-to-interpret prenatal CNVs.

P1-12 Prenatal diagnosis of a de novo, interstitial duplication of 10q24.32, associated with split hand and foot malformation (SHFM3) by array comparative genomic hybridization Piotr Wegrzyn, Marta Smyk, Marta Kedzior, Ewa Obersztyn, Beata Nowakowska 1st Department of Obstetrics and Gynecology of the Medical University of Warsaw, Warsaw, Poland OBJECTIVES: Split hand and foot malformation (SHFM) is a condition characterized by hypoplasia or aplasia of central digital rays, clefts of hands and feet and variable fusion of remaining digits, and occurs in 1:20,000 newborns. In 2004 Roscioli et al. characterized a large autosomal dominant pedigree segregating split-hand/foot linked to chromosome 10q24. Since then only three reports appeared describing patients with duplication of this region and SHFM phenotype. Herein we present for the first time prenatal cases of a de novo interstitial duplication of 10q24.32 detected by array CGH in a fetus with SHFM. METHODS: A 33-year-old woman underwent amniocentesis at 16 + 2 weeks of gestation, due to the presence of fetal hands and foods malformations, detected on ultrasound at 12 + 4 wks. Chromosome analysis was performed on cultured amniotic cells at approximately 350 band resolution. Array CGH was performed using commercially available array (CytoSure, ISCA 8x60K v2.0, Oxford Gene Technology, Oxfordshire, UK). Confirmatory FISH analyses were performed in cultured amniotic cells and peripheral blood lymphocytes from parents, using BAC clone (RP11-264H19) specific for the 10q24.32 region. RESULTS: Conventional G-band chromosome analysis revealed normal karyotype, whereas the array CGH detected ~450 kb duplication at chromosome 10q24. In the duplicated region 3 genes are mapped; BTRC, POLL and FBXW4. FISH analysis confirmed array result and revealed the duplication occurred de novo. CONCLUSIONS: The detailed second trimester ultrasound scan can detect major fetal malformations and is offered for routine prenatal care. With the advent of high-resolution ultrasound numerous fetal malformations have become feasible to diagnose at “nuchal scan” performed between 11–14 wks. In majority of these cases fetal karyotyping is advised. However the standard G-band chromosome analysis is not always sufficient to determine the aberration and its gene content. This is particularly true in rare congenital anomalies. Array-CGH allowed a precise chromosomal diagnosis, confirming suitability of this analysis Prenatal Diagnosis 2015, 35, 27–109

as a first tier approach to clarify molecular basis of fetal malformations. This work was supported by Ministry of Science and Higher Education grant (3942/E-215/S/2014 to BAN). P1-13 Prenatal SNP microarray analysis in cases with minor karyotype aberration Hiroyasu Ohashi1, Masayoshi Takeda2, Ritsuko K. Pooh3 1

Ritz Medical Co, Ltd., Osaka, Japan Individual, Ikoma, Japan 3 CRIFM Clinical Research Institute of Fetal Medicine PMC, Osaka, Japan OBJECTIVES: Recently SNP microarray has often been utilized after karyotype analysis in a field of prenatal diagnosis. SNP microarray can compensate in this study, SNP microarray analysis has been utilized for further investigation in women whose fetus had a high probability of having chromosome structural abnormalities with unknown origin. METHODS: Between January 2014 and February 2015, 2849 prenatal samples were analyzed in our laboratory. As a routine examination, QF-PCR using ANEUFAST (Genomed AG)and karyotyping using Msearch &AutoCapt / Ikaros (Carl Zeiss & MetaSystems) were performed and as an additional examination for identification of aberration origin, FISH was performed with SNRPN and ToTelVysion (Vysis/ Abbott) probes. In 4 cases with marker chromosome, de novo inversion and de novo balanced translocation, SNP microarray was performed with CytoScan HD or 750 k (Affymetrix). RESULTS: Case1: Karyotype was 46,XX,inv(8)(p11.2q13)dn and SNP array result was normal. Case2: Karyotype was 46,XY,t(2;7)(p13;q11.23) dn and SNP array result was 5.4 Mb deletion of 2p13.3p12. Case3: Karyotype was 47,XX,+mar dn[20] and SNP array result was normal. This marker chromosome was derived from chromosome 15 and negative for the SNRPN by FISH. UPD was not found in chromosome 15 by SNP array and confirmed by methylation analysis. Case4: Karyotype was 47,XY,+mar dn[42]/46,XY[18] and SNP array result was normal. This marker chromosome was unidentified by FISH. UPD was not found in any chromosome. CONCLUSIONS: SNP array is useful for the cases marker chromosome (sSMC) and de-novo balanced translocation. In some sSMC cases, SNP array cannot detect genomic gain. However, in other words, it is that clinically significant genes are not contained in the sSMC and it is indicated that do not affect phenotypes if UPD is not found any chromosome. 2

P1-14 Improved identification for 5p deletion syndrome and partial trisomy 11q presented in a fetus by SNP array Shanshan Shi, Weijing Li Fetal Medicine Center, The First Affiliated Hospital of Jinan University, Guangzhou, China OBJECTIVES: Employing single nucleotide polymorphism array (SNP array) to identify unknown abnormality of chromosome 5 in a fetus and accurately locate the breakpoints. METHODS: A pregnant woman with a fetus of multiple malformations underwent amniocentesis for G-banded karyotyping. And then SNP array was used to identify unknown abnormality of © 2015 John Wiley & Sons, Ltd.


chromosome 5. Furthermore, G-banded karyotyping was performed on the parental peripheral blood specimens to ascertain the origin of chromosome abnormalities and then fluorescence in situ hybridization (FISH) was also utilized to confirm the results. RESULTS: Karyotype of amniocyte showed 46,XY,der(5)(?::p15 → qter). And SNP array revealed a 13.907 Mb deletion at 5p15.33p15.2(113,576-14,020,561), overlapping the region of 5p deletion syndrome (Cri-du-chat syndrome), and a 18.254 Mb duplication at 11q23.3q25 (116,684,627-134,938,470), overlapping no known syndrome. Karyotype of the parents showed a normal 46,XX in mother and 46,XY,t(5;11)(p15;q23) in father. Three-color metaphase FISH analysis on paternal peripheral blood specimens also confirmed the paternal karyotyping result. CONCLUSIONS: SNP array could uncover chromosome abnormality unidentified by G-banded karyotyping and accurately locate the genomic breakpoints, facilitating the mapping of pathogenic critical regions and the identification of candidate genes, also accumulating research data for genotype-phenotype study.

P1–14 Image.

P1-15 Withdrawn. P1-16 Does increase in genomic microarray resolution result in increased diagnostic yield?

clinical significance (VOUS). Therefore, the aim of this study was to investigate whether an increase in genomic microarray resolution result in an increased diagnostic yield. METHODS: In our database we started the analysis of the microarray results from 1,404 children with unexplained develop-mental delay/ intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). The patients were analyzed using whole-genome array com-parative genomic hybridization (a-CGH) or single nucleotide polymorphism (SNP) based microarray with different resolutions. We used the platforms Agilent 44 K, 60 K and 180 K (Agilent Technologies/Oxford GeneTechnology), and CytoSNP-850 K BeadChip (Illumina Inc.), according to the recommendations of the manufacturers. Data were analyzed with the Genomic Workbench (Agilent) and with the BlueFuse Multi-software (BlueGnome). CNVs detected on different platforms were compared to those found in databases such as DECIPHER and DGV, reflecting the current interpretation of classification and interpretation of CNVs, according to The International Collaboration for Clinical Genomics. RESULTS: Of the 1,404 cases analyzed by CGH and oligo-SNP arrays, 910 cases (65.2%) revealed only common benign CNVs, 364 cases (25.9%) CNVs explain the phenotype and 118 cases (8.9%) had copy number variants of unclear significance (VOUS). Table 1 summarizes the diagnostic yield of this study and it shows the distribution of patients across different platforms used. CONCLUSIONS: In our experience with postnatal diagnosis, we observed an increased frequency of alterations with increase in resolution. Nonetheless, this increase was due mainly to higher detection of VOUS, without a corresponding increase in the detection of variants known to be pathogenic. The next step of our study is to analyze the microarray results from prenatal diagnosis, but based on our knowledge the interpretation of CMA for prenatal diagnosis is complicated both by limited phenotypic information and by the potential of driving immediate and irreversible decisions. Interpreting these variants of unknown significance has become a bottleneck and we question the benefits obtained by higher resolution arrays not only in postnatal but mainly in prenatal diagnosis. Thus, we thought that customized arrays that are designed to target regions associated with deletion/duplication syndromes and focus on CNVs of known pathogenicity seems to be a better strategy on prenatal testing instead of interrogating the entire genome. P1–16 Table.

Darine Villela, Silvia Costa, Ana Krepischi, Carla Rosenberg University of São Paulo, São Paulo, São Paulo, Brazil OBJECTIVES: Chromosomal microarray analysis (CMA) used to assess DNA copy number throughout the human genome is a powerful clinical diagnostic tool. However, implementation of this method for prenatal testing continues to be extensively debated and no international consensus has been reached. Although the primary advantage of CMA over the conventional karyotype is the higher resolution, which yields more genetic information and allows identifying clinically important copy number variations (CNVs) not detectable at microscope level, this method also raises the detection of variants of uncertain Prenatal Diagnosis 2015, 35, 27–109

P1-17 Prenatal Chromosomal Microarray Analysis (CMA) in routine clinical practice Ronald Wapner1, Julia Zachary2, Rebecca Clifton2 1

Columbia University, New York, New York, United States George Washington University, Washington, DC, United States


© 2015 John Wiley & Sons, Ltd.


P1–17 Table. Pathogenic CNV

Variant of uncertain significance (VOUS)

Ultrasound anomaly (N=608)



AMA (N=1477)



Positive aneuploidy screen (N=659)



Family history (N=140)



Parental choice (N=264)



Other (N=350)



Primary Indication

OBJECTIVES: Initial studies have shown that pathogenic copy number variants (CNVs) occur in over 6% of euploid pregnancies with structural anomalies and in over 1.2% of structurally normal euploid pregnancies. This has led to CMA transitioning to clinical practice and being performed at multiple labs. We now report the use of CMA in routine clinical care. METHODS: From January 2013 through November 2014, ten prenatal diagnostic centers offering CMA as part of routine prenatal diagnosis logged all diagnostic procedures in which CMA was performed. The classification of copy number variants (CNVs) was at the discretion of the clinical laboratory performing the array and results were reported to the patient per local counseling policies. RESULTS: 3,669 arrays were performed, of which 2,023 (55%) samples were villi and 1641 (45%) amniotic fluid (5 on fetal blood or tissue). Results for the 3,498 karyotypically normal fetuses are seen in the Table below, tabulated by primary indication for procedure. A total of 187 copy number variants were identified. CONCLUSIONS: In clinical practice pathogenic CNVs occur in 1.38% of structurally normal cases and in 4.11% of those with a structural anomaly; a rate similar to that in initial studies. In structurally normal cases VOUS results occurred in 1.73% of cases but varied significantly (0.19% - 5.52%, p < .0001) by center; probably related to differences in laboratory calling policies.

P1-18 Withdrawn. P1-19 Genetic counseling of prenatal diagnosis in client of unknown additional chromosome with SNP array Masatoshi Yamaguchi1, Toshiro Ikeda2, Hiroki Kurahashi3, Hidehiko Miyake4, Hiroshi Sameshima1, Masanao Ohhashi1 1

Department of Obstetrics and Gynecology, University of Miyazaki, Miyazaki, Japan 2 Division of Medical Genetics, Kagoshima University Medical and Dental Hospital, Kagoshima, Japan 3 Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Medical University , Nagoya, Japan 4

Kyoto University hospital, Kyoto-city, Kyoto, Japan

Prenatal Diagnosis 2015, 35, 27–109

OBJECTIVES: After initiation of non-invasive prenatal diagnosis (NIPT), number of the client wishes to prenatal diagnosis is increasing. In our department, since NIPT is not introduced, number of the amniocentesis is increasing that we reported in last ISPD congress. In contrast to NIPT, any kind of the chromosomal abnormality will detected with amniocentesis. We want to report our experience of de novo addition of the chromosome 17 with amniocentesis. METHODS: Our case is 35 years old women. She was pointed out increased nuchal translucency in 12 weeks of gestation. After genetic counseling, she wished to perform amniocentesis. Two weeks later, we received the report describing addition of unknown fragment to chromosome 17 (46, XY, add(17)(?;;p13.3 → q ter)). Chromosomal analysis of the parents was normal suggesting fetal chromosomal abnormality was a de novo arrangement. After informed choice, parents wished re-amniocentesis for SNP array analysis. RESULTS: SNP array indicated additional fragment was Down syndrome critical region (DSCR) of the chromosome 21. After genetic counseling, parents wished to termination of the pregnancy. CONCLUSIONS: Our experience indicated importance of SNP array in diagnosis of the prenatal chromosomal abnormality. In Japan, SNP array of the prenatal diagnosis is not common procedure. Therefore multi center communication is important for prenatal diagnosis.

P1-20 Withdrawn. P1-21 Confirmation of a maternal cryptal balanced translocation through analysis of a fetus using microarray Jianzhu Wu, Qun Fang, Yi Zhou, Yanmin Luo, Shaobin Lin Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: To analyze a fetus with heart defects and to assess the recurrence risk for her family. METHODS: Single nucleotide polymorphism-based arrays(SNP-Array) analysis using Affymetrix Genome-Wide Human SNP CytoHD was performed to analyze the fetus and her parents. Karyotype analysis was also carried out. RESULTS: SNP-Array has detected a 14.5 Mb duplication at 9p and a 14.7 Mb deletion at 11q. Karyotype analysis indicated that the fetus’s mother has a karyotype of 46,XX,t(9;11)(p23;q24). Therefore, the fetus has inherited a derivative chromosome 11 derived from the maternal translocation, and her karyotype was 46,XX,der(11)t(9;11)(p23;q24)mat. CONCLUSIONS: SNPArray combined with high resolution GTG banding has confirmed that the fetus has a derivative chromosome 11 derived from her mother’s balanced translocation, resulting in partial 9p trisomy and partial 11q monosomy. This couple therefore have a high recurrence risk. SNPArray is capable of detecting small chromosomal imbalance in abnormal fetuses and can pinpoint the breakpoints. It therefore has the advantage for the © 2015 John Wiley & Sons, Ltd.


detection of unbalanced translocation which is difficult to detect with GTG banding, which is important for assessment the recurrence risk for cryptic balanced translocation carriers. P1-22 Single nucleotide polymorphism-based arrays analysis misdiagnosis of a mosaic tetrasomy 9p in a fetus Jianzhu Wu, Qun Fang, Yi Zhou, Yanmin Luo, Shaobin Lin Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: To analyze the reason for discordant results of karyotyping and microarray analysis in a fetus with a mosaic tetrasomy 9p. METHODS: Amniocentesis was done for an older pregnant woman whose ultrasonic result indicated fetal ventricular expansion, intrauterine growth retardation and persistent upper venous cavity. GTG banding karyotyping and single nucleotide polymorphism-based arrays(SNP-Array) analysis were performed at the same time. RESULTS: Amniotic fluid karyotype of the fetus was mosaic tetrasomy 9p: 47,XX, +psu idic(9)(q21)[23]/46,XX[27]. While SNP-Array detected a non-mosaic trisomy 9p with a 68.7 Mb duplication at 9p24.3q21.11. The results of karyotyping and microarray analysis were discordant. CONCLUSIONS: SNP-Array analyzed the data of genetic material in the form of numbers, not in the form of morphology. For chimeras containing two kinds of cell lines, when the mosaic rate was close to 50% and the average amount of genetic material of the chimeras was exactly equivalent to the amount of genetic material of a nonchimeras, microarray analysis would come to the result of a non-mosaic heteroploid. Therefore, microarray results of large segments of chromosome abnormalities should be combined with the results of GTG banding karyotyping for genetic counseling. P1-23 Comparison of the success rate of cultivation of different specimens used for karyotype analysis of fetuses suffering from alpha thalassemia Jianzhu Wu, Qun Fang, Yi Zhou, Yanmin Luo Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: To compare the success rate of cultivation when karyotype analysis were taken for fetuses who were suffering from alpha thalassemia using different specimens and to analyze the reasons. To provide the basis for choosing the most appropriate specimen. METHODS: 670 couples were the carriers of the alpha thalassaemia and the pregnant women were done fetal karyotyping due to various indications of prenatal diagnosis. Villus, amniotic fluid or umbilical cord blood was gathered for cultivation according to different gestational age. The success rate of them were compared. Normal fetus was used for control group. Nucleated red blood cell count, white blood cell count, lymphocyte count and Prenatal Diagnosis 2015, 35, 27–109

hemoglobin of alpha thalassemia fetus with different degree of anemia were compared. Reasons for failure of umbilical cord blood culture were analyzed. RESULTS: The success rate of fetal karyotyping of intermediate type was significantly lower in umbilical cord blood group than in amniotic fluid group (P < 0.05). The success rate of fetal karyotyping of severe type was significantly lower in umbilical cord blood group than in villus and amniotic fluid group(P < 0.05). White blood cell count, lymphocyte count and hemoglobin of intermediate type group were significantly lower than that of normal group(P < 0.05), there was no statistically significant difference in nucleated red blood cell count between them(P > 0.05). Nucleated red blood cell count of severe type group was significantly higher than that of normal group(P < 0.05), while white blood cell count, lymphocyte count and hemoglobin of severe type group were significantly lower than that of normal group(P < 0.05). The conversion rate of lymphocyte of intermediate type or severe type fetus was low. CONCLUSIONS: In order to improve the success rate of cultivation, if the couples are the carriers of the alpha thalassaemia and the pregnant women want to carry out fetal karyotyping, the preferred specimen will be amniotic fluid or villus, especially when fetal edema is found by ultrasound. P1-24 Prevalence of 22q11.2 microdeletion and microduplication in over 9.500 pregnancies Francesca Romana Grati1, Denise Molina Gomes2, Jose Carlos Pinto B. Ferreira3, Celine Dupont4, Viola Alesi5, Laetitia Gouas6, Nina Horelli-Kuitunen7, Kwong Wai Choy8, Jose Antonio Martínez-Conejero9, Alberto Gonzales de la Vega10, Krzysztof Piotrowski11, Rita Genesio12, Gloria Queipo13, Barbara Malvestiti2, Bérénice Hervé2, Brigitte Benzacken4, Antonio Novelli5, Philippe Vago6, Kirsi Piippo7, Tak Yeung Leung8, Francesca Malvestiti14, Thibault Quibel15, Anne Claude Tabet4, Giuseppe Simoni1, François Vialard2 1

TOMA Advanced Biomedical Assays, S.p.A., Busto Arsizio, Italy CHI Poissy St Germain, Département de Cytogénétique, Obstétrique et Gynécologie, Poissy, France 3 1st Department of Obstetrics and Gynecology, Medical University of Warsaw, Warsaw, Poland 4 Hôpital Robert Debré-AP-HP, GHU Nord, Unité de Cytogénétique-Département de Génétique, Paris, France 5 Laboratorio di Genetica, Ospedale Pediatrico del Bambino Gesù, Polo di Ricerca, Rome, Italy 6 CHU de Clermont Ferrand, Unit of Cytogenetics, Clermont Ferrand, Clermont Ferrand, France 7 United Medix Laboratories Ltd., Department of Genetics, Helsinki, Finland 8 Chinese University of Hong Kong, Department of Obstetrics and Gynecology, Hong Kong, China 9 Iviomics SL, Laboratory of Cytogenetics, Valence, Spain 10 CGC Genetics, Laboratory of Cytogenetics, Madrid, Spain 11 University of Medicine Pomeranian, Unit of Cytogenetics, Dept. of Pathology, Szczecin, Poland 2

© 2015 John Wiley & Sons, Ltd.



Dipartimento di Medicina Molecolare e Biotecnolgie Mediche Università di Napoli Federico II, Napoli, Italy 13 Hospital General de México Eduardo Liceaga-Facultad de Medicina UNAM and NanoLab "Next generation diagnostic," Mexico City, Mexico OBJECTIVES: 22q11.2 region is involved in DiGeorge (DGS, #188400) and 22q11.2 microduplication (#608363) syndromes. Most patients (85%) have a 3Mbp deletion/duplication; a subset of patients present with “atypical” deletions/duplications within the 3 Mb region. DGS phenotypic findings include congenital heart and palate defects, dysmorphisms, developmental delay and compromised immune system. 22q11.2 duplication. ranges from no abnormality or mild learning disabilities to severe mental retardation (MR) with multiple congenital malformations some of which are reminiscent of the DGS. Prenatal screening for DGS currently is done by targetted FISH analysis of fetal cells in cases with congenital heart disease and/or palate anomalies at US. A recent study on DGS neonates showed that neonatal hypocalcemia and seizures are significantly associated with more severe MR. The early recognition of 22q11.2 microdeletion by neonatal or prenatal screenings could prompt meaningful anticipatory care and help improve long-term outcome in DGS. The value of prenatal screening for copy number variations in general prenatal population by microarray is still subject to debate. However, some propose, in low-risk/general population pregnancies, in addition to karyotype, Prenatal BACs-onBeadsTM as a transitional test investigating 9 criticalregions associated with dominant well characterized microdeletion syndromes, including 22q11.2. The 22q11 deletion and duplication estimated prevalences are 1/30006000. METHODS: 9327 prenatal samples analyzed by karyotype + PNBoBs. All indications for invasive prenatal diagnosis were included. RESULTS: In our general cohort the prevalence of DGS is 0.34% (95%CI:1/458-1/186) and of microduplication is 0.16% (95%CI:1/1195-1/324); in the low risk population it is 1/992 (95%CI: 1/ 2719-1/362) and 1/850 (95%CI: 1/2174-1/333), respectively. CONCLUSIONS: A prevalence higher than previously estimated by pediatric studies but in concordance with previous prenatal data was found for 22q11.2 microdeletion; duplication shows a superimposable prevalence. This data have important implications for the evolution of cffDNA screening assay targets and justify the proposed prenatal screening since early recognition of 22q11.2 deletions/duplications improves its prognosis.

OBJECTIVES: BACs-on-BeadsTM (BoBsTM) is a new emerging technology based on a modification of comparative genomic hybridisation, and can be used to detect rapidly DNA copy number gains and losses. Unlike rapid FISH or QF-PCR, PNBoBs also has the ability to detect 9 common microdeletion syndromes aside of the common trisomies, which may be easily missed by prenatal sonography. We describe a retrospective application of Prenatal BoBs (CE-IVD) to detect the most frequent microdeletion syndromes as well as the trisomy 13, 18 and 21 and sex chromosome aneuploidies. Herein we present a comparison of the diagnostic performance of the PNBoBsTM and QF-PCR assays for prenatal detection of chromosomal abnormalities. METHODS: Retrospective monocentric study of 2768 prenatal samples (CVS and AF) analyzed by karyotype + PNBoBs. All indications for invasive prenatal diagnosis were included. Based on QF-PCR performances described by previous studies the theoretical number of chromosome abnormalities expected to be detected by QF-PCR was calculated based on the karyotype results. RESULTS: For the full cohort the detection rate for chromosome abnormalities was reported as 2.96% (95% CI: 2.39%-3.66%) for karyotype, 2.67% (95%CI:2.13%-3.34%) for PNBoBs and 2.02% for QF-PCR (95%CI:1.56%-2.62%); for low risk population samples only, this equated to 2.41% (95%CI:1.87%3.09%), 1.93% (95%CI:1.46%-2.54%) and 1.52% (95%CI:1.11%2.09%), respectively. PNBoBs generally showed a higher diagnostic yield compared to QF-PCR in each prenatal indication due to the additional detection ability of microdeletions/ duplications (7 cases in low risk prenatal population samples, 8 cases in high risk prenatal population samples). In addition, mosaics for chromosomes other than 13,18,21,X/Y and targetted by the assay for microdeletions can be detected by PNBoBs (3 cases in low risk prenatal population). Two cases (1 triploid case in a pregnancy with high risk US abnormality and 1 mosaic T21 < 30% in a pregnancy with advanced maternal age) detectable by QF-PCR were not detected by PNBoBs. CONCLUSIONS: Although PNBoBsTM may not have the breadth and scope to replace chromosomal microarrays in the prenatal setting, particularly when a fetal anomaly has been detected by US analysis, it is a well suited alternative for QF-PCR because PNBoBsTM is superior in terms of sensitivity.

P1-26 Investigation for fetal lateral ventriculomegaly chromosomal microarray analysis abnormally


Zhiqiang Zhang1, Xiaodan Chen2, Qun Fang3, Jianzhu Wu4, Shaobin Lin4 1

P1-25 Prenatal BACs-on-BeadsTM versus QF-PCR for prenatal detection of chromosomal abnormalities Francesca Romana Grati, Barbara Malvestiti, Francesca Malvestiti, Simona De Toffol, Livia Marcato, Francesca Dulcetti, Anna Maria Ruggeri, Valentina Zanatta, Beatrice Grimi, Elena Niada, Dario Poltronieri, Lorenza Martinoni, Federico Maggi, Giuseppe Simoni TOMA Advanced Biomedical Assays, S.P.A., Busto Arsizio, Italy Prenatal Diagnosis 2015, 35, 27–109

The First Affiliated Hospital, Sun Yat-Sen University, GuangZhou, China 2 Department of Medical Genetics, Zhongshan Medical College, Sun Yat-Sen University, GuangZhou, Guangdong, China 3 Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-Sen University, GuangZhou, Guangdong, China OBJECTIVES: To investigate the relationship between fetal lateral ventriculomegaly and chromosomal microarray analysis (CMA) abnormally. METHODS: Chose 50 cases who were © 2015 John Wiley & Sons, Ltd.


detected by ultrasound for fetal lateral ventriculomegaly but karyotype analysis were normal. These cases were collected from Prenatal Diagnosis Center of the First Affiliated Hospital of Sun Yat-sen university between April 2013 to October 2014. The width of fetal lateral ventricularatrium larger than 10 mm was considered as the criteria of ventriculomegaly. These fetuses were divided into two groups:the group of mild ventri-culomegaly (MVM) had 44 cases, in which the lateral ventricularatrium was 10 ~ 15 mm. The group of severe ventriculomegaly (SVM) had 6 cases, the lateral ventricularatrium was ≥15 mm. All fetuses were divided into isolated and nonisolated subgroups according to whether they were associated with other anomalies.The isolated group had 21 cases,the non-isolated group had 29 cases. RESULTS: Of 44 cases in MVM group, 9 (20.9% )cases were CMA abnormally,while 4 cases (66.7%) were CMA abnormally in 6 SMV fetuses, P > 0.05. CMA abnormally were found in 2 of 21(9.5%) in isolated ventriculmegaly group,while 11 of 29(37.9%) in nonisolated ventriculmegaly group, P < 0.05. CONCLUSIONS: Chromosome microdeletion and microduplication are the most common abnormal in the cases of fetal lateral ventriculomegaly. When ventriculomegaly is associated with other anomalies,the incidence of CMA abnormally is much higher. Therefore,prenatal diagnosis is necessary for the fetuses with lateral ventriculomegaly.

P1-27 An improved method to culture amniotic fluid cells in the middle-late trimester of pregnancy Zhiqiang Zhang1, Qun Fang1, Jianzhu Wu1, Shaobin Lin1, Xiaodan Chen2 1

The First Affiliated Hospital, Sun Yat-Zen GuangZhou, Guangdong, China 2 Zhongshan Medical College, Sun Yat-Zen Guangdong, China

University, University,

OBJECTIVES: To improve the success rate of amniotic fluid cells culture in the middle-late trimester of pregnancy. METHODS: A total of 340 cases of amniotic fluid samples were collected from the gravidas at 24 to 36 + 5 weeks of gestation. They were obtained from Prenatal Diagnosis Center of The First Affiliated Hospital of Sun Yat-sen University between January 2010 to December 2014. We adjusted the method of inoculation according to the amount of precipitation after centrifugation and kept the cell concentration at 1 ~ 2 * 108 cells per liter. We also adjusted the time for exchange culture medium according to the growth status of cells. RESULTS: 131(38.5%) cases of amniotic fluid samples were collected from the gravidas at 24 ~ 24+6 weeks of gestation, 79(23.2%) cases at 25 ~ 25+6 weeks of gestation, 54(15.9%) cases at 26 ~ 26+6 weeks of gestation, 34(10.0%) cases at 27 ~ 27+6 weeks of gestation, 16(4.7%) cases at 28 ~ 28+6 weeks of gestation, 8(2.4%) cases at 29 ~ 29+6 weeks of gestation, 5 (1.5%) cases at 30 ~ 30+6 weeks of gestation, 3(0.9%) cases at 31 ~ 31+6 weeks of gestation, 6 (1.8%) cases at 32 ~ 32+6 weeks of gestation, 3(0.9%) cases at 34 ~ 34+6 weeks of gestation, 1(0.3%) cases at 36 ~ 36+5 weeks Prenatal Diagnosis 2015, 35, 27–109

of gestation. A cytogenetic karyotypes analysis was performed in all samples. The success rate was improved to 100%. We found 5 cases of trisomy 18 and 2 cases of trisomy 21. The detection rate of abnormal karyotype was 2.0%. We also found 18 cases of chromosome polymorphism. The detection rate of chromosome polymorphism was 5.3%. CONCLUSIONS: We get more cells island and chromosome karyotypes by the improved method, which can meet the clinical demand for prenatal diagnosis of gravidas in the middle-late trimester of pregnancy.

P1-28 Rates of all type of fetal karyotype defects for each year of maternal age in women presenting for NIPT counseling Francesca Romana Grati1, Komal Bajaj2, Francesca Malvestiti1, Elisa Gaetani1, Cristina Agrati1, Beatrice Grimi1, Lorenza Martinoni1, Federico Maggi1, Susan Gross2, Giuseppe Simoni1, Jose Carlos Pinto B. Ferreira3 1

TOMA Advanced Biomedical Assays, S.p.A., Busto Arsizio, Italy Albert Einstein College of Medicine, Bronx, NY, United States 3 1st Department of Obstetrics and Gynecology, Medical University of Warsaw, Warsaw, Poland 2

OBJECTIVES: Screening for fetal aneuploidy have focused on autosomal trisomies (13, 18, 21) whose incidence is maternal age (MA) dependent. The MA-related risks of fetal aneuploidy are based on seminal work by Hook and others. Their risk predictions were based on prenatal data for older women and liveborn data for younger women and were limited to the most frequent defects. Our aims is to re-assess the basal incidence of each fetal microscopic chromosome aberration for each year of MA, at two gestation periods, based on actual prenatal data. METHODS: Fetal karyotype data from women of ages ranging from 15 to 50, undergoing prenatal diagnosis, without any prior re-assessment of their age attributable risk, were analysed retrospectively. The frequency of each karyotype anomaly, with respective 95% CI, was predicted for each gestational age period analysed (10 to 15 weeks and 15 to 20 weeks) and for each year of MA. Logistic regression smoothing or simple ratios were calculated, respectively, for lesions whose frequency was found to be, or not, associated with MA. RESULTS: Data from 129,263 women collected from 1994 to 2012 was analysed. The frequency of each of the most common aneuploidies confirmed prior estimates for older women but was lower for younger ones. Estimates of each rare type of karyotype anomaly, mostly found to be MA independent, were also calculated. CONCLUSIONS: The proportion of the genomic defects that are targeted by screening methods varies substantially with MA. The combination of the frequency estimates of karyotype defects not targeted by such screening, with the frequency of submicroscopic defects, estimated from prior microarray studies, reaches values that many patients, considered as low risk by screening protocols, would regard has non-tranquilizing. Proper prenatal counselling must make sure that patients understand the limitations of the non-invasive screening strategies in comparison with invasive based diagnostic methods. © 2015 John Wiley & Sons, Ltd.


P1-29 Application of fluorescence in situ hybridization combined with karyotyping on detection of the causes for spontaneous abortion Jianzhu Wu, Qun Fang, Yi Zhou, Yanmin Luo, Shaobin Lin Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: To investigate the effect of fluorescence in situ hybridization (FISH) combined with karyotyping in increasing the detection rate of abnormal karyotypes from spontaneous abortion specimens. METHODS: 216 cases of spontaneous abortion specimens were performed karyotype analysis and FISH detection at the same time, then advantages and disadvantages of the two methods were compared. RESULTS: The cultivation success rate of abortion specimens was 85.2% (184/216), the detection rate of abnormal karyotypes was 46.7% (86/184), among which 56 cases were autosomal aneuploidies, 13 sex chromosome aneuploidies, 12 triploids, two tetraploids and three chromosome structural aberrations. The mainly abnormal karyotypes were trisomy 16, trisomy 22, monosomy X, trisomy 21 and triploid. 216 cases of abortion specimens obtained FISH test results. The success rate was 100.0%. The detection rate of abnormal karyotypes was 42.1% (91/216), among which 59 cases were autosomal aneuploidies, 13 sex chromosome aneuploidies, 13 triploids, four tetraploids and two trisomic chimeras. Among the 32 samples which failed by conventional method, FISH found seven abnormal samples. In one specimen of 47,XY,+16, FISH showed mosaicism of trisomy 16 with XY and normal karyotype with XX. In one specimen of 47,XY,+18, FISH showed mosaicism of trisomy 18 with XY and normal karyotype with XY. In one specimen of trisomy 2 and one specimen of inversion 9, FISH showed normal results. In two specimens of trisomies of Robertsonian translcation type, FISH showed trisomies. CONCLUSIONS: FISH can quickly and easily detect number abnormalities of aneuploid and polyploid in spontaneous abortion specimens, combined with karyotype analysis can improve the detection rate of abnormal karyotypes in spontaneous abortion specimens. FISH has a smaller detection range of abnormal karyotypes than gene array chip, so it will be replaced by gene array chip on detection of the causes for spontaneous abortion.

P1-30 Research on prenatal diagnosis of villi chimera Jianzhu Wu, Qun Fang, Yi Zhou, Yanmin Luo Fetal Medicine Center, Department of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: To explore the formation mechanisms of prenatal diagnosis of villi chimeras and the processing methods. METHODS: In 560 cases of early pregnancy villus karyotype analysis, 27 fetuses with villi chimeras were performed amniotic fluid karyotype analysis which was combined with ultrasound results to assess the risk of the fetuses. RESULTS: In 27 cases Prenatal Diagnosis 2015, 35, 27–109

of villi chimeras, 16 cases of bisexual chimeras, one case of 45, X/46,XX chimera and one case of trisomic chimera were proved to be having maternal cells contamination by the results of amniotic fluid karyotype and ultrasound. The villi karyotype of two cases of bisexual chimeras, one case of 45,X/46,XX chimera, one case of ring chromosome chimera and four cases of trisomic chimera were accordant with amniotic fluid karyotype. One case of trisomic chimera had normal amniotic fluid karyotype and ultrasound result, but it was proved to be confined placental mosaicism. CONCLUSIONS: Villi chimeras are mostly caused by maternal cells contamination, partialy accordant with fetal karyotype and fewly having confined placental mosaicism in prenatal diagnosis. Amniotic fluid karyotype combined with ultrasound results can confirm that villi chimeras are derived from maternal cells contamination, confined placental mosaicism or accordant with fetal karyotype, which provides the basis for prenatal counseling. P1-31 Differences in the background factors and awareness of noninvasive prenatal testing between pregnancy achieved with assisted reproductive technology and spontaneous pregnancy Akimune Fukushima1, Kayono Yamamoto1, Yumiko Kobayashi1, Tomonobu Kanasugi2, Fumiharu Miura2 1

Department of Clinical Genetics, School of Medicine, Iwate Medical University, Morioka, Iwate, Japan 2 Department of Obstetrics and Gynecology, Iwate Medical University, Morioka, Iwate, Japan OBJECTIVES: In Japan, the proportion of pregnancies at ≥35 years is increasing annually due to changes in lifestyle and advances in assisted reproductive technology (ART). Accordingly, the number of ≥35-year-old individuals receiving genetic counseling following fertility treatment is also tending to increase, in particular cases in which the individual wishes to undergo noninvasive prenatal testing (NIPT) after achieving pregnancy with ART. We herein investigated the hypothesis that the background behind undergoing NIPT and the awareness thereof differ between cases of pregnancy achieved with ART and those of spontaneous pregnancy. METHODS: During the one-year period between April 1, 2013 and March 31, 2014, 113 women underwent NIPT at our department. After excluding 14 women receiving standard fertility treatment, 103 women were divided into the ART group (Group A; n = 25) and the spontaneous pregnancy group (Group N; n = 78) for comparison of items including background factors such as maternal age and gestational age at time of NIPT, and the results of a questionnaire survey used in clinical research conducted by the NIPT Consortium. RESULTS: The mean age at NIPT was higher in Group A (39.8 years) compared to Group N (38.5 years); the mean gestational age at NIPT was 12 weeks in both groups. Additionally, compared to Group N, Group A had a greater proportion of women from neighboring prefectures that do not conduct NIPT or from the Greater Tokyo Area. In the comparison of household income, a bimodal distribution was observed in Group A with peaks at 5–7 million yen (40.0%) and ≥10 million yen (28.0%), while in Group N the mode was 5–7 million yen (34.6%). As for © 2015 John Wiley & Sons, Ltd.


questionnaire survey results (based on mean scores on a five-point scale), Group A scored higher than Group N for all data as follows: Items regarding knowledge of NIPT, 4.90 vs. 4.79; items regarding recognition of NIPT, 4.39 vs. 4.27; items regarding understanding of explanations on NIPT, 4.77 vs. 4.75; and items regarding requests for genetic counseling, 4.72 vs. 4.64. CONCLUSIONS: Compared to Group N, Group A had a greater awareness of NIPT and higher levels of understanding and expectations regarding genetic counseling. P1-32 Clinical and cytogenetic study of molar tissues from patients with recurrent molar pregnancy Monica Aguinaga, Carolina Galaz, Guadalupe Razo, Elsa Moreno Instituto Nacional de Perinatologia, Mexico City, Mexico OBJECTIVES: 1.-Know the frequency, family history, age and complications of patients with recurrent molar pregnancy (RMP) in a hospital in Mexico City. 2.-Determine if molar tissues from patients with RMP are diploid or triploid. 3.-Correlate cytogenetic FISH analysis with the histopathological results from molar pregnancies. METHODS: We performed a retrospective study to identify patients with recurrent molar pregnancy during the period of 1998 to 2013 treated at the Instituto Nacional de Perinatología, Mexico City. We included patients with two or more molar pregnancies with at least one mole analyzed by the Pathology Department. We performed FISH analysis for chromosomes X,Y and 18 (Cytocell) in molar tissues embedded in paraffin. RESULTS: During a period of 16 years, 363 patients were diagnosed with a molar pregnancy. 29 (7.9%) cases presented two or more molar pregnancies. Two patients were excluded because they had a

normal pregnancy. We identified 5 familiar cases, 3 were included in the analysis. Of the 27 patients, we obtained 14 molar tissues from 12 patients. Patient´s age at their first molar pregnancy was between 16 and 29 years old, 6 (50%) of the patients had at least one first pregnancy loss different from the RMP. The number of pregnancies was between 2 and 7 with an average of 4.5. The 12 patients had a total of 38 molar pregnancies and 9 abortions. Four (11%) molar pregnancies progressed to persistent trophoblastic disease. Pathology classified the tissues as: 8 (57%) mole, 4 (28.5%) as complete mole, 1 (7%) partial mole and 1 (7%) invasive mole. FISH study showed diploidy in all 14 tissues analyzed, 10 (71.5%) moles were XX and 4 (28.5%) were XY. CONCLUSIONS: Our study showed that the frequency of a second molar pregnancy was 7.9% which is higher than other studies. We also found more XY tissues in moles. All tissues analyzed were diploid which confirms that RMP is a diploid condition. This study allowed a correct classification of molar tissues. The determination of parental molar origin is necessary to know if molar tissues are biparental. P1-33 Women’s experience of genetic counseling for non-invasive prenatal genetic screening Megan Allyse1, Marsha Michie1, Stephanie Alessi2, Melissa Constantine3, Cherie Rao4 1

University of California San Francisco, San Francisco, California, United States 2 Stanford University, Stanford, California, United States 3 University of Minnesota, Minneapolis, Minnesota, United States 4 Kaiser Permanente, San Diego, California, United States OBJECTIVES: To understand the effect of genetic counseling for non-invasive prenatal genetic screening (NIPGS) on

P1–32 Table. Age at first mole

No. Moles

No. Abortions














Familiar case

Patient karyotype

Partner karyotype

No. of tissues









Family G




Complete Mole












Pathology report

FISH result













Family M
























Partial Mole
















Family A














Invasive Mole






Family E
















WS: without study, D: Diploid.

Prenatal Diagnosis 2015, 35, 27–109

© 2015 John Wiley & Sons, Ltd.


women’ decision-making ability, emotional status, self-reported understanding of NIPS, and objective knowledge of NIPGS and the conditions for which it screens. METHODS: Survey methods and medical record review were used for data collection with pregnant women eligible for NIPGS referred to one of two genetic counseling practices. Women were asked to complete surveys post GC, and women who accepted the offer of NIPGS were also surveyed post return of NIPGS results. RESULTS: Survey respondents averaged 37.0 years old and 11.5 weeks gestation. Nearly all women found genetic counseling extremely or very helpful in deciding whether to have NIPGS. All agreed strongly or somewhat that they understood enough after counseling to make a decision. Women reported that genetic counseling made things more clear than confusing and made them feel more calm than anxious. Over half of women reported no or “a little” prior understanding of the conditions for which NIPGS screens. Women who reported no or little prior knowledge about NIPGS more often reported that counseling helped their understanding of NIPGS “a lot,” compared to women who reported a lot of prior knowledge. Women who reported no prior understanding of the conditions for which NIPGS screens found that counseling helped their understanding, compared to women who reported a little, some, and a lot of prior understanding. Women’s objective knowledge of NIPS after counseling was high overall. Women who selfreported some prior understanding had slightly higher objective knowledge scores than women who reported no or a little understanding (p ≤ 0.05); there was no difference in objective knowledge across self-reported understanding of NIPGS after counseling or across self-reported prior knowledge CONCLUSIONS: Women report that genetic counseling helps with decision-making about NIPS, improves their emotional experience of screening, and makes them feel more knowledgeable. Genetic counseling also improves women’s objective knowledge of NIPGS and the conditions tested. Women who believe they understand NIPGS without genetic counseling scored lower on objective knowledge measures and were more likely to report that genetic counseling was less helpful.

1988–2013 at a single tertiary care center were identified using a comprehensive ultrasound database. We recorded patient demographics, relevant obstetric, fetal and neonatal data, indication and success of PUBS and any complications. Data were analyzed using SAS, 9.3. RESULTS: 455 PUBS were performed on 208 pregnant women, 97.8% of which were successful. The average gestational age at the time of PUBS was 26.5 weeks (SD 5.6 weeks). Indications were available for 441: 245 (55.6%) for isoimmunization, 77 (17.5%) for nonimmune hydrops, 98 (22.2%) chromosomal diagnosis and 21 (4.8%) for other indications. Isoimmunization was a less common indication for PUBS in 2008–2013 as compared to 1988–1992 (66.2% vs. 51.7%; p = 0.06). Amongst PUBS in the setting of hydrops, isoimmunization was much less common in the later time period (61% vs 0%, respectively; p < 0.01). The procedure complication rate was 2.5%, was stable over the study period, and most common with non-immune hydrops (2% without hydrops, 0% with immune hydrops and 6.3% with non-immune hydrops; p = 0.04). Of the 208 women with a PUBS performed, 74 had more than one PUBS performed (mean 2.2, max 18). Transfusions were performed in 233/455 procedures (51.2%). Overall, 14.8% of the pregnancies had an IUFD, which was most common in pregnancies with non-immune hydrops (8.1% without hydrops, 6.3% with immune hydrops and 23.3% with nonimmune hydrops; p < 0.01) (Table). CONCLUSIONS: PUBS has a high likelihood of success with a relatively low complication rate. PUBS performed in the setting of nonimmune hydrops is more likely to have a complication when compared to non-hydropic patients; the complications rate is highest in pregnancies with non-immune hydrops, and these pregnancies are also at a significantly higher risk of IUFD. Our findings can be used when counseling patients who are considering PUBS for diagnostic or therapeutic purposes.

P1-34 Withdrawn.

Elysia Davis1, Jun Liao2, Neeta Vora3

P1-35 Percutaneous umbilical blood sampling (PUBS): Current trends and outcomes Catherine Bigelow1, Christina Cinelli2, Sarah Little1, Carol Benson1, Mary Frates1, Louise Wilkins-Haug1 1

Brigham & Women’s Hospital, Boston, Massachusetts, United States 2 Spectrum Medical Group, S. Portland, Maine, United States OBJECTIVES: To report our experience with percutaneous umbilical cord blood sampling (PUBS), including indications and complications. METHODS: All PUBS performed between

Prenatal Diagnosis 2015, 35, 27–109

P1-36 Withdrawn. P1-37 Atypical HexA enzyme activity complicating reproductive genetic counseling and molecular testing


University of North Carolina, Chapel Hill, North Carolina, United States 2 Icahn School of Medicine at Mount Sinai, New York, New York, United States 3 University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States OBJECTIVES: Tay-Sachs and Sandhoff diseases are lethal, autosomal recessive lysosomal storage diseases caused by deleterious mutations in the HEXA and HEXB genes, respectively. HEXA encodes the alpha subunit of hexosaminidase A (HexA, α/β), and HEXB encodes the beta subunit of HexA (α/β) and hexosaminidase B (HexB, β/β). The American College of Obstetrics and Gynecology recommends screening for Tay-Sachs © 2015 John Wiley & Sons, Ltd.


P1–35 Table. Percutaneous Umbilical Cord Blood Sampling (PUBS): Patient information All Pregnancies (N = 208) Transfused at any time IUFD

68/208 (32.7%) 26/176* (14.8%)

GA delivery (wks) Mean (SD) Neonatal death

33.8 (5.1) 12/175 (6.9%)

Immune Hydrops (N = 11)

Nonimmune Hydrops (N = 48)

No Hydrops (N = 149)


9/11 (81.8%)

19/48 (39.6%)

40/149 (26.9%)

99/100 for T13 (fetal DNA fraction 10.4%). Amniocentesis confirmed disomy for chromosome 13 (100 cells examined by interphase FISH; SNP microarray normal), suggesting confined placental mosaicism for T13. The baby delivered at 32 + 1/40 by emergency caesarean section. Growth was normal for gestation. Physical examination revealed very minor hypospadias and a left hydrocoele, mild micrognathia and a systolic murmur. Echocardiogram confirmed a small patent ductus arteriosus that closed spontaneously. Cranial and abdominal ultrasound and ophthalmological examination were normal. SNP microarray on umbilical cord-derived DNA was normal. Peripheral blood demonstrated disomy for chromosome 13 by interphase FISH (200 cells). Two placental biopsies confirmed high level mosaic T13 (interphase FISH: 75/100 and 84/100 cells with T13). SNP microarray from placental biopsies was consistent with non-mosaic T13, concordant with NIPS results. The baby was diagnosed with left unicoronal craniosynostosis at 7 weeks corrected age, no syndromic features were present. Common mutations in the FGFR and TWIST genes were negative. SNP microarray analysis using DNA derived from skin and soft tissue over the left unicoronal region demonstrated no evidence for T13 mosaicism. Conclusions: CFTS is well-recognised as a screening test, with fairly poor capacity to differentiate between T13 and T18. The very high level of placental mosaicism in this case, as established by cytogenetic investigations on placental biopsy samples post-partum, produced the high risk T13 result on NIPS. NIPS utilises cellfree fetal DNA derived from maternal plasma, the origin of this DNA is placental trophoblast. Therefore the possibility of confined placental mosaicism is critical when interpreting NIPS results. The most common mechanism for confined placental mosaicism is post-zygotic rescue of a trisomic conception, usually due to a maternal meiosis I error. SNP genotyping data confirmed this mechanism; the parental origin of the trisomy was not investigated. Four post-natal samples (peripheral blood, umbilical cord, skin and soft tissue) and the amniocentesis demonstrated disomy for chromosome 13, supporting the diagnosis of confined placental mosaicism. We considered that residual T13 mosaicism in the baby may have contributed to the development of craniosynostosis, but the normal chromosome results from these five sources make that unlikely. Nevertheless, the association of craniosynostosis with both non-mosaic, and rarely, mosaic T13, mean that this remains a possibility. P2-5 Implementation of NIPT in India-first insights from a qualitative study of provider experiences Subhashini Chandrasekharan1, Shilpa Sridhar2, Dechen Lama2 1

Duke Global Health Institute, Duke University, Durham, North Carolina, United States 2 Trinity College of Arts and Sciences, Duke University, Durham, North Carolina, United States Prenatal Diagnosis 2015, 35, 27–109

OBJECTIVES: To examine the use of new non-invasive prenatal genetic screening (NIPT) within the first two years of its availability in India. METHODS: We interviewed key informants including medical geneticists and obstetricians/gynecologists in India, who were identified through literature and web searches. We analyzed interviews to identify experiences with NIPT and views about barriers to broad implementation in India. RESULTS: NIPT is available in India through at least six local distributors/laboratories, and six international companies market tests in India. Blood samples are sent to US or Chinese laboratories for testing. Physicians and medical geneticists in both public and private facilities knew several commercial tests and had ordered NIPT to variable degrees. Test costs ranged from INR 23,000 (~$350) to INR 35,000(~$550). Most informants expressed concern with high price limiting NIPT usage. Other concerns included improper use, lack of independent quality control, and false positive/negative rates associated with different tests. Lack of physician genetics education and formal genetic counseling services were considered major challenges for proper implementation. Informants acknowledged that NIPS is an important technology for delivering prenatal genetic services broadly in India, since it only requires a blood sample. However opinions differed about clinical utility in the Indian context. State and federal politics and issues surrounding India’s Pre-conception and Pre-natal Diagnostic Techniques (PCPNDT) Act which prohibits reporting fetal sex for non-medical reasons, were other barriers reported. CONCLUSIONS: NIPT use is limited largely to private-pay patients due to cost and availability only in urban hospitals. In the short-term, Indian professional societies must expediently inform clinicians on appropriate uses of NIPT. Clarification is also required on procedures surrounding the PCPNDT act such as registration of NIPT providers and distributors. In the longterm, public sector introduction will require capacity building for in–country testing, cost-effective products, and integration with other community genetic services, such as newborn screening. Establishing additional national programs for medical genetics education and genetic counseling will be essential for assuring appropriate and ethical use. In the interim, publicprivate partnerships with national medical genetics centers would allow broader access to NIPT through referral systems and national/state funded antenatal care programs. P2-6 Non-invasive prenatal diagnosis for congenital adrenal hyperplasia in a clinically accredited laboratory Suzanne Drury1, Kitty Lo2, Christopher Boustred1, Naomi Gibson1, Fiona McKay1, Sarah Mason1, Samantha Edwards3, Melissa Hill4, Rebecca Daley3, Lucy Jenkins1, Lyn Chitty4 1

NE Thames Regional Genetics Laboratory, London, United Kingdom 2 UCL Genetics Institute, London, United Kingdom 3 University College London Hospital & NE Thames Regional Genetics Laboratory, London, United Kingdom 4 University College London Hospital, NE Thames Regional Genetics Laboratory & UCL Institute for Child Health, London, United Kingdom © 2015 John Wiley & Sons, Ltd.


OBJECTIVES: Our laboratory offers definitive non-invasive prenatal diagnosis (NIPD) for a number of disorders, but currently this is restricted to paternal exclusion and de novo mutations. Introduction of an assay for congenital adrenal hyperplasia (CAH) was used as a model disorder for expanding our NIPD service for autosomal recessive disease and disorders for which NIPD is complicated by the presence of a pseudogene. METHODS: A Sureselect Custom Enrichment assay (Agilent Biosystems) targeting heterozygous SNPs flanking ~7 Mb around CYP21A2 andHLA-B, HLA-C, ZFX, ZFY and SRY was designed. Cell-free DNA (cfDNA) was prepared for enrichment using the KAPA Hyper Prep Kit for Illumina (Kapa Biosystems). DNA from parents and proband were prepared using the Sureselect enrichment protocol. Libraries were sequenced on an Illumina MiSeq using 75 bp pairedend reads. Data was aligned using BWA and variants called using Varscan. Parental haplotypes were constructed and fetal inheritance deduced. RESULTS: In unaffected family A, parental haplotypes were constructed from CVS material. CfDNA analysis from the same pregnancy had an estimated fetal fraction of 11%. The fetus had inherited paternal haplotype 1. Relative haplotype dosage analysis showed the fetus inherited maternal haplotype 1. These results were consistent with haplotype of the CVS sample. Parents from family B were consanguineous and carriers of the chimeric CYP21A2 deletion/conversion (involving exons 1–3). The affected proband was used to construct parental haplotypes. A shared haplotype, identical by descent along ~10 Mb, was found between the parents, Analysis of cfDNA suggested the fetus inherited maternal haplotype 2 and paternal haplotype 1, resulting in a heterozygous carrier of the mutation. Followup data from the pregnancy confirmed this result. CONCLUSIONS: Disorders caused by mutations in genes with known pseudogenes cannot be directly sequenced using cffDNA. A haplotyping assay using next generation sequencing as implemented here has enabled our accredited public sector laboratory to expand the clinical service in this field. We are developing this approach for disorders such as spinal muscular atrophy, whilst using direct sequencing and with estimation of fetal fraction to deliver NIPD for other autosomal recessive conditions, including the haemoglobinopathies and cystic fibrosis. P2-7 Withdrawn. P2-8 Leveraging targeted sequencing of paired homologs for noninvasive detection of fetal aneuploidies Chris Ellison, Youting Sun, Grant Hogg, Jesse Fox, Helen Tao, Erin McCarthy, Bright Sagoe, Mostafa Abdellatef, Amin Mazloom, John Tynan, Tim Burcham, Dirk van den Boom, Mathias Ehrich, Sung Kim, Taylor Jensen Sequenom Laboratories, San Diego, California, United States OBJECTIVES: Current commercial offerings for noninvasive prenatal testing (NIPT) ascertain fetal aneuploidies using direct or relative counts of mapped sequencing reads or Prenatal Diagnosis 2015, 35, 27–109

single nucleotide polymorphism frequencies. Alternatively, the ratios of homologous sequence pairs located on the chromosome of interest and an alternative autosome were predicted to reflect fetal aneuploidy through changes in relative abundance. The objective of this study was to develop and evaluate a NIPT assay which specifically enriched for a selected set of paired homolog targets to detect fetal trisomy 21 (T21) and trisomy 18 (T18). METHODS: Homolog target pairs in which one homolog was located on either chromosome 18 or 21 were identified and filtered for assay design acceptance criteria. A total of 1060 amplicons were designed, and included targets to determine fetal aneuploidy status, fetal sex, and the proportion of fetal DNA present in a sample. Circulating cell free (ccf) DNA was extracted from the plasma of pregnant female donors with a whole genome NIPT result. Each library was prepared by co-amplifying all targets together in a single reaction well. Amplified products were evaluated using massively parallel sequencing (Illumina) and deviations from expected (euploid) homolog ratios were determined based on the read depth from each homolog. RESULTS: We evaluated the performance of the developed assay in a blinded set of 480 ccfDNA samples with fetal genotypes orthogonally validated by the MaterniT21® PLUS assay. Samples were sequenced at a mean depth of only 2.3 million reads per sample with 432 assayed samples returning a result. Using MaterniT21 PLUS assay results for paired plasma aliquots from the same individuals as a reference, all 385 euploid samples, all 31 T21 samples, and 14 of 16 T18 samples were correctly classified with no false positive results observed. CONCLUSIONS: This study introduces a novel NIPT aneuploidy detection approach using targeted sequencing of paired homolog motifs and establishes proof of concept for a low-cost, highly scalable method for the identification of selected fetal aneuploidies with performance and non-reportable rate similar to some other published methods.

P2-9 Clinical utility of NextSeq 500 sequencing for noninvasive prenatal testing Kornelia Neveling, Djie Tjwan Thung, Lean Beulen, Wendy Buijsman, Ingrid Gomes, Simone van den Heuvel, Hanneke Mielo, Ellen Kater-Baats, Brigitte Faas Radboud University Medical Center, Nijmegen, Netherlands OBJECTIVES: In April 2014, whole genome NIPT was implemented in our center, using the 5500XL next generation sequencing (NGS) platform (Life Technologies) and the WISECONDOR algorithm for data analysis. The mean reporting time (=number of days between blood collection and reporting) was 15.5 days. This relatively long reporting time was mainly due to sample preparation and sequencing time. The NextSeq 500 platform (Illumina) is a new benchtop NGS machine, and for NIPT, library preparation takes ~1½ days and an NGS run (1x75bp) ~11 hours. Because of these advantages, we validated this platform for whole genome © 2015 John Wiley & Sons, Ltd.


NIPT. METHODS: To compare the performance of the NextSeq 500 to that of the 5500XL, a technical validation was carried out. For this, plasma samples that had previously been sequenced on the 5500XL were now tested on the NextSeq 500. In total, 155 samples were sequenced on seven different sequencing runs, with a maximum of 24 samples per run. 107 euploid samples were used to assemble a new reference pool for WISECONDOR. In addition five T21, three T18, three T13, and samples with known partial aberrations were tested, with each run containing at least one aneuploid sample. In addition, a DNA dilution series of two T21 samples (10, 5, 2 and 1 ng input) was performed, as had previously been done on the 5500XL with the same samples. RESULTS: When pooling 24 samples (4 out of 7 sequencing runs) and using a duplicate read and MAPQ filter of > =30, the mean number of reads per sample was 14.9 M (± SD 1.6 M). All aneuploid samples were reported correctly using WISECONDOR, and no false positive T13, T18 or T21 were encountered. Moreover, all known partial aberrations were detected correctly. The additionally performed dilution series demonstrated that an input amount as low as 1 ng is technically still sufficient. CONCLUSIONS: Our results show that NIPT can reliably be performed using the NextSeq 500 platform. Switching the NIPT process from the 5500XL to the NextSeq 500 platform enabled us to reduce our mean reporting time from 15.5 to 7.8 days. P2-10 Quality assessment scheme for NIPT in the Netherlands: A national initiative Heleen Schuring-Blom1, Els Voorhoeve2, Karin Huijsdens-van Amsterdam3, Merryn Macville4, Diana van Opstal5, Femke de Vries6, Elles Boon7, Ron Suijkerbuijk8, Shama Bhola9, Erik Sistermans10, Brigitte Faas11 1

UMC Utrecht, Utrecht, Netherlands 2 Department of Clinical Genetics, VU University Medical Center Amsterdam, Amsterdam, Netherlands 3 Academic Medical Center, Amsterdam, Netherlands 4 Maastricht University Medical Center, Maastricht, Netherlands 5 Erasmus Medical Center, Rotterdam, Netherlands 6 Department of Clinical Genetics, Erasmus MC, Rotterdam, Netherlands 7 Leiden University Medical Center, Leiden, Netherlands 8 Department of Genetics, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands 9 Dept. of Clinical Genetics, VU University Medical Center Amsterdam, Amsterdam, Netherlands 10 Free University Medical Center, Amsterdam, Netherlands 11 Radboud University Medical Center, Nijmegen, Netherlands OBJECTIVES: Since April 1st 2014, NonInvasive Prenatal Testing (NIPT) is introduced in the Netherlands as part of a nationwide implementation study (TRIDENT: Trial by Dutch laboratories for Evaluation of NIPT). It is offered in all eight academic centers. In six out of eight (accredited) laboratories (labs A-F) the complete laboratory procedure is performed, two laboratories (labs G-H) outsource the laboratory procedures and only perform data analysis and Prenatal Diagnosis 2015, 35, 27–109

reporting. Up to now, no external quality assessment scheme (EQA) for NIPT is available. Therefore, we aimed to organize a national scheme to assess the technical and reporting performance of the participating laboratories and to gain insight in local procedures, in order to achieve as much harmonization as possible between the eight Dutch laboratories. METHODS: Labs A-F were requested to distribute three plasma samples to one of the other five laboratories: at least one normal and one T13, 18 or 21 sample (as established by the distributing laboratory) should be included. Relevant anonymized patient information was provided. Labs A-F performed the complete NIPT procedure for these three plasma samples and labs G-H received data for data analysis and reporting. Reports had to be submitted to the organizers within 3 weeks after distribution. Labs A-F were also requested to send their original reports. Results from the original reports were compared to those of the EQA reports and all results were discussed in a final meeting with all participants. RESULTS: All 24 reports were submitted in time. No technical failures were reported. Of the 18 different samples, seven were aneuploid: six T21 and one T13. There were no discrepancies between the results in the EQA reports and the original reports. CONCLUSIONS: Although not fully compliant with ISO standards yet, we have set up a simple and effective national EQA scheme for NIPT. By distributing plasma samples, both the technical and analytical proficiency of each participating laboratory could be evaluated. The subsequent meeting provided the opportunity to discuss both technical data and reporting, and fosters in achieving further nationwide harmonization.

P2-11 Non-invasive prenatal testing for fetal submicroscopic copy number variations by next generation sequencing Xuyang Yin1, Ya Gao1, Jun Wang1, Hongtai Liu1, Fuman Jiang2, Fang Chen3, Wei Wang1 1

BGI, Shenzhen, China BGI Diagnostics, Shenzhen, China 3 BGI Research, Shenzhen, China 2

OBJECTIVES: Next generation sequencing (NGS)-based noninvasive prenatal testing (NIPT) has become a powerful tool in prenatal screening for fetal aneuploidies. Chromosome submicroscopic copy number variations (CNVs), which are associated with many birth defects, are more challenging for NIPT to detect due to their small size. The objective of this study was to develop a NIPT method to identify fetal submicroscopic CNVs, and validate it using artificial and actual plasma samples. Fetal fraction and sequencing depth for identifying submicroscopic CNVs were assessed. METHODS: We developed a method for noninvasive prenatal detection of fetal submicroscopic CNVs by whole-genome sequencing using the BGISEQ1000 platform. The performance of this method was evaluated in silico firstly, followed by testing in artificial and actual plasma samples. Artificial © 2015 John Wiley & Sons, Ltd.


samples were created by mixing the sonicated male DNA with plasma from non-pregnant female at ratios of 5%, 10% and 15% fetal fractions, and with fetal CNVs of 2.5 Mb, 5 Mb and 10 Mb sizes. Actual plasma samples were collected from the cases with fetal submicroscopic CNVs confirmed by invasive procedures. RESULTS: In silico analysis showed that 30 M unique reads (~0.5X sequencing depth) and 10% fetal fraction were required to detect 5 M CNV with above 90% detection power. With artificial samples, CNVs of 5 Mb at 5% fetal fraction, and CNVs of 2.5 Mb at 15% fetal fraction were able to be detected by NGS-based NIPT using 30 M unique reads. CNVs of 2.5 Mb at 5% fetal fraction can be detected with approximately 100 M unique reads (depth of ~1.5X). An actual plasma sample with fetal CNVs of 2 Mb size was detected correctly using our method, consistent with the diagnostic result. CONCLUSIONS: Our method can accurately identify submicroscopic CNVs as small as 2 Mb in a noninvasive way. Future clinical application of NIPT may involve testing for fetal sub-chromosomal abnormalities after validation in larger sample size.

studies investigating its accuracy and sensitivity. Studies suggest reasons for discordant NIPT result with fetal karyotype to include: placental mosaicism, maternal mosaicism, co-twin demise, or maternal malignancy. Since the primary source of cell-free fetal DNA is believed to be from the placenta, in a dichorionic twin gestation it would be difficult to ensure adequate sampling from both placentas. Therefore, this case is of interest as it reports a rare occurrence of co-twins with discordant aneuploidies with a false-negative NIPT result for trisomy 18, and this will be an important issue for providers to be aware of in future patient counseling.

P2-13 Noninvasive prenatal diagnosis of Down syndrome with arrayCGH Tuba Gunel, Mohammad Kazem Hosseini, Ece Gumusoglu, Hayri Ermis, Kilic Aydinli Istanbul, Turkey

P2-12 Case of a twin gestation affected with discordant aneuploidies, and a false-negative noninvasive prenatal test result Catherine Griswold, Allison Durica, Larry Dennis Ann Jewell Carilion Clinic, Roanoke, Virginia, United States OBJECTIVES: To present a twin gestation case, affected with discordant aneuploidies, with a false-negative noninvasive prenatal test (NIPT) result. METHODS: Case report, and review of the PubMed literature. RESULTS: We present a 35-year old G2P1 female with a dichorionic, diamniotic twin gestation. The patient was referred secondary to advanced maternal age. Twin A was observed to have multiple ultrasound abnormalities including choroid plexus cysts, ventricular septal defect, small stomach, ectopic kidney, second trimester intrauterine growth restriction, and polyhydramnios. The patient was counseled about her testing options, and she declined the amniocentesis but elected NIPT. NIPT showed the expected representation of chromosomes 21, 18 and 13 (low-risk result). Sex chromosome analysis was not able to be performed using NIPT in a twin gestation. The patient delivered at 33 weeks gestation by cesarean section secondary to non-reactive fetal monitoring. Postnatal karyotypes were performed showing 47,XX,+18 for twin A and 47,XXX for twin B. This is consistent with co-twins having discordant aneuploidies (trisomy 18 and triple X syndrome), and the trisomy 18 was not detected using NIPT. CONCLUSIONS: Co-twins with different aneuploidies are rarely observed and reported in the literature. Previously published tables regarding age-related risks suggests the risk for a 35-year old to have one or both fetuses with any chromosome anomaly at live-birth is 1 in 114, and to have both fetuses affected with any chromosome anomaly at live-birth is 1 in 1,000. Furthermore, NIPT is a newer technology with ongoing Prenatal Diagnosis 2015, 35, 27–109

OBJECTIVES: To develop a DNA comparative genomic hybridisation or Array CGH method for non-invasive prenatal testing (NIPT) using cell-free fetal DNA (cffDNA) for early detection of Down syndrome (DS). This project was supported TUBITAK (The Scientific and Technological Council of Turkey). METHODS: Maternal plasma from 100 pregnant women was evaluated for trisomy risk and fetal sonographic abnormality. cfDNAs isolated from maternal plasma and fragmentation has been done to increase of cfDNA ratio in isolated cfDNA. The fragments sized ≤300 bp were separated. Obtained cfDNAs from fragmentation evaluated using by arrayCGH method. We divided the study into two steps: the first step is human total oligonucleotide microarray profiling; the second step is design of custom sequences on 21st choromosome according to microarray results. RESULTS: Total oligo-nucleotide microarray analysis was performed in 24 patients and custom array was performed in 76 patients from 100 abnormal pregnancy. Targeted chromosomes were analyzed using Agilent CytoGenomics Edition Microarrays classified all trisomy and nontrisomy cases. CONCLUSIONS: Using cfDNA obtained from maternal plasma and serum has been shown to improve the NIPT for screening of fetal aneuploidy risk [1] Quantitative comparisons between many different samples with oligonucleotide arrays (DNA microarrays) could be an important tool for prenatal diagnosis, consequently DNA arrays have provided a platform for high throughput detection of biomolecules in aneuploidies[2].

P2-14 22q11.2 deletion via non-invasive prenatal screening (NIPS) unconfirmed by karyotype, FISH or microarray: A case report Ginger Hocutt, Emily Hardisty, Kathleen Kaiser-Rogers, Neeta Vora University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States © 2015 John Wiley & Sons, Ltd.


OBJECTIVES: Non-invasive prenatal screening (NIPS) is widely used as a screening test for fetal aneuploidy in high risk populations. Recently, microdeletion syndromes, including 22q11.2 deletion syndrome, have been added to this screening. Wapner, et al. (2014) published data indicating a detection rate for 22q11.2 deletion of 97.8% (45/46) with a false-positive rate of 0.76% (3/397) in a sample set of 358 pregnancies and 111 artificial mixtures. Of these samples, only 6 were from pregnancies with microdeletions, 3 of which had 22q11.2 deletion syndrome. For rare conditions, even with high sensitivity and specificity, the positive predictive value (PPV) remains low. METHODS: We present a case of a positive NIPS for 22q11.2 deletion syndrome that was not confirmed through invasive testing and demonstrate the importance of counseling patients that NIPS is a screening test. RESULTS: A 36-year-old G2P1001 received the Panorama (SNP-based) NIPS at 10w5d through her referring provider due to her advanced maternal age. Results of this screen indicated a “high risk” for 22q11.2 deletion syndrome with a positive predictive value of 1/19. Upon referral to our institution, a cystic hygroma was noted at 12w6d gestation. Amnio-centesis was performed and karyotype was normal (46,XY) with normal FISH for 22q11.2 deletion syndrome in 50 interphase and 30 metaphase cells. Given the finding of a cystic hygroma, a microarray was performed which was consistent with a normal male fetus. A targeted anatomy scan did not detect any structural anomalies. Fetal echocardiogram is scheduled. Results were not available at the time of abstract submission. Maternal karyotype and FISH for 22q11.2 deletion have been declined by the patient. CONCLUSIONS: NIPS is currently being used as a screening test for common aneuploidies in a high risk pregnancy population, however its expanding use for microdeletions is controversial because of the low prevalence of these rare disorders. Because the risk of having a clinically significant copy number variant in a structurally normal fetus is 1.7% (Wapner et al., 2013) regardless of maternal age, some would argue that microdeletion screening should be offered. This case demonstrates that genetic counseling and invasive testing are critical in the setting of a positive NIPS, especially in the setting of microdeletions, given the low positive predictive value.

P2-15 Recent bioinformatic advances of non-invasive prenatal detection to enhance diagnostic accuracy and aneuploidy discovery Wera Hofmann, Sebastian Grömminger, Matthias Sachse, Joachim Bonnet, Ulrike Schöck LifeCodexx AG, Konstanz, Germany OBJECTIVES: In recent years multiple independent studies have shown the ability of random massively parallel sequencing (MPS) of maternal plasma DNA to accurately detect common fetal aneuploidies. Here, we present the results of a new algorithmic approach improving the test performance Prenatal Diagnosis 2015, 35, 27–109

of PraenaTest®. METHODS: Un-blinded study data was reanalyzed by a new bioinformatics algorithm based on a reference mappability score following GC normalization to correct for genomic library preparation and sequencing bias. A MAD-based z-score equation was used to identify fetal aneuploidies. RESULTS: Results of test performance (including sensitivity and specificity) based on advanced bioinformatics algorithm will be presented and compared with those from previously certified algorithm. CONCLUSIONS: Due to the improved bias correction followed by a significant reduction of statistical variance, the PraenaTest® even allowed the detection of sub-chromosomal events confirming an even more reliable detection of common autosomal trisomies; furthermore it enables the potential of decreasing read coverage without reducing detection accuracy and sequencing at lower costs and higher sample performance. P2-16 Non-invasive prenatal test (NIPT) for trisomy 21 by quantitative real-time PCR Wera Hofmann, Matthias Sachse, Joachim Bonnet, Ulrike Schöck, Sebastian Grömminger LifeCodexx AG, Konstanz, Germany OBJECTIVES: Current NIPT assays for T21 in clinical application use massively parallel sequencing (MPS) based strategies which are quite costly and hence limited to restricted patient groups. Here we present data of an affordable T21 specific NIPT assay based on quantitative real-time PCR (qPCR) which might have the potential to become the first universally/globally applied NIPT method. METHODS: The method has been developed to use qPCR to determine T21 positive cases. The test performance of this T21 assay is compared with the results from our MPS-based PraenaTest® laboratory routine. RESULTS: In a proof-ofconcept study it was shown that a qPCR-determined quantitative variable is clearly distinguishable in the investigated T21 positive cases. CONCLUSIONS: Our results suggest that the technical requirements for an affordable PCR test for the non-invasive detection of fetal trisomy 21 are fulfilled. Further studies are required to improve the quality of this new test system for the detection of other fetal aneuploidies. P2-17 Evaluation of array Comparative Genomic Hybridisation for non-invasive prenatal screening for common aneuploidies Evangelia Karampetsou1, Natalie Milner2, Victoria Demetriou2, James Reid2, Douglas Hurd2, Lyn Chitty3 1

NE Thames Regional Genetics Service, Great Ormond Street Hospital for Children NHS Foundation Trust and University College London, London, United Kingdom 2 Oxford Gene Technology, Oxford, United Kingdom 3 University College London Hospital, NE Thames Regional Genetics Laboratory & UCL Institute for Child Health, London, United Kingdom © 2015 John Wiley & Sons, Ltd.


OBJECTIVES: To evaluate array Comparative Genomic Hybridisation (CGH) for the non-invasive prenatal detection of common aneuploidies based on analysis of cell-free DNA (cfDNA) in maternal plasma. METHODS: Pregnant women attending maternity units in England for invasive diagnostic testing due to high risk for aneuploidy were consented to donate a blood sample for use in the development of Non-Invasive Prenatal Testing (NIPT). Maternal blood was collected in EDTA or Streck tubes, double spun and plasma stored at 80 °C before cfDNA extraction. Results of invasive testing were known in all cases. Samples were tested on the array CGH based NIPT assay and a variety of statistical analyses and methods were evaluated for results analysis. The effect of fetal fraction, sample haemolysis and DNA extraction method will also be reported. RESULTS: At the time of submission, 107 samples (including controls) were collected, of which 24 were excluded due to apparent haemolysis of samples. Using a statistical method, of the remaining 83 samples tested on the array CGH NIPT assay, 65 were truenegatives, 9 were true-positives, and 9 were no calls due to high Derivative Log Ratio (DLR) scores. Whilst results were largely consistent between samples for different bioinformatics approaches, there were some differences. Here we will report the effect of varying statistical analyses and other factors, such as fetal fraction, degree of haemolysis and sample collection methodology. CONCLUSIONS: This array CGH NIPT assay offers promise for a low cost, rapid and easily transferrable test based on analysis of cfDNA in maternal plasma. The test requires a standard array platform which is available in most genetics laboratories. This approach should allow in-house testing in most genetics laboratories offering prenatal testing with minimal capital outlay and running costs compared to Next Generation Sequence based technologies. The results of this study are informing the development of an array CGH based NIPT assay.

RESULTS: A total of 6,684 samples were drawn and 6,608 samples were resulted. Only 4 of the 76 “cancellations” were due to technical reasons. Mean maternal age at delivery was 36.9 years. There were 221 unique positive results from 219 positive specimens (3.3%) for T21 (n = 104), T18 (n = 38), T13 (n = 15), 45X (n = 41), other sex chromosome abnormalities (SCA, n = 21), monosomy 21 (n = 1), and monosomy 18 (n = 1). Two cases were double positives. Prenatal or neonatal karyotypes were available for 174/219 of positive specimens (79.5%). Of 6,389 negative results, pregnancy outcomes were available for 5,180 (81.1%). Karyotypes were available for 205/6,389 negative NIPT specimens (3.2%); clinical features at birth did not warrant a karyotype on the others. Known false positive results occurred 51 times (6 T21, 11 T18, 6 T13, 19 45X, 7 SCA, 2 monosomies) for an overall false positive rate of 0.7%. In two false positive 45X cases, maternal karyotype revealed mosaic 45X. Known false negative results occurred in 4 cases (1 T21, 2 T18, 1 XXX). Positive predictive values (PPV) were 93.2% for T21, 59.3% for T18, and 50.0% for T13. Negative predictive values (NPV) were ≥99.97% for T21, T18, and T13. CONCLUSIONS: The limited data received for pregnancy outcomes by commercial NIPT laboratories may result in underestimates of false negative/false positive rates, overestimates of sensitivity/specificity, and preclude calculations of PPV and NPV. KPSC’s closed health care system and access to outcome information in the EMR provided a unique opportunity to obtain outcome data for >80% of NIPT tests performed.

P2-19 Case report: Discrepancy between NIPT results and invasive test of Korea Youngju Kim1, Sookryung Kim2, Eunjung Choi2, Borum Suh3, Miyoung Lee3, Anna Nam4, Teayoon Kim4 1

P2-18 Positive and negative predictive values of NIPT in an integrated health care system: Summary of NIPT outcomes in Kaiser Permanente Southern California Hilary Kershberg, Jeff Greenberg, Amy Liou, Aki Logg, Monica Alvarado, Jaime Natoli, George Tiller Southern California Kaiser Permanente Medical Group, Pasadena, California, United States OBJECTIVES: Since June 2013, Kaiser Permanente Southern California (KPSC) has offered non-invasive prenatal testing (NIPT) to women with high-risk singleton pregnancies. The objective of this study is to report our first year of clinical experience with NIPT. METHODS: We queried our electronic medical record (EMR) for prenatal diagnostic testing and/or postnatal chromosome testing for pregnancies that had NIPT (Illumina “verifi”) from 6/10/ 13-6/30/14. A result of “aneuploidy detected” or “aneuploidy suspected” was considered a positive screen. Prenatal Diagnosis 2015, 35, 27–109

Mizmedi Hospital iDream Research Center, Anyangsi, Kyungkido, Republic of Korea 2 Mizmedi Hospital iDream Research Center, Seoul, Republic of Korea 3 Department of Laboratory Medicine, Seoul, Republic of Korea 4 Department of Obstetrics and Gynecology, Seoul, Republic of Korea OBJECTIVES: Recently non-invasive prenatal tests (NIPT) are gaining popularity in Korea. Many pregnant women prefer non-invasive prenatal screening to the invasive alternative. And most patients hold misconceptions that NIPT results are virtually the same test as the invasive tests. But our NIPT sample with a high risk for trisomy 21 shows that this is not always the case. METHODS: The NIPT results were reported from BGI-Shenzhen. And the high risk samples were confirmed by invasive amniocentesis. The analysis of the invasive prenatal studies were conducted by karyotyping and FISH (fluorescence in situ hybridization) at our iDream Research Center © 2015 John Wiley & Sons, Ltd.


RESULTS: We performed a total of 80 cases of NIPT from May 2013 to December 2014, and among them, 3 cases revealed an increased risk of trisomy 21. Case 1. A 36year-old patient with fetal nuchal translucency of 4.0 mm performed the NIPT was conducted at 12.3 weeks gestation. This patient terminated her pregnancy when she received positive results without confirmation through invasive testing. Case 2. Patient is a 34-year-old woman screened positive for Down syndrome from the integrated multiple marker test(1:69). NIPT was performed at 17.6 weeks gestation and the high risk result of NIPT was confirmed by invasive prenatal testing from amniotic fluid. The results of FISH and karyotype were both trisomy 21 mosaicism. Case 3. Advanced maternal age and short fetal nasal bone demonstrated on ultrasound examination were indications for amniocentesis in a 38-year-old patient. This case was analyzed by NIPT at 14 weeks gestation. We carried out invasive prenatal tests on amniotic fluid. Amniocentesis karyotype was normal 46,XX with a normal FISH result. CONCLUSIONS: The accuracy of NIPT results is higher than the conventional multiple marker screening test. But our analysis shows that the NIPT results are still not diagnostic in value. For this reason, patients who want NIPT should be offered counseling about the various possibilities of NIPT results, such as false positive or false negative results. The discordant results of NIPT and invasive test arose from a variety of biomedical reasons. So patients who have positive results of NIPT should always be confirmed by invasive analysis. It is essential to counsel before and after NIPT and confirm the positive results of NIPT by invasive tests.

P2-20 Three year clinical experience with noninvasive prenatal testing in 3000 high risk cases in the United States Sebastian Larion, Steven Warsof, Letty Romary, Margaret Mlynarczyk, Alfred Abuhamad Eastern Virginia Medical School, Norfolk, Virginia, United States OBJECTIVES: The objective of this study is to report on theclinical experience of noninvasive prenatal testing (NIPT) forscreening of fetal aneuploidy in a single, large referral centerin the United States. METHODS: A retrospective analysis of aprospectively maintained database was performed of allconsecutive high risk patients who underwent noninvasiveprenatal testing for fetal aneuploidy including trisomy 21, 13,18 and sex chromosome abnormalities between March 2012and December 2014 at a single, large referral center in theUnited States. NIPT assays were performed by 5 commerciallaboratories. Positive screening results were correlated withultrasound information, diagnostic testing and/or neonataloutcomes, whenever possible. RESULTS: A total of 3,054NIPT’s were performed in 2,992 patients during the studyperiod. The mean patient age was 34.9 years, with 71% referredfor advanced maternal age. A total of 109 patients had positiveor indeterminate screening results for fetal aneuploidy, withfollowup information available in 105 (96%) Prenatal Diagnosis 2015, 35, 27–109

patients. Of 53screen positive cases for trisomy 21, 53 were confirmed withfollowup testing (100% positive predictive value [PPV]; 95%confidence interval [CI]: 93.3-100.0%), 10/12 for trisomy 13(83.3% PPV; CI: 51.6-97.9%), 8/10 for trisomy 18 (80.0% PPV;CI: 44.4-97.5%), and 8/14 for sex chromosome abnormalities(57.1% PPV; CI: 28.9-82.3%). Suspected or indeterminatescreening results were less predictive of aneuploidy. Sampleprocessing issues including low fetal fraction occurred in0.74% of results. No false negative screening results werereported during the study period. CONCLUSIONS: NIPT hadhigh positive predictive value for trisomy 21, but was lesspredictive for trisomy 13, 18 and fetal sex chromosomeabnormalities. Sample processing issues were relatively rare.Patient counseling and diagnostic testing should be offered toall patients with non-negative screening results beforeundergoing definitive management.

P2-21 Clinical implementation of non-invasive prenatal testing worldwide – results from a global survey Celine Lewis Genetics and Genomic Medicine, London, United Kingdom OBJECTIVES: Non-invasive prenatal testing (NIPT) is marketed in nearly 90 countries worldwide and is being considered for implementation within many public health systems. However, data on clinician experiences with implementation outside the US and UK is limited. Here we present a survey examining NIPT use worldwide. METHODS: An online survey to examine current practice regarding prenatal screening, invasive testing and NIPT was developed. The final instrument contained 34 questions with either multiple choices and/or option to enter open text and comments. A link to the survey was sent to 78 health professionals in 46 different countries, who were either colleague known to the authors, or members of the International Federation of Gynaecology and Obstetrics. RESULTS: Forty-nine individuals (63%) from 28 countries responded. All reported that screening and invasive testing for chromosomal aneuploidies was available in their country. Forty three participants reported that NIPT is currently available in their country, four said NIPT is not available, one was unsure and one respondent was not familiar with NIPT. The majority of respondents (n = 40) reported NIPT was available through the private sector; 10 respondents reported public sector availability. Eighteen respondents from 14 countries reported that there were plans to introduce NIPT into routine antenatal care in their country, while 11 reported there were no such plans. Notably, where individuals from the same country responded, there was variation in responses with regard to current use of NIPT and plans for routine implementation. The cost of NIPT reported by respondents varied widely from about $350 USD (Australia) to $2900 USD (United States). Many respondents commented that cost limited the use of NIPT. CONCLUSIONS: This study provides the first insight into current uses and experiences © 2015 John Wiley & Sons, Ltd.


with NIPT globally. It highlights the disparity in service provision that exists both across and within countries, emphasizing the importance of developing national and international guidelines. High cost of NIPT appears to be a barrier for broad implementation in many countries. Further research on stakeholders’ experiences is needed to explore how particular legal and socio-economic systems influence the use of, and access to NIPT, and its effect on prenatal care globally.

P2–23 Table.

Results 100% 90% 80% 70% 60% 50% 40% 30% 20% 10%

P2-22 Withdrawn.

0% Total sent 629

P2-23 One clinic’s experience with cell free fetal DNA testing in Argentina 1



Jose Pablo Marchili , Aristides Chaulet , Oscar Guetmonovitch , Norberto Daniel Margulies2, Victor Solarz2 1

Hospital Juan A Fernández Buenos Aires, Argentina Buenos Aires, Argentina


OBJECTIVES: We have offered cell free fetal DNA (cffDNA) testing in our clinic in Buenos Aires, Argentina since July 2013, We wanted to highlight our experience with cffDNA testing. METHODS: Samples were shipped to Illumina’s CLIA Laboratory in USA to perform the verifi® prenatal test. We offered testing for chromosomes 21/18/13/X/Y for singletons and chromosomes 21/18/13/Y for twins. In July 2014, we now offer additional testing for all eligible singletons, including chromosomes 9/16 and a microdeletion panel. RESULTS: Outcome information was reviewed for a total of 629 cases. Results were reported in 614 cases (97.6%) with an average turn-around-time of 2.95 business days from date of sample receipt in lab. There were no technical cancellations; of 15 (2.40%) administrative cancellations, all were due to sample arrival in lab beyond stability date (>5 days from date of draw). Of 614 cases in which results were reported, 583 (94.95%) cases were singletons; 31 (5.05%) cases were twins. Initially, we offered cffDNA testing to high risk patients (i.e. patients ≥35 years at delivery, positive first trimester combined test and/or ultrasound findings); however, we recently have noticed a trend in increased use of cffDNA testing in the average risk population. Overall, aneuploidy was detected (AD) in 16 (2.6%) samples; with 12 detected T21 (1.95%), 2 detected T18 (0.33%), 1 detected MX (0.16%) and 1 detected XXX (0.16%). Interestingly, and as expected, we saw an increased prevalence of AD cases (16/18) in patients with abnormal ultrasound findings. Outcome information was available in 12/16 (75%) abnormal cases. No false negative cases were reported; one (0.16%) putative false positive case (for T18) reported. Two (0.33%) cases of XY/XX discordance were reported. CONCLUSIONS: This is the first report of a single clinic’s experience with cffDNA testing in South America, which are consistent with other publications. As cffDNA testing expands globally and to average risk population, continual monitoring of its performance is important for assuring accurate and reliable results. Prenatal Diagnosis 2015, 35, 27–109

Detected 16

>35 years old 463

Singletons 385

P2-24 Noninvasive prenatal test, one year of experience in the genetics clinic at the Hospital Angeles Lomas in Mexico City Dora Gilda Mayen, Mariana Hernandez-Gomez, Eva Ramirez, Ricardo Meléndez, Luz Maria Garduño Genetics Clinic, Hospital Angeles Lomas, Mexico OBJECTIVES: The discovery of free fetal DNA in maternal blood (ccf DNA) in 1997 by Lo et al., has opened the possibility of a noninvasive prenatal test (NIPT). The percentage of ccf DNA represents a fraction 3–13 % of total free DNA in maternal blood and is rapidly eliminated after termination of pregnancy. Initially, it was clinically applied to monogenic disorders, but currently it is employed in the analysis of aneuploidies and fetal sex determination. Massive parallel sequencing detects the origin of each amplified sequence, and analyses over-representation of sequences or any decrease in the fetal chromosomes in maternal plasma. This technique has been validated and allows assessment of trisomy 13, 18 and 21, obtaining the result in about a week from week 10 of gestation. By using NIPT, we expect a reduction in the number of invasive studies and the risk of fetal loss. The objective is to present the experience of a Genetics Clinic in Mexico, in the use of NIPT as a method of prenatal screening for aneuploidies and fetal sex determination METHODS: Development of a database of patients to which NIPT was applied from August 2013 to date. RESULTS: There have been 42 patients, averaging 37.4 years of maternal age, 13.4 weeks and 12.5 % of fetal fraction. Two cases of high risk for trisomy 18 and two with high risk for X monosomy were obtained. In the first case of trisomy 18 was not possible to make a confirmatory cytogenetic study, because of pregnancy loss, but the ultrasound data and biochemical screening of the first trimester was according with the diagnosis and in the second case was confirmed by karyotype in amniotic fluid. In the first case of X monosomy, amniocentesis was performed and later in peripheral blood with a result 46,XX, registering as the only false positive so far. In the second case the result obtained was 46,XX, in the amniotic fluid, but maternal contamination was confirmed in the sample. The rest of © 2015 John Wiley & Sons, Ltd.


the results are low risk and have confirmed the result by following the birth, as well as the fetal sex determination. CONCLUSIONS: NIPT is currently the screening test with the highest detection rate (greater than 98 % with a false negative rate less 0.5 %, with a sensitivity and specificity close to 100 %), although it can vary from one chromosome to another. It is indicated for: 1) Women aged 35 or older at delivery, 2) ultrasound data that increase the risk for any of the aneuploidies mentioned, 3) First or second trimester screening test with a result of high risk for trisomy 13, 21 and 18, 4) History of a previous child with one of these three trisomies, 5) If a parent is a carrier of a Robertsonian translocation involving chromosomes 13 or 21. This test has not been validated for low risk women or multiple pregnancies. In our series, the most frequent indication was advanced maternal age. The weight of the patients is important because it is a factor related to the percentage of fetal DNA. Among the 42 cases, only in one patient with 114 kg of weight, we did not obtain the results. In all other cases the results were obtained by first intention, because in all of them the percentage of DNA remained above 4%.

P2-25 Comparison of results from noninvasive prenatal screening and diagnostic cytogenetic testing Jeanne Meck, Elizabeth Kramer Dugan, Daniel PinedaAlvarez, Andrea Wray, Gabriele Richard, Ludmila Matyakhina GeneDx, Gaiithersburg, Maryland, United States OBJECTIVES: (1) Compare results from noninvasive prenatal screening (NIPS) and diagnostic cytogenetics to determine the accuracy of NIPS. (2) Determine the referral reasons for cytogenetic testing in pregnancies with negative NIPS results. METHODS: This study is based on follow-up cytogenetic testing of specimens (n = 108) referred to our laboratory, for which prior NIPS results were available. Using cytogenetic results as the gold standard, NIPS results were classified as true positive (TP), false positive (FP), true negative (TN) or false negative (FN). RESULTS: 110 prenatal specimens (CVS or amniotic fluid) from women with prior NIPS testing revealed the following outcomes: 62 (56%) TP, 25 (23%) FP, 20 (18%) TN, 2 (2%) FN. One sample was inconclusive by NIPS and positive for T18 by karyotype. Trisomy 21 (T21) was the most frequent abnormal finding by NIPS: all 47 cases with positive NIPS for T21 were confirmed by cytogenetics; however, there was one FN result for T21. Results for other aneuploidies were as follows: Monosomy X - 7FP:4TP; T13- 4FP:2TP; T18- 2FP: 3TP. All NIPS results indicating increased risk for multiple aneuploidies (n = 3) or for a microdeletion syndrome (n = 6) were FP. Twenty-two cases were referred after a negative NIPS result, including 14 with fetal sonographic abnormalities (average maternal age 34.5 years), and the others with the sole provided indication of advanced maternal age (average maternal age 41 years). Twenty of the twenty-two cases were TN and 2 were FN (T21, triploidy). CONCLUSIONS: NIPS is a highly accurate screen for T21 with no FP and only one FN in our cohort. Prenatal Diagnosis 2015, 35, 27–109

Although the sample size is small, NIPS was found to be much less accurate when a result shows increased risk for non-T21 aneuploidies, for multiple aneuploidies, or for microdeletion syndromes. A negative NIPS result was accurate in 20 of 22 (91%) cases. Women who receive negative NIPS results are most often referred for diagnostic testing when there is an ultrasound abnormality and/or advanced maternal age.

P2-26 NIPT in unusual aneupoidy cases: Partial trisomies and low-level mosaicism Rachel Michaelson Shaare Zedek Medical Center, Hebrew University of Jerusalem, Jerusalem, Israel OBJECTIVES: Non-invasive prenatal testing for common fetal aneuploidies by massively parallel sequencing of cell-free DNA is extremely efficient, in particular for trisomy 21 and 18, with sensitivity and specificity of over 99%. Clinical utilization of NIPT for screening patients for aneuploidies has grown extensively over the last few years in many countries. Since the endorsement of NIPT by the Israeli Society of Medical Geneticists, its use is rapidly growing in Israeli centers. METHODS: In our referral center, women are offered NIPT for several indications. We have collected clinical and laboratory data for all cases and describe performance in 3 atypical cases. Three rare aneuploidies were diagnosed combining NIPT and karyotyping in our center. NIPT was done by a different platform in each of these cases (all USAbased laboratories). The first case was a 42-year-old woman whose integrated first and second trimester screening test indicated that the pregnancy was at high risk of trisomy 18 (1/ 3). Ultrasound scan revealed an atrioventricular septal defect and hyronephrosis. NIPT at 17 weeks demonstrated a great probability of trisomy 18. The second was a 36-year-old primigravida who had conceived through IVF-ICSI, and had a mildly increased nuchal-translucency. NIPT was consistent with a normal fetus with two sex chromosomes (XY). A later sonogram demonstrated a thickened nuchal fold with two nuchal cysts. The third case was a 28-year-old with normal first and second trimester screening. Ultrasound scan at 15 weeks showed two soft signs of aneuploidy (cardiac-echogenic-focus, choroidplexus-cyst). NIPT was consistent with trisomy 21. RESULTS: The first patient, with high trisomy 18 risk, declined invasive testing and delivered near term. The baby showed several signs of trisomy 18, however lacked many classic features of this syndrome. The neonate was treated in the intensive care unit, and died of respiratory complications at 3 weeks of age. Postnatal cord-blood karyotype result was 46,XX,der(14)t (14;18)(p10;q10), indicative of an unbalanced translocation of long arms of chromosome 14 and 18, causing partial trisomy 18 (see figure, patient 1). This partial trisomy explains the atypical phenotype. The second patient, with a normal NIPT but a thickened nuchal fold, underwent amniocentesis. Karyotype result was 47,XY,iso(X)(q10q10), confirmed by CMA, indicating atypical Klinefelter syndrome, caused by an extra chromosome consisting of two long arms of the X © 2015 John Wiley & Sons, Ltd.


chromosome (isochromosome Xq) (see figure, patient 2)). The third patient, with trisomy 21 according to NIPT, had an amniocentesis for confirmation of NIPT results. Rapid (QF-PCR) testing was normal, including disomy of all chromosome 21 markers. However, full-karyotyping of amniocytes revealed trisomy 21 mosaicism, with a relatively low level of trisomy-21 cells (50%, 19/38). CONCLUSIONS: There are many factors that limit the accuracy of NIPT. One main limitation is the genetic discrepancy between fetal and placental tissue. This discordance can potentially lead to inaccurate NIPT results. This is true in simple aneuploidies cases, moreover in incomplete and mosaic trisomies. For incomplete trisomies: In the first case described, although chromosome 18 was only partially duplicated, it was correctly identified as trisomy 18 by NIPT. However, in the second patient, NIPT was unable to detect a partial X duplication. This rare cause of Klinefelter, isochromosome Xq, induced a false negative NIPT result. This is consistent with lower performance of NIPT for detection of rare sex aneuploidies. In this case only the long arm of chromosome X was duplicated, which probably causes an even lower detection rate than complete X duplication in the more common 47, XXY. For low-level mosaicism, NIPT performed well: NIPT was able to detect a low-level mosaicism of trisomy 21, that was even below the threshold of QF-PCR detection, in the third case. These three cases are clinical examples that demonstrate the strengths and weaknesses of NIPT, in unusual aneuploidy cases.

P2–26 Image.

P2-27 Non-invasive prenatal screening for trisomies – 2 years experience in a large Health Maintenance Organization (HMO) Carol Norem1, Elise Obolensky1, Emma Bijesse1, John Turocy2, Bruce Blumberg1, Peggy Fehlen-Quizon1, Robyn Krieger1, Monica Wohlferd1 1

Kaiser Permanente Northern California, Oakland, CA, United States Kaiser Permanente Northern California, Modesto, CA, United States


OBJECTIVES: To assess the performance of Non-Invasive Prenatal Testing (NIPT) for Trisomy 21 (T21), Trisomy 18 (T18), and Trisomy 13 (T13) in a high risk population within Kaiser Permanente Northern California (KPNC) from October 2012 through December 2014. METHODS: Pregnancy outcomes were obtained by review of relevant mother/baby ICD-9 codes, cytogenetic results, and molecular results within the KPNC system, and correlated with NIPT results. All cytogenetic and molecular studies for this cohort were performed by a single internal laboratory. Women were offered NIPT per ACOG guidelines using a single outside laboratory. Final NIPT results were reported as low risk, high risk, or no result. RESULTS: From Oct 2012 through Dec 2014, NIPT was drawn in a total of 7588 pregnancies: 7214 results were low risk (95.1%), 271 high risk (3.6%), and 103 (1.3%) showed no final results. Of the 271 high risk results, 129 were high risk for T21, 40 for T18, 11 for T13, 89 for Sex Chromosome Aneuploidy (SCA), and 2 for more than one condition (T21/45,X and T18/45,X). SCA results were reported previously (Obolensky et al. ACMG 2015). Of the 7588 pregnancies, 114 cases of T21, 41 of T18, and 7 of T13 were confirmed by cytogenetic/molecular analysis or clinical features seen at US or birth. High risk NIPT cases that resulted in termination, miscarriage, or are ongoing, without confirmation were excluded (16 T21, 2 T18, 1 T13). Results are listed in Table 1. CONCLUSIONS: While NIPT performed as expected for Down syndrome (T21) in our high risk population, our experience with screening for T18 and T13 revealed lower DR and PPV than those quoted in most large studies to date. This illustrates the importance of continued studies in clinical practice and further supports the recommendation that NIPT be presented as a screening test with confirmatory studies strongly encouraged.

P2–27 table.

P2-28 Introduction of a cell free (cf) DNA test for aneuploidy as part of routine prenatal care: Results of a structured patient survey in women having testing Geralyn Messerlian, Edward Kloza, Barbara O’Brien, Glenn Palomaki Prenatal Diagnosis 2015, 35, 27–109

© 2015 John Wiley & Sons, Ltd.


Women & Infants Hospital, Providence, Rhode Island, United States OBJECTIVES: Evaluate information sources, decision-making, understanding, satisfaction, reactions to test failures and sex aneuploidy testing among an unbiased group of women from the general pregnancy population of Rhode Island offered cf DNA testing by their primary care providers. METHODS: Rhode Island prenatal care providers were offered orientation to cf DNA testing as part of routine prenatal care. New educational materials were validated, translated and made available to patients in conjunction with the offer of testing. Women accepting testing (at no charge) could provide permission to be contacted on the test requisition. A subset of women completed a structured telephone interview to assess adequacy of education in the primary care setting. RESULTS: Women were eligible if they provided permission to contact (65% did) and had testing at 10–20 weeks’ through a practice offering cf DNA for >2 months. Test indication could not be a positive screen, family history or abnormal ultrasound (15 minutes). Preliminary results indicate that 30 of 33 women felt they received sufficient information for decision-making and had their questions answered. However, 30% could not remember receiving the patient pamphlet. Three of 33 women felt the provided information was insufficient, usually due to a short time with the provider, not receiving a pamphlet or a specific issue with the test (one with test failure). CONCLUSIONS: Issues of concern are being addressed via newsletters and follow-up visits. Preliminary findings suggest that cf DNA testing for aneuploidy can be implemented in a comprehensive programmatic environment similar to that used in academic laboratories offering serum/ultrasound screening for Down syndrome.

This paper aims to counter these arguments by making an analogy to access to amniocentesis and making a claim that a highly sensitive test for Down syndrome in particular cannot be justifiably withheld from low-risk women. METHODS: In the past 10 years, ACOG and ACMG have changed their stance on amniocentesis and recommended that diagnostic testing be made available to all women who desired it regardless of risk status. This policy change was made based on arguments that the excess cost of amniocentesis should not be part of the calculus in deciding who should have access to this diagnostic test. An analogous argument can be made for allowing low-risk women access to NIPS. A review of the literature was performed to assess the sensitivity of these new technologies in low-risk women and the types of aneuploidies that NIPS will miss in these women. RESULTS: The sensitivity of NIPS for detecting fetal aneuploidy varies based on the patient population being studied. For Down syndrome, however, sensitivities are fairly consistent in the 97-99% range. The prevalence of fetal trisomies in younger, low-risk women, is much lower therby affecting the positive predictive value of these technologies, but the sensitivity remains the same. Many aneuploidies missed by NIPS in low-risk women will either be lethal or be detected on prenatal ultrasound. CONCLUSIONS: Because of its high sensitivity for detecting Down syndrome, in particular, excluding low-risk women from informed access to this technology cannot be supported just as limiting access to amniocentesis cannot be justified. While questions of payment and burdens on the healthcare system need to be addressed by insurance carriers and public health authorities, for the individual patient, the availability of this technology should not be denied.

P2-31 What will be missed if Noninvasive Prenatal Testing (NIPT) is the only assessment for “Ultrasound Indicated” genetic studies? Rebecca Reimers1, Heather Mason-Suares1, Sarah Little1, Emily Reiff1, Bryann Bromley1, Lori Dobson1, Louise Wilkins-Haug1, Vineeta Agarwala2 1

P2-29 Withdrawn. P2-30 Non-invasive prenatal screening should be made available to all pregnant women Steven Ralston Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States OBJECTIVES: Arguments have been made against making noninvasive prenatal screening (NIPS) available to all women because of poor positive predictive capabilities of the test in low-risk women, the costs of adding this testing as a screening tool in all pregnancies, and the inappropriate emphasis on screening for the common aneuploidies in low-risk women. Prenatal Diagnosis 2015, 35, 27–109

Brigham and Women’s Hospital, Boston, Massachusetts, United States 2 Harvard Medical School, Boston, Massachusetts, United States OBJECTIVES: Examine cytogenetic results following “ultrasound indicated” invasive genetic assessment to establish the distribution of chromosome anomalies, the contribution of microarray anomalies, and the potential for baseline change pre and post NIPT introduction. METHODS: Retrospective review of pathology records utilizing ICD-9 codes, correlated with electronic medical records and radiology results from 2005–2014. Aneuploidy of whole chromosome changes of 13, 18, 21, X, and Y was considered detectable by NIPT without correction for sensitivity in this study. Mosaics, deletions, duplications, triploidy, and balanced inversions and translocations were assumed to not be detected by NIPT. Apparently balanced rearrangements were retained in the abnormal population for the initial assessment. “Ultrasound © 2015 John Wiley & Sons, Ltd.


indicated” included anomalies defined as those representing major structural defects requiring surgery, those with known long-term morbidity, and soft markers associated with aneuploidy. Giemsa banded karyotypes were obtained by standard protocols. Microarray investigations were initiated in 2014 utilizing an Affymetrix CytoScan HD platform. Statistical analyses included parametric and nonparametric assessment with T test, chi square and Fisher exact correction. RESULTS: There were 532 genetic abnormalities detected by FISH and karyotype amongst the 4725 women who underwent ultrasound-indicated invasive testing from 2005–2014. By the stated assumptions, abnormalities in 437 (82.1%) of these patients likely would have been detectable by NIPT and 95 (17.9%) would have been missed. In three fetuses (0.6%) with karyotype anomalies detectable by NIPT, there was an additional clinically-relevant genetic abnormality not detectable by NIPT: 46,X,+21[16]/47,XX,+21[4], 47,XX,+21[16]/48,XX,+12, +21[4]), and 48,XY,+10,+18. Of the 95 abnormalities not detectable by NIPT: 14 (14.7%) involved inherited balanced translocations, 21 (22.1%) were triploid, 21(22.1%) involved mosaicism of any chromosome, 11 (11.6%) involved mosaicism of chromosomes 13, 18, 21, X or Y, and 17 (17.9%) involved duplications, deletions, markers, rings, or unbalanced translocations. Microarray assessment of the “ultrasound indicated” cohort since 2014 revealed 16.7% (6/36) of fetuses with a microdeletion or duplication not detected by routine karyotype examination. When the ultrasound indicated cohorts were assessed before and after the implementation of NIPT for high-risk women, the proportions of major aneuploidy (NIPT detectable) versus non-major aneuploidy (non-NIPT detectable) karyotype abnormalities were not statistically different (p = 0.665). There has been a reduction in overall invasive procedures by over 57% in the years since the introduction of NIPT. CONCLUSIONS: Analysis of a pregnancy in which ultrasound has identified a malformation or soft marker requires careful consideration of the limitations of screening. In a cohort ascertained due to “ultrasound indications” we found an 18% rate of karyotype changes of clinical significance that were likely not to be detected by NIPT. This rate lowers to 15.5% if one forgoes identification of inherited balanced translocations and to 11.3% if triploidy assessment is possible using SNP-based platforms. However, this rate is likely higher than our estimate as NIPT has less than 100% sensitivity especially for trisomies 13 and 18. Additionally, microarray analysis further contributes important clinical information with a wider distribution of deletion and duplication aberrancies than available currently through NIPT. P2-32 Nationwide project using next-generation sequencing of cellfree DNA in maternal plasma in Japan: One and a half years of experience Osamu Samura1, Haruhiko Sago2, Akihiko Sekizawa3, Japan NIPT consortium4 1

The Jikei University School of Medicine, Minato-ku, Tokyo, Japan Prenatal Diagnosis 2015, 35, 27–109


National Center for Child Health and Development, Tokyo, Japan 3 Nagasaki University, Nagasaki, United States 4 Japan NIPT consortium, Minato-ku, Tokyo, Japan OBJECTIVES: To report one and a half years of experience of a nationwide demonstration project aimed to introduce noninvasive prenatal testing of aneuploidy using maternal plasma and discuss ways to implement this program in Japan. METHODS: We conducted tests to detect aneuploidy in high-risk pregnant women with adequate genetic counseling. The clinical data, test results, and pregnancy outcomes were recorded. RESULTS: Of 12,320 women tested, 210 (1.7%) had positive results, 12102 (98.2%) had negative results, and eight (0.1%) had results that were not reportable. Of the 210 women who tested positive, 25 women experienced intrauterine fetal death, and four women refused to undergo a more invasive testing procedure Of the 186 positive cases confirmed karyotyping, a normal karyotype was observed for trisomy 21 in five cases, trisomy 18 in ten cases, and trisomy 13 in two cases. In the follow-up study of the negative cases (n = 1,638), only one false negative case of trisomy 18 was detected. CONCLUSIONS: This study describes the findings of a nationwide demonstration project aiming to introduce noninvasive prenatal testing for fetal aneuploidies using maternal plasma in Japan, based on the historical background of the prenatal diagnosis. It is expected that the present data will stimulate a debate regarding prenatal genetic testing and hopefully lead to improvements in the perinatal care system with respect to genetic counseling in Japan.

P2-33 Clinical laboratory experience in a general obstetrical population of noninvasive prenatal screening for fetal aneuploidy from cell free DNA Christina Settler, Lindsay Dohany Progenity, Ann Arbor, Michigan, United States OBJECTIVES: Noninvasive prenatal screening (NIPS) analyzes cell free DNA to assess the fetal risk for aneuploidy. Our laboratory receives samples for the verifi® by Progenity NIPS test from clients who offer this testing to a general obstetrical population. Here we report our experience with NIPS, including ordering patterns of clinicians for optional content. METHODS: NIPS was performed by Illumina via massively parallel sequencing in a general obstetrical patient population from June to December 2014. Results included analysis of aneuploidy for chromosomes 21, 18, and 13 on all samples. Sex chromosome aneuploidy detection was an “opt-out” test. Detection for expanded aneuploidy content (trisomies 9 and 16) was an “opt-in” test. Outcomes were collected when our laboratory was notified of a discordant result. RESULTS: The median gestational age in this cohort was 12 weeks. Aneuploidy was detected in 1.67% of samples and aneuploidy was suspected in 0.2% of samples. Overall, 1.23% of results revealed an autosomal aneuploidy, while 0.63% of results detected a sex chromosome aneuploidy. The reported false positive and false negative rates were 0.12% and 0.01% respectively. Gender discordancy was © 2015 John Wiley & Sons, Ltd.


reported in .07% of samples. The total cancellation rate was 1.87%, with 1.6% cancelled by the referring physician for administrative reasons. Only 0.21% of cancellations were technical; including failed QC metrics, too little cell free DNA, and cell free DNA concentration above the range. Approximately 24% of samples had expanded aneuploidy content ordered and approximately 97% of samples had sex chromosome aneuploidy requested. CONCLUSIONS: This summary suggests that our experience with NIPS for aneuploidy detection is within the performance parameters established in validation studies. Additionally, data indicates when expanded content is optional, a significant number of clinicians choose to “opt-out”. Clinicians do “opt-in” for sex chromosome aneuploidy content, which reveals fetal gender, in a majority of cases. P2-34 Discordant NIPT and standard cytogenetic results following amniocentesis: Clinical case management Sophia Stewart, Vg Dev, Henry Chamberlin, Christy Cotten Genetics Associates, Inc., Nashville, Tennessee United States OBJECTIVES: To detail the clinical case management strategy in a case with discordant NIPT and cytogenetic results. To characterize the structural abnormality detected and compare genetic content to the NIPT assay design in an attempt to explain the discrepancy To inform regarding the possiblility of such a discrepancy. To reinforce the necessity to confirm/visualize NIPT results, especially unusual or unexpected ones, with another technology such as cytogenetics or FISH. METHODS: Amniotic fluid was submitted for standard cytogenetics and FISH following NIPT analysis suggestive of monosomy 21. Microarray was initiated on the cultured cells to further charactrerize the structural abnormality detected. Parental blood chromosome analysis was performed on maternal blood. RESULTS: The FISH result revealed three signals for the chromosome 21 probe cocktail, suggestive of trisomy 21 in some form. Standard cytogenetics revealed the presence of one normal chromosome 21 and an idic(21)(q22.3), resulting in functional trisomy 21. Metaphase FISH confirmed that the idic(21) has two separated signals. Microarray analysis to further characterize the abnormality is pending. Comparison of the genetic content with the NIPT assay coverage map id pending. Maternal chromosome analysis was normal; paternal studies are pending. CONCLUSIONS: The pregnancy is scheduled for elective termination, a request for confirmation studies has been made and is pending receipt. It is assumed that the probe coverage of the chromosome 21 region in the NIPT assay was lost in formation of the idic(21), resulting in the discordant, in fact converse, results. This case highlights the continued need for confirmation of abnormal NIPT results, especially those with unusual results. P2-35 An unusual case of fetal trisomy 21, detected by NIPT and missed by QF-PCR but verified by all other molecular cytogenetic methods 1



Ron Suijkerbuijk , Gineke Drok-Woltjer , Martin Elferink , Heleen Schuring-Blom3, Marjan van Kempen2, Hans Beekhuis4, Lieke Berger1, K. Bouman1, Richard Sinke1, Birgit Sikkema-Raddatz1 Prenatal Diagnosis 2015, 35, 27–109


Department of Genetics, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands 2 Department of Medical Genetics, University Medical Center, Utrecht, Netherlands 3 UMC Utrecht, Utrecht, Netherlands 4 Department of Gynaecology, Isala Klinieken, Zwolle, Netherlands OBJECTIVES: With Non-Invasive Prenatal Testing (NIPT) becoming increasingly available in many countries, the number of discrepancies between NIPT and prenatal diagnosis are raising as well, mostly due to placental mosaicism. Here, we describe an unusual case of a fetal trisomy 21, detected by NIPT but missed by QF-PCR analysis of various prenatal tissues. METHODS: A 35-year old woman presented at our hospital at 12+4 weeks of gestation. She was at risk (1:43) for having a fetus with trisomy 21 calculated after first trimester combination screening (FTCS), with a nuchal translucency (NT) of 4.1 mm. Initially, the patient opted for non-invasive prenatal testing (NIPT) by massively parallel shotgun sequencing. At 13+3 weeks gestation, she also went for a chorionic villi biopsy because of the increased NT. QF-PCR was performed, followed by FISH, karyotyping and array analysis. The results were confirmed in amniotic fluid taken at 15+5 weeks of gestation and placental tissue, when the parents decided to terminate the pregnancy. RESULTS: NIPT analysis indicated a positive result for trisomy 21, with Z-scores of 3.2 and 4.9 for two consecutive NIPT runs, with concomitant a posteriori risks of 12.5% and 40.3%. After invasive procedures, QF-PCR did not confirm trisomy 21 in both (un)cultured chorionic villi and amniotic fluid. In contrast, FISH, karyotyping and array analysis unequivocally demonstrated a full trisomy 21 in the fetus, in (un)cultured chorionic villi and amniotic fluid. Follow-up FISH studies in placental samples revealed a mosaic trisomy 21 pattern. Remarkably, all QF-PCR markers showing the relatively highest peaks (but within the normal range) for chromosome 21 were of maternal origin in chorionic villi, while being of paternal origin in other tissues. CONCLUSIONS: We speculate that - next to normal male cells - two cell lines of trisomy 21 were present in the placenta, each with the extra chromosome 21 of different parental origin. With both lines being present in all fetal tissues, the net result was trisomy 21 as shown by NIPT and most cytogenetic methods, but missed by QF-PCR. We therefore suggest FISH analysis to rule out (mosaics of) aneuploidies, in case QF-PCR test is negative after a positive NIPT result.

P2-36 Whole genome maternal plasma DNA sequencing for fetal aneuploidy accurately detects autosomal and sex chromosome abnormalities: A validation study with independent fetal fraction observations Brendan Tarrier, Matthew Sekedat, Tobias Mann, Jay Stoerker, Allan Bombard Progenity, Ann Arbor, Michigan, United States OBJECTIVES: The overall goal of noninvasive prenatal screening (NIPS) is to reduce the number of invasive procedures by decreasing the false positive rates and © 2015 John Wiley & Sons, Ltd.


minimizing anxiety surrounding multi-step screening. The demand for NIPS is high, and a need for well validated assays exists. Recently concern has been raised regarding whole genome plasma DNA sequencing and the quality of findings in the absence of fetal fraction determination. Our objective was to compare results from our validated whole genome plasma sequencing assay with an independent index for fetal fraction in consecutive cases. Our experimental hypothesis is that the normalized chromosome value (NCV) is not influenced by the fetal fraction. METHODS: Plasma samples were collected in a prospective blinded study from 3,000 women undergoing prenatal diagnostic procedures. Independent classifications for chromosomes 21, 18,13, male, female, and other sex chromosome anomalies were made for each sample and compared to a reference method (verifi ™). NCVs were used to establish whether fetuses were affected or unaffected for each trisomy. A fetal fraction index was developed using single nucleotide polymorphisms (SNPs) with a high minor allele frequency (MAF). The independently validated index is applied to a subset of the validation cohort. RESULTS: Within an analysis cohort of 3000 samples including > 150 T-21, > 80 T-18, and > 50 T-13 cases, we report specificity, sensitivity, and 95% CI. In addition, we report on fetal fraction determinations in a subset of consecutive samples, and its relationship to NCV scores. CONCLUSIONS: Our clinically validated whole genome maternal plasma DNA sequencing NIPS is used to confirm or deny the hypothesis that fetal fraction in plasma affects the NCV statistic. Additionally, we examine specifically the trisomic chromosomes for this affect.

P2-37 Karyotype level non-invasive prenatal testing by sequencing of circulating cell-free DNA from maternal plasma Amin Mazloom, Tong Liu, Roy Lefkowitz, Grant Hogg, John Tynan, Tim Burcham, Dirk van den Boom, Mathias Ehrich Sequenom Laboratories, San Diego, California, United States OBJECTIVES: Non-invasive prenatal testing (NIPT) has become an accepted method for the detection of trisomy 21, 18 and 13 in patients at high risk to carry a fetus with one of these chromosomal abnormalities. NIPT can also be used to detect other aneuploidies and sub-chromosomal copy number variations. With the inevitable progress of sequencing technologies, it is likely that sequencing-based NIPT will offer greater diagnostic capabilities in the same way that karyotypinglevel resolution was eventually superseded by microarray analysis of invasively acquired fetal cells or tissue. We present studies that support the concept of NIPT delivering genome-wide unbalanced copy number results, analogous to what can be detected by cytogenetic G-band karyotyping. METHODS: We analyzed a set of maternal plasma samples for which karyotype confirmation of fetal anomalies were available. Next, we constructed a comprehensive in-silico system of maternal plasma DNA sequencing results modeled to contain sub-chromosomal events at genomic coordinates reported in the ISCA database. Finally, we constructed an analytical model system with both karyotype Prenatal Diagnosis 2015, 35, 27–109

and microarray confirmed samples with genome wide subchromosomal deletion and duplication events. RESULTS: In karyotype confirmed samples, we detected 17 of 18 samples with a sub-chromosomal deletion or duplication. In-silico modeling of 587 genome wide ISCA sub-chromosomal deletion and duplication events ≥7 Mb showed that sensitivity could reach 94% with high specificity. CONCLUSIONS: Using whole genome sequencing results, we were able to model in-silico genome wide events and estimate sensitivity metrics for their detection. Measurement of analytical performance in mixture models with confirmed deletions/duplications is currently ongoing.

P2-38 Two cases of discrepancy between ultrasound assigned fetal sex and cell free DNA (cfDNA) screening Wendy White, Kristi Borowski, Myra Wick Mayo Clinic, Rochester, Minnesota, United States OBJECTIVES: The determination of fetal sex can often be made in the late first trimester with cfDNA screening, but problems can arise at the time of the anatomic survey, when the fetal sex noted on ultrasound is discrepant from the cfDNA result. We report two cases with a discrepancy between ultrasound assigned sex and results on cfDNA, with two different likely explanations. METHODS: Two cases were identified at one academic center where the cfDNA sex result was called into question at the time of anatomic survey. Fetal sex was resolved with amniocentesis, FISH and chromosomal microarray, and the likely etiology of discrepancy was determined from thorough review of medical history and serial imaging. RESULTS: Case 1. was an IVF pregnancy with two embryos transferred, but only one gestational sac ever identified, that had cfDNA screening performed at 11 weeks showing the presence of Y chromosome material. At a 20 weeks anatomic ultrasound, normal female genitalia was seen. Amniocentesis revealed a normal female prenatal FISH, and 46 XX was confirmed on array. Case 2. was the result of a singleton pregnancy with no Y chromosome material detected on cfDNA at 11 weeks. At the time of ultrasound at 19 weeks, a mass was present on the perineum resulting in suspicion for ambiguous genitalia. However, amniocentesis confirmed a normal female via prenatal FISH, confirmed 46 XX on array. Serial ultrasound and fetal MRI at a later gestational age suggested the mass was part of an anorectal malformation closely adjacent to normal female genitalia. CONCLUSIONS: Due to the sensitivity of cfDNA to detect Y chromosome material in the setting of an XX mother, screening early in pregnancy may result in a false positive male fetal sex assignment due to the residual presence of male trophoblast tissue even in the absence of an identifiable second gestational sac. Masses in the area of the perineum or sacrum can complicate visual assignment of fetal sex at the time of ultrasound. In cases where cfDNA sex is discordant from ultrasound assigned sex, additional imaging and amniocentesis can clarify both the true fetal sex, and the cause. © 2015 John Wiley & Sons, Ltd.


P2-39 Discordant NIPT results due to complex confined mosaic of placenta and cord blood Saknan Manotaya1, Yuen Tannirandorn1, Huixin Xu2, Fang Chen2 1

Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 2 BGI Research, Shenzhen, Guangdong, China OBJECTIVES: Nowadays several studies reported that reduced accuracy of NIPT for detecting sex chromosomal abnormalities (SCAs) were mostly caused by confined placental mosaicism, maternal mosaicism or fetal mosaicism. In such confined placental mosaic cases, discordant karyotypes of 45, XO for placenta and 46, XX for amniotic fluid cells or cord blood were always observed and could explain the false positive or negative results. Herein, we reported a very unique case where discordant karyotypes of [45, X (45/49) /46,X, +15(4/49)] in placenta and [45, X (19/100) /46, XY (56/100) /47,XYY (25/100)] in cord blood were identified by full karyotyping, which may originate from mitosis in early stage during embroyo development and result in a false positive result for SCA detection and wrong fetal sex determination in NIPT. METHODS: Plasma samples were isolated from five milliliters of maternal blood and sent to the clinical laboratory of BGI DX for low coverage whole genome sequencing. RESULTS: The NIPT result showed low risks of fetal trisomy 21, 18 and 13, but high risk of XO. The cell-free fetal DNA fraction was estimated by T-score of the X chromosome to be 9.6%. Since the patient rejected invasive prenatal diagnosis, ultrasound examinations at 16’s and 21 weeks’ gestation were recommended, showing a singleton live male fetus of a normal size and no gross abnormality. After careful examination and post-test counseling, the patient decided to continue pregnancy. A healthy male fetus with no obvious abnormality was delivered at 41 weeks’ gestation. Full karyotyping of maternal peripheral blood, cord blood and placenta revealed that complex mosaicism was observed in both cord blood [45,X (19/100) /46, XY (56/100) /47,XYY (25/100)] and placenta tissues [(45, X (45/49) /46,X, +15(4/49)), showing genetic discrepancy between the fetus and placenta on sex determination. CONCLUSIONS: Based on the assumption that the cell-free fetal DNA that NIPT assesses is mostly originated from trophoblast cells in placenta, confined placental and fetal mosaicism in this case may be the main reason leading to discordant SCA result in NIPT. Thus, we suggest that for SCA cases, more careful considerations, such as a series of ultrasound scanning, could be an alternative option if the patient reject invasive procedures, with proper post-test genetic counseling to ensure well- informed decisions and full understanding of NIPT.

P2-40 Withdrawn. P2-41 Nationwide evaluation on noninvasive prenatal testing through maternal plasma DNA sequencing under governmental regulation in China Jingmei Ma1, Huixia Yang1, Fang Chen2, Yicong Wang2 Prenatal Diagnosis 2015, 35, 27–109


Peking Univeristy First Hospital, Beijing, China BGI Research, Shenzhen, China


OBJECTIVES: Noninvasive prenatal testing (NIPT) for fetal chromosomal aneuploidy with maternal plasma DNA sequencing has been commercialized since 2011 worldwide including China. Whether or not should NIPT be regulated by official Food and Drug Administration (FDA) is controversial. In February 2014, NIPT was suspended by Chinese FDA (cFDA) until the quality and reliability was evaluated. METHODS: A multicenter clinical trial was conducted with cFDA oversight. At 20 medical centers across China, perspective plasma from February to May 2014 and retrospective samples from February 2013 to May 2014 were recruited. All perspective samples were from women with singleton pregnancy before invasive procedure, while subsequent fetal karyotypes or birth outcomes were collected as the gold standard for respective samples. Maternal plasma DNA sequencing was performed with BGISEQ-1000 sequencer under the regulation of cFDA. RESULTS: Totally 10,598 cases were recruited, including 2,439 perspectively recruited samples (median age, 32 years) and 8159 retrospective samples. After 19 cases were excluded, 2.03% (215/10579) cases were classified as test positive, including 1.55% (164/10579) cases of trisomy 21, 4.54%(48/ 10579) cases of trisomy 18 and 0.02% (3/10579) cases of trisomy 13. Compared with fetal karyotyping results and fetal outcome data, two false positive case of trisomy (T) 21, two false positive case of T18 and no false negative cases indicated 100% sensitivity and 99.96% specificity for the detection of T 21, 18 and 13. For NIPT, the positive predictive value and the negative predictive value was 98.14% [95% confidence interval] and 100.04% [95% confidence interval]), respectively. CONCLUSIONS: The nationwide evaluation of NIPT in China under the regulation cFDA showed that in the high-risk population, noninvasive prenatal testing had significantly lower false positive rates and higher positive predictive values than current prenatal screening.

P2-42 Birth of a child with mosaic trisomy 9 syndrome associated with paternal uniparental disomy 9: A case of discordant results between non-invasive and invasive prenatal testing Jingmei Ma, Huixia Yang, Hong Pan Peking Univeristy First Hospital, Beijing, China OBJECTIVES: In China, non-invasive prenatal testing (NIPT) is commonly used as a frontline screening test to identify fetuses with trisomy 21, 18 or 13. Occasionally, incidental NIPT results are reported suggestive of a fetus with a rare chromosome disease syndrome. We describe a child with mosaic trisomy 9 where the prenatal history reported a positive NIPT result for trisomy 9 that was unconfirmed by conventional prenatal diagnosis. METHODS: NIPT was performed by low coverage whole genome plasma DNA sequencing. Karyotyping and FISH analysis with chromosome 9p-ter and 9q-ter probes was used to determine the somatic cell level of trisomy 9 mosaicism in the fetus and child. Quantitative fluorescent PCR (Q-PCR) of highly polymorphic short tandem repeat markers was also © 2015 John Wiley & Sons, Ltd.


performed to investigate the nature of the trisomy 9 mosaicism and the parental origin. RESULTS: A 22 month old girl presented at our genetic counseling clinic at the Peking University First Hospital with severe motor and intellectual disability, recurrent respiratory infection and failure to thrive, consistent with the phenotype of trisomy 9 mosaicism syndrome. Review of the prenatal testing history revealed a positive NIPT result for chromosome 9. However, follow up confirmatory karyotyping and FISH analysis of blood cells returned a normal karyotype. Post-natal studies of somatic cell trisomy 9 mosaicism by FISH detected levels of 20% in blood and buccal cells. Q-PCR analysis of family DNA samples suggested that the trisomy 9 mosaicism probably originated by trisomic rescue of a paternal meiotic I chromosome 9 non-disjunction error resulting in the formation of two distinct somatic cell lines in the proband, one with paternal uniparental disomy 9 and one with trisomy 9. CONCLUSIONS: This study shows that NIPT may also be a useful screening technology to increase prenatal detection rates of rare fetal chromosome disease syndromes. P2-43 Organ transplantation from a male donor as a cause of discrepant sex results on noninvasive prenatal testing: A case report Kathryn Gray, Erin Thorpe, Marie Discenza, Katherine Economy, Louise Wilkins-Haug Brigham & Women’s Hospital, Boston, Massachusetts, United States OBJECTIVES: To report prior organ transplantation from a male donor as an additional source of discrepant sex results on cell-free DNA testing. METHODS: A 38 yo G3P1011 at 12.1 weeks presented for genetic counseling due to advanced maternal age. She opted for prenatal testing by cell-free DNA (Verifi). She had a history of Henoch-Schönlein Purpura (HSP) and was status post living related renal transplant from her brother in 2008. RESULTS: Cell-free DNA testing returned as “No aneuploidy detected” for chromosomes 13, 18, and 21 and predicted male sex. Prenatal ultrasound performed at 17.1 weeks demonstrated female genitalia. Given this discrepancy, the patient underwent amniocentesis. Karyotype performed on the amniotic fluid was normal and confirmed female sex (46, XX). The patient was counseled that the cell-free DNA testing may have detected the presence of a Y chromosome due to her renal transplant from her brother. Confirmation of fetal karyotype and repeat cell-free DNA analysis are planned following delivery. CONCLUSIONS: Discrepancies between genetic sex reported on cell-free DNA testing and that of the fetus confirmed by karyotype of amniotic fluid prenatally or peripheral blood after birth are often due to biologic phenomena. Thus, it is important to obtain a thorough medical and family history from patients undergoing cell-free DNA testing. Patients with a history of organ transplantation from a male donor should be counseled regarding the possibility of erroneous sex results when undergoing noninvasive prenatal testing with cell-free DNA. Prenatal Diagnosis 2015, 35, 27–109

P2-44 An HNF1B mutation as a cause of familial renal dysplasia: A case report Kathryn Gray, Katherine Economy, Joel Krier Brigham & Women’s Hospital, Boston, Massachusetts, United States OBJECTIVES: To describe a patient with a personal and family history of renal dysplasia who was found to have an HNF1B mutation and subsequently underwent preimplantation genetic diagnosis (PGD). METHODS: A 31 yo G0 presented for adult genetics consultation after referral from maternalfetal medicine due to her personal and family history of renal dysplasia. The patient had a history of bilateral echogenic kidneys on prenatal ultrasound and developed end-stage renal disease in late adolescence. She had a living related renal transplant from her mother at age 20, which she ultimately rejected. Six years later, she had a second living related kidney transplant from a paternal uncle. She developed insulindependent diabetes following her initial kidney transplant. Of note, the patient’s mother developed an elevated creatinine of 1.7 following kidney donation that never returned to prior normal baseline. The patient also had a brother who had bilateral renal agenesis and died shortly after birth. Following consultation with adult genetics, the patient underwent HNF1B gene sequencing. The HNF1B gene encodes a transcription factor critical for the development of the kidney and the pancreas and mutations in HNF1B can lead to congenital renal anomalies, pancreas atrophy, and maturityonset diabetes of the young (MODY). RESULTS: HNF1B gene sequencing identified a heterozygous nonsense mutation in HNF1B, c.232G > T (p.Glu78X). This mutation has been reported in at least two other patients with renal dysplasia. Testing of the patient’s first-degree relatives revealed that the patient’s mother has the same mutation, while her healthy sister tested negative. The patient is now pursuing PGD to select embryos negative for the mutation for in vitro fertilization (IVF). CONCLUSIONS: Patients with a history of familial renal dysplasia may benefit from adult genetics evaluation prior to attempting pregnancy, as the genetic basis for inherited renal disease is increasingly understood. When causative mutations are identified, patients can be offered PGD to prevent transmission of inherited kidney disease. This particular case also underscores the extremely variable phenotype possible for HNF1B mutations within a family.

P2-45 The changing prenatal diagnosis pattern of single gene disorders Jingmei Ma, Hong Pan, Huixia Yang Peking University First Hospital, Beijing, China OBJECTIVES: To evaluate the changing patterns of prenatal diagnosis for single gene disorder (SGD) with the application of multiple discipline referral workflow. METHODS: From January, 2012 the workflow of referral for SGD prenatal diagnosis was established, involving departments of Obstetrics, © 2015 John Wiley & Sons, Ltd.


Pediatrics, Neurology, Dermatology and Central Laboratory. Each pregnancy should be guaranteed the diagnosis of index case in the relevant department, then have prenatal diagnosis by Obstetrician. All of them were registered and followed for the test result and pregnancy outcome. Till December 2014, there were 279 prenatal cases with SGD indication (Total prenatal diagnosis, 6558), 76 of them was CVS (11–14 gestational weeks),and 203 of them was amniocentesis (AC)(16–22 gestational weeks). The proportion of and safety of each procedure were compared with Chi-square analysis. RESULTS: The proportion of SGD indication in AC group stayed at the same level (2012:2.3%, 47/2054, 2013:3.9%, 78/2023, 2014:3.4%, 78/2278, total 3.2%, 203/6355), no significant difference between 2013 and 2014 (χ2 = 0.571,P = 0.463). While in CVS group, the proportion increased (2012:18.2%, 8/44, 2013:17.6%, 19/108, 2014:28.2%, 49/174, total 23.3%, 76/326).There was significant difference between 2013 and 2014(χ2 = 4.067,P = 0.046). During the three years, the proportion of SGD indication in CVS group was higher than AC group (2012, χ2 = 42.626, 2013, P = 0.000, χ2 = 44.531, P = 0.000, 2014, χ2 = 201.400, P = 0.000). There was No other procedure related complication from both prenatal diagnosis approaches in the follow-up registration other than one Intra-uterine fetal death after 6 month of CVS procedure. 3 chromosome anomaly were found with one trisomy 18, one 45,X, and one mosaicism of 45,X/46,XY (then determined to be normal in AC). CONCLUSIONS: SGD is one of important indications in prenatal diagnosis, following established referral workflow with multiple discipline management, SGD prenatal diagnosis as early as first trimester with CVS is feasible.

P2-46 Preimplantation genetic screening of blastocysts by multiplex qPCR followed by fresh embryo transfer: Validation and verification Ming Chen, Shun-Ping Chang, Gwo-Chin Ma, Wen-Hsiang Lin Changhua Christian Hospital, Changhua, Taiwan OBJECTIVES: Aneuploidy is an important etiology of implantation failure and quantitative real-time polymerase chain reaction (qPCR) seems a promising preimplantation genetic screening (PGS) technology to detect aneuploidies. This verification study aimed at verifying the impact on reproductive outcomes in IVF cycles using fresh embryos transfer in which the embryos were selected by blastocyst biopsy with qPCRbased PGS in our settings. METHODS: A total of 13 infertile couples with more than once failed in vitro fertilization were enrolled during July to October of 2014. PGS was conducted by qPCR with selectively amplified markers to detect common aneuploidies (chromosomes 13, 18, 21, X, and Y). The design of the qPCR molecular markers adopted the locked nucleic acid (LNA) strategy. The blastocyst biopsy was performed on day 5/ 6 and the PGS was done on the same day, which enabled fresh embryo transfer (FET). RESULTS: A total of 72 blastocysts were biopsied. Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100%. The aneuploidy rate was 38.9% (28/72). 28 embryos were transferred. The clinical pregnancy rate was 61.5% (8/13) per cycle. Early first Prenatal Diagnosis 2015, 35, 27–109

trimester abortion was encountered in 1 and the ongoing pregnancy rate was 53.8% (7/13) per cycle. CONCLUSIONS: This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting. Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted. P2-47 Withdrawn. P2-48 Pre-implantation genetic diagnosis in the Netherlands: Experience of a national committee assessing disease eligibility Martin Breuning1, Ineke Bolt2, Annelien Bredenoord3, Robert Jan Galjaard2, Jacques Giltay3, Jan van Lith1, Cor Oosterwijk4, Hajo Wildschut5 1 Leiden University Medical Center, Leiden, Netherlands 2 Erasmus MC, Rotterdam, Netherlands 3 UMC Utrecht, Utrecht, Netherlands 4 VSOP, Soest, Netherlands 5 West-Fries Gasthuis, Hoorn, Netherlands OBJECTIVES: Evaluate 5 years of PGD eligibility assessment by Dutch expert committee. In 2008 a heated debate took place within the Dutch coalition government concerning the eligibility of pre-implantation genetic diagnosis for inherited breast and- ovarian cancer due to a BRCA mutation. The Dutch parliament reached consensus on criteria for the application of PGD. The fetus should be at high risk for a serious condition for which there is no treatment. Eventually, the debate led to the formation of a national committee of experts to assess the eligibility of PGD requests for diverse genetic disorders and establish guidelines for PGD eligibility. METHODS: A committee existing of representatives of patient organisations, ethicists, obstetricians, and clinical geneticists assessed each PGD request for a novel disease indication. Individual case histories were taken as examples of a particular genetic disease or syndrome, and combined with the information provided by the literature. Criteria were discussed at length, taking into account the burden of the procedure itself as well. RESULTS: In the past 5 years the committee assessed 58 inherited conditions from all fields of medicine, with diverse inheritance risk and disease penetrance. In clinical practice it remains extremely difficult to define concepts such as ‘serious’ and ‘treatable’ due to the wide variability in phenotype of inherited diseases, and the currently limited efficacy of the few treatments available. The majority of conditions submitted to the committee are characterized by substantial morbidity, inconvenient or ineffective therapy and/or risk of early death. In those cases the committee issued a positive indication, provided that extensive genetic counseling be given in every case. Regarding a small subset of conditions the committee issued a ‘no, unless’, leaving some leeway for the counsellor to make exceptions. Examples of these are: some variable forms of cardiomyopathy, such as Long QT Syndrome (HERG stop mutation), and Cleidocranial Dysostosis (RUNX3 © 2015 John Wiley & Sons, Ltd.


mutation). On some requests for PGD, the committee has not reached agreement. An example is the so-called susceptibility loci (submicroscopic deletions and duplications) for mental retardation. CONCLUSIONS: The majority of couples can now be offered PGD for a wide spectrum of genetic disorders. P2-49 Whole exome sequencing as a diagnostic tool for cases of fetal demise or termination Deborah Copenheaver, Jane Juusola, Patrik Vitazka, Bethany Friedman GeneDx, Gaithersburg, Maryland, United States OBJECTIVES: We examine the use of whole exome sequencing in determining a molecular genetic basis in cases of fetal demise or termination for fetal anomalies. METHODS: Whole exome sequencing was performed on a total of 31 fetal demise/termination samples. Clinical findings for the samples included multiple congenital anomalies, hydrops, skeletal anomalies, brain malformations, hydrocephalus, congenital nephrosis, diaphragmatic hernia, renal agenesis, anemia and facial edema. 28 of the cases were performed on the proband, maternal and paternal samples and for 7 of these 28 cases, additional family member samples were submitted. Three were performed as singleton cases. RESULTS: A definitive result was detected in nine of the cases (29%). Three of the definitive cases involved autosomal recessive conditions. Ten of the cases (32%) revealed a variant(s) of unknown clinical significance. Four of the cases (13%) yielded indeterminate results with variants in candidate genes that may be associated with the phenotype. Eight cases had negative results (26%). CONCLUSIONS: Three cases reported having a previous negative microarray result while another 17 cases reported that the pregnancy had previous chromosome analysis and chromosomal microarray analysis that was normal. Three of the cases had genetic testing for specific disorders that were negative. These data of a definitive diagnosis in 30% of our fetal demise/ termination cases support the clinical utility of whole exome sequencing as a valuable tool for genetic diagnosis in cases of fetal demise or termination for fetal anomalies. Whole exome sequencing is a promising method with which to identify genetic variants that cause fetal abnormalities and thereby give families closure as well as improve the reproductive decisions and clinical management of future pregnancies. P2-50 Study of a single-nucleotide polymorphism; rs 1799884 of glucokinase gene with gestational diabetes mellitus in a sample of Egyptian patients Dalal Elkaffash Alexandria University, Alexandria, Egypt OBJECTIVES: The aim of the study was to detect the role of variation of enzyme glucokinase (GCK) gene in determination of fasting glucose in pregnant women and birth weight using a tagging single-nucleotide polymorphism (tSNP) approach of rs 1799884 in a sample of Egyptian patients. The study was a Prenatal Diagnosis 2015, 35, 27–109

cross sectional study carried on 50 patients with definite gestational Diabetes Mellitus (GDM). METHODS: The glucose tolerance status was assessed with OGTT using the WHO criteria at 24–28 weeks of pregnancy. Determination of blood glucose level was done on Dimension analyzer using Hexokinase (HK) method. All pregnant women with positive screen test for GDM (O’ Sullivan test) and positive confirmatory test Oral glucose tolerance test, will be subjected to detection of rs 1799884 in enzyme glucokinase gene by Light Cycler– PCR by melting point detection. RESULTS: All genotype frequencies were in the Hardy-Weinberg equilibrium analysis. (X2 = 2.29, p > .05). There was no significant difference in fasting glucose (mg/dl) between patients with GG genotype (median = 116, ranging from 95 to 150) and those with GA genotype (median = 109, ranging from 95to 160). (t = 0.526, p = .601). There was no significant difference in birth weight between patients with GG genotype (median = 3750, ranging from 2800 to 4500) and those with GA genotype (median = 3800, ranging from 3650 to 4700). (Z = 1.017, p = .309). CONCLUSIONS: No apparent statistically significance was found between the -30G/A polymorphism with fasting glucose in GDM patients or offspring birth weight in our sample patients.

P2-51 Preimplantation genetic diagnosis in Sweden: Patients’ experience and attitudes Charlotta Ingvoldstad, Katarina Haapaniemi Kouru, Helena Malmgren, Elisabeth Syk Lundberg Karolinska University Hospital, Stockholm, Sweden OBJECTIVES: Couples at high risk of having affected offspring are in a difficult reproductive situation. Preimplantation genetic diagnosis (PGD) is a reproductive alternative for these couples with the advantage that pregnancy termination of an affected foetus can be avoided. The aim of this study was to learn more about patient’s psychological and physical experience of PGD in Sweden. METHODS: A retrospective cohort study among 222 couples that went through PGD in Stockholm between June 2005 and December 2011 was performed. A study-specific questionnaire was used together with a validated selfassessment scale for detecting states of depression and anxiety (HAD Scale). A pilot study had been performed during the first years of PGD activity in Sweden (1998 to May 2005). The following questions were explored a) What was the reason to opt for PGD? b) How did the couples experience the procedure and which part was most stressful? c) Did factors like previous reproductive history or experience of previous traditional prenatal diagnosis affect the experience of PGD? d) What reproductive option was considered after closure of PGD? RESULTS: The results from this study indicate that both physical and psychological stress was experienced during PGD, but the psychological stress was lower compared to traditional prenatal diagnosis (PND). Previous reproductive history did not significantly affect the couple’s experience of PGD. The main reason to choose PGD was objection to pregnancy termination (carriers of monogenic disorders) or avoidance of miscarriage (carriers of chromosome abnormalities). In spite of the extensive © 2015 John Wiley & Sons, Ltd.


experienced stress, PGD is a preferred reproductive alternative for couples at high-risk of having an affected child. CONCLUSIONS: In conclusion, the results from this survey show that, despite an extensive experienced stress, PGD is a preferred alternative among the different reproductive options that are available for couples at high risk of having an affected child. This was supported by the fact that 94% of our patients would recommend PGD to other couples in the same situation. P2-52 Exome sequencing on a family with 3 pregnancies affected by central nervous system malformation identified a novel stop mutation in WDR81 Anita S.Y. Kan1, Jiaen Deng2, Patrick K.C. Au3, Miaoxin Li2, Kelvin Y.K. Chan3, Elizabeth T. Lau3, Chun-Yiu Law4, Chin-Peng Lee1, Mary H.Y. Tang1, Pak-Chung Sham2, Ching-Wan Lam4 1

Prenatal Diagnostic and Counselling Division, Tsan Yuk Hospital, Hong Kong, China 2 Centre for Genomic Sciences, the University of Hong Kong, Hong Kong, China 3 Prenatal Diagnostic Laboratory, Tsan Yuk Hospital, Hong Kong, China 4 Department of Pathology, the University of Hong Kong, Hong Kong, China OBJECTIVES: Exome sequencing was performed on a Chinese non-consanguineous family with 3 previous pregnancies affected by central nervous system malformation. All three affected pregnancies showed 46,XY karyotype and DNA samples were retrieved for analysis. METHODS: We performed whole exome sequencing (WES) on the second pregnancy, along with the unaffected parents using Illumina Hiseq sequencing platform. The sequence variants are called by GATK ( WES data filtering was performed using VariantStudio (v2.2.1, Illumina). The damaging effects of SNVs on gene products were assessed by PolyPhen-2 and SIFT. RESULTS: A total number of 201503 variants were identified from exome sequencing result and 19632 of them passed the family-based filtering. After further filtering the variants with minor allele frequency A (p.Ser1665Arg) inherited from mother and a novel stop-gain mutation c.4626G > A (p.Trp1542*) inherited from father. The findings were confirmed by Sanger sequencing. This novel stop-gain mutation removed the important WD domains of WDR81. CONCLUSIONS: Our exome sequence analysis suggests two promising mutations in WDR81 gene which is associated with central nervous system malformation.

P2-53 COL7A1 gene mutation analysis of dystrophic epidermolysis bullosa and prenatal diagnosis Xiangdong Kong, Liu Ning Prenatal Diagnosis 2015, 35, 27–109

The First Affiliated Zhengzhou, China





OBJECTIVES: To analyze the mutations of COL7A1 gene in two dystrophic epidermolysis bullosa pedigrees and to make prenatal diagnosis on the high-risk fetus which has been pregnant for eleven weeks. METHODS: COL7A1 gene was first analyzed by next-generation sequencing for seeking suspicious gene mutations of the two probands. And then the mutations were confirmed by polymerase chain reaction and Sanger sequencing in the probands and the parents and unrelated healthy individuals. Prenatal genetic diagnosis for the highrisk fetus was performed by chorionic villus sampling after the genotypes was identified. RESULTS: The four mutations were detected in the two pedigrees:c.5230G > T (p.E1744X),c.5932C > T (p.R1978X),c.5605-10 T > G(IVS6610 T > G),c.8305-1G > A(IVS110-1G > A),among which p.E1744X IVS66-10 T > G and IVS110-1G > A mutations were first reported. The proband in the No.1 family carried p.E1744X and p.R1978X nonsense mutations,and her parents were respectively carriers. The proband in the No.2 family carried IVS66-10 T > G and IVS110-1G > A splicing mutations,and her parents were respectively carriers.All the four mutations were not found in the 100 healthy individuals. Prenatal diagnosis in the No.1 family indicated that the fetus also carried p.E1744X and p.R1978X nonsense mutations as the proband. The fetal parents decided termination of the pregnancy, and the result of gene analysis for the aborted tissue was consistent with that of prenatal diagnosis. CONCLUSIONS: COL7A1 gene mutation is causative of the two dystrophic epidermolysis bullosa families. Nextgeneration sequencing combine Sanger sequencing is an effective and accurate method to make gene diagnosis and prenatal diagnosis.Avoiding the birth of DBE fetuses is the best choice.

P2-54 A new strategy for controlling birth defect: Pregestational screening recessive hereditary disease by next generation sequencing Can Liao1, Wei-yue Gu2 1

Prenatal Diagnostic Center, Guangzhou Women & Children Medical Center, Guangzhou City, Guangdong Province, China 2 Beijing DE Yi Oriental Translational Medicine Research Center, Beijing, China OBJECTIVES: The birth defect rate of low-income countries, middle-income countries and high-income countries around the world is 6.42%, 5.57% and 4.72%, respectively. And the incidence of monogenic disease is approximately 3%, which composed by the vast majority of recessive genetic disease. This study aimed to pregestational screening recessive hereditary disease by next generation sequencing. METHODS: A total of 100 normal male and 100 normal female individuals were sequencing with specialized Panel including 1515 genes covering all 1800 autosomal recessive inherited diseases within OMIM database. The sequencing was run on Hiseq2500 by a sequencing strategy 2 * 100, with an average depth of 100X and coverage of 99%. High-throughput sequencing data were step by step analysed by clinical levels © 2015 John Wiley & Sons, Ltd.


analysic system and screened four kinds of precisely detrimental mutation forms (harmful mutations reported in literatures, terminator codon mutations, frame shift mutations and the classic splice site mutations). Finally, computer programming made all 100 male and 100 female mutational datas paired randomly. RESULTS: 100 male and 100 female can constitute 10000 kinds of random combination. Among total of 10000 pairs, high risk of mutations in the same disease-causing genes were detected in 214 pairs, leading to 2.14% of recessive hereditary disease screening positive rate. CONCLUSIONS: Our results demonstrated the value of pregestational screening recessive hereditary disease by next generation sequencing. Even if just screening the above four kinds of mutations, there are up to 2.14% of couples exist high risk of having offspring with recessive genetic disease, which constitute 0.5% of population birth defect. The screening results will provide useful information to fetal medicine doctors.

in exon 03 of PTPN11 was identified. In neonatal cases 3 and 4, a c.34G > A (p.Gly12Ser) transition in exon 02 of NRAS was identified. Cases 3 and 4 each passed away due to NSDassociated complications within the neo-natal period, while cases 1 and 2 had multiple severe abnormalities detected by prenatal ultrasound and were electively terminated. Parental testing identified that the variants were de novo in case 1, 2, and 4. Parents were unavailable for case 3. CONCLUSIONS: JMML-associated pathogenic variants cause a severe form of NS incompatible with life past the neonatal period when inherited as germline changes.

P2-55 JMML-associated variants cause neo-natal lethal Noonan syndrome

Progenity, Ann Arbor, Michigan, United States

Heather Mason-Suares1, Diana Toledo2, David Sharpe3, M. Cristina Pacheco4, Katherine Lafferty2, Jean Gekas5, Naomi Meeks6, Thomas Mullen2, Heidi Rehm2, Matthew Lebo7 1

Brigham Women’s Hospital, Cambridge, Massachusetts, United States 2 Laboratory for Molecular Medicine, Partners Personalized Medicine, Cambridge, Massachusetts, United States 3 Riverside Methodist Hospital, Columbus, Ohio, United States 4 Children’s Hospitals & Clinics of Minnesota, Saint Paul, MN, United States 5 Le CHU de Québec, Québec, Canada 6 University of Colorado Anschutz Medical Campus , Aurora, Colorado, United States 7 Laboratory for Molecular Medicine, Partners Personalized Medicine, Cambridge, Massachusetts, United States OBJECTIVES: Germline gain-of-function mutations in the Ras– MAPK pathway genes PTPN11 and NRAS are associated with Noonan syndrome (NS) whereas somatic gain-of-function mutations cause isolated Juvenile myelomonocytic leukemia (JMML). In general, JMML-associated variants do not overlap with NS-associated variants. This is attributed to a stronger gain-of-function activity for the JMML-associated variants and has led to the suggestion that these variants would be embryonic lethal. METHODS: Two prenatal and two neonatal cases were tested for a NSD by next generation sequencing of the PTPN11, SOS1, RAF1, KRAS, NRAS, BRAF, MAP2K1, MAP2K2, HRAS, SHOC2 exon 02, CBL and SPRED1 genes, with confirmation testing performed using Sanger sequencing. Parental samples were tested by Sanger sequencing when available. RESULTS: Three variants, previously only observed as somatic changes in isolated JMML, were identified in four cases with a severe preor neo-natal presentation of NS. In prenatal case 1, a c.227A > G (p.Glu76Gly) transition in exon 03 of PTPN11 was identified. In prenatal case 2, a c.214G > A (p.Ala72Thr) transition Prenatal Diagnosis 2015, 35, 27–109

P2-56 Yield of carrier screening for preconception individuals of Ashkenazi and Persian Jewish ancestry at community based events by targeted mutation panels Tessa Niemchak, Lindsay Dohany, Christina Settler, Stephanie Farner, Carol Holland, Jeana DaRe

OBJECTIVES: The Jewish Genetic Disease Prevention Program aims to identify carriers of common diseases in the Ashkenazi and Persian/Mizrahi/Sephardic Jewish populations. Screening is performed through community events. This study describes the results from preconception community-based clinical sites. METHODS: Samples from community-based clinical sites were obtained during November 2014. Individuals were provided an opportunity to discuss testing, give informed consent, and determine the testing most appropriate for their ancestry. Ancestry-based panel testing was then offered. The Ashkenazi Jewish Panel (AJP) screens for carrier status for twenty conditions while the Persian Jewish Panel (PJP) consists of nine different tests. RESULTS: Carrier testing was performed for 320 individuals and a total of 363 tests; 259 AJP tests and 104 PJP tests were performed. Forty-three individuals underwent testing for both panels due to mixed Jewish ancestry. The AJP identified 103 mutations in 259 individuals for a carrier frequency of 40.8%, while the PJP identified 27 mutations in 104 individuals for a carrier frequency of 24.0%. In total, 130 mutations were identified in 115 individuals for an overall carrier frequency of 37.7%. The most common conditions detected by the AJP were Gaucher disease (25 carriers; 9.7%), Tay-Sachs disease (15 carriers; 5.8%), and Cystic Fibrosis (11 carriers; 4.3%). One carrier of Tay-Sachs disease was identified solely based on hexosaminidase A enzyme analysis. The PJP detected carriers of three conditions: Glucose-6-Phosphate Dehydrogenase Deficiency (12 carriers; 11.5%), Familial Mediterranean Fever (6 carriers; 5.8%), Inclusion Body Myopathy 2 (5 carriers; 4.8%). In addition, individuals of Persian Jewish ancestry had hemoglobin electrophoresis performed which identified four potential carriers (3.9%). CONCLUSIONS: Population based screening with testing panels targeted to both the Ashkenazi and Persian Jewish populations yields a high number of carriers. This also highlights the importance of offering combination testing for Tay-Sachs disease to maximize carrier detection. Finally, providing at-risk individuals with an opportunity for screening in their community is well received based on feedback provided by participants in these events. © 2015 John Wiley & Sons, Ltd.


P2-57 Non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies by relative haplotype dosage Michael Parks, Samantha Court, Siobhan Cleary, Samuel Clokie, Julie Hewitt, Denise Williams, Trevor Cole, Fiona MacDonald, Mike Griffiths, Stephanie Allen Birmingham Women’s NHS Foundation Trust, Birmingham, United Kingdom OBJECTIVES: As part of the NIPSIGEN project (Non-Invasive Prenatal diagnosis for Single Gene disorders), we are aiming to develop and validate a clinical test for the non-invasive prenatal diagnosis (NIPD) of Duchenne and Becker muscular dystrophies (DMD/BMD) in at-risk pregnancies. METHODS: cfDNA was extracted from maternal blood; DNA sample libraries were prepared for massively parallel sequencing on an Illumina MiSeq by enriching specific target regions (200 Kb in total) on chromosome X with a custom built capture probe library; sequencing data was analysed by relative haplotype dosage (RHDO) on 300–400 heterozygous SNPs across a 2.4 Mb long region containing the dystrophin gene. RESULTS: cfDNA extracted from peripheral blood samples of four healthy pregnant donors and two pregnant DMD carriers all bearing a male fetus and undergoing invasive prenatal testing was tested. Fetal genomic DNA from the CVS obtained from the healthy donors was used to identify the haplotype of interest on chromosome X. For the DMD pregnancies, the affected haplotype was identified from genomic DNA obtained from a previously affected sibling. Using RHDO analysis, the allelic imbalance observed in heterozygous SNPs was used to determine over or under-representation of independent haplotype blocks (containing ≥25 SNPs) across the region of interest. The combined outcomes of the haplotype blocks determined the final result. For all healthy pregnancies, over-representation of the expected fetal haplotype was observed for all identified haplotype blocks. Both DMD pregnancies were correctly diagnosed and a single recombination event was also detected. These results suggest a very high sensitivity and specificity for this test based on the samples tested so far. The fetal portion of cfDNA inputted in the RHDO analysis was also calculated using the same allelic imbalance. CONCLUSIONS: This new assay for NIPD of DMD has shown great promise in the initial stages of validation and is highly affordable (2–3 patients can be multiplexed in a single sequencing run). It is also capable of detecting recombination events within the DMD gene by splitting the RHDO analysis into multiple statistically independent haplotype blocks across the targeted region. P2-58 Prenatal molecular genetic diagnosis of phenylketonuria in Kazakhstan Damilya Salimbayeva Scientific Center of Obstetrics, Gynecology and Perinatology, Almaty, Kazakhstan OBJECTIVES: Prenatal molecular genetic diagnosis is an important method of preventing monogenic diseases such as Prenatal Diagnosis 2015, 35, 27–109

phenylketonuria (PKU). But for unknown mutations in a patient`s family member prenatal molecular genetic diagnosis is impossible. METHODS: Analyze of mutations in PAH gene by PCR method in 79 families. VNTR1 (211–239 p.n.), VNTR2 (380–650 p.n.) polymorphisms of DNA-locuses of RAH gene in 10 PKU families by PCR method were studied. RESULTS: The National Register of patients with PKU in Kazakhstan includes information about 114 patients, out of them 79 children with PKU and their families members were investigated by PCRmethod. There are the spectrum and frequency of mutations in PAH gene: R408W 0.367, R261Q 0.063, P281L 0.044, IVS12 + 1G > A 0.019, IVS4 + 5G 0.019, IVS10-11G > A 0.013, R252W 0.060, R158Q 0.060, R243Q 0.095, W187X 0.060, Y387N 0.060, IVS10nt546 0.019, IVS12nt1 0.060, IVS2 + 5G > C 0.060, IVS1014C > G 0.060. The frequency of unknown mutation was 0.316. 42 patients (53.2%) had known widespread mutations, 28 (35.4%) were heterozygous for known mutations and 9 (11.4%) mutations were not identified by common PAH mutations spectrum. Thus, 37 (46.8%) PKU families in RK need indirect prenatal molecular genetic diagnosis by linkage analysis of the markers linked to the PAH gene. In 12 families with known PAH gene’s mutations was direct prenatal molecular genetic diagnosis of fetus DNA. In order to determine the information of studied polymorphic system for indirect diagnosis was examined 10 complete families (including PKU – fetus, proband, mother and father). CONCLUSIONS: Thus, in Kazakhstan families can have prenatal molecular genetic diagnosis for PKU by direct and indirect methods depending on spectrum of PAH gene mutations.

P2-59 Withdrawn. P2-60 Prenatal diagnosis of autosomal reccessive Larsen syndrome Hana Sroka, David Chitayat Mount Sinai Hospital, Toronto, Ontario, Canada OBJECTIVES: We report the prenatal diagnosis of a skeletal dysplasia causing shortening of all long bones associated with bi-allelic inheritance of two mutations in the CHST3 gene causing autosomal recessive Larsen syndrome. METHODS: Prenatal findings were associated with a non-specific from of skeletal dysplasia with shortening of all long bones (humerus, tibia, radius and femur lengths at approximately the 5th percentile); however, post-natal studies, which we describe, were suggestive of autosomal Larsen syndrome RESULTS: Postmortem Faxitron Film Studies demonstrated mild narrowing of the interpedicular spaces of the lumbar spine along with platyspondyly and coronal clefting. The chest, ribs and shoulders were described as normal. There were small acute hypoplastic acetabular notches. The arms were described as micromelic, with mild bowing of the proximal ulna. There was an impression that the fetus had dislocated elbows. The legs were also micromelic with the fibula being longer than the tibia with mild bowing of femurs and tibias. The hands and feet were grossly normal; however, © 2015 John Wiley & Sons, Ltd.


question was raised about the possibility of a modelling defect in the proximal phalanges, particularly the 2nd and 3rd of the left hand. Based on these findings, molecular analysis for CHST3 was performed and revealed compound heterozygous mutations (c.334G > T (Glu 132 Term and c.475 T > A). Parental DNA analysis revealed that the mother is heterozygote for the c.334G > T mutation and the father is heterozygote for the c.475 T > A mutation. These results enabled prenatal diagnosis by CVS in two subsequent pregnancies with one affected and the other not. CONCLUSIONS: We present a case of autosomal recessive Larsen syndrome made following termination of pregnancy for an unspecified skeletal dysplasia. Molecular testing revealed the presence of compound heterozygous mutations in the CHST3 gene.

P2-61 Prenatal diagnosis of severe inherited coagulation factor VII deficiency Yi Zhou, Aiqing Zhang, Zhi-ming He, Qun Fang Fetal Medicine Center, Deparment of Gynaecology and Obstetrics, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China OBJECTIVES: To establish a reference ranges for levels of FVII:C in cord blood in healthy human fetus. To provide prenatal diagnosis for fetus in a family with rare congenital factor VII deficiency base on a combination of molecular genetic diagnosis and comparing the FVII:C levels of the fetus with the normal fetal. METHODS: (1). Fetal blood samples obtained and the FVII:C levels detected in 31 healthy human fetus with the gestational age of 22–32 weeks, excluding the fetus with unusual chromosome, thus, a reference range was built. (2). DNA sequencing was performed to detect mutations in the parents, fetal FVIIgene examined by Sanger method and MLPA to exclude maternal cell contamination through linkage analysis. (3). FVII:C levels of the target fetus was assayed at 26 weeks of gestation and at birth. RESULTS: (1). FVII:C mean levels in 30 normal fetus is 36.8% ranging from 21.5% to 64.6%. The FVII:C levels of fetus in study is 27.5%. (2). Paternal FVII gene was found a splice mutation c.681 + 1G > T, a missense mutation c.1028G > A was detected in maternal FVIIgene and the target fetus did not carry the familiar mutation. CONCLUSIONS: Establish a reference ranges of FVII:C levels in normal fetus(21.5%—64.6%), and fetal FVII:C levels increase slightly with the increasing gestational age. The FVII:C level of the fetal in study lie in normal reference, mutation was not detected in the fetal FVII gene. This is the first report of prenatal diagnosis of inherited coagulation factor VII deficiency in our country.

P2-62 Association of factor V Leiden and factor II G20210A mutations with recurrent pregnancy loss in Iranian women Minoo Rajaei1, Pooneh Nikuei2, Majid Yavarian3, Mehrdad Mohtarami4 Prenatal Diagnosis 2015, 35, 27–109


Hormozgan Fertility and Infertility Research Center, Hormozgan University of Medical Sciences , Bandar Abbas, Hormozgan, Iran 2 Molecular Medicine Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Hormozgan, Iran 3 Shiraz University of Medical Sciences, Bandar Abbas, Hormozgan, Iran 4 Long Island University, Brookville, New York United States OBJECTIVES: Various genetic and non-genetic factors are reported as causes of recurrent pregnancy loss (RPL). After chromosome abnormality, thrombophilia is known as the most important genetic factor for RPL. Thrombophilia gene mutations like Factor V Leiden and factor II G20210A are more common in women with recurrent pregnancy loss than normal population. The purpose of this study was to determine the frequency of factor V Leiden and prothrombine gene mutations in women with RPL compared with women without history of abortion. METHODS: This case control study evaluated the frequency of factor V-Leiden and factor II G20210 mutations in 125 women with two or more pregnancy losses, compared with 315 women who had at least one normal pregnancy without abortion. The mutations were assessed by PCR-RFLP. RESULTS: Frequency of the factor V Leiden mutation among cases was 9.6% which was higher than controls (1.9%). The difference between two groups was significant (p = 0.001)(OR = 0.18 CI = 0.06-0.49). Frequency of prothrombine G20210A, was (23.2%) among cases and (3.8%) in controls. The difference between two groups was significant (p 0.001)(OR = 0.13 CI = 0.06-0.26 ), CONCLUSIONS: These data showed that factor V Leiden and factor II G20210 mutations might play a role in recurrent pregnancy loss in Iranian women.

P2-63 Review article: The imbalance in expression of angiogenic and antiangiogenic factors could be as candidate predictive biomarker in preeclampsia Minoo Rajaei1, Pooneh Nikuei2, Kianoosh Malekzadeh3, Azim Nejatizadeh3 1

Hormozgan Fertility and Infertility Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Hormozgan, Iran 2 Molecular Medicine Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Hormozgan, Iran 3 Molecular Medicine Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Hormozgan, Iran OBJECTIVES: Preeclampsia is an important pregnancy disorder with serious maternal and fetal complications which its etiology has not been completely understood yet. Preeclampsia diagnosis remains a challenge, because it is based on non-specific signs of the disease. Early diagnosis and management of disease could reduce its potential side effects. The VEGF family including VEGF-A, most potent endothelial growth which induces angiogenesis and endothelial cell proliferation and has basic role in vasculogenesis. VEGF and its tyrosine kinase receptors (Flt-1 and KDR) are major factors for fetal and placental angiogenic development. Finding mechanisms involved in expression of angiogenic factors maybe lead to new prognostic and therapeutic points in management of preeclampsia. METHODS: In order to © 2015 John Wiley & Sons, Ltd.


collect papers on preeclampsia and angiogenic and antiangiogenic factors, a comprehensive literature review was conducted in Pubmed, Medline, Science Direct, Cochrane and Google Scholar. We used the following keywords to retrieve related publications: "Pre-eclampsia” OR “Preeclamsia” OR “PE” AND "Aniogenesis” OR “Antiangiogenesis” OR “Anti-angiogenesis” OR “Angiogenic factors “ OR “VEGF” OR “PIGF” OR “VEGF receptor” OR “VEGF-R” OR “FLT” OR “ sFLT1” OR “soluble fms-like tyrosin kinase-1” OR “KDR” OR “FLK” OR “VEGFR-2". RESULTS: Recent researches, has shown capability of some antiangiogenic factors as potential candidate to be used as early predictors for preeclampsia. An imbalance between angiogenic factors and their inhibitors can be considered as reliable biomarker. It is also observed that the ratio of sFlt1 to PlGF is valuable as prognostic marker, yet. CONCLUSIONS: In this review study, VEGF family members role in angiogenic evaluated as biomarker to be used in early clinical diagnostics in preeclampsia.

P2-64 Cerebroplacental ratio as a prediction for adverse perinatal outcomes Inas Babic University of Ottawa, Ottawa, Ontario, Canada OBJECTIVES: To investigate whether cerebroplacental ratio (CPR) is a useful tool in a prediction of poor perinatal

outcomes in intrauterine growth restriction (IUGR) pregnancies METHODS: A prospective cohort study was conducted over a period of 1 year. 177 IUGR pregnancies were followed (IUGR defined as antenatal ultrasound EFW 24 hr (p = 0.001) (Table 1.). Sensitivity, specificity, positive predictive value and negative predictive values for adverse perinatal outcomes were calculated and were 53, 78, 63 and 71, respectively for at least one adverse outcome CONCLUSIONS: MCA Doppler should be routinely used in assessment of IUGR pregnancies. Cerebroplacental Doppler can be used as an important screening tool in IUGR pregnancies for the prediction of adverse perinatal outcomes

P2–64 Table. Table 1. Demographic characteristics and Perinatal Outcomes IUGR with Low CPR 32 (40 %) Median Age (yr) Parity


IUGR with Normal CPR 48 (60%) Median

P value









Onset of IUGR(wk)




GA at delivery (wk)




Duration b/w onset of IUGR & delivery (days) Duration b/w last US & delivery (days) AFI (cm) AEDF/REDF












7 (14.5 %)


2 (6%)

C/S Emergency

17 (53 %)

NRFH/ Dopplers (indication for CS)

17 (53 %)

Birth weight (grams)


15 (31 %) 2610


19 (59 %)

20 (42 %)


14 (43.7 %)

27 (56 %)

Apgar score (5 min) IUFD/NND


9 3 (9 %)

0.0527 0.0001 0.145 0.145





NICU admission

18 (56%)

10 (20%)

NICU stay > 24 hr

16 (50 %)

8 (16 %)

NICU stay (days)

13 (1–47)

12 ( 1–30)

0.0016 0.0021 0.365

* P value < 0.05 is considered statistically significant Prenatal Diagnosis 2015, 35, 27–109

© 2015 John Wiley & Sons, Ltd.


P2-65 Trends in cell-free fetal DNA levels during pregnancy in patients affected by recurrent pregnancy loss

P2-66 Withdrawn.

Angela Barrett1, Henna Thadani1, Amelia Tan1, Cecille Laureano Asibal1, Cynthia Zapata Tagarino1, Lin Lin Su2, Mahesh Choolani1

P2-67 False positive non-invasive prenatal screening (NIPS) results with subsequent diagnosis of a hypertensive disorder of pregnancy

1 2

National University of Singapore, Singapore, Singapore National University Hospital, Singapore, Singapore

OBJECTIVES: Maternal cell-free DNA (cfDNA) contains an average of 10-15% cell-free fetal DNA (cffDNA) in a background of maternal DNA. Studies have shown that cffDNA levels increase by 1% per week in the first and second trimesters, rising more rapidly in the third trimester; however no one has measured cffDNA from an individual patient over the duration of the pregnancy. We aimed to follow cffDNA levels in women with ‘normal’ uncomplicated pregnancies as well as those who have suffered recurrent pregnancy loss (RPL) to determine whether there is a difference in early rate of release of cffDNA in women with early pregnancy complications involving the placenta. Our hypothesis is that there will be a sudden increase in cffDNA immediately prior to a miscarriage, and so if this can be monitored in these patients, an early intervention may be possible. METHODS: One woman with a normal pregnancy as well as ten women suffering from RPL gave a 10 ml blood sample at twoweekly intervals (median starting gestation 10 + 0 weeks (IQR: 8 + 3- 10 + 5 days)). Plasma was extracted from 2 mL of plasma at each time point using the Qiagen Circulating Nucleic Acid kit. At delivery, a sample of cord blood was collected from each woman, and genomic DNA (gDNA) was extracted from cord blood and maternal blood cells. A panel of insertion/deletion polymorphisms (indels) was used to genotype the maternal and fetal gDNAs, and five to seven informative indels, present in the fetus and absent in the mother, were selected for each pregnancy. Regions between 70 and 120 bp around these indels were amplified for each sample, sequenced using 100 bp single end sequencing on the Illumina MiSeq, and each allele was counted using a Visual Basic script. The fetal fraction was calculated as the geometric mean percentage of the fetal specific indels relative to the maternal allele. RESULTS: We have confirmed data from previous studies showing that there appears to be very little change in the percentage of fetal DNA during the first and second trimester of pregnancy using a time course of two weekly intervals in our normal control. During the third trimester there is a sharp increase in fetal fraction. None of the patients recruited to the study so far have miscarried over the duration of the study, and so we have not observed any increased levels of cffDNA immediately prior to a miscarriage, instead seeing only the same trend as observed in the normal control. CONCLUSIONS: Previous studies have indicated that there appears to be a slow change in fetal fraction during early stages of pregnancy, but have not measured fetal fraction from the same woman over time. None of the patients with previous RPL have miscarried during our study, and not observed any sudden rises in cffDNA levels in any of our patients. We are continuing to recruit normal patients to try to establish a definitive baseline for fetal fraction in a ‘normal’ pregnancy, and are also have recently recruited a further twenty women with RPL to our study. Data on the fetal fractions obtained from fortnightly samples from these women will be presented. Prenatal Diagnosis 2015, 35, 27–109

Erin Eaton, Emily Hardisty, Neeta Vora University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States OBJECTIVES: A false-positive NIPS result may be indicative of confined placental mosaicism. Confined placental mosaicism (CPM) is identified in 1-2% of all pregnancies in which CVS is performed and has been associated with an increased incidence of placental insufficiency, intrauterine growth restriction, fetal loss, and gestational hypertension. As noted in a recent review article by Taglauer, et al., NIPS may lead to increasing diagnoses of CPM with the potential to recognize previously unknown etiologies for gestational hypertension, fetal loss, or fetal growth restriction (2014). We present these cases to add to the literature regarding incidental findings and potential clinical concerns with false-positive NIPS results. METHODS: In this report we present two women with a false positive NIPS result consistent with trisomy 13 who later developed preeclampsia or gestational hypertension. RESULTS: Case 1. was a 36 year old primigravida who underwent NIPS in the setting of advanced maternal age. Results were reported as “aneuploidy detected” and were consistent with trisomy 13. The patient returned for genetic counseling and elected to proceed with amniocentesis. Final fetal karyotype was consistent with a normal male fetus (46, XY). The patient developed gestational hypertension and delivered via primary cesarean section at 37 weeks because of fetal malpresentation. Case 2 was a 35 year old multigravida who also underwent NIPS in the setting of advanced maternal age. Her results were reported as “aneuploidy suspected” for chromosome 13. The patient elected to proceed with chorionic villus sampling and final karyotype results were mosaic for trisomy 13 (mos 47,XX,+13[3]/46,XX[27]). Followup amniocentesis was performed and final fetal karyotype results were consistent with a normal female fetus (46, XX). Targeted anatomy ultrasound was normal. The patient subsequently developed preeclampsia with severe features and delivered via spontaneous vaginal delivery at 34 weeks. CONCLUSIONS: Gestational hypertension and preeclampsia are sources of maternal morbidity and mortality. We present these cases to add to the literature regarding incidental findings and potential clinical concerns with false-positive NIPS results.

P2-68 Circulating levels of C19MC-cluster microRNAs in pregnant women with severe pre-eclampsia Naoki Fuchi1, Yuri Hasegawa1, Ai Higashijima1, Masanori Kaneuchi1, Hideaki Masuzaki1, Kiyonori Miura1, Shoko Miura1, Yuko Murakami2 © 2015 John Wiley & Sons, Ltd.



Nagasaki University, Nagasaki, Japan Departments of Obstetrics and Gynecology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan 2

OBJECTIVES: The aim of this study was to clarify the association between circulating C19MC microRNAs in maternal plasma and severe pre-eclampsia. METHODS: Maternal blood samples (7 ml) at 27–34 weeks of gestation were obtained from 20 pregnant women with severe preeclampsia (sPE group) and 20 uncomplicated pregnant women (NP group). Twenty cases of severe pre-eclampsia were classified into 14 cases of late-onset (sPELO group) and six cases of early-onset (sPEEO group). The plasma concentrations of C19MC microRNAs (miR-518b, 1323, 516b, 516a-5p, 525-5p, 515-5p, 520 h, 520a-5p, 519d and -526b) were measured by quantitative real-time RT-PCR. The study protocol was approved by the IRB for Ethical, Legal and Social Issues in Nagasaki University. RESULTS: The circulating levels of all 10 C19MC microRNAs in maternal plasma were significantly increased in the sPE group compared with the NP group. Plasma concentrations of all 10 C19MC microRNAs tested were significantly increased in the sPEEO group compared with the NP group, while plasma concentrations of nine miRNAs, except for miR-519d, were significantly increased in the sPELO group compared with the NP group. Of the 10 C19MC microRNAs measured, plasma concentrations of eight miRNAs, except for miR-518b and miR-519d, were significantly increased in the sPEEO group compared with the sPELO group. CONCLUSIONS: Increased levels of C19MC microRNAs in maternal plasma are a characteristic phenomenon of established severe pre-eclampsia. This study demonstrated for the first time that circulating C19MC microRNAs in maternal plasma might play a role in the onset of pre-eclampsia.

immunohistochemical assay and Western Blot. RESULTS: In sIUGR group, placental CDKN1C mRNA of larger twin and smaller twin were 1.58 ± 0.89 and 1.79 ± 0.89,(P > 0.05), while KCNQ1OT1 mRNA expression was significantly higher in larger twin than smaller twin (0.72 ± 0.43 vs. 0.36 ± 0.19); the ratio of CDKN1C mRNA and KCNQ1OT1 mRNA was lower in larger twin than smaller twin (2.92 ± 1.97 vs. 6.01 ± 3.54) (P < 0.05). In normal control group, placental CDKN1C mRNA of larger twin and smaller twin were 1.56 ± 89 and 1.45 ± 0.53,(P > 0.05), while KCNQ1OT1 mRNA expression was significantly lower in larger twin than smaller twin (0.63 ± 0.32 vs. 1.08 ± 0.67); the ratio of CDKN1C mRNA and KCNQ1OT1 mRNA was higher in larger twin than smaller twin (3.07 ± 1.95 vs. 1.73 ± 1.07) (P < 0.05). Immunohistochemistry showed that the mean light density of CDKN1C protein was higher in smaller twin than larger twin (0.071 ± 0.044 vs. 0.025 ± 0.016) (P < 0.05) in sIUGR group, while no difference was showed in control group. Western Blot also showed that CDKN1C protein expression was higher in smaller twin than larger twin (1.81 ± 1.06 vs. 1.27 ± 0.95) (P < 0.05) in sIUGR group and no difference was showed in control group. CONCLUSION: KCNQ1OT1 mRNA expression was significantly decreased and CDKN1C protein expression was significantly increased in smaller twins of sIUGR. The pathogenesis of sIUGR may be related to the co-effect of the down-regulated KCNQ1OT1 mRNA expression and up-regulated expression of CDKN1C protein in placenta.

P2–69 Image.

P2-69 Placental expression of CDKN1C and KCNQ1OT1 in monozygotic twins with selective intrauterine growth restriction Chenyu Gou1, Xiaomei Shi2, Zhi-ming He3, Xuan Huang3, Lanzhen Zhang1, Qun Fang3 1

Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 2 Prenatal Diagnosis Centre, Guangdong Women and Children Hospital, Guangzhou, Guangzhou, China 3 Fetal Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China OBJECTIVE: CDKN1C and KCNQ1OT1 are imprinted genes which might be potential regulators of placental development. This study is to investigate placental expression of CDKN1C and KCNQ1OT1 in monozygotic (MZ) twins with and without selective intrauterine growth restriction (sIUGR). METHODS: 17 sIUGR cases and 15 normal monochorionic twins were included. All the cases were confirmed monozygotic twins by micro-satellite markers. Placental mRNA expression of CDKN1C and KCNQ1OT1 were detected by real-time fluorescent quantitative PCR. CDKN1C protein expression was detected by Prenatal Diagnosis 2015, 35, 27–109

© 2015 John Wiley & Sons, Ltd.


P2-70 Withdrawn. P2-71 The results of biochemical prenatal screening in Kazakhstan Republic: Results of 2014 Meruert Kirikbayeva, Damilya Salimbayeva Scientific Center of Obstetrics, Gynecology and Perinatology, Almaty, Kazakhstan OBJECTIVES: The national genetic prenatal screening (PS) program was performed in RK in 2007. METHODS: The national genetic prenatal screening (PS) Program was performed in RK in 2007. In 2014 in RK 257.517 pregnant women were screened, in totally, the coverage of PS was 64.8%. In different regions of RK the PS coverage ranged from 36% to 99%. Out of them, the 1st trimester test was 40.8% , 2nd test - 45.6% and 13.6% of pregnant women passed both tests. RESULTS: The prenatal invasive diagnosis procedures (IPD) were performed to 1.718 pregnant women of “high group risk” due abnormal serum markers (SM), age factor (AF) and ultrasound markers (USM). In common, chromosomal abnormalities (CA) of the fetus were diagnosed in 87 cases - 5.1%. The most informative indications for IPD were combination of SM + AF + USM, detectability of CA was the highest – 13.9% out of all IPD procedures. MoM value for pregnancies with confirmed fetal diagnosis of Down syndrome was in 1st trimester PAPPA- 0.60 MoM, β-hCG - 2.16 MoM; in 2nd trimester - AFP 0.91 MoM, β-hCG - 2.56 MoM and Estriol 1.02 MoM. For Edwards syndrome of fetus PAPP-A was 0.40 MoM, β-hCG - 0.59 MoM, in the 2nd trimester - AFP 0.81 MoM, β-hCG - 0.43 MoM and Estriol - 0.97 MoM. CONCLUSIONS: Thus, in 2014 the most informative for fetal CA detection was biochemical screening test of 1st trimester and the most diagnostically important was SM - β-hCG. The highest detection rate of CA was due combination of indications SM + AF + USM for IPD procedures. P2-72 Plasma biomarkers for early identification of women at risk for early onset preeclampsia Aggeliki Kolialexi1, George Tsangaris2, Stavros Sifakis3, Aggeliki Katsafadou2, Athanasios Anagnostopoulos2, Alexandra Lykoudi1, Danai Mavreli1, Gorge Papaioannou4, Ariadni Mavrou1 1

Department of Medical Genetics, Athens University, Athens, Greece 2 Proteomics Research Unit, Biomedical Research Foundation of the Academy of Athens, Athens, Greece 3 Department of Obstetrics & Gynecology, University of Crete, Heraklion, Greece 4 3rd Department of Obstetrics & Gynecology, Athens University, Athens, Greece OBJECTIVES: To identify in the plasma of pregnant women, in the 1st trimester of pregnancy, biomarkers that may allow reliable, non-invasive early detection of women at risk for early onset Preeclampsia (ePE), using high throughput proteomic technology. METHODS: Blood samples for this case–control proteome study were collected prospectively from pregnant Prenatal Diagnosis 2015, 35, 27–109

women at approximately 11 weeks of gestation. After delivery, 10 samples from cases that subsequently developed early PE (ePE) and 10 from women with uncomplicated pregnancies were analysed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in order to identify biomarkers for ePE. The differential expression of selected proteins was further verified using Western Blot analysis. RESULTS: Gel comparison revealed 12 proteins differentially expressed in maternal plasma in women that subsequently developed ePE. Six proteins, [Alpha-1-antitrypsin (A1AT), CD5 antigen-like molecule (CD5L) Keratin, type I cytoskeletal 9 (K1C9), Myeloid cell nuclear differentiation antigen (MNDA), Serotransferrin (TRFE) and Vitamin D-binding protein (VTDB)] were up-regulated and six, [Alpha-2-HSglycoprotein (FETUA), Beta-2-glycoprotein 1 (APOH), Complement factor B (CFAB), Haptoglobin (HPT), Vitronectin (VTNC) and Zinc-alpha-2-glycoprotein (ZA2G)] were downregulated. The differential expression of ZA2G, VTNC and FETUA was confirmed by Western blot and densitometric analysis. CONCLUSIONS: This pilot study indicates that proteomics could be a useful tool for the identification of new ePE screening markers. However, as these are preliminary findings, follow-up experiments are needed for their evaluation. The study was funded by «Aristeia», through the operation program «Education and lifelong learning» cofinanced by EU and the Greek state.

P2-73 Expression profile of miRNAs in human placentas from pregnancies complicated by preeclampsia Alexandra Lykoudi1, Aggeliki Kolialexi1, Georgia Tounta1, Thanasis Papanikolaou2, Theodora Tsokaki3, George Spyrou4, Marilena Bourdakou4, Emmanouil Athanasiadis4, Ariadni Mavrou1, Nikolaos Papantoniou3 1

Department of Medical Genetics, Athens University, Athens, Greece 2 1st Department of Obstetrics and Gynecology, Athens University, Athens, Greece 3 3rd Department of Obstetrics and Gynecology, Athens University, Athens, Greece 4 Center of Systems Biology, Biomedical Research Foundation, Academy of Athens, Athens, Greece OBJECTIVES: MicroRNAs (miRNAs) have emerged as posttranscriptional regulators implicated in key cellular processes, such as cell proliferation, differentiation and apoptosis. Their aberrant expression is also linked with pregnancy-associated disorders. The present study aims to access the miRNA expression profile in placentas complicated by preeclampsia (PE) and compare it that of normal pregnancies. METHODS: In this pilot study, 10 placentas from women with PE and 10 from uncomplicated pregnancies were analyzed. For RNA extraction, samples were mechanically homogenized in TRIzol and enriched for miRNAs using the mirVana miRNA extraction kit. Following labeling, samples were analyzed for miRNA expression © 2015 John Wiley & Sons, Ltd.


profiling on the miRlink Bioarray 300054–169 3PK platform which contains 1211 human miRNAs. Hybridization was performed at 37 °C for 16 h. Images were scanned with a microarray Scanner and raw data were normalized with GeneSpring 11.2 software. Corrected background intensities were normalized to the positive control count values, followed by log2 transformation. Ratio intensity plot, using “lowess normalization” was applied in order to extract the most significant differentially expressed miRNAs. Two target gene lists for the most significant up and down-regulated miRNAs were created with 3 different online tools. The differential expression of selected miRNAs was further confirmed using Real-Time qRT-PCR. RESULTS: In PE cases 13 miRNAs (miR-34a*, miR-3187, miR-767-5p, miR-let7d*, miR-33a, miR-3187, miR-144, miR1908, miR-548aa, miR1193, miR-633, miR-3920, miR-4310, miR-378*) were significantly up-regulated (p < 0.02), whereas 9 (miR-485-3p, miR- 494, miR-875-3p, miR-3184, miR-552, miR-378b, miR-513c, miR-299-5p, miR-429) were down-regulated. Using 3 computational algorithms, target genes of differentially expressed miRNAs were identified. Predicted target genes are known to be involved in several signaling pathways associated to PE. CONCLUSIONS: The results provide further evidence that placentas affected by PE display an altered miRNA expression profile. Further studies are needed to identify miRNA biomarkers for early diagnosis and monitoring of the disease.

P2-74 The role of FOXM1 in early placentation in normal pregnancy Lara Monteiro, Marianela Sanches, Ignacio Valenzuela, Macarena Palominos, Maria José Torres, Aneta Dobierzewska, Sebastian Illanes Universidad de los Andes, Santiago, Chile OBJECTIVES: The development of placenta plays a central role in determining the outcome of pregnancy. Extravillous trophoblast invasion is a fundamental component of human placentation. However, the transcriptional networks responsible for controlling the extent of trophoblast invasion are not well defined. Trophoblast cells are often compared to highly invasive carcinoma cells due to their capacity to proliferate and invade and to exhibit similar gene expression signatures. Thus, the transcriptional regulation of genes associated with cancer metastasis and trophoblast invasion may also be shared. The transcription factor forkhead box M1 (FOXM1), which is one of a few genes shown to be upregulated during early cancer development, possibly fits this scenario. FOXM1 has been shown to play a crucial role in the regulation of cell proliferation, cell cycle progression, angiogenesis, metastasis and DNA damage repair. Taking this, we aim to evaluate the role of FOXM1 in normal placental development. METHODS: To evaluate the expression of FOXM1 throughout pregnancy Sprague Dawley dams were sacrificed at E14, E15, E18, E19 and E20 and protein was isolated from one placenta per dam. Changes in placental protein expression were analyzed by western blot. The influence of FOXM1 on trophoblast proliferation was evaluated by Sulforhodamine B Prenatal Diagnosis 2015, 35, 27–109

colorimetric (SRB) assay in JEG and BeWo cell lines following silencing of FOXM1 via RNA interference. RESULTS: The transcription regulator FOXM1 was expressed during earlymid gestation (E14) but its expression decreased as gestational age increased (E14 to E18 there was a decrease in 0.29 fold and E14 to E21 0.04 fold). Compared to trophoblast cells expressing control non-specific siRNA, knockdown of FOXM1 resulted in a significant reduction of cell proliferation by 36% and 86% at 48 and 72 h, respectively in JEG-3 cells and by 29% and 58% at 48 and 72 h, respectively in BeWo cells. CONCLUSIONS: Understanding the regulation of trophoblast proliferation and invasion is crucial for determining the etiology of several placental-associated obstetrical complications. Our data is suggestive that the transcription factor FOXM1 modulates trophoblast proliferative potential and the results of the rat placenta indicate that FOXM1 may play a role in early placentation events. However, more studies are required to fully understand the function of FOXM1 in angiogenesis and metastasis in the placenta.

P2-75 Plasma concentrations of pregnancy-associated microRNAs showed a different transition during pregnancy and after delivery Yuko Murakami1, Kiyonori Miura2, Ai Higashijima2, Naoki Fuchi2, Shuhei Abe2, Yuri Hasegawa2, Atsushi Yoshida2, Masanori Kaneuchi2, Hideaki Masuzaki2 1

Departments of Obstetrics and Gynecology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan 2 Nagasaki University, Nagasaki, Japan OBJECTIVES: The aim of this study is to investigate how the plasma concentrations of pregnancy-associated microRNAs (miRNAs) change during pregnancy and after delivery METHODS: Maternal blood samples were collected at 11 weeks of gestation, 23 weeks of gestation, 30 weeks of gestation, 36 weeks of gestation, 1 day after delivery and 7 days after delivery. As the pregnancy associated miRNAs we investigated, placenta specific miRNAs (miR-518b, miR517a, miR-517c and miR-515-3p) on the chromosome 19 miRNA cluster (C19MC) region and fetus-specific miRNAs (miR-654-5p, miR-323-3p, miR-409-3p and miR-370) on the chromosome 14 miRNA cluster (C14MC) region were measured by real-time quantitative PCR. U6 snRNA was used for internal control miRNA. RESULTS: The median (minimum-maximum) of cell-free miR-518b level in maternal plasma was 0.1446 (0.08-0.33), 0.2657 (0.11-0.54), 0.3849 (0.24 2.45), 0.6709 (0.24-2.12), 0.2796 (0.17-0.60) and 0.1446 (0.03 0.34). Plasma concentrations of other placenta specific miRNAs on C19MC also increased during pregnancy and decreased after delivery significantly (p < 0.01). Wile, the median (minimum-maximum) cell free miR-654-5p level in maternal plasma was 0.0291 (0.01-0.59), 0.539 (0.01-0.35), 0.0890 (0.02-0.37), 0.1245 (0.02-0.29), 0.2225 (0.01-1.41) and 0.819 (0.00-0.50). Plasma concentrations of other fetus specific miRNAs on C14MC also showed a “not significantly © 2015 John Wiley & Sons, Ltd.


changed” during pregnancy and after delivery (p = 0.558). CONCLUSIONS: Circulating levels of placenta-specific miRNAs on C19MC and fetus-specific miRNAs on C14MC level in maternal plasma showed a different transition during pregnancy and after delivery. P2-76 Predictive performance of a seven-plex antibody array in prenatal serum screening Jeroen Pennings1, Sandra Imholz1, Ilse Zutt1, Maria Koster2, Jacqueline Siljee1, Annemieke de Vries1, Peter Schielen1, Wendy Rodenburg1 1

National Institute for Public Health and the Environment, Bilthoven, Netherlands 2 Wilhelmina Children’s Hospital, Utrecht, Netherlands OBJECTIVES: To evaluate the use of multiplex antibody array methodology for simultaneous measurement of serum protein markers for first trimester screening of Down Syndrome (DS) and other pregnancy outcomes such as pre-eclampsia. METHODS: We constructed an antibody array for indirect (‘sandwich’) measurement of seven serum proteins: pregnancy-associated plasma protein A (PAPP-A), free beta subunit of human chorionic gonadotropin (fβ-hCG), alphafetoprotein (AFP), angiopoietin-like 3 (ANGPTL3), epidermal growth factor (EGF), insulin-like growth factor 2 (IGFII), and superoxide dismutase 1 (SOD1). This array was tested using 170 DS cases and 510 matched controls drawn during the 8th-13th week of pregnancy. Data were used for prediction modelling and compared to previously obtained AutoDELFIA data for PAPP-A and fβ-hCG. RESULTS: PAPP-A and fβ-hCG serum concentrations obtained using antibody arrays were highly correlated with AutoDELFIA data. Moreover, prediction modeling using (log-MoMmed) antibody array and AutoDELFIA data gave comparable results. Of the other markers, AFP and IGFII concentrations showed significant changes, but adding these markers to a prediction model based on prior risk, PAPP-A and fβ-hCG did not substantially improve the predictive performance. CONCLUSIONS: Our study shows that multiplex antibody array methodology gives quantitative PAPP-A and fβ-hCG measurement. We also demonstrate for the first time that for DS risk prediction, using multiplex antibody array data performs as accurately as when using data

from current immunoassay methods. Implementation of antibody arrays in a wider prenatal screening setting will require additional first trimester screening markers. P2-77 Is the ratio of maternal serum to amniotic fluid AFP superior to serum levels as a predictor of pregnancy complications? Reuven Sharony Meir Medical Center, Kfar Saba, Israel OBJECTIVE: The use of maternal serum alpha fetoprotein (MSAFP) levels as a predictor of pregnancy complications is well-established. We hypothesized that the ratio between the MSAFP/AFAFP levels will predict placenta-associated pregnancy complications (PAPC) more accurately than MSAFP levels alone. METHODS: Women who had a MSAFP test and an amniocentesis were divided into two groups. Those who had PAPC comprised the study group and those who had an uneventful pregnancy served as the control group. Data regarding pregnancy and delivery course were collected. The MSAFP/AFAFP ratio between the study and the control groups was compared RESULTS: A total of 166 women were included in the study, of which 24 had complications. A significant correlation was found between the MSAFP/AFAFP ratio and intrauterine growth restriction (IUGR) and week of delivery. Four pregnancies had elevated MSAFP levels, but MSAFP/AFAFP ratio was below 2. All had uneventful pregnancies CONCLUSION: Our data suggest that the MSAFP/ AFAFP ratio is a better predictor of IUGR and week of delivery. Larger studies are needed to confirm this finding. P2-78 First trimester screening of preeclampsia in Taiwan S.W. Steven Shaw1, Yao-Long Chang1, SD Chang1, Chris Tsia2, Po-Jen Cheng1 1

Chang Gung Memorial Hospital, Taoyuan, Taiwan Bionet Corp. Taiwan, Taipei, Taiwan


OBJECTIVES: To set up a standard protocol for preeclampsia (PE) screening by examining biochemical and Doppler indices during the first trimester of pregnancy. METHODS: The software provided by Fetal Medicine Foundation was used for risk calculation. The risks of intrauterine growth retardation (IUGR) and preterm labor (PTL) were also recorded. A total of

P2–77 table. Table 1: MSAFP/AFAFP ratio according to the pregnancy complications* N (%) Complications

Mean ± SD**









11 (6.6)

155 (93.4)







8 (4.8)

158 (95.2)























*Some cases had more than one complication **Geometric mean ± log 10 standard deviation ***p in Mann–Whitney test IUGR-intrauterine growth restriction; PE-pre-eclampsia; PTL-preterm labor; PA-placental abruption

Prenatal Diagnosis 2015, 35, 27–109

© 2015 John Wiley & Sons, Ltd.


202 cases were enrolled and the results of uterine artery pulsatility index (PI), placental growth factor (PlGF) and PAPP-A were all expressed in multiples of the median (MoM). RESULTS: There were 10 cases of confirmed preeclampsia at delivery with 90.9% detection rate at 5% false positive rate, including 8 early and 2 late onset PE. Under the current platform of first trimester screening, additional 3.12 minutes were needed for uterine artery flow scanning. The median PlGF (0.56 MoM) and PAPP-A (0.71 MoM) were lower and uterine artery PI was higher (1.63 MoM) in the preeclampsia cases than in non-PE cases (PlGF: 1.061 MoM; PAPP-A: 1.070 MoM; uterine artery PI: 1.030 MoM). There were no significant difference in maternal body height, weight, parity, and mean arterial pressure in PE and non-PE groups. CONCLUSIONS: The setting of first trimester PE screening under current Down syndrome screening platform was feasible for early and late preeclampsia detection in Taiwan. P2-79 Knowledge of gestational diabetes among pregnant women in Ogun-State Nigeria Olubayode Akinbi1, Oluwatosin Akinbi2 1

All Saints University Medical School, Roseau, Dominica Olabisi Onabanjo University Teaching Hospital, Ogun, Nigeria 2

OBJECTIVES: To know the knowledge of pregnant women about gestational diabetes and how it affects pregnancy. METHODS: The study is retrospective cross sectional design, the survey was carried out in sagamu Ogun State Nigeria where about two hundred and ten, (210) pregnant women was asked to answer a well-designed questionnaire to determine their knowledge on gestational diabetes and risk factors which they are predispose to such as race, age, physical activities, diets, family history, previous history of diabetes related pregnancy complications and the effect of early pregnancy diagnose. RESULTS: A total of two hundred and ten (210) pregnant women was interviewed and 38% of them has knwledge of gestational diabetes, 25% had gestational diabetes in previous pregnancy with complications such as macrosomic baby,cephalopelvic disproportion and obstructed labour. 60% of them has no knowledge of GDM. 15% of those with GDM in previous pregnancy has had family history of diabetes in close relatives and 10% were obese based on there basal metabolic index (BMI) and do not engage in regular exercise. CONCLUSIONS: It was discovered that majority of the women has no knowledge of GDM and few of those with family history of diabetes and obesity had complication in previous pregnancy. Creation of awareness and educational programs about GDM will reduce the risk and prompt early diagnose.


Leiden University Medical Center, Department of Obstetrics and Fetal Medicine, Leiden, Netherlands OBJECTIVES: Congenital heart disease is present in 44-56% of fetuses with Down syndrome (DS). There are, however, signs that hearts in Down syndrome without apparent structural heart defects, also differ from the normal population. Sonic hedgehog signaling may be involved in the pathogenesis of AVSD in Down syndrome, although currently data is limited. We aimed to compare the atrioventricular septum and valves in 3 groups: Down syndrome without atrioventricular septal defect (‘DS noAVSD’), Down syndrome with AVSD (‘DS AVSD’) and control hearts. METHODS: The ventricular septum, (atrioventricular and interventricular) membranous septum and atrioventricular valves were examined and measured in histological sections of 15 ‘DS no-AVSD’, 8 ‘DS AVSD’ and 34 ‘control’ hearts. In addition, the ventricular septum length was measured on ultrasound images of fetal (6 ‘DS AVSD’, 9 ‘controls’) and infant (10 ‘DS no-AVSD’, 10 ‘DS AVSD’, 10 ‘controls’) hearts. RESULTS: The membranous septum volume was 3 times larger in ‘DS no-AVSD’ fetuses compared to control fetuses (panel B) and valve dysplasia (panel D) was frequently (64%) observed. In the Down syndrome fetuses with a (complete) AVSD, fibrous tissue was observed at the top of the ventricular septum in 3 cases. The ventricular septum was shorter in patients with Down syndrome both with (0.7 times the length of controls, p = 0.001) and without AVSD (0.78 times the sizes of the control, p < 0.001). In contrast to controls, in ‘DS no-AVSD’ fetuses, clear expression of Gli1, an effector of sonic hedgehog signaling, was present in the dysplastic plump atrioventricular valves and membranous septum in 2 out of 4 cases. CONCLUSIONS: Down syndrome no-AVSD hearts are not normal as they have a larger membranous septum, shorter ventricular septum and dysplasia of the atrioventricular valves as compared to control hearts. Findings indicate that a careful cardiac follow-up may be warranted in these patients.

P3–1 Image.

P3-1 How normal is a ‘normal’ heart in fetuses with Down syndrome? Bauke Adriaanse1, Monique C Haak2 1

VU University Medical Center, Amsterdam, Netherlands

Prenatal Diagnosis 2015, 35, 27–109

© 2015 John Wiley & Sons, Ltd.


P3-2 Stomach in contact with the bladder: A sonographic sign of left congenital diaphragmatic hernia Horacio Aiello, César Meller, Lucía Vázquez, Lucas Otaño Hospital Italiano de Buenos Aires, Buenos Aires, Argentina OBJECTIVES: The evaluation of the characteristics of the fetal stomach should be part of every routine prenatal ultrasound after 11 weeks. The abnormal position of the stomach may represent a key sign of different anomalies: when it is in the thorax it is the main sign of left congenital diaphragmatic hernia (CDH); if it is in the abdomen but right-sided, it may represent an heterotaxy syndrome; it may be near an abdominal wall-defect or in the amniotic fluid in gastroschisis; and recently described, when it is in the abdomen but with a horizontal shape it may represent a right-sided CDH. However, a position that has not been previously described in the literature is the stomach in contact with the bladder, an ultrasonographic finding that we have observed in association with left-sided CDH. The purpose of this paper is to describe a series of fetuses with the stomach in contact with the bladder and left-sided CDH. METHODS: Retrospective chart review of a series of consecutive patients with prenatal diagnosis of left-sided CDH with the stomach in the abdomen in contact with the bladder (fig. 1), managed between July 2009 and July 2014 in our Fetal Medicine Unit. Ultrasound examinations were performed by the same specialist in Fetal Medicine (H.A.), with a Toshiba Xsario (Toshiba Medical Corporation, Japan), or a MedisonSamsung Accuvix V20, with a 3.5 MhZ probe. For all fetuses, the following data were stored in a computerized database (Astraia software, Munich, Germany, and/or Microsoft Excel 2011): cause of referral to our Unit, gestational age at ultrasound, presence of liver herniation, severity of lung hypoplasia expressed as observed/expected lung-to-head ratio (O/E LHR), presence of associated anatomical or chromosomal anomalies, and position of the stomach. RESULTS: During the study period 62 cases of antenatally diagnosed CDH were managed in our Unit, 53 left-sided, 7 right-sided and 2 bilateral. In left-sided CDH the stomach was in the abdomen in 9/53 (17 %) patients. Among these 9 cases, the stomach was in contact with the bladder in 6/9. The median gestational age at diagnosis was 32 weeks (range 25–33). The median O/E LHR was 50% (range 43–72), and in all cases the liver was in the abdomen. The cause of referral was CDH in 4 cases, congenital cystic adenomatoid malformation (CCAM) in one, and routine third trimester scan in the other one. The median gestational age at delivery was 38 weeks (range 36–39), and the 6 neonates survived the surgery and are alive and well. CONCLUSIONS: We describe six cases with the ultrasonographic finding of the stomach in contact with the bladder, all of them in fetuses with left-sided CDH. We have not seen this feature in any other fetal condition and we are not aware that it has been described in the literature. In our series 2/6 fetuses the diagnosis was not CDH at referral: one was a CCAM and the other was Prenatal Diagnosis 2015, 35, 27–109

referred for a routine third trimester evaluation. Aware of the association, the position of the stomach contributed to reach the correct diagnosis of left CDH in both fetuses. The gastric chamber in contact with a normal bladder may be counterintuitive with the concept of visceral herniation into the thorax in CDH. We cannot explain the reasons of this position of the stomach. It may be related to the gastric distension and/or malrotation observed in newborns with CDH. Even though the reasons of this striking position of the stomach are not clear, our series highlights the fact that the visualization of the stomach in contact with the bladder may be a specific sonographic marker of CDH.

P3–2 image.

P3-3 Spontaneous chorioamniotic separation and its impact on pregnancy outcomes Carolina Bibbo, Sarah Little, Jad Bsat, Carol Benson, Julian Robinson Brigham and Women’s Hospital, Boston, Massachusetts, United States OBJECTIVES: There is a paucity of literature on the outcome of spontaneous chorioamniotic separation diagnosed from 17 weeks’ gestation onward. Our goal was to evaluate pregnancy outcomes with this diagnosis. METHODS: This is a case series of women with a singleton gestation who were diagnosed with chorioamniotic membrane separation from 17 weeks’ gestation onward from January 1987 to January 2013. SAS Statistical Software was used to compare pregnancy outcomes between spontaneous cases of chorioamniotic separation that were diagnosed before and after 24 weeks of gestation. RESULTS: A total of 106 singleton pregnancies had chorioamniotic separation from 17 weeks’ gestation onward. There were 74 cases (69.8%) of spontaneous chorioamniotic separation and 32 cases (30.2%) of iatrogenic chorioamniotic separation. Within the spontaneous group, there were 25 (33.8%) cases diagnosed before 24 weeks, 49 (66.2%) cases diagnosed after 24 weeks, and there was no information available about the pregnancy outcome of 9 cases. There were two cases of aneuploidy; one pregnancy with trisomy 18 and one with monosomy x. There were two cases of previable © 2015 John Wiley & Sons, Ltd.


rupture of membranes, and two in-utero demises, each at 22 and 25 weeks. The median gestational age at diagnosis was 30 weeks and the median gestational age at delivery was 36.7 weeks. There were 13 (20%) cases of preterm rupture of membranes and there were 26 (40%) cases of spontaneous preterm birth. When comparing pregnancy outcomes between cases of spontaneous chorioamniotic separation diagnosed before and after 24 weeks, there was a higher number of demises in the group diagnosed before 24 weeks (4 vs.0 p = 0.0057). The demises consisted in the cases of intrauterine fetal demises and the previable preterm rupture of membranes that lead to a neonatal demise. CONCLUSIONS: Our case series showed that when spontaneous chorioamniotic separation occurs from 17 weeks’ gestation onward, there is a higher risk of preterm rupture of membranes and preterm birth. However, there were no cases of spontaneous chorioamniotic separation diagnosed after 24 weeks that were associated with an abnormal karyotype, previable preterm rupture of membranes, or stillbirth.

P3-4 Withdrawn. P3-5 Decrease of perinatal mortality associated with congenital anomalies after prenatal screening was introduced in the Netherlands H.H. Faber1, K. Bouman1, H.E.K. Walle1, M. Haeusler2, E. Garne3, A. Rissmann4, M. O’Mahony5, C. Lynch6, M. McDonnell7, J. Rankin8, A. Pierini9, O. Zurriaga10, M-C. Addor11, D. Tucker12, N. Zymak-Zakutnia13, H. Groen1 1

University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands 2 University of Graz, Austria 3 Pediatric Department, Hospital Lillebaelt, Kolding, Denmark 4 Medical Faculty Otto von Guericke University, Magdeburg, Germany 5 Department of Public Health, St Finnbar’s Hospital, Cork, Ireland 6 Department of Public Health, Health Service Executive South East Lacken, Kilkenny, Ireland 7 Health Information Unit, Dr Steevens Hospital, Dublin, Ireland 8 Institute of Health and Society, Newcastle University, Newcastle upon Tyne, United Kingdom 9 Unit of Epidemiology, CNR Institute of Clinical Physiology, Pisa, Italy 10 Centro Superior de Investigacion en Salud Publica, Valencia, Spain 11 Registre Vaudois des Malformations, Lausanne, Switzerland 12 Congenital Anomaly Register & Information Service, Singleton Hospital, Swansea, United Kingdom 13 Khmelnytsky Regional Medical Center, Khmelnytsky, Ukraine OBJECTIVES: There has been much discussion about the relatively high perinatal mortality seen in the Netherlands (Buitendijk 2004, Europeristat 2009), for which congenital anomalies (CA) are known to be one of the four main risk factors. There was no nationwide routine prenatal screening for CA in the Netherlands until 2007. We have analysed data for a 14-year period Prenatal Diagnosis 2015, 35, 27–109

from the EUROCAT registries to investigate the effect of the introduction of screening for CA on the perinatal mortality rate in the Netherlands and compared the results with those from other European registries. METHODS: We used data from the European Surveillance of Congenital Anomalies (EUROCAT) database covering the period 1998 to 2011. We included registries that had correctly coded the date of death for more than 80% of their cases. Perinatal mortality was defined as: deaths in the first week after birth plus late foetal deaths and stillbirths from 20 weeks’ gestation onwards, excluding terminations of pregnancy for foetal anomalies (TOPFA). RESULTS: A total of 84.832 cases of CA were included from 13 European registries covering a total of 3.1 million births. In Europe the perinatal mortality associated with CA decreased from an average of 1.35 per 1000 births in the period 1998–2006 to 1.15 per 1000 births in the period 2007–2011. In the northern Netherlands, it dropped from 1.73 per 1000 births in the period 1998–2006 to 1.00 per 1000 births in the period 2007– 2011. In 2011, the perinatal mortality associated with CA in the northern Netherlands was on the same level as the rest of Europe. Our data also showed that, since the introduction of prenatal screening, more parents have chosen to terminate a pregnancy if congenital anomalies are discovered. CONCLUSIONS: Perinatal mortality associated with congenital anomaly (CA) has decreased in the northern Netherlands since 2005. The introduction of prenatal screening in 2007 markedly contributed to this trend: pregnancies involving CA were terminated more often, leading to a decrease in the perinatal mortality rate. By 2011, the perinatal mortality rate associated with CA in the northern Netherlands was equal to that for the rest of Europe. P3-6 Postnatal mortality, growth failure and heart defects in the Ts1Cje mouse model of Down syndrome Millie Ferres1, Faycal Guedj2, Diana Bianchi3 1

Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States 2 Tufts Medical Center, Boston, Massachusetts, United States 3 Tufts University School of Medicine, Boston, Massachusetts, United States OBJECTIVES: Several mouse models have been developed to better understand the pathophysiology of Down syndrome and design new treatments. The Ts1Cje model is of particular interest for prenatal therapy, because affected males are fertile, allowing affected pups to be carried in wild-type females, similar to the situation in humans. Here we sought to establish natural history and growth profiles in Ts1Cje fetal and neonatal mice, and to determine if heart defects are present in this strain. METHODS: Pups were studied either on embryonic (E) day 15, or postnatally (P) on days 3 to 21. PCR amplification targeting the neomycincassette (present in Ts1Cje) and Sry gene (present in males) was used to analyze genotypes and sex ratios. Histological evaluations to investigate for the presence of heart defects were performed on day E15. Postnatal body weights and lengths in Ts1Cje mice were recorded and compared to wildtype littermates. RESULTS: Genotyping of embryos revealed Mendelian transmission of the derivative chromosome. In © 2015 John Wiley & Sons, Ltd.


embryos, 50% were Ts1Cje. Heart defects (ASD, VSD) were observed in Ts1Cje embryos (5 out of 15). Ts1Cje pups showed significant growth delays, manifesting as two different phenotypes, I and II (Figure 1). The type II phenotype was more severely affected, with smaller size and neonatal lethality by day P10. Postnatal mortality was significantly higher in Ts1Cje compared to WT (69% vs. 31%, p < 0.05), and was associated with intrauterine growth restriction. After weaning, only 24% of pups were Ts1Cje with a male predominance in survivors (ratio = 1.6:1). CONCLUSIONS: In this study we have identified two different neonatal phenotypes that are associated with the Ts1Cje genotype. The equal proportions of Ts1Cje and WT embryos observed in utero suggest that this genotype is not associated with antenatal lethality. There is, however, significant postnatal mortality, which may be due to the presence of cardiac defects and growth failure. The presence of congenital heart disease and growth restriction are additional endpoints against which to assess the effects of prenatal therapy.

P3–6 Image. Figure 1.

following an ultrasound (US) diagnosis of fetal lung masses. METHODS: A retrospective analysis of all fetal lung masses at Lucile Packard Children’s Hospital Stanford, between 2009 and 2014 with both prenatal US and MRI exams. Prenatal, intrapartum, and postnatal data were extracted from medical records including assessment of lesion characteristics and the Congenital Pulmonary Airway Malformation (CPAM) Volume Ratio (CVR) across gestation. MRI data were compared with US data for both second trimester and third trimester studies. Obstetric and neonatal outcome data were compared between those with 1 MRI exam before 28 weeks gestation and those with 2 exams, the second performed after 30 weeks gestation. RESULTS: 40 fetuses with lung masses, with both US and MRI data were included in the analysis. 32 patients (80 %) had first MRI before 28 weeks gestation, 29 (73%) of whom had a second MRI after 30 weeks. Of those who had their first MRI before 28 weeks (n = 29), MRI provided added information in 12 cases (41%) including: discovery of a feeding vessel (n = 4), presence of a bronchiocele (n = 1), confirmation of a fetal lung mass (n = 1), finding of a microcystic component (n = 1), diagnosis of a hypoplastic right lung (n = 1), bronchial atresia (n = 2) and mucous plug (n = 2). Of those who had a second MRI performed (n = 29), 4 cases (14%) had additional findings including: persistence of the lung lesion no longer visualized by US (n = 2), diagnosis of a diaphragmatic hernia previously unseen (n = 1), and mild pericardial effusion (n = 1). The performance of a second MRI exam following 30 weeks was not associated with common adverse perinatal and neonatal outcomes, although those with a second MRI delivered at later gestational ages and had less frequent preterm birth (3 vs. 46%, p < 0.005) (table). CONCLUSIONS: Adjunct prenatal MRI before 28 weeks gestation provided additional information not obtained by US in 41% of cases. The additional value of MRI after 30 weeks was lower but assisted in clarifying the ongoing presence of the lung mass when visualization by US can be limited. P3-8 Factors associated with neonatal respiratory distress in cases of prenatally diagnosed lung masses

P3-7 Added value of prenatal MRI in cases of fetal lung masses Anna Girsen1, Rami Sammour2, Susan Hintz3, Alexis Davis4, Kristin Riley3, Richard Barth5, Erika Rubesova5, Karl Sylvester6, Yair J Blumenfeld1 1

Department of Obstetrics & Gynecology, Stanford University School of Medicine, Stanford, California, United States 2 The Fetal Medicine Foundation, London, United Kingdom 3 Department of Pediatrics, Division of Neonatal and Developmental Medicine, Stanford University School of Medicine, Stanford, California, United States 4 Pediatrix Medical Group , San Jose, California, United States 5 Department of Radiology, Stanford University School of Medicine, Stanford, California, United States 6 Department of Surgery, Stanford University School of Medicine, Stanford, California, United States OBJECTIVES: To investigate the benefit of adjunct fetal MRI Prenatal Diagnosis 2015, 35, 27–109

Anna Girsen1, Rami Sammour2, Susan Hintz3, Alexis Davis4, Kristin Riley3, Richard Barth5, Erika Rubesova5, Karl Sylvester6, Yair J Blumenfeld1 1

Department of Obstetrics & Gynecology, Stanford University School of Medicine, Stanford, California, United States 2 The Fetal Medicine Foundation, London, United Kingdom 3 Department of Pediatrics, Division of Neonatal and Developmental Medicine, Stanford University School of Medicine, Stanford, California, United States 4 Pediatrix Medical Group , San Jose, California, United States 5

Department of Radiology, Stanford University School of Medicine, Stanford, California, United States 6

Department of Surgery, Stanford University School of Medicine, Stanford, California, United States OBJECTIVES: To investigate the association between prenatal and intrapartum factors and neonatal respiratory distress at birth in cases of congenital lung masses. METHODS: A © 2015 John Wiley & Sons, Ltd.


P3–7 Table. Comparison of lung mass characteristics, perinatal and neonatal outcomes between cases that had 1 vs. 2 MRI exams during pregnancy 2nd MRI performed at >30 weeks N = 29 Maternal age (y)

29.9 (6.2)

No 2nd MRI performed N = 11 29.8 (4.8)

Maternal race/ethnicity Non-hispanic white Hispanic Other


0.97 0.18

5 (17 %)

5 (46 %)

15 (52 %)

4 (36 %)

9 (31 %)

2 (18 %)

Gestation at diagnosis (wks)

21.0 (2.9)

22.8 (5.1)


CVR at first US

0.37 (0.07 - 2.40)

0.23 (0.001 - 1.7)


Lesion appearance at first ultrasound: Complex (cystic and solid) Macrocystic Microcystic Unspecified Maximum CVR CVR change during pregnancy (%) Last CVR Maximum Lesion Volume Last Lesion Volume

0.16 8 (28 %)

0 (0.0 %)

4 (14 %)

4 (36 %)

11 (38 %)

5 (46 %)

6 (21 %) 0.59 (0.15 - 3.4) 56 (0–606) 0.3 (0.02 - 1.3) 14.2 (4.1 - 85.7) 9.1 (0.6 - 82)

2 (18 %) 0.50 (0.05 - 1.82)


76 (15–400)


0.3 (0.05 - 1)


13.2 (1.4 - 48.9)


9.3 (1.4 - 31.9)


Hydrops at any point

3 (10%)

1 (9 %)


Corticosteroid treatment

4 (14 %)

2 (18 %)


Gestational age at delivery (wks) Preterm delivery 1 year to conceive, had utilised Assisted Reproductive Technology (ART), had a personal or family history of miscarriage and/or aneuploidy and were more likely to consider termination of an affected fetus. In a future pregnancy, 95% said they would use NIPT again. CONCLUSIONS: Low risk women choosing to have NIPT have similar levels of trait anxiety to high risk women. Both groups benefit from a decrease in state anxiety after NIPT. High risk women, who initially have higher levels of state anxiety, continue their pregnancy with similar levels of anxiety to low risk women who have a ‘negative’ NIPT result. Both populations considered NIPT a valuable addition to prenatal care.

P3-66 The gift of time: The integration of perinatal palliative care Ginny Silva Brigham & Women’s Hospital, Boston, Massachusetts, United States OBJECTIVES: This presentation will provide key concepts regarding the perceptions of the term palliative care, discuss the ethical dilemmas faced by the health care team when offering perinatal palliative care, and identify barriers faced when dealing with families experiencing a life-limiting fetal diagnosis. METHODS: Two international surveys given to healthcare providers found the majority of respondents either never heard of or were confused about the term palliative care. This presentation will provide the challenging concepts of perinatal palliative care (PPC) by presenting two contrasting cases of families whose fetus was diagnosed with Trisomy 18. Within the case studies, a discussion of the concepts and misconceptions of palliative care will be explored. RESULTS: In our highly technical world where intensive, invasive, and often aggressive life-saving or life-prolonging interventions have become the norm, palliative care has an important role that is often overlooked. Families facing an uncertain diagnosis for their unborn babies deserve and can benefit from multidisciplinary care that addresses pain and symptom management, ethical, spiritual, and emotional support, and optimization of quality of life. Palliative care in the perinatal setting can provide all these aspects of care while at the same time providing support and guidance to the multidisciplinary care team. CONCLUSIONS: The goal of a Perinatal Palliative Care team is to provide a seamless approach to the family facing a lifelimiting diagnosis for their unborn baby and the entire care team. © 2015 John Wiley & Sons, Ltd.


Introducing the PPC team early on in discussions with the care team will give families the information and support they need and desire without viewing the term palliative care negatively. Acknowledging Perinatal Palliative Care as a necessary component and stackholder with a multidisciplinary approach will enhance planning and care for not only these very challenging and deserving families but also the entire care team. This will in turn provide parents with meaning, respect, and honor in their babies’ lives. P3-67 Patient and professional experiences with non invasive prenatal diagnosis (NIPD) and testing (NIPT): Social and ethical issues raised Heather Strange Cardiff University, Abergavenny, Monmouthshire, United Kingdom OBJECTIVES: The aim of this study is to gain a rich understanding of early experiences with emerging non-invasive prenatal diagnosis (NIPD) and non-invasive prenatal testing (NIPT) testing technologies. The study aims to identify issues of common interest and concern to both patients and professionals located in a wide variety of settings. METHODS: This qualitative PhD study is based on data gathered through a total of forty-three in-depth (narrative) interviews with patients and professionals in the UK. Twenty-three experts with direct professional experience of NIPD or NIPT (including scientists, fetal medicine consultants, policy makers and genetic counsellors) were interviewed, along with ten patients with direct experience of NIPD or NIPT, and ten parents with more general experience of prenatal screening or diagnosis. Clinicians involved in both the NHS-based provision of NIPD and the private provision of NIPT participated in the study. The interviews yielded a rich and diverse dataset, and were analysed for crosscutting themes. RESULTS: A number of core themes were identified as being of significance across the data set. These included: test accuracy; the boundary between NIPD and NIPT; the boundary between ‘high risk’ and ‘low risk’ populations; hopes around NIPT as a possible replacement technology; anxieties around the possible routinisation of selective termination; tensions between the aims of public health screening programmes and individualised care. CONCLUSIONS: The rapid development of NIPD and NIPT has very quickly given rise to a diverse and rich set of patient and professional experiences. The issues of interest and concern identified within patient and professional accounts of this new testing technology present a complex ethical, legal, political and social landscape. Knowledge of these

Prenatal Diagnosis 2015, 35, 27–109

early experiences with NIPD and NIPT may enable a better understanding of the advantages these tests may offer, as well as practical and ethical problems that are likely to arise in future, as both NIPD and NIPT become further embedded in specialist clinical practice and routine prenatal care. P3-68 Physician utilization and interpretation of non-invasive prenatal screening (NIPS) Andrea Wray1, Helain Landy2, Elizabeth Kramer Dugan1, Jeanne Meck1 1

GeneDx, Gaithersburg, Maryland, United States Georgetown University Hospital, Washington, DC, United States 2

OBJECTIVES: To evaluate how obstetrical providers utilize non-invasive prenatal screening (NIPS) and interpret results METHODS: MFM and Ob providers were invited to take an on-line survey asking about criteria for offering NIPS and other adjunctive screening/diagnostic tests. Four different NIPS test results extracting wording from actual reports were included: (1) high risk (>99/100) for Trisomy 18; (2) low risk (

Poster Abstracts of the ISPD 19th International Conference on Prenatal Diagnosis and Therapy, Washington, DC, USA, 12-15 July 2015.

Poster Abstracts of the ISPD 19th International Conference on Prenatal Diagnosis and Therapy, Washington, DC, USA, 12-15 July 2015. - PDF Download Free
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