Case Report Accepted: October 17, 2014 by M. Schmid Published online: February 20, 2015
Cytogenet Genome Res DOI: 10.1159/000370256
Prenatal Diagnosis of a Female Fetus with Ring Chromosome 9, 46,XX,r(9)(p24q34), and a de novo Interstitial 9p Deletion Vanessa Penacho c Francisco Galán a Tina-A. Martín-Bayón b Sonia Mayo d Irene Manchón a Alfonso Carrasco b Francisco Martínez-Castellano d Luis A. Alcaraz c
c
Centro de Genética Humana, b Unidad de Diagnóstico Prenatal, Hospital General Universitario de Alicante, and Bioarray, S.L., Elche, Alicante, and d Unidad de Genética, Hospital Universitario La Fe, Valencia, Spain
Key Words Array CGH · De novo · Interstitial 9p deletion · Prenatal diagnosis · Ring chromosome 9
Abstract Ring chromosomes are circular structures formed as a result of breaks in the chromosome arms and the fusion of the proximal broken ends with a loss of distal material, or by fusion of dysfunctional telomeres without any loss. The mechanism underlying this process has not yet been sufficiently explained. Commonly, rings occur as acquired genetic abnormalities; however, sometimes they are found as constitutional aberrations with a prevalence of around 1:50,000 live births. Here, we present a new case of r(9) in a female fetus with intrauterine growth retardation and slight craniofacial dysmorphisms. Both parents had a normal phenotype. Amniotic fluid karyotype showed r(9)(p24q34). An array CGH revealed 3 deletion segments: a ring chromosome with a 2.57Mb deletion at 9pterp24.2 (chr9:163,131–2,729,722), a 2.60Mb deletion at 9q34.3qter (chr9: 138,523,302–141,122,055), and also a 0.15-Mb interstitial deletion at 9p24.1 (chr9: 5,090,443–5,235,765). These deletions overlap with proposed regions for the 9p24.3 deletion and Kleefstra syndrome. Segregation analysis revealed a maternal origin of
© 2015 S. Karger AG, Basel 1424–8581/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/cgr
the rearranged chromosome. We conclude that both the ring chromosome and the interstitial deletion occurred de novo. This last deletion has not been reported before. Prenatal array CGH, combined with fine mapping of breakpoints contributes to the assessment of genotype-phenotype correlations. © 2015 S. Karger AG, Basel
Within structural defects of chromosomes, the ring chromosome 9 [r(9)] is a rarely diagnosed anomaly at prenatal level. Up to now, only 2 prenatal cases have been reported in the literature [Stumm et al., 2000; Chen et al., 2006]. Even at the postnatal level, only 28 cases described [Aldemir et al., 2013; la Cour Sibbesen et al., 2013] showed a variable phenotype depending on the size and locations of deletions, with common features such as psychomotor retardation; craniofacial dysmorphisms; skeletal malformations as well as cardiovascular, gastrointestinal and genitourinary abnormalities, and infectious complications. However, in some cases, intelligence may be in the low normal range [Purandare et al., 2005]. Constitutional ring chromosomes occur in 1/50,000 human fetuses [Jacobs et al., 1975]. Principally, ring Dr. Francisco Galán Centro de Genética Humana Plaza de Los Luceros, No. 3, E-C ES–03001 Alicante (Spain) E-Mail fgalan @ centrodegeneticahumana.com
Downloaded by: UCSF Library & CKM 169.230.243.252 - 4/11/2015 1:45:15 PM
a
Patient and Methods A pregnant 31-year-old woman was referred to the Prenatal Diagnosis Unit for Genetic Amniocentesis (University General Hospital of Alicante) due to an abnormal serum screening performed at 12 weeks. For this publication, written informed consent was obtained from the parents. According to the standards of the Fetal Medicine Foundation, the patient showed a fetal nuchal translucency thickness of 2.7 mm (90th percentile) and the presence of a hypoplastic nasal bone. Combining the results of the nuchal scan with the results of maternal blood tests [102 mU/ ml (2.46 MoM) of human chorionic gonadotropin (hCG), 0.54 mU/ml (0.26 MoM) of pregnancy-associated plasma protein A (PAPP-A)], the Down’s syndrome risk increased more than 1/50. Additionally, an amniocentesis was performed in the 15th pregnancy week, observing no evidence for a trisomy 13, 18 or 21 with the rapid FISH testing (X, Y, 13, 18, and 21 probes). The confirmation of this pattern was more reliable with the longterm culture. After 2 weeks, first the amniotic cell culture was processed for fetal karyotyping and a total of 60 metaphases were analyzed (GTC-banding) showing 46,XX,r(9)(p24q34) (fig. 1A). Also, 16% of the metaphases had a chromosome 9 monosomy (45,XX,–9). This result was confirmed by subculturing and processing a second primary culture. In this context, to better understand the structure of chromosome 9, we decided to perform array CGH to identify CNVs for both fetus and parents. In the first case, we used an array Agilent Human Genome 244K with fetal DNA extracted from cultured cells (Agilent Technologies, Inc., Santa Clara, Calif., USA); for the parents, we used Agilent Human Genome 105K with genomic DNA from peripheral
2
Cytogenet Genome Res DOI: 10.1159/000370256
blood cells. Labeling and hybridization were set up following the manufacturer’s instructions. Arrays were scanned with Agilent G2565CA Microarray Scanner System, and the array data were extracted from the images using Feature Extraction 11.5 software (Agilent Technologies, Inc.). All arrays passed quality control checks performed by the Feature extraction software to ensure uniform signals in spots and low background-to-signal ratios. We imported the data into Agilent’s Genomics Workbench 7.0 and, using ADM-2 algorithm, threshold of 6, bin of 10, and a centralization threshold of 6, aberrations were identified. The parameters for defining copy number alterations were as follows: significance threshold = 5.0E–4; maximum contiguous probe spacing (kb) = 1,000; minimum number of contiguous probes per CNA segment = 5; threshold of signal intensity ratio >0.2 on log2 scale for gains and
Cytogenet Genome Res DOI: 10.1159/000370256
Prenatal Diagnosis of a Female Fetus with Ring Chromosome 9, 46,XX,r(9)(p24q34), and a de novo Interstitial 9p Deletion Vanessa Penacho c Francisco Galán a Tina-A. Martín-Bayón b Sonia Mayo d Irene Manchón a Alfonso Carrasco b Francisco Martínez-Castellano d Luis A. Alcaraz c
c
Centro de Genética Humana, b Unidad de Diagnóstico Prenatal, Hospital General Universitario de Alicante, and Bioarray, S.L., Elche, Alicante, and d Unidad de Genética, Hospital Universitario La Fe, Valencia, Spain
Key Words Array CGH · De novo · Interstitial 9p deletion · Prenatal diagnosis · Ring chromosome 9
Abstract Ring chromosomes are circular structures formed as a result of breaks in the chromosome arms and the fusion of the proximal broken ends with a loss of distal material, or by fusion of dysfunctional telomeres without any loss. The mechanism underlying this process has not yet been sufficiently explained. Commonly, rings occur as acquired genetic abnormalities; however, sometimes they are found as constitutional aberrations with a prevalence of around 1:50,000 live births. Here, we present a new case of r(9) in a female fetus with intrauterine growth retardation and slight craniofacial dysmorphisms. Both parents had a normal phenotype. Amniotic fluid karyotype showed r(9)(p24q34). An array CGH revealed 3 deletion segments: a ring chromosome with a 2.57Mb deletion at 9pterp24.2 (chr9:163,131–2,729,722), a 2.60Mb deletion at 9q34.3qter (chr9: 138,523,302–141,122,055), and also a 0.15-Mb interstitial deletion at 9p24.1 (chr9: 5,090,443–5,235,765). These deletions overlap with proposed regions for the 9p24.3 deletion and Kleefstra syndrome. Segregation analysis revealed a maternal origin of
© 2015 S. Karger AG, Basel 1424–8581/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/cgr
the rearranged chromosome. We conclude that both the ring chromosome and the interstitial deletion occurred de novo. This last deletion has not been reported before. Prenatal array CGH, combined with fine mapping of breakpoints contributes to the assessment of genotype-phenotype correlations. © 2015 S. Karger AG, Basel
Within structural defects of chromosomes, the ring chromosome 9 [r(9)] is a rarely diagnosed anomaly at prenatal level. Up to now, only 2 prenatal cases have been reported in the literature [Stumm et al., 2000; Chen et al., 2006]. Even at the postnatal level, only 28 cases described [Aldemir et al., 2013; la Cour Sibbesen et al., 2013] showed a variable phenotype depending on the size and locations of deletions, with common features such as psychomotor retardation; craniofacial dysmorphisms; skeletal malformations as well as cardiovascular, gastrointestinal and genitourinary abnormalities, and infectious complications. However, in some cases, intelligence may be in the low normal range [Purandare et al., 2005]. Constitutional ring chromosomes occur in 1/50,000 human fetuses [Jacobs et al., 1975]. Principally, ring Dr. Francisco Galán Centro de Genética Humana Plaza de Los Luceros, No. 3, E-C ES–03001 Alicante (Spain) E-Mail fgalan @ centrodegeneticahumana.com
Downloaded by: UCSF Library & CKM 169.230.243.252 - 4/11/2015 1:45:15 PM
a
Patient and Methods A pregnant 31-year-old woman was referred to the Prenatal Diagnosis Unit for Genetic Amniocentesis (University General Hospital of Alicante) due to an abnormal serum screening performed at 12 weeks. For this publication, written informed consent was obtained from the parents. According to the standards of the Fetal Medicine Foundation, the patient showed a fetal nuchal translucency thickness of 2.7 mm (90th percentile) and the presence of a hypoplastic nasal bone. Combining the results of the nuchal scan with the results of maternal blood tests [102 mU/ ml (2.46 MoM) of human chorionic gonadotropin (hCG), 0.54 mU/ml (0.26 MoM) of pregnancy-associated plasma protein A (PAPP-A)], the Down’s syndrome risk increased more than 1/50. Additionally, an amniocentesis was performed in the 15th pregnancy week, observing no evidence for a trisomy 13, 18 or 21 with the rapid FISH testing (X, Y, 13, 18, and 21 probes). The confirmation of this pattern was more reliable with the longterm culture. After 2 weeks, first the amniotic cell culture was processed for fetal karyotyping and a total of 60 metaphases were analyzed (GTC-banding) showing 46,XX,r(9)(p24q34) (fig. 1A). Also, 16% of the metaphases had a chromosome 9 monosomy (45,XX,–9). This result was confirmed by subculturing and processing a second primary culture. In this context, to better understand the structure of chromosome 9, we decided to perform array CGH to identify CNVs for both fetus and parents. In the first case, we used an array Agilent Human Genome 244K with fetal DNA extracted from cultured cells (Agilent Technologies, Inc., Santa Clara, Calif., USA); for the parents, we used Agilent Human Genome 105K with genomic DNA from peripheral
2
Cytogenet Genome Res DOI: 10.1159/000370256
blood cells. Labeling and hybridization were set up following the manufacturer’s instructions. Arrays were scanned with Agilent G2565CA Microarray Scanner System, and the array data were extracted from the images using Feature Extraction 11.5 software (Agilent Technologies, Inc.). All arrays passed quality control checks performed by the Feature extraction software to ensure uniform signals in spots and low background-to-signal ratios. We imported the data into Agilent’s Genomics Workbench 7.0 and, using ADM-2 algorithm, threshold of 6, bin of 10, and a centralization threshold of 6, aberrations were identified. The parameters for defining copy number alterations were as follows: significance threshold = 5.0E–4; maximum contiguous probe spacing (kb) = 1,000; minimum number of contiguous probes per CNA segment = 5; threshold of signal intensity ratio >0.2 on log2 scale for gains and
