CLINICAL REPORT
Segmental Uniparental Isodisomy of Chromosome 6 Causing Transient Diabetes Mellitus and Merosin-Deficient Congenital Muscular Dystrophy Raissa Coelho Andrade,1 Julia´n Nevado,2 Maria Ange´lica de Faria Domingues de Lima,3,4 Taˆnia Saad,5 Lucia Moraes,6 Leila Chimelli,7 Pablo Lapunzina,2 and Fernando Regla Vargas1,3,8* 1
Genetics Division, Instituto Nacional de Caˆncer, Rio de Janeiro, Brazil INGEMM, Instituto de Gene´tica Me´dica y Molecular, IdiPAZ-CIBERER, Universidad Auto´noma de Madrid, Madrid, Spain
2 3
Genetics and Molecular Biology Department, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil Programa de Internato em Gene´tica, Universidade UnigranRio, Rio de Janeiro, Brazil
4 5
Neuropediatrics Service, Instituto Fernandes Figueira, Fundac¸˜ao Oswaldo Cruz, Rio de Janeiro, Brazil Medical Genetics Center, Instituto Fernandes Figueira, Fundac¸˜ao Oswaldo Cruz, Rio de Janeiro, Brazil
6 7
Pathology Department, Medical School, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Birth Defects Epidemiology Laboratory, Fundac¸˜ao Oswaldo Cruz, Rio de Janeiro, Brazil
8
Manuscript Received: 13 March 2014; Manuscript Accepted: 7 July 2014
Segmental uniparental isodisomy (iUPD) is a rare genetic event that may cause aberrant expression of imprinted genes, and reduction to homozygosity of a recessive mutation. Transient neonatal diabetes mellitus (TNDM) is typically caused by imprinting aberrations in chromosome 6q24 TNDM differentiallymethylated region (DMR). Approximately, 15.12 Mb upstream in 6q22-q23 is located LAMA2, the gene responsible of merosindeficient congenital muscular dystrophy type 1A (MDC1A). We investigated a patient diagnosed both with TNDM and MDC1A, born from a twin dichorionic discordant pregnancy. Parents are first-degree cousins. Methylation sensitive-PCR of the imprinted 6q24 TNDM CpG island showed only the non-methylated (paternal) allele. Microsatellite markers and SNP array profiling disclosed normal biparental inheritance at 6p and a segmental paternal iUPD, between 6q22.33 and 6q27. Sequencing of LAMA2 exons showed a homozygous frameshift mutation, c.7490_7493dupAAGA, which predicts p.Asp2498GlufsX4, in exon 54. Her father, but not her mother, was a carrier of the mutation. While segmental paternal iUPD6 causing TNDM was reported twice, there are no previous reports of MDC1A caused by this event. This is a child with two genetic disorders, yet neither is caused by the parental consanguinity, which reinforces the importance of considering different etiological mechanisms in the genetic clinic. Ó 2014 Wiley Periodicals, Inc.
Key words: LAMA2; MDC1A; TNDM; 6q24 segmental uniparental disomy
INTRODUCTION Segmental uniparental disomy is a rare genetic event, characterized by the inheritance of both homologues of part of a chromosome
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Andrade RC, Nevado J, de Faria Domingues de Lima MA, Saad T, Moraes L, Chimelli L, Lapunzina P, Vargas FR. 2014. Segmental uniparental isodisomy of chromosome 6 causing transient diabetes mellitus and merosin-deficient congenital muscular dystrophy. Am J Med Genet Part A. 9999:1–6.
from one parent only, while the remaining part of the chromosome shows normal biparental inheritance. The chromosomal segment involved can present both homologues from one parental pair (i.e., heterodisomy), or two copies can be originated from one homologue only (i.e., isodisomy). Unfavorable phenotypes can arise from both hetero and isodisomy when the involved segment encompasses imprinted regions, resulting in aberrant genomic expression. Conflict of interest: none. Grant sponsor: INCA/MS; Grant sponsor: Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq); Grant number: 476808/2010-3; Grant sponsor: Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ); Grant number: E26/110.535/2012. Correspondence to: Fernando R. Vargas, Birth Defects Epidemiology Laboratory, Pav Leonidas Deane, sala 617, Fundac¸a˜o Oswaldo Cruz, Av. Brasil, 4365, Rio de Janeiro 21040-900, Brazil. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2014 DOI 10.1002/ajmg.a.36716
AJMGA-14-0186.R1(36716)
1
2 Isodisomy can also lead to reduction to homozygosity of a recessive mutation, with consequent expression of a recessive phenotype [Kotzot, 2008; Liehr, 2010; Yamazawa et al., 2010]. Transient neonatal diabetes mellitus (TNDM), characterized by hyperglycemia in the first weeks of life and intrauterine growth retardation, is a disorder typically caused by imprinting aberrations in chromosome 6q24 TNDM differentially methylated region (DMR), which can originate from paternal uniparental disomy [Temple et al., 2000; Temple and Shield, 2002; Mackay et al., 2005]. Close to this imprinted locus, 6q22-q23 encompasses the Lamininalpha 2 gene (LAMA2), the gene that is mutated in patients with merosin-deficient congenital muscular dystrophy type 1A (MDC1A), a recessively inherited disease characterized by early onset muscular dystrophy, feeding difficulties, progressive scoliosis, and white matter hypodensity. Seizures and cardiac involvement may also be observed. Patients do not achieve independent ambulation, and intellect is usually normal [Allamand and Guicheney, 2002; Quijano-Roy et al., 2012; Bo¨nnemann et al., 2014]. We here report on a discordant twin pair, where the proband is a girl who presented in the neonatal period both TNDM and MDC1A, caused by a segmental paternal isodisomy of the long arm of chromosome 6 and a homozygous mutation in LAMA2.
MATERIAL AND METHODS Clinical Report The patient was born at 37 weeks from a twin dizygotic dichorionic pregnancy. Both twins were female and their parents are first-degree cousins. The proband had intra-uterine growth retardation (birth weight of the proband 1,470 g; of her twin sister 2,800 g). Macroglossia was present at birth, and due to high blood glucose levels, neonatal diabetes mellitus was diagnosed on day 3, demanding NPH insulin administration for two months, with subsequent spontaneous regression. Hypotonia and severe muscle weakness were present since day 1, with swallowing difficulty that required gastrostomy. Her weight is currently on the 10th centile, and height on the 25th centile, while her twin sister’s height is in the 50th centile. She had delayed developmental milestones in the first years of life, but currently she shows apparently normal congnition. Electromyographic findings were suggestive of myopathy. Cranial MRI showed diffuse periventricular and subcortical white matter changes, mainly in the temporal regions. Immunohistochemistry of skeletal muscle biopsy was negative for merosin. Due to the diagnosis of both TNDM and MDC1A, cytogenetic investigation was performed in search of chromosomal rearrangements involving 6q, and a 46,XX karyotype was reported.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A protocol, and submitted to a duplex methylation-specific PCR with a set of three primers, as described by Mackay et al., [2005]: A forward primer, non-selective for methylation; and two reverse primers, one that targets highly methylated CpG sequences, and the other targets completely unmethylated CpG sequences located between positions 52,808 and 53,005 (AL109755). For a normal individual, each reaction using this set of primers should amplify a maternal (methylated) allele of 175 bp, and a paternal (nonmethylated) allele of 187 bp. Products were genotyped in a ABI Prism 3130 Genetic Analyzer (Applied Biosystems), and analyzed using Peak Scanner software (Life Technologies).
Microsatellite and SNP Array Analysis of Chromosome 6 To investigate the possibility of UPD6, microsatellite profiling was performed using genomic DNA from all family members. Seventeen markers along chromosome 6 were chosen (D6S1713, D6S1610, D6S434, D6S287, D6S407, D6S2437, D6S1620, D6S1705, D6S292, D6S314, D6S311, D6S1654, D6S1581, D6S305, D6S1719, D6S281, D6S446). The PCR products were genotyped in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems) and analyzed using Peak Scanner software (Life Technologies). For a further refinement of the region of interest, a SNP array was applied in the proband and her parents. Briefly, 200 ng of genomic DNA was used into a CytoSNP-850K BlueGnome BeadChip (Illumina, San Diego, USA), subsequently scanned with an Illumina iScan system and analyzed with the Illumina’s KaryoStudio software.
LAMA2 Sequencing All 65 exons of LAMA2 were amplified from the patient’s blood DNA, and sequenced with Big Dye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems1), according to manufacturer’s protocol, using the intronic primer sequences described by Geranmayeh et al., [2010]. Exons containing variants observed in the proband were also genotyped in her parents and sisters.
RESULTS 6q24 Imprinted TNDM CpG Island Methylation-specific PCR showed both maternal and paternal alleles in the patient’s parents and sisters. The patient’s DNA, however, exhibited only the 187 bp product, corresponding to the paternal, non-methylated allele of the imprinted TNDM CpG island (Fig. 1).
Analysis of 6q24 Imprinted TNDM CpG Island
Microsatellite Analysis
Peripheral blood genomic DNA was extracted from all family members (the proband, her twin sister, her older sister, and her parents). For each sample, 1 mg of DNA was treated with bisulphite using EpiTect Bisulphite Kit (Qiagen), according to manufacturer’s
Microsatellite profiling of chromosome 6 was informative for ten of the 17 markers analyzed. Two of them (D6S1713 and D6S1610), located respectively at 6p25.1 and 6p21.2, showed normal biparental inheritance. However, the other eight informative markers
ANDRADE ET AL.
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FIG. 1. Pedigree of the proband’s family. Each individual is followed by a schematic representation of chromosome 6 (red for paternal and blue for maternal chromosome), symbolizing the segmental uniparental disomy in the patient, and normal biparental inheritance in the patient’s siblings. The electropherograms corresponding to the c.7490_7493dupAAGA (p.Asp2498GlufsX4) mutation in exon 54 of LAMA2, followed by the electropherograms of MS-PCR products in the 6q24 TNDM DMR, are also shown for each individual. Duplication c.7490_7493dupAAGA (shown as a red underline in the electropherograms) is homozygous in the proband, was heterozygous in her father and siblings, and absent in her mother. For MS-PCR products, the patient exhibited only the 187 bp product, corresponding to the paternal, nonmethylated allele of the imprinted TNDM CpG island, while the remaining family members exhibited both maternal (175 bp) and paternal (187 bp) alleles.
(D6S407, D6S1620, D6S292, D6S311, D6S1654, D6S1581, D6S281, and D6S446) exhibited only the paternal allele (Table I). These results confirmed the patient had a segmental paternal UPD in the long arm of chromosome 6, encompassing the region between 6q22.33 and 6q27. Both sisters of the patient exhibited normal biparental inheritance for all informative markers (data not shown).
SNP Array Analysis SNP array confirmed the microsatellite findings, and allowed a more accurate estimation of the size of the UPD region, as well as its proximal limit. The UPD region in the proband spans from 6q22 to 6qter, and is 43.76 Mb in size (chr6: 127,158,296-170,919,470)
(GRCh37/hg19) (Fig. 2). The SNP array analysis did not disclose any other genomic rearrangement in the proband (data not shown).
LAMA2 Sequencing Given the patient’s segmental paternal UPD6 of a region containing the LAMA2 locus, and the absence of merosin in the skeletal muscle biopsy, we hypothesized the patient’s father carried a heterozygous mutation in LAMA2, and that the isodisomic state of the inherited mutated allele would have caused a reduction to homozygosity, resulting in the MDC1A phenotype in the proband. Sequencing of LAMA2 exons in the patient showed a homozygous frameshift mutation, c.7490_7493dupAAGA, which predicts p. Asp2498GlufsX4, located in exon 54. This mutation was reported
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
TABLE I. Genotypes of Markers of Chromosome 6 in the Patient and her Parents Marker D6S1713 D6S1610 D6S434 D6S287 D6S407 D6S2437 c.7490_7493dupAAGA rs1049476 D6S1620 D6S1705 D6S292 D6S314 D6S311 D6S1654 D6S1581 D6S305 D6S1719 D6S281 D6S446
pter-qtera 6p25.1 6p21.2 6q16.3 6q22.31 6q22.33 6q22.33 6q22.33 6q22.33 6q22.33 6q23.1 6q23.3 6q24.1 6q24.3 6q25.1 6q25.3 6q26 6q27 6q27 6q27
Father 2,4 1,1 1,3 2,2 2,3 1,1 wt,dup TC 2,3 1,2 2,3 1,1 1,2 1,3 1,2 1,1 1,2 1,2 1,3
Patient 1,2 1,3 1,3 1,2 2,2 1,1 dup,dup CC 2,2 2,2 3,3 1,1 2,2 1,1 1,1 1,1 2,2 2,2 3,3
Mother 1,3 2,3 2,3 1,2 1,3 1,2 wt,wt TT 1,3 1,2 1,1 1,2 3,4 2,3 2,2 1,1 2,3 1,3 1,2
Result Biparental Biparental Uninformative Uninformative Paternal UPD Uninformative Paternal UPD Paternal UPD Paternal UPD Uninformative Paternal UPD Uninformative Paternal UPD Paternal UPD Paternal UPD Uninformative Uninformative Paternal UPD Paternal UPD
a Genotypes from 17 microsatellite markers, LAMA2 mutation (c.7490_7493dupAAGA) and one informative SNP (rs1049476) are arranged from the distal end of the short arm to the distal end of the long arm of chromosome 6, according to information from the National Center for Biotechnology Information (NCBI) database. Wt: wild-type allele; dup: duplication present.
FIG. 2. SNP array of chromosome 6 of the patient and her parents. A: Father, B: Patient, C: Mother. Note that patient’s sample shows a large region of neutral copy-number block or loss of heterozygosity corresponding to uniparental isodisomy. It has 43.76 Mb of extent, from chr6: 127,158,296-170,919,470 (GRCh37/hg19; 6q22.33 to telomere) (green shaded). Maternal sample also exhibits some smaller copy-neutral blocks of homozygosity (green shaded), which are usually seen in normal population.
ANDRADE ET AL. once before in a Portuguese male infant with MDC1A [Oliveira et al., 2008]. As expected, the father, but not the mother, is the heterozygous carrier of the mutation. Both of her sisters are also heterozygotes for the mutation. Additionally, five SNPs with no clinical relevance were visualized in homozygosis in the patient, four of them uninformative for parental origin (data not shown), and one was present in heterozygosis in the father, and absent in the mother (Table I).
DISCUSSION The proband developed TNDM and MDC1A associated with segmental paternal uniparental disomy of chromosome 6. To our knowledge, there are no previous reports of MDC1A caused by uniparental disomy. This observation, together with the fact that the parents are first-degree cousins and thus, at higher risk of carrying the same mutation segregating in the family, could have led us to the assumption that both were probably carriers of a mutation in the LAMA2 gene, and thus, the recurrence risk in forthcoming offspring would be 25%. On the other hand, bearing in mind that the real cause of the disease is reduction to homozygosity of a mutation in LAMA2 by the paternal uniparental disomy, the risk of another sibling carrying the same disease is negligible, since uniparental disomy in cells with normal karyotype is considered a de novo event [Yamazawa et al., 2010], and it is estimated to happen in 1:3,500 live births in any chromosome [Robinson, 2000]. Likewise, the recurrence risk of transient neonatal diabetes mellitus will depend on the genetic mechanism causing the disorder. Approximately 70% of cases are caused by abnormal imprinting in 6q24 [Flanagan et al., 2007]. The DMR involved lies within the shared promoter of the imprinted, paternally expressed genes PLAGL1 [ZAC] and HYMAI. At least three different mechanisms are associated with overexpression of these two genes, and cause TNDM: (1) segmental or complete paternal UPD6; (2) chromosomal duplication of paternal 6q24; and (3) hypomethylation of the maternal 6q24 DMR [Mackay et al., 2005; Docherty et al., 2010]. While the first mechanism carries insignificant recurrence risk, the second type may recur in an autosomal dominant manner if the duplication is inherited from the father. The third mechanism, hypomethylation of maternal 6q24 DMR, when associated with mutations in the ZFP57 gene, may recur in an autosomal recessive manner [Mackay et al., 2005]. Segmental paternal UPD of the long arm of chromosome 6 as the cause of transient neonatal diabetes has been reported twice: in a Hispanic male patient that died 14 days after birth [Das et al., 2000], and in a pair of phenotypically concordant monozygotic twin sisters conceived after in vitro fertilization [Suzuki et al., 2010]. As distinct from the previously reported affected twins, the proband and twin sister reported here were not conceived by in vitro fertilization. It is also noteworthy that in the patient reported here, the parents were first-degree cousins. Still, we could not find any evidence that parental consanguinity is associated with the occurrence of UPD. It has been stated that segmental isodisomy happens as a postzygotic event, when an aberrant mitotic recombination followed by random segregation of sister chromatids produces different cell types, resulting in variable levels of mosaicism, depending basically on two factors: the developmental stage when the recombination
5 occurred, and the preferential death of specific cell types [Das et al., 2000]. Analysis of blood DNA did not show any evidence of mosaicism in our patient. Although there is the possibility of a low percentage of mosaicism undetectable by the methods we used, the absence of detected mosaicism indicates that the mechanism forming the uniparental disomy in this case was most probably a meiotic/mitotic error: a non-disjunction meiotic event, followed by mitotic crossing over between the maternal and paternal homologues, with subsequent trisomy rescue [Suzuki et al., 2010]. To sum up, we report a consanguineous family with a child with two genetic disorders, neither of which was caused by homozygosity due to identity by descent associate with consanguinity. This fact reinforces the importance of considering alternative genetic mechanisms, such as uniparental disomy, as possible causes of developmental diseases, resulting in a better understanding of the patient’s phenotype and a more accurate prediction of recurrence risks.
ACKNOWLEDGMENTS RCA is recipient of INCA/MS grant. FRV is recipient of Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) grant 476808/2010-3 and Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) grant E26/110.535/2012. PL is part of the EUCID network of imprinting disorders.
REFERENCES Allamand V, Guicheney P. 2002. Merosin-deficient congenital muscular dystrophy, autosomal recessive (MDC1A, MIM#156225, LAMA2 gene coding for alpha2 chain of laminin). Eur J Hum Genet 10:91–94. Bo¨nnemann CG, Wang CH, Quijano-Roy S, Deconinck N, Bertini E, Ferreiro A, Muntoni F, Sewry C, Be´roud C, Mathews KD, Moore SA, Bellini J, Rutkowski A, North KN. 2014. Diagnostic approach to the congenital muscular dystrophies. Neuromuscul Disord 24:289–311. Das S, Lese CM, Song M, Jensen JL, Wells LA, Barnoski BL, Roseberry JA, Camacho JM, Ledbetter DH, Schnur RE. 2000. Partial paternal uniparental disomy of chromosome 6 in an infant with neonatal diabetes, macroglossia, and craniofacial abnormalities. Am J Hum Genet 67:1586– 1591. Docherty LE, Poole RL, Mattocks CJ, Lehmann A, Temple IK, Mackay DJ. 2010. Further refinement of the critical minimal genetic region for the imprinting disorder 6q24 transient neonatal diabetes. Diabetologia 53:2347–2351. Flanagan SE, Patch A, Mackay DJG, Edghill EL, Gloyn AL, Robinson D, Shield JP, Temple K, Ellard S, Hattersley AT. 2007. Mutations in ATPsensitive K+ channel genes cause transient neonatal diabetes and permanent diabetes in childhood or adulthood. Diabetes 56:1930–1937. Geranmayeh F, Clement E, Feng LH, Sewry C, Pagan J, Mein R, Abbs S, Brueton L, Childs AM, Jungbluth H, De Goede CG, Lynch B, Lin JP, Chow G, Sousa Cd, O’Mahony O, Majumdar A, Straub V, Bushby K, Muntoni F. 2010. Genotype-phenotype correlation in a large population of muscular dystrophy patients with LAMA2 mutations. Neuromuscul Disord 20:241–250. Kotzot D. 2008. Complex and segmental uniparental disomy updated. J Med Genet 45:545–556. Liehr T. 2010. Cytogenetic contribution to uniparental disomy (UPD). Mol Cytogenet 3:8.
6 Mackay DJ, Callaway JL, Marks SM, White HE, Acerini CL, Boonen SE, Dayanikli P, Firth HV, Goodship JA, Haemers AP, Hahnemann JM, Kordonouri O, Masoud AF, Oestergaard E, Storr J, Ellard S, Hattersley AT, Robinson DO, Temple IK. 2008. Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57. Nat Genet 40:949–951. Mackay DJ, Temple IK, Shield JP, Robinson DO. 2005. Bisulphite sequencing of the transient neonatal diabetes mellitus DMR facilitates a novel diagnostic test but reveals no methylation anomalies in patients of unknown aetiology. Hum Genet 116:255–261. Oliveira J, Santos R, Soares-Silva I, Jorge P, Vieira E, Oliveira ME, Moreira A, Coelho T, Ferreira JC, Fonseca MJ, Barbosa C, Prats J, Arı´ztegui ML, Martins ML, Moreno T, Heinimann K, Barbot C, Pascual-Pascual SI, Cabral A, Fineza I, Santos M, Bronze-da-Rocha E. 2008. LAMA2 gene analysis in a cohort of 26 congenital muscular dystrophy patients. Clin Genet 74:502–512. Quijano-Roy S, Sparks S, Rutkowski A. 2012. LAMA2-related muscular dystrophy. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR,
AMERICAN JOURNAL OF MEDICAL GENETICS PART A Fong CT, Smith RJH, Stephens K, editors. GeneReviews. Seattle (WA): University of Washington. Seattle; 1993-2014. Available from http:// www.ncbi.nlm.nih.gov/books/NBK97333/ Robinson WP. 2000. Mechanisms leading to uniparental disomy and their clinical consequences. BioEssays 22:452–459. Suzuki S, Fujisawa D, Hashimoto K, Asano T, Maimaiti M, Matsuo K, Tanahashi Y, Mukai T, Fujieda K. 2010. Partial paternal uniparental disomy of chromosome 6 in monozygotic twins with transient neonatal diabetes mellitus and macroglossia. Clin Genet 78:580–584. Temple IK, Gardner RJ, Mackay DJ, Barber JC, Robinson DO, Shield JP. 2000. Transient neonatal diabetes: Widening the understanding of the etiopathogenesis of diabetes. Diabetes 49:1359–1366. Temple IK, Shield JP. 2002. Transient neonatal diabetes, a disorder of imprinting. J Med Genet 39:872–875. Yamazawa K, Ogata T, Ferguson-Smith AC. 2010. Uniparental disomy and human disease: An overview. Am J Med Genet C Semin Med Genet 154C:329–334.
Segmental Uniparental Isodisomy of Chromosome 6 Causing Transient Diabetes Mellitus and Merosin-Deficient Congenital Muscular Dystrophy Raissa Coelho Andrade,1 Julia´n Nevado,2 Maria Ange´lica de Faria Domingues de Lima,3,4 Taˆnia Saad,5 Lucia Moraes,6 Leila Chimelli,7 Pablo Lapunzina,2 and Fernando Regla Vargas1,3,8* 1
Genetics Division, Instituto Nacional de Caˆncer, Rio de Janeiro, Brazil INGEMM, Instituto de Gene´tica Me´dica y Molecular, IdiPAZ-CIBERER, Universidad Auto´noma de Madrid, Madrid, Spain
2 3
Genetics and Molecular Biology Department, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil Programa de Internato em Gene´tica, Universidade UnigranRio, Rio de Janeiro, Brazil
4 5
Neuropediatrics Service, Instituto Fernandes Figueira, Fundac¸˜ao Oswaldo Cruz, Rio de Janeiro, Brazil Medical Genetics Center, Instituto Fernandes Figueira, Fundac¸˜ao Oswaldo Cruz, Rio de Janeiro, Brazil
6 7
Pathology Department, Medical School, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Birth Defects Epidemiology Laboratory, Fundac¸˜ao Oswaldo Cruz, Rio de Janeiro, Brazil
8
Manuscript Received: 13 March 2014; Manuscript Accepted: 7 July 2014
Segmental uniparental isodisomy (iUPD) is a rare genetic event that may cause aberrant expression of imprinted genes, and reduction to homozygosity of a recessive mutation. Transient neonatal diabetes mellitus (TNDM) is typically caused by imprinting aberrations in chromosome 6q24 TNDM differentiallymethylated region (DMR). Approximately, 15.12 Mb upstream in 6q22-q23 is located LAMA2, the gene responsible of merosindeficient congenital muscular dystrophy type 1A (MDC1A). We investigated a patient diagnosed both with TNDM and MDC1A, born from a twin dichorionic discordant pregnancy. Parents are first-degree cousins. Methylation sensitive-PCR of the imprinted 6q24 TNDM CpG island showed only the non-methylated (paternal) allele. Microsatellite markers and SNP array profiling disclosed normal biparental inheritance at 6p and a segmental paternal iUPD, between 6q22.33 and 6q27. Sequencing of LAMA2 exons showed a homozygous frameshift mutation, c.7490_7493dupAAGA, which predicts p.Asp2498GlufsX4, in exon 54. Her father, but not her mother, was a carrier of the mutation. While segmental paternal iUPD6 causing TNDM was reported twice, there are no previous reports of MDC1A caused by this event. This is a child with two genetic disorders, yet neither is caused by the parental consanguinity, which reinforces the importance of considering different etiological mechanisms in the genetic clinic. Ó 2014 Wiley Periodicals, Inc.
Key words: LAMA2; MDC1A; TNDM; 6q24 segmental uniparental disomy
INTRODUCTION Segmental uniparental disomy is a rare genetic event, characterized by the inheritance of both homologues of part of a chromosome
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Andrade RC, Nevado J, de Faria Domingues de Lima MA, Saad T, Moraes L, Chimelli L, Lapunzina P, Vargas FR. 2014. Segmental uniparental isodisomy of chromosome 6 causing transient diabetes mellitus and merosin-deficient congenital muscular dystrophy. Am J Med Genet Part A. 9999:1–6.
from one parent only, while the remaining part of the chromosome shows normal biparental inheritance. The chromosomal segment involved can present both homologues from one parental pair (i.e., heterodisomy), or two copies can be originated from one homologue only (i.e., isodisomy). Unfavorable phenotypes can arise from both hetero and isodisomy when the involved segment encompasses imprinted regions, resulting in aberrant genomic expression. Conflict of interest: none. Grant sponsor: INCA/MS; Grant sponsor: Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq); Grant number: 476808/2010-3; Grant sponsor: Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ); Grant number: E26/110.535/2012. Correspondence to: Fernando R. Vargas, Birth Defects Epidemiology Laboratory, Pav Leonidas Deane, sala 617, Fundac¸a˜o Oswaldo Cruz, Av. Brasil, 4365, Rio de Janeiro 21040-900, Brazil. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2014 DOI 10.1002/ajmg.a.36716
AJMGA-14-0186.R1(36716)
1
2 Isodisomy can also lead to reduction to homozygosity of a recessive mutation, with consequent expression of a recessive phenotype [Kotzot, 2008; Liehr, 2010; Yamazawa et al., 2010]. Transient neonatal diabetes mellitus (TNDM), characterized by hyperglycemia in the first weeks of life and intrauterine growth retardation, is a disorder typically caused by imprinting aberrations in chromosome 6q24 TNDM differentially methylated region (DMR), which can originate from paternal uniparental disomy [Temple et al., 2000; Temple and Shield, 2002; Mackay et al., 2005]. Close to this imprinted locus, 6q22-q23 encompasses the Lamininalpha 2 gene (LAMA2), the gene that is mutated in patients with merosin-deficient congenital muscular dystrophy type 1A (MDC1A), a recessively inherited disease characterized by early onset muscular dystrophy, feeding difficulties, progressive scoliosis, and white matter hypodensity. Seizures and cardiac involvement may also be observed. Patients do not achieve independent ambulation, and intellect is usually normal [Allamand and Guicheney, 2002; Quijano-Roy et al., 2012; Bo¨nnemann et al., 2014]. We here report on a discordant twin pair, where the proband is a girl who presented in the neonatal period both TNDM and MDC1A, caused by a segmental paternal isodisomy of the long arm of chromosome 6 and a homozygous mutation in LAMA2.
MATERIAL AND METHODS Clinical Report The patient was born at 37 weeks from a twin dizygotic dichorionic pregnancy. Both twins were female and their parents are first-degree cousins. The proband had intra-uterine growth retardation (birth weight of the proband 1,470 g; of her twin sister 2,800 g). Macroglossia was present at birth, and due to high blood glucose levels, neonatal diabetes mellitus was diagnosed on day 3, demanding NPH insulin administration for two months, with subsequent spontaneous regression. Hypotonia and severe muscle weakness were present since day 1, with swallowing difficulty that required gastrostomy. Her weight is currently on the 10th centile, and height on the 25th centile, while her twin sister’s height is in the 50th centile. She had delayed developmental milestones in the first years of life, but currently she shows apparently normal congnition. Electromyographic findings were suggestive of myopathy. Cranial MRI showed diffuse periventricular and subcortical white matter changes, mainly in the temporal regions. Immunohistochemistry of skeletal muscle biopsy was negative for merosin. Due to the diagnosis of both TNDM and MDC1A, cytogenetic investigation was performed in search of chromosomal rearrangements involving 6q, and a 46,XX karyotype was reported.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A protocol, and submitted to a duplex methylation-specific PCR with a set of three primers, as described by Mackay et al., [2005]: A forward primer, non-selective for methylation; and two reverse primers, one that targets highly methylated CpG sequences, and the other targets completely unmethylated CpG sequences located between positions 52,808 and 53,005 (AL109755). For a normal individual, each reaction using this set of primers should amplify a maternal (methylated) allele of 175 bp, and a paternal (nonmethylated) allele of 187 bp. Products were genotyped in a ABI Prism 3130 Genetic Analyzer (Applied Biosystems), and analyzed using Peak Scanner software (Life Technologies).
Microsatellite and SNP Array Analysis of Chromosome 6 To investigate the possibility of UPD6, microsatellite profiling was performed using genomic DNA from all family members. Seventeen markers along chromosome 6 were chosen (D6S1713, D6S1610, D6S434, D6S287, D6S407, D6S2437, D6S1620, D6S1705, D6S292, D6S314, D6S311, D6S1654, D6S1581, D6S305, D6S1719, D6S281, D6S446). The PCR products were genotyped in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems) and analyzed using Peak Scanner software (Life Technologies). For a further refinement of the region of interest, a SNP array was applied in the proband and her parents. Briefly, 200 ng of genomic DNA was used into a CytoSNP-850K BlueGnome BeadChip (Illumina, San Diego, USA), subsequently scanned with an Illumina iScan system and analyzed with the Illumina’s KaryoStudio software.
LAMA2 Sequencing All 65 exons of LAMA2 were amplified from the patient’s blood DNA, and sequenced with Big Dye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems1), according to manufacturer’s protocol, using the intronic primer sequences described by Geranmayeh et al., [2010]. Exons containing variants observed in the proband were also genotyped in her parents and sisters.
RESULTS 6q24 Imprinted TNDM CpG Island Methylation-specific PCR showed both maternal and paternal alleles in the patient’s parents and sisters. The patient’s DNA, however, exhibited only the 187 bp product, corresponding to the paternal, non-methylated allele of the imprinted TNDM CpG island (Fig. 1).
Analysis of 6q24 Imprinted TNDM CpG Island
Microsatellite Analysis
Peripheral blood genomic DNA was extracted from all family members (the proband, her twin sister, her older sister, and her parents). For each sample, 1 mg of DNA was treated with bisulphite using EpiTect Bisulphite Kit (Qiagen), according to manufacturer’s
Microsatellite profiling of chromosome 6 was informative for ten of the 17 markers analyzed. Two of them (D6S1713 and D6S1610), located respectively at 6p25.1 and 6p21.2, showed normal biparental inheritance. However, the other eight informative markers
ANDRADE ET AL.
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FIG. 1. Pedigree of the proband’s family. Each individual is followed by a schematic representation of chromosome 6 (red for paternal and blue for maternal chromosome), symbolizing the segmental uniparental disomy in the patient, and normal biparental inheritance in the patient’s siblings. The electropherograms corresponding to the c.7490_7493dupAAGA (p.Asp2498GlufsX4) mutation in exon 54 of LAMA2, followed by the electropherograms of MS-PCR products in the 6q24 TNDM DMR, are also shown for each individual. Duplication c.7490_7493dupAAGA (shown as a red underline in the electropherograms) is homozygous in the proband, was heterozygous in her father and siblings, and absent in her mother. For MS-PCR products, the patient exhibited only the 187 bp product, corresponding to the paternal, nonmethylated allele of the imprinted TNDM CpG island, while the remaining family members exhibited both maternal (175 bp) and paternal (187 bp) alleles.
(D6S407, D6S1620, D6S292, D6S311, D6S1654, D6S1581, D6S281, and D6S446) exhibited only the paternal allele (Table I). These results confirmed the patient had a segmental paternal UPD in the long arm of chromosome 6, encompassing the region between 6q22.33 and 6q27. Both sisters of the patient exhibited normal biparental inheritance for all informative markers (data not shown).
SNP Array Analysis SNP array confirmed the microsatellite findings, and allowed a more accurate estimation of the size of the UPD region, as well as its proximal limit. The UPD region in the proband spans from 6q22 to 6qter, and is 43.76 Mb in size (chr6: 127,158,296-170,919,470)
(GRCh37/hg19) (Fig. 2). The SNP array analysis did not disclose any other genomic rearrangement in the proband (data not shown).
LAMA2 Sequencing Given the patient’s segmental paternal UPD6 of a region containing the LAMA2 locus, and the absence of merosin in the skeletal muscle biopsy, we hypothesized the patient’s father carried a heterozygous mutation in LAMA2, and that the isodisomic state of the inherited mutated allele would have caused a reduction to homozygosity, resulting in the MDC1A phenotype in the proband. Sequencing of LAMA2 exons in the patient showed a homozygous frameshift mutation, c.7490_7493dupAAGA, which predicts p. Asp2498GlufsX4, located in exon 54. This mutation was reported
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
TABLE I. Genotypes of Markers of Chromosome 6 in the Patient and her Parents Marker D6S1713 D6S1610 D6S434 D6S287 D6S407 D6S2437 c.7490_7493dupAAGA rs1049476 D6S1620 D6S1705 D6S292 D6S314 D6S311 D6S1654 D6S1581 D6S305 D6S1719 D6S281 D6S446
pter-qtera 6p25.1 6p21.2 6q16.3 6q22.31 6q22.33 6q22.33 6q22.33 6q22.33 6q22.33 6q23.1 6q23.3 6q24.1 6q24.3 6q25.1 6q25.3 6q26 6q27 6q27 6q27
Father 2,4 1,1 1,3 2,2 2,3 1,1 wt,dup TC 2,3 1,2 2,3 1,1 1,2 1,3 1,2 1,1 1,2 1,2 1,3
Patient 1,2 1,3 1,3 1,2 2,2 1,1 dup,dup CC 2,2 2,2 3,3 1,1 2,2 1,1 1,1 1,1 2,2 2,2 3,3
Mother 1,3 2,3 2,3 1,2 1,3 1,2 wt,wt TT 1,3 1,2 1,1 1,2 3,4 2,3 2,2 1,1 2,3 1,3 1,2
Result Biparental Biparental Uninformative Uninformative Paternal UPD Uninformative Paternal UPD Paternal UPD Paternal UPD Uninformative Paternal UPD Uninformative Paternal UPD Paternal UPD Paternal UPD Uninformative Uninformative Paternal UPD Paternal UPD
a Genotypes from 17 microsatellite markers, LAMA2 mutation (c.7490_7493dupAAGA) and one informative SNP (rs1049476) are arranged from the distal end of the short arm to the distal end of the long arm of chromosome 6, according to information from the National Center for Biotechnology Information (NCBI) database. Wt: wild-type allele; dup: duplication present.
FIG. 2. SNP array of chromosome 6 of the patient and her parents. A: Father, B: Patient, C: Mother. Note that patient’s sample shows a large region of neutral copy-number block or loss of heterozygosity corresponding to uniparental isodisomy. It has 43.76 Mb of extent, from chr6: 127,158,296-170,919,470 (GRCh37/hg19; 6q22.33 to telomere) (green shaded). Maternal sample also exhibits some smaller copy-neutral blocks of homozygosity (green shaded), which are usually seen in normal population.
ANDRADE ET AL. once before in a Portuguese male infant with MDC1A [Oliveira et al., 2008]. As expected, the father, but not the mother, is the heterozygous carrier of the mutation. Both of her sisters are also heterozygotes for the mutation. Additionally, five SNPs with no clinical relevance were visualized in homozygosis in the patient, four of them uninformative for parental origin (data not shown), and one was present in heterozygosis in the father, and absent in the mother (Table I).
DISCUSSION The proband developed TNDM and MDC1A associated with segmental paternal uniparental disomy of chromosome 6. To our knowledge, there are no previous reports of MDC1A caused by uniparental disomy. This observation, together with the fact that the parents are first-degree cousins and thus, at higher risk of carrying the same mutation segregating in the family, could have led us to the assumption that both were probably carriers of a mutation in the LAMA2 gene, and thus, the recurrence risk in forthcoming offspring would be 25%. On the other hand, bearing in mind that the real cause of the disease is reduction to homozygosity of a mutation in LAMA2 by the paternal uniparental disomy, the risk of another sibling carrying the same disease is negligible, since uniparental disomy in cells with normal karyotype is considered a de novo event [Yamazawa et al., 2010], and it is estimated to happen in 1:3,500 live births in any chromosome [Robinson, 2000]. Likewise, the recurrence risk of transient neonatal diabetes mellitus will depend on the genetic mechanism causing the disorder. Approximately 70% of cases are caused by abnormal imprinting in 6q24 [Flanagan et al., 2007]. The DMR involved lies within the shared promoter of the imprinted, paternally expressed genes PLAGL1 [ZAC] and HYMAI. At least three different mechanisms are associated with overexpression of these two genes, and cause TNDM: (1) segmental or complete paternal UPD6; (2) chromosomal duplication of paternal 6q24; and (3) hypomethylation of the maternal 6q24 DMR [Mackay et al., 2005; Docherty et al., 2010]. While the first mechanism carries insignificant recurrence risk, the second type may recur in an autosomal dominant manner if the duplication is inherited from the father. The third mechanism, hypomethylation of maternal 6q24 DMR, when associated with mutations in the ZFP57 gene, may recur in an autosomal recessive manner [Mackay et al., 2005]. Segmental paternal UPD of the long arm of chromosome 6 as the cause of transient neonatal diabetes has been reported twice: in a Hispanic male patient that died 14 days after birth [Das et al., 2000], and in a pair of phenotypically concordant monozygotic twin sisters conceived after in vitro fertilization [Suzuki et al., 2010]. As distinct from the previously reported affected twins, the proband and twin sister reported here were not conceived by in vitro fertilization. It is also noteworthy that in the patient reported here, the parents were first-degree cousins. Still, we could not find any evidence that parental consanguinity is associated with the occurrence of UPD. It has been stated that segmental isodisomy happens as a postzygotic event, when an aberrant mitotic recombination followed by random segregation of sister chromatids produces different cell types, resulting in variable levels of mosaicism, depending basically on two factors: the developmental stage when the recombination
5 occurred, and the preferential death of specific cell types [Das et al., 2000]. Analysis of blood DNA did not show any evidence of mosaicism in our patient. Although there is the possibility of a low percentage of mosaicism undetectable by the methods we used, the absence of detected mosaicism indicates that the mechanism forming the uniparental disomy in this case was most probably a meiotic/mitotic error: a non-disjunction meiotic event, followed by mitotic crossing over between the maternal and paternal homologues, with subsequent trisomy rescue [Suzuki et al., 2010]. To sum up, we report a consanguineous family with a child with two genetic disorders, neither of which was caused by homozygosity due to identity by descent associate with consanguinity. This fact reinforces the importance of considering alternative genetic mechanisms, such as uniparental disomy, as possible causes of developmental diseases, resulting in a better understanding of the patient’s phenotype and a more accurate prediction of recurrence risks.
ACKNOWLEDGMENTS RCA is recipient of INCA/MS grant. FRV is recipient of Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) grant 476808/2010-3 and Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) grant E26/110.535/2012. PL is part of the EUCID network of imprinting disorders.
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