Parental Balanced Chromosomal Rearrangement Leading to Major Genomic Imbalance and an Autosomal Trisomy Resulting in Consecutive Pregnancy Loss: a Case Report

Journal of Genetics (2021)100:54 Ó Indian Academy of Sciences

https://doi.org/10.1007/s12041-021-01304-3 (0123456789().,-volV)(0123456789().,-volV)

RESEARCH NOTE

Parental balanced chromosomal rearrangement leading to major genomic imbalance and an autosomal trisomy resulting in consecutive pregnancy loss: a case report

ANUSHKA SHRIVASTAVA1,2, SEEMA THAKUR3, TARA NATH2, ABHIPSA V. F. DEBNATH1 and SONAL R. BAKSHI1*

1Institute of Science, Nirma University, Ahmedabad 382 481, India 2Advanced Genomic Institute and Laboratory Medicine (Labassure), New Delhi 110 003, India 3Department of Clinical Genetics and Fetal Medicine, Fortis Hospital, New Delhi 110 088, India *For correspondence. E-mail: [email protected].

Received 8 November 2020; revised 9 February 2021; accepted 23 March 2021

Abstract. Chromosomal aberrations such as parental balanced translocation contribute to a significant proportion of recurrent pregnancy losses. These have extreme genetic implications on the foetus which can either cause physical and/or mental retardation or early death. In this study, we report a unique clinical case of a couple with three consecutive pregnancy losses and we aim to determine the genetic abnormalities causing the miscarriages. Conventional cytogenetic and molecular genetic analysis were performed on the products of conception as well as for the parents. Chromosomal analysis was performed based on the ISCN 2016 guidelines. This was followed by Chromosomal microarray analysis carried out using ISCA consortium probe set (8X60K). Genetic testing for the 1st product of conception was not performed. However, the 2nd and 3rd products of conception indicated an autosomal trisomy 22 and a 3.7 Mb deletion of 2p (cytoband p25.3) along with 13.6 Mb duplication of 16p (cytoband p13.3p13.12), respectively. The paternal karyotype was normal but mother showed a balanced translocation 46,XX,t(2;16)(p25.3;p13.3). This was parallel to the products of conception microarray findings, unbalanced chromosomal abnormality in the foetus. Balanced translocation carriers are susceptible for meiotic nondisjunction processes and early detection of genetic anomalies can be informative to parents trying to conceive. Genetic analysis of the abortus after the 1st loss can be helpful to understand the cause of miscarriage. A combined approach with microarray and karyotyping of the products of conception can be important to determine the specific aberrations in the foetal chromosomes leading to abortion.

Keywords. spontaneous abortions; balanced translocation; chromosomal analysis; microarray; trisomy.

Introduction recurrent abortions (Stern et al. 1999). The translocation carrier have a risk to have a child with birth defects in terms Recurrent spontaneous abortion is defined as two or more of intellectual development and other phenotypic anomaly consecutive loss of clinically established intrauterine preg- due to a segmental aneusomy (Gardner et al. 2012). nancy before 20 weeks of gestation. Early pregnancy loss In case of couples with balanced translocation carrier, the occurs in 15% of all clinically recognized pregnancies. The reproductive risk of an abnormal foetus depends on the chromosomal abnormalities make a significant contribution extent of genetic imbalance caused, also on the number and in recurrent spontaneous abortions, accounting for *50% of breakpoints of the chromosome involved (Mackie and first trimester foetal loss (Van den Berg et al. 2012). Studies Scriven 2004). of couples who are balanced translocation carriers indicate Balanced translocation is defined as rearrangement of that as many as 80% of the pregnancies may end up in segments of chromosomes with no loss of critical genetic spontaneous miscarriage (Sheth et al. 2013). Balanced material or no lethal fusion gene formation, resulting in a translocations occur in 0.2% of the neonatal population, in phenotypically normal individual whereas, unbalanced 0.6% of infertile couples and in up to 9.2% of patients with translocation is defined as chromosomal rearrangement 54 Page 2 of 6 Anushka Shrivastava et al. resulting in loss or gain of genetic material which produces (GIBCO). Harvesting of cells was done after the proper an altered phenotype (Sadler and Langman 2011). confluency of the culture appeared using standard cytoge- Balanced chromosomal translocation carriers may have netic methods (Jobanputra et al. 2002). Karyotype analysis more risk of recurrent spontaneous abortion and/or children was done using Cytovision software (Leica Microsystems) with serious birth defects due to unbalanced chromosome as per ISCN 2016. complements (Kunwar and Bakshi 2016). For further analysis, chromosomal microarray CMA (Agilent) (whole genome microarray-based hybridization) was performed on the DNA isolated from product of con- Materials and method ception. This array contains *60,000 CGII probes, which consists of the entire ISCA (International standards for Case report Cytogenetic Arrays) consortium 8X60K version probe set. The array can detect gain and losses at a minimum of 400 kb The present study reports a case of a young couple presented to 200 kb, respectively across the genome, or smaller (C50 with a history of three recurrent abortions in the first tri- kb) for clinically relevant deletion/duplication syndromes in mester of pregnancy (figure 1). The couple themselves did the subtelomere and pericentromere regions or targeted not show any evidence of genetic abnormality. Genetic genes. testing of the 1st product of conception (POC) was not performed, and thus analysis of genetic basis of the next two recurrent abortions was necessary. A conventional chromo- Results some analysis of the 2nd POC was done first followed by Chromosomal microarray for 3rd POC specifically to find Table 1 combines the genetic findings from chromosomal out the genetic defects in the abortus to determine the reason analysis and microarray analysis of the products of con- behind previous pregnancy failures. The results from ception. The karyotype of the 2nd POC showed the presence microarray lead to the cytogenetic study of the parents. The of trisomy 22 (47,XX,?22) (figure 2). The product of con- couple did not approach us for the 4th pregnancy, so it is ception from the couple was collected for CMA studies at 7 assumed that it would have been a successful pregnancy. weeks of gestation from the 3rd abortion. The array report The POC samples were transported in sealed and sterile from this POC [arr[hg19] 2p25.3(17019-3729241)x1, sample collection vial containing RPMI media with antibi- 16p13.3p13.12(106271-13792996)x3] showed a clinically otics at room temperature. The samples were then processed significant genomic deletion of 3.7 Mb on short arm of for culture. Abortus material (preferably villi) was washed, chromosome 2p25.3 and a clinically significant genomic enzyme treated and cultured in T-Flask in Amniomax media duplication of 13.6 Mb on short arm of chromosome 16 p13.3p13.12 (figure 3). Parental karyotyping revealed a normal karyotype for the father (46,XY) but the mother was a carrier of balanced translocation involving chromosomes 2 and 16 [46,XX,t(2;16)(p25.3;p13.3)] (figure 4).

Discussion

A 25 year old female presented with a history of three consecutive spontaneous abortions, clinically indicated as G4P0A3. The cytogenetic studies were carried out on Figure 1. Pedigree showing three consecutive abortions and one products of conception to identify any constitutional genetic live pregnancy. anomaly in the aborted foetus. Genetic testing was not

Table 1. Details of recurrent abortions and clinically signiﬁcant genetic ﬁndings in POCs.

Gravida Status Gestation (weeks) Ultrasound ﬁnding Genetic ﬁnding

First Spontaneous abortion 7 No Gestational sac seen – Second Spontaneous abortion 8 Cardiac activity not seen Trisomy 22 Third Spontaneous abortion 7 Cardiac activity present but 3.7 Mb deletion on chromosome 2p disappeared after 2 days and 13.6 Mb duplication on chromosome 16p Fourth Current pregnancy 7 Anembryonic conception, – no yolk sac seen Balanced translocation causing recurrent pregnancy loss Page 3 of 6 54

Figure 2. Karyotype from 2nd POC showing trisomy 22, 47,XX,?22. POC, product of conception.

Figure 3. Graph showing genomic deletion on short arm of chromosome 2 and genomic gain on short arm of chromosome 16. 54 Page 4 of 6 Anushka Shrivastava et al.

Figure 4. Karyotype from the mother showing balanced chromosome translocation 46,XX,t(2;16)(p25.3;p13.3).

Table 2. List of genes involved in the critical region of chromosome 2p25.3 (as listed in www.Omim.org).

Gene Location Gene Gene name number Phenotype

2:38,813 FAM110C Family with sequence 611395 2p25.3 similarity 110, member C 2:218,135 SH3YL1 SH3 domain- and SYLF 617314 2p25.3 domain-containing protein 1 2:663,876 2p25.3 TMEM18 Transmembrane protein 18 613220 2:1,631,886 PXDN, D2S448E, KIAA0230, Peroxidasin 605158 2p25.3 PRG2, PXN, COPOA, ASGD7 2:3,497,365 ADI1, SIPL Acireductone dioxygenase 1 613400 2p25.3 2:1,789,112 MYT1L, KIAA1106, MRD39 Myelin transcription factor 1-like 613084 Mental retardation, autosomal dominant 39 2p25.3 2:1,413,287 TPO, TPX, TDH2A Thyroid peroxidase 606765 Thyroid dyshormonogenesis 2A 2p25.3 2:3,188,924 EIPR1, TSSC1 EARP complex and GARP 608998 2p25.3 complex interacting protein 1 2:3,379,244 TRAPPC12, TTC15, PEBAS Trafﬁcking protein particle 614139 Encephalopathy, progressive, early-onset, 2p25.3 complex, subunit 12 with brain atrophy and spasticity 2:3,594,831 COLEC11, CLK1, 3MC2 Collectin 11 612502 3MC syndrome 2 2p25.3 2:3,531,812 RNASEH1, PEOB2 Ribonuclease H1 604123 Progressive external ophthalmoplegia 2p25.3 with mitochondrial DNA deletions, autosomal recessive 2 2:3,575,259 RPS7, DBA8 Ribosomal protein S7 603658 Diamond-Blackfan anemia 8 2p25.3 performed on the 1st POC. The second consecutive abortion autosomal trisomy of chromosome 22 (47,XX,?22). The occurred at 8 weeks of gestation with no cardiac activity miscarriages occurring before 10 weeks of gestation are observed on ultrasound. The karyotype revealed an majorly due to chromosomal aneuploidies arising from new Balanced translocation causing recurrent pregnancy loss Page 5 of 6 54

Table 3. List of a few genes involved in the critical region of chromosome 16p (as listed in www.Omim.org).

Gene Location Gene Gene name number Phenotype

16:0 DEL16p13.3, RSTSS Chromosome 16p13.3 deletion 610543 Chromosome 16p13.3 16p13.3 syndrome (Rubinstein–Taybi deletion deletion syndrome syndrome) 16:0 DUP16p13.3, C16DUPq13.3 Chromosome 16p13.3 duplication 613458 Chromosome 16p13.3 16p13.3 syndrome duplication syndrome 16:0 HBHR, ATR1 Alpha-thalassaemia/mental retardation 141750 Alpha-thalassaemia/mental 16pter-p13.3 syndrome, type 1 retardation syndrome, type 1 16:0 MCOPCT1 Microphthalmia with cataract 1 156850 Microphthalmia with cataract 1 16p13.3 16:0 PKDTS Polycystic kidney disease, infantile 600273 Polycystic kidney disease, 16p13.3 severe, with tuberous sclerosis infantile severe, with tuberous sclerosis 16:1,984,192 GFER, ERV1, ALR Growth factor, erv1, S. cerevisiae, 600924 Myopathy, mitochondrial 16p13.3 homolog of (augmenter of liver progressive, with congenital regeneration) cataract, hearing loss, and developmental delay 16:2,155,777 TRAF7, RFWD1, CAFDADD TNF receptor-associated factor 7 606692 Cardiac, facial and digital anomalies 16p13.3 with developmental delay

nondisjunctional events, which are even more frequent in translocation carriers had two or more pregnancy losses very early miscarriages (Sierra and Stephenson 2006). Apart (Sheth et al. 2013). Various reports of cytogenetic studies from the common autosomal trisomies involving chromo- show 2 to 8% cases with recurrent spontaneous abortions somes 13, 18 and 21 reported in early pregnancy loss, tri- had chromosomal abnormalities (Ghazaey et al. 2015). somy 22 is also reported along with other uncommon Parental chromosomal abnormalities play an important role autosomal trisomies (Choi et al. 2014; Pylyp et al. 2018). in the pathogenesis of recurrent abortion (Flynn et al. 2014). Chromosomal microarray studies are informative regard- A chromosomal break can cause loss of function of gene ing sub-microscopic deletions and duplications commonly if it disrupts the coding sequence (Kunwar and Bakshi missed by conventional cytogenetic analysis. The product of 2016). According to OMIM database, results from conception from this couple was collected for CMA studies microarray are suggestive of 2p25.3 deletion syndrome and at 7 weeks of gestation for the 3rd pregnancy. The cardiac 16p duplication syndrome involving multiple genes. The activity was recorded at 7th week on ultrasound scan which genomic imbalance detected using chromosomal microarray stopped after two days. The deletion of 3.7 Mb on short arm can be responsible for the foetal loss. The earlier miscarriage of chromosome 2p25.3 and duplication of 13.6 Mb on short in this case was found to be associated with trisomy of arm of chromosome 16 p13.3p13.12 was observed. chromosome 22. The autosomal trisomy is reported in Both these regions on chromosomes 2 and 16 are gene 43–54% of cases in recurrent miscarriages (Choi et al. rich and hence can be critical. There are numerous genes 2014). Autosomal trisomies commonly associated with involved in genomic deletion of 3.7 Mb on chromosome recurrent spontaneous abortions are trisomy 13, 16, 18 and 2p25.3 (table 2) which are reported to be associated with 21, apart from these trisomy, 15 and 22 have also been various clinical conditions including mental retardation, reported. The mispairing of translocated chromosomes dur- encephalopathy with brain atrophy and spasticity. ing the first meiotic division can give rise to different forms The genomic duplication of 13.6 Mb on chromosome 16p of segregation, resulting in aneuploidy of the translocated also involves multiple genes (table 3). The clinical features chromosomes (Pourjafari et al. 2012). Couples with bal- of duplication 16p syndrome includes critical region of ter- anced reciprocal translocation have 50% chance of recurrent minal 16p13.1-p13.3 reported to be associated with specific spontaneous abortions and 20% risk of progeny with genetic facial anomalies, mental retardation, congenital cardiac and imbalance (De et al. 2015). The formation of balanced, vascular defects, urinary malformations and hypoplastic unbalanced, and normal gametes depends on the breakpoints distal phalanges of hands (Digilio et al. 2009). of the chromosomes involved. The findings of CMA from third POC prompted parental Chromosomal abnormality is one of the major causes of karyotype study for which the mother was a carrier of a recurrent abortions; however, genetic testing is routinely balanced translocation 46,XX,t(2;16)(p25.3;p13.3). It has indicated after two or three consecutive abortions. 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