Molecular Human Reproduction, Vol.22, No.5 pp. 364–372, 2016 Advanced Access publication on January 28, 2016 doi:10.1093/molehr/gaw008 ORIGINAL RESEARCH Whole exome sequencing in recurrent early pregnancy loss Ying Qiao1, Jiadi Wen2, Flamingo Tang1,SallyMartell1, Naomi Shomer1, Peter C.K. Leung3, Mary D. Stephenson4, and Evica Rajcan-Separovic1,* 1Department of Pathology, BC Child and Family Research Institute (CFRI), University of British Columbia (UBC), Vancouver, BC, Canada 2University of Texas, Dallas, TX, USA 3Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, BC, Canada V6Z 2 K5 4University of Chicago and University of Illinois at Chicago, Chicago, IL, USA *Correspondence address. Department of Pathology (Cytogenetics), BC Child and Family Research Institute, University of British Columbia, 950 West 28th, Room 3060, Vancouver, BC, Canada V5Z 4H4. Tel: +1-604-875-3121; Fax: +1-604-875-3601; E-mail: [email protected] Submitted on December 11, 2015; resubmitted on January 18, 2016; accepted on January 25, 2016 study hypothesis: Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). study finding: We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in mis- carriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. what is known already: Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in 2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. study design, samples/materials, methods: Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platformon seveneuploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. main results and the role of chance: Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in mis- carriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in ‘complement and coagulation cascades pathway’, and ‘ciliary motility disorders’. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. limitations, reasons for caution: The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. wider implications of the findings: This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. study funding and competing interest(s): The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. Key words: recurrent pregnancy loss / whole exome sequencing / euploid miscarriages / compound heterozygous mutation / copy number variants / SNVs & The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected] Genetic causes of recurrent pregnancy loss 365 Introduction Filges et al., 2015). In all of these studies, the abnormal pregnancies inher- ited different mutations in a same gene from each parent, rendering the Recurrent pregnancy loss (RPL), defined as two or more pregnancy losses, gene defective in the conceptus (compound heterozygous mutations). occursin 5%ofcouplestryingtoconceive(Sierra and Stephenson, 2006). The affected genes havebeenfound to participatein avarietyofbiological Both parental and embryo/fetal factors are associated with RPL. Parental processes important for embryonic/fetal development and fertility, such factors include parental translocations, maternal thyroid or prolactin as ciliogenesis (kinesin family member 14, KIF14 (Filges et al., 2014) and disease or diabetes mellitus, endometrial changes, intrauterine abnormal- intraflagellar transport 122, IFT122 (Tsurusaki et al., 2014)), RNA trans- ities and antiphospholipid antibodies (Stephenson and Kutteh, 2007). port and processing (nucleoporin, GLE1 (Ellard et al., 2015)), signal trans- Numericchromosomeerrorsintheconceptusarethemostcommon duction (ryanodine receptor 1, RYR1 (Ellard et al., 2015) and cholinergic cause of miscarriage, particularly in miscarriages occurring before 10 receptor, nicotinic alpha 1, CHRNA1 (Shamseldin et al., 2013)) and weeks of gestation (early miscarriages) (Kolte et al., 2015). Association of maternal meiosis II (phospholipase C delta 4, PLCD4 and oxysterol single-gene or multi-genic changes with miscarriage has been proposed binding protein-like 5, OSBPL5)(Filges et al., 2015). (Larsen et al., 2013), however, for the vast majority of the 40–50% of In this study, we have used exome sequencing to look for pathogenic miscarriages with a normal karyotype (euploid miscarriage), the genetic variants (single nucleotide variants, SNVs, and small insertions and etiology remains uncertain (Sierra and Stephenson, 2006). deletions, indels) in euploid early miscarriages from couples with RPL, The recent advent of genome-wide high-resolution technologies, who previously had high-resolution CMA analysis. Our expectation including chromosome microarray analysis (CMA) and next generation was that we will identify rare and deleterious SNVs or indels affecting sequencing (NGS), have opened new possibilities for discovery of both the maternal and paternal allele which would make the gene dys- genetic changes that are too small to be detected by traditional miscar- functional when both parental alleles are simultaneously transmitted to riage chromosome analysis. To date, reportedly over 3000 miscarriages the conceptus resulting in miscarriage. Recurrent inheritance of the have been studied using CMA technology, including those studied by our mutated alleles from both parents in miscarriages would support their group (Rajcan-Separovic et al., 2010a, b) and others (for review, see significance in abnormal development. In addition to analyzing single Bagheri et al., 2015). Clinically relevant copy number variants (CNVs) genes for mutations which could cause miscarriage, we further assessed were identified in 1.6% of cases (Levy et al., 2014), while rare variants the mutation ‘burden’ (i.e. sum of putatively pathogenic mutations in all of uncertain clinical significance (VUS) were seen in 1–40% of cases. genes) in the couples and miscarriages. Homozygosity affecting chromosomal segments, whole chromosomes and genomes occurred in 1–30% of cases (Robberecht et al., 2012; Bug et al., 2014; Levy et al., 2014). Although the impact of CNVs on Materials and Methods the function of affected genes in miscarriage was demonstrated for iso- lated CNVs (Wen et al., 2015), and CNVs as a group (Nagirnaja et al., Subjects 2014; Bagheri et al., 2015), the role of the majority of predominantly Couples with RPL and their miscarriages previously assessed by CMA (Rajcan- inherited miscarriage VUS remains uncertain (Rajcan-Separovic et al., Separovicetal., 2010a)wereselectedforexomesequencingbasedonavailabil- 2010a, b; Robberecht et al., 2012; Viaggi et al., 2013; Bug et al., 2014; ityofsufficientDNAfrom atleasttwo miscarriagesand thecoupleand absence Levy et al., 2014; Bagheri et al., 2015), leaving the cause of euploid of clearly pathogenic CNVs at the time of exome analysis. Seven miscarriages miscarriages unresolved. were subjectedfor WES: two miscarriageseach from threefamilies(Family 4,6 and 9) and one miscarriage from Family 12. The presence of candidate variants More recently, the development and application of NGS, including determined in exome sequenced miscarriage from Family 12 was then tested whole genome or whole exome sequencing (WES), allowed screening by Sanger sequencing in the second miscarriage and the couple. Both partners the whole genome for new genetic causes of human disease at the nu- from three families (4, 6 and 9) had their exomes sequenced. cleotide level (Koboldt et al., 2013). Thus far,