Whole Exome Sequencing in Recurrent Early Pregnancy Loss

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 miscarriages 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 miscarriages 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 inherited 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 isolated 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, over 100 candidate mono- The couples’ obstetric histories and clinical findings for miscarriages were genic disease genes have been identified by WES in a variety of disorders reported previously (Rajcan-Separovic et al., 2010a) and summarized in Sup- (Rabbani et al., 2014), while identification of genes for complex disorders plementary data, Table SI, while the CMA results are shown in Table I. There is more challenging. The use of NGS is reported in larger cohorts (i.e. were no multiple miscarriages (twins, triplets) in the study cohort. The study thousands of cases) of subjects with neurodevelopmental disorders to was approved by the Committee for Ethical Review of Research involving identify known and new gene mutations (Boycott et al., 2013), as well Human Subjects, University of British Columbia and Institutional Research as to look for a role of all identified variants in relevant disease pathways Board at the University of Chicago. All subjects gave written informed consent. in more complex diseases (Stamou et al., 2015). However, the application of NGS in finding the cause of prenatal lethal or abnormal development, including miscarriages, has been reported in a very limited number Whole exome sequencing and data analysis of studies (for review, see Filges and Friedman, 2014; Hillman et al., DNA was extracted from peripheral blood of parents and miscarriage chori- 2015). The largest WES study of 30 fetuses with morphologic abnormal- onic villi as previously described (Rajcan-Separovic et al., 2010a). Maternal ities identified de novo pathogenic mutations in two genes in two sporadic cell contamination in miscarriage tissues was excluded using microsatellite analysis. WES was performed by Oxford gene technology (OGT) (Oxford- cases (fibroblast growth factor receptor 3 [FGFR3] and collagen, type II, shire, UK). Agilent SureSelect All Exon Plus v4 + UTR was used for exome alpha 1 [COL2A1]) and a very small CNV in oral-facial-digital syndrome 1 capture and Illumina HiSeq 2000 minimum (San Diego, CA, USA) for (OFD1) gene in one case. Thus far, five studies applied WES in recurrent 100 bp paired end sequencing. We obtained 9.97 GB of data per individ- pregnancy losses. One study included a cohort of eight couples (Ellard ual, with .95.8% of exome covered more than 20 times. In order to stream- et al., 2015) while the other studies used single family-based strategies line the analysis and reduce the amount of false-positive variants in the (Shamseldin et al., 2013; Filges et al., 2014; Tsurusaki et al., 2014; samples, a multiple sample vcf file is created using individual raw bam files. 366 Qiao et al.

Table I Genomic data of the euploid miscarriages and couples with a history of RPL.

Chromosome microarray study (Rajcan-Separovic et al., 2010a) Results: WES ...... Samples used Resultsa CNVs gene Sample Results Gene mutations content ...... Family 4 Two miscarriages: 04-3A Normalb No genes Two miscarriages Compound DYNC2H1 (M) and 04-3B (M) and (04-3A and 04-3B) heterozygous mutations (p.Tyr2016Cys; couple and couple in DYNC2H1 in both p.Asp2184Val) miscarriages Family 6 Five miscarriages: 06-3A (1) 86 kb maternal (1) TIMP2c; Two miscarriages Normal Not detected (F), 06-3B (M), 06-3C (F), duplication at 17q25.3 in (2) LIPC (06-3A and 06-3E) 06-3D (F) and 06-3E (M) four miscarriages (06-3A, and couple and couple 06-3B, 06-3C, 06-3E)c (2) 76 kb paternal deletion at 15q22.1 in one miscarriage (06-3D) Family 9 Two miscarriages: 09-3A 330 kb maternal EGFL6, TCEANC, Two miscarriages Normal Not detected (M) and 09-3B (F) and duplication atXp22.2inone RAB9A, TRAPPC2c, (09-3A and 09-3B) couple miscarriage (09-3B)c OFD1c, and GPM6B and couple Family 12 Two miscarriages: 12-3A (1) A 1.6 Mb maternal (1) HDHD1A, STS, One miscarriage Compound ALOX15 (F) and 12-3B(M) and duplication at Xp22.31 in VCX, and PNPLA4d (12-3B) heterozygous mutations (p.Tyr139Cys; couple one miscarriage (12-3B). d in ALOX15 in 12-3A and p.Thr560Met) (2) A 62 kb paternal (2) NPAS3 (intron) 12-3B deletion at 14q13.1 in two benign miscarriages (12-3A and 12-3B)

Note: All miscarriages were embryonic except 6-3D which was a yolk sac miscarriage. aSee Rajcan-Separovic et al. (2010a). bThe small deletion (a 155 kb maternal loss at 16q23.1) reported previously is currently described as a benign variant in the database of genetic variants (DGVs). cWith further gene functional assessment. See Wen et al. (2015). dVariant of unknown signiﬁcance also found in controls, although at lower frequency. See Liu et al. (2011a, b).

Raw bam files were generated from the sequencer and contained the whole determined using Indel SIFT webtool (http://sift.bii.a-star.edu.sg/www/ sequenced data aligned to the reference genome (hg19) from an individual SIFT_indels2.html) and Indel conservation score using PhastCons46wayPla- sample. The multi-sample vcf file contains the annotated variant calls from cental and PhyloP46wayPlacental from University of California Santa Cruz a collection of samples such that all samples are subject to the same level (UCSC) (https://genome.ucsc.edu/). In total, nine prediction tools were of stringency of filtering pipeline and that the analysis is performed uniformly used for evaluation of variant pathogenicity (SIFT, Polyphen2, LRT, Mutation- between families. This .vcf files were generated using the Seven Bridges Taster, MutationAssessor, FATHMM, RadialSVM, LR and Indel SIFT) and pipeline (https://www.sbgenomics.com/). Briefly, this included raw read conservation scores were determined using GERP++ RS, PhastCons46- mapping to reference using Burrows–Wheeler Aligner (BWA) and wayPlacental and PhyloP46wayPlacental. mapping duplicates and sorting using Picard program. Genome Analysis Coding, non-synonymous variants identified in miscarriages as deleterious Toolkit (GATK; Broad Institute) was used for data quality assurance as to normal gene function (causing frameshift or gain/loss of stop codon, and well as variant discovery. affecting the initial codon or splicing) and conserved with at least one of the Variant characterization, including filtering, annotation, classification, pri- above prediction and conservation tools were assessed for inheritance pat- oritization and inheritance pattern analysis, was performed using SNP & Vari- terns. Those that fitted the autosomal recessive, compound heterozygous or ation Suite (SVS) v8.1.5(Golden Helix, Inc., Bozeman, MT,www.goldenhelix. X-linked inheritance patterns (when both miscarriages were male) and were com). Briefly, quality-control metrics (QC) were set to only retain variants in presentin both miscarriages wereconsidered candidates for the cause of RPL the positions with Read Depth ≥10, Genotype Quality ≥20 and Alternate and further evaluated manually using other data resources for determining allele read ratio ≥0.25. Variants were classified, annotated and functionally their role in the recurrent miscarriage, e.g. human gene mutation data profiled in SVS v8.1.5 using multiple publicly available databases. Variants (HGMD; https://portal.biobase-international.com/cgi-bin/portal/login.cgi), with .1% minor allele frequency were excluded after analysis using Genome Browser (http://www.goldenhelix.com/GenomeBrowse/; Golden Exome Variant Server (EVS) [NHLBI Exome Sequencing Project (ESP) Helix, Bozeman, MT), PubMed, GeneCards (http://www.genecards.org/) (NHLBI ESP6500SI, Seattle, WA. http://evs.gs.washington.edu/EVS/)], and mouse knock-out database (MGI, http://www.informatics.jax.org/). 1000 Genomes project (http://www.1000genomes.org/data) and ExAc Selected candidate pathogenic variants were confirmed using Sanger sequencing (http://exac.broadinstitute.org/). The impact of the remaining SNVs on in both miscarriages and couples. Primers were designed using Primer Express protein function was evaluated by database of non-synonymous functional 3.0. Traditional PCR was preformed according to the Invitrogen protocol predictions (dbNSFP) NS Functional Predictions track of SVS (Liu et al., for native Taq DNA polymerase and cleaned products run on an ABI 3130xl 2013) while Indel (generally ,20 bp in this study) function prediction was sequencer. Genetic causes of recurrent pregnancy loss 367

Enrichment analysis human genome as described previously (Qiao et al., 2010; Supplementary data, Table SII). WebStalts (http://bioinfo.vanderbilt.edu/webgestalt/; Wang et al., 2013) was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and disease association enrichment analysis of all variants in miscarriages identified as being deleterious (as defined above) and conserved with Results at least one database, regardless of the inheritance (termed as putatively pathogenic variants). This list included variants transmitted from one or In this study, WES was performed in seven miscarriages: two miscarriages the other parent, or de novo in one but not the other miscarriage. Sixty-six each fromthree families (Family 4, 6 and 9) and one miscarriage fromFamily genes with putative pathogenic variants detected in all seven miscarriages 12 (Table I). Couples from Family 4, 6 and 9 also had WES. After filtering with WES data were assessed as a pool for pathway and disease association and consideration of inheritance pattern (Supplementary data, Fig. S1), enrichment using Webgestalt. Similarly, genes with variants predicted to be we found candidate pathogenic compound heterozygous variants in mis- deleterious with at least one prediction and conservation tool detected in carriages from two families. In Family 4, both miscarriages had mutations the three couples with WES data were assessed for KEGG pathway and inherited fromthe mother and father (compound heterozygous mutations) disease association enrichment (72 genes). The findings in miscarriages and in DYNC2H1 (MIM 603297) at chromosome 11q22.3 (p.Tyr2016Cys; and couples were compared with positive and negative control groups of p.Asp2184Val). In Family 12, both miscarriages had mutations inherited genes. Positive control group of genes consisted of 141 genes in which mutations were reportedto be implicated in miscarriage or pregnancy loss accord- from the mother and father (compound heterozygous mutations) in ing to PubMed literature and HGMD database search (Supplementary data, ALOX15 (MIM 152392) at 17p13.2 (p.Tyr139Cys, rs113604586; and Table SII). The negative control genes included two groups of genes: (a) 44 p.Thr560Met, rs34210653) (Table I). The minor allele frequencies of genes with deleterious variants detected by WES in three normal subjects these variants are ,1%.TheywereconfirmedbySangersequencingin (our internal control) and (b) 50 genes randomly selected from whole both miscarriages and the couple from Family 4 and 12 (Figs 1 and 2).

Figure 1 Putatively pathogenic compound heterozygous mutations in DYNC2H1 in euploid miscarriages from a couple (Family 4) with a history of RPL. (A) Protein domains and mutation locations of DYNC2H1.(B) Mutation p.Tyr2016Cys with Genome Browser image (left) and Sanger sequencing validation (right). (C) Mutation p.Asp2184Val with Genome Browser image (left) and Sanger sequencing validation (right). DHC, dynein heavy chain; AAA, ATPase associated; ABC, ATPase superfamily; MT, microtubule-binding stalk of dynein motor. 368 Qiao et al.

Figure 2 Putatively pathogenic compound heterozygous mutations in ALOX15 in euploid miscarriages from Family 12. (A) Protein domains and mutation locations of ALOX15.(B) Mutation p.Tyr139Cys with Genome Browser image and Sanger sequencing validation (C). Mutation p.Thr560Met with Genome Browser image and Sanger sequencing validation. Red arrows indicate the mutation found in our study. Blue arrows indicated heterozygous missense/ nonsense mutations ALOX15 found in HGMD related to cardiovascular, hyperproliferative and neurodegenerative diseases. HGMD, human gene mutation database.

The DYNC2H1 variant positions in miscarriages from Family 4 were were similar (Table II), with genes from both miscarriages and couples conserved and predicted to be damaging by SIFT and disease causing enriched in pregnancy-related pathways, such as ‘Complement and by MutationTaster prediction tools. In two miscarriages from couple coagulation cascades pathway’ and ‘Steroid hormone biosynthesis’ 12, the compound heterozygous mutations in ALOX15 (arachidonate (P , 0.01). ‘Complement and coagulation cascades pathway’ was also 15-lipoxygenase) were conserved and predicted to be damaging and/ found to be enriched for positive control genes (P , 0.01) but not for or disease causing by ﬁve out of eight prediction tools used in Golden genes from any of the two negative control gene groups. No pathway en- Helix SVS. Exome sequencing of miscarriages from the remaining two richment was found for genes from two negative control group genes. couples 6 and 9 did not reveal candidate pathogenic mutations using Notably, genes with putatively pathogenic variants detected in the our criteria. couples and miscarriages were enriched in ciliary motility disorders Enrichment in KEGG pathways and associated disease was compared disease category (in both groups, P , 0.01) while genes from the positive among pools of genes with putatively pathogenic SNVs detected by control gene group were also enriched for ‘pregnancy complications’, exome sequencing in seven miscarriages (66 genes) and three couples and ‘recurrent and spontaneous abortion’ disease categories. Genes (72 genes) in comparison to positive control group genes (141 genes from the two negative control groups were enriched for diseases not with mutations reported to be implicated in miscarriage) and two nega- associated with reproduction (P , 0.01) (Table II). tive control gene groups (44 and 50 genes, detected by WES in three normal subjects and randomly selected from whole human genome, re- spectively; See Materials and Methods for detail and Supplementary data, Discussion Table SII for list of genes). We performed WES analysis to identify recurrent pathogenic variants in The majority of the putatively pathogenic variants (.90%) found in miscarriages from couples with RPL and to look for the overall character- miscarriages were familial in origin and the variants in miscarriages com- istics of rare variants predicted to be deleterious and conserved in mis- parable to variants detected in couples. Consequently, the pathways and carriages from RPL couples. Compound heterozygous mutations were diseases enriched for genes with variants in miscarriages and couples identiﬁed in recurrent early miscarriages from 50% of couples. Genetic causes of recurrent pregnancy loss 369

Table II Enrichment of pathways and disease categories from different groups of genes.

Origin of genes Number Pathwaya Associated diseasea of genes ...... WES of 7 SA_POCsb 66 Metabolism of xenobiotics by cytochrome P450 Ciliary motility disorders Complement and coagulation cascades Glycerophospholipid metabolism Steroid hormone biosynthesis WES of 6 SA parentsb 72 Complement and coagulation cascades Brenner tumor of ovary Steroid hormone biosynthesis Agranulocytosis Metabolism of xenobiotics by cytochrome P450 Ciliary motility disorders Drug metabolism—other enzymes Leukopenia RIG-I-like receptor signaling pathway Neutropenia Candidate RPL genes from PubMed and HGMD 141 Complement and coagulation cascades Pregnancy complications Malaria Abortion, habitual Cytokine–cytokine receptor interaction Disease susceptibility African trypanosomiasis Pathologic processes Amoebiasis Abortion, spontaneous WES of three normal subjectsb 44 None Tangier disease Tourette syndrome Tic disorders Fibroma Antisocial personality disorder Randomly selected genes from genome 50 None Hernia, diaphragmatic Retinoblastoma Adhesion Osteosarcoma

POC, product of conception; RPL, recurrent pregnancy loss; WES, whole exome sequencing; SA, spontaneous abortion; HGMD, human gene mutation database. aTop ﬁve ranking pathways and associated diseases shown with adjusted P-value ,0.01. Pathways in italics indicate those related to RPL. bGenes with variants that have deleterious mutations, conservation scores ≥1 out of 3 tools, and pathogenicity score ≥1 out of 9 prediction tools.

Recurrent miscarriages from one couple (Family 4) carried compound and cardiovascular disease (Ivanov et al., 2015). Recently, association heterozygousmutations(p.Tyr2016Cysandp.Asp2184Val)inDYNC2H1. of 12/15-LOX with endoplasmic reticulum stress response was DYNC2H1 (Dynein Cytoplasmic 2 Heavy Chain 1) encodes a large cyto- reported (Cole et al., 2012), which is of interest due to association of plasmic dynein protein (4307 amino acid) that is involved in ciliary intrafla- ER stress response with early pregnancy loss (Francisco et al., 2010; gellar transport (IFT), a process that is essential for the generation and Liu et al., 2011a; Gao et al., 2012). The overexpression of ALOX15 in maintenance of mammalian cilia (Grissom et al., 2002; Perrone et al., transgenic mice showed reduced fertility with increased abortion rate 2003; Goetz and Anderson, 2010). Primary cilia and IFT proteins are im- (Dar et al., 2001). This gene has a role in spermatogenesis and ovulation, portant components in Hedgehog and Wnt pathways and are essential for and it is up-regulation was associated with preeclamsia (Yuan et al., 2014). early development in zebrafish (Gouttenoire et al., 2007; Haycraft et al., Three missense/nonsense mutations in ALOX15 have been listed in 2007). Homozygous and compound heterozygous mutations in the HGMD associated with phenotypes, including heart disease and schizo- DYNC2H1 gene have been frequently found in patients with Asphyxiating phrenia. One of the mutations, g.4535314G.A (p.Thr560Met), asso- thoracic dystrophy or Short rib-polydactyly syndrome (OMIM 613091) ciated with coronary artery disease (Schurmann et al.,2011), is the (Dagoneau et al.,2009; Schmidts et al.,2013), which can be prenatally same variant as in the miscarriages from Family 12. This variant was classi- lethal (Ellard et al.,2015). More than 80 damaging mutations of fied in HGMD as disease-associated polymorphism with additional sup- DYNC2H1 have been reported in HGMD, mostly associated with this porting functional evidence. In miscarriages from Family 12, we did not syndrome. Mutations in both alleles of Dnchc2 (mouse orthologue of identify CNVs that could explain their recurrence by CMA (Rajcan- DYNC2H1) resulted in midgestation lethality and abnormal dorsoventral Separovic et al., 2010a). A maternal Xp22.31 duplication was detected patterning of the forebrain (Huangfu and Anderson, 2005; May et al., in one miscarriage (Table I), however, the role of this duplication 2005). In miscarriages from Family 4, previous CMA study did not remains uncertain due to its occurrence in healthy controls (although detect any unique CNVs, suggesting that the compound heterozygous their reproductive history is unknown) and in subjects with developmental mutations in DYNC2H1 could have a role in RPL. delay (Liu et al.,2011b). Recurrent miscarriages in Family 12 were found to carry compound Based on the information from the literature, the SNVs in ALOX15 (or heterozygous variants in ALOX15 (arachidonate 15-lipoxygenase) DYNC2H1) could be contributory to pregnancy loss, however, functional (p.Tyr139Cys and p.Thr560Met). ALOX15 encodes human 12/15- analysis in cells from miscarriages would be needed as a solid proof of this lipoxygenases (12/15-LOXs), an oxidizing enzyme capable of producing possibility (e.g. cilia function for DYNC2H1 or biochemical assays for reactive lipid hydroperoxides. ALOX15 and its products are involved in ALOX15). Unfortunately, such studies in the case of miscarriage are many pathological conditions, such as inflammation, carcinogenesis challenging as they require viable cells in culture. 370 Qiao et al.

Exome sequencing of miscarriages from Family 6 did not identify Acknowledgements clearly pathogenic SNVs. However, subsequent expression analysis of the previously detected recurrent CNV of maternal origin in four out We appreciate the support of the family members involved in this study. of five miscarriages in this family showed altered expression of the dis- rupted TIMP2 gene. This CNV could therefore be associated with Authors’ roles RPL, considering the role of TIMP2 in vascular remodeling (Wen et al., 2015). Miscarriages from Family 9 did not have a common genetic abnor- Y.Q. analyzed the data and wrote the manuscript. J.W. performed mality, either at the single nucleotide or CNV level. Of note, one of the experiments and analyzed part of the data. F.T. performed experiments miscarriages from couple 9 had a maternal CNV affecting the expression and analyzed the data. S.M. and N.S. performed experiments. M.S. of integral genes OFD1 (oral-facial-digital syndrome 1) and TRAPPC2 (traf- recruited families, provided clinical data and revised the manuscript. ficking protein particle complex 2) (Wen et al., 2015), so the CNV E.R.S. designed and supervised the study and revised the manuscript. remained of unclear relevance to RPL. The possibility that other genetic changes (e.g. mutations in non-coding parts of the genome not Funding detectable by exome sequencing) or that non-genetic factors contribute The study was funded by Canadian Institutes of Health Research (grant to the RPL in this family cannot be excluded. In summary, the recurrence MOP 106467) and Michael Smith Foundation of Health Research Career of genetic lesions of different nature (SNV or CNV) was detected in re- Scholar salary award to E.R.S. current early miscarriages in majority (75%) of studied families (putatively causative SNVs in families 4 and 12, and CNV in Family 6). Numerous studies of CNVs and SNVs in patients with neurodevelop- Conflict of interest mental disorders have revealed that pathways associated with brain de- None declared. velopment and function, such as synaptic transduction and chromatin remodeling are enriched in genes affected by CNVs or deleterious mutations (O’Roak et al., 2012; Epi et al., 2013; Pinto et al., 2014). This sug- References gested that in addition to isolated genetic mutations, the sum of Bagheri H, Mercier E, Qiao Y, Stephenson MD, Rajcan-Separovic E. Genomic genetic variations affecting relevant pathways could also contribute to characteristics of miscarriage copy number variants. Mol Hum Reprod the susceptibility of diseases. We were therefore interested in the 2015;21:655–661. overall role of all putatively pathogenic mutations detected by exome se- Boycott KM, Vanstone MR, Bulman DE, MacKenzie AE. 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