Identifying the causes of recurrent pregnancy loss in consanguineous couples using whole exome sequencing on the products of miscarriage with no chromosomal abnormalities

Recurrent miscarriages occur in about 5% of couples trying to conceive. In the past decade, the products of miscarriage have been studied using array comparative genomic hybridization (a-CGH). Within the last decade, an association has been proposed between miscarriages and single or multigenic changes, introducing the possibility of detecting other underlying genetic factors by whole exome sequencing (WES). We performed a-CGH on the products of miscarriage from 1625 Iranian women in consanguineous or non-consanguineous marriages. WES was carried out on DNA extracted from the products of miscarriage from 20 Iranian women in consanguineous marriages and with earlier normal genetic testing. Using a-CGH, a statistically significant difference was detected between the frequency of imbalances in related vs. unrelated couples (P < 0.001). WES positively identified relevant alterations in 11 genes in 65% of cases. In 45% of cases, we were able to classify these variants as pathogenic or likely pathogenic, according to the American College of Medical Genetics and Genomics guidelines, while in the remainder, the variants were classified as of unknown significance. To the best of our knowledge, our study is the first to employ WES on the products of miscarriage in consanguineous families with recurrent miscarriages regardless of the presence of fetal abnormalities. We propose that WES can be helpful in making a diagnosis of lethal disorders in consanguineous couples after prior genetic testing.

quantity of fetal samples that have proved problematic in routine chromosomal study. CMA has its limitations including its inability to detect polyploidy, low-grade mosaicism and balanced rearrangements 7 .
In 2013, Larsen et al. proposed an association between miscarriage and single or multigenic changes 14 , introducing the possibility of being able to detect other underlying genetic factors by exome sequencing. Today, next-generation sequencing (NGS) is a crucial tool for pathogenic variants discovery in research and diagnostic settings 15,16 . Exome sequencing has a diagnostic yield of about 25-40% in patients with suspected Mendelian diseases in western populations [17][18][19][20] . Carss et al. performed exome sequencing on 30 non-aneuploid fetuses and neonates with diverse structural abnormalities detected by prenatal ultrasound. They identified candidate pathogenic variants and concluded that exome sequencing may substantially increase the detection rate of underlying etiologies of prenatal abnormalities. In 3 out of 30 fetuses, they found highly likely causative variants 15 . In 2018, Mengu Fu et al. performed exome sequencing on 19 products of miscarriage of unrelated couples and reported 36 rare variants associated with miscarriage 21 .
Studies of this kind have led to the use of NGS in prenatal diagnosis for the detection of pathogenic and causal genetic variants below the resolution of CMA 15,22 . The application of NGS in identifying the causes of lethal or abnormal prenatal development, including miscarriages, has been reported since 2012. An estimate of 30% of all mammalian genes are vital for life. Dickinson et al. (2016) identified 410 lethal genes in mice. In general, the diagnosis of lethal genes is challenging for the following reasons: the difficulties of phenotype-genotype correlation, the many potential genes, and the variable phenotypes associated with the same genetic causes 23 . There is controversy as to whether there is any correlation between recurrent pregnancy loss (RPL) and consanguinity. In studies conducted in the Middle East, where consanguineous marriage is culturally prevalent, some showed that the prevalence of RPL is higher in consanguineous couples. Kuntla et al. (2013) concluded that the occurrence of spontaneous miscarriages is higher among women in related vs. unrelated couples in India. In 2010, Rad conducted a study on recurrent miscarriage in Iran and concluded that the prevalence of RPL was higher in the consanguineous group 24 . On the other hand, in 2002, Saad and Jauniaux reported no association between consanguinity and recurrent miscarriage in Qatar and concluded that this finding could be because autosomal recessive alleles are uncommon in the Qatari population or because of the absence of any relationship between consanguinity and recurrent miscarriage 25 . In 2011, Gowri et al. reported that consanguinity does not appear to play a significant role in the etiology of recurrent miscarriage and is not related to recurrent miscarriage based on a study conducted in Oman 26 .
Since 2010, about 1625 cases with pregnancy loss (recurrent or single) have been studied in our center using quantitative fluorescent polymerase chain reaction (QF-PCR) and a-CGH, in which about 20% of clinically relevant CNVs and aneuploidies were detected. About 35% of the cases were from consanguineous couples. The inbreeding coefficients in consanguineous couples were between F = 0.125 and F = 0.0156. We realized that the detection rate in unrelated couples was higher. The difference between the frequency of imbalances in related vs. unrelated couples was significant (χ2 = 11.4926, P < 0.001) 27 . We proposed that it is plausible through the same mechanism by which single gene disorders have a higher prevalence of manifesting disease in consanguineous couples, they can cause lethal genetic disorders leading to pregnancy loss. As miscarriage is etiologically heterogeneous, the selection criteria for the evaluation of this postulate are very important. Whole exome sequencing (WES) on cases that present with a strong Mendelian inheritance pattern is more likely to be successful 28 .
Here we have used WES to detect the causes of miscarriage in consanguineous Iranian families with recurrent miscarriages, in whom oligoarray CGH was normal and the maternal causes of miscarriage had been ruled out.

Materials and methods
Signed informed consent was obtained from couples for participation in this study and publication of data. The research was performed under the National Institute for Medical Research Development, Tehran, Iran (IR. NIMAD.REC.1396.355).
Subjects. Twenty consanguineous Iranian couples were selected from a pool of 1625 couples in whom oligoarray CGH was normal and maternal causes of miscarriage had been ruled out by Kariminejad-Najmabadi Pathology & Genetics Center. The couples had a history of two or more pregnancy losses (RPL) and the results of previous a-CGH and QF-PCR were normal. Five of the cases had fetal autopsy reports.
The cases were selected from consanguineous couples with spontaneous loss of a pregnancy at less than 20 weeks of gestation and whose fetuses had sufficient quantity of DNA with appropriate quality. To minimize the effect of maternal causes of miscarriage, we chose families who had a history of one normal child, or one pregnancy beyond the second trimester or whose fetus showed evidence of fetal abnormalities (non-immune hydrops fetalis (NIHF), cardiac anomalies, etc.) 29 . None of the cases had induced abortion or prior abnormality detected in ultrasound. (Table 1).
All experimental protocols were approved by National Institute for Medical Research Development committee.
Whole exome sequencing. We used archival genomic DNA from the products of spontaneous miscarriage and extracted DNA from the peripheral blood of parents. All DNA was extracted using the conventional salting out method 30 . For fetuses or the products of miscarriage, DNA was extracted from chronic villi of the products of miscarriage and where fetal tissue was available, from the quadriceps muscle. Where possible, we saved some fetal tissue in a freezer at -80 °C. We checked the quality and quantity of DNA using a Nanodrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA) and gel electrophoresis. Samples were enriched using the Agilent SureSelectXT Human All Exon V6 platform (Agilent Technologies Inc, Santa Clara, CA, USA). Whole exome sequencing was performed using an Illumina HiSeq2000 platform (Illumina www.nature.com/scientificreports/ Inc., San Diego, CA, USA) 31 . All methods were carried out in accordance with ACMG guidelines and regulations.
We confined our analysis to the primary list of genes reported to be lethal in animal models, lethal genes from earlier articles, and lists of genes incorporated in NIHF and fetal development collected by examining earlier publications and databases. In cases where no candidate variants were found, we then used Tru Sight's gene list for inherited diseases 40 and finally, we used all Online Mendelian Inheritance in Man (OMIM) genes (https:// omim. org/ downl oads/).

Results
We collected DNA from the products of miscarriage from 20 Iranian women in consanguineous marriages and with a history of recurrent pregnancy loss. QF-PCR and a-CGH had previously been performed and reported as normal. None of the women had a specific diagnosis for pregnancy loss. As all cases were of loss of a pregnancy at less than 20 weeks of gestation, most (70%, n = 14) had no phenotype other than lethality. An autopsy was available in five cases and clinical data for these five cases are shown in Table 2. WES positively identified relevant alterations in 65% of cases (n = 13). In 45% (n = 9) of cases, we were able to classify these variants as pathogenic or likely pathogenic, while in the remaining 20% (n = 4), the candidate variants were classified as variants of unknown significance (VUS) according to the American College of Medical Genetics and Genomics www.nature.com/scientificreports/ guidelines (ACMG) 49 . In contrast, we were able to reach a precise diagnosis in all 5 cases (100%) with autopsies. In total, 13 rare variations including 2 de novo heterozygous, 9 inherited homozygous, and 2 inherited compound heterozygous variants were found in 11 genes; 2 genes were autosomal dominant (COL1A1, SCN5A). In silico predictions of the sequence variants are included in Supplementary Data I.

1.
PIEZOs are large transmembrane ion channel proteins, and mutations in this gene have recently been reported to be a cause of hydrops fetalis 50 . PIEZO1 has a role in urine osmolarity regulation, blood pressure control and blood vessel formation [50][51][52][53][54] . Mutations of this gene are associated with two diseases: (1) dehydrated hereditary stomatocytosis with/without pseudohyperkalemia and/or perinatal edema (DHS; OMIM 194380); this is an autosomal dominant disease caused by gain of function mutations; (2) hereditary lymphedema III, the generalized lymphatic dysplasia of Fotiou with non-immune fetal hydrops (GLDF; OMIM 616843), an autosomal recessive disease caused by loss of function mutations 50 . In a non-immune hydrops fetalis sample (case 82169), we detected a homozygous c.30_31delAC alteration in PIEZO1. This variant is a frame shift mutation in the first exon that causes nonsense-mediated decay (NMD) in translated mRNA. In this case, the biallelic loss of function of PIEZO1 and the fetal phenotype give credence to GLDF. The parents were both carriers of this variant. According to ACMG, this variant is considered pathogenic. 2. Mutations in FRAS1 are associated with Fraser syndrome, a rare autosomal recessive disorder. Thomas et al. (1986) proposed criteria for clinical diagnosis: major features are cryptophthalmos, syndactyly, siblings with cryptophthalmos and abnormal genitalia, and minor features include congenital malformations of the nose, ear and larynx, skeletal defects, umbilical hernia, renal agenesis and mental retardation. For diagnosis, two major or one major and four minor criteria are needed 55 58 . This complex is abundant in fetal brain, skeletal www.nature.com/scientificreports/ muscles and testes 57 . Case 91414 with four ventricular hydrocephaly, atrophic cortex, hypoplastic cerebellum, severe general cortical dysplasia type II (lissencephaly type II), cerebellar cortex dysplasia and clenched hands was found to have a homozygous variation in POMT1 (c.490C > T). This variation has been reported in the HGMD database (CM022978) and is a stop-gain resulting in NMD. Disruption of this gene in mice is lethal 59 . This variation was considered to be pathogenic. 5. Another homozygous variant in intron 3 of DIS3L2 gene (2q37.1) in case 97406 (Family 13) was predicted to be likely pathogenic from ACMG guidelines. DIS3L2 is associated with Perlman syndrome (#267000), an autosomal recessive disorder. Perlman syndrome is a congenital overgrowth syndrome 60 characterized by distinctive facies, visceromegaly, abdominal wall hypoplasia, bilateral renal hamartomas, nephroblastomatosis, Wilms tumor and neonatal lethality 60,61 . A 15-week fetus (case 97406) with limb atrophy, body weight at the 91 percentile, abdominal muscle agenesis, mild hydrocephaly, choroid plexus cysts, muscular and corticospinal tract atrophy , and kidney weight at the 99 percentile (observed to expected ratio of kidney weight to heart weight: 3.38, 1.99 in that order) was found to have a homozygous alteration in DIS3L2 (c.211-1G > A) which is predicted to disrupt the highly conserved acceptor splice site of axon 4. This alteration was classified as likely pathogenic according to ACMG guidelines.
In the other 15 cases, we have no phenotype other than lethality, and possible justifying variants were found in eight cases. (Table 3) In four cases, we could classify the variant as pathogenic/likely pathogenic. These variants were found in genes SCN5A, FUCA1, GBE1 and COL1A1 (Table 5).
1. SCN5A encodes a membrane protein, which is an α-subunit of the predominant cardiac sodium channel isoform. This protein is responsible for the initial upstroke of the action potential in the heart. In case 94947, a homozygous alteration was detected in SCN5A (c.3749C > T). The parents were both carriers with a history of cardiac events in the family. This variant has been reported to cause long QT syndrome 3 (LQT3) (#603830) in the heterozygous state 62,63 .
Homozygous mutations in SCN5A in mice cause intrauterine lethality mostly during organogenesis due to heart defects 64 . 2. FUCA1 expresses a lysosomal enzyme. Fucosidosis (#230000), an autosomal recessive disorder, is a lysosomal storage disease caused by homozygous or compound heterozygous mutations in FUCA1. Cardinal features are coarse facies, neurological signs, visceromegaly, intellectual disability and dysostosis multiplex. There are two types of fucosidosis: type 1 is more severe and signs are seen around 6 months of age with a lifespan of a decade; type 2 is milder with longer survival 65   www.nature.com/scientificreports/ Osteogenesis imperfecta, type II, OMIM #166210, AD; Osteogenesis imperfecta, type III, OMIM #259420, AD; Osteogenesis imperfecta, type IV, OMIM #166220, AD. This mutation is in exon 68 and does not affect the triplet repeats and Glu has been replaced by Gly at the amino acid level. It was classified as likely pathogenic. www.nature.com/scientificreports/ In four cases, we found variations that are classified as variants of uncertain significance (VUS) because of the lack of phenotypic information. (Table 6) These variations were present in genes BBS12, STIL, COG6 and PIEZO1.

A mutation in BBS12
causes Bardet-Biedl syndrome 12 (OMIM #615989). Bardet-Biedl is a heterogeneous ciliopathy disorder with an autosomal recessive pattern of inheritance. The BBS12 mutation is more common in the Iranian population than reported in other populations 67 . Case 84904 was found to have a homozygous mutation in BBS12 gene (c.2014G > A). Bardet-Biedl 12 is associated with obesity and mental impairment that cannot be detected in fetal stages. This alteration was considered to be of uncertain clinical significance. 2. STIL is expressed in proliferating cells during early embryonic development and is necessary for mitotic spindle organization in the human cell cycle [68][69][70][71] . Alteration of this gene is associated with Primary microcephaly (#612703), an autosomal recessive disorder 71  and Shaheen syndrome (#615328). Shaheen syndrome is an autosomal recessive disorder characterized by intellectual disability and microcephaly. Congenital disorder of glycosylation III (CDG2L) is also autosomal recessive, characterized by intrauterine growth retardation, gastrointestinal abnormalities, infection and hematologic abnormalities. CDG2L usually results in death in infancy 72 . In case 94162, a homozygous mutation with uncertain clinical significance was detected in COG6 (c.1884 T > G). 4. Case 95136 was found to have two mutations in PIEZO1 (c.C6584T and c.G2764T). As discussed earlier, mutations in this gene are associated with GLDF and DHS. In this case, we have no phenotype to correlate it with our findings and thus it was considered to be of uncertain clinical significance.  www.nature.com/scientificreports/

Discussion
Whole exome sequencing can detect the underlying genetic causes of disease in 25-40% of patients (pediatric and adult) with clinical indications such as congenital anomalies or developmental delay 74 . Before WES, array CGH and targeted genetic testing were performed in sporadic cases. The Middle East has the highest rate of consanguineous marriage globally due to socio-cultural factors. The prevalence of consanguineous marriage in Iran is about 38.6% 75 . Consanguineous marriage is preferred in Arab countries, particularly first cousin marriages with a rate of 20-50% 76 .
Since 2012, the possible benefit of WES for detection of the cause of miscarriage has been discussed and the results so far are promising 29 . A number of studies have been published with sample sizes of over 7 fetuses (7 to 84 cases) and these have reported a 10% to 54.5% detection rate of variants using WES in prenatal and fetal samples. In these studies, 30% to 70% of detected variants were autosomal recessive 15,74,[77][78][79][80][81][82][83] . In our study, 84% of variants were autosomal recessive. These studies have variable inclusion criteria such as (1) single versus multiple anomalies or specific organ anomalies, or (2) singleton WES vs. trio WES. The highest diagnostic yield and pathogenic variants reported are in a study where fetuses with specific organ anomalies were studied 83 . In other studies, it was reported that trio WES increases the detection rate 84,85 . The advantages of trio WES have been reported in postnatal and prenatal studies. Trio WES allows reviewing variants with a higher probability of being pathogenic and detecting de novo variants 83 . In these studies, all fetuses had anomaly/anomalies and most cases were from unrelated couples.
To the best of our knowledge, our study is the first to employ WES on the products of miscarriage in consanguineous families with recurrent miscarriages regardless of fetal anomalies. The main difference between our study and others is in the inclusion criteria. We chose consanguineous families with spontaneous recurrent pregnancy loss (RPL) and studied the products of loss of a pregnancy at less than 20 weeks of gestation. In most cases, we had no phenotype other than lethality, and in 65% of cases, we found a possible variant. In only 45% of cases could we classify the variant as pathogenic/likely pathogenic according to ACMG guidelines. Since all fetuses were under 20 weeks gestation and some were under 10 weeks, there was no phenotype, and this made it more challenging to interpret the findings. Although all couples had RPL, we only had one product of miscarriage per family so segregation analysis was not possible due to lack of DNA samples from the other affected cases in each family. What makes interpretation of WES findings on the products of miscarriage more challenging is that Mendelian disorders in severe cases can be present as embryonic lethality, out of which many have no prenatal phenotype so mutations may not be suspected in fetuses 85 (80).
WES on the products of miscarriage is helpful to verify lethal genes, and genes essential for embryonic development, and it expands our knowledge of prenatal phenotypes of many Mendelian disorders.
This study and others of this kind show that WES can assist in the diagnosis of the cause of miscarriage. Positive results and having a diagnosis can be useful in preconception genetic counselling for future successful pregnancies. Preimplantation genetic testing may be possible and the results may provide families with closure. After diagnosis, it is important to advise families on the risks of recurrence and their options for future pregnancies 78,86 . Identification of the cause of miscarriage will determine the risk for future pregnancies, and enable prenatal diagnosis or preimplantation genetic diagnosis for the given family. In addition, we will be able to identify lethal genes and their role in pregnancy loss. This and other studies of this kind will provide information that can assist in the assessment of repeated pregnancy loss.

Conclusion
WES can be helpful in making a diagnosis of lethal disorders (especially autosomal recessive disorders) in consanguineous couples after prior genetic testing (QF-PCR and a-CGH). www.nature.com/scientificreports/