Discussion
Our pilot WGS study identified 87 SNVs involving 75 genes in embryonic
loss (n=1), 370 SNVs involving 228 genes in fetal death (n=3), and 122
SNVs involving 122 genes in stillbirth (n=2) samples, as potentially
related to pregnancy loss, across three families. The SNVs included
twenty-two de novo , six autosomal dominant and one X-linked
recessive mutation(s) that had high pathogenicity scores
(pLI>0.9; LOUEF<0.36). In addition, our findings
for higher counts of de novo SNVs in losses compared with live
births, excess of genes with >1 loss-of-function de
novo SNVs (p-value=0.01), and occurrence of multiple de novoevents in a single gene in samples from losses, implicate de novoSNVs in the pathogenesis of pregnancy loss. Furthermore, several of the
identified SNVs impact genes (e.g., DICER1,25FBN2,22 FLT4,26HERC1,27,28 and TAOK129 )
that were known to be involved in the development of the embryo and
fetus, and that are associated with congenital abnormalities,
highlighting the potential role of SNVs in phenotypes that may share a
common pathway with recurrent pregnancy loss. Lastly, we identified
missense compound heterozygous SNVs impacting genes (e.g.,VWA5B2 ) in two fetal death samples that were absent from live
births and population controls, providing evidence for haplosufficient
genes relevant to recurrent pregnancy loss.
Previous genetic studies of pregnancy losses are limited for several
reasons, including (1) lack of access to paternal DNA samples, which
would make interpretations difficult without distinguishing inherited
form from de novo variants,30 (2)
unavailability of pedigrees with products of conception from
chromosomally normal losses and live-births, or (3) unavailability of
high-quality data and protocols for DNA restoration and variant
detection.31-33 Loss-of-function risk variants and
inherited variants in intolerant genes (i.e., genes that are critical
for human development, conditions incompatible with life resulting in
fetal demise)16,23,34 were not identified, possibly
due to limited sample size and focus on families with recurrent, rather
than sporadic losses.
Recently, whole-exome sequencing of stillbirth in maternal-offspring
duos was conducted to identify variants in intolerant genes that were
impossible to ascertain with karyotype or
microarray.23 Though the study was limited in
ascertaining de novo from inherited variants, due to
unavailability of paternal DNA, genes were reported by the authors that
are either lethal, known to cause disease, or increase stillbirth risk
(e.g., CCR5 , FAT1 , FLNB , INPP5K ,MYO1C , PLOD2 ). Importantly, these genes overlap with
findings in our data. For example, we identified a de novomissense chr3:58141895:C:T in FLNB , a gene known for its role in
atelosteogenesis type 1, in a stillbirth of Family 3. In addition, we
identified autosomal dominant missense chr17:1471262:C:T inMYO1C , a gene linked to deafness and cytoskeletal development, in
a stillbirth of Family 1. Given that the SNVs were not identified in
live births, the findings warrant validation to confirm potentially
lethal variants in FLNB and MYO1C genes causing
chromosomally normal stillbirths.
Recently, Kline et . al . similarly hypothesized that
chromosomally normal losses are caused by rare variants in several
different genes, some of which are incompatible with development to the
fetal stage.22 The authors reported damaging variants
in several genes that are relevant to recurrent pregnancy loss,
including FBN2 (Fibrillin 2). Notably, we identified a de
novo in-frame deletion involving FBN2 in fetal death in Family 4
that was not identified in any of the live births across families.
Although Kline et . al . identified compound heterozygous
variants of FBN2 in embryonic loss, our SV analysis in stillbirth
in Family 4 (see Online Supplement ) confirmed a de novoSVs (chr5:128335405) impacting FBN2 , suggesting variants
disrupting the FBN2 gene may be incompatible with development to
the fetal stage. Furthermore, FBN2 is well described for its role
in congenital contractual arachnodactyly and embryonic and fetal
development, and may be a potential candidate worth investigating in
larger studies.35,36
Given the small participant sample with WGS data in our pilot study, it
is noteworthy that we identified variants in several genes (e.g.,DICER1,25 FBN2,22FLT4,26 HERC1,27,28 andTAOK129 ) that were previously identified by
genetic studies of pregnancy loss. To confirm our findings, we conducted
several validation and confirmatory analyses. First, we compared our
data to a population of healthy controls (gnomAD) and identified rare
(gnomAD AF<0.006) compound heterozygous SNVs in four genes
(TM2D1, MUC16, VWA5B2 ) across two families that were not observed
as homozygotes in healthy gnomeAD controls. This finding suggested that
variants in haplosufficient genes may contribute to fetal demise in
offspring of two healthy parent carriers. Given that our filtering
approach is cantered on allele frequencies and predicted impact, and is
agnostic to the phenotype of interest, the identification of gene
candidates associated with congenital and developmental phenotypes is
notable. Although we demonstrated some sharing of SNVs across families
(e.g., compound heterozygous SNVs in four TM2D1 , MUC16 ,VWA5B2 were shared across two families), losses may not have
common etiologies.22,37 As such, this finding suggests
that different genes may play a role at different developmental epochs
and across families.16 Second, we explored validation
of VWA5B2 gene by Sanger sequencing and confirmed that WGS in our
sample confidently called its compound heterozygous SNV
(chr3:184236380:T:C). However, further interpretations from our Sanger
sequencing results were hindered by the DNA extraction quality and
require sequencing of additional samples with higher DNA quality.
Compound heterozygous variants have been previously implicated in
pregnancy loss38 and present a scenario in which each
parent is purportedly healthy but carries variants in the same gene(s)
that may be incompatible with life. As such, functional validation of
compound heterozygous variants may provide a clearer picture of the
genetic landscape of recurrent pregnancy loss, especially recurrent
cases. De novo variants in highly conserved or constrained genes
also may lead to pregnancy loss. However, a de novo mutation has
a much lower recurrence rate than recessive or dominant inherited
disorders.39,40 Impactful X-linked recessive variants,
for example, a missense X-linked SNV (chrX:108591181:C:A) impactingCOL4A5 (Alport syndrome 1 gene) and possibly relevant to fetal
death (Online Supplement Table S3 ), may also serve as
candidates for validation. Importantly, genetic diagnoses based on
impactful variants following various modes of inheritance may be used to
provide a prognosis based on data from other families with similar
mutations.41,42 Confirmation of genes relevant to
pregnancy loss will also identify critical pathways and novel
therapeutic targets for improving pregnancy outcomes.
Our study has several limitations. The higher counts of de novoSNVs we observed in pregnancy losses compared with live births could
result from sequencing error, reflected from degradation of placenta
samples due to FFPE. FFPE samples have small fragment sizes and very
uneven coverage, contributing to false positive SNVs/SVs. For example,
low quality libraries (high DNA degradation) from two samples may have
contributed to the large number of de novo SNVs observed in
losses in our data. To validate SNVs in our data, we conducted
exploratory Sanger sequencing analysis. Results showed poor validation
for de novo (data not shown) but confirmed several compound
heterozygous calls (Table 3 ) that were not confidently called
in our samples. Furthermore, we used Slivar, a method that is strictly a
filtering strategy, and the utility of the output relies on high-quality
input variants. Future studies utilizing freshly obtained placenta
samples for WGS may address elevated sequencing error potentially
contributed by FFPE.
Strengths of our study include some prospective collection of samples
from losses and live births. This may improve strategies for determining
the ‘intolerome’, conditions incompatible with life resulting in fetal
demise, and potential to improve database of lethal genes and
phenotypes, which are poorly represented. Although our study is
underpowered to compare rates of SNVs/SVs between losses and live
births, our study serves as a requisite feasibility step in exploring
genes relevant to pregnancy loss. Thus, the findings from our pilot
study will provide justification for conducting WGS using larger
parent-offspring families with potential to identify SNVs causing
pregnancy loss.
In summary, the findings reported herein provide evidence for genetic
variants (including several in previously recognized genes) relevant to
unexplained pregnancy loss in families. WGS of DNA from larger numbers
of families (including parent-offspring DNA from affected and unaffected
pregnancies) may help identify lethal genes contributing to sporadic and
recurrent pregnancy loss. Elucidating pregnancy loss causing genes may
lead to biomarkers useful for risk stratification, the identification of
genes relevant to normal and abnormal pregnancy, and novel therapeutic
targets.