Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage

Abstract

Study question: Can whole exome sequencing (WES) followed by trio bioinformatics analysis identify novel pathogenic genetic causes of first trimester euploid miscarriage?

Summary answer: We identified genetic variants in six candidate genes that indicated plausible underlying causes of first-trimester euploid miscarriage.

What is known already: Previous studies have identified several monogenic causes of Mendelian inheritance in euploid miscarriages. However, most of these studies are without trio analyses and lack cellular and animal models to validate the functional effect of putative pathogenic variants.

Study design, size, duration: Eight unexplained recurrent miscarriage (URM) couples and corresponding euploid miscarriages were included in our study for whole genome sequencing (WGS) and WES followed by trio bioinformatics analysis. Knock-in mice with Rry2 and Plxnb2 variants and immortalized human trophoblasts were utilized for functional study. Additional 113 unexplained miscarriages were included to identify the mutation prevalence of specific genes by multiplex PCR.

Participants/materials, setting, methods: Whole blood from URM couples and their <13 weeks gestation miscarriage products were both collected for WES, and all variants in selected genes were verified by Sanger sequencing. Different stage C57BL/6J wild-type mouse embryos were collected for immunofluorescence. Ryr2N1552S/+, Ryr2R137W/+, Plxnb2D1577E/+, and Plxnb2R465Q/+ point mutation mice were generated and backcrossed. Matrigel-coated transwell invasion assays and wound-healing assays were performed using HTR-8/SVneo cells transfected with PLXNB2 small-interfering RNA and negative control. Multiplex PCR was performed focusing on RYR2 and PLXNB2.

Main results and the role of chance: Six novel candidate genes, including ATP2A2, NAP1L1, RYR2, NRK, PLXNB2, and SSPO, were identified. Immunofluorescence staining showed that ATP2A2, NAP1L1, RyR2, and PLXNB2 were widely expressed from the zygote to the blastocyst stage in mouse embryos. Although compound heterozygous mice with Rry2 and Plxnb2 variants did not show embryonic lethality, the number of pups per litter was significantly reduced when backcrossing Ryr2N1552S/+ ♂ with Ryr2R137W/+ ♀ or Plxnb2D1577E/+ ♂ with Plxnb2R465Q/+ ♀ (P < 0.05), which were in accordance with the sequencing results of Family 2 and Family 3, and the proportion of Ryr2N1552S/+ offspring was significantly lower when Ryr2N1552S/+ female mice were backcrossed with Ryr2R137W/+ male mice (P < 0.05). Moreover, siRNA-mediated PLXNB2 knockdown inhibited the migratory and invasive abilities of immortalized human trophoblasts. Besides, additional 10 variants of RYR2 and PLXNB2 were detected in 113 unexplained euploid miscarriages by multiplex PCR.

Limitations, reasons for caution: The relatively small number of samples is a limitation of our study which may result in the identification of variants in unique candidate genes with no definitive although plausible causal effect. Larger cohorts are needed to replicate these findings and additional functional research is needed to confirm the pathogenic effects of these variants. Moreover, the sequencing coverage restricted the detection of low-level parental mosaic variants.

Wider implications of the findings: For first-trimester euploid miscarriage, variants in unique genes may be underlying genetic etiologies and WES on trio could be an ideal model to identify potential genetic causes, which could facilitate individualized precise diagnostic and therapeutic regimens in the future.

Study funding/competing interests: This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700604), National Natural Science Foundation of China (31900492, 82101784, 82171648), Basic Science Center Program of the National Natural Science Foundation of China (31988101), Key Research and Development Program of Shandong Province (2021LCZX02), Natural Science Foundation of Shandong Province (ZR2020QH051), Natural Science Foundation of Jiangsu Province (BK20200223), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201812154) and Young Scholars Program of Shandong University. The authors declare no conflicts of interest.

Trial registration number: N/A.

Keywords: candidate gene; euploid; miscarriage; single-nucleotide variant; whole exome sequencing.

Figure 1.

Flowchart for identifying putatively pathogenic variants and candidate genes by WGS and WES in URM families. URM, unexplained recurrent miscarriage; WES, whole exome sequencing; WGS, whole genome sequencing.

Figure 2.

Sanger sequencing of putatively pathogenic variants in six candidate genes and corresponding structural mapping. (A) Sanger sequencing validated the existence of putatively pathogenic genetic variants in six genes from four families. (B) Structures of RyR2, ATP2A2, PLXNB2, and NAP1L1 are shown. Structural mapping of the mutation T357I onto the structure of ATP2A2. T357 forms four hydrogen bonds with K352, G354, and D600, whereas only one is left after changing to I357. The variant p.N1551S (located in the SPRY3) and p.R137W (located in the NTD) are indicated on the structure of RyR2. Structural mapping of the mutation E54G onto the structure of NAP1L1, and this mutation changes a negatively charged residue to a non-charged residue. Structural mapping of the variant p.R463Q and p.D1573E onto the structure of PLXNB2. R463Q and D1573E are located on the extracellular and intracellular sides, respectively. The reference structures were downloaded from the PDB and AlphaFold protein structure databases. All the figures were prepared using PyMOL.

Figure 3.

Reproductive performance of backcrossing Ryr2^N1552S/+ with Ryr2^R137W/+ parents and Plxnb2^D1577E/+ with Plxnb2^R465Q/+ parents. The number of pups per litter derived from pairs of Ryr2^N1552S/+ with Ryr2^R137W/+, Plxnb2^D1577E/+ with Plxnb2^R465Q/+ and WT with WT at the age of 8 weeks for 3 months. The results are shown as the mean ± SD, both female and male mice n = 8/group. *P < 0.05.

Figure 4.

PLXNB2 knockdown inhibited the migration and invasion ability of HTR-8/SVneo cells. (A and B) Transfection efficiency was examined by RT-qPCR and western blot analysis respectively. The results showed that PLXNB2 knockdown attenuated PLXNB2 expression at both mRNA and protein levels. (C and D) Representative images of scratch wound-healing assays at 0, 12, 24, 36, and 48 h. The migration ability of HTR-8/SVneo cells was significantly inhibited 48 h after siPLXNB2 transfection. Scale bars = 200 μm. (E) Representative images of transwell invasion and migration assays. Cell invasion ability was inhibited after transfection with siPLXNB2. Scale bars = 100 μm. The results are shown as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001. RT-qPCR, reverse transcription-quantitative polymerase chain reaction.