Biological Effects Of Variants In RYR2 And PLXNB2 In First-trimester Euploid Miscarriage

Chapter Ⅰ Trio-WES in Unexplained Recurrent Miscarriage Families Identified Novel Pathogenic Genetic Causes of First-trimester Euploid MiscarriageBackground:Miscarriage is one of the most common adverse pregnancy outcomes.Approximately 80%of miscarriages occur before 12 weeks of gestation,also called firsttrimester miscarriage.Women with a history of miscarriage have an increased risk of pregnancy loss during next pregnancy.1%～5%of couples suffer from two or more consecutive miscarriages,a scenario that is diagnosed as recurrent miscarriage(RM),who are the main clinical target of treatment.Nearly 50%of RM cases lack a clear etiology and are termed unexplained RM(URM).For URM patients,there is an urgent need for etiologic diagnosis and effective early interventions.Next generation sequencing(NGS),including whole-genome sequencing(WGS)and whole-exome sequencing(WES),can detect variants at the single nucleotide level and has been widely utilized in the diagnosis of genetic diseases.Genetic factors are one of the hotspots in first-trimester miscarriage studies.WES could detect the potential genetic etiology of URM efficiently and has been recommended for the evaluation of fetuses with structural anomalies after normal karyotype and CMA results.The Trio-WES,containing samples from both embryo/fetus and parents,helps to identify the hereditary feature of variants and significantly increases the diagnostic yield.Moreover,patients with a definitive genetic diagnosis can choose preimplantation genetic testing(PGT)as a treatment for the next pregnancy,which helps the primary prevention of miscarriage.For first-trimester euploid miscarriage,WES has not yet been investigated since there are more challenges in bioinformatic analysis without detailed fetal imaging reports and a database of pathologic genes and variant loci.Although previous studies have identified candidate genetic causes of euploid miscarriages,the interpretation of these findings was primarily based on WES results in miscarriage products per se or female risks,and few studies based on trio analysis.Furthermore,most studies lack functional verification,such as phenotypes of animal models and biological functions of cell lines.Thus,further research in trio is still needed to enrich the database of genes and variants related to euploid miscarriage which could facilitate individualized precise diagnostic and therapeutic regimens in the future.Objective:This chapter aimed to identify novel pathogenic genetic causes of firsttrimester euploid miscarriage based on Trio-WES.Materials and methods:Eight URM couples and corresponding euploid miscarriages after PGT for aneuploidies were included in our study for WGS and WES followed by trio bioinformatics analysis.All putative pathogenic variants were confirmed by Sanger sequencing.Variant mapping and all structure figures were generated by PyMOL.Immunofluorescence staining in mouse preimplantation embryos was used to examine the expression of candidate genes.In addition,multiplex PCR was performed focusing on RYR2 and PLXNB2 in 113 unexplained euploid miscarriages by multiplex PCR.Results:Six novel candidate genes,ATP2A2,NAP1L1,RYR2,NRK,PLXNB2,and SSPO,were identified from four URM families.Variant mapping of ATP2A2(p.T357I)and NAP1L1(p.E54G)showed hydrogen bond reduction and changes in electric potential,respectively.Immunofluorescence staining showed that ATP2A2,NAP1L1,RyR2,and PLXNB2 were widely expressed from the zygote to the blastocyst stage in mouse embryos.In addition,an additional ten variants of RYR2 and PLXNB2 were detected in 113 unexplained euploid miscarriages by multiplex PCR.Conclusion:We identified genetic variants in six candidate genes that indicated plausible underlying causes of first-trimester euploid miscarriage,including ATP2A2,NAP1L1,RYR2,NRK,PLXNB2,and SSPO.All these genes play essential roles in embryonic development.The current findings supported the application of Trio-WES in identifying genetic etiologies for first-trimester euploid miscarriage.Chapter Ⅱ Biological Effects of Variants in RYR2 and PLXNB2Background:The results of WES in the first trimester of euploid miscarriage usually lack biological function verification.Based on our Trio-WES results,we identified the variants p.R137W and p.N 1551S in RYR2,as well as p.D 1573E and p.R463Q in PLXNB2.The functions of these loci have not been reported.RYR2 encodes ryanodine receptor 2,which are voltagedependent Ca2+channels in the sarcoplasmic reticulum.A deficiency of RyR2 causes catecholaminergic polymorphic ventricular tachycardia(CPVT),sudden cardiac death,etc.Ryr2 knock-out and some point mutant mice showed cardiac dysfunction or embryonic lethality.PLXNB2,an axon guide molecule,belongs to a transmembrane family of plexin proteins that are widely expressed in various mammalian organs.Plxnb2-/-mice showed cephalic neural tube closure defects,accompanied by abnormal development of the cerebellum and urinary system,leading to perinatal lethality.Previous studies have reported that PLXNB2 plays an essential role in regulating cell migration and invasion and influences the biological behavior of microglia,neuroblasts,macrophages,and many other cells.During the first trimester,the migratory and invasive abilities of extravillous trophoblasts(EVTs)are essential for pregnancy establishment and maintenance.Many pregnancy complications are associated with insufficient trophoblast invasion and migration,such as miscarriage and preeclampsia.PLXNB2 has not been reported in the regulation of trophoblast biological function.Objective:In this chapter,we aimed to investigate the biological effects of the variant loci in RYR2(p.R137W and p.N1551S)and PLXNB2(p.D1573E and p.R463Q),and the regulatory effects of PLXNB2 on trophoblast invasion and migration.Materials and methods:Ryr2N1552S/+,Ryr2R137W*+,Plxnb2D1577E/+ and Plxnb2R465Q/+knock-in mice were generated by the CRISPR/Cas9 technique.Ryr2N1552S/+×Ryr2R137W/+，Plxnb2D1577E/+×Plxnb2R465Q/+,and WT × WT pairs were formed,and the genotype of progeny and litter size were recorded.Hematoxylin-eosin(HE)and WGA staining were performed on the myocardial tissue from mice at 6 weeks and 6～8 months postpartum,to observe the pathological changes in myocardial tissue,while ultrasound was used to evaluate cardiac function.Neural tube closure development was captured at E10.5 and E17.5 in Plxnb2D1577E/D1577E,Plxnb2R465Q/R465Q,and Plxnb2D1577E/R465Q mice.The survival risk at 6 weeks in Plxnb2D1577E/D1577E mice was calculated.Matrigel-coated transwell invasion assays,woundhealing assays,and tube formation assays were performed using HTR-8/SVneo cells transfected with PLXNB2 small-interfering RNA and negative control.EdU,TUNEL,and flow cytometry were performed to test cell proliferation and apoptosis,and RNAseq was utilized to test differential expression at the mRNA level.Results: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/+♀,which was 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.Ryr2N1552S/R137W mice showed normal cardiac structure and function.Plxnb2D1577E/D1577E,Plxnb2R465Q/R465Q and Plxnb2D1577E/R465Q mice showed normal cranial development;partial Plxnb2D1577E/D1577E mice showed post-partum developmental delay,and the survival ratio decreased.siRNA-mediated PLXNB2 knockdown inhibited the migratory and invasive processes of HTR-8/SVneo cells and improved cell apoptosis.Conclusion:In mice,Ryr2 p.N1552S and p.R137W,as well as Plxnb2 p.D1577E and p.R465Q,did not cause severe abnormalities during the embryonic development process,but reduced reproductive performance was observed under specific genotype mice mating,indicating that critical steps in pregnancy establishment and maintenance were influenced.In immortalized human trophoblasts cell line,PLXNB2 is involved in regulating cell invasion and migration processes,and knockdown of PLXNB2 inhibited trophoblast invasion and migration ability.