Etiological Analysis Of Zygote Cleavage Failure Caused By Maternal Effect Gene Mutations

Purpose:Zygotic cleavage failure(ZCF)refers to the failure of the zygote with normal morphology to complete the first mitosis,resulting in embryonic arrest at the 1-cell stage,which is an early embryo development arrest type with the earliest onset and worst consequences.The translation products of maternal effect genes(MEGs)accumulate during follicular development and participate in key events such as meiotic maturation and early embryonic development.In recent years,with the mature application of high-throughput sequencing technology,combined with abundant domestic cases,the pathogenic mechanisms of some MEG mutations leading to oocyte meiotic arrest,fertilization disorder and embryonic arrest have been elucidated successively.Until2020,the relationship between MEG mutation and ZCF had been explained,however,only two MEGs,BTG4 and CHEK1,have been identified so far,explaining about 10%-15% of ZCF cases.This study intends to explore other potential candidate genes or mutations and elucidate their possible pathogenesis based on 35 ZCF patients in Reproductive and Genetic Hospital of CITIC-XIANGYA.Methods: In this study,whole exome sequencing was performed for recruited patients to screen potential mutations based on mutation frequency,location and type,combining with the transcriptome data of oocyte and/or early embryo.The pathogenesis was further explored by mutation site analysis,functional verification in-vitro and oocyte/embryo transcriptomic data analysis.Results: We identified three missense mutations of TRIP13 in two ZCF patients followed the recessive inheritance pattern: homozygous mutation,c.G1141 A,p.E381K;Compound heterozygous mutations,c.A77 G,p.H26 R and c.A1258 G,p.K420 E.These three mutations all cause changes in hydrogen bonding of the three-dimensional protein structure prediction model,protein abundance and up-regulation of DNA damage levels.In addition,single cell transcriptome sequencing for GV oocytes and arrested 1-cell embryos showed a larger number of differentially expressed transcripts in the oocyte than that in the embryo,a down-regulation level in energy metabolism and cell cycle events,while an up-regulation level in DNA repair.Additionally,we identified8 TUBB8 mutations in 10 ZCF patients,including 4 novel mutations(2missense mutations and 2 frameshift mutations),and found 3 hot spots from 6 ZCF patients: recessive pathogenic mutations,c.G322 A,p.E108 K and c.G922 A,p.G308 S,dominant pathogenic mutation,c.G1057 A,p.V353 I.Moreover,we found that the abnormal shape and position of spindle in oocyte may be the possible etiology.In addition,we performed meta-analysis for the existing 102 TUBB8 mutations combined with 20 our novel TUBB8 mutations,from the perspectives on pathogenic grade evidence and genotype-phenotype correlationConclusions: Human TRIP13 mutation related to the DNA damage repair defect and ZCF,followed an autosomal recessive pathogenic pattern.TUBB8 mutation resulted in abnormal spindle assembly,followed autosomal dominant or recessive pathogenic patterns based on the damage degree to the protein function.Our findings provide additional molecular diagnostic markers in genetic counseling for ZCF patients,and greatly expand the mutation and phenotype spectrum of TUBB8,contributing to the genetic diagnosis of female infertility in future.