Identification Of Disease Genes Of Primary Female Infertility And Analysis Of The Pathogenic Mechanism Of Mutations In WEE2 Gene

Primary female infertility is a special reproductive disorder,in which women of child-bearing age have never been pregnant,have normal sex life more than one year after marriage and have used any contraception,but they are still not pregnant.About 5-10% of infertile women are caused by genetic factors such as single or multiple gene mutations.In this paper,5 women who were diagnosed with primary infertility were screened to do genetic analysis.The disease-causing gene of one of the female patients was identified by whole exome sequencing bioinformatic analysis and Sanger sequencing.To decipher of the mechanisms of the mutation of the disease-causing gene,methods of biochemistry and molecular biology were used to elucidate the related pathogenic mechanism.Through whole exome sequencing and sanger DNA sequencing,a novel compound heterozygous mutations: c.598C> T(p.Arg200Ter)and c.1319G> C(p.Trp440Ser)was identified in WEE2 gene in one patient,the proband’s father carries the c.1319G> C(p.Trp440Ser)heterozygous mutation,and her mother carries the c.598C> T(p.Arg200Ter)heterozygous mutation,the sister of the proband carries a c.1319G> C(p.Trp440Ser)heterozygous mutation.By analyzing the conservation of amino acids of WEE2 protein and structure of the WEE2 protein,we found that the p.Trp440 amino acids highly conserved during evolution,and the mutation site is located in the PKinase kinase region of WEE2 protein.In order to further assess the effect of these mutations on the function of WEE2 protein,wild-type and mutant WEE2 c DNAs were cloned into the p EGFP-C1 / p3 x Flag-CMV-10 vector,and they are transfected into HEK293 T cells,the expression level of WEE2 protein was detected by western blot.The Arg200 Ter mutant WEE2 gene produce a truncated protein,the expression level of Trp440 Ser mutant WEE2 gene is decreased significantly.Meanwhile,in order to detect phosphorylation of Cdc2 which mediated by WEE2 protein,HEK293 T cells were transfected with wild and mutant WEE2 gene constructs.Western blot was used to test the effect of WEE2 mutations on phosphorylation of Cdc2 tyrosine 15(p Y15)in vitro.The results showed that the downstream Cdc2 phosphorylation mediated by mutant WEE2 was reduced significantly when compare with that of wild type WEE2 protein.To assess the effect of the mutations on protein localization,wild-type and mutant WEE2 expression plasmids were transfected into He La cells.By check the GFP-tagged WEE2 localization,we found that wild-type and p.Arg200 Ter mutant WEE2 proteins are located in the nucleus,while the expression of the Trp440 Ser mutant WEE2 protein was decreased in the nucleus,and the p.Trp440 Ser WEE2 proteins are located both in the nucleus and cytoplasm.In this study,we do the genetic study on 5 primary infertile women and her family members,and a novel compound heterozygous mutation of the disease gene WEE2 was identified.The molecular mechanisms of WEE2 mutataions were analyzed by biochemical and molecular experiments.We found that the expression of WEE2 gene was severely affected by these mutations.The level of phosphorylation of Cdc2 tyrosine 15 was reduced by mutant WEE2 protein,then leads to failure of oocyte intracytoplasmic sperm injection(ICSI)and fertilization failure.Our work provides genetic evidence for pronucleus formation failure,and provides novel theoretical basis for genetic counseling for female infertility.