Genetic Analysis Of Infertility And Pathogenic Mechanism Of New Mutations In PATL2、DNAH1、PLCZ1 Genes

Objective:(1)To establish a standardized infertility associated biobank of clinical data and samples,and to collect and collate rare clinical cases of infertility,to perform multiplatform genetic sequential detection as a diagnostic system so that to determine the cause of specific cases,thus providing the necessary basis for further guidance on genetic counseling and reproduction.(2)To clarify the pathogenicity of some variants in specific cases of infertility via further experimental study,and to discuss their molecular mechanism.Methods:(1)Infertility cases were recruited from assisted reproduction center.A genetic sequential detection including chromosome karyotyping analysis,chromosome microarray analysis,whole-exome sequencing was carried out on all the samples to screen the pathogenic variation,and then to provide genetic counseling and reproductive guidance to the affected families.(2)The molecular mechanism was explored by means of Quantitative PCR,ELISA,papaniclaoll staining,immunofluorescence assay,Transmission electron microscopy technique,Scanning electron microscopy techniqueand bioinformatic structural analysis and multi-omics study on specific variants in three genes,with samples from affected patient.Results:(1)We recruited and collected 82 cases of infertility from several centers,by sequential detection,47 reproduction-related pathogenic/likely pathogenic/variants of uncertain pathogenic were detected in 31 cases,and 47 variants involved 17 different genes,namely BUB1B,DIAPH2,EIF2B2,INSL3,NLRP7,ZP1,ZP3,TUBB8,PA TL2,NLRP5,PAD16,TLE6,CCDC39&CCDC40,DNAH1,CFTR and PLCZ1 genes.(2)Bioinformatics analysis was performed on PA TL2 gene mutation to predict its impact on the protein stability of the molecule;sperm expression and morphological tests were performed on patient with DNAH1 gene mutation,and the protein was detected in the inner-arm dynein heavy-chain of sperm flagella,mutations lead to abnormal sperm flagella structure;the expression of PLCZ1 gene mutations was detected and revealed with abnormal low expression level.The results of multi-omics study of sperm showed that PLCZ1 gene mutations caused the oocyte maturation defect by affecting the calcium signal pathway.Conclusion:(1)The multi-platform genetic sequential detection has a good efficiency for infertility which is difficult to distinguish in clinical and imaging diagnosis.(2)Variations in the PATL2 gene cause oocyte maturation defect.(3)Variations in the DNAH1 gene cause multiple morphological abnormalities of the sperm flagella.(4)Variations in the PLCZ1 gene causeoocyte activation deficiency by affecting the calcium signal pathway,ELISA detection of PLCZ1 can be a rapid clinical screening program,and PLCZ1 gene sequencing can be used as a diagnostic program to provide a basis for recommending AOA.