The Role Of DNA Repair Related Genes CSB-PGBD3, MSH5 And FMR1 Played In Premature Ovarian Failure

Chapter Ⅰ. DNA Repair Deficiency of CSB-PGBD3 Fusion Protein Causes Premature Ovarian FailurePart Ⅰ. Whole Exome Sequencing in Non-consanguineous Family with Premature Ovarian FailureOBJECTIVE:Premature ovarian Failure (POF), defined as cessation of menstruation before the expected age of menopause, is one of the commonest hypergonadotropic disorders resulting in infertility. Approximately 1% of women are affected, and the genetic factors are responsible for 25% of cases. Progress has been made in identifying causative genes with the use of animal models and Sanger sequencing in sporadic POF patients, but moves slowly. In this study, we will perform the whole exome sequencing (WES) in the non-consanguineous familiy with POF to identify novel genes responsible for POF.METHODS:WES on the Illumina platform was performed in the POF patients, genetically obligate carriers and normal family members in the POF pedigree. Reads were aligned to hgl9 databases. All unreported variants following autosomal dominant inheritance model were confirmed by Sanger sequencing. Candidate variations for POF were further validated in the other individuals of the family and 432 sporadic cases by Sanger sequencing.RESULTS:After being "blasted" with public databases and verified by Sanger sequencing, only the heterozygous variant c.2237G>A (p.G746D) in PiggyBac transposable element derived 3 (PGBD3) was observed to follow autosomal dominant inheritance model in the family with POF. We further performed mutation screening of CSB-PGBD3 in 432 sporadic POF patients and 400 matched controls, and identified one additional heterozygous missense mutation c.3166G>A (p.V10561) and one heterozygous nonsense mutation c.643G>T (p.E215X) in CSB-PGBD3.CONCLUSION:With the use of WES in the non-consanguineous familiy with POF, we identified CSB-PGBD3 as the candidate causative gene for POF, which shed a new light on the study of POF pathogenesis. Moreover, family-based WES could be a powerful means in revealing novel genes responsible for POF.Part Ⅱ. The Adverse Effects of CSB-PGBD3 Mutations on DNA RepairOBJECTIVE:Through the whole exome sequencing performed in a POF pedigree, we identified CSB-PGBD3 as a candidate causative gene for POF. The PGBD3 transposon integrated into intron 5 of the CSB gene about 43 Mya in marmoset, resulting in abundant CSB-PGBD3 fusion protein. CSB-PGBD3 was reported to participate in transcription-coupled DNA repair (TCR), while its function in oogenesis was not clear. The objective of our study was to 1) locate the CSB-PGBD3 fusion protein in human ovary,2) illustrate whether and how did the CSB-PGBD3 mutations adversely affect DNA repair, which might result in POF.METHODS:Histologic tests with the use of human and rhesus ovarian tissues were performed to locate the CSB-PGBD3 fusion protein. Laser irradiation and other DNA damage tests were performed to illustrate the role of CSB-PGBD3 played in DNA repair. Immunoprecipitation and Real-time PCR were used to identify the interaction between CSB-PGBD3 and other DNA repair factors.RESULTS:Mutated CSB-PGBD3 fusion protein was functionally impaired in response to DNA damage, including delayed or absent recruitment to damaged sites and failure to up-regulate downstream DNA-repair factors, which was mediated by dysfunctional interaction with RNAPol Ⅱ. In addition, CSB-PGBD3 is expressed in nuclei of oocytes, indicating the potential role of CSB-PGBD3 in maintenance of ovarian function mediated by functional DNA repair.CONCLUSION:CSB-PGBD3 is an important DNA repair factor in human oocytes. Functionally impaired mutations in CSB-PGBD3 are associated with both familial and sporadic POF, mediated by impeded DNA damage repair. These studies provide evidence for a pivotal role of DNA repair in POF.Chapter Ⅱ. MSH5 D487Y Causes Premature Ovarian Failure by Dysfunctional Homologous Recombination RepairOBJECTIVE:MSH5 is involved in DNA mismatch repair and meiotic recombination, facilitates crossover between homologs by stabilizing Holliday Junction, which was important for oogenesis and ovarian function. The objective of this study was to illustrate whether MSH5 mutation identified by WES in POF-2 pedigree could explain the disease by dysfunctional recombinational double-strand break (DSB) repair.METHODS:We performed whole exome sequencing (WES) in a POF pedigree and Sanger sequencing in 432 sporadic POF patients and 400 controls, and conducted histologic and functional studies to investigate further the highly plausible gene discovered through WES.RESULTS:A homozygous missense mutation c.1459G>T(p.D487Y) in MSH5 was identified in the two affected family members, inherited in autosomal recessive fashion. With the protein structural analysis, we found that p.D487Y located in the DNA binding domain of MSH5, which was conserved from saccharomyces to human. The homologous mutation in Yeast results in diminished spore viability. MSH5 was not only highly expressed in human fetus ovary, but also in the oocytes, granulosa cells and theca cells from primordial, primary, secondary to antral follicles. Cells overexpressing mutant MSH5 failed to repair DSBs as wild type did.CONCLUSION:MSH5 not only participates in the meiotic recombination in oocytes, but also is involved in the homologous recombination repair for DSBs in granulosa cells and theca cells. Dysfunctional DNA repair caused by MSH5 mutation might lead to a greater rate of follicle loss through atresia, finally causing POF.Chapter Ⅲ. FMR1 Premutation Is an Uncommon Explanation for Premature Ovarian Failure in Han ChineseOBJECTIVE:Premature ovarian failure (POF) is a rare, heterogeneous disorder characterized by cessation of menstruation occurs before the age of 40 years. FMRl premutation was reported to be responsible for up to 3.3%-6.7% of sporadic POF and 13% of familial cases in Caucasians, while the data was absent in Chinese population. Therefore, the impact of FMR1 CGG repeat on ovarian reserve is needed to be investigated in large Chinese cohort.METHODS:FMR1 CGG repeat was determined in 379 Han Chinese women with sporadic POF and 402 controls. With the genomic DNA extracted from peripheral blood lymphocytes, the FMR1 gene was amplified by PCR with fluorescently labeled primers, and the CGG repeats were counted by capillary electrophoresis (CE). The age of menopause onset in respect to CGG repeats was further analyzed.RESULTS:The frequency of FMR1 premutation in Han Chinese POF was only 0.5%(2/379), which was much lower than that reported in Caucasian with POF (3.3%-6.7%). The prevalence of intermediate FMRl (41-54) was not increased significantly in sporadic POF than that in controls (2.9% vs.1.7%, P=0.343). However, POF patients more often carried a single additional CGG repeat in a single allele than did fertile women (allele-1:29.7 vs.28.8, P<0.001; allele-2:32.6 vs.31.5, P<0.001). POF patients with sub-genotype hom-high/high and hom-low/high showed an earlier age of cessation of menses than those with norm genotype (20.4±4.8 vs. 24.7±6.4, p<0.01; 18.7±1.7 vs.24.7±6.4, p<0.01, respectively).CONCLUSION:FMR1 premutation seems to be an uncommon explanation for POF in Han Chinese. However, having both alleles with CGG repeats outside the normal range might still adversely affect ovarian aging.