The Role Of DNA Damage Repair Gene MCM8 Mutation Played In Primary Ovarian Infufficiency

BACKGROUND:Primary ovarian insufficiency(POI),also known as premature ovarian failure(POF[MIM311360]),is one of the common female reproductive endocrine diseases affecting women reproductive health.Clinical characteristics including primary or secondary amenorrhea prior to 40 years old,accompanied with hypergonadotrophic and hypoestrogenemia symptoms.POF is a heterogeneous disorder.The reported etiology includes chromosomal abnormalities,gene mutations,autoimmune,iatrogenic and environmental factors.Genetic factors accounted for about 20%to 25%of patients.Evidence for genetic factors has been provided by population and candidate gene studies.However,up to date,only a few candidate genes have been proved to be causative:Genes on the X chromosome including BMP15,PGRMC1 and FMR1;genes on autosomes including FSHR,GDF9,NR5A1,NOBOX,FIGLA,and FOXL2.Recently,more and more researches discovered the correlation between DNA repair genes and women early menopause,declined ovarian reserve and POI.Minichromosome maintenance complex component 8(MCM8)is one of the research hot spots.Minichromosome maintenance complex component 8(MCM8),is a member of evolutionarily conserved MCMs protein family,involved in DNA replication,meiosis and homologous recombination repair.In mouse models,Mcm8 knockout resulted in diminished follicle and female infertility.Recently,a causative homozygous mutation has been found in consanguineous family with POI.Meanwhile,in vitro functional experiments proved that the MCM8 mutations destroyed the stability of chromosome.But the relationship between MCM8 mutation and sporadic POI has not been reported.Therefore,we performed MCM8 mutation screen in 192 patients with sporadic POI,further explored the adverse effect of identified mutations on the DNA repair function through in vitro functional experiments.Objective:To investigate the relationship between MCM8 mutation and sporadic POI in Chinese Han population,and explore the pathogenic mechanism.Methods:In the study,192 patients with sporadic POI and 312 control women were recruited.Recruitment creteria of POI included primary or secondary amenorrhoea,serum FSH>40IU/L,46,XX karyotype and no family history of POI.Known causes,such as autoimmune diseases,pelvic infection,ovarian surgery,and chemoradiotherapy treatment were excluded.The 312 control women with regular menstruation included 192 age matched women and 120 women older than 45 years,whose inclusion criteria were:1)serum FSH<12 IU/L,2)three or more antral follicles remaining in bilateral ovaries.The genomic DNA was extracted fromperipheral blood,and 19 exons and corresponding flanking sequences of MCM8 gene were amplified by polymerase chain reaction(PCR).Then,the result was compared with the control sequence provided by Ensembl.The corresponding functional studies were performed to illustrate its adverse effect on DNA repair.Results:Two novel missense variations and six known single-nucleotide polymorphism(SNP)were identified in patients with sporadic POI.Among the known SNPs,five were nonsynonymous,including rs236110 in exon3,rs116926921 and rs58487183 in exon5,rs16991591 in exon6,rs61752028 in exon8 and one synonymous rs3761873 in exon7.The functional studies have found that HeLa cells overexpressing mutant p.H317L and p.H601R were more sensitive to MMC compared with wild type.Furthermore,mutant p.H317L showed decreased repair capacity after MMC treatment with much more yH2AX remaining after 2 hours recovery.Conclusion(s):This study for the first time identified MCM8 mutations in Chinese Han patients with sporadic POI.The functional experiments confirmed that the mutant p.H317L and p.H601R disrupted the DNA repair capacity of MCM8,suggesting that the diminished DNA repair induced by MCM8 mutation might be one of the causations for POI.