The Role Of C10ORF2 And HELQ,PR1M1 Genes Played In Primary Ovarian Insufficiency

Chapter Ⅰ.The Effects and Mechanisms of Mitochondria-related gene C10ORF in the Pathogenesis of Perrault SyndromePart Ⅰ.Whole Exome Sequencing in Perrault Syndrome FamilyOBJECTIVE:Primary Ovarian Insufficiency is a highly heterogeneous complex disease. It is one of the pathogenesis for female infertility which characterized by secondary amenorrhoea, elevated gonadotrophin level (FSH> 40 IU/L) and declined estrogen to menopausal level. It is the end phase of primary ovarian insufficiency. The overall presumption of POI prevalence in general population was reported to be 1-2%. The disorder is heterogeneous, with a wide spectrum of causes, including genetic, autoimmune, metabolic, infectious and iatrogenic. Genetic causes are highly heterogeneous and include both isolated (nonsyndromic) POI and syndromic forms. However, the etiology remains to be elucidated in most cases. The development of genomics technology for illuminate disease susceptibility loci and candidate genes provides a more efficient and effective way. Perrault syndrome (PS) is an autosomal recessive ovarian dysgenesis associated with sensorineural deafness. The patients showed a typical primary ovarian insufficiency (POI) phenotype. In this study, we will perform the whole exome sequencing (WES) in the Perrault Syndrome Family with ovarian POI to identify the innovative genes responsible for POI.METHODS:The study participants comprised the perrault syndrome patients and virulence genes suspected carriers and direct family members suspected carriers, then sequencing the whole genome exons by the way of Agilent V5. We checked out all the single-base changes, insertion/deletion and compared with public databases, then eliminate known polymorphism. After the detection of amino acid change with non-synonymous mutations, then find out candidate loci according to the rules of Mendelian inheritance. We made the genetic screening by the rule of autosomal recessive heredity, and then find out the candidate loci lacated on C10ORF2 gene. RESULT:By directly sequence the whole genome exome, and analysis the result according to the phenotype and Mendelian inheritance. We find out a novel homozygous mutations C10ORF2 c.1388G>A (p.R463Q).CONCLUSION:With the application of WES in the PS famlily with POI, we identified a new loci in C10ORF2 gene. As C10ORF2 is closely related to the function of mitochondria, mutations of the gene may made an obstacles in the cell mitochondrial energy metabolism of ovaries of oocyte and its support cells. Resulting in most patients accompanied with a typical primary ovarian premature aging signs. The new discovery provide a new research orientation in the mechanism research of POI.Part Ⅱ. Functional Study of C10ORF2 gene in Primary Ovarian InsufficiencyOBJECTIVE:In a familial Aggregation Analysis of Perrault syndrome with a typical phenotype of POI, we found a new gene closely related to function of mitochondria through the whole exome sequencing (WES) and identified a new mutation loci in C10ORF2 c.1388G>A(p.R463Q). This gene encodes a hexameric DNA helicase which unwinds short stretches of double-stranded DNA in the 51 to 3’ direction, along with mitochondrial single-stranded DNA binding protein and mtDNA polymerase gamma, is thought to play a key role in mtDNA replication. Mutations in this gene cause infantile onset spinocerebellar ataxia (IOSCA) and progressive external ophthalmoplegia (PEO) and are also associated with several mitochondrial depletion syndromes. Alternative splicing results in multiple transcript variants encoding distinct isoforms. Perrault syndrome is characterized by POI in females and progressive hearing loss in both females and males. The objective of our study was to investigate the effect of mutation in the immortalized cell line, and illustrate the gene function in the mitochondrial, which may offer a new thought in searching the mechanism of POI.METHODS:Genomic DNA samples from the probands with POI and from their unaffected parents were evaluated in our laboratory by the whole genome exome sequence according to previously published methods, extract the blood of recessive hereditary probands, and create immortalized cell line. The proband presented with primary amenorrhea and postmenopausal levels of follicle-stimulating hormone and luteinizing hormone. Her ovaries were not visualized on abdominal ultrasound. We test the function of mitochondria in the mutant immortalized cell, including the mtDNA copy number, detect the transcripts of mitochondrial genes, ATP and ROS level was measured in conducted immortalized cell line,and test the mitochondria membrane potential.RESULT: Compared with other control group, the function of mitochondria in mutant cell was functionally impaired, the mutant cell showed a turbulence of OXPHOS in the cell, accompanied with decrease ATP and increased ROS production, decreased transcripts of mitochondrial genes and mtDNA copy number. However, mitochondria exist in the cell plentifully, and possess a vital role in the cell energy supply, especially in oocyte, the potential role of C10ORF2 gene is unneglectable in the maintenance of female fertility mediated by functional of mitochondria.CONCLUSION:C10ORF2 gene plays a key role in mitochondrial replication, it functions as an energy supplier factor in oocyte cells. Mutations in C10ORF2 gene are associated with familial POI with Perrault Syndrome, which mediated by the impaired function of mitochondria. These studies provide evidence for the pitoval role of mitochondria in POI.Chapter II. The Screening of HELQ Gene in Chinese Patients with Primary Ovarian InsufficiencyOBJECTIVE:A meta-analysis of 22 genome-wide association studies has reported 13 novel loci associated with age at menopause. In the following biological pathway analysis, eight candidate genes (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG, PRIM1) identified are related to DNA damage repair and replication. Among all of these genes, HELQ encodes the protein HEL308, which is a DNA-dependent ATPase and DNA helicase and required for repair of DNA interstrand crosslinks through the association with RAD51 paralogs. Helq-/- mouse displayed subfertility and dysgenesis or atrophy of ovary was observed in histological sections, mimicking the human POI phenotype and suggesting HELQ’s critical role in germ cell maintenance. Therefore, in this study, the potential role of HELQ mutations in women with POI was explored by sequencing the coding region of HELQ gene.METHODS:The study comprised 192 Chinese Han women with secondary amenorrhoea who were recruited. All the patients recruited were non-syndromic and idiopathic. Genomic DNA was extracted from peripheral blood samples. The 18 exons and exon-intron boundaries of HELQ gene were amplified by using polymerase chain reaction (PCR). The PCR products were purified using the method of polyethylene glycol precipitation and sequenced directly on an automated sequencer (ABI3100; Applied Biosystems) using the ABI-Prism big-dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). All sequence variants were confirmed in triplicate with forward and reverse sequencing.RESULT: Six known single-nucleotide polymorphisms (SNP) were identified in the HELQ gene. Three synonymous SNP located in the exon regions, including rs1494961(exon 2), rs13141136 (exon 3) and rs7665103 (exon6). The other three SNP were in the exon-intron boundaries, including rs 11099600 (intron 4), rs2047210 (intron 10) and rs 12645412 (intron 11). For genotype frequencies of these 6 SNP, no significant difference was found between cases and controls.CONCLUSION:Although difference in the allele frequency was shown in the other four SNP, rs13141136, rs7665103, rs11099600 and rs12645412, no amino acid was altered and thus they are unlikely to be causative for POI. In conclusion, although no mutations were found in the HELQ gene, genes pivotal for DNA repair could not be fully ruled out in the candidates for POI, which is characterized by genetic heterogeneity with distinct loci in different ethnicities. A specific molecular perturbation in a given gene may vary, even within a given thnic group. Therefore, future studies in different ethnic populations are necessary to determine the role of HELQ in POI.Chapter Ⅲ. Variation of PRIM1 Gene in Chinese Patients with Primary Ovarian Insufficiency and Reproductive Phenotype Analysis in Prim1+/- MousePart Ⅰ. Variation Analysis of PRIM1 Gene in Chinese Patients with Primary Ovarian InsufficiencyOBJETIVE: A meta-analysis of 22 genome-wide association studies reported 13 novel loci associated with AANM, many of which have been replicated in other ethnicities. Candidate genes located at these loci are implicated in either DNA damage repair and replication or immune function. According to the Meta analysis, the nonsynonymous SNP rs2277339 in the PRIM1 gene is closely related to AANM. EM and POI in European women. During discontinuous DNA replication, PRIM1 (primase) plays a key role in the process of DNA synthesis initiation by synthesizing RNA primers for Okazaki fragments. However, the potential role of PRIM1 in POI pathogenesis has not been determined. Therefore, we examined 192 Han Chinese women with idiopathic POI by sequencing the coding region of PRIM1 gene to determine whether variants in this gene contribute to human POI.METHODS:Between June 2014 and September 2015, a number of 192 Han Chinese women with secondary amenorrhea were recruited. All the patients recruited were non-syndromic and idiopathic. Genomic DNA was extracted from peripheral blood samples. All the 13 exons and exon-intron boundaries of PRIM1 gene were sequenced. The sequence variants were confirmed by three independent PCR runs, followed by sequencing in both forward and reverse directions.RESULT: Three known SNPs, rs2277339 in exon 1, rs1131514 in exon 10, and rs1026565 in intron 6, were identified. Comparisons of genotype and allele frequencies showed no significant differences between POI cases and the general population. No plausible mutations were identified.CONCLUSION:Mutations in the coding region of PRIM1 are not common in Chinese women with POI. The exact role of PRIM1 in POI pathogenesis needs to be further explored in larger cohorts from Chinese and other ethnic populations.Part II.Reproductive Ability Analysis in Prim1+/- MouseOBJECTIVE: as a candidate gene in the POI, Priml was reported to be implicated in either DNA repair or replication according to the prior genome-wide association studies. It is functions as a primase in the process of DNA synthesis initiation by synthesizing RNA primers for Okazaki fragments. It involved the process of DNA repair but the functions in the oocyte not clear, the study aimed to construct Prim1 mutant mouse to investigate the phenotype of fertility and illustrate how C10ORF2 gene defects lead to the pathogenesis of POI.METHODS:Use the CRISPR/Cas9 technique to construct Prim1+/- mouse, after the mating test with wild type to ensure the reproductive capacity of Prim1+/- mouse, we keep the record of the offspring of every female mutant mouse, make ovarian tissue section with 3 weeks old mutant mouse to study the difference of oocyte at different levels.RESULT: Obviously there is no Prim1+/- offsprings after the mating of Prim1+/- mouse, E5, E1, E14, E16 days’embryos included, we guess the homozygous genes lead to an embryonic death in early days especially in the cleavage stage. But the Prim1+/-mouse shows a normal reproductive phenotype compared with the wild type.CONCLUSION:Priml gene participate in the DNA replication, impaired function of the gene are associated with the DNA repair pathway, the loss of the function would induce cell to death, theses study provided us the lethality of the systemic knockout gene in the embryogenesis, and showed us a further study with a purpose knockout in oocyte maturation process.