Pedigree Study On Patients With Nonobstructive Azoospermia And Genetic Variation Analysis Associated With PiRNA Biogenesis Pathway

Background and Objective: Male fertility has been decreased remarkably due to genetic factors,living envioronment pollutions and bad diet structure and unhealthy life style.It not only has been a serious disease affecting human reproductive healthy,but broke harmony and stability of the society.Thus,mechanisms of spermatogenesis and male infertility are required to be solved urgently.In our previous studies,the gene MOV10L1 mutation,one gene associated with piRNA biogenesis pathway,seemed to have contributions to the pathogenesis of IHH by whole genome exon sequencing and linkage analysis.Many studies on the animal models harboring gene deletions have shown that the genes involved in piRNA biogenesis and their defective expression can result in the spermatogenic dysfunction.Meanwhile,the presence of piRNAs have identified in neurons.So,we speculated that the mutation of MOV10L1 gene may account for IHH in this family.Based on our previous study,our project plan to map candidate genes related to the disease of NOA coupled with progressive limbs weakness by pedigree analysis and molecular biology technologies,and explore the associations between genetic variants of important genes involved in piRNA biogenesis and male infertility with spermatogenic impairment.Materials and Methods: Genome DNA was extracted from peripheral blood.Whole exome sequencing was used to analyze the genome DNA of the proband and his uncle.11 STR markers and linkage analysis were done by MERLIN software.After application of whole genome exon sequencing and linkage analysis,combined with bioinformatics analysis,we found the candidate genes in the family.Subsequently,the other members of this family were verified the new gene variants by Sanger sequencing.Pathological study of quadriceps femoris was performed in the proband.RT-PCR and immunohistochemistry were applied to study the candidate gene expressions and productions.106 Chinese NOA patients(47 hypogonadotropic hypogonadism and 56 hypergonadotropic hypogonadism)and 60 matched controls(donors)were recruited and divided into three groups.Genome DNA was extracted from peripheral blood for DNA library construction.14 genes associated with piRNA biogenesis pathway were selected as target genes to prepare for RNA probe.Target capture sequencing was performed for all the 106 samples and 14 genes mutation frequency were determined in three groups.Compared with mutation frequencies in Han Chinese in Beijing(CHB)and Han Chinese South(CHS)populations in China,we explored the difference between the patients presenting with spermatogenetic failure and the controls.Eight SNPs in the ASZ1,MOV10L1,PIWIL1,TDRD1,and TDRD9 genes were genotyped in 342 cases with idiopathic non-obstructive azoospermia(NOA)and 493 healthy controls by using an improved multiplex ligation detection reaction(iMLDR)technique developed by GENESKY Biotechnologies Inc.(Shanghai,China).Plink1.03 and SPSS13.0 softwares were employed for statistical analysis,P<0.05 was considered statistically significant.Bonferroni analysis was performed to correct the P value.Results: By whole genome exon sequencing,linkage analysis and verification in the family,we found that the gene PLP2(c.265 C > T;p.L 89 F)and the gene CCDC22(c.977 C > T;p.T 326 M)were homozygous mutations in the male patients and heterozygous mutations in the obligate female carriers,while the other normal male were wild type homozygous.The points were conserved among orthologs,and functional prediction shows “disease damaging” by some softwares of protein function prediction.The two genes had no reports to associate with the disease of NOA or the symptom of progressive limbs weakness.Pathological study of quadriceps femoris in proband showed skeletal muscular dystrophies,and the mRNA expressions of CDCC22 in patient group increased than the control group(p<0.05),whereas PLP2 decreased significiantly in patient group(p<0.01)by Q-PCR.The protein expressions of CDCC22 and PLP2 showed no signaificant changes by immunohistochemistry.The frequency of rare gene mutation in MOV10L1,TDRD9,ASZ1,PIWIL3,PIWIL4,TDRD1,MAEL in the patients group was significantly greater than the control group,the results indicated that these genes variants might be associated with the spermatogenesis disorder in the Chinese Han population.Because of too small sample size,the result reliability needs to be further confirmed.10 SNPs in 7 genes were found with higher frequencies in NOA patients than in control group,we speculated that these SNPs might have special functions in the Chinese population.The further sthdy showed that TDRD1 variant rs77559927 was significantly associated with lower risk of spermatogenic impairment and played a protective role against the disease(P=0.032).We combined the rs77559927 TC and CC genotype,assuming a dominant model effect,the combined rs77559927 TC+CC variant genotypes were associated with a 26.6%(OR = 0.73,95% CI=0.56-0.97),the genotype TC and TC+CC showed odds ratios and 95% confidence intervals of 0.73(0.55-0.98,P = 0.034)and 0.73(0.56-0.97,P = 0.030),respectively in the patients with NOA compared with the controls.The OR values suggested that the variants rs77559927 T>C may be protective factors against the risk of spermatogenic impairment.Haplotype analysis of eight SNPs sites of genes in combination exhibited that haplotype AG(rs4555053,rs378331)in TDRD9 might play a protective role against the occurrence of NOA comparing with the controls(P=0.016,OR=1.598,95%CI:1.088-2.346).Conclusion: This study confirms a new X-linked recessive inherited disease of NOA coupled with progressive both lower limbs asthenia in a Chinese family and the candidate gene is mapped to Xp11.23.PLP2 and CDCC22 may be candidate genes.It is the first report about genetic researches of X-linked recessive disease of NOA coupled with progressive limbs weakness in our country.It is important to contribute to the etiology and diagnosis of the disease,however the elucidation of the molecular mechanism still need further study.The rare mutation in MOV10L1,TDRD9,ASZ1,PIWIL3,PIWIL4,TDRD1,MAEL genes might be associated with the spermatogenesis disorder in the Chinese Han population.Our results provided epidemiological evidence supporting the involvement of genetic polymorphisms of the piRNA processing genes TDRD1 and TDRD9 in modifying the risk of spermatogenic impairment in a Han Chinese population.