Transcriptome-wide Association Study For Non-obstructive Azoospermia Susceptibility In Chinese Males

Background:Infertility,a severe reproductive health issue,is affecting approximately 10-15%of the couples in the world and becomes the third largest disease following cancer and cardiovascular diseases.Male factor is responsible for about 50%of the cases,including severe oligospermia,asthenospermia,teratozoospermia and azoospermia.Clinically,azoospermia is defined as no sperm after three-time routine semen examination,and occurs in 15-20%of infertile men.According to whether there are obstructions in seminal ducts,azoospermia can be categorized into two major categories:obstructive azoospermia（OA）versus non-obstructive azoospermia（NOA）.Compared to OA,NOA is more complicated with congenital dysfunction in spermatogenesis,with a prevalence of 1%in all adult men.Therefore,it is very important to identify NOA susceptibility loci and explore the molecular mechanism,which may identify high-risk individuals and adopt preventive measures or treatments.The etiology of NOA is complex and multifactorial,possibly being a result of interaction between environmental and genetic factors.As reported,known environmental factors include high temperature,radiation,toxic gases,long-term exposure to drugs/crude cottonseed oil.And well known genetic causalities of NOA involve chromosomal alterations,Y chromosome microdeletions,epigenetic alterations,autosomal polymorphisms and mutations.Recently,as a powerful tool in identifying the significant susceptible single nucleotide polymorphisms（SNPs）associated with complex diseases,genome-wide association study（GWAS）has been applied into the exploration of genetic etiology of NOA.It is based on high throughput genotyping platform and tests millions of the whole genome SNPs in a large sample size of cases and controls.However,most of the GWAS identified SNPs are located in noncoding regions or“gene desert regions”,the mechanisms beyond the genetic variants identified by GWAS are largely unknown.And multiple researches have shown that many GWAS identified variants exert their effects on complex trait by modulating gene expression,thus altering the abundance of relevant proteins.With the rapid development of high-throughput sequencing technology,theoretically,we can use RNA-seq technology to comprehensively detect the overall transcriptional level for specific tissue and systematically identify genes associated with the disease.However,large-scale studies in individuals have been hampered because of difficult tissue availability.To address this problems,Gusev and Ko developed a new approach,leveraging expression imputation from large-scale GWAS data by using a small set of individuals with both genotype and gene expression data as a reference panel,to perform a transcriptome-wide association study（TWAS）.Methods:Based on Genotype-Tissue Expression（GTEx）database,we included157 individuals with both gene expression data in testis and SNP array data in blood as a reference panel.We first estimated the cis SNP heritability（cis-hg²）for each gene in 157 testis tissues using genome-wide complex trait analysis（GCTA）software,and kept cis-heritable genes for subsequent analysis.Then,we estimated the effect sizes of cis-SNPs on gene expression using genome-wide efficient mixed-model association（GEMMA）algorithm.Using the effect sizes trained from the reference panel,we imputed the gene expression levels for our existing NJMU NOA GWAS cohort（981 NOA patients and 1,657 controls）and correlated the imputed gene expression values with NOA trait.Results:The cis-hg² estimate was significantly nonzero for 1,296 genes after accounting for multiple-testing burden(P<3.01×10^-6).The average cis-hg²value was0.64 in these 1,296 cis-heritable genes.Using GTEx database as a reference panel,we successfully imputed expression levels of 1,296 cis-heritable genes for our existing NJMU NOA GWAS data and correlated the imputed gene expression levels with NOA trait.After FDR correction,we identified two potentially NOA-associated genes:PILRA（paired immunoglobin like type 2 receptor alpha）(P_TWAS=8.54×10^-6)and ZNF676（zinc finger protein 676）(P_TWAS=5.01×10^-5).PILRAislocalizedonchromosome7q22.1,andencodesan immunoglobulin-liketransmembranereceptor,whichcontainsan immunoglobulin-like V-type domain in the extracellular region and two cytoplasmic tyrosines positioned within an immunoreceptor tyrosine-based inhibitory motif（ITIM）in the intracellular region.By using the STRING database,PILRA was predicted to interact with PTPN11（protein-tyrosine phosphatase non-receptor type 11）.Of note,PTPN11 was known to be essential for the proliferation of spermatogonial stem cells（SSCs）,as well as the integrity of blood-testis barrier（BTB）and attachments of SSCs to their niche.ZNF676 is localized on chromosome 19p12.Since the ZNF676 protein contains classical Cys₂His₂ zinc finger domains（ZnFs）,by binding to specific DNA sequences in the promoter or enhancer regions of target genes it might alter the expression of these genes.GO enrichment analysis showed that genes co-expressed with ZNF676 gene in testis tissues were enriched in pathways of spermatogenesis and male gamete generation.In addition,our TWAS approach further supported the involvement of 6p21.32 in susceptibility to NOA,prioritizing 10 genes for follow-up studies.Since TWAS approach increased power over standard GWAS,the best GWAS SNP at TWAS-selected gene loci was probably novel disease-associated signal.In the7q22.1 loci,the best GWAS SNP was rs6945952,and in the 19p12 loci was rs808373.These two best GWAS SNPs found by our NOA TWAS approach were probably novel NOA-associated signals(rs6945952:OR=2.04,95%CI=1.64-2.54,P=2.64×10^-10,rs808373:OR=1.26,95%CI=1.11-1.42,P=3.18×10^-4).Conclusions:In conclusion,we carried out a“TWAS”strategy to pinpoint disease-associated genes,both genome and transcriptome levels information was considered,cis-heritable genes were explored and evaluated efficiently.Our results provided novel insight into the etiology of NOA,and pinpointed fruitful targets for follow-up functional studies,which might have an impact on screening,diagnosis and treatment of NOA.