Clinical Genetic Analysis And Whole-exome Sequencing With Rare Variant Association Study Of Nonobstructive Azoospermia

BackgroundInfertility affects approximately 10?15% of couples,and male factor is responsible for 30%–50% of all infertility.The causes of male infertility include endocrine factors,genetic factors,testicular trauma,cryptorchidism,varicoceles,infection,iatrogenic injury,metabolic diseases,etc.Genetic abnormalities are thought to be more serious impact.The most severe form of male infertility is non-obstructive azoospermia(NOA).The typical phenotype of NOA are severely impaired spermatogenesis and testicular failure.The NOA patients with genetic abnormalities who are received medication,surgery and other therapeutic interventions,have no significant effects.NOA is a more complex disease caused by multiple factors,which had highly heterogeneous clinical phenotypes.Therefore,the genetic diagnosis of NOA is very difficult.Chromosomal abnormality is an important genetic etiology of NOA.Chromosomal karyotypic analysis is a routine method which can diagnose the etiology for partial NOA patients(e.g.Klinefelter's syndrome).However,the detection rate,clinical phenotypes and mechanisms of different chromosomal abnormalities in NOA patients are still unclear.AZF(azoospermia factor)microdeletion on the Y chromosome is also the main genetic cause of NOA,which is second only to Klinefelter's syndrome.At present,sequence tagged site(STS)-PCR is mainly used for AZF microdeletions.However,only a part of AZF microdeletions could be detected and other variations in AZF region could not be detected due to the limited number of STS sites.In addition to chromosomal abnormalities and AZF microdeletions,the reason for infertility in 60-80% of NOA patients can still not be defined and they are ultimately defined as idiopathic NOA patients,most of whom could be resulted from unknown genetic factors.Hence,clarifying the genetic etiology of NOA would be beneficial to offering appropriate genetic counseling and clinical therapy for these patients.In clinic,a sufficient number of usable sperm could be discovered in the testis of some NOA patients,who have the opportunity to have their own offsprings by the combination of testicular biopsy and intracytoplasmic sperm injection(ICSI).With the rapid development of sequencing platforms,next generation sequencing(NGS)has been gradually applied to the clinical detection of cancer,intelligence disability,genetic diseases,cardiovascular and cerebrovascular diseases,diabetes and other diseases.In recent years,the research on the molecular mechanism of NOA patients has also made great progress.Some susceptibility loci for NOA and new pathogenic genes have been reported continuously.However,most of the newly discovered pathogenic genes have not been further verified in sporadic NOA patients or pedigrees.Although chromosomal karyotypic analysis and AZF microdeletion anlaysis have been widely used in clinic,the effects of special chromosomal abnormalities,chromosomal breakpoints and diverse types of AZF subregions on male fertility have not been delineated in detail,which would cause difficulties in genetic counseling in clinic.The application of new high-throughput sequencing technology can discover new pathogenic genes,but a large number of gene mutations need to be analyzed by the combination of clinical phenotypes and pedigree verification.Therefore,in order to provide more accurate genetic counseling,the genetic factors of NOA patients remain to be further studied and discussed.Objective1.1.To investigate the types and detection rates of chromosomal abnormalities in NOA patients and their effects on clinical phenotypes.2.2.To investigate the types,detection rates and subregional variation types of AZF region in NOA patients.3.3.To analyze the detection efficiency of spermatogenic gene capture and sequencing and the detected pathogenic variation.4.40 To explore the results of whole-exome sequencing(WES)and rare variant association study(RVAS)analysis were verified by pedigree verification.To screen and and analysis of NOA-related pathogenic genes,and provides an experimental basis for further research on NOA pathogenic genes.MethodsThis study included infertile male patients who visited the Reproductive Medicine & Prenatal Diagnosis Center of the First Hospital of Jilin University from Sep 2014 to Dec 2018.Obstructive azoospermia was excluded(using by cytological examination after routine semen analysis,Semen cytology analysis,testicular fine-needle puncture or microscopic testicular sperm extraction).Clinical information and infertility relevant test results were collected from 1739 patients diagnosed with NOA.This study was approved by the Ethics Committee of the First Hospital of Jilin University.The research is carried out from the following four parts:Part 1: Karyotype analysis was performed on all 1739 NOA patients.Peripheral blood lymphocyte culture,chromosome preparation and G banding analysis were performed in this part.Chromosome microarray analysis(CMA),specific probe-fluorescence in situ hybridization(FISH)were used to diagnose abnormal karyotypes.The detection rate of different types of chromosomal abnormalities in NOA patients,clinical phenotypes and related influencing mechanisms were analyzed.Part 2: AZF microdeletion analysis were performed on those NOA patients without chromosomal abnormalities.The detection rates of STS-PCR method and NGS method were compared.The relevant mechanisms of AZF subregion variation were analyzed.The effect on NOA spermatogenic disorders was also discussed.Part 3: After the exclusion of chromosomal abnormalities and AZF microdeletions,the spermatogenic gene capture sequencing(Panel)screening analysis was performed on 136 patients who agreed to participate in the spermatogenic gene study.Spermatogenic gene Panel list is according to the previous research of our research group.Genetic variation screening and pathogenicity analysis were performed in this section.Part 4: WES was performed on 133 NOA patients without related mutations after spermatogenic gene Panel and 343 patients in the fertility control group.RVAS analysis and protein interaction association analysis were performed to identified genes with significant differences.Further,11 families were recalled for Sanger sequencing verification and family association analysis.The results of RVAS and protein interaction association analysis were combined to check the possible NOA-related genes,and the relationships between these genes and phenotypes were discussed.Results1.Chromosomal abnormalities were detected in 299 cases of 1739 NOA patients,X accounting for 17.19%.Among them,sex chromosome aneuploidy(duplication or deletion of whole or part of sex chromosome)were found in 247 cases,which was the highest proportion in NOA patients with chromosomal abnormalities,accounting for82.61%(247/299).2.The testicular volume and testosterone level whose in X chromosome complete or partial duplication group were lower than those in Y chromosome complete or partial duplication group and Y chromosome complete or partial deletion group,with statistical differences(P<0.05);Follicle-Stimulating Hormone(FSH)and Luteinizing Hormone(LH)were higher than those in the other two groups,with statistical difference(P<0.05).Sex chromosome aneuploidy leads to aneuploidy in the pseudochromatin regions(PARs),and 14 out of 16 types of abnormal karyotypes have aneuploidy in the PARs,accounting for 93.93%(232/247)of all sex chromosome aneuploidy.3.There were founded 87.5%(7/8)NOA patients with AZF microdeletions in abnormal karyotypes which 45,X/46,XY and derivatives.The type of Y chromosome microdeletions in these abnormalities was usually of AZF b+ c region deletion.4.Xp-Yp translocation was contributed to the formation of XX-male syndrome,resulting in sex determining gene(SRY)translocation to X chromosome,accounting for 92.86%(13/14).5.CMA,FISH and AZF microdeletions analysis were performed on the abnormal which cannot be diagnosis by conventional karyotype analysis.2 cases with t(Y;D/G group)chromosome translocation,1 case with X/Y chromosome imbalance translocation,1 case with mosaic Y chromosome structure abnormal and 2 cases of small marker chromosome of Y origin were diagnosed.6.The levels of testicular volume,FSH,LH and testosterone in NOA patients with chromosomal balanced structure abnormality were close to those of normal men.A total of 31 breakpoints were detected in 16 cases with balanced structure abnormality,which were distributed on 18 chromosomes.There were two breaks at5q15,11q25 and 22q13 respectively,suggesting that the related spermatogenic genes at the breakpoints might lead to NOA.7.STS-PCR method were used to detect 109 cases with AZF microdeletions in868 NOA patients,with a detection rate of 12.56%.NGS method were used to detect172 cases with AZF region variants in 572 NOA patients,with a detection rate of30.07%.In this study,multiple abnormal types were detected,not only included the traditional microdeletion types,but also included a variety of AZF subregion deletion patterns.There were 7 types of pure deletions,in which partial deletion of AZFb + c region and partial deletion of AZFc region were further divided into 12 deletion modes.There were 3 types of pure repeats,in which the partial repeats of AZFc region were divided into 2 repeating modes.Duplication + deletion were totally 9modes.Deletion + inversion was 1 mode.Multiple AZFc subregion variants lead to increased Y chromosome instability.8.2 cases of TEX11 gene mutation and 1 case of AR gene mutation were detected by spermatogenic gene Panel screening.TEX11(c.1985T>C,p.Leu662Pro)and AR(c.528C>A,p.Ser176Arg)were possibly the pathogenic for NOA.Spermatogenesis mature arrest in the early stages of spermatogonia or primary spermatocytes were founded in 2 cases patients with TEX11 mutation.The clinical phenotype of AR gene mutation is heterogeneous,not completely manifested as androgen insensitivity and has heterogeneity.The detection rate of spermatogenic gene Panel screening in our previous study was 2.21%(3/136).9.WES was performed in 133 NOA patients and 343 reproductive controls.Six known and validated NOA-related genes(TEX15?BRCA2?FAM47C?MEIOB?SYCP3?TDRD7)were screened in the NOA patients.Through RVAS analysis of NOA group and control group,five most significant(P<0.0001,OR>1)genes were screened out: DHRS4?WARS?PICK1?RRBP1?ENTPD2,187 confirmed genes(P=0.0001? 0.05,testicular normalized expression>6,OR>6 or not estimable)were screened out.These included three genes known to be associated with NOA: BRCA2?TDRD7?SYCP3.10.The most significant genes(5 genes),verified genes(184 genes),verified NOA-related genes(3 genes)and known verified NOA-related genes(28 genes)in the literature were analyzed by protein interaction association.A total of 117 associated proteins were identified,and the results showed that these associated proteins were concentrated in meiotic protein-interaction pathways.11.A totally 11 families were recalled.After Sanger sequencing and Trios pedigree association analysis,combined with RVAS analysis and protein interaction association analysis.At last,six genes were screened out though Trios pedigree association analysis: ACTL8?TRA2B?IDE?FIBP?NUP37?PIGT.12.In addition,three known validation genes,BRCA2,TDRD7 and SYCP3.A total of 9 NOA-related genes were detected by WES.The detection rate of NOA-related pathogenic genes by WES and RVAS was 6.76%(9/133).Conclusion1.Sex chromosome aneuploidy is the main type of chromosomal abnormality in NOA.Whole or partial X chromosome duplication were contributed to more severe spermatogenic dysfunction effects.Chromosome breakpoints 5q15,11q25 and 22q13 may be related to spermatogenesis,and there were spermatogenic genes existed.2.AZF subregion has many forms of variation(partial deletion,partial duplication,partial deletion + partial deletion,partial deletion + inversion,etc.),which can lead to NOA by affecting Y chromosome stability.3.TEX11(c.1985T>C,p.Leu662Pro)and AR(c.528C>A,p.Ser176Arg)were the potentially pathogenic for NOA.The detection rate of spermatogenic gene Panel screening was 2.21%(3/136).4.ACTL8?TRA2B?IDE?FIBP?NUP37?PIGT identified by pedigree association analysis were potential to be NOA related gene.In addition,three known validation genes from literature: BRCA2,TDRD7 and SYCP3 were detected in our study.The detection rate of NOA-related genes by WES and RVAS was 6.76%.