Mutation Screening And Function Exploring Of Human Idiopathic Non-Obstructive Azoospermia Associated Genes

Background: Infertility has become a global health problem in the 21 st century,affecting approximately 8%-12% of couples at childbearing age.About 48 million couples worldwide are suffering from infertility.In the cause of infertility,both men and women account for about half of them.Among them,infertility caused by male factors is called male infertility.Among male infertility,non-obstructive azoospermia(NOA)is considered to be the most serious type,accounting for about 3%-5% of the causes of male infertility.As the idiopathic NOA with the highest incidence among NOA patients,it is generally difficult to find clear pathogenic factors,and its occurrence may be affected by environmental and genetic factors.Some single-gene mutations have also been found to lead to NOA,such as TEX11,FANCM,STAG3,etc.Purpose: Patients with idiopathic NOA(NOA1-NOA60)were enrolled as the research object.Whole exome sequencing(WES)and bioinformatics analysis were applied to screen and identify related variants of known pathogenic genes related to NOA,and and screening unknown candidate genes of NOA.We aim to provide theoretical basis for genetic diagnosis,genetic counseling and individualized treatment for patients with idiopathic NOA.Methods: Peripheral blood samples of 60 patients with idiopathic NOA(NOA1-NOA60)from the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University were collected for whole exome sequencing(WES).Firstly,screening variants and performing bioinformatics analysis on the data obtained by WES to screen known disease-causing gene variants and new candidate disease-causing gene variants: the first step is to compare the currently known NOA disease-causing genes and inherited patterns,and screen known pathogenic gene variants;the second step is to focus on rare and harmful testicular-specific/highly expressed genes in cases where the known pathogenic genes cannot be explained,in order to discover new candidate genes associated with idiopathic NOA.Secondly,for the discovered known disease-causing gene variants and candidate disease-causing gene variants,Sanger sequencing is used to verify the accuracy of the sequencing results and conduct family co-segregation analysis to determine the family source of the gene variants.Finally,through HE staining,immunofluorescence co-staining different spermatogenic cell markers to analyze the pathological types of testicular tissues of patients with idiopathic NOA,and through immunofluorescence,immunohistochemistry,real-time fluorescent PCR and Western Blot,etc.to detect the location and quantitative expression of candidate pathogenic genes in human testis tissues.Results: First,through mutation screening and bioinformatics analysis,we found 3 new NOA candidate pathogenic genes in 5 patients—HFM1,FER1L6 and SRRM5 :(1)In patients NOA5 and NOA26,two homozygous variants of the HFM1 gene were identified,respectively.NOA5 patient carries a homozygous stop-gain mutation of HFM1 gene(NM?001017975: exon32: c.3490C>T: p.Q1164X);NOA26 patient carries a homozygous missense variants of HFM1 gene,and the softwares predict that the variant is harmful.The histopathological type of the testis of two patients was both identified as spermatocyte arrested NOA.Immunofluorescence experiments showed that HFM1 protein was mainly expressed in the nucleus of spermatocytes in the seminiferous tubules of the testis in the control group,while the expression of HFM1 was significantly reduced in the testis tissues of patients NOA5 and NOA26.QRT-PCR experiments and Western Blot experiments indicated that the expression levels of HFM1 m RNA and protein in two patients were also significantly reduced.(2)The biallelic variants of FER1L6 were found and identified in patients NOA8 and NOA44.The patient NOA8 carries a homozygous missense variant of the FER1L6 gene(NM?001039112:exon9:c.722C>T:p.P241L),and the softwares predict that the variant is harmful.Testicular histopathological type is identified as Sertoli-cell-only syndrome type NOA in patient NOA8.In patient NOA44,we found compound heterozygous variants in the FER1L6 gene(NM?001039112: exon13: c.1693G>A: p.G565R/ exon23:c.2905 del C: p.P969fs),and the patient's testicular tissue pathological analysis suggested a spermatocyte arrested NOA.Through immunofluorescence experiments,we found that FER1L6 protein was mainly expressed in the seminiferous tubules of the testis in the control group,while the expression of NOA5 and NOA26 in the testis of the patients were significantly reduced.(3)A homozygous variant of SRRM5 gene(NM?001145641: exon1: c.1531C>T: p.Q511X)was found and identified in patient NOA10.The patient's pathological phenotype was identified as spermatocyte arrested NOA.Secondly,we analyzed the currently known NOA pathogenic genes.We found multiple harmful variants in 7 known pathogenic genes in 10 patients,including 3recessive genes and 2 X-linked genes and 2 dominant genes.(1)Three recessive pathogenic genes:(1)A rare homozygous mutation of MSH4gene(MSH4: NM?002440: exon12:c.1552C>T:p.Q518X)was discovered and identified in the patient NOA2,and the patient's testicular tissue pathology type was identified as spermatocyte arrested NOA.Immunofluorescence experiments showed that MSH4 protein was mainly expressed in spermatocytes in the seminiferous tubules of the testis in the control group,while no clear expression was found in the testis of NOA2 patients.QRT-PCR experiments also indicated that the expression level of MSH4-m RNA in the patient NOA2 was significantly reduced.(2)The homozygous fragment-missing of MEI1 gene was found in patient NOA9(MEI1: NM?152513.3: exon19-intron19:c2262?2268+9del ACGTGAGGTATGGACC).(3)In the patient NOA16,we found and identified a homozygous missense variant in the FANCA gene(FANCA:NM?000135:exon33:c.3263C>T:p.S1088F),and the patient's testicular tissue pathological phenotype was identified as Sertoli-cell-only syndrome;In the patient NOA50,we found and identified two missense variants of FANCA gene(FANCA:NM?000135: exon19: c.1729C>G: p.P577A/ exon33: c.3263C>T: p.S1088F),the patient's testis histopathological phenotype was also identified as Sertoli-cell-only syndrome.(2)Two X-linked pathogenic genes:(1)A hemizygous missense variant of AR gene was found in patient NOA7(AR:NM?000044: exon5: c.2263T>C: p.F755L),and the patient's testicular tissue pathological type was identified as Sertoli-cell-only syndrome.(2)A hemizygous missense variant(NM?031276: exon7: c.466A>G: p.M156V)and a hemizygous frameshift mutation(NM?031276: NM?031276: exon8: c.559?560del:p.M187fs)of TEX11 gene were found in patient NOA39 and patient NOA49,respectively.The phenotypes of the testicular tissues of these two patients were hypospermatogenesis NOA and spermatocyte arrested NOA,respectively.(3)Two dominant pathogenic genes:(1)In patients NOA22 and NOA25,we respectively discovered and identified a heterozygous missense variant in a known NOA dominant pathogenic gene-DMRT1(NOA22: NM?021951: exon2: c.425C>T:p.A142V;NOA25: exon1: c.340G>A: p.V114M).The pathological phenotype of testis in these two patients were identified as Sertoli-cell-only syndrome.(2)In the patient NOA42,we discovered and identified a heterozygous deleterious missense mutation in PLK4 gene(NM?001190799: exon15: c.2785A>G: p.M929V),and the patient's testicular tissue pathology is also Sertoli-cell-only syndrome.Conclusion: Through whole exome sequencing combined with Sanger sequencing technology,the known pathogenic genes of NOA can be efficiently identified.Among60 NOA patients,the known pathogenic genes can explain for 16.67% of the cases.Among them,the proportion of idiopathic NOA patients from consanguineous families with known gene mutations is as high as 30%.Therefore,these patients have a higher genetic risk,and in-depth genetic testing is recommended to guide clinical treatment.In addition,we also identified HFM1 and FER1L6 as new candidate pathogenic genes for NOA,and their homozygous or compound heterozygous mutations may lead to NOA.Our findings provide some new theoretical basis for the genetic diagnosis and genetic counseling of patients with idiopathic NOA,and also provide references for the individualized treatment of such patients with NOA.