The Detection And Significance Of Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutations In Chinese Congenital Bilateral Absence Of The Vas Deferens

Purpose:10-15%of couples are infertile after the first year of marriage in the word.Involve in 60-80 millions couples and more than 50%are caused by males.Congenital bilateral absence of the vas deferens?CBAVD?is a kind of commonly autosomal recessive disease and morbidity is 1/1000.2-6%of infertility are caused by CBAVD which it is the main reason.Clinical symptom include bilateral absence of the vas deferens,decrease of semen,absence or dysagenesis of seminal vesicle or cauda of epididymis.The primary treatment method is introcytoplasmic sperm injection combined with percutaneous testis or epididymis sperm extraction for birth.CFTR gene is closely related to CBAVD.Variants types of CFTR gene are 2009 which are published by NCBI.It have highly polymorphyism,the vast majority of variants frequency are less than 0.1%,and are apparently ethic specificity.The research about CFTR gene variants in CBAVD is limited and late in China.This study is aimed at discuss of the variants of CFTR gene in chinese CBAVD through sequencing the CFTR gene and discuss the affect on spermatogenesis through observing the CFTR protein expression level in testis.Methods:Immunohistochemical method detect CFTR protein expression level and spermatogenesis stage in testis on 66 cases CBAVD and 68 cases normal males respectivly.To discuss the expression level of CFTR protein and spermatogenesis.Sequenceing the whole exon and 1.4kb promoter before ATG,capillary electrophoresis detected IVS10-11?TAAA?n STR of 66 cases CBAVD.Compared with60 normal males and blast CFTR gene sequence with NCBI database online.To research the variants of CFTR gene in chinese CBAVD.To discuss the function of variants through phylogenetic footprinting and Tranfac method.Contruct the variants plasmid and detect the its influnce on CFTR gene transcription through Dual luciferase reporter gene assay.Results:?1?Immunohistochemical results reveal CFTR protein express in sertoli cell and spermatogenic epithelium cell of testis,mainly in cytoplasm and cytomenbrane. Expression level of CFTR protein in CBAVD is positive and weak positive ?87.87%,58/66?,3 of 66 are negative.Wheras control group is strongly positive and positive?95.59%,65/68?,none of them is negative.There is significant statistic difference?Fisher's exact test=37.792,P<0.01?.OD of CBAVD and control group are 0.150±0.022and 0.194±0.079 respectively.There is significant statistic difference also?t=4.305,P<0.01?.Johnsen score of CBAVD group are from 7 to 10 and the proportion is 4.45%,40.9%,42.42%and 12.23%respectively according to score,Johnsen score of control group are from 8 to 10,and the proportion is 29.41%,55.88%and 14.71%respectively according to score,There is no significant statistic difference?Fisher's exact test=5.355,P=0.127?.The correlation coefficient rs is 0.808 between spermatogenesis and CFTR protein expression level.There are 9 cases who have exon mutations in 66 CBAVD patients and mutation proportion is 13.63%?9/66?.There are 6 types of mutation which including 5 kinds of missence mutaion?p.A107D,p.E476K,p.W496R,p.H949P and p.M1140K?and 1?p.T854T?kind of synonymy mutation.All of the missence mutation are new kinds mutation which are not found either in NCBI database and 1000 Genome Project database.There are no deletion,insertion or large fragment rearrangement mutation.There is no p.F508del,p.R117H and p.D1152H mutation which are very commongly in Caucasian.Main genotype of CFTR IVS10-11?TAAA?n is?TAAA?₉/?TAAA?₁₀0 in CBAVD group,the proportion is 50.00%and the genotype of?TAAA?₉/?TAAA?₉ is rare which proportion is 21.21%.Whereas in normal males the genotype of CFTR IVS10-11 ?TAAA?n is?TAAA?₉/?TAAA?₉ and proportion is 79.33%.The other allele types ?TAAA?_10,?TAAA?₁₁and?TAAA?₁₂2 only seen in CBAVD group.The OR of genotype?TAAA?₉/?TAAA?₁₀0 is 9.22 compared with?TAAA?₉/?TAAA?₁₀0 according to logistic regression analysis.There are higher OR if combined with allele?TAAA?_10,?TAAA?₁₁and?TAAA?₁₂.We find 6 kinds of point mutation and 1kind of deletion mutation which are c.-150G>T,c.-205T>C,c.-245C>T,c.-1062G>C,c.-871G>T,c.-966T>G and c.-861delT respectively through CFTR gene 1.4kb promotor sequencing in CBAVD.c.-966T>G have the highest variants rate which including heterozygous and homozygous mutation type.The other type only have heterozygous mutation type and is not seen in study before,but variants propotion is lower,none of the variants is seen in normal people.The proportion of c.-966T>G homogenous variant is 50%?33/66?in CBAVD group,the genotype proportion of T/G and T/T are 27.27%and 22.73%.The correspondent proportion in control group is 3.33%,50.0%and 46.67%.There is significant statistic difference between two group?X²=34.179,P<0.01?.The OR of G/G homozygous variant is 30.8 compared with T/T wild type.c.-966T>G locate in conserved sequence through DNAman software compared with several homospecies and phylogenetic footprinting method.We find the domain which including this necleotide can combine with EHF and STAT3 tanscriptor through transfac online prediction.So that we construct G/G and T/T plasmid and transfect Hela cell,HEK-293 cell and SW480 cell respectively.The results suggest that G/G variant decrease the CFTR gene transcription level 18.75-35.50%which have significant statistic difference and have prominent tissue specificity.Conclusions:?1?CFTR protein express in sertoli cell and spermatogentic cell.It has lower expressiong level in CBAVD than normal people and has lower spermatogensis function also.CFTR potein expression level has positive correlation with spermatogensis function.?2?CFTR gene exon mutation in Chinese CBAVD is less than in Causian CBAVD and have significant ethic specificity.Genotype of CFTR IVS10-11?TAAA?n in CBAVD mainly is?TAAA?₉/?TAAA?₁₀0 whereas is?TAAA?₉/?TAAA?₉ mainly in normal people.Relative OR is 9.22.?3?CFTR gene promotor variants type is different between Chinese and Causian.We find 6 types point variant and 1 types deletion variant,most of the variant are not seen in Causian.The frequency of c.-966T>G homozygous variant is higher than the others and the OR is 30.8.The element which including this neclotide is located in conserved sequence and combine with EHF or STAT3 transcriptor which is have an important role in fertility.c.-966T>G homozygous variant will decrease the CFTR gene transcription level 18.75-35.50%and have predominant tissue specificity.