The Change Of Expression Profiles And DNA Methylation In Klinefelter Syndrome

Objective:In order to find the relevance between the change of RNA expression and DNA methylation on the extra X chromosome,Y chromosome and autosome.In our study,RNA expression level and DNA methylation level were measured in Klinefelter’s syndrome patients(47,XXY),normal males(46,XY),and females(46,XX).Then,we explored the internal relations between the change of RNA expression and DNA methylation status in Klinefelter’s syndrome with varied considerable phenotype.Methods:Seven Klinefelter syndrome patients whose karyotye is 47,XXY after karyotype analysis were selected as the experimental group.Seven health males and seven health females were selected as the control group.All of the control individuals had normal karyotype,normal fertility and no diseases.Genomic DNA and RNA were extracted from peripheral blood lymphocytes using the standard(QIAGEN)isolation protocol.Using RNA deep sequencing and Reduced Representation Bisulfite Sequencing to analyze gene expression profile and gene methylation profile.Result:1.According to the criterion FDR≤0.001 and |log2Ratio≥1|,the result of expression profile indicated that:(1)Compared with normal male controls,there were 216 differentially expression genes(DEGs)in KS group.① The DEGs included 100 upregμlated genes(4 X chromosome genes and 96 autosomal genes),in which,the most significant over-expressed gene was XIST(X chromosome)that is thought to be the major regulator of the X chromosome inactivation(XCI)process and the key component in the initiation stage.② There were 116 down-regμlated genes(5 X chromosome genes and 111 autosomal genes),in which,the most significant down-regμlated gene was NR4A3(autosome)that associated with the type 2 diabetes.③ There was no DGEs on Y chromosome.(2)Compared with normal female controls,there were 243 DEGs in KS group.① The DEGs included 118 upregulated genes(3 X chromosome genes,11 Y chromosome genes and 104 autosomal genes),in which,there was no significant changes in gene XIST.② There were 125 down-reμlated genes including 3 X chromosome genes and 122 autosomal genes.(3)Compared with female controls,there were 30 DEGs in normal male group.① The DEGs included 16 upregμlated genes including 10 Y chromosome genes and 6 autosomal genes.② There were 14 downregulated genes including 2 X chromosome genes and 12 autosomal genes.The most significant downregulated genes wasXIST.2.The whole genome methylation profile analysis results showed that:(1)Compared with normal male controls,there were 358 genes including 252 X chromosome genes were methylated in KS group.(2)Compared with normal female controls,there were 80 genes including 5 X chromosome genes were methylated in KS group.(3)Compared with normal female controls,there were 337 genes including 257 X chromosome genes were methylated in normal male group.(4)In the three groups,there were no methylation changed genes on the Y chromosome.Conclusion:1.The expression of XIST is related to the number of X chromosome.Two comparisons between KS(2 X chromosomes)and the male control group(1 X chromosme),normal female control group(2 X chromosomes)and normal male control group(1 X chromosme)showed that XIST was the most significant up-regulated gene.Comparing female control group with KS,the XIST log2Ratio is too small to show the significant difference.These results indicate that,as the same as the female,the extra X chromosome in KS can be inactive normally,and then the expression of XIST is related to the number of X chromosome.2,Because the change of DEGs to less to influence phenotypic diversification of KS,there maybe have other effect mechanisms.The results showed that comparing between the two control group,there were 2 DEGs on X chromosome;comparing between the male control group and KS,there were 9 DEGs on X chromosome;comparing between the female control group and KS,there were 6 DEGs on X chromosome.From these results,we can find that the number of DEGs on X chromosome was less.The main funtions of the DEGs products were DNA binding and contact with immune system.However,except the change of immune system,the phenotype of KS is diverse.The change of DEGs on X chromosome can’t explain adequately the phenotypic diversification.As a conclusion,except the extra X chromosome,other mechanisms maybe influence the change of phenotype of KS.3.The phenotype of KS is influenced by autosomal genes expression.The results showed that most of the DEGs were on the autosome,the most significant down-regulated gene was NR4A3 which associated with the type 2 diabetes.NR4A3 is an Insulin Resistance-Linked Gene which down-regμlated expression leads to type 2 diabetes and metabolic syndrome.What has been discussed above leading to the conclusion that the change of NR4A3 expression level may be the cause of type 2 diabetes in KS and the change of autosomal genes expression level may influence the phenotype of KS.4.The Y chromosome gene expression is not influenced by the extra X chromosome.The Y chromosome gene expression was normal in KS,which was not influenced by the extra X chromosome.Other mechanisms maybe exist that lead to azoospermia or oligospermatism.5.The change of gene methylation may influence the phenotype of KS.The gene expression is influenced by gene methylation status.Methylation leads to gene silencing.There were less DEGs on X chromosome,but most genes of X chromosome were methylated.On the contrary,There were more DEGs on autosome,but less genes of autosome were methylated.These results suggest that the change of methylation may influence the phenotype of KS.