The Correlation Of X Chromosome Inactivation And The Clinical Manifestions Of DMD Female Carriers

Objective This study was aimed at analyzing X chromosome inactivation pattern and the correlation of X chromosome inactivation and the clinical manifestions of DMD female carriersand by methylation enzyme digestion experiment to explore the possible mechanisms of different clinical manifestions of DMD female carriers.Method1.Collected the clinical data of DMD probands and female relatives with DMD mutations in Guangxi Medical University First Affiliated Hospital of pediatrics clinics or admission office,from September in 2011 and August in 2017.According to the CK levels,DMD female carriers were divided into high CK group and normal CK group.2.Extracted the DMD probands and DMD female carriers’ DNA of peripheral blood lymphocytes and measured the concentration and purity.Amplified HUMARA gene fragments of all samples after enzyme/non-enzyme method by methylation sensitive restriction enzyme(HpaII),then observed the digestion experiment results under Image Lab software after 2% agarose gel electrophoresis and analyzed whether the digestion experiment was successful.3.Amplified HUMARA gene fragments of enzyme/non-enzyme samples by PCR with primer labeled by Fam fluorescent dye,then separated amplification products by capillary electrophoresis in ABI373 automatic sequencer,and analyzed fluorescent markup fragments by Peak Scanner 2.0.Identified the inactive X chromosome and maternal/paternal origin.According to the fluorescent peak of fragments,calculated the X chromosome inactivation ratios by formula.Values ≥80% was classified as skewed.4.Used SPSS software(Version 22.0)to process the experiment data by Wilcoxon rank sum test and Spearman analysis to compare the difference of maternal and paternal X chromosome inactivation,high CK group and normal CK group and the correlation of X chromosome inactivation ratios and CK levels of DMD female carriers.P<0.05 was considered statistically significant.Results1.38 DMD families were included in this study,including 40 cases of male DMD probands,57 cases of female carriers with DMD gene mutations.There were 36 cases(63.2%)of DMD female carriers with high CK,who were divided into high CK group.And there were 21 cases(36.8%)of DMD female carriers with normal CK,who were divided into normal CK group.The maximum value of CK in high CK group was 7012 U/L,the minimum value was 183 U/L,the average value was 1072.4 U/L.2.The DNA extractions of DMD probands and DMD female carriers was successful(The maximum concentration was 125.8ng/ul,the minimum concentration was 21.1ng/ul,and the range of purity wass from 1.63 to 2.1).Methylation enzyme digestion experiments of all samples were successful.The electrophoresis results of DMD probands in enzyme group were lack of bands,while the results of non-enzyme group were shown as one clear band.There was only one band of DMD female carriers in enzyme group and non-enzyme group,and the concentration of non-enzyme group was higher than enzyme group3.The results of ABI373 automatic sequencer capillary electrophoresis showed that the enzyme group of DMD probands were lack of significant peak,and the results of non-enzyme group were one peak.The enzyme and non-enzyme group of DMD female carriers were shown as two peaks,the fragment size of one peak was consistent to the size of DMD probands in the non-enzyme group.There were 30 cases that the origin of X chromosome inactivation was maternal(52.6%).There were 27 cases that the origin of X chromosome inactivation was paternal(47.4%).4.No significant difference was observed in the levels of CK between maternal chromosome inactivation group and paternal chromosome inactivation group(Z=-0.176,P=0.86>0.05).No significant difference was observed in the X chromosome ratios between high CK group and normal CK group(Z=-0.24,P=0.81>0.05).There was no significant correlation of X chromosome inactivation ratios and CK levels of DMD female carriers statistically(P =0.858> 0.05).Conclusion1.Although the majority of DMD female carriers manifests as high CK,the diagnosis of DMD female carriers cannot be determined exclusively by CK levels,which is depending on genetic diagnosis,ultimately.2.No significant difference was observed in the CK levels between maternal chromosome inactivation group and paternal chromosome inactivation group.No significant difference was also observed in the X chromosome ratios between high CK group and normal CK group.And there was no significant correlation of X chromosome inactivation ratios and CK levels of DMD female carriers.It is suggested that the origin of X chromosome inactivation and X chromosome inactivation ratios may not be the cause of various clinical manifestions of DMD female carriers.