Association Of Phenotype-Genotype And Methylation In 4q35-D4Z4/PAS Region Of FSHD

Background and ObjectivesFacioscapulohumeral Muscular Dystrophy(FSHD,OMIM 158900)is the most common muscular dystrophy after Duchenne muscular dystrophy(DMD)and Myotonic dystrophy(DM),with an incidence of 1:20,000.In general,the onset of adolescent diseases shows asymmetrical muscle weakness,mainly involving the facial,scapularis and upper brachial muscles,and progresses to the trunk and lower extremity muscles.FSHD can be divided into FSHD1 and FSHD2 according to different genetic mechanism with indistinguishable phenotype.FSHD1 is accounts for 95%,with the contraction of the D4Z4 repeats on 4q35(<10 units),which causes the hypomethylation of D4Z4 and mis-expression of DUX4.FSHD2 is about 5% with a normal D4Z4 sequence,due to the mutation of SMCHD1,which also leads to the abnormal expression of DUX4 and widespread hypomethylation of D4Z4.Both FSHD1 and FSHD2 are closely linked with a FSHD-permissive haplotype,which contains a simple sequence length polymorphism(SSLP)located proximal to the D4Z4 repeat and a special polyadenylation signal(PAS,ATTAAA)located on the distal,only the specific SSLP-4q A genotype can lead to FSHD.Based on the standardized FSHD diagnosis process,patients in the family were observed with significant phenotypic heterogeneity,the simple contracted number of D4Z4 repeats have been unable to explain the severity of phenotype,whether the degree of methylation may imply the phenotypic differences.This research analyzed the relation of clinical phenotype?genotype and methylation in 330 Chinese FSHD1 patients and a FSHD2 family based on the established standardized FSHD diagnosis and treatment process.To confirm the correlation of methylation level and clinical phenotype severity,the methylation detection to become a new auxiliary diagnosis,which can help improve the molecular diagnosis of FSHD and evaluate the severity of phenotype.Methods1.Study subjects: 330 FSHD1 patients,130 normal controls,50 other myopathy patients and one FSHD2 family.The clinical examination is consistent with the diagnostic criteria proposed by Padberg GW,and the clinical evaluation of CSS,FSHD clinical score and phenotype classification are conducted.The study is in accordance with the provisions of the hospital ethics committee,and informed consent is obtained.2.Research methods: based on the pulse gel electrophoresis of Southern Blot and probe hybridization method,D4Z4 copy number and haplotype were detected.By the target region methylation sequencing method,the primers in the PAS region were selected according to the literature.Bioinformatics analysis,Sanger sequencing,family separation,the validation in transcription,protein and methylation levels were performed to verify the pathogenicity.The statistical method was described by mean ± standard deviation(SD),Two independent samples t-test,Pearson correlation and ROC curve,P<0.05 was statistically significant,and statistical software was SPSS16.0Results1.FSHD1 patients showed significant hypomethylation compared with controls in 4q35-D4Z4/PAS region,the data of 4q A/B patients were more sensitive than 4q A/A patients(P < 0.05);Based on the correlation analysis of onset age,clinical score and phenotype classification,the methylation was negatively related to the clinical severity.The specificity and stability of hypomethylation of FSHD patients were confirmed by comparison with other myopathies patients,and DNA from different tissues,peripheral blood DNA extracted from different periods.The optimal threshold value(Cp G/6 = 81.62%,Mean = 52.64%)was calculated by ROC curve.2.A typical FSHD2 family,the PFGE electrophoresis revealed a 41.5-kb fragment.A mutation of SMCHD1(c.1 A > G)on initiation codon position was founded,confirmed the pathogenicity through the family separation,bioinformatics prediction,furtherly validated in transcription,protein and DNA methylation level.Conclusion1.FSHD1 patients showed obvious low methylation in PAS region,negatively related to clinical severity,with high specificity and stability,the ROC curve calculating optimal threshold between patients and controls,make the methylation detection is expected to become a new diagnosis index,which can help improve the molecular diagnosis of FSHD and evaluate the severity of phenotype.2.FSHD2 family was first found in the Chinese population,and the family emphasized three important factors of FSHD2: pathogenicity SMCHD1 mutation,4q A allele and D4Z4 low methylation.