Relationship Between DR1 Methylation, SMCHD1 Mutationand Clinical Variety Of Facioscapulohumeral Muscular Dystrophy

Background and Objectives Facioscapulohumeral muscular dystrophy is a common myopathy inherited in autosomal dominant pattern, clinically characterized by progressive weakness of facial, shoulder girdle, upper extremities, descending to abdominal, pelvic girdle and lower extremities. FSHD patients generally become symptomatic in the second decade. FSHD is highly variable in progression and severity with 20% affected individuals becoming wheelchair dependent while an equal proportion of gene carriers remain asymptomatic throughout their lives. Life expectancy is barely shortened in FSHD patients. FSHD could be classified as FSHD1 and FSHD2 according to different genetic defect. FSHD1 and FSHD2 are clinically indistinguishable, but both associated with chromatin relaxation of the D4Z4 repeat array on chromosome 4 and transcriptional de-repression of the D4Z4-encoded DUX4 gene in skeletal muscle. The common form, FSHD1（>95%） is caused by contraction of D4Z4 repeat array on 4q35 to 1-10 units（11-150 in healthy individuals）. The less common form, termed as FSHD2, is often caused by heterozygous mutations in the structural maintenance of chromosomes flexible hinge domain containing 1（SMCHD1） gene. Both situations lead to a partial opening of the D4Z4 chromatin structure, and transcription of D4Z4-encoded polyadenylated DUX4 m RNA in muscle. DUX4 is normally expressed in testis and repressed in somatic tissue, and its inappropriate expression in muscle will introduce cell apoptosis. SMCHD1 is a member of the SMC gene superfamily, a family of proteins that is involved in chromosome condensation and cohesion, genome maintenance and gene regulation. SMCHD1 has a critical role in X inactivation and epigenetic gene silencing through Cp G methylation. SMCHD1 could act as a chromatin modifier by binding to D4Z4, blocking the expression of DUX4. Haploinsufficiency of SMCHD1 may be a cause of D4Z4 hypomethylation in FSHD2. SMCHD1 mutation could solely lead to FSHD2; moreover, it’s a modifier of disease severity in families affected by FSHD1. The study is aim at discussing the relationship between SMCHD1 genotype and clinical variety of facioscapulohumeral muscular dystrophy in Chinese population by using the Sanger sequencing and pyrosequencing technique.Methods To investigate the link between FSHD clinical variety and SMCHD1 genotype, we explored 36 cases（32 FSHD1, 4 FSHD1+FSHD2） by two techniques. We use Age-corrected Clinical Severity Score（ACSS） to evaluate patients’ phenotype. 1. Screening the SMCHD1 gene coding sequence and flanking sequence by using Sanger sequencing. 2. Detect the methylation ratio of DR1 region at 5’ of DUX4 ORF. Grouping: Cases are classified as control, FSHD1 and FSHD1+2 according to SMCHD1 genotype.Results 1. Detect two SMCHD1 gene mutation, NM₀15295.2:c.5308C>T and NM₀15295.2: c.1040+1G>A. Both are predicted to be pathological mutation according to several bioinformatics analyses. NM₀15295.2: c.1040+1G>A has been reported. We did not find NM₀15295.2:c.5308C>T in 300 healthy controls. 2. The average DR1 region methylation level in control, FSHD1 group and FSHD2 group are 52.42±8.105（%）, 43.36±7.687（%） and 22.12±8.405（%） respectively. FSHD1 group methylation level is lower than control（p=0.000480）, FSHD1+2 group methylation level is lower than FSHD1 group（p=0.000014） and control（p=0.000000）. 3. Age-corrected clinical severity score in FSHD1 group and FSHD1+2 group are 200.00（37.04-500.00） and 241.69（75.00-375.00） respectively. Age-corrected clinical severity score showed no significant between FSHD1 group and FSHD1+2 group.Conclusion 1. Detect two SMCHD1 gene mutation, NM₀15295.2:c.5308C>T and NM₀15295.2: c.1040+1G>A, the variant NM₀15295.2:c.5308C>T is not yet reported. 2. D4Z4 repeat contraction leads to hypomethylation in DR1 region, and methylation level in FSHD1 patients is lower than asymptomatic carriers, supporting that epigenetically modification is important in FSHD mechanism. 3. SMCHD1 mutation causes more demethylation in D4Z4 unit but didn’t directly modify the clinical sign. FSHD1 and FSHD1+2 show no difference. 4. Detection of methylation level could help the diagnosis of FSHD.