Diagnostic approach for FSHD revisited: SMCHD1 mutations cause FSHD2 and act as modifiers of disease severity in FSHD1

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder with a wide clinical variability. Contractions of the D4Z4 macrosatellite repeat on chromosome 4q35 are the molecular basis of the pathophysiology. Recently, in a subset of patients without D4Z4 repeat contractions, variants in the SMCHD1 gene have been identified that lead to hypomethylation of D4Z4 and thus DUX4 transcription, which causes FSHD type 2. In this study, we have screened 55 FSHD1-negative and 40 FSHD1-positive patients from unrelated families for potentially pathogenic variants in SMCHD1 by nextgeneration sequencing (NGS). We identified variants in SMCHD1 in 11 index patients, including missense, splice site and nonsense mutations. We developed a pyrosequencing assay to determine the methylation status of the D4Z4 repeat array and found significantly lower methylation levels for FSHD2 patients than for healthy controls and FSHD1 patients. Two out of eleven SMCHD1 mutation carriers had moderately contracted D4Z4 alleles thus these patients are suffering from FSHD1 and 2. Comparing the phenotype of patients, all FSHD2 patients were relatively mildly affected while patients with FSHD1+2 were much more severely affected than expected from their D4Z4 copy number. Our findings confirm the role of SMCHD1 mutations in FSHD2 and as a modifier of disease severity. With SMCHD1 variants found in 16.4% of phenotypic FSHD patients without D4Z4 repeat contractions, the incidence of FSHD2 is rather high and hence we suggest including sequencing of SMCHD1, haplotyping and methylation analysis in the workflow of molecular FSHD diagnostics.

Allele-specific DNA hypomethylation characterises FSHD1 and FSHD2

“…However, FseI efficiently identifies FSHD2 individuals, which are clustered in the lower part of the graph with a methylation level<25% 14 26 28. Therefore, the combination of PAS-specific methylation test and other D4Z4 methylation assays (eg, FseI ) may provide the identification of all three classes of individuals 15 17–19 21…”

Leg Muscle Involvement in Facioscapulohumeral Muscular Dystrophy: Comparison between Facioscapulohumeral Muscular Dystrophy Types 1 and 2

“…In our clinic, 7 FSHD2 patients are registered of which 5 individuals from 3 families had been assessed by MRI. The genetic characteristics of our FSHD2 patients have been described previously [5] . In short, they all had a mutation in the SMCHD1 gene and severe hypomethylation of the D4Z4 fragment.…”

Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2

“…Deletions in the 5′ splice site of exon 25 were previously reported in eight other FSHD2 families suggesting it to be a mutation hotspot. 21,24 Previous RNA analysis of an independent FSHD2 family with a c.3276_3276 +1del variant showed that this variant results in both cryptic splicing and in complete skipping of exon 25, both with retention of the ORF. 21 The same splice effect is expected for the c.3276_3276+4del variant in individual 1414-201.…”

“…Splice donor site variants in exon 25 have already been reported in eight other FSHD2 families to segregate with D4Z4 hypomethylation and disease presentation, confirming the functional consequences of this variant. 21,24 In individual 385-203, two variants on different alleles were detected. Both variants are predicted to affect function by SIFT, MutationTaster and Align GVGD.…”

“…Heterozygous diseasecausing SMCHD1 variants are distributed over the entire SMCHD1 locus and include splice site, insertion-deletion, missense and nonsense variants. 14,[20][21][22][23][24] We have shown that a combination of the size of the permissive D4Z4 array and the type of SMCHD1 variant together determine the epigenetic susceptibility to disease presentation. 21 Until now, only heterozygous SMCHD1 variants, which are dominant in combination with a permissive haplotype, have been reported.…”