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. 2015 Jun;23(6):808-16.
doi: 10.1038/ejhg.2014.191. Epub 2014 Nov 5.

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

Mirjam Larsen  1 , Simone Rost  1 , Nady El Hajj  1 , Andreas Ferbert  2 , Marcus Deschauer  3 , Maggie C Walter  4 , Benedikt Schoser  4 , Pawel Tacik  5 , Wolfram Kress  1 , Clemens R Müller  1
Affiliations

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

Mirjam Larsen et al. Eur J Hum Genet. 2015 Jun.

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 next-generation sequencing (NGS). We identified variants in SMCHD1 in 11 index patients, including missense, splice site and non-sense 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.

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Figures

Figure 1
Figure 1
Presentation of a typical FSHD2 patient. (a) Muscular weakness of the shoulder girdle with scapulae alatae in an FSHD2 patient. (b) Muscle histology (M. gastrocnemius, HE stain) of an FSHD2 patient with inflammatory infiltration of non-necrotic muscle fibers, mildly increased endomysial connective tissue and internal nuclei.
Figure 2
Figure 2
Methylation analysis of the D4Z4 locus. (a) Genomic arrangement of the D4Z4 locus. D4Z4 repeat units are depicted as triangles. The binding site of the Southern blot probe p13E11, the region of extreme demethylation (DR1) and the open reading frame (ORF) of DUX4 are depicted as boxes. Restriction enzyme cutting sites are marked with arrows. By the pyrosequencing assay, nine CpGs within the DR1 region on every repeat unit are analyzed (reference sequence shown, analyzed CpGs underlined). The test therefore represents mainly the methylation status of internal repeats. In the methylation-sensitive Southern blot, genomic DNA is digested with restriction enzymes EcoRI (cutting site outside the shown region), BglII and the methylation-sensitive enzyme FseI. Hybridization is performed with the probe p13E11. This test shows the methylation level of the most proximal repeat unit only. Both tests cannot distinguish between chromosomes 4 and 10. (b) Boxplot diagram of methylation levels determined by pyrosequencing. The boxplot is created using the mean methylation level of nine CpGs for each individual sample (sample size: 48 healthy controls, 49 FSHD-like (FSHD phenotype, no contraction in D4Z4, no pathogenic variant in SMCHD1), 37 FSHD1, 13 FSHD2). Outliers are depicted as ‘○'. **P<0.01, ***P<0.001. (c) Methylation-sensitive Southern blot. Methylated (M) and unmethylated (UM) D4Z4 fragments are indicated on the right. For each index patient and available family members methylation data (%) are given as estimated by Southern blotting (SB) and pyrosequencing (Pyro), respectively.
Figure 3
Figure 3
Genetic characterization of four families with FSHD2. (a) Pedigrees with FSHD2. Affected individuals are depicted in black, index patients are marked with an arrow. For each individual, the following information is given if available: year of birth, variant in SMCHD1 (protein position based on NP_056110.2) and methylation level of D4Z4 in percent as determined by pyrosequencing. For family FSHD2-89, the number of repeat units on a contracted 4qA allele is given. All individuals with no indicated repeat number showed ≥11 repeat units. (b) Sequencing traces of the SMCHD1 variants from families shown above, generated by the software GenSearchNGS. The diagram shows reference and consensus sequence of protein and nucleotide, respectively (indicated as ‘AS seq.', ‘transcript' and ‘nucleotide seq.' on the right), exon/intron structure of the selected transcript (NM_015295.2) as well as base exchanges or deletions found in the displayed reads (marked by vertical red lines). The inset ‘base info' gives data on frequency and balance of the viewed base (‘A C T G' bases, ‘-' deletion, ‘N' base not defined, ‘I' insertion) and the coverage at the genomic position (hg19). In addition to the heterozygous variant, patient FSHD2-38 shows a homozygous SNP (c.1851A>G, rs635132) at g.2705700.
Figure 4
Figure 4
Mutations in SMCHD1. All mutations in the gene SMCHD1 known to date are compiled. Exons are indicated with boxes (5′ splice site I +0, >+1, <+2. 3' splice site I +0, >−1, <−2), the SMCHD1 conserved protein domains are marked with arrows. The listed mutations were published by 1Lemmers et al, 2Mitsuhashi et al, 3Sacconi et al and 4the present study. Mutations are spread all over the gene, not sparing any of the functional domains of the protein.
Figure 5
Figure 5
Diagnostic workflow for FSHD. Suggestion for a diagnostic workflow for patients with a typical FSHD phenotype. Up to 95% of FSHD cases can be explained by contractions in the D4Z4 repeat array, for the remaining cases FSHD2 should be investigated. The dotted lines indicate the suggested diagnostic workflow for FSHD1 patients with a severe phenotype investigating FSHD1 plus FSHD2. Special cases like p13E-11 deletions or hybrid alleles are not considered, but should always be kept in mind.
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