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Case Reports
. 2016 Sep 15;17(1):66.
doi: 10.1186/s12881-016-0328-9.

Segregation between SMCHD1 mutation, D4Z4 hypomethylation and Facio-Scapulo-Humeral Dystrophy: a case report

Marie-Cécile Gaillard  1 , Francesca Puppo  1 , Stéphane Roche  1 , Camille Dion  1 , Emmanuelle Salort Campana  1   2 , Virginie Mariot  3 , Charlene Chaix  4 , Catherine Vovan  4 , Killian Mazaleyrat  1 , Armand Tasmadjian  1 , Rafaelle Bernard  1   4 , Julie Dumonceaux  3 , Shahram Attarian  1   2 , Nicolas Lévy  1   4 , Karine Nguyen  1   4 , Frédérique Magdinier  5 , Marc Bartoli  1   4
Affiliations
Case Reports

Segregation between SMCHD1 mutation, D4Z4 hypomethylation and Facio-Scapulo-Humeral Dystrophy: a case report

Marie-Cécile Gaillard et al. BMC Med Genet. .

Abstract

Background: The main form of Facio-Scapulo-Humeral muscular Dystrophy is linked to copy number reduction of the 4q D4Z4 macrosatellite (FSHD1). In 5 % of cases, FSHD phenotype appears in the absence of D4Z4 reduction (FSHD2). In 70-80 % of these patients, variants of the SMCHD1 gene segregate with 4qA haplotypes and D4Z4 hypomethylation.

Case presentation: We report a family presenting with neuromuscular symptoms reminiscent of FSHD but without D4Z4 copy reduction. We characterized the 4q35 region using molecular combing, searched for mutation in the SMCHD1 gene and determined D4Z4 methylation level by sodium bisulfite sequencing. We further investigated the impact of the SMCHD1 mutation at the protein level and on the NMD-dependent degradation of transcript. In muscle, we observe moderate but significant reduction in D4Z4 methylation, not correlated with DUX4-fl expression. Exome sequencing revealed a heterozygous insertion of 7 bp in exon 37 of the SMCHD1 gene producing a loss of frame with premature stop codon 4 amino acids after the insertion (c.4614-4615insTATAATA). Both wild-type and mutated transcripts are detected.

Conclusion: The truncated protein is absent and the full-length protein level is similar in patients and controls indicating that in this family, FSHD is not associated with SMCHD1 haploinsufficiency.

Keywords: DNA combing; DNA methylation; DUX4; Facio-Scapulo-Humeral Dystrophy; Haploinsufficiency; SMCHD1.

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Figures

Fig. 1
Fig. 1
Clinical diagnosis and pedigree. a Pedigree of the family. For each individual year of birth is indicated together with the presence of SNP or mutation of the SMCHD1 gene and D4Z4 methylation level (%) at D4Z4 the DR1 (left) and 5’ (right) proximal regions. Individuals I1 and II1 carry the c.4614_4615insTATAATA heterozygous SMCHD1 mutation and display a low methylation levels compared to II2 and II3. b Presentation of a typical FSHD phenotype in the proband (II1) with characteristic asymmetrical scapulo humeral weakness and facial involvement
Fig. 2
Fig. 2
Molecular diagnosis by DNA Combing. Combed DNA from the different family members using specific probes and bar code for the 4q and 10q regions. The chromosome, haplotype and D4Z4 array size estimation (in kb) is indicated for each allele. The bar-code used to distinguish the three different alleles is based on a combination of three different colors and different DNA probes encompassing the distal regions up to the telomeric sequence [19]. The 3-color barcode comprises 2 probes detected in blue for the proximal region common to chromosomes 4 and 10, one 6 kb probe (red), which hybridizes the telomere, and a red probe that hybridizes the qA-specific β-satellite region, with a variable length (1–5 kb). The qB-specific probe, immediately adjacent to D4Z4, is detected in blue (Additional file 1: Figure S1)
Fig. 3
Fig. 3
DNA methylation analysis in peripheral blood and tissues. a Four regions within D4Z4 were amplified by PCR after sodium bisulfite treatment of genomic DNA. Amplicons were cloned and at least 10 individual clones were analyzed by Sanger sequencing. Each clone is representative of a molecule of DNA of the initial sample. The position of the four sets or primers used is indicated with black lines below schematic D4Z4. b Histogram bars represent the percentage of methylated (black) or unmethylated (white) CpG for each position in the DR1 (31 CpGs), 5’ (21 CpGs), Mid (31 CpGs), and 3’ (14 CpGs) regions in genomic DNA from PBMCs for each individual. c DNA methylation analysis in genomic DNA from a quadriceps muscle biopsy and primary fibroblasts from the II1 index case
Fig. 4
Fig. 4
DUX4 expression and characterization of the SMCHD1 mutation by RT-PCR and western blotting. a Expression of the DUX4 gene in total RNA from muscle (M) and primary fibroblasts (F) of the proband (II1). A positive control expressing DUX4-fl was used (+) and amplification was performed without reverse transcriptase (-). The ß2microglobulin gene was used as a standard of amplification. b Analysis of the SMCHD1 transcript on cDNA obtained from II1 PBMCs, muscle biopsy and primary fibroblasts. The wild-type and transcript carrying the (r.4614_4615 insTATAATA) insertion have been amplified using primers encompassing exons 36-38. PBMCs, muscle or primary fibroblasts from healthy individual (CT) were used as controls. The XNP gene was used as a positive control. c SMCHD1 western blot on whole cell extracts from PBMCs and fibroblasts from the index case (II1) compared to control cells from healthy donors (CT5 and CT7 PBMC; CT1, CT2 and CT3 primary fibroblasts) with antibodies against either the N- or C-terminal epitope. The lamin B2 protein was used as loading reference. d Primary fibroblasts were treated for 2 h with a final concentration of 50 μM NMDi14 or mock treated with DMSO. SMCHD1 transcripts were amplified by RT-QPCR in the different conditions. The ATF3 gene was used as a positive control [25]. Samples were amplified in triplicates
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