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

International audienceBackground: 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

Tricho-Hepato-Enteric Syndromemutation update: Mutations spectrum of TTC37 and SKIV2L, clinical analysis and future prospects

International audienceTricho-Hepato-Enteric syndrome (THES) is a very rare autosomal recessive syndromic enteropathy caused by mutations of either TTC37 or SKIV2L genes. Very little is known of these two gene products in mammals nor of the pathophysiology of the disease. Since the identification of the genes, we have set up the molecular diagnostic of THES in routine, gathering a large cohort with clinical and molecular data. Here, we report the phenotype and genotype analysis of this cohort together with an extensive literature review of THES cases worldwide, that is, 96 individuals harboring mutations in one gene or the other. We set up locus-specific databases for both genes and reviewed the type of mutation aswell as their localization in the proteins. No hot spot is evidenced for any type of mutation. The phenotypic analysis was first made on the whole cohort but is limited due to heterogeneity in clinical descriptions. We then examined the lab diagnostic cohort in detail for clinical manifestations. For the first time, we are able to suggest that patients lacking SKIV2L seem more severely affected than those lacking TTC37, in terms of liver damage and prenatal growth impairment

A new mutation in the C-terminal end of TTC37 leading to a mild form of syndromic diarrhea/tricho-hepato-enteric syndrome in seven patients from two families

International audienceSyndromic diarrhea/tricho-hepato-enteric syndrome (SD/THE) is a rare congenital enteropathy with seven main clinical features: intractable diarrhea of infancy, hair abnormalities, intrauterine growth restriction (IUGR), facial dysmorphism, immune dysfunction, and liver and skin abnormalities. SD/THE is caused by mutations in TTC37 or SKIV2L, two genes encoding components of the human SKI complex. To date, approximately 50 SD/THE patients have been described with a wide spectrum of mutations, and only one recurrent mutation has been identified in independent families. We present a detailed description of seven patients of Turkish origin with the same new mutation in TTC37: c.4572 G>A p.(Trp1524X). All seven patients were homozygous for this mutation and presented the typical clinical features of SD/THE, but with a milder presentation than usual. All seven patients were alive at the last follow-up. Four out of seven patients had no IUGR, and four patients never required parenteral nutrition. All patients presented a better growth rate than previously described in patients with SD/THE, with 4/7 above the 3rd percentile. The mutation is localized only forty amino acids from the end of TTC37, and as TTC37 is longer than the yeast SKI3, it is possible that a truncated protein is expressed and plays a reduced role in the SKI complex

Complex 4q35 and 10q26 Rearrangements

International audienceBackground and Objectives After clinical evaluation, the molecular diagnosis of type 1 facioscapulohumeral dystrophy (FSHD1) relies in most laboratories on the detection of a shortened D4Z4 array at the 4q35 locus by Southern blotting. In many instances, this molecular diagnosis remains inconclusive and requires additional experiments to determine the number of D4Z4 units or identify somatic mosaicism, 4q-10q translocations, and proximal p13E-11 deletions. These limitations highlight the need for alternative methodologies, illustrated by the recent emergence of novel technologies such as molecular combing (MC), single molecule optical mapping (SMOM), or Oxford Nanopore-based long-read sequencing providing a more comprehensive analysis of 4q and 10q loci. Over the last decade, MC revealed a further increasing complexity in the organization of the 4q and 10q distal regions in patients with FSHD with cis -duplication of D4Z4 arrays in approximately 1%–2% of cases. Methods By using MC, we investigated in our center 2,363 cases for molecular diagnosis of FSHD. We also evaluated whether previously reported cis -duplications might be identified by SMOM using the Bionano EnFocus FSHD 1.0 algorithm. Results In our cohort of 2,363 samples, we identified 147 individuals carrying an atypical organization of the 4q35 or 10q26 loci. Mosaicism is the most frequent category followed by cis -duplications of the D4Z4 array. We report here chromosomal abnormalities of the 4q35 or 10q26 loci in 54 patients clinically described as FSHD, which are not present in the healthy population. In one-third of the 54 patients, these rearrangements are the only genetic defect suggesting that they might be causative of the disease. By analyzing DNA samples from 3 patients carrying a complex rearrangement of the 4q35 region, we further showed that the SMOM direct assembly of the 4q and 10q alleles failed to reveal these abnormalities and lead to negative results for FSHD molecular diagnosis. Discussion This work further highlights the complexity of the 4q and 10q subtelomeric regions and the need of in-depth analyses in a significant number of cases. This work also highlights the complexity of the 4q35 region and interpretation issues with consequences on the molecular diagnosis of patients or genetic counseling

L’apport du peignage moléculaire pour révéler la variabilité génétique et la complexité du diagnostic moléculaire dans la dystrophie Facio-Scapulo Humérale.

International audienceLa dystrophie Facio Scapulo Humérale (FSHD) est la troisième dystrophie neuromusculaire par ordre de fréquence. Cette maladie autosomale dominante est liée au locus subtélomérique 4q35 et est associée à différents variants génétiques de régions non codantes. Dans 95% des cas, une réduction du nombre d’unités du macrosatellite D4Z4 sur un allèle de type qA a été associé à la FSHD. La perte de ces éléments d’ADN répété est associée à une baisse de la méthylation de l’ADN. Pour 5% des patients, la réduction du nombre de D4Z4 n’est pas observée (FSHD2). Une large proportion de patients FSHD2 présente cependant une baisse de la méthylation de D4Z4 associée dans certains cas à une mutation du gène SMCHD1.Dans la plupart des laboratoires, le diagnostic moléculaire est réalisé par la technique de Southern blot qui permet de déterminer le nombre d’unités répétées D4Z4. Toutefois, cette technique ne permet pas toujours de conclure, soit en raison de limitations techniques soit raison de l’existence de variants complexes. Afin de contourner cette difficulté, nous avons développé une approche diagnostique alternative basée sur la technique de peignage moléculaire (Nguyen et al., 2010). Grâce à cette méthodologie hautement résolutive, nous avons exploré 895 individus pour diagnostic de confirmation ou d’exclusion de la FSH. Dans 9% des cas, nous avons identifié l’existence de nouveaux remaniements complexes de la région 4q35 liée à la maladie ou de la région 10q26 homologue à 98%. Chez ces patients, nous avons systématiquement analysé les variants génomiques associés par la FSHD (allèles subtélomériques qA ou qB), la méthylation de l’ADN de D4Z4 et recherché des mutations du gène SMCHD1. Nos résultats qu’il n’y a pas de corrélation systématique entre variation de SMCHD1, hypométhylation de D4Z4 et présence des signes cliniques de la maladie et chez certains patients, ces remaniements complexes sont les seules anomalies associées à la pathologie. L’ensemble de ces résultats soulève donc de nombreuses questions sur le diagnostic moléculaire et le conseil génétique en particulier dans le cadre de la FSHD2

Inflammatory facioscapulohumeral muscular dystrophy type 2 in 18p deletion syndrome

International audienceFacioscapulohumeral muscular dystrophy (FSHD) has been shown to be related to genetic and epigenetic derepression of DUX4 (mapping to chromosome 4), a gene located within a repeat array of D4Z4 sequences of polymorphic length. FSHD type 1 (FSHD1) is associated with pathogenic D4Z4 repeat array contraction, while FSHD type 2 (FSHD2) is associated with SMCHD1 variants (a chromatin modifier gene that maps to the short arm of chromosome 18). Both FSHD types require permissive polyadenylation signal (4qA) downstream of the D4Z4 array

Segregation between a frameshift SMCHD1 mutation, D4Z4 hypomethylation and Facio-Scapulo-Humeral Dystrophy

International audienceFacio-­Scapulo-­Humeral muscular Dystrophy (FSHD) is linked to a copy number reduction (n<10) of the 4q D4Z4 subtelomeric macrosatellite, in association with a DUX4-­permissive haplotype. This main form of the disease is indicated as FSHD1. FSHD‐like phenotypes may also appear, in 5% of cases, in the absence of D4Z4 copy number reduction (FSHD2). In 70‐80% of these FSHD2 patients, variants of the SMCHD1 gene have been reported to segregate with DUX4 -­compatible haplotypes and associate with D4Z4 hypomethylation. Here, we describe a family presenting neuromuscular symptoms reminiscent of FSHD but without D4Z4 copy number reduction. Bisulfite sequencing showed significant hypomethylation in a proximal region within D4Z4 in symptomatic cases of the family. Exome sequencing revealed a heterozygous insertion of 7 bp in exon 37 of the SMCHD1 gene (c.4614_4615 insTATAATA) segregating with clinical signs and producing a frameshift in the protein with a premature stop codon 4 amino acids after the insertion (p.A1539Yfs*4),potentially leading to a truncated protein with a putative dominant negative effect. At the transcript level both wild‐type and mutated alleles were detected although the mutated allele was weakly expressed in all patients’ tissues analyzed. In western blot full length protein was expressed at the same level than controls and truncated protein was not detected, excluding aploinsufficiency as pathological mechanism. Ongoing analysis of nonsense mediated degradation of the mutated transcript and quantification of relative allele expression will allow elucidating the cause of truncated protein bsence. By this work, we aim to sheds light on SMCHD1 mutated protein complex contribution to FSHD pathogenesis

Molecular combing reveals complex 4q35 rearrangements in Facioscapulohumeral dystrophy

International audienceFacioscapulohumeral dystrophy (FSHD), one of the most common hereditary neuromuscular disorders, is associated with a complex combination of genetic variations at the subtelomeric 4q35 locus. As molecular diagnosis relying on Southern blot (SB) might be challenging in some cases, molecular combing (MC) was recently developed as an additional technique for FSHD diagnosis and exploration of the genomic organization of the 4q35 and 10q26 regions. In complement to the usual SB, we applied MC in a large cohort of 586 individuals with clinical FSHD. In 332 subjects, the two 4q alleles were normal in size, allowing exclusion of FSHD1 while we confirmed FSHD1 in 230 patients. In 14 patients from 10 families, we identified a recurrent complex heterozygous rearrangement at 4q35 consisting of a duplication of the D4Z4 array and a 4qA haplotype, irresolvable by the SB technique. In five families, we further identified variations in the SMCHD1 gene. Impact of the different mutations was tested using a minigene assay and we analyzed DNA methylation after sodium bisulfite modification and NGS sequencing. We discuss the involvement of this rearrangement in FSHD since all mutations in SMCHD1 are not associated with D4Z4 hypomethylation and do not always segregate with the disease

Methylation hotspots evidenced by deep sequencing in patients with facioscapulohumeral dystrophy and mosaicism

International audienceObjective To investigate the distribution of cytosine-guanine dinucleotide (CpG) sites with a variable level of DNA methylation of the D4Z4 macrosatellite element in patients with facioscapulo-humeral dystrophy (FSHD). Methods By adapting bisulfite modification to deep sequencing, we performed a comprehensive analysis of D4Z4 methylation across D4Z4 repeats and adjacent 4qA sequence in DNA from patients with FSHD1, FSHD2, or mosaicism and controls. Results Using hierarchical clustering, we identified clusters with different levels of methylation and separated, thereby the different groups of samples (controls, FSHD1, and FSHD2) based on their respective level of methylation. We further show that deep sequencing-based methylation analysis discriminates mosaic cases for which methylation changes have never been evaluated previously. Conclusions Altogether, our approach offers a new high throughput tool for estimation of the D4Z4 methylation level in the different subcategories of patients having FSHD. This methodology allows for a comprehensive and discriminative analysis of different regions along the macro-satellite repeat and identification of focal regions or CpG sites differentially methylated in patients with FSHD1 and FSHD2 but also complex cases such as those presenting mosaicism

Differential DNA methylation of the D4Z4 repeat in patients with FSHD and asymptomatic carriers

International audienceObjective: We investigated the link between DNA hypomethylation and clinical penetrance in facioscapulohumeral dystrophy (FSHD) because hypomethylation is moderate and heterogeneous in patients and could not thus far be correlated with disease presence or severity. Methods: To investigate the link between clinical signs of FSHD and DNA methylation, we explored 95 cases (37 FSHD1, 29 asymptomatic individuals carrying a shortened D4Z4 array, 9 patients with FSHD2, and 20 controls) by implementing 2 approaches: methylated DNA immunoprecipitation and sodium bisulfite sequencing. Results: Both methods revealed statistically significant differences between asymptomatic carriers or controls and individuals with clinical FSHD, especially in the proximal region of the repeat. Absence of clinical expression in asymptomatic carriers is associated with a level of methylation similar to controls. Conclusions: We provide a proof of concept that the targeted approaches that we describe could be applied systematically to patient samples in routine diagnosis and suggest that local hypomethylation within D4Z4 might serve as a modifier for clinical expression of FSHD phenotype. Classification of evidence: This study provides Class III evidence that assays for hypomethylation within the D4Z4 region accurately distinguish patients with FSHD from individuals with D4Z4 contraction without FSHD