Claude Desnuelle

Digenic inheritance of an SMCHD1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2

Facioscapulohumeral dystrophy (FSHD) is characterized by chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4 and expression of the D4Z4-encoded DUX4 gene in skeletal muscle. The more common form, autosomal dominant FSHD1, is caused by contraction of the D4Z4 array, whereas the genetic determinants and inheritance of D4Z4 array contraction–independent FSHD2 are unclear. Here, we show that mutations in SMCHD1 (encoding structural maintenance of chromosomes flexible hinge domain containing 1) on chromosome 18 reduce SMCHD1 protein levels and segregate with genome-wide D4Z4 CpG hypomethylation in human kindreds. FSHD2 occurs in individuals who inherited both the SMCHD1 mutation and a normal-sized D4Z4 array on a chromosome 4 haplotype permissive for DUX4 expression. Reducing SMCHD1 levels in skeletal muscle results in D4Z4 contraction–independent DUX4 expression. Our study identifies SMCHD1 as an epigenetic modifier of the D4Z4 metastable epiallele and as a causal genetic determinant of FSHD2 and possibly other human diseases subject to epigenetic regulation.

Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.

A double-blind, placebo-controlled randomized clinical trial of α-tocopherol (vitamin E) in the treatment of amyotrophic lateral sclerosis

INTRODUCTION: Increasing evidence suggests that oxidative stress may be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). The antioxidant vitamin E ( f -tocopherol) has been shown to slow down the onset and progression of paralysis in transgenic mice expressing a mutation in superoxide dismutase found in certain forms of familial ALS. The current study was designed to determine whether a-tocopherol (500 mg b.i.d.) may be efficacious in the treatment of ALS. METHODS: Two hundred and eighty-nine patients with ALS of less than 5 years duration, treated with riluzole, were enrolled in this study, and were randomly assigned to receive either a-tocopherol or placebo daily for one year. The primary outcome measure was the rate of deterioration of function assessed by the modified Norris limb scale. Patients were assessed at entry, and every 3 months thereafter during the study period. Survival was also recorded. Biochemical markers of oxidative stress were measured in a subset of patients on entry and after 3 months of treatment. RESULTS: After 12 months of treatment, a-tocopherol had no effect on the primary outcome measure. Survival was not influenced by treatment. Among secondary outcome measures, patients given a-tocopherol were less likely to progress from the milder state A to the more severe state B ( P = 0.046) of the ALS Health State scale. After 3 months treatment, analysis of oxidative stress markers showed an increase in glutathione peroxidase activity in plasma ( P = 0.0389) and a decrease in plasma levels of thiobarbituric acid reactive species ( P = 0.0055) in the group of patients given f -tocopherol in combination with riluzole. CONCLUSION: Although f -tocopherol did not appear to affect the survival and motor function in ALS, patients receiving riluzole plus f -tocopherol remained longer in the milder states of the ALS Health State scale and showed, after 3 months, changes in biochemical markers of oxidative stress. Further studies are required to confirm the greater sensitivity of the ALS Health State scale over other clinical endpoints. (ALS 2001; 2: 9-18)

Clinical features of facioscapulohumeral muscular dystrophy 2.

Objective: In some 5% of patients with facioscapulohumeral muscular dystrophy (FSHD), no D4Z4 repeat contraction on chromosome 4q35 is observed. Such patients, termed patients with FSHD2, show loss of DNA methylation and heterochromatin markers at the D4Z4 repeat that are similar to patients with D4Z4 contractions (FSHD1). This commonality suggests that a change in D4Z4 chromatin structure unifies FSHD1 and FSHD2. The aim of our study was to critically evaluate the clinical features in patients with FSHD2 in order to establish whether these patients are phenotypically identical to FSHD1 and to establish the effects of the (epi-) genotype on the phenotype. Methods: This cross-sectional study studied 33 patients with FSHD2 from 27 families, the largest cohort described to date. All patients were clinically assessed using a standardized clinical evaluation form. Genotype analysis was performed by pulsed field gel electrophoresis and PCR; D4Z4 methylation was studied by methylation-sensitive Southern blot analysis. Results: FSHD2 is identical to FSHD1 in its clinical presentation. Notable differences include a higher incidence (67%) of sporadic cases and the absence of gender differences in disease severity in FSHD2. Overall, average disease severity in FSHD2 was similar to that reported in FSHD1 and was not influenced by D4Z4 repeat size. In FSHD2, a small effect of the degree of hypomethylation on disease severity was observed. Conclusions: Clinically, patients with FSHD2 are indistinguishable from patients with FSHD1. The present data suggest that FSHD1 and FSHD2 are the result of the same pathophysiologic process.

The D4Z4 Macrosatellite Repeat Acts as a CTCF and A-Type Lamins-Dependent Insulator in Facio-Scapulo-Humeral Dystrophy

Both genetic and epigenetic alterations contribute to Facio-Scapulo-Humeral Dystrophy (FSHD), which is linked to the shortening of the array of D4Z4 repeats at the 4q35 locus. The consequence of this rearrangement remains enigmatic, but deletion of this 3.3-kb macrosatellite element might affect the expression of the FSHD-associated gene(s) through position effect mechanisms. We investigated this hypothesis by creating a large collection of constructs carrying 1 to >11 D4Z4 repeats integrated into the human genome, either at random sites or proximal to a telomere, mimicking thereby the organization of the 4q35 locus. We show that D4Z4 acts as an insulator that interferes with enhancer–promoter communication and protects transgenes from position effect. This last property depends on both CTCF and A-type Lamins. We further demonstrate that both anti-silencing activity of D4Z4 and CTCF binding are lost upon multimerization of the repeat in cells from FSHD patients compared to control myoblasts from healthy individuals, suggesting that FSHD corresponds to a gain-of-function of CTCF at the residual D4Z4 repeats. We propose that contraction of the D4Z4 array contributes to FSHD physio-pathology by acting as a CTCF-dependent insulator in patients.

The FSHD2 Gene SMCHD1 Is a Modifier of Disease Severity in Families Affected by FSHD1

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4 to a size of 1-10 units. The residual number of D4Z4 units inversely correlates with clinical severity, but significant clinical variability exists. Each unit contains a copy of the DUX4 retrogene. Repeat contractions are associated with changes in D4Z4 chromatin structure that increase the likelihood of DUX4 expression in skeletal muscle, but only when the repeat resides in a genetic background that contains a DUX4 polyadenylation signal. Mutations in the structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier of D4Z4, also result in the increased likelihood of DUX4 expression in individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may act as a genetic modifier in FSHD1. We describe three unrelated individuals with FSHD1 presenting an unusual high clinical severity based on their upper-sized FSHD1 repeat array of nine units. Each of these individuals also carries a mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4 expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which the FSHD2 gene can act as modifier for disease severity in families affected by FSHD1.

Causes of death amongst French patients with amyotrophic lateral sclerosis: a prospective study

Background and purpose:  To prospectively investigate causes of death and the circumstances surrounding death in 302 patients with amyotrophic lateral sclerosis (ALS). The functional status of patients immediately before death was also determined.

Can malondialdehyde be used as a biological marker of progression in neurodegenerative disease

Abstract There is a large body of evidence that free radical-mediated oxidative damage is involved in the pathogenesis of neurodegenerative disease. Although it is unlikely that markers of such damage will have any diagnostic value, they might be of considerable interest in following disease progression and monitoring the efficacy of different treatments. Among such markers, there is evidence for the elevation of peripheral malondialdehyde levels in several neurodegenerative diseases, including Parkinsons disease, amyotrophic lateral sclerosis and Alzheimers disease. The measurement of malondialdehyde levels, which is both simple and cheap to perform, can and should be incorporated into future clinical trials. This will allow a clearer picture to emerge as to whether malondialdehyde can be considered as a marker for the evolution of these diseases.

A functionally dominant mitochondrial DNA mutation

Mutations in mitochondrial DNA (mtDNA) tRNA genes can be considered functionally recessive because they result in a clinical or biochemical phenotype only when the percentage of mutant molecules exceeds a critical threshold value, in the range of 70-90%. We report a novel mtDNA mutation that contradicts this rule, since it caused a severe multisystem disorder and respiratory chain (RC) deficiency even at low levels of heteroplasmy. We studied a 13-year-old boy with clinical, radiological and biochemical evidence of a mitochondrial disorder. We detected a novel heteroplasmic C>T mutation at nucleotide 5545 of mtDNA, which was present at unusually low levels (<25%) in affected tissues. The pathogenic threshold for the mutation in cybrids was between 4 and 8%, implying a dominant mechanism of action. The mutation affects the central base of the anticodon triplet of tRNA(Trp) and it may alter the codon specificity of the affected tRNA. These findings introduce the concept of dominance in mitochondrial genetics and pose new diagnostic challenges, because such mutations may easily escape detection. Moreover, similar mutations arising stochastically and accumulating in a minority of mtDNA molecules during the aging process may severely impair RC function in cells.

Null mutations causing depletion of the type 1 ryanodine receptor (RYR1) are commonly associated with recessive structural congenital myopathies with cores

Mutations of the ryanodine receptor cause dominant and recessive forms of congenital myopathies with cores. Quantitative defects of RYR1 have been reported in families presenting with recessive forms of the disease and epigenic regulation has been recently proposed to explain potential maternal monoallelic silencing of the RYR1 gene. We investigated nine families presenting with a recessive form of the disease and showing a quantitative defect of RYR1 expression. Genetic analysis allowed the identification of a mutation on both alleles of the RYR1 gene for all patients, 15 being novel variants. We evidenced for all patients an alteration of the expression of the RYR1 gene caused by amorphic mutations responsible either for mRNA or protein instability. In seven families the variant present on the second allele was a missense mutation. In the remaining two families the second variant led to a hypomorphic expression of the RYR1 gene and was associated with a severe neonatal phenotype, pointing out the minimal amount of RYR1 needed for skeletal muscle function. Noticeably, a novel additional exon 3b was characterized in the most severely affected cases. This study showed that all cases presenting with a quantitative defect of RYR1 expression in our panel of patients affected by recessive core myopathies were caused by the presence of one recessive null allele and that variability of the phenotype depended on the nature of the mutation present on the second allele. Our study also indicated that presence of a second mutation must be investigated in sporadic cases or in dominant cases presenting with a familial clinical variability. Hum Mutat 29(5), 670–678, 2008.