Clinical and Molecular Spectrum Associated with COL6A3 c.7447A>G p.(Lys2483Glu) Variant: Elucidating its Role in Collagen VI-related Myopathies.

BACKGROUND: Dominant and recessive autosomal pathogenic variants in the three major genes (COL6A1-A2-A3) encoding the extracellular matrix protein collagen VI underlie a group of myopathies ranging from early-onset severe conditions (Ullrich congenital muscular dystrophy) to milder forms maintaining independent ambulation (Bethlem myopathy). Diagnosis is based on the combination of clinical presentation, muscle MRI, muscle biopsy, analysis of collagen VI secretion, and COL6A1-A2-A3 genetic analysis, the interpretation of which can be challenging. OBJECTIVE: To refine the phenotypical spectrum associated with the frequent COL6A3 missense variant c.7447A>G (p.Lys2483Glu). METHODS: We report the clinical and molecular findings in 16 patients: 12 patients carrying this variant in compound heterozygosity with another COL6A3 variant, and four homozygous patients. RESULTS: Patients carrying this variant in compound heterozygosity with a truncating COL6A3 variant exhibit a phenotype consistent with COL6-related myopathies (COL6-RM), with joint contractures, proximal weakness and skin abnormalities. All remain ambulant in adulthood and only three have mild respiratory involvement. Most show typical muscle MRI findings. In five patients, reduced collagen VI secretion was observed in skin fibroblasts cultures. All tested parents were unaffected heterozygous carriers. Conversely, two out of four homozygous patients did not present with the classical COL6-RM clinical and imaging findings. Collagen VI immunolabelling on cultured fibroblasts revealed rather normal secretion in one and reduced secretion in another. Muscle biopsy from one homozygous patient showed myofibrillar disorganization and rimmed vacuoles. CONCLUSIONS: In light of our results, we postulate that the COL6A3 variant c.7447A>G may act as a modulator of the clinical phenotype. Thus, in patients with a typical COL6-RM phenotype, a second variant must be thoroughly searched for, while for patients with atypical phenotypes further investigations should be conducted to exclude alternative causes. This works expands the clinical and molecular spectrum of COLVI-related myopathies.

Effect of enzyme replacement therapy with alglucosidase alfa (Myozyme®) in 12 patients with advanced late-onset Pompe disease.

BACKGROUND: The efficacy of enzyme replacement therapy (ERT) in patients at an advanced stage of Pompe disease has only been addressed in a few studies. Our objective was to assess the long term effects of ERT in a cohort of patients with severe Pompe disease. METHODS: We identified patients from the French Pompe Registry with severe respiratory failure and permanent wheelchair use (assisted walk for a few meters was allowed) when starting ERT. Patients' medical records were collected and reviewed and respiratory and motor functions, before ERT initiation and upon last evaluation were compared. RESULTS: Twelve patients (7 males) were identified. Median age at symptom onset was 24years [IQR=15.5; 36.0]. At baseline ventilation was invasive in 11 patients and noninvasive in one, with a median ventilation time of 24h [IQR=21.88; 24.00] (min 20; max 24). ERT was initiated at a median age of 52.5years [IQR=35.75; 66.50]. Median treatment duration was 55months [IQR=39.5; 81.0]. During observational period no adverse reaction to ERT was recorded, five patients (41.67%) died, three decreased their ventilation time by 30, 60 and 90min and two increased their assisted walking distance, by 80 and 20m. CONCLUSION: Some patients at a very advanced stage of Pompe disease may show a mild benefit from ERT, in terms of increased time of autonomous ventilation and of enlarged distance in assisted walk. ERT can be initiated in these patients in order to retain their current level of independence and ability to perform daily life activities.

PNPLA2 mutation: a paediatric case with early onset but indolent course.

Neutral lipid storage disease (NLSD) due to PNPLA2 mutation is a rare disorder with a severe muscular and cardiac outcome. All but one reported cases have been diagnosed during adulthood. It is thus ordinarily distinguished from Chanarin-Dorfman syndrome, a paediatric NLSD with a more widespread symptomatology. We report the case of a young child incidentally diagnosed with significant and persistent hyperCKemia. At 3 years, muscle biopsy showed marked lipid storage. A homozygous mutation in PNPLA2 was found. Fourteen years later, the noticeable outcome is the absence of muscle weakness at rest, a normal muscular MRI, and no cardiac involvement. Yet the patient exhibits some systemic features, notably hearing loss. This paediatric case of NLSD with myopathy indicates that important lipid accumulation may occur very early in the absence of patent clinical and imaging muscle involvement. Furthermore, PNPLA2 mutations may be associated with multisystem features more frequently encountered in Chanarin-Dorfman syndrome.

Mutation spectrum in the large GTPase dynamin 2, and genotype-phenotype correlation in autosomal dominant centronuclear myopathy.

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT.

Rural environment and risk factors of amyotrophic lateral sclerosis: a case-control study.

The aetiology of sporadic ALS is still unknown. Links with several environmental factors have been suggested, and some epidemiological studies have shown an increased incidence of ALS in rural populations. This study was designed to investigate risk exposures in a well-delimited rural population and to assess whether rural residency or occupations, such as farming, were associated with an increased risk of developing ALS. A prospective case-control-study of 108 sporadic ALS cases matched by age and sex to 122 controls was performed in Brittany from 2006 to 2008. A strong association was found between agricultural activity and ALS (odds ratio: 2.919; p = 0.01), while rural residence itself did not influence the risk of the disease. Bulbar forms of onset prevailed among agricultural workers as compared with other occupations (55 vs. 26%; p = 0.009). These results suggest a potential role of exposure to agricultural chemicals or contact with animals linked to agricultural work. The prevalence of bulbar forms of onset in agricultural workers has not been reported before. In addition to variable methodological approaches, differences in agricultural practices could explain the discrepancy between these findings and other studies.

OPA1 mutations induce mitochondrial DNA instability and optic atrophy 'plus' phenotypes.

Mutations in OPA1, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA). We here report on eight patients from six independent families showing that mutations in the OPA1 gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPA1 protein in mtDNA stability. The five OPA1 mutations associated with these DOA 'plus' phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLG1, PEO1 (Twinkle) and SLC25A4 (ANT1) were ruled out. Our results show that certain OPA1 mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability.

Clinical, electrophysiological and molecular genetic studies in a family with X-linked dominant Charcot-Marie-Tooth neuropathy presenting a novel mutation in GJB1 Promoter and a rare polymorphism in LITAF/SIMPLE.

Charcot-Marie-Tooth disease is a genetically heterogeneous group of neuropathies. In the demyelinating form of Charcot-Marie-Tooth disease with dominant inheritance, five genes have been incriminated: PMP22, MPZ, LITAF/SIMPLE, EGR2 (CMT1A to D), and GJB1 (CMTX). Here, we report clinical, electrophysiological and molecular genetic studies in a family with a Charcot-Marie-Tooth disease variable phenotype, ranging from asymptomatic to moderately affected. The absence of male-to-male transmission as well as the results of systematic electrophysiological studies suggested a CMTX secondary to a GJB1 mutation. Screening for mutations in the coding regions of PMP22, MPZ, EGR2 and GJB1 was negative. We identified (1) a LITAF/SIMPLE substitution (T49M), absent in 1000 control chromosomes, but which was thought to be a polymorphism because of discrepancies of segregation when considering the results of electrophysiology; and (2) a novel substitution T>C in the P2 promoter of GJB1 at position -529, in the SOX10 binding site S2. The transmission of this second mutation was consistent with the electrophysiological data. We emphasise the role of electrophysiological studies that help to discriminate between asymptomatic subjects and that bring some additional valuable data to the genetic approach.

In vivo and in vitro functional characterization of Andersen's syndrome mutations.

The inward rectifier K(+) channel Kir2.1 carries all Andersen's syndrome mutations identified to date. Patients exhibit symptoms of periodic paralysis, cardiac dysrhythmia and multiple dysmorphic features. Here, we report the clinical manifestations found in three families with Andersen's syndrome. Molecular genetics analysis identified two novel missense mutations in the KCNJ2 gene leading to amino acid changes C154F and T309I of the Kir2.1 open reading frame. Patch clamp experiments showed that the two mutations produced a loss of channel function. When co-expressed with Kir2.1 wild-type (WT) channels, both mutations exerted a dominant-negative effect leading to a loss of the inward rectifying K(+) current. Confocal microscopy imaging in HEK293 cells is consistent with a co-assembly of the EGFP-fused mutant proteins with WT channels and proper traffick to the plasma membrane to produce silent channels alone or as hetero-tetramers with WT. Functional expression in C2C12 muscle cell line of newly as well as previously reported Andersen's syndrome mutations confirmed that these mutations act through a dominant-negative effect by altering channel gating or trafficking. Finally, in vivo electromyographic evaluation showed a decrease in muscle excitability in Andersen's syndrome patients. We hypothesize that Andersen's syndrome-associated mutations and hypokalaemic periodic paralysis-associated calcium channel mutations may lead to muscle membrane hypoexcitability via a common mechanism.