ClC-1 mutations in myotonia congenita patients: insights into molecular gating mechanisms and genotype-phenotype correlation.

KEY POINTS: Loss-of-function mutations of the skeletal muscle ClC-1 channel cause myotonia congenita with variable phenotypes. Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right-shifting the slow gating of ClC-1 channel, without exerting a dominant-negative effect on the wild-type (WT) subunit. Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H-bond formation between E232 in helix F and Y578 in helix R. Three other myotonic ClC-1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT-like behaviour (electrophysiology data only). Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function. ABSTRACT: Myotonia congenita is an inherited disease caused by loss-of-function mutations of the skeletal muscle ClC-1 chloride channel, characterized by impaired muscle relaxation after contraction and stiffness. In the present study, we provided an in-depth characterization of F484L, a mutation previously identified in dominant myotonia, in order to define the genotype-phenotype correlation, and to elucidate the contribution of this pore residue to the mechanisms of ClC-1 gating. Patch-clamp recordings showed that F484L reduced chloride currents at every tested potential and dramatically right-shifted the voltage dependence of slow gating, thus contributing to the mild clinical phenotype of affected heterozygote carriers. Unlike dominant mutations located at the dimer interface, no dominant-negative effect was observed when F484L mutant subunits were co-expressed with wild type. Molecular dynamics simulations further revealed that F484L affected the slow gate by increasing the frequency and stability of the H-bond formation between the pore residue E232 and the R helix residue Y578. In addition, using patch-clamp electrophysiology, we characterized three other myotonic ClC-1 mutations. We proved that the dominant L198P mutation in the channel pore also right-shifted the voltage dependence of slow gating, recapitulating mild myotonia. The recessive V640G mutant drastically reduced channel function, which probably accounts for myotonia. In contrast, the recessive L628P mutant produced currents very similar to wild type, suggesting that the occurrence of the compound truncating mutation (Q812X) or other muscle-specific mechanisms accounted for the severe symptoms observed in this family. Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.

Expression of transforming growth factor-beta 1 in dystrophic patient muscles correlates with fibrosis. Pathogenetic role of a fibrogenic cytokine.

Duchenne muscular dystrophy is a fatal disorder characterized by progressive muscular weakness, wasting, and severe muscle contractures in later disease stages. Muscle biopsy reveals conspicuous myofiber degeneration and fibrosis substituting muscle tissue. We quantitatively determined mRNA of the potent fibrogenic cytokine transforming growth factor-beta 1 by quantitative PCR in 15 Duchenne muscular dystrophy, 13 Becker muscular dystrophy, 11 spinal muscular atrophy patients, and 16 controls. Higher transforming growth factor-beta 1 expression was greater in Duchenne muscular dystrophy patients than controls (P = 0.012) and Becker patients (P = 0.03). Fibrosis was significantly more prominent in Duchenne muscular dystrophy than Becker muscular dystrophy, spinal muscular atrophy, and controls. The proportion of connective tissue in muscle biopsies increased progressively with age in Duchenne muscular dystrophy patients, while transforming growth factor-beta 1 levels peaked at 2 and 6 yr of age. Transforming growth factor-beta 1 protein was also detected by immunocytochemistry and immunoblotting. Our findings suggest that transforming growth factor-beta 1 stimulates fibrosis in Duchenne muscular dystrophy. Expression of transforming growth factor-beta 1 in the early stages of Duchenne muscular dystrophy may be critical in initiating muscle fibrosis and antifibrosis treatment could slow progression of the disease, increasing the utility of gene therapy.

Identification of three novel mutations in the major human skeletal muscle chloride channel gene (CLCN1), causing myotonia congenita.

Myotonia congenita (MC) is a genetic disease characterized by mutations in the CLCN1 gene (OMIM*118425) encoding the skeletal muscle voltage-gated chloride channel (ClC-1). Autosomal dominant and recessive forms are observed, characterized by impaired muscle relaxation after forceful contraction (myotonia), which is more pronounced after inactivity and improves with exercise. We report three novel and one known mutations of the CLCN1 gene in four unrelated MC families. In two families the mutations were missense: 803C>T (T268M) and 1272C>G (I424M) in exons 7 and 12, respectively. The third was a splice mutation in intron 5 (696+2T>A), which induced a frame shift with a stop codon in exon 6 (fs213X). In the fourth family the previously-reported missense mutation 689G>A (G230E) was found. We also report two known polymorphisms: 261C>T (T87T) and 2154T>C (D718D) in exons 2 and 17 of two MC families; also found in 14 (33%) and 28 (67%) of 42 healthy controls, respectively. These findings expand our knowledge of mutations responsible for myotonia congenita, reducing the proportion of MC patients in whom genetic alterations have not been found.

DMD and BMD in the same family due to distinct mutations.

We report on a family with a boy affected by Duchenne muscular dystrophy (DMD) and an asymptomatic cousin with a Becker-type dystrophin abnormality, diagnosed by chance. Dystrophin gene analysis showed that these conditions were caused by two distinct deletions with breakpoints in different exons. In Xp21 families, DNA analysis and dystrophin testing of asymptomatic males with high CK plasma levels might detect different dystrophin mutations in separate haplotypes as in our family, although we stress there should be clear clinical or familial indications for such testing.

A new non-radioactive method for the screening and prenatal diagnosis of myotonic dystrophy patients.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease with an estimated incidence of 1 in 8000 and is the most common form of muscular dystrophy affecting adults. An unstable, untranslated part of the myotonic dystrophy protein kinase gene on the long arm of chromosome 19, composed of CTG repeats, is a genetic marker for DM. We have developed a fast non-radioactive polymerase chain reaction (PCR) procedure to detect the (CTG)n repeat expansion in DM patients and their relatives. Genomic DNA extracted from peripheral blood lymphocytes was amplified by PCR using specific primers to flank the region containing the triplets. To improve the amplification of this CG-rich region, either 10% glycerol or rTth DNA polymerase XL (extra long) was added to the reaction mixture, allowing amplification of huge expansions otherwise not polymerized by PCR. The PCR products were Southern blotted and the expansion revealed using a fluorescein-labelled (CTG)10 probe. We compared our results with those obtained in 24 patients and relatives using genomic digestion followed by radioactive Southern blot; in all cases the results overlapped. The same technique was used for prenatal diagnosis in pregnant DM mothers. We conclude that this new method is reliable for the genetic testing of DM patients.

Dystrophin characterization in BMD patients: correlation of abnormal protein with clinical phenotype.

We have investigated protein expression and genotype in 59 Becker muscular dystrophy (BMD) patients. The aim was to identify possible causes of the marked variability in phenotype in patients with similar deletions/mutations. The patients were examined neurologically and functionally and underwent Manual Muscle Testing. Dystrophin expression was analysed by immunohistochemistry and western blot using antibodies against six different segments of the protein. DNA mutations were investigated by PCR amplification of 30 exons. Based on dystrophin expression at the sarcolemma, two groups of patients were identified: group A (29 patients) with the classic patchy distribution of dystrophin and group B (30 patients) with absence or reduction of one or more dystrophin portions and variable, although mostly normal, expression of the other portions of the protein. Dystrophin molecular weight was normal or slightly reduced in group A and was variably reduced, generally conspicuously so, in group B. The quantity of dystrophin expressed varied markedly in both groups. The pattern of immunohistochemical staining in group B patients correlated with milder clinical phenotype, suggesting that small dystrophin molecules lacking a portion in the N-terminus or in the rod domain, are more functional than proteins with normal or slightly reduced molecular weight that display the BMD-typical patchy distribution at the sarcolemma.

Is the CACNA1A gene involved in familial migraine with aura?

The discovery of mutations in the neural calcium channel (CACNA1A) gene in familial hemiplegic migraine (FHM), variant of migraine with aura, led to the suggestion that this gene might be involved in familial migraine with aura (FMA). We investigated whether the mutations in FHM are present in FMA patients, analyzing genomic DNA by PCR, single stranded conformation polymorphism, sequencing and restriction enzyme. No mutations were found. A known polymorphism (5682-14C>T) was found in exon 36. These findings suggest that the mutations found in FHM and the other known mutations of the CACNA1A gene are not the genetic basis of FMA. Genetic alterations in FMA patients may be localized on chromosome 19 but not in the CACNA1A exons we investigated.

Disruption of heart sarcoglycan complex and severe cardiomyopathy caused by beta sarcoglycan mutations.

Two young males with limb-girdle muscular dystrophy (LGMD) resulting from sarcoglycan deficiency died at 27 (patient 1) and 18 years (patient 2) of severe cardiomyopathy. Genetic analysis showed that they were compound heterozygotes for mutations in the beta sarcoglycan gene. One of these mutations, an 8 bp duplication in exon 3, was common to both patients. The second mutation in patient 2 was a 4 bp deletion at the splice donor site of intron 2, not reported previously. Patient 2 had more severe heart and skeletal muscle defects with faster deterioration; no sarcoglycans were detected in his skeletal muscle. The second mutation in patient 1, inferred because the unaffected father carries the 8 bp duplication, was not found. In patient 1, both heart and skeletal muscle were analysed and showed reduction of all sarcoglycans in both tissues and incorrect localisation of alpha and gamma sarcoglycans in heart. Therefore mutations in one sarcoglycan gene can disrupt the entire sarcoglycan complex in both skeletal and cardiac muscle. Differing expression patterns of sarcoglycan components in heart and skeletal muscle could be the result of alternatively spliced transcripts in these tissues. By sequencing an alternative transcript, highly expressed in the heart and skeletal muscle of patient 1, we found an 87 bp cryptic exon not previously reported. Although cardiomyopathy can result from mutations in alpha and gamma sarcoglycans, we show for the first time that the condition can also be caused by mutations in the beta sarcoglycan gene. This report therefore expands the phenotype of sarcoglycanopathies and suggests that cardiac function in LGMD patients with defective sarcoglycan expression should be monitored.