New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics

Abstract

Objective: The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so.Methods: An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies.Results: This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing. Conclusions:We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia. Neurology â 2012;79:2194-2200 GLOSSARY DM1 5 myotonic dystrophy type 1; DM2 5 myotonic dystrophy type 2; GFP 5 green fluorescent protein; HEK 5 human embryonic kidney; NDM 5 nondystrophic myotonia.We have previously reported an increased frequency of coexisting recessive CLCN1 mutations in patients with currently diagnosed myotonic dystrophy type 2 (DM2), 1 because patients with DM2 heterozygous for a recessive CLCN1 mutation have more pronounced myotonia.1 With the aim of showing this modifying effect of the cosegregating CLCN1 mutations on the protein level, we developed an immunohistochemical assay for ClC-1 protein expression. The method proved to be efficient in the molecular diagnostic clarification of nondystrophic myotonias (NDMs) caused by mutations in CLCN1 and SCN4A genes.Autosomal recessive Becker (OMIM #255700) and dominant Thomsen (OMIM #160800) congenital myotonia are NDMs caused by mutations in CLCN1 on chromosome 7q35.2 More than 100 different CLCN1 mutations have been identified.3 Some CLCN1 mutations are clearly more common than others. R894X (c.2680C.T) has an estimated carrier frequency of about 1% in the European population. In the Finnish population the mutation F413C (c.1238T.G) is almost as frequent, at least in northern Finland. 4 However, these 2 mutations explain only about half of the congenital myotonias in the studied population, and many myotonia patients remain with just one mutation identified when screening for these 2 common mutations.In this study, we focused on the validation of an immunohistochemical assay for the diagnosis of NDM. With this method combined with molecular genetics, we were able to clarify all undetermined myotonia patients, identify new recessive mutations, and verify normal protein expression with dominant CLCN1 mutations.

Predominantly myalgic phenotype caused by the c.3466G>A p.A1156T mutation in SCN4A gene

“…Both had normal CLC-1 protein expression as judged by immunohistochemical staining, indicating no additional defect in the corresponding gene. 11 …”

“…In addition, chloride channel CLC-1 immunohistochemistry was performed in 4 samples with a published method. 11 …”

Chloride Channels are Involved in the Development of Atrial Fibrillation – A Transcriptomic and proteomic Study

“…Chloride channels are ubiquitously expressed, being localized both in plasma membrane and in intracellular organelles 10 . The chloride intracellular channel (CLIC) proteins are highly conserved in vertebrates and successive analysis of human CLIC isoforms demonstrates that nine CLICs have been found in humans 11 – 16 . Like other ion channels, CLICs function in the plasma membrane or in membranes of intracellular organelles and the function of CLICs may involve enzymatic activity in the soluble form and anion channel activity in the integral membrane form 17 .…”

Defective Gating and Proteostasis of Human ClC-1 Chloride Channel: Molecular Pathophysiology of Myotonia Congenita

“…Direct evidence supporting the latter hypothesis was first demonstrated for three diseasecausing mutations located at the distal C-terminal region (A885P, R894X, and P932L): Upon heterologous expression in Xenopus oocytes, they all manifested significantly decreased ClC-1 protein expression at the surface membrane (104). Immunohistochemical examinations of muscle tissues from human patients carrying the R849X mutation further confirmed a dramatic loss of human ClC-1 staining in the sarcolemma (105). Importantly, despite the presence of a notable reduction in whole-cell Cl − current amplitude, only A885P, but not R894X and P932L, is associated with a positive shift of the steadystate voltage-dependent activation property [ Table 1; (35,84,104)].…”