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. 2010 Feb 12;86(2):213-21.
doi: 10.1016/j.ajhg.2009.12.013. Epub 2010 Jan 21.

Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies

Véronique Bolduc  1 , Gareth Marlow, Kym M Boycott, Khalil Saleki, Hiroshi Inoue, Johan Kroon, Mitsuo Itakura, Yves Robitaille, Lucie Parent, Frank Baas, Kuniko Mizuta, Nobuyuki Kamata, Isabelle Richard, Wim H J P Linssen, Ibrahim Mahjneh, Marianne de Visser, Rumaisa Bashir, Bernard Brais
Affiliations

Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies

Véronique Bolduc et al. Am J Hum Genet. .

Abstract

The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.

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Figures

Figure 1
Figure 1
Identification of ANO5 Mutations in French Canadian LGMD2L Families (A) Pedigrees of LGMD2L families IX and XXXI. Parents of the two families are first-degree cousins. Genotypes for the c.1295C>G variant are indicated below the individuals for whom DNA was available. Family IX has been described earlier.21 (B) Genomic sequence chromatogram of exon 13 in patient IX-II-9 reveal an exonic substitution (c.1295C>G). Sequencing of ANO5 in the two families in (A) confirmed that affected individuals IX-II-3, IX-II-4, IX-II-5, XXXI-II-3, and XXXI-II-11 were also homozygous for the mutation. (C) cDNA amplicons of ANO5 exons 10 to 15 from muscle RNA samples for patient IX-II-9 and a control individual. (D) Sequence chromatograms of the cDNA amplicons in (C) in the region of the nucleotide c.1295. (E) Pedigree of LGMD2L family XXIX. Genomic sequence chromatograms show that patient XXIX-II-1 is compound heterozygote for two exonic variants: a 1 bp duplication in exon 5 (c.191 dupA) and a missense mutation in exon 8 (c.692G>T/G231V). Her unaffected brother is a carrier for the c.191 dupA. (F) Protein alignment of the G231 residue in ANO5 via Multiz.
Figure 2
Figure 2
Identification of ANO5 Mutations in Non-Dysferlin Miyoshi Myopathy Families (A) Genomic sequence chromatograms of exon 20 of ANO5 show that in the non-dysferlin MM Finnish family H that is linked with defective membrane repair, there is a homozygous nucleotide substitution that would result in an amino acid substitution (c.2272C>T/R758C). (B) In the MMD3 Dutch family IV, the mutation identified is c.191 dupA in exon 5 inherited as homozygous. The same mutation is present as heterozygous in the LGMD2L Family XXIX. (C) EM micrograph of patient II-6 muscle from Family H highlights multifocal loss of sarcolemmal membrane. Magnification ×5000. Scale bar represents 5 μm. (D) Predicted structure of human ANO5 and the relative position of the GDD1, LGMD2L, and MMD3 mutations. ANO5 is predicted to contain eight transmembrane (TM) domains with both the N-terminal and the C-terminal regions located intracellularly. The DUF590 domain contains at last three TM domains (TM6-8). The MMD3 R758C mutation that is associated with defective membrane repair is located extracellularly as are the two GDD mutations. The LGMD2L mutations and one MMD3 mutation are located to intracellular regions.
Figure 3
Figure 3
Analysis of ANO5 Expression after Cycloheximide Treatment in Patients Carrying Mutations Producing Premature Terminations (A) ANO5 relative expression in lymphoblastoid cell lines treated with 100 μg/ml of the translation inhibitor cycloheximide for 9 hr as compared to cells treated with vehicle (0.1% DMSO). A greater increase was observed for all three patients (9-, 8-, and 5.5-fold) compared to controls (2-fold). Data represent means ± standard errors of the experimental triplicates. Patients IX-II-9 and XXXI-II-11 are homozygous for c.1295C>G, causing an aberrant splicing in exon 13, a frameshift, and a premature termination codon. Patient XXIX-II-1 is heterozygote for c.191 dupA, causing a frameshift and a premature stop codon, and for c.692G>T, predicted to result in a missense. (B) The differential expression represents the ANO5 expression ratio of cycloheximide-treated versus untreated cells. Cases homozygous for c.1295C>G (n = 2) showed an 8-fold increase, which was significantly greater than the 2-fold differential increase observed in controls (n = 3, p < 0.001, Student's t test). Data represent the mean differential expression ± standard error of, respectively, three control samples and two case samples.
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