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Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

ABSTRACT: Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.

SUBMITTER: Vermeer S

PROVIDER: S-EPMC2997370 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

Vermeer Sascha S Hoischen Alexander A Meijer Rowdy P P RP Gilissen Christian C Neveling Kornelia K Wieskamp Nienke N de Brouwer Arjan A Koenig Michel M Anheim Mathieu M Assoum Mirna M Drouot Nathalie N Todorovic Slobodanka S Milic-Rasic Vedrana V Lochmüller Hanns H Stevanin Giovanni G Goizet Cyril C David Albert A Durr Alexandra A Brice Alexis A Kremer Berry B van de Warrenburg Bart P C BP Schijvenaars Mascha M V A P MM Heister Angelien A Kwint Michael M Arts Peer P van der Wijst Jenny J Veltman Joris J Kamsteeg Erik-Jan EJ Scheffer Hans H Knoers Nine N

American journal of human genetics 20101118 6

Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation ...[more]

PMID: 21092923

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Project description:Background and objectivesThe term autosomal recessive cerebellar ataxia (ARCA) encompasses a diverse group of heterogeneous degenerative disorders of the cerebellum. Spinocerebellar ataxia autosomal recessive 10 (SCAR10) is a distinct classification of cerebellar ataxia caused by variants in the ANO10 gene. Little is known about the molecular role of ANO10 or its role in disease. There is a wide phenotypic spectrum among patients, even among those with the same or similar genetic variants. This study aimed to characterize the molecular consequences of variants in ANO10 and determine their pathologic significance in patients diagnosed with SCAR10.MethodsWe presented 4 patients from 4 families diagnosed with spinocerebellar ataxia with potential pathogenic variants in the ANO10 gene. Patients underwent either clinical whole-exome sequencing or screening of a panel of known neuromuscular disease genes. Effects on splicing were studied using reverse transcriptase PCR to analyze complementary DNA. Western blots were used to examine protein expression.ResultsOne individual who presented clinically at a much earlier age than typical was homozygous for an ANO10 variant (c.1864A > G [p.Met622Val]) that produces 2 transcription products by altering an exonic enhancer site. Two patients, both of Lebanese descent, had a homozygous intronic splicing variant in ANO10 (c.1163-9A > G) that introduced a cryptic splice site acceptor, producing 2 alternative transcription products and no detectable wild-type protein. Both these variants have not yet been associated with SCAR10. The remaining patient was found to have compound heterozygous variants in ANO10 previously associated with SCAR10 (c.132dupA [p.Asp45Argfs*9] and c.1537T > C [p.Cys513Arg]).DiscussionWe presented rare pathogenic variants adding to the growing list of ANO10 variants associated with SCAR10. In addition, we described an individual with a much earlier age at onset than usually associated with ANO10 variants. This expands the phenotypic and allelic heterogeneity of ANO10-associated ARCA.

Dataset Information

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

Publications

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

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