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.

Figure 1

Identifcation of ANO10 Mutation in the Dutch Family A with Autosomal-Recessive Cerebellar Ataxia (A) Pedigree for the Dutch family A together with the segregation of the mutation identified in this family. M1/M1 indicates homozygous carriers of the p.Leu510Arg mutation, and M1/+ indicates heterozygous carriers. The proband is indicated by an arrow. Unfortunately, DNA was not available for individual A:V.3. (B) Coverage histogram of ANO10. Upper part: Sequence-read histograms uploaded to the UCSC Genome Browser display the sequencing depth of all exons of ANO10. Tracks displayed: scale, chromosomal position, read depth histogram per bp (between 0 and 60-fold coverage; higher than 60-fold coverage is not displayed), RefSeq gene track, highly conserved elements (PhastCons Placental Mammal Conserved Elements, 28-way Multiz Alignment). Lower part: Next-generation sequencing-read coverage of ANO10 exons 8–10. Tracks displayed: scale, chromosomal position, individual 454 sequencing reads, RefSeq gene track. (C) Mutation visualization in the IGV browser. Individual reads overlapping with the mutation are displayed. Seventeen of eighteen reads show the homozygous mutation at genomic position chr3:43571913 (the reads are mapped and displayed on the + strand). (D) Verification by Sanger sequencing of missense mutation c.1529T>G (p.Leu510Arg) in ANO10 (accession number NM_018075.3). (E) Sequence comparison of ANO10 in different species. The amino acid that is mutated in the Dutch family is depicted, indicated by an arrow. The amino acid is highly conserved throughout different species, including the fruit fly.

Figure 2

Pedigree Structure of Two Additional Families, B and C Pedigrees for family B, from Serbia, and family C, from France, together with the segregation of the mutations identified in these families. M2/M2 indicates homozygous carriers of the p.Leu384fs mutation, and M2/+ indicates heterozygous carriers, whereas +/+ indicates individuals with two wild-type alleles. M3/M4 indicates compound-heterozygous carriers for the c.1476+1G>T and the p.Leu535X mutations, and M4/+ indicates heterozygous carriers for the p.Leu535X mutation. For family B, the degree of consanguinity is not known. Unfortunately, DNA was not available for individual C:I.1.

Figure 3

Expression of ANO10 in Different Human Tissues, Seen by qPCR (A) Expression of ANO10 in different adult tissues, with the highest expression seen in the brain. (B) Expression of ANO10 in different brain tissues, with the highest expression seen in the adult cerebellum and frontal and occipital cortices.