Targeted Next-Generation Sequencing of a 12.5 Mb Homozygous Region Reveals ANO10 Mutations in Patients with Autosomal-Recessive Cerebellar Ataxia
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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DOI：
10.1016/j.ajhg.2010.10.015
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发表时间：
2010-12-10
影响因子：
9.8
通讯作者：
Knoers, Nine
中科院分区：
文献类型：
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作者：
Vermeer, Sascha;Hoischen, Alexander;Knoers, Nine
常染色体隐性小脑性共济失调包括一组临床和遗传上异质性的神经退行性疾病。与它们的主要对应物相比，解开这些共济失调的分子背景已被证明更加复杂，并且目前已知的突变为患者的基因分型提供了不完整的覆盖。通过结合基于SNP阵列的连锁分析和连锁区间内相关序列的靶向重测序以及新一代测序技术，我们鉴定了一个基因突变，并证明了其与常染色体隐性小脑性共济失调的关联。在一个有 3 个受影响兄弟姐妹的荷兰近亲家庭中，基于阵列的序列捕获针对 3 号染色体上的纯合 12.5 Mb 区域进行了定位。所有检测到的序列变体的优先级排序产生了四个候选基因，其中一个包含具有高碱基对保守得分（phyloP得分：5.26）的变体。该变体是 16K 跨膜蛋白的 DUF 590 结构域中亮氨酸替换为精氨酸，16K 跨膜蛋白是由 anoctamin 10 (ANO10) 编码的推定钙激活氯离子通道。通过桑格测序对 ANO10 进行的分析揭示了三个额外的突变：塞尔维亚家族中的纯合突变 (c.1150_1151del [p.Leu384fs]) 和法国家族中的复合杂合剪接位点突变 (c.1476+1G>T) 和移码突变 (c.1604del [p.Leu535X])。这说明了使用初始纯合性作图和下一代测序技术来识别与常染色体隐性疾病有关的基因的力量。此外，鉴定出与小脑共济失调有关的推定的钙依赖性氯离子通道为可能导致小脑共济失调的病理生理机制列表添加了另一条途径。
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.