Genetic etiology of Amyotrophic Lateral Sclerosis

在上一个财政年度，我们完成了两个与了解肌萎缩性侧索硬化症的遗传学直接相关的主要项目。在第一个项目中，我们使用外显子组测序在一个常染色体显性遗传肌萎缩性侧索硬化症（ALS）的意大利家庭中鉴定了含缬氨酸蛋白（VCP）基因的编码突变。VCP突变先前已在包涵体肌病、Paget病和额颞叶痴呆（IBMPFD）家族中发现。对210例家族性ALS病例和尸检证实的ALS病例进行VCP筛查，发现了4个额外的突变，包括一个病理证实的ALS病例中的突变。VCP蛋白对含泛素自噬体的成熟至关重要，突变型VCP的毒性部分是通过其对TDP-43蛋白的作用介导的，TDP-43蛋白是肌萎缩性侧索硬化神经病理学特征中泛素包涵体的主要成分。我们的数据扩大了IBMPFD的表型，包括运动神经元变性，表明VCP突变可能占家族性ALS的1%-2%，并提供了直接暗示运动神经元变性中泛素化/蛋白质降解途径缺陷的证据。此外，我们的工作表明，有可能应用下一代测序技术成功地找到迟发性衰老神经退行性疾病的致病基因。

During the last fiscal year, we have completed two major projects that are directly relevant to understanding the genetics underlying amyotrophic lateral sclerosis. In the first project, we used exome sequencing to identify a coding mutation in the valosin-containing protein (VCP) gene in an Italian family with autosomal dominantly inherited amyotrophic lateral sclerosis (ALS). Mutations in VCP have previously been identified in families with Inclusion Body Myopathy, Paget disease, and Frontotemporal Dementia (IBMPFD). Screening of VCP in a cohort of 210 familial ALS cases and autopsy-proven ALS cases identified four additional mutations including a mutation in a pathologically proven case of ALS. VCP protein is essential for maturation of ubiquitin-containing autophagosomes, and mutant VCP toxicity is partially mediated through its effect on TDP-43 protein, a major constituent of ubiquitin inclusions that neuropathologically characterize ALS. Our data broaden the phenotype of IBMPFD to include motor neuron degeneration, suggest that VCP mutations may account for ∼1%-2% of familial ALS, and provide evidence directly implicating defects in the ubiquitination/protein degradation pathway in motor neuron degeneration. Furthermore, our work shows that it is possible to apply next generation sequencing techniques to successfully find causative genes in late-onset neurodegenerative diseases of aging. In the second project, we published the results of our genome-wide association study of ALS in Finland. Finland is an ideal location for a genome-wide association study of ALS because the incidence of the disease is one of the highest in the world, and because the genetic homogeneity of the Finnish population enhances the ability to detect risk loci. We identified two association peaks that exceeded genome-wide significance. One was located on chromosome 21q22, which corresponds to the autosomal recessive D90A allele of the SOD1 gene. The other was detected in a 232kb block of linkage disequilibrium in a region of chromosome 9p that was previously identified in linkage studies of families with ALS. Within this region, we defined a 42-SNP haplotype that was associated with significantly increased risk of ALS, and which overlapped with an association locus recently reported for frontotemporal dementia. For the 93 patients with familial ALS, the population attributable risk for the chromosome 9p21 locus was 37.9% (95% CI 27.7-48.1) and that for D90A homozygosity was 25.5% (16.9-34.1). These data clearly show that the chromosome 9p21 locus is a major cause of familial ALS in the Finnish population. Furthermore, the overlap with the risk haplotype recently reported for frontotemporal dementia provides further evidence of a shared genetic cause for these two neurodegenerative diseases. This paper was published in Lancet Neurology in October 2010. Our ongoing major projects include: (1) exome sequencing of additional familial ALS samples to look for causative genes underlying motor neuron degeneration. DNA for these cases were obtained from our collaborators, Adriano Chi, Michael Benatar, Michael Sendtner, and Ekaterina Rogaeva, as well as our own efforts to recruit subjects locally and nationally; (2) We are continuing our efforts to identify the precise genetic lesion that underlies chromosome 9p21-linked ALS-FTD. To achieve this, we have reached out to other laboratories interested in studying this locus, and formed an international consortium involving the University of Manchester, University of Cardiff, University College London, VU University Medical Center in Amsterdam, and the University of Helsinki in Finland. In particular, my laboratory is applying next generation sequencing techniques to identify variants and structural changes in the core region. The high frequency of the chromosome 9p21 risk haplotype in the Finnish ALS population indicates that this locus is particularly important to understanding the disease, and may also be relevant to familial disease outside of Finland. In summary, the current year has been successful in identifying genetic variants important in the pathogenesis of ALS using exome sequencing and genome-wide association approaches. Each of these studies employed large cohorts of research subjects, and utilized the sequencing and genotyping facilities available within the Laboratory of Neurogenetics, NIA. By understanding the cellular mechanisms underlying late-onset motor neurodegeneration, we also hope to shed light on the role of aging in the CNS and in age-related decline in mobility.