The impact of rare variants in lysosomal genes in the pathogenesis of Parkinson disease

帕金森病(PD)是最常见、最严重的神经退行性疾病之一。帕金森病的病因疗法并不存在。帕金森病是一种遗传复杂的疾病，在大多数患者中，只有少数患者是单基因的。GWAS的研究局限于每个基因相对较少的常见变异。最近的大型外显子组测序项目表明，大多数编码稀有变异的蛋白质的频率在1：1000到1：10.000的等位基因范围内。评估稀有变异体作用的最好方法是DNA测序。GWAS的发现只解释了帕金森病估计遗传率的一小部分。剩下的部分缺失的遗传性将是由于罕见的变异。罕见变异对包括阿尔茨海默氏症在内的许多疾病的影响已经被证明。然而，由于Sanger测序的高成本，在大的患者和对照样本中还没有系统地评估罕见变异的影响。下一代测序技术(NGS)为获取海量DNA序列数据提供了一种更为经济的手段，溶酶体在PD的发病机制中起着极其重要的作用。α-突触核蛋白(a-syn)是散发性帕金森病的关键致病蛋白。α-SYN自发聚集，是病理性神经元内包涵体的主要成分，称为路易小体。溶酶体通过不同途径降解可溶型和聚集型a-syn。聚集或其他病理改变的a-syn会损害溶酶体功能。大量研究表明，编码溶酶体酶葡萄糖脑苷酶(GBA)的基因中罕见的杂合变异与帕金森病密切相关。这些突变会干扰a-syn的降解。溶酶体参与去除有缺陷的线粒体，这一功能在帕金森病患者中也受到干扰。此外，一些导致单基因PD的基因(如LRRK2、VPS35、PINK1、PARK2、ATP13A2)与溶酶体功能有关。我们建议对2500例PD患者和相同数量的对照组中精心挑选的18个溶酶体候选基因进行测序。已经测试过的DNA池策略保证了非常经济而又彻底的序列分析。这些数据不仅使我们能够详细了解溶酶体基因在帕金森病中的作用，而且还将提供一个独特的数据集来测试和建立新的统计工具，用于分析或罕见变异关联。由于罕见的变异更有可能对基因功能产生重大影响，这一发现也可能使我们能够改进溶酶体基因故障在帕金森病中的生物学研究。基尔大学是此类研究的理想地点，因为申请者拥有帕金森病(G.Deuschl)、神经遗传学(G.Kuhlenbäumer)、神经遗传学(A.Franke)和统计遗传学(M.Krawcak)方面的专业知识。此外，我们拥有德国最大的NGS设施之一。

Parkinson disease (PD) is one of the commonest and most serious neurodegenerative disorders. A causative therapy for PD does not exist. PD is a genetically complex disorder in most patients and monogenic in a minority.GWAS studies are limited to a relatively small number of common variants per gene. Recent large exome sequencing projects have demonstrated that the majority of protein coding rare variants have frequencies in the range of 1:1000 to <1:10.000 alleles. The best method to assess the role of rare variants is DNA sequencing. GWAS findings explain only a small part of the estimated heritability of Parkinson disease. Part of the remaining missing heritability will be due to rare variants. The impact of rare variants has already been shown for numerous disorders including Alzheimers disease. However, the impact of rare variants has not been systematically assessed in large patient and control samples due to the high cost of Sanger sequencing. Next-Generation-Sequencing (NGS) now offers a much more economic means to obtain very large amounts of DNA-sequence data.Lysosomes play an extremely important role in the pathogenesis of PD. Alpha-Synuclein (a-syn) is the key pathogenic protein in sporadic PD. Alpha-syn aggregates spontaneously and is a major constituent of the pathognomonic intraneuronal inclusions termed Lewy bodies. Lysosomes degrade soluble and aggregated a-syn using different pathways. Aggregated or otherwise pathologically altered a-syn impairs lysosomal function. Large studies showed that rare heterozygous variants in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA) are strongly associated with Parkinson disease. These mutations interfere with a-syn degradation. Lysosomes are involved in the removal of defective mitochondria and this function is also disturbed in PD. In addition, a number of genes causing monogenic PD are implied in lysosomal function (e.g. LRRK2, VPS35, PINK1, PARK2, ATP13A2).We propose to sequence 18 carefully selected lysosomal candidate genes in 2500 PD patients and the same number of controls. An already tested DNA pooling strategy guarantees a very economic, yet thorough sequence analysis. These data will not only allow us to obtain a detailed view of the role of rare variants in lysosomal genes in PD but will also provide a unique dataset to test and establish novel statistical tools for the analysis or rare variant associations. Since rare variants are more likely to have a large impact on gene function the findings might also enable us to improve biological studies of lysosomal gene malfunction in PD. Kiel University is an ideal location for such a study because the applicants dispose of expertise in Parkinson disease (G. Deuschl), neurogenetics (G. Kuhlenbäumer), NGS (A. Franke) and statistical genetics (M. Krawcak). In addition we have one of the largest NGS facilities in Germany.