Genomic characterisation of Alzheimer's disease risk genes using long-read sequencing

阿尔茨海默病(AD)是一种慢性神经退行性疾病，以进行性神经病理和认知能力下降为特征。AD开始出现在60多岁的人群中，并随着年龄的增长而增加。英国约有50万人患有阿尔茨海默病，随着人口老龄化，这种疾病将给公共卫生带来越来越大的负担。目前还没有针对AD的疾病修饰疗法，尽管一些药物可以提供一段时间的症状缓解。可能需要几种不同的治疗方法才能成功治疗AD，糖尿病和其他常见疾病就是如此。阿尔茨海默病在大脑中的典型特征是淀粉样蛋白沉积到不溶性斑块中，并在神经元中形成tau蛋白缠结，导致脑组织丢失。人们还认为，大脑中存在广泛的炎症。虽然与阿尔茨海默病相关的脑内病理改变已经被很好地描述，但涉及疾病发生和发展的具体机制仍不清楚。了解这些过程对于开发治疗AD的新药将是重要的。治疗阿尔茨海默氏症的方法试图去除或减缓淀粉样蛋白沉积，目前正在对患者进行评估，尽管目前还不知道这些方法是否有效。为了开发更多的治疗方法，我们必须专注于与阿尔茨海默氏症的发展和进展相关的悬而未决的问题，例如了解为什么有些人无法清除大脑中的淀粉样蛋白和tau蛋白，从而导致它们的堆积，这些蛋白如何对神经元产生毒性，以及大脑中的炎症在疾病中所起的作用。在这四年的博士生学习中，这些问题将通过分析大脑中已经牵涉到AD的特定基因的表达来解决，使用一种新的基因组测序技术，该技术能够对基因的整个表达形式进行测序，而不是像目前标准的RNA测序那样只对小片段进行测序。这一点很重要，因为已知存在不同形式的基因，称为亚型，具有不同的蛋白质序列和功能。例如，一种版本的tau蛋白在稳定微管方面做得更好，但也更容易在AD患者的大脑中聚集。选择性剪接和RNA亚型可能极大地增加人类基因组的蛋白质编码潜力；有证据表明，95%的人类基因存在选择性剪接。通过使用太平洋生物科学公司(PacBio)开发的名为小分子实时测序(SMRT)的长读测序方法，这名学生将检查AD患者去世时大脑中的这些长mRNA亚型，并更好地了解它们在疾病中的作用。利用我们的MRC临床研究基础设施计划奖，我们的实验室最近优化了这种‘iso-seq’方法，使其能够从人脑组织样本中生成全长cDNA序列。这名学生将使用1)由志愿者捐赠给MRC伦敦神经退行性疾病脑库的大量人类死后大脑样本，以及2)由礼来公司的工业合作伙伴提供的具有良好特征的淀粉样蛋白和tau病理啮齿动物模型的组织来执行这些实验，以确定与AD密切相关的基因。ISO-SEQ分析将对内嗅皮层组织(AD早期受影响的大脑区域)和小脑(在很大程度上受到AD病理的保护)进行，在代表AD病理的所有个体中进行。相同基因转录异构体的后续变化将在具有良好特征的淀粉样蛋白和tau病理的啮齿动物模型中进行检查，以确定与AD神经病理的发生和发展相关的变异。这种对阿尔茨海默病潜在分子机制的理解可能导致识别潜在的新的治疗靶点。

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is characterised by progressive neuropathology and cognitive decline. AD starts to appear in the population in people in their 60s, and increases in incidence as people age. Around 500,000 people in the UK have AD, and the condition will make an increasing public health burden as the population ages. There are currently no disease modifying treatments for AD, although some drugs can provide a period of symptomatic relief. Several different therapies may be needed to successfully treat AD, as is the case for diabetes and other common diseases. The classical signatures of AD in the brain are the deposition of amyloid protein into insoluble plaques, and the formation of tau protein tangles in neurons, leading to loss of brain tissue. There is also thought to be extensive inflammation in the brain. Although the pathological changes in the brain associated with AD have been well described, the specific mechanisms involved in the onset and progression of the disease are still unknown. Understanding these processes will be important for the development of novel drugs to treat AD. Therapies for Alzheimer's which try and remove or slow down amyloid protein deposits are currently being evaluated in patients, although it is not yet know if these will work. In order to develop additional therapies, we must focus on the unanswered questions relating to the development and progression of Alzheimer's, such as an understanding of why some people fail to clear amyloid and tau protein from the brain, thus allowing their build-up, how these proteins become toxic to neurons, and the role that inflammation in the brain plays in the disease. In this 4 year PhD studentship these problems will be approached by analysing the expression of specific genes in the brain, already implicated in AD, using a novel genomic sequencing technology that is able to sequence the entire expressed form of the gene, not just small fragments as is currently the case with standard RNA sequencing. This is important as different forms of genes, known as isoforms, with different protein sequence and functions, are known to exist. For example one version of the tau protein is better at stabilising microtubules but also more prone to aggregate in the brain in AD. Alternative splicing and RNA isoforms may dramatically increase the protein-coding potential of the human genome; there is evidence for alternative splicing at >95% of human genes. By using a long-read sequencing method known as small molecule real time sequencing (SMRT), developed by the company Pacific Biosciences (PacBio), the student will examine these long mRNA isoforms in the brains of people who had AD when they died, and better understand their role in disease. Capitalising on our MRC Clinical Research Infrastructure Initiative award, our lab has recently optimised this 'iso-seq' method to enable the generation of full-length cDNA sequences from human brain tissue samples. The student will perform these experiments for genes robustly implicated in AD using 1) a large collection of human post-mortem brain samples donated by volunteers to the MRC London Brainbank for Neurodegenerative Diseases and 2) tissue from well-characterised rodent models of amyloid and tau pathology provided by our industrial partners at Eli Lilly. Iso-seq analysis will be performed on entorhinal cortex tissue (an area of the brain affected early in AD) and cerebellum (which is largely protected from AD pathology) in a large collection of individuals representing the full range of AD pathology. Subsequent changes in transcript isoforms at the same genes will be examined in well-characterised rodent models of amyloid and tau pathology to identify variation associated with the onset and progression of AD neuropathology. This improved understanding of the molecular mechanisms underlying AD may lead to the identification of potential new targets for treatment.