Modernising the diagnosis of mucocutaneous bleeding disorders: next generation sequencing of novel loci associated with platelet dysfunction.

轻度和中度出血性疾病相对常见，比血友病等严重出血性疾病普遍得多。由于症状通常是皮肤粘膜出血，如流鼻血和月经过多，它们通常被归因于原发性止血缺陷：特别是血小板功能异常。实验室检查中血小板功能异常常支持这一观点。然而，尽管血小板功能检测是复杂而费力的，但它在识别或排除血小板功能特异性缺陷方面的能力有限。此外，这种检测方法不能识别血管壁或其他血液成分中的其他致病因素。因此，这些患者中的许多人可能接受不适当或不必要的治疗，通常是血小板输注，这是一种稀缺资源。检测和诊断血小板和其他可能有缺陷的血管或血液成分缺陷的另一种方法是在遗传水平上检测缺陷。迄今为止，这将是一项极其困难和昂贵的任务。过去几年的技术进步使得在几天内快速而廉价地对个人表达的全部基因进行测序成为可能。因此，这提供了对单个基因的非常全面的分析。值得注意的是，这种方法不需要对血小板生理学或它们与其他组织的相互作用有任何理解或假设。在这项研究中，我们建议确定在哈默史密斯医院就诊的轻度出血性疾病患者，并对他们进行基因分析。这项分析将在剑桥附近的威康信托桑格研究所进行。人类基因组所有表达片段的测序将产生大量的信息。需要进行仔细的分析，以确定那些可能对确定患者所遭受的临床问题具有重要意义的变化。这些分析将与计算机专家一起进行，这些专家已经对数千名健康个体的基因分析结果进行了分析，这些分析可用于比较。最后，当候选基因改变被确定时，有必要证明它们能够产生感兴趣的临床表型。因此，该培训奖学金的最后一部分将需要在实验产生的血小板或其他系统中复制候选基因变化，并检查从出血性疾病患者身上提取的培养血小板生成细胞，以确定血小板功能或生成的任何关键方面是否可以复制。因此，我希望精通三个不同但互补的领域：出血性疾病患者的临床和实验室评估，高通量基因分析的性能和分析，体外操作和血小板生成和功能分析。在奖学金期间获得的技能和能力将使我成为未来在NHS及其他领域应用现代dna检测的专家之一。我还预见到，从拟议的研究中获得的新见解将最终为出血性疾病患者带来新的治疗方法，这些治疗方法可以更好地根据疾病的遗传原因或心脏病发作或中风患者量身定制。后者似乎违反直觉，通过调查出血性疾病患者，可能会开发出更好、更安全的药物来治疗我们社会中的头号杀手疾病。然而，血小板驱动的凝块形成是心脏病发作和中风的部分原因。

Mild and moderate bleeding disorders are relatively common and much more prevalent than severe bleeding disorders such as haemophilia. Because the symptoms are usually of mucocutaneous bleeding such as nosebleeds and menorrhagia, they are frequently attributed to defects of primary haemostasis: in particular abnormalities of platelet function. This is often supported by abnormalities of platelet function in laboratory tests. However, although platelet function testing is complicated and laborious, it has a limited ability to identify or to exclude, specific defects in platelet function. In addition this approach to testing will not identify alternative or additional causative factors in the vascular wall or other blood components. As a consequence many of these patients may receive inappropriate or unnecessary treatment, usually platelet transfusions, which are a scarce resource.An alternative approach to detecting and diagnosing defects in platelets and other vessel or blood components that may be defective is to detect the defects at the genetic level. Hitherto this would have been a prohibitively difficult and expensive undertaking. Technological advances over the last few years have made it possible to rapidly and cheaply sequence the entirety of an individual's expressed genes in days. This therefore provides a very comprehensive analysis of the individual genes. It is important to note that this approach does not require any understanding or assumptions about platelet physiology or their interaction with other tissues. In this study we propose to identify patients with mild bleeding disorders who are attending clinics at the Hammersmith Hospital and subject them to genetic analysis. This analysis will be performed at the Wellcome Trust Sanger Institute near Cambridge. Sequencing of all expressed segments of the human genome will generate an enormous amount of information. Careful analysis will be required to identify those changes that are likely to be of significance in determining the clinical problems from which the patients suffer. These analyses will be carried out in conjunction with computer experts who have already analysed the results of the genetic analysis of thousands of healthy individuals that can be used for the purpose of comparison. Finally, when candidate genetic changes have been identified it will be necessary to prove that they are capable of producing the clinical phenotype of interest. The last part of this training fellowship will therefore entail reproducing the candidate genetic changes in experimentally produced platelets or other systems and examining cultured platelet producing cells, which have been derived from patients with bleeding disorders, to determine whether any critical aspects of platelet function or production can be reproduced. I therefore expect to become proficient in three different but complementary areas: clinical and laboratory assessment of patients with bleeding disorders, performance and analysis of high throughput genetic analysis and in vitro manipulation and analysis of platelet production and function. The skills and competencies gained during the fellowship will allow me to become one of the future experts in the application of modern DNA-based tests in the NHS and beyond. I also foresee that new insights gained from the proposed study will eventually lead to new treatments for patients with bleeding disorders, which are better tailored on basis of the genetic cause of their disorder or for patients with heart attacks or strokes. The latter seems counter-intuitive that by investigating patients with bleeding disorders better and safer drugs for the treatment of the Number 1 killer diseases in our society may be developed. However platelet-driven clot formation is in part the cause of heart attacks and strokes.