Mitochondrial diseases - long read genome and transcriptome sequencing in cases unresolved after short-read genomics

线粒体疾病包括由丙酮酸氧化途径的酶缺陷引起的临床和遗传异质性疾病组。广泛的测序方法，如外显子组甚至全基因组测序，显着提高了诊断效率。然而，大约一半的患者仍然没有得到明确的诊断。这种情况阻碍了对患者及其家人的充分咨询以及有针对性的治疗策略的发展。我们在BMBF资助的JuniorVerund‘mitOmics’中的一个多学科团队中进行了广泛的努力，以确定线粒体疾病患者的分子基础。该项目包括全基因组三联分析，以及对患者来源的成纤维细胞系进行RNAseq分析，以显示疾病的生化特征。尽管我们确实能够在另外5%-10%的以前未解决的病例中准确地找到分子缺陷，但绝大多数仍然不清楚。在其他潜在的解释(例如非遗传原因)中，一种是我们根本没有检测到疾病原因的变异，因为我们使用了错误的方法。目前绝大多数完整的外显子组或基因组数据集是在Illumina平台上产生的，这通常允许确定约75-150个核苷酸的DNA或RNA序列。虽然这项技术很好地检测单核苷酸变异(SNV)，但它基本上对其他类型的遗传变异(如重复扩增或收缩以及结构变异(SVS，例如倒位))是盲目的。所谓的长读取技术的最新进展允许对长度为数千个碱基对的读出进行测序。这些方法解决了重复序列或其他短阅读基因组学无法获得的基因组区域。此外，纳米孔技术有可能检测到表观遗传标记。然而，目前的缺点是更高的基础呼叫错误率和成本。在这项研究中，我们希望建立在我们之前建立的资源、数据和数据解释方面的专业知识。我们应用长读和亚硫酸氢盐测序方法，对30名经过短读全基因组和转录组测序后仍未解决的未解决索引患者进行高精选。数据整合和分析将被量身定做，以确定遗传和表观遗传因素的优先顺序，这些因素以前在短文分析中被忽视。患者的细胞系随后可以作为验证性功能研究或治疗干预评估的有价值的模型。生成的数据将在当地和国际罕见疾病网络中共享。这项研究的结果有望为疾病机制和细胞生物学提供新的见解，使个性化治疗方案的评估成为可能，改善罕见疾病患者的分子诊断，并指导未来常规诊断策略的发展。

Mitochondrial disorders comprise clinically and genetically heterogeneous group of disorders arising from enzymatic defects of the pyruvate oxidation route. Broad sequencing approaches such as exome or even full genome sequencing significantly increased the diagnostic yield. However, about half of the patients remain without a firm diagnosis. This situation prevents adequate counseling of the patients and their families as well as the development of targeted therapeutic strategies.We have performed extensive efforts to identify the molecular bases of mitochondrial disease patients in a multidisciplinary team in the BMBF-funded Juniorverbund 'mitOmics'. The project included full genome trio analyses as well as RNAseq on patient-derived fibroblast cell lines displaying the biochemical signature of the disease. Although we were indeed able to pinpoint the molecular defect in an additional 5-10 % of previously unsolved cases the vast majority had remained unclear.Amongst other potential explanations (e.g. non-genetic cause) one is that we simply don’t detect the disease-causal variation because we use the wrong method. The vast majority of full exome or genome datasets is currently produced on Illumina platforms which usually allow to determine the DNA or RNA sequence of about 75-150 bp. While this technology works well to detect single nucleotide variants (SNVs) it is basically blind for other types of genetic variation such as repeat expansions or contractions as well as structural variants (SVs, e.g. inversions).Recent advances in so-called long read technologies allow the sequencing of reads with a length of several thousand base pairs. These methods resolve repetitive sequences or other genomic regions not accessible to short read genomics. In addition, the Nanopore technology has the potential to detect epigenetic marks. However, current disadvantages are higher base calling error rates and costs.In this study, we want to build on our previously established resources, data, and expertise in data interpretation. We apply for long read and bisulfite sequencing on a highly selected cohort of 30 unresolved index patients which had remained unsolved after short read whole genome and transcriptome sequencing. Data integration and analysis will be tailored to prioritize genetic and epigenetic factors previously overlooked with short read analyses. Patients’ cell lines can subsequently serve as a valuable model for confirmatory functional studies or evaluation of therapeutic interventions.Generated data will be shared within local and international rare disease networks. The results of the study are expected to provide new insights into disease mechanisms and cellular biology, to enable the evaluation of personalized therapeutic options, to improve molecular diagnostics of rare disease patients, and guide the development of tomorrow’s routine diagnostic strategies.