Searching for genetic cause of neurological disorderes by nextgeneration sequencing/ exome sequencing

Resumen

1. INTRODUCTION Exome sequencing is one of the applications of Next Generation Sequencing (NGS) that is based on an entirely new principle of sequencing technology following Sanger (first generation) sequencing. NGS enables massively parallel sequencing and automation, thereby increasing the sequencing capacity from a few hundred base pairs to several billions of them within a single analysis. To date, NGS is the best available tool to elucidate disease-causing mutations. It is not only useful in large extended families, where linkage information provides information about the disease locus, but may also be applied to detect disease-causing de novo mutations in sporadic patients when parent-offspring trio exome sequencing is performed, a research impossible to address by conventional Sanger sequencing without having a candidate gene. The objective of this thesis is to identify and sort novel potential genetic causes in different complex neurological disorders through NGS, such us exome sequencing. With this aim, the project has been divided in three different parts that will be discussed in greater depth in the next sections. - In part I of this thesis, I investigated eight Canadian families that I followed in clinic during my two-year-research/training stay at the Toronto Western Hospital in Toronto, Canada. All of them suffered from complex movement disorders of an unknown etiology and all of them had been previously tested with CES and results were negative. Re-analysis of WES data, looking at all genes, was performed with the objective of identifying new pathogenic variants in novel disease-causing genes. - In part II of the thesis, children affected by complex neurological disorders, in whom the genetic cause has not been identified yet, were investigated. As pointed out in Part I, parent-offspring trio exome sequencing is a powerful way to detect disease causing variants, especially de-novo variants in newly elucidated disease genes. However, comprehensive studies from diagnostic sequencing laboratories have demonstrated diagnostic yields of only about 50%. A reason that could at least partially explain this gap is that in most cases, parents of the families under study are assumed to be genetically unaffected for the filtering strategies. However, (heterozygous) disease-causing mutations can also be transmitted through a healthy parent who shows reduced penetrance, and this is often an underestimated phenomenon. Here, we hypothesized that an inherited mutation in a known or novel imprinted gene could explain the disease in at least a subset of these trios. In order to identify such variants that could be disease causing, a strategy that looked for candidate variants in potentially imprinted genes in the index patients that may explain the lack of disease in their parents was designed. The strategy followed and the potential identification of one new imprinted gene is described in this part. - In part III, X-linked dystonia-parkinsonism (XDP), a neurodegenerative disorder endemic to Panay Island, the Philippines, was extensively assessed with the objective of including systematic clinical and genetic data about this disorder in the MDSGene database. This disorder is associated with an antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of the TAF1 gene, which is located on the X chromosome. Another aim of this study was to assess the benefit of including previously unpublished clinical data in the MDSGene workflow. To accomplish this aim, clinicians from the Philippines as well as authors of two genetic-focused cohort studies were contacted and invited to contribute relevant phenotypic information that were neither included in the text nor in the supplement of their original articles. Further, in a set of cases where comprehensive phenotypic data was available to us, we assessed the benefit of standardized data collection in revealing novel aspects of the XDP phenotype. 2. METHODS - Part I - Index cases and family members with complex movement disorders, mainly dystonia, tremor and parkinsonism were recruited from the Movement Disorders Clinic at the Toronto Western Hospital in Toronto, Canada. All cases underwent a comprehensive phenotyping and results from additional clinical investigations have been included when available: neuroimaging with brain magnetic resonance imaging (MRI), electromyogram or nerve conduction studies, and videography. Details on the genetic testing, the strategy for variant filtering and the process of validation are further analized though the text of the thesis. - Part II - In this part, 38 German families were included in the study (trios: index patient/mother/father) in which the index patient presented with a combination of diverse movement disorders and developmental delay. All parents were asymptomatic. The 38 families have been sequenced as part of a clinical diagnostic evaluation in the Center for Rare diseases Lübeck. Sequencing was performed at Centogene AG, Rostock, Germany. Patients ́ phenotypes and details on filtering strategies and variant prioritization are described throughout the text of the thesis. The phenomenon of genomic imprinting has been explored as a potential strategy to follow in those cases where candidate genes for non-penetrant inherited variants in trios with unaffected parents are searched for (rare variants that are shared between index patients and unaffected parents are often excluded in conventional filtering strategies). - Part III - The MDSGene database systematically links reported genetic mutations with movement disorder phenotypes and other demographic and clinical information. The content of MDSGene is based on genetic as well as demographic and clinical data extracted from the relevant literature by an experienced team of movement disorders specialists, geneticists, and epidemiologists following systematic and regularly updated literature screens. To reduce data missingness, we (1) contacted authors and engaged the research community to provide additional clinical and genetic information, and (2) revisited previously unpublished data from a cohort of XDP patients seen at the Institute of Neurogenetics in Lübeck, Germany. 3. RESULTS - Part I: I provide detailed phenotypical data of eight families affected with combined movement disorders of an unknown cause. I also carefully described the strategies I followed for the identification of potential disease-causing variants. I was able to find potential candidate genes in 4 out of the 8 families. All variants were confirmed by Sanger sequencing in the laboratory and all of them had a CADD score higher than 15. Pathogenicity of all the reported variants remains uncertain and currently, all of them continue to be followed-up awaiting another hit from the worldwide community in the GeneMatcher. Lack of matching hits but presence of non-matching ones questions a pathogenic role for variants in some of these genes, at least for the respective movement disorder. Nevertheless, data from exome sequencing can be reevaluated as new data are incorporated into mutation and disease databases, which would be expected to enhance the chances for discovering a potential diagnosis. - Part II: We identified 264 known imprinted or possibly imprinted genes. Within the 38 trios, one trio carried a potentially pathogenic variant in these genes. Results of Part II provide some evidence on the importance of going a step further when looking at patients with rare neurological disorders and take reduced penetrance due to imprinted genes into account. This strategy may guide future discoveries of new genes implicated in rare disorders. Indeed, a potential pathogenic variant in a likely new imprinted gene has been reported in this work: NM_001039948.3:c.670A>G; p.Ile224Val in the SGMS1 was found in a patient with a complex movement disorder, hypotonia and epilepsy. To confirm imprinting, it needs to be shown that there is parent-dependent expression of only one allele. However, the gene is not expressed in easily accessible material/tissue such as blood or fibroblasts. It shows exclusive neuronal expression. Thus, further validation is needed. - Part III: Contributes to an innovative, user-friendly, and expandable tool that systematically relates genotypes and phenotypes to movement disorder clinicians and basic scientists. A standardized minimal data set additionally improves data quality and is thus advocated for deeper and more consistent phenotyping across studies, institutions, and disorders. We screened 233 citations and curated phenotypic and genotypic data for 414 cases. Using the aforementioned approaches, we expanded the cohort to 577 cases and increased information available for important clinical and genetic features such as age at onset, initial manifestation, predominant motor symptoms, functional impairments, and repeat size information. We established the use of mining unpublished data to expand the MDSGene workflow and present an up-to-date description of the phenomenology of XDP using an extensive collection of previously reported and unreported data. 4. CONCLUSIONS All the advances in molecular genetic research will provide further insights into the pathogenesis of movement disorders and will open new perspectives for hypothesis- driven research to develop effective treatments. Taken together, within this thesis I identified novel candidate disease genes and contributed to the systematic collection of genotypic and phenotypic data to enable meaningful phenotype-genotype correlations to better serve our patient