Molecular genetic dissection of disease in the era of next-generation sequencing
26 January 2017
UAntwerp, Campus Drie Eiken, Building Q, Promotiezaal - Universiteitsplein 1 - 2610 WILRIJK (route: UAntwerpen, Campus Drie Eiken
Nikhita Ajit Bolar
Prof B. Loeys & Prof L. Van Laer
PhD defence Nikhita Ajit Bolar - Faculty of Medicine and Health Sciences
Since the completion of the human genome in 2003, the tremendous growth and advancement in sequencing technologies, has led to decreased costs per mega base, enabling researchers to study and understand genome complexity and biological systems. These technical advances and the decreasing cost of sequencing technologies has propelled the generation and refinement of a number of sample preparation and enrichment methods, data analysis pipelines and tools to effectively sequence and analyze genomes with maximum sensitivity and coverage. NGS approaches generate a large amount of data in a relatively short time, and this, along with tailored strategies for data analysis, can be extremely advantageous in both research and clinical diagnostics to provide insights into the genetic and molecular basis of phenotype and disease. As such, NGS technology aids not only in the characterization and classification of novel disorders but also in disease prognosis and tailored treatment. Furthermore, it provides the possibility to offer prenatal genetic diagnosis to affected families. The chapters that follow, explore the ability of NGS combined with different analysis strategies to successfully identify genes responsible for disorders with different genetic backgrounds and inheritance patterns.
While a classical linkage strategy followed by candidate gene sequencing was used to identify a novel premature stop codon in the GFI1B gene implicating it for the first time as a causative gene for the autosomal dominant form of the grey platelet syndrome, combinatorial strategies involving linkage analysis, WES, and targeted resequencing in specific autosomal dominant tubulo-interstitial kidney disease (ADTKD) cohorts aided in the identification of causative mutations in SEC61A1.
The importance of biochemical and metabolic profiling in highly heterogeneous autosomal recessive mitochondrial disorders coupled with the use of homozygosity mapping/and or WES to identify homozygous regions and the causative variants in genes located within these regions has also been highlighted via two studies: one identified mutations in the IBA57 gene, which is involved in the ISC biosynthesis pathway and is responsible for the assembly of the iron-sulphur cluster, essential for mitochondrial complex assembly implicating it for the first time in a novel mitochondrial disorder characterized by severe myopathy and encephalopathy. The other study revealed recessive mutations in SLC25A26, encoding a mitochondrial S-adenosylmethionine (mtSAM) transporter essential for methylation of a number of mitochondrial components including those involved in respiratory chain function confirming a novel disorder caused by an intra-mitochondrial deficiency, as a consequence of the identified mutations.
While a large number of disorders follow truly Mendelian patterns of inheritance with complete penetrance, a number of disorders display more complex inheritance patterns, which are usually confounded by incomplete penetrance and variable expressivity. In order to understand the genetic basis of one such disorder, namely Bicuspid Aortic Valve associated Thoracic Aortic Aneurysm (BAV-TAA), an in depth WES effort on a cohort of 220 BAV-TAA patients coupled with discrete filtering and specific gene stratification approaches was carried out. This was further followed by targeted resequencing in a replication cohort of 441 BAV-TAA individuals leading to the identification of six mutations in the Semaphorin 3C (SEMA3C) gene in total. SEMA3C is responsible for the guidance of cardiac neural crest cells upon entry into the outflow tract facilitating its septation. Assessment of the effects of the mutations on particular roles of SEMA3C, revealed dysregulation of the pathways resulting in a novel hypothesis of possible gene regulation via nuclear localization.
Although the vast advantages of NGS, there still remain several challenges with regard to gene identification in research and clinical diagnostics and ethical considerations that need to be taken into account during the return and management of results in clinic.