The next generation of targeted mutation discovery

Sequencing technologies (NGS) now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. In Chapter 2, we present our results with a genomic DNA pooling strategy for rare variant discovery on a NGS platform. The high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples are the limiting factors for successful variant confirmation. Alternatively, in Chapter 3, we present a detailed protocol for a new strategy for barcoded (indexed) targeted re-sequencing of multiple samples. We demonstrated the effectiveness of our method on a wide range of applications starting from the enrichment and re-sequencing of 96 patients in a single assay for a limited number of genes to the full human exome projects. In Chapter 4, thanks to trio exome sequencing we show that Cantu syndrome is caused by mutations in ABCC9. Electrophysiologic studies demonstrated that the mutant channels reduce ATP-mediated potassium channel inhibition, resulting in channel opening. Given the availability of ABCC9 antagonists (e.g. glibenclamide), our findings may have direct implications for the treatment of patients with Cantu syndrome. In Chapter 5, we characterized a novel chromosomal instability syndrome and using family-based exome sequencing complemented with homozygosity mapping we detected a damaging variant in NDNL2. For the first time, NDNL2 gene was linked to human disease in spite of the fact, that function of the structural maintenance of chromosomes complex SMC5/6 was already known. In Chapter 6, we show the results of a comprehensive analysis of the largest series to date on ablepharon-macrostomia syndrome (AMS) which could, however, not identify a causal variant. Although this is essentially a negative result, it expanded our knowledge about the distinctness of AMS as a genetic entity. In Chapter 7, we describe a large Dutch family with seven males affected by a novel syndrome of X-linked intellectual disability. By performing a highly multiplexed next-generation sequencing on the exome of the chromosome X, we discovered a novel variant in HDAC8 introducing a premature stop at the beginning of its catalytic domain. In Chapter 8, we detected a missense germline mutation in PIGA gene causing an X-linked syndrome of accelerated growth, severe developmental delay, elevated alkaline phosphatase levels and CNS abnormalities. In Chapter 9, we discovered two large families with thoracic aortic aneurysm and patent ductus arteriosus with an incomplete segregation of the disease with a diagnostically-determined damaging variant in a known gene (MYH11). Caution should be exercised when family members who do not carry a family-specific MYH11 variant are counseled. In Chapter 10, we identified a novel candidate locus for thoracic aortic aneurysm and dissection (TAAD) on chromosome 7p15 in one of the previously described families in Chapter 9. In spite of a deep resequencing of the exome in this locus using our targeted re-sequencing method we failed to identify the causal variant