Next generation sequencing analysis : from monogenic to polygenic disorders

Abstract

Since the introduction of Next Generation Sequencing(NGS), it has quickly become a popular tool for studying monogenic and polygenic disorders. Genetic architecture between monogenic and polygenic disorders differs significantly, posing unique analysis challenges. By studying4 different disorders in this thesis, I have attempted to demonstrate the strength of NGS and the analysis approaches towards NGS data across very different disorders.

Diffuse oesophageal leiomyomatosis(DOL) is a monogenic disorder displaying an X-Link dominant inheritance. In the case study described, I have studied 4 members of the family with mother and son affected. The whole exome sequencing analysis coupled with genotyping array resulted in the identification of a new COL4A5/6 deletion shared by2 affected individuals. The difficulty in detecting gonosomal mosaicism on the affected mother revealed the weakness of current analysis methods. A similar deletion was described in previous studies and could destabilize the collagen IV monomer and induce leiomyomatosis.

The three disorders discussed after, including choledochal cyst(CDD), mesial temporal lobe epilepsy related to hippocampal sclerosis(MTLE-HS),and hepatocellular carcinoma(HCC)are examples of polygenic disorders. Polygenic disorders often present a complex genetic architecture affected by a combination of genetic and environmental factors. In the cases of MTLE-HS and CDD, both demonstrated a complex and heterogeneous genetic profile where a vast number of genes liable to the disorder have been discovered. For the MTLE-HS study, 23 trios were studied to investigate inherited rare recessive mutations and de novo mutations. As aresult,PKD1 and CEP170Bwerefound to be recurrently mutated across patients, multiple listed de novo variants were also found on genes liable to psychiatric disorder. The study on 33 CDD trios suggested amore complex genetic architecture of the disorder. Three different analysis approaches to detection of candidate mutations resulted in finding double hit rare variants, de novo mutations, and an overall enrichment of rare variants compared to the normal population. The3 approaches identified 31 genes recurrently mutated in the double hit setting, 21 genes carrying de novo damaging variants, and significant enrichment in the interacting gene pair TRIM28/ZNF382(p<3.6x10-6). To further understand the biology behind the vast number of genes involved, multiple functional databases on pathways, mouse knockout phenotypes, and human disease were queried.

The capability of NGS is beyond simple mutations on DNA level. As demonstrated by the HCC study, a malicious and recurrent intronic Hepatitis B Virus(HBV) integration was uncovered. Although this interesting event did not fall within the coding region, the effect is only observable after post-transcriptional splicing. By coupling transcriptome sequencing, Sanger sequencing and functional studies, a large number of HCC patients affected by a non-degradable CCNA2 protein encoded by a 177 b.p. long insertion from HBV have been identified. The study highlighted capability NGS in studying the transcriptome, which could help understanding complex mutations in diseases.

This thesis has demonstrated the unprecedented capability of NGS in understanding various types of disorders by the analysis of 4 diseases with the substantial difference in their genetic architecture and the underlying type of variants.