Hirschsprung disease as a model to determine the contribution of rare variants to polygenic diseases

Understanding complex or polygenic diseases has become a major topic in the field of human genetics. Most research groups in this field work with the hypothesis of “common disease, common variant”, which suggests that complex diseases are caused by a large set of common but weak disease-associated variants. To find these common variants, Genome-wide association studies (GWAS) are being undertaken to successfully detect the genomic loci and genes therein in many common, complex diseases. However, the identified loci usually only account for a fraction of the total genetic risk. In this project we will work with a complementary hypothesis, namely that besides many common weak mutations, complex diseases also need several rare, and probably stronger, mutations. To test this hypothesis we will work on a highly heritable, complex disorder called Hirschsprung disease (HSCR). This is the most common form of congenital obstruction of the bowel. HSCR results from a failure of the enteric nervous system progenitors, the neural crest stem cells, to migrate, proliferate, differentiate or survive in the bowel wall, resulting in an aganglionic part of the colon. This results in clinically severe and sometimes even life-threatening constipation. The reasons for these failures in neural crest cell development are not yet well understood. As HSCR is almost 100% heritable, genetic variation (mutations) in the genomes of patients will largely contribute to disease development. Although we and others have so far identified 10 HSCR susceptibility genes and 6 linkage regions and a GWAS study by our consortium has revealed a few common disease-associated variants, the mutations and variations identified explain no more than 25% of the overall genetic risk. Thus, the vast majority of cases (mostly patients with only HSCR) cannot genetically be explained by the identified mutations or associations yet. These findings led us to hypothesize that, in patients with HSCR, the majority of the disease risk may be explained by combinations of rare coding or non-coding variants in the identified and other unknown genes, variants that cannot be detected by genome-wide association studies, but need direct sequencing for detection. Sequencing large numbers of candidate genes or entire genomes in patient populations was almost impossible until recently. However, new technological breakthroughs now make it is possible to sequence entire genomes or entire exomes, i.e. all the coding sequences of a genome. For this project we will combine this new technology together with expression data generated on enteric nervous system progenitors (in both man and mouse) and use the functional analysis of potential candidate disease genes in vitro and in vivo to prove our hypothesis. Not only will we be able to unravel the genetic background of HSCR but, more significantly, it might also serve as a model for other complex diseases and establish the importance of combinations of rare coding and non-coding variants in complex disease. We have two major aims: -Our first aim is to identify combinations of rare coding mutations in not yet identified disease genes. To do this, we will perform exome-sequencing of all the coding DNA sequences (~37 million base pairs/patient). We will apply this to a large group of 50 familial, non-syndromic HSCR cases, which do not have a mutation in one of the known HSCR susceptibility genes. As we will find many DNA variants, we need a method to select the most promising candidates. This will include the in silico prediction of the mutations found; the presence of mutations in multiple families; the expression of the gene in enteric nervous system progenitors; the function of the encoded proteins, and segregation analysis. All candidate-HSCR genes that pass all these criteria will be validated in an in vitro and an in vivo differentiation screen. Candidate HSCR genes that also pass the last experiments will then be sequenced in a cohort of 500 non-syndromic HSCR patients. -Our second aim is to find out whether rare, disease-associated non-coding sequence variants make a major contribution to disease development. For this we will focus on the major HSCR gene RET. To find these non-coding DNA variants we will apply next generation sequencing on all the highly conserved sequences in the RET region in a cohort of 500 HSCR patients. If we have strong indications from in silico analysis and statistical work for an involvement of these variants in HSCR, we will characterize these variants functionally. If we can show that non-coding RET variants play a major role in the disease development, we will screen for such mutations in the other HSCR-associated genes, if time allows. Our project will establish the role of rare variants in HSCR and, more generally, provide insight into the role these rare variants may play in complex diseases.