Integrative genomics reveals that cis-regulatory mutations cause isolated pancreatic agenesis

CEBOLA, I.; WEEDON, M.N.; PATCH, A.M.; FLANAGAN, S.; DE FRANCO, E.; CASWELL, R.; RODRÍGUEZ SEGUÍ, S.A.; SHAW-SMITH, C.; CHO, C.; ALLEN, H.L.; HOUGHTON, J.; ROTH, C.L.; CHEN, R.; HUSSAIN, K.; MARSH, P.; VALLIER, L.; MURRAY, A.; ELLARD, S.; FERRER, J.; HATTERSLEY, A.T.

Sitges

Encuentro; EASD-Islet Study Group Annual Meeting 2013; 2013

EASD-Islet Study Group

Most of the disease-causing mutations described to the date are located in protein-coding sequences and promoters. Nevertheless, recent initiatives from the ENCODE and others have pinpointed that the non-coding fraction of the genome, in particular distal regulatory elements, may comprise a large collection of functional variants.Recessive mutations in PDX1 are the only known cause of non-syndromic pancreatic agenesis, but the cause of the great majority of cases remains unknown. In this work, whole genome sequencing was performed in 2 probands from consanguineous families with non-syndromic pancreatic agenesis. This failed to show candidate recessive coding mutations, but revealed 6,024 non-coding novel or rare variants (< 1% frequency in the 1000 Genomes Project) . To annotate non-coding regions that might harbour functional variants relevant to human pancreas development, we created a cis-regulatory map of hESC-derived pancreatic progenitor cells. Remarkably, only one recessive variant from both individuals mapped to an annotated element. This variant lies in a putative distal enhancer in the PTF1A locus, and matches a region previously mapped by homozygosity mapping in 3 unrelated consanguineous families. In posterior analyses, 4 additional point mutations and a 8kb deletion encompassing this element were identified in 9 out of 12 patients with isolated pancreas agenesis. We determined that this element truly acts as an enhancer in human pancreatic progenitor cells using chromosome conformation capture analysis and episomal reporter assays. Furthermore, all of the point mutations disrupt sequence-specific binding of pancreatic progenitor nuclear proteins, and impair the activity of the enhancer in reporter assays. These results show that cis-regulatory mutations are the most common cause of non-syndromic pancreatic agenesis and demonstrate how the integration of whole genome sequencing, linkage and epigenetic annotation in a disease-relevant cell type can unveil novel and important functional variants for human development and disease.