Identificazione di loci di suscettibilitá ed interazioni epistatiche associate allo sviluppo di schisi orofacciali isolate

Orofacial clefts (OFCs) are the most common congenital malformations (1/700 live births), second only to clubfoot. OFCs have a very significant impact on healthcare and knowing their etiology is essential for genetic counseling, setting prevention strategies and improving therapeutic interventions. OFCs include cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO). CL/P and CPO can be part of a number of monogenic and chromosomal syndromes, but in most cases occur as isolated birth defects (non-syndromic), assignable to complex diseases such as multifactorial etiology. Their causes are still largely unknown and are believed to be due to an interplay of environmental and genetic factors. The specific aim of this research is to track down genetic variants with susceptibility to OFCs and to identify the network of gene-gene and gene-environment interactions at the base of the etiology of non-syndromic OFCs. This research was carried out using a large dataset of European triads from the EUROCRAN and ITALCLEFT projects. I initially applied a combined positional/candidate gene approach studying the IRF6 gene. This association study resulted in the identification of the rs642961G> A gene variant, which causes the loss of a AP-2α site and associates with an increased risk for cleft lip. rs642961 has been considered the first functional variant involved in the pathogenesis of non-syndromic cleft lip. In an independent study on European CPO trios, using a candidate gene strategy, I identified a significant association between variants in the FAF1 gene and the risk for isolated Cleft Palate. FAF1 is so far the only gene associated with CPO. Next, I set up a genome-wide approach, studying a panel of approximately 550,000 SNPs covering the entire genome. This study of genome-wide association (GWA) study resulted in the identification of a region of 640 kb on chromosome 8q24.21, among which the rs987525 variant was identified as the SNP with maximum risk for CL/P (p = 3.34*10-24). rs987525 is located in a gene desert and its function completely unknown. Its relevance was later confirmed by two other independent GWA studies. The GWA analysis was later extended to identify other major susceptibility loci for CL/P. By replicating the study on a dataset of parental triads I identified other 4 gene variants associated with significant risk for CL/P. These polymorphisms are located near genes invlved the TGFβ-pathway, in the craniofacial development and cancerogenesis. Thereafter, to look for gene-gene and gene-environment interactions, I used a strategy of data-mining (MDR). It came out that the variant located near NOG gene was interacting with maternal exposure to tobacco smoke during the first trimester of pregnancy. The MDR analysis also identified a remarkable multiplicative interaction between variants in SPRY2 and GREM1 genes. This is the first evidence ever documented of an epistatic interaction in the etiology of isolated CL/P. These experimental results pave the way for translation into genetic counseling practice, and the base for new strategies of primary prevention of non-syndromic OFCs.