2. Pathophysiology of cleft lip and palate
Since IRF6 causes VWS that has a phenotype strongly overlapping with isolated CL/P and it showed strong association to nonsyndromic CL/P in an American study (Zucchero et al., 2004), we studied its role in a set of 195 trios with isolated CL/P from the Belgian population. Transmission disequilibrium analysis confirmed that the IRF6 locus is associated with nonsyndromic CL/P in Northern Europe. It is likely that association with this locus can be identified in various populations and that the IRF6 locus represents an important genetic modifier for clefts.
Since candidate gene approach has given limited results in unraveling major causative genes for isolated nonsyndromic CL/P, additional approaches are needed. We conducted a genome-wide scan combining karyotyping and Affymetrix 50K SNP arrays. Interestingly, one patient and his affected father showed a reciprocal translocation. Both had a Pierre Robin sequence (PRS), characterized by CPO, micro/retrognathia, malar hypoplasia, microstomia and dental anomalies (Ghassibe et al, unpublished). Fine mapping of the breakpoints showed the disruption of a new gene. We tested association of the locus with isolated nonsyndromic clefts and found that it is associated to CPO andmay contribute to as much as 2.4-3.75% of all CPO patients, including PRS. By in situ hybridization on mouse tissues, we showed high levels of the mRNA along the medial edge epithelium of the fusing palate, at the fusing superior lips, the palatal blastemata and the tongue. The same approach allowed us to detect expression in the pharyngeal cartilages of the zebrafish larvae. Finally, a knockdown of the gene expression in zebrafish larvae showed that the only anomaly that the larvae develop is an orofacial defect. We are currently trying to elucidate the mechanism by which the disruption of this gene would lead to a developmental craniofacial defect in zebrafish, mice and men.
In parallel, association studies are being conducted on a large series of patients and their parents. The aim is to investigate all the known candidate genes/loci and to determine at which percentage each locus contributes to the cleft occurrence in our cohort. TDT and case control studies would allow us to unravel contributing predisposing loci. This technique, coupled with high-through put sequencing will allow us to dissect complex regulatory, conserved regions and determine what is the genomic alteration behind the phenotypic expression.
Besides the ongoing projects, collecting DNA and tissue samples, as well as clinical data continues in parallel. Karyotyping and/or molecular karyotyping is performed for familial and syndromic cases. Clinical details are recorded to enable genotype-phenotype comparisons. This approach has been very successful and will keep on giving us new avenues for further research and investigations.
To know more... (pdf chapter of the last de Duve Institute report)
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