Identification of methylation epialleles conferring susceptibility to cleft lip and palate using patient samples and mouse models for cleft palate

Abstract

Nonsyndromic cleft lip/palate (NSCL/P) is the most common congenital craniofacial malformation, with a prevalence of 1 affected individual to 700 live-births worldwide. NSCL/Ps are clinically variable, ranging from affected lip only to the dental alveolum and palate, uni or bilaterally. Heritability studies show a genetic contribution ranging from 40% to 85% and NSCL/P is classified as a complex and heterogeneous disease. Even though GWAS (Genome-Wide Association Study) was applied for NSCL/P genetic factors elucidation by several studies, the loci found barely explain the observed heritability. Meanwhile epidemiologic studies show an association of low socioeconomic status to NSCL/P, pointing out an important environmental contribution. Those data suggest that alternative mechanisms might be involved in the NSCL/P etiology, as dysregulation of epigenetic markers triggered by environmental factors. Aiming to better identify the NSCL/P etiologic factors, we propose the investigation of DNA methylation differences, as DNA methylation is one of the most common epigenetic mechanisms, between NSCL/P and control samples using an EWAS (Epigenome-Wide Association Study) approach. We hypothesized that DNA methylation sites act as epialleles (epigenetic alleles) in relevant genes for the craniofacial development and differences in their methylation levels predispose to NSCL/P. To test those hypotheses we initiate a methylome analysis with 67 NSCL/P and 59 control samples (peripheral blood DNA), using the Infinium Human Methylation 450K platform (Illumina), as a part of the current doctorate, in order to find differentially methylated regions (DMRs). Our preliminary results evidenced a significantly distinct epigenetic signature in NSCL/P samples, with 1000 DMRs, in which 28 belong to NSCL/P reported candidate genes and 128 belong to differentially methylated genes in murine palatal development. To generate conclusive results, sample size expansion and validation in an independent sample is fundamental. Therefore, our BEPE proposal is to investigate the candidate DMRs methylation levels in an expanded sample (N<500, peripheral blood DNA) and to validate the results in an independent sample with European origin (English). Next we intend to correlate the methylation level and their respective gene expression levels in palatal muscle samples of NSCL/P individuals in whom the peripheral blood DNA methylome were also studied. Further, as an evaluation of the methylation epialleles relevance to the clefting etiology, we will use a clefting animal model, the Chd7 mutant mouse, in which the penetrance of cleft palate is 35%. We will quantify the DMRs methylation levels in palatal shelves from penetrant and non-penetrant mutant Chd7 mice embryos to test whether those candidate DMRs could act as clefting modifier epialleles. In this step we will also correlate the methylation levels with gene expression levels. If our hypotheses are corroborated, our next step will be to identify the environmental and/or genetic factors capable of interfering in the epialleles, besides the verification whether those epigenetic alterations could be inherited and explain, at least partially, the high NSCL/P heritability. (AU)