Investigation of the etiological relevance of novel candidate genes to orofacial clefts

• Grant number: 16/23648-4
• Support type: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): May 01, 2017
• Effective date (End): April 30, 2020
• Field of knowledge: Biological Sciences - Genetics
• Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Maria Rita dos Santos e Passos Bueno
• Grantee: Luciano Abreu Brito
• Home Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/08028-1 - CEGH-CEL - Human Genome and Stem Cell Research Center, AP.CEPID

Abstract

Orofacial clefts comprehend a heterogeneous group of defects of the lip and palate, of which the most prevalent subgroup is nonsyndromic cleft lip with or without cleft palate (NS CL/P). NS CL/P is a complex disease highly genetically determined. However, the major associated loci are common variants that do not account for the disease's heritability. Given the burden on health system represented by NS CL/P, research groups have sought for its missing heritability. In this context, the role of rare, high-effect variants has been explored, mainly by exome sequencing studies. However, these studies often result in a group of variants of uncertain significance ("VUS") and mining the disease-causing ones is not trivial. This project aims to study the etiological contribution of rare variants located at new candidate genes for NS CL/P, using, as a starting point, VUS previously obtained by exome analysis conducted by our group. To achieve this, we will perform sequencing analysis of VUS-containing genes in an expanded sample of patients and functional analysis in zebrafish (Danio rerio). We herein propose 3 specific objectives:1- To determine the etiological contribution of these new candidate genes by sequencing them in 300 patients and looking for novel possibly pathogenic rare variants. In addition, we will perform rare-variant association tests, comparing the aggregation of rare variants in these genes between patients and 609 local controls. 2- To functionally determine the pathogenicity of the missense VUS uncovered in our patients, using zebrafish embryos. In this regard, we will overexpress human mRNAs harboring the VUS in the embryos, and analyze possible developmental alterations during the first 48h. We are confident that this approach can result in developmental impairment or embryo lethality, since the VUS are located at genes expressed since early embryogenesis.3- To investigate the genetic mechanisms underlying NS CL/P caused by rare variant in CDH1 (E-cadherin gene). CDH1 been subject of study by our group, and an etiological in NS CL/P role has been recently described by us. Here, we will evaluate the two-hit hypothesis (one germline and another somatic hit occurring during the palatogenesis) to explain the cleft risk associated to CDH1 mutations. To test this hypothesis, we will use a zebrafish lineage harboring a mutation that truncates one allele of cdh1 (orthologous of human CDH1). As this mutation is lethal in homozygosity, we will overexpress cdh1 wild-type mRNA in the offspring of an intercross of heterozygous fish, in order to postpone the double-knockout death, and analyze their craniofacial development (from the fourth day post-fertilization on). We will also evaluate the hypothesis of a second hit due to exposure to environmental susceptibility factors. In this regard, we will induce methylation of cdh1 promoter using growth medium treated with interleukin 1-beta - a factor highly correlated with cdh1 promoter methylation - and the phenotypes will be analyzed by staining for bone and cartilage. Finally, in the case of the functional approaches above proposed prove unsuccessful, we will perform a craniofacial-specific genome editing approach mediated by CRISPR/Cas9 (in order to avoid the possible lethality of double-knockout fish, as observed for cdh1). At the end of this project, we will contribute for expanding the landscape of known genes contributing with rare variants to NS CL/P etiology, as well as characterizing the respective genetic mechanisms involved. In addition, our results can help to elaborate predictive diagnosis, i.e., identifying, with molecular tools, individuals with higher risk of having children with NS CL/P, as well as evaluate the contribution of epigenetic in the penetrance of NS CL/P. Finally, we intend to establish a straightforward method for screening of missense VUS of craniofacial disorder, which can be useful in the routine of genomic analyses on these diseases. (AU)