Significance of Genetic Mutations in the Molecular Pathogenesis of Human Orofacial Clefts in Ghana

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Human orofacial clefts (OFCs) are congenital craniofacial dysmorphologies, with a global prevalence of 1 per 700 live births. OFCs may be syndromic or nonsyndromic, with the syndromic forms presenting with extra congenital anomalies. Nonsyndromic OFCs (NSOFCs) account for about 70% of all OFCs. The aetiology of the more common NSOFCs is complex, with both genes and environmental factors playing vital roles. The main objective of the present study was to demonstrate association between genetic variants and the pathogenesis of OFCs in Ghanaians. The specific objectives were fivefold: (i) determine pattern of inheritance of OFCs, (ii) establish the role of environmental and other risk factors in OFC aetiology, (iii) ascertain genetic susceptibility loci for OFCs, (iv) detect rare aetiologic mutations in affected individuals and, (v) subsequently validate the pathogenic mechanism of action of some selected rare variants in zebrafish embryos. A questionnaire was administered to participating families mainly at KATH to collect environmental and phenotypic data. Saliva and cheek swab samples were collected from participants using Oragene DNA collection kits. DNA was extracted from samples using Oragene saliva processing protocol and Quibit assay was used to quantify extracted DNA, with subsequent validation of sexes of participants through XY-Genotyping that employed Real Time PCR. A total of 3,585 individuals (872 cases, 1635 relatives and 1078 unrelated controls) were genotyped at 48 SNPs using Fluidigm SNP Genotyping Protocol. Eight genes were also directly sequenced in 184 NSOFC cases and IRF6 gene only on 80 individuals with multiple congenital anomalies (MCAs), using Sanger sequencing technology. Primers used for DNA sequencing were designed with Primer3 software based on human genome assembly GRCh37/hg19, 2009 (, followed by ascertainment of their optimal PCR conditions through Gradient PCR. Initial PCR was used to amplify exons and flanking intronic sequences as well as 5’ and 3’ untranslated regions (UTRs) of the eight genes. PCR amplicons were examined by gel electrophoresis. The Initial PCR product was then sequenced with ABI 3730XL DNA Sequencer. Employing genetic engineering, site-directed mutagenesis and developmental biology techniques, two selected variants of IRF6 were also functionally validated in zebrafish embryos. The study established multifactorial pattern of inheritance for OFCs in Ghana. Low socio-economic status, delayed antenatal attendance and folate deficiency were significantly associated with families with OFCs by increasing OFC susceptibility. In SNP association studies, case-control meta-analyses demonstrated that PAX7 (rs742071, p=5.10×10-03), 8q24 (rs987525, p=1.22×10-03) and VAX1 (rs7078160, p=0.04) were nominally associated with NSCL/P. MSX1 (rs115200552, p=0.01), TULP4 (rs651333, p=0.04), CRISPLD2 (rs4783099, p=0.02) and NOG (rs17760296, p=0.04) were nominally associated with NSCP. Many other loci exhibited threshold association with NSOFCs in TDT and DFAM analyses. Coding, splice site and/or regulatory region variants were observed in all eight sequenced genes. Novel pathogenic mutations were observed in both NSOFCs (p.Glu69Lys, p.Asn185Thr and c.175-2A>C) and MCA (p.Gly65Val, p.Lys320Asn and c.379+1 G>T) cases in IRF6, including probable genetic modifiers. In functional validation of p.Glu69Lys and p.Gly65Val, these variants, through dominantnegative effect, disrupted craniofacial structures, such as pharyngeal arches, in zebrafish. These observations are relevant for prenatal diagnosis of high risk families, understanding the genetic architecture of OFCs and genetic counseling. This is the first ever genetic study on OFCs in Ghana and also the most extensive done in African populations on the African continent to date. The study has for the first time, demonstrated associations between the studied genetic loci and NSOFCs among continental Africans, with striking racial differences.
A Thesis submitted to the Department of Biochemistry and Biotechnology, College of Science, in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY