Abstract
BACKGROUND: Orofacial clefts (OFCs) are the most frequent congenital craniofacial anomalies that occur during embryonic development. The incidence is ~1 in 700 live births; it may occur in isolation or with other abnormalities, such as limb deformities. Congenital limb malformations are the second most prevalent birth defect, affecting 1 per 500 to 1000 live births. It can also occur in isolation or as part of a syndrome. This study investigated the genetic aetiology of OFCs co-occurring with limb abnormalities in a Sub-Saharan African cohort. METHODS: Nine unrelated probands with concurrent OFC and limb anomalies were recruited, including one multiplex family involving an affected mother and proband. Whole exome sequencing (WES) was performed at 100X on the DNA samples obtained from affected families, utilising paired-end configuration on the Illumina HiSeq platform. Variant calling utilized the Sentieon workflow. Rare, deleterious variants were identified in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines on variant classification. De novo and other variants predicted as pathogenic were prioritized based on all possible Mendelian inheritance patterns, including variable penetrance and expressivity. Pathway enrichment analysis, protein-protein interactions, and gene expression analysis were undertaken to decipher the biological functions of implicated genes. RESULTS: All cases were syndromic, presenting with preaxial and postaxial limb anomalies along with other craniofacial features. WES revealed plausible pathogenic variants in pleiotropic genes (TP63, NIPBL, MYH3, FGFR2) in four simplex cases. In four other simplex probands, multiple rare variants were identified in developmentally relevant genes (e.g., RGPD5, FAM90A26, FOXD4L1, FAM170A, DLG1, ANKRD1, TRIM74, TRIM73, PRDM9) necessary for normal craniofacial and limb development. The multiplex family had two affected individuals (the mother and the proband), both carrying a TP63 variant, consistent with autosomal dominant inheritance with variable expressivity. Most of the observed variants were de novo, with some being novel. CONCLUSION: While some cases can be attributed to single-gene syndromes (e.g., NIPBL-associated Cornelia de Lange Syndrome), others may result from multiple co-occurring syndromes. These findings will inform recurrence risk estimates, genetic counselling, and clinical management.