Whole-genome sequencing analyses of Africans with clefts 2.0-insights gained

Background: Harmful genetic mutations are known to contribute to the risk of the most common craniofacial anomaly, orofacial clefts (OFCs). However, even within families known to carry these mutations, the resulting manifestations can vary from no overt clefting to the presence of different cleft types that vary in severity and laterality. To unravel the genetic etiology of OFCs, which will inform diagnosis and genetic counseling, we conducted the second whole-genome sequencing (WGS) analysis on a large cohort of families with nonsyndromic cleft lip with or without cleft palate (nsCL/P) from Africa, the most genetically diverse human population.Methods: We sequenced the genomes of 148 case-parent trios, each including a child affected by nsCL/P. We investigated pathogenic protein-altering variants with CADD scores ≥ 20 and REVEL scores ≥ 0.5 as well as computed their clinical significance using criteria from the American College of Medical Genetics and Genomics / American Association of Molecular Pathology (ACMG/AMP). Genotype-phenotype analyses were performed using mouse gene expression and human genomics databases to link those pathogenic variants with cleft pathogenesis. Results: Our analyses revealed novel pathogenic protein-altering mutations in several key genes associated with lip and palate development including: KIF1B, QSOX1, MEGF6, FAP, SLC8A1, LRP2, TTN, FLNB, COL6A6, COL7A1, and CPO. Notably, mutations in LRP2 and TTN were previously reported in Africans with cleft following the first WGS analysis. While we identified variants in CTNNA2 (CADD scores > 20)which were reported as cleft risk gene in the first African genome-wide association study, they were non-pathogenic based on the ACMG criteria. The presence of these mutations in some unaffected parents suggests a multifaceted causative mechanism underlying OFC phenotypes. Conclusion: This study identifies new pathogenic variants contributing to OFCs, reinforcing the complex causative mechanism for the clinical manifestation of OFCs. These findings enhance our understanding of the mechanisms driving the most prevalent craniofacial birth defect, paving way for improved diagnosis and genetic counseling strategies.