Association of DOT1L histone methyltransferase with non-syndromic orofacial clefts in a cohort of children of African and Asian ancestry

Non-syndromic orofacial clefts (nsOFCs), such as cleft lip, cleft lip with or without cleft palate (CL/P), and cleft palate only, are among the most common craniofacial malformations in humans. We identified a range of rare and de novo structural variants (SVs) associated with nsOFCs in a cohort of children of African and Asian ancestry from the Gabriella Miller Kids First Data Resource Center. Most of these novel candidate SVs are located in non-coding regions. More specifically, we characterized SVs associated with nsOFCs across the DOT1L (disruptor of telomeric silencing 1-like) genomic locus. DOT1L is the sole histone methyltransferase that catalyzes the mono-, di-, and trimethylation of histone H3 at lysine 79 (H3K79). In addition, our data revealed associations between specific SVs and genes encoding DOT1L complex subunits as well as downstream target genes. Single-nuclear RNA sequencing analysis demonstrated that mouse Dot1L and its associated targets are highly expressed in neural crest-derived mesenchyme enriched for key osteogenic genes essential for palate formation. Depletion of a Dot1L allele resulted in cleft palate and micrognathia in mice. Furthermore, we identified several families carrying SVs in the DOT1L locus and downstream DOT1L targets genes. These findings support an oligogenic model in which the concurrent presence of several SVs enhances susceptibility to the nsOFC phenotype. We propose that DOT1L and its targets act within the same genetic pathways to influence CL/P such that the combined effects of variants affecting these genes may be more substantial than their individual effects. Collectively, these studies establish a pivotal role for DOT1L-mediated H3K79 methylation in craniofacial development and palate formation and identify this pathway as a key contributor to the etiology of CL/P.