A non-syndromic orofacial cleft risk locus links tRNA splicing defects to neural crest cell pathologies [RNA-seq]
Orofacial clefts are the most common form of congenital craniofacial malformations worldwide. The etiology of these birth defects is multifactorial, involving genetic and environmental factors. In most cases, however, the underlying causes remain unexplained, precluding molecular understanding of disease mechanisms. Here, we integrated genome-wide association data, targeted re-sequencing of case and control cohorts, cell type-specific epigenomic profiling, and genome architecture analyses, to functionally and molecularly dissect a genomic locus associated with an increased risk of non-syndromic orofacial cleft. We found that common and rare risk variants associated with orofacial cleft intersect with a conserved enhancer (e2p24.2) that becomes activated in cranial neural crest cells—the embryonic cell type responsible for sculpting the craniofacial complex. We mapped e2p24.2 long-range interactions to a topologically associated domain harboring MYCN and DDX1 and demonstrated that both MYCN and DDX1 are required for craniofacial development in chicken embryos. Molecularly, we found that e2p24.2 regulates the expression of MYCN, but not DDX1, in cranial neural crest cells. In turn, DDX1 is a target of the MYC family of transcription factors and a component of the tRNA splicing complex. The loss of DDX1 in cranial neural crest cells resulted in the accumulation of unspliced tRNA fragments, and impaired both global protein synthesis and cranial neural crest cell migration. We further showed that the induction of tRNA fragments is sufficient to disrupt craniofacial development. Together, these results uncovered a molecular mechanism in which impaired tRNA splicing, and the concomitant accumulation of tRNA fragments, affect neural crest and craniofacial development and positioned MYCN, DDX1, and tRNA processing defects as risk factors in the pathogenesis of orofacial clefts. Overall design: Bulk-RNA-sequencing experiments were performed in triplicates for DDX1 KO cNCCs cultured with 0.4 ng/mL DOX (DDX1 ON) and without DOX for 4 days (DDX1 OFF). cDNA libraries were prepared using the RNA sample preparation kit (TruSeq v2; Ilumina) as previously described (Respuela et al., 2016) and sequenced with a 2x 100-bp strand-specific protocol on a HiSeq 2500 sequencer (Illumina).  Basic read quality control was performed using FastQC (Babraham Bioinformatics) and read statistics were obtained with SAMtools. Reads (61-73 million per sample) were mapped to the human reference assembly (GRCh38.p13) using STAR Aligner (Dobin et al., 2013). Aligned reads were sorted with featureCounts (Liao et al., 2014), specifying for paired end reads. Differential expression analysis was performed using DESeq2 (Love et al., 2014), excluding low-count transcripts (<5).