Identification of conserved skeletal enhancers associated with craniosynostosis risk genes

Abstract

AbstractCraniosynostosis (CS) is a common congenital defect affecting more than 1/2000 infants. Infants with CS have a premature fusion of one or multiple cranial sutures resulting in restricted brain expansion. Single gene mutations account for 15-20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. Two noncoding genomic regions contributing to CS risk were previously identified by GWAS, one near BMP2 and one within BBS9. We hypothesized that the region within BBS9 contains distal regulatory elements controlling the neighboring gene encoding BMPER, a secreted modulator of BMP signaling. To identify regulatory sequences that might underlie disease risk, we surveyed conserved noncoding sequences from both risk loci identified from the GWAS for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous gene expression. Importantly, for each locus, we found a skeletal enhancer that also contains a sequence variant associated with CS risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. We used an enhanced yeast one-hybrid assay to identify transcription factor interactions with several identified enhancers, implicating multiple signaling pathways in their regulation. In a targeted screen focused on risk-associated SNPs, we further identified differential binding to alternate and reference alleles. Additionally, we found that the risk allele of the BMPER enhancer directs significantly broader expression than the reference allele in transgenic zebrafish. Our findings support a specific genetic mechanism to explain the contribution of two risk loci to CS. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.Author SummaryGenome–wide association studies (GWASs) provide a wealth of information implicating regions of the genome in disease risk. The great challenge is linking those regions to specific genetic mechanisms. We used complementary approaches in zebrafish and yeast to evaluate the genetic risk of craniosynostosis (CS), a craniofacial birth defect affecting 1/2000 infants where two or more skull bones are fused prematurely. Using transgenic zebrafish, we identified sequences regulating expression of two genes in the BMP signaling pathway that had been previously implicated by GWAS. These included one from each region containing a sequence variant linked to disease risk. We used an assay in cultured yeast to detect proteins binding to identified DNA sequences that could alter expression of the target genes, including changes in protein binding caused by the sequence variants. Finally, we found that transgenic fish carrying one of the variant sequences showed broader and more sustained activity throughout the skeleton. Taken together, our results support a model where variant sequences lead to increased gene expression and BMP pathway activity, contributing to aberrant skull growth in CS. Importantly, our paradigm is broadly applicable to other complex genetic diseases, potentially illuminating many connections between genome variation and disease risk.