A variant associated with sagittal nonsyndromic craniosynostosis alters the regulatory function of a non‐coding element

Abstract

Craniosynostosis presents either as a nonsyndromic congenital anomaly or as a finding in nearly 200 genetic syndromes. Our previous genome-wide association study of sagittal nonsyndromic craniosynostosis identified associations with variants downstream from BMP2 and intronic in BBS9. Because no coding variants in BMP2 were identified, we hypothesized that conserved non-coding regulatory elements may alter BMP2 expression. In order to identify and characterize noncoding regulatory elements near BMP2, two conserved noncoding regions near the associated region on chromosome 20 were tested for regulatory activity with a Renilla luciferase assay. For a 711 base pair noncoding fragment encompassing the most strongly associated variant, rs1884302, the luciferase assay showed that the risk allele (C) of rs1884302 drives higher expression of the reporter than the common allele (T). When this same DNA fragment was tested in zebrafish transgenesis studies, a strikingly different expression pattern of the green fluorescent reporter was observed depending on whether the transgenic fish had the risk (C) or the common (T) allele at rs1884302. The in vitro results suggest that altered BMP2 regulatory function at rs1884302 may contribute to the etiology of sagittal nonsyndromic craniosynostosis. The in vivo results indicate that differences in regulatory activity depend on the presence of a C or T allele at rs1884302.

SMAD6 variants in craniosynostosis: genotype and phenotype evaluation

“…13,14,17 To seek an explanation for the unpredictable penetrance, Timberlake et al 4,18 genotyped a single-nucleotide polymorphism (SNP), rs1884302, previously reported in a genome-wide association study (GWAS) of nonsyndromic SS to be the most significant associated SNP, which may differentially regulate the most proximal gene BMP2. 19,20 The risk-conferring C allele (prevalence in non-Finnish Europeans of 32.7%, gnomAD), 21 was found to be present in 15/21 individuals with CRS but only 1/20 unaffected relatives heterozygous for the SMAD6 variant but without CRS, suggesting a two-locus mechanism to account for variable manifestation of CRS. 18 Although the studies described above 4,18 represent an important advance in delineating the contribution of singlegene variants to nonsyndromic midline CRS, they raise several questions.…”

Identification of conserved skeletal enhancers associated with craniosynostosis risk genes

“…This region includes the risk locus in BBS9 identified from the GWAS for sagittal NCS (4) and the entire intergenic region between BBS9 and BMPER ( Figure 1A ). We also selected sequences in the risk locus downstream of BMP2 , including one containing rs18843302, which was previously suggested to act as an enhancer (19) ( Figure 1B ). The primary criterion for selection was conservation across species.…”

The genetic overlap between osteoporosis and craniosynostosis

“…This study identified novel, strong associations to BMP2 and BBS9 ( 88 ). Functional studies using zebrafish demonstrated that rs1884302 in the vicinity of BMP2 acts as an enhancer ( 89 ). In 2020, Justice et al.…”

“…This permits in vivo cell traceability while the skeleton is formed using reporter lines labeling specific cell types (i.e., osteoblasts, osteoclasts, and chondrocytes) ( 14 , 126 ). Zebrafish cranial bones and sutures are homologous to those in mammals, representing the only vertebrates enabling non-invasive in vivo visualization of cranial bones growth and suture formation ( 55 , 128 ), therefore of great interest for models of craniosynostosis ( 89 , 142 , 143 ). Zebrafish develop the same sutures as humans and mice ( 55 , 128 ) ( Figures 4A, B ).…”