Copy number variation analysis in single-suture craniosynostosis: multiple rare variants including RUNX2 duplication in two cousins with metopic craniosynostosis

Abstract

Little is known about genes that underlie isolated single-suture craniosynostosis. In this study, we hypothesize that rare copy number variants (CNV) in patients with isolated single-suture craniosynostosis contain genes important for cranial development. Using whole genome array comparative genomic hybridization (CGH), we evaluated DNA from 186 individuals with single-suture craniosynostosis for submicroscopic deletions and duplications. We identified a 1.1 Mb duplication encompassing RUNX2 in two affected cousins with metopic synostosis and hypodontia. Given that RUNX2 is required as a master switch for osteoblast differentiation and interacts with TWIST1, mutations in which also cause craniosynostosis, we conclude that the duplication in this family is pathogenic, albeit with reduced penetrance. In addition, we find that a total of 7.5% of individuals with single-suture synostosis in our series have at least one rare deletion or duplication that contains genes and that has not been previously reported in unaffected individuals. The genes within and disrupted by CNVs in this cohort are potential novel candidate genes for craniosynostosis.

FIG. 1

Duplication of chromosome 6p21 encompassing RUNX2 in two affected cousins. A: Oligonucleotide array CGH results for cases 1007 and 1019 for chr6:44,700,000–46,500,000 (NCBI Build 36). For each individual, deviations of probe log₂ ratios from zero are depicted by gray/black lines, with those exceeding a threshold of 1.5 standard deviations from the mean probe ratio colored green and red to represent relative gains and losses, respectively. Yellow shading represents extent of duplication based on follow-up high-density array CGH validation. Red bars show the mapped location of fosmids used for FISH. B: Pedigree showing relationship of cases 1007 and 1019 and phenotypic features. The father of 1019 is a presumed carrier of the duplication but DNA was not available for analysis. C: FISH analysis using two fosmid probes shows that the duplication is tandem (see metaphase spread, upper left) and inverted (see interphase nucleus, lower right). Results are shown for patient 1019; similar results were obtained for patient 1007. White arrows indicate chromosome 6 homolog carrying the inverted duplication. The normal chromosome 6 in the metaphase spread is indicated by the “>.”

FIG. 2

Oligonucleotide array CGH results for (A) case 4038 with 3.9 Mb deletion of 9q22; (B) case 2082 with 2.5 Mb duplication of 5p15, (C) case 1056 with 3.3 Mb duplication of 3p25 and (D) case 1061 with a 1.6 Mb duplication of 1q43. Results are presented as in Figure 1.