Two Unrelated Families With Noncoding Duplications Upstream of MSX2 Refine the Critical Regulatory Region Likely Involved in Cranial Bone Development and a Cleidocranial Dysplasia‐Like Phenotype

Abstract

Cleidocranial dysplasia (CCD) is a genetic disorder characterized by delayed cranial suture closure, hypoplastic clavicles, and dental anomalies, with varying severity. Most cases are linked to RUNX2 variants; however, rare CCD‐like phenotypes can arise from other genetic alterations, including variants in MSX2, a critical skeletal development gene. Here, we describe two patients with persistent anterior fontanelles and microduplications upstream of MSX2, a region previously associated with CCD‐like phenotypes. Microarray analysis revealed narrower duplication ranges in these patients than in two earlier reported cases. To elucidate the underlying mechanisms, we performed in silico analyses using publicly available datasets. Epigenetic characterization of the minimal overlapping duplicated region (chr5:173848716–173888281) identified two transcriptionally active subregions showing high chromatin accessibility in osteoblasts. These findings suggest a regulatory role in osteogenic differentiation. Differential chromatin accessibility analysis using ATAC‐seq data demonstrated preferential accessibility in osteoblasts over chondrocytes in both human and mouse models, further implicating this region in bone development. The duplicated region was fully contained within a single topologically associating domain (TAD), suggesting that intra‐TAD duplications may disrupt spatial and temporal MSX2 regulation, contributing to the CCD‐like phenotype. Phenotypic variations, such as synpolydactyly in a previously reported patient, likely arise from additional regulatory elements outside the minimal overlapping region. Our findings establish a causal link between MSX2 upstream duplications and CCD‐like phenotypes, emphasizing the role of epigenetic mechanisms and TAD structures in regulating skeletal development. Further studies, including long‐read sequencing and ChIP‐seq, are needed to clarify the precise regulatory pathways involved.