A novel RUNX2 missense mutation predicted to disrupt DNA binding causes cleidocranial dysplasia in a large Chinese family with hyperplastic nails

Cleidocranial dysplasia [CCD, Min 119600] is a skeletal dysplastic disorder. Clinical features include delayed closure of skull sutures, hypoplastic or aplastic clavicles, and dental anomalies [1]. Patients often present with wide-open fontanels and middle face dysplasia, such as depressed nasal bridges. Clavicular hypoplasia can cause narrow, sloping shoulders that are often opposed at the mid line. Other clinical features include brachydactyly, tapering fingers, and short, broad thumbs and toes. Dental malformations include delayed eruption of secondary dentition and failure to shed primary teeth, which results in supernumerary teeth with dental crowding and malocclusion [2, 3].

A variety of mutations in RUNX2 cause CCD [4‐11], but no clear genotype-phenotype correlation has been established in CCD patients [8]. RUNX2 is an osteoblast-specific transcription factor that is a member of the core binding family (CBF) [12]. RUNX2 interacts with a non DNA-binding CBFβ subunit and the resulting complex binds to cis-acting elements in the promoters of genes required for skeletal formation such as osteocalcin [13]. The RUNX2-CBFβ complex serves as a master regulator of osteoblast differentiation and a scaffold that controls the assembly of transcription factors regulating skeletal gene expression [14]. In addition to osteocalcin, RUNX2 regulates vascular endothelial growth factor (VEGF) [15], COL10A1, and matrix metalloproteinase 13 (MMP13) [16]. COL10A1 is crucial for chondrocyte hypertrophy. VEGF and MMP13 are critical for hypertrophic chondrocytes, and mutations in RUNX2 result in a markedly diminished hypertrophic zone in long bone cartilage [17]. Additionally, RUNX2 and phosphatidylinositol 3-kinase (PI3K) regulate osteoblast and chondrocyte differentiation and migration in a mutually dependent manner [18]. Signal transducer and activator of transcription 1(STAT1) also interacts with RUNX2 and attenuates its transcriptional activity through cytoplasmic sequestration [19]. It has been shown that overexpression of RUNX2 results in growth arrest through p27 (KIP1)-induced inhibition of the s-phase cyclin complex, followed by dephosphorylation of the RB1 protein [20]. Since osteoblast proliferation and differentiation are inversely correlated, RUNX2 inhibits osteoblast proliferation. Here, we report a large Chinese family with CCD. We predict that a novel mutation of RUNX2 disrupts the protein-DNA interaction.

We detected a single base-pair variant in exon 1 that was verified by sequencing both strands (Figure 4). This variant in the cDNA at position 407 results in the substitution of leucine 136 with proline (Left panel). It co-segregates with affected individuals of the family. There is no such a sequence variant at that position in unaffected individuals (Right panel). More than 200 population-matched chromosomes were sequenced and no sequence variations were detected, suggesting that the sequence aberration is pathogenic. A leucine at that position is conserved in the Runt domain proteins of all vertebrates, suggesting it is functionally important (Figure 5A). The analogous residue in RUNX1 is L85, which in the crystal structures of the highly conserved RUNX1 Runt domain-CBFβ-DNA ternary complex is located in the βA'-B loop [21]. Two adjacent residues in the βA'-B loop, R131 and K134 are predicted to directly contact the DNA based on the RUNX1 structure (Figure 5B). The L136 side chain is oriented away from the DNA interface. Substitution of L136 with proline is predicted to alter the structure of the DNA-binding βA'-B loop, and thereby impair DNA binding by RUNX2. Several mutations at the DNA binding interface in RUNX1 were shown to also affect binding of CBFβ to the opposite side of the Runt domain because of their overall effect on the Runt domain structure [22] (Matheny CJ et al, unpublished data). One of these mutations was R80C, which is also located in the RUNX1 βA'-B loop (corresponding to R131 in RUNX2). Therefore, it is possible that the L136P mutation in RUNX2 would also affect CBFβ binding, but this was not directly tested.

Thus far, more than 70 mutations have been identified in the RUNX2 gene. The mutations are clustered in the Runt domain but occur throughout the RUNX2 protein. RUNX2 binds to DNA in a sequence-specific manner and CBFβ enhances its DNA binding affinity. Here, we identified a novel missense mutation in the Runt domain in a large Chinese family. Clinically, all affected individuals have hyperplastic nails. We show for the first time an unusual phenotype, hyperplastic nails, the molecular basis of which is unknown. This additional phenotype is most likely caused by RUNX2 mutation. However, we cannot rule out the possibility that another genetic alternation is co-segregated with the RUNX2 gene and associated with this phenotype. The most likely mechanism by which the L136P mutation affects RUNX2 function is through impaired DNA binding caused by structural alterations in the DNA-binding βA'-B loop. The identification of the L136P mutation in this family further expands the clinical phenotypes and mutation spectrum, and will contribute to prenatal molecular diagnosis and preimplantation genetic diagnoses.

Thus far, genotype-phenotype correlations have not been established for the different RUNX2 mutations found in CCD, although Zhou et al.[11] suggested that a T200A mutation found in a family with mild CCD might be a hypomorphic mutation. The hypomorphic nature of the T200A mutation was recently confirmed in mice [11] (Matheny CJ et al, unpublished data). There is variable expressivity of RUNX2 CCD mutations. For example, in one Italian family with a RUNX2 mutation, the reported clinical features ranged from minimal or absent clavicle to the presence of hypoplastic clavicles and delayed closure of the anterior fontanel in addition to classic craniofacial features. The family we describe here also displays significant intrafamilial phenotypic variation, indicating that there are modifying alleles at other loci. It is uncommon for CCD patients to display clinical features other than skeletal abnormalities. Two Italian CCD patients were reported to exhibit shoulder muscle abnormalities [23]. Quack et al. reported that an R225Q mutation affects nuclear RUNX2 accumulation and results exclusively in dental phenotypes. One patient with mutations resulting in an amino acid substitution at position 702 in sequences C-terminal to the Runt domain had osteoporosis, suggesting that RUNX2 not only functions in skeletal development, but also in maintaining adult bone density [6]. Extensive mutagenesis experiments have shown that many RUNX2 mutations impair DNA binding, while others affect CBFβ binding or the Runt domain structure [9, 11, 21, 24‐29] (Matheny CJ et al., unpublished data).

L136P is a novel mutation that most likely interferes with DNA binding by RUNX2 and would result in a nonfunctional or strongly hypomorphic allele.