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Alternative titles; symbolsCORE-BINDING FACTOR, RUNT DOMAIN, ALPHA SUBUNIT 1; CBFA1AML3 GENE; AML3PEBP2-ALPHA-AOSF2HGNC Approved Gene Symbol: RUNX2Cytogenetic lo...

Alternative titles; symbols

  • OSF2

HGNC Approved Gene Symbol: RUNX2

Cytogenetic location: 6p21.1 Genomic coordinates (GRCh38): 6:45,328,329-45,551,081 (from NCBI)

▼ Description
The RUNX2 gene encodes a Runt-related transcription factor, which is part of the RUNX gene family (see RUNX1, 151385 and RUNX3, 600210). The RUNX transcription factors are composed of an alpha subunit, encoded by the RUNX1, RUNX2, and RUNX3 genes, which binds to DNA via a Runt domain, and a beta subunit, encoded by the CBFB gene (121360), which increases the affinity of the alpha subunit for DNA but shows no DNA binding by itself. These proteins have a conserved 128-amino acid Runt domain, so called because of its homology to the pair-rule gene runt, which plays a role in the segmented body patterning of Drosophila. RUNX2 has a primary role in the differentiation of osteoblasts and hypertrophy of cartilage at the growth plate, cell migration, and vascular invasion of bone; is expressed in vascular endothelial cells, breast cancer cells, and prostate cancer cells; is linked to vascular calcification in atherosclerotic lesions; and is expressed in adult bone marrow, thymus, and peripheral lymphoid organs (review by Cohen, 2009).

▼ Cloning and Expression
Levanon et al. (1994) isolated and characterized cDNAs corresponding to 3 human 'runt domain' containing genes, AML1 (RUNX1; 151385), CBFA3 (RUNX3; 600210), and CBFA1. In addition to homology in the highly conserved runt domain, extensive sequence similarities were also observed in other parts of the proteins. They found that CBFA1 is the human homolog of one component of mouse PEBP2, i.e., PEBP2A (see Ogawa et al. (1993)). In the mouse, PEBP2 (also known as core-binding factor) is a heterodimer consisting of 2 polypeptides: alpha, the DNA-binding subunit, and beta (PEBP2B; 121360), which associates with the alpha subunit and enhances its affinity for DNA. Zhang et al. (1997) cloned the PEBP2A gene.

Ducy et al. (1997) cloned the cDNA encoding Cbfa1, which encodes a protein that binds to an osteoblast-specific cis-acting element, termed OSE2, in the promoter of osteocalcin (112260). They showed that Cbfa1 is an osteoblast-specific transcription factor and a regulator of osteoblast differentiation. The CBFA1 gene is also symbolized OSF2. Geoffroy et al. (1998) found 2 OSF2/CBFA1 cDNAs due to an alternative splicing event around exon 8 that affects the transcriptional activity of the protein. Northern blot analysis demonstrated that the expression of human OSF2/CBFA1 is restricted to osteoblastic cells.

Terry et al. (2004) identified mouse and human RUNX2 splice variants encoding up to 12 RUNX2 isoforms. They reported 2 alternate promoter regions and start codons, alternative splicing following the exons encoding the central invariant DNA-binding Runt domain, and 2 alternate 3-prime exons encoding different C-terminal domains. One C-terminal domain of 180 amino acids contains a nuclear matrix targeting signal (NMTS), a repression domain, and a C-terminal conserved motif. The alternate C-terminal domain of 200 amino acids contains a proline-rich sequence and a leucine zipper-like motif. A destabilizing PEST sequence is encoded by both alternate 3-prime terminal exons. Northern blot analysis and RT-PCR detected differential utilization of the 2 alternate promoters and both 3-prime terminal exons in mouse and human tissues and cells and in early mouse embryos.

▼ Gene Function
Ziros et al. (2002) examined the relationship between mechanical load and osteoblast differentiation and function. They found that low-level mechanical deformation (stretching) of cultured human osteoblastic cells directly upregulated the expression and DNA binding of CBFA1 to osteoblast-specific cis-acting element-2 (OSE2), which is found in the promoter region of osteoblast-specific genes. There was a stretch-triggered activation of the mitogen-activate protein kinase (MAPK) cascade that included a rapid induction of both JNK1 (601158) and JNK2 (602896) and a more sustained induction of ERK1 (601795) and ERK2 (176948). Ziros et al. (2002) found evidence that CBFA1 and ERK2 physically interact, resulting in phosphorylation of CBFA1 and potentiation of its transcriptional activity.

Kim et al. (2003) found enhanced bone formation and accelerated osteoblast differentiation in Stat1 (600555)-deficient mice, resulting in increased bone mass. Runx2 DNA-binding activity was upregulated in Stat1 mutant osteoblasts. Kim et al. (2003) determined that Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2 and its nuclear transcriptional activity. They showed that the Stat1-Runx2 interaction does not require phosphorylation of Stat1 on tyr701, which is necessary for Stat1 transcriptional activity, and it does not require interferon (see IFNG; 147570) signaling. Kim et al. (2003) concluded that bone remodeling by RUNX2 is attenuated by its sequestration in the cytoplasm by latent STAT1.

Zheng et al. (2003) identified multiple functional RUNX2-binding sites within the promoter region of the human, mouse, and chicken COL10A1 genes (120110). In transgenic mouse cells, Runx2 contributed to the transactivation of the Col10a1 promoter. Also, decreased Col10a1 expression and altered chondrocyte hypertrophy were observed in Runx2 heterozygous mice, whereas Col10a1 was barely detectable in Runx2 null mice.

Stein et al. (2004) reviewed the function of mammalian Runx proteins in osteogenesis. They stated that Runx2 is the principal osteogenic master switch, while Runx1 and Runx3 are expressed in bone cells and appear to support bone cell development and differentiation. Stein et al. (2004) described the role of Runx2 in the structural modification of the osteocalcin gene promoter during osteoblast development. They concluded that RUNX2 acts as a scaffold that controls the integration, organization, and assembly of nucleic acids and regulatory factors for skeletal gene expression.

Bialek et al. (2004) determined that the Twist proteins transiently inhibit Runx2 function during skeletal development in mice. Twist1 (601622) and Twist2 (607556) were expressed in Runx2-expressing cells throughout the skeleton early during development, and osteoblast-specific gene expression occurred only after their expression decreased. Double heterozygotes for Twist1 and Runx2 deletion showed none of the skull abnormalities observed in Runx2 +/- mice, a Twist2 null background rescued the clavicle phenotype of Runx2 +/- mice, and Twist1 or Twist2 deficiency led to premature osteoblast differentiation. The antiosteogenic function of the Twist proteins was mediated by a domain Bialek et al. (2004) called the Twist box, which interacted with the Runx2 DNA-binding domain to inhibit its function.

Fujita et al. (2004) investigated the role of Runx2 in the differentiation of mouse osteoblasts and mesenchymal stem cells. They presented evidence suggesting that Runx2 and phosphatidylinositol 3-kinase (see PIK3CG; 601232)-Akt (see 164730) signaling are mutually dependent on each other in the regulation of osteoblast and chondrocyte differentiation and migration.

Terry et al. (2004) determined that RUNX2 isoforms containing either the 180- or 200-amino acid C-terminal domain were able to bind canonical Runx DNA target sequences.

The inverse relationship between proliferation and differentiation in osteoblasts has been well documented. Thomas et al. (2004) found that Runx2, a master regulator of osteoblast differentiation in mammalian cells, was disrupted in 6 of 7 mammalian osteosarcoma cell lines. Immunohistochemical analysis of human osteosarcomas indicated that expression of p27(KIP1) (CDKN1B; 600778) was also lost as tumors lost osteogenic differentiation. Thomas et al. (2004) found that ectopic expression of Runx2 induced growth arrest through p27(KIP1)-induced inhibition of S-phase cyclin complexes, followed by dephosphorylation of the RB1 protein (614041) and G1 cell cycle arrest. They concluded that RUNX2 establishes a terminally differentiated state in osteoblasts through RB1- and p27(KIP1)-dependent mechanisms that are disrupted in osteosarcomas.

Hassan et al. (2004) found that Msx2 (123101), Dlx3 (600525), Dlx5 (600028), and Runx2 regulated the expression of osteocalcin (OC) (BGLAP; 112260) in mouse embryos and therefore are implicated in the control of bone formation. Msx2 associated with transcriptionally repressed OC chromatin, and Dlx3 and Dlx5 were recruited with Runx2 to initiate OC transcription. In a second regulatory switch, Dlx3 association decreased and Dlx5 recruitment increased coincident with the mineralization stage of osteoblast differentiation. The appearance of Dlx3 followed by Dlx5 in the OC promoter correlated with increased transcription represented by increased occupancy of RNA polymerase II.

Young et al. (2007) established that mammalian RUNX2 not only controls lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II (see 180660) but also acts as a repressor of RNA Pol I (see 602000)-mediated ribosomal RNA (rRNA) synthesis. Within the condensed mitotic chromosomes, Young et al. (2007) found that RUNX2 is retained in large discrete foci at nucleolar organizing regions where rRNA genes reside. These RUNX2 chromosomal foci are associated with open chromatin, colocalize with the RNA Pol I transcription factor UBF1 (600673), and undergo transition into nucleoli at sites of rRNA synthesis during interphase. Ribosomal RNA transcription and protein synthesis are enhanced by RUNX2 deficiency that results from gene ablation or RNA interference, whereas induction of RUNX2 specifically and directly represses rDNA promoter activity. RUNX2 forms complexes containing the RNA Pol I transcription factors UBF1 and SL1 (see 604903), co-occupies the rRNA gene promoter with these factors in vivo, and affects local chromatin histone modifications at rDNA regulatory regions. Thus, RUNX2 is a critical mechanistic link between cell fate, proliferation, and growth control. Young et al. (2007) suggested that lineage-specific control of ribosomal biogenesis may be a fundamental function of transcription factors that govern cell fate.

Young et al. (2007) showed that RUNX2 protein was stable during cell division and remained associated with chromosomes during mitosis via sequence-specific DNA binding. Using small interfering RNA, mitotic cell synchronization, and expression profiling, they identified RUNX2-regulated genes that were modulated postmitotically. During mitosis, RUNX2 interacted directly with promoters of cell fate- and cell cycle-regulated target genes that exhibited distinct RUNX2-dependent modification in histone acetylation and methylation.

Zaidi et al. (2007) stated that RUNX2 may function as a tumor suppressor in some cell types and have oncogenic potential in others. They showed that Runx2 deficiency and defective subnuclear targeting in primary mouse osteoblasts promoted immortalization and tumorigenic phenotype.

▼ Gene Structure
Terry et al. (2004) determined that the mouse and human RUNX2 genes contain 9 alternatively spliced exons. Exons 1 and 2 contain alternatively utilized promoter regions and an ATG translational start codon. There are 3 alternate exons 5 (exons 5, 5.1, and 5.2) and 2 alternate exons 6 (exons 6 and 6.1). Exon 6.1 is rich in CpG dinucleotides.

Stein et al. (2004) described the key regulatory elements contained within the promoter region of exon 1 of the RUNX2 gene.

▼ Mapping
By FISH, Levanon et al. (1994) mapped the CBFA1 gene to 6p21. The AML1, CBFA1, and CBFA3 genes all map to chromosomal regions involved in translocations underlying leukemia or myelodysplastic syndrome and, in the case of AML1, a fusion gene has been demonstrated as the basis of leukemia.

Zhang et al. (1997) mapped the PEBP2A gene to 6p21.1-p12.3 by FISH.

▼ Molecular Genetics
Cleidocranial Dysplasia

That the CBFA1 gene is the site of mutations responsible for cleidocranial dysplasia (119600) was established by Mundlos et al. (1997), who found that heterozygous deletions of the gene are present in some families, and that in other families, insertion, deletion, or missense mutations lead to translational stop codons in the DNA-binding domain or in the C-terminal transactivating region. In-frame expansion of a polyalanine stretch segregated in an affected family with brachydactyly and minor clinical findings of CCD. They concluded that CBFA1 mutations cause CCD and that heterozygous loss of function is sufficient to produce the disorder.

Quack et al. (1999) analyzed the CBFA1 gene in 42 unrelated patients with CCD. In 18 patients, they detected mutations in the coding region, including 8 frameshift, 2 nonsense, and 9 missense mutations, as well as 2 novel polymorphisms. A cluster of missense mutations at arginine-225 (R225) identified this residue as crucial for CBFA1 function. In vitro green fluorescent protein fusion studies showed that R225 mutations interfere with nuclear accumulation of CBFA1 protein. There was no phenotypic difference between patients with deletions or frameshifts and those with other intragenic mutations, suggesting that CCD is generally caused by haploinsufficiency. However, Quack et al. (1999) were able to extend the CCD phenotypic spectrum. A missense mutation (600211.0006) identified in a patient with supernumerary teeth and a radiologically normal skeleton indicated that mutations in the CBFA1 gene can be associated exclusively with a dental phenotype. (In another place in their report, Quack et al. (1999) stated that the mutation was a frameshift and that the patient, in fact, showed a gap in the most lateral part of the clavicle bilaterally, as well as the supernumerary teeth.) In addition, a patient with severe CCD and a frameshift mutation at codon 402 (600211.0007) had osteoporosis leading to recurrent bone fractures and scoliosis, providing the first evidence that CBFA1 may help maintain adult bone in addition to its function in bone development.

Rodan and Harada (1997) gave a comprehensive review of the role of the 3 CBFA genes and specifically the role of CBFA1 in normal and abnormal bone development. They pointed out that a difference between the heterozygous CBFA mutations in the human and in mice is the supernumerary teeth in humans, the basis of which remained to be determined.

In an extensive review of the genetics of craniofacial development and malformation, Wilkie and Morriss-Kay (2001) provided a useful diagram of the molecular pathways in cranial suture development with a listing of all craniofacial disorders caused by mutations in the corresponding genes. Four proteins were indicated as having strong evidence for existing in the pathway, with successive downstream targets as follows: TWIST (601622)--FGFR2 (176943)--FGFR1--CBFA1.

Bergwitz et al. (2001) reported 2 new mutations in RUNX2 causing cleidocranial dysplasia.

Otto et al. (2002) tabulated a large number of mutations in the RUNX2 gene that cause CCD; 20 of them were previously unreported. Missense mutations that cluster in the runt domain had been reported in 26 CCD patients. Only 1 missense mutation was found to be located outside the runt domain. The authors stated that R225 mutations arg225 to gln (R225Q; 600211.0008) and arg225 to trp (R225W; 600211.0009) had been identified in 7 unrelated patients. R225 resides within a stretch of basic amino acids at the carboxy terminus of the runt domain. This motif acts as a nuclear localization signal and mutations affecting R225 inhibit the nuclear accumulation of RUNX2 protein. Moreover, at least the R225Q mutation seems to abolish DNA binding (Zhou et al., 1999).

Zheng et al. (2005) observed growth plate abnormalities in a patient with a 1-bp insertion (600211.0017) in the RUNX2 gene. Histologic analysis of the rib and long-bone cartilages showed a markedly diminished zone of hypertrophy; analysis of limb cartilage RNA revealed a 5- to 10-fold decrease in the hypertrophic chondrocyte molecular markers VEGF (192240), MMP13 (600108), and COL10A1. Zheng et al. (2005) concluded that humans with CCD have altered endochondral ossification due to altered RUNX2 regulation of hypertrophic chondrocyte-specific genes during chondrocyte maturation.

Fernandez et al. (2005) described a case of a 20-year-old woman with features of both holoprosencephaly and cleidocranial dysplasia. She showed premaxillary agenesis, which is part of the holoprosencephaly spectrum, as well as skeletal abnormalities and impacted teeth reminiscent of cleidocranial dysplasia. She was found to carry a de novo 6;7 reciprocal translocation, with breakpoints at 6p21.1 and 7q36. The 7q36 breakpoint maps 15 kb telomeric to the 5-prime end of the Sonic hedgehog gene (SHH; 600725), which appeared to explain the patient's holoprosencephaly phenotype (Belloni et al., 1996). Using fluorescence in situ hybridization, Fernandez et al. (2005) identified a P1 artificial chromosome clone 800 kb upstream of the RUNX2 gene that spans the 6p breakpoint. Fernandez et al. (2005) proposed that the patient's complex phenotype was due to 2 position-effect mutations, 1 at each translocation breakpoint, which altered the expression of the SHH and RUNX2 genes. Fernandez et al. (2005) gave a listing of examples of position-effect mutations in human disease.

El-Gharbawy et al. (2010) studied a 7-year-old boy with CCD who also displayed features of hypophosphatasia (see 241500) and in whom no RUNX2 mutation was found by sequencing. Using array CGH, the authors identified a 50- to 70-kb deletion that predicted a disruption of the C terminus of RUNX2, encompassing the coding sequence for amino acids 327 to 521 and involving the SMAD 1,2,3,5 binding sites and the nuclear matrix targeting signal regions. El-Gharbawy et al. (2010) emphasized the need to search for deletions when sequencing of the target gene is normal, and noted that the C terminal region of RUNX2 appears to play an integral role in human osteogenesis and osteoblast differentiation.

Metaphyseal Dysplasia with Maxillary Hypoplasia with or without Brachydactyly

In affected members of a 4-generation French Canadian family with metaphyseal dysplasia with maxillary hypoplasia with or without brachydactyly (MDMHB; 156510), Moffatt et al. (2013) identified heterozygosity for a 105-kb duplication containing exons 3 to 5 of the RUNX5 gene (600211.0014) that was absent in unaffected family members. Moffatt et al. (2013) noted that the clinical findings of MDMHB and mechanistic studies were in accordance with the notion that duplication of RUNX2 exons 3 to 5 leads to a gain of function in RUNX2. This gain of function may result from increased cellular levels of mutated RUNX2 protein, as suggested by transfection experiments. The authors pointed out that MDMHB affects similar skeletal sites as CCD but in some way represents the mirror image of CCD. Clavicles are enlarged in MDMHB but are hypoplastic or absent in CCD. In MDMHB the cranial vault is thickened, whereas there is lack of skull mineralization in CCD. Persons with MDMHB present with dystrophic teeth, whereas CCD is associated with supernumerary teeth.

In a 20-year-old Finnish woman with MDMHB, Avela et al. (2014) identified heterozygosity for an intragenic duplication in RUNX2 encompassing exons 3 to 5. Similar to the duplication reported by Moffatt et al. (2013), the duplication breakpoints were in intron 2 and intron 5; the location of the breakpoints differed, but the exact breakpoints in the Finnish patient were not identified.

In 3 affected members of a 3-generation family with MDMHB, Al-Yassin et al. (2018) identified heterozygosity for an intragenic tandem duplication of RUNX2 exons 3 to 6 (600211.0015). Further analysis showed that exon 3 was spliced to exon 6, confirming a tandem duplication, which was predicted to be in-frame.

Somatic Mutation in Osteosarcoma

Sadikovic et al. (2009) performed integrative whole-genome analysis of DNA copy number, promoter methylation, and gene expression using 10 pediatric osteosarcoma tissue samples. Hypomethylation, copy number gain, and overexpression were identified for the histone cluster 2 genes (see 142750) on chromosome 1q21.1-q21.3. They also found loss of chromosome 8p21.3-p21.2 and underexpression of DOCK5 (616904), TNFRSF10A (603611), and TNFRSF10D (603614) genes, as well as copy number gain of chromosome 6p21.1-p12.3 and amplification-related overexpression of RUNX2. Amplification and overexpression of RUNX2 could disrupt G2/M cell cycle checkpoints, and downstream osteosarcoma-specific changes, such as failure of bone differentiation and genomic polyploidization. Failure of DOCK5 signaling, together with p53 (191170) and TNFRSF10A/D-related cell cycle and death pathways, may play a critical role in abrogating apoptosis. Sadikovic et al. (2009) hypothesized that the RUNX2 interactome may be constitutively activated in osteosarcoma, and that the downstream intracellular pathways may be associated with the regulation of osteoblast differentiation and control of cell cycle and apoptosis in osteosarcoma.

▼ Genotype/Phenotype Correlations
To correlate CBFA1 mutations in different functional domains with the CCD clinical spectrum, Zhou et al. (1999) studied 26 independent cases of CCD, and a total of 16 new mutations were identified in 17 families. Most mutations were de novo missense mutations that affected conserved residues in the runt domain and completely abolished both DNA binding and transactivation of a reporter gene. These, and mutations that resulted in premature termination in the runt domain, produced a classic CCD phenotype by abolishing transactivation of the mutant protein with consequent haploinsufficiency. Zhou et al. (1999) further identified 3 putative hypomorphic mutations that resulted in a clinical spectrum including classic and mild CCD, as well as an isolated dental phenotype characterized by delayed eruption of permanent teeth (600211.0010). Functional studies showed that 2 of the 3 mutations were hypomorphic in nature and 2 were associated with significant intrafamilial variability in expressivity, including isolated dental anomalies without the skeletal features of CCD. Together these data showed that variable loss of function due to alterations in the runt and C-terminal proline/serine/threonine-rich (PST) activation domains of CBFA1 may give rise to clinical variability, including classic CCD, mild CCD, and isolated primary dental anomalies.

Yoshida et al. (2002) performed mutation analysis of RUNX2 on 24 unrelated patients with CCD. In 17 patients, 16 distinct mutations were detected in the coding region of RUNX2: 4 frameshift, 3 nonsense, 6 missense, and 2 splicing mutations, and 1 polymorphism. The missense mutations were all clustered around the runt domain, and their protein products were severely impaired in DNA binding and transactivation. In contrast, the runt domain was intact in 2 RUNX2 mutants, with partial competence for transactivation remaining. One criterion of CCD, short stature, was much milder in the patients with the intact runt domain than in those without. Furthermore, there was a significant correlation between short stature and the number of supernumerary teeth. On the one hand, these genotype-phenotype correlations highlighted a general, quantitative dependency of skeletal/dental development on gene dosage of RUNX2. On the other hand, the classic CCD phenotype, hypoplastic clavicles or open fontanels, was invariably observed in all patients, including those of normal height. Thus, cleidocranial bone formation, as mediated by intramembranous ossification, may require a higher level of RUNX2 than does skeletogenesis (mediated by endochondral ossification), as well as odontogenesis (involving still different complex processes). These results suggested that CCD could result from much smaller losses in RUNX2 function than envisioned by the conventional haploinsufficiency model.

In 29 patients with CCD from 19 unrelated families, Baumert et al. (2005) sequenced the RUNX2 gene and identified 12 different RUNX2 mutations. They examined phenotypic data using homogeneity analysis and observed mild to full-blown expression of the CCD phenotype, with intrafamilial clinical variability (see also Baumert et al., 2006). Baumert et al. (2005) commented that homogeneity analysis simplified grouping the patients into distinct entities, but noted that the analysis separated individuals with the same mutation, emphasizing the clinical variability within the patient cohort.

Moffatt et al. (2013) noted that the apparent gain-of-function duplication causing metaphyseal dysplasia with maxillary hypoplasia and brachydactyly (MDMHB) results in a phenotype that is in some ways the mirror image of cleidocranial dysplasia, which is associated with loss-of-function mutations in RUNX2: clavicles are enlarged in MDMHB, whereas they are hypoplastic or absent in CCD; in MDMHB, the cranial vault is thickened whereas there is a lack of skull mineralization in CCD; and individuals with MDMHG have dystrophic teeth, whereas CCD is associated with supernumerary teeth.

▼ Evolution
Green et al. (2010) published a draft sequence of the Neandertal genome. Comparisons of the Neandertal genome to the genomes of 5 present-day humans from different parts of the world identified a number of genomic regions that may have been affected by positive selection in ancestral modern humans, including genes involved in metabolism and in cognitive and skeletal development. Green et al. (2010) identified a total of 212 regions containing putative selective sweeps. One of the 20 widest regions contains the RUNX2 gene. Mutations in this gene cause cleidocranial dysplasia, and some of the features associated with cleidocranial dysplasia are more common among Neandertals including cranial malformations such as frontal bossing. The clavicle, which is affected in cleidocranial dysplasia, differs in morphology between modern humans and Neandertals and is associated with a different architecture of the shoulder joint. Finally, a bell-shaped rib cage is typical of Neandertals and other archaic hominins. Green et al. (2010) suggested that a reasonable hypothesis is thus that an evolutionary change in RUNX2 was of importance in the origin of modern humans and that this change affected aspects of the morphology of the upper body and cranium. Green et al. (2010) also showed that Neandertals shared more genetic variants with present-day humans in Eurasia than with present-day humans in sub-Saharan Africa, suggesting that gene flow from Neandertals into the ancestors of non-Africans occurred before the divergence of Eurasian groups from each other.

▼ Animal Model
Komori et al. (1997) generated mice with a mutated Cbfa1 locus and found that mice homozygous for the mutation died just after birth without breathing. Examination showed complete lack of ossification of the skeleton. Although immature osteoblasts, which expressed alkaline phosphatase weakly but not osteopontin (OPN; 166490) or osteocalcin, and a few immature osteoclasts appeared at the perichondrial region, neither vascular nor mesenchymal cell invasion was observed in cartilage. The data suggested that both intramembranous and endochondral ossification were completely blocked and demonstrated that Cbfa1 plays an essential role in osteogenesis. Otto et al. (1997) likewise generated Cbfa1-deficient mice and found that homozygotes died of respiratory failure shortly after birth. Absence of osteoblasts and bone was demonstrated in homozygotes. Heterozygotes showed specific skeletal abnormalities characteristic of cleidocranial dysplasia (CCD). The same structural defects are observed in the murine mutant (Ccd), a CCD-like phenotype described by Selby and Selby (1978) as a gamma-ray-induced dominant mutation. Otto et al. (1997) demonstrated that the Cbfa1 gene is deleted in the Ccd mutation.

Ducy et al. (1999) studied the postnatal expression of Cbfa1 in mice. The perinatal lethality occurring in Cbfa1-deficient mice had hitherto prevented study of its function after birth. To determine if Cbfa1 plays a role during bone formation, they generated transgenic mice overexpressing Cbfa1 DNA-binding domain in differentiated osteoblasts only postnatally. The Cbfa1 DNA-binding domain has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. Mice expressing this form of the gene product had a normal skeleton at birth but developed an osteopenic phenotype thereafter. Dynamic histomorphometric studies showed that this phenotype was caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts, thus indicating that once osteoblasts are differentiated, Cbfa1 regulates their function. The study demonstrated that beyond its differentiation function, Cbfa1 is a transcriptional activator of bone formation (the first to be identified to that time) and illustrated that developmentally important genes control physiologic processes postnatally. In light of the absence of reported juvenile or more severe osteoporosis in patients with cleidocranial dysplasia, the observations in mice were unexpected. Ducy et al. (1999) thought that this is probably because of the more severe decrease of expression of the genes encoding bone extracellular matrix proteins, notably type I collagen, in the transgenic mice compared to the heterozygous Cbfa1-deficient mice. During embryonic development, Cbfa1 controls cell differentiation along the osteoblastic pathway; postnatally Cbfa1 has an additional function, directly controlling bone matrix deposition by differentiated osteoblasts.

Geoffroy et al. (2002) examined bone marrow stromal cells and cocultures of primary osteoblasts and spleen cells from wildtype and transgenic Cgfa1-overexpressing mice. Primary osteoblasts and bone marrow stromal cells from transgenic mice had stronger osteoclastogenic properties than cells derived from wildtype animals. Expression of Rankl (602642) and collagenase-3 (MMP13; 600108), factors involved in bone formation-resorption coupling, was markedly increased in transgenic cells. Geoffroy et al. (2002) concluded that overexpression of Cbfa1 enhances osteoclast differentiation in vitro and bone resorption in vivo.

Zhou et al. (2000) showed that mice carrying a pro250-to-arg mutation in Fgfr1 (136350), which is orthologous to the Pfeiffer syndrome mutation pro252 to arg (136350.0001) in humans, exhibit anterio-posteriorly shortened, laterally widened, and vertically heightened neurocrania. Cranial sutures of early postnatal mutant mice exhibited multiple premature fusions, accelerated osteoblast proliferation, and increased expression of genes related to osteoblast differentiation, suggesting that bone formation at the sutures is locally increased in Pfeiffer syndrome. Markedly increased expression of Cbfa1 accompanied premature fusion, suggesting that Cbfa1 may be a downstream target of Fgf/Fgfr1 signals. This was confirmed in vitro by demonstrating that transfection with wildtype or mutant Fgfr1 induced Cbfa1 expression. The induced expression was also observed using Fgf ligands Fgf2 and Fgf8 (600483).

D'Souza et al. (1999) reported a unique phenotype involving dentition in mice lacking a functional Runx2 gene. The markedly hypoplastic tooth organs as well as defects in the maturation of ameloblasts and odontoblasts pointed to an important nonredundant role for RUNX2 in both tooth morphogenesis and cytodifferentiation. To identify genes that are affected by the absence of Runx2, Gaikwad et al. (2001) generated a cDNA library from Runx2 -/- and Runx2 +/+ first molar organs. They found several tooth-specific downstream target genes of Runx2 that included extracellular matrix proteins, kinases, receptors, growth factors, mitochondrial proteins, and transcription molecules. Sequence analysis of 61 differentially expressed genes showed that 96% of the clones matched previously described genes in the GenBank/EBML database. Expression analysis of one of the differentially expressed clones that encodes a zinc finger transcription factor showed that the gene is temporally regulated during tooth development. Gaikwad et al. (2001) noted that the zinc finger transcription factor, which they called Zfp, shares 96% homology with Zfp64 (618111).

In studies of Runx2 mutants, Aberg et al. (2004) found that developing teeth failed to advance beyond the bud stage and that mandibular molar organs were more severely affected than maxillary molar organs. Molecular analyses showed differential effects of the absence of Runx2 on tooth extracellular matrix gene expression.

Yoshida et al. (2004) found that Runx2 knockout mice expressed reduced levels of Ihh (600726), which regulates chondrocyte proliferation and maturation. Adenoviral introduction of Runx2 into Runx2-deficient mice restored Ihh expression. Runx2 directly bound to the promoter region of the Ihh gene and induced expression of a reporter gene driven by the Ihh promoter. Runx2/Runx3 double-knockout mice displayed a complete absence of chondrocyte differentiation and a complete lack of Ihh expression. Single- or double-heterozygous mice showed intermediate degrees of chondrocyte differentiation depending upon the dosages of Runx2 and Runx3 expressed. Limb length was also reduced depending on the dosages of Runx2 and Runx3.

Napierala et al. (2008) found that mice homozygous for deletion of the Trps1 (604386) DNA-binding GATA domain (delta-GT mutation) showed elongation of the growth plate due to delayed chondrocyte differentiation and abnormal mineralization of perichondrium. These abnormalities were accompanied by increased Runx2 and Ihh expression and increased Ihh signaling. Cotransfection experiments showed that wildtype Trps1 bound Runx2 and repressed Runx2-mediated activation of a reporter plasmid. Double heterozygosity for Trps1 delta-GT and a Runx2-null mutation rescued the opposite growth plate phenotypes found in single mutants. Napierala et al. (2008) concluded that TRPS1 and RUNX2 interact to regulate chondrocyte and perichondrium development.

Lou et al. (2009) generated a mouse model of CCD using a hypomorphic Runx2-mutant allele (neo7), in which only part of the transcript is processed to full-length Runx2. Runx2 neo7/neo7 mice expressed a reduced level of wildtype transcript (55 to 70%) and protein and had grossly normal skeletons with no abnormalities observed in the growth plate, but exhibited developmental defects in calvaria and clavicles that persisted through postnatal growth. Clavicle defects were caused by disrupted endochondral bone formation during embryogenesis. These hypomorphic mice had altered calvarial bone volume, as observed by histology and micro-CT imaging, and decreased expression of osteoblast marker genes. Runx2 neo7/+ mice had 79 to 84% of wildtype transcript and exhibited a normal bone phenotype. Lou et al. (2009) concluded that there is a critical gene dosage requirement of Runx2 for the formation of intramembranous bone tissues during embryogenesis and that a decrease to 70% of wildtype Runx2 levels results in the CCD phenotype, whereas levels above 79% produce a normal skeleton, suggesting that the range of bone phenotypes in CCD patients is attributable to quantitative reduction in the functional activity of RUNX2.

▼ ALLELIC VARIANTS ( 15 Selected Examples):

In an isolated case of cleidocranial dysplasia (CCD; 119600), Mundlos et al. (1997) found heterozygosity for insertion of 16 bp within the polyglutamine-encoding CAG repeat region of the CBFA1 gene. The shift in reading frame produced a stop codon at nucleotide 435-437, in the middle of the 'runt' domain.

A sporadic case of cleidocranial dysplasia (CCD; 119600) was found to be caused by heterozygosity for a G-to-A transition at codon 283 in exon 5 of CBFA1 (Mundlos et al., 1997). This nucleotide change converted a TGG (trp) codon to a TGA (stop) codon.

Mundlos et al. (1997) found an unusual CBFA1 mutation in a family in which multiple members in 3 generations had a phenotype distinct from classic cleidocranial dysplasia (CCD; see 119600): minor craniofacial features of CCD were associated with brachydactyly of hands and feet. As illustrated by radiographs, the clavicles showed a distal gap in the continuity of the bone. Distal phalanges were hypoplastic and middle phalanges had cone-shaped epiphyses. Metacarpals exhibited pseudoepiphyses and shortening of metacarpals IV and V. The affected individuals in this family were found to have an in-frame duplication within the polyalanine stretch, leading to a total of 27 alanine residues instead of 17 residues as found in the wildtype sequence. Some unaffected members of the family had an allele with 11 alanine residues rather than 17; this appeared to be an uncommon but normal variant of CBFA1. (CBFA1 contains a region of 23 uninterrupted glutamine residues followed by 17 uninterrupted alanine residues on the N-terminal side of the 'runt' domain.)

Lee et al. (1997) described the first missense mutations in the CBFA1 gene in cleidocranial dysplasia (CCD; 119600): met175 to arg and ser191 to asn (600211.0005). These 2 mutations result in substitution of highly conserved amino acids in the DNA-binding domain. In DNA-binding studies with the mutant polypeptides they showed that these amino acid substitutions abolish the DNA-binding ability of CBFA1 to its known target sequence.

Lee et al. (1997) described the first missense mutations in the CBFA1 gene in cleidocranial dysplasia (CCD; 119600): met175 to arg (600211.0004) and ser191 to asn. These 2 mutations result in substitution of highly conserved amino acids in the DNA-binding domain. In DNA-binding studies with the mutant polypeptides they showed that these amino acid substitutions abolish the DNA-binding ability of CBFA1 to its known target sequence.

RUNX2, 1-BP INS, 1380C
In a patient with a very mild form of cleidocranial dysplasia (CCD; 119600), Quack et al. (1999) identified a 1-bp insertion (1380C) at the very 3-prime end of the coding region of the RUNX2 gene, resulting in a frameshift. The patient came to medical attention only because of supernumerary teeth. He was of normal height and excellent physical health. The clinical signs of CCD were restricted to supernumerary teeth and a gap in the most lateral part of the clavicle bilaterally.

RUNX2, 1-BP INS, 1206C
Quack et al. (1999) identified a 1-bp insertion (1206C) at codon 402 of the RUNX2 gene, resulting in a frameshift, in a patient who, in addition to very severe manifestations of cleidocranial dysplasia (CCD; 119600), had severe osteoporosis with both prenatal and antenatal fractures and severe scoliosis. At birth, the skull was almost unossified, both clavicles were absent, and distal hypoplasia of phalanges with partial absence of the nails was noted. The patient suffered from partial deafness due to conduction hearing impairment. A number of supernumerary teeth were extracted. At the age of 23 years, the patient had a body height of 129 cm.

Quack et al. (1999) identified missense mutations in the RUNX2 gene in 8 patients with cleidocranial dysplasia (CCD; 119600). In 4 of these patients, arg225 (R225), which is located at the C-terminal end of the runt domain, was mutated. The exchange of glutamine for arginine, due to a G-to-A transition at nucleotide 674, occurred in 3 unrelated patients. A replacement of arginine by tryptophan (600211.0009), caused by a C-to-T transition at nucleotide 673, occurred in 1 patient. Both amino acid exchanges abolished the positive charge of the residue at this position. The high frequency of mutations affecting R225 identified this codon as either especially prone to mutagenic events or of unusual relevance for the normal function of RUNX2.

In a patient with cleidocranial dysplasia (CCD; 119600), Quack et al. (1999) identified a 673C-T transition in the RUNX2 gene, resulting in an arg225-to-trp (R225W) substitution. See 600211.0008 for another mutation at the same codon in patients with CCD.

In a Mennonite family, Zhou et al. (1999) found classic cleidocranial dysplasia (CCD; 119600) in 2 of 4 children, while the father, who also harbored the mutation, had only dental anomalies, including delayed eruption of permanent teeth, misalignment, and multiple dentures. He did not have evidence of CCD on skeletal radiographs. All affected members shared the same mutation in the RUNX2 gene, resulting in a thr200-to-ala change in the runt domain. Even though the mutation affected a highly conserved amino acid immediately adjacent to a previously described mutation, the T200A mutation was not found in 100 unrelated control chromosomes and 50 Mennonite control chromosomes. The 2 affected brothers had dental anomalies, delayed closure of fontanel, and hypoplastic clavicles; the father, a brother, and 2 of the brother's children had only dental anomalies.

In a classic case of cleidocranial dysplasia (CCD; 119600), Machuca-Tzili et al. (2002) found heterozygosity for a stop codon mutation, 1565G-C (ter522 to ser; X522S), which theoretically resulted in a longer protein with 23 additional amino acids.

In a mother and daughter with cleidocranial dysplasia (CCD; 119600), Morava et al. (2002) identified a heterozygous 506G-C transversion in the RUNX2 gene, resulting in an arg169-to-pro (R169P) substitution within the highly conserved DNA-binding domain of the protein. In addition to the characteristic CCD phenotype, both patients had biochemical signs of hypophosphatasia (see 241500; 146300), including decreased levels of alkaline phosphatase (171760). Morava et al. (2002) noted that RUNX2-knockout mice show decreased alkaline phosphatase, and suggested that the clinical findings of hypophosphatasia in these patients was secondary to the RUNX2 mutation affecting early bone maturation and turnover.

RUNX2, 1-BP INS, 1228C
In a 20-week fetus with cleidocranial dysplasia (CCD; 119600), Zheng et al. (2005) identified a 1-bp insertion (1228insC) in exon 9 of the RUNX2 gene, resulting in a frameshift at codon 410 and premature termination. RUNX2 mRNA was downregulated by approximately 50% in the patient's cartilage, suggesting that the mutation causes haploinsufficiency.

RUNX2, 105-KB DUP, EX3-5
In affected members from a 4-generation French Canadian family with metaphyseal dysplasia with maxillary hypoplasia without brachydactyly (MDMHB; 156510), Moffatt et al. (2013) identified heterozygosity for a 105-kb duplication (chr6:45,308,920-45,413,885, GRCh37) containing exons 3 to 5 of the RUNX2 gene. The duplication of exons 3 to 5 was confirmed with cDNA derived from a patient fibroblast line, and was not found in unaffected family members. Functional analysis of the corresponding duplication in mouse Runx2 in HEK293 cells demonstrated markedly increased protein levels for the mutant compared to wildtype, as well as increased transactivation activity for mutant Runx2.

In all 3 affected members of a 3-generation family with metaphyseal dysplasia with maxillary hypoplasia with brachydactyly (MDMHB; 156510), Al-Yassin et al. (2018) identified heterozygosity for an intragenic tandem duplication of exons 3 to 6 (c.58+1_59-269_859+1_860-1dup, NM_001024630.3). Further analysis showed that exon 3 was spliced to exon 6, confirming a tandem duplication, which was predicted to be in-frame.

Tags: 6p21.1