Disease mutations in RUNX1 and RUNX2 create nonfunctional, dominant-negative, or hypomorphic alleles

Monoallelic RUNX1 mutations cause familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML). Sporadic mono- and biallelic mutations are found at high frequencies in AML M0, in radiation-associated and therapy-related myelodysplastic syndrome and AML, and in isolated cases of AML M2, M5a, M3 relapse, and chronic myelogenous leukemia in blast phase. Mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Most hematopoietic missense mutations in Runx1 involve DNA-contacting residues in the Runt domain, whereas the majority of CCD mutations in Runx2 are predicted to impair CBFbeta binding or the Runt domain structure. We introduced different classes of missense mutations into Runx1 and characterized their effects on DNA and CBFbeta binding by the Runt domain, and on Runx1 function in vivo. Mutations involving DNA-contacting residues severely inactivate Runx1 function, whereas mutations that affect CBFbeta binding but not DNA binding result in hypomorphic alleles. We conclude that hypomorphic RUNX2 alleles can cause CCD, whereas hematopoietic disease requires more severely inactivating RUNX1 mutations.     

A case of Japanese cleidocranial dysplasia with a CBFA1 frameshift mutation

Cleidocranial dysplasia (CCD), which is caused by mutations of the core binding factor alpha 1 (CBFA1)/runt-related gene 2 (Runx2), is an autosomal, dominantly inherited disorder of high penetrance affecting skeletal ossification and tooth development. Recently, we found a novel frameshift mutation 383-T-insertion (S128F) in exon 3 in the CBFA1 gene of a Japanese classic CCD patient. We describe our detailed investigation of the patient with CCD associated with the CBFA1 mutation. The patient showed the characteristic expression of CCD, such as dysplasia of the clavicles, patent fontanelles, short stature, impacted supernumerary teeth, and delayed eruption of the permanent teeth. In addition to these characteristics, orthopantomography delayed ossification of the mandibular symphysis and a three-dimensional computed tomograph (3D-CT) analysis showed hypoplasia of the zygomatic arch. Furthermore, the acellular cementum of an impacted supernumerary tooth was absent in this patient. Thus, the CBFA1 mutation was critical for the pathogenesis of CCD in this patient.     

A novel RUNX2 mutation in exon 8, G462X,in a patient with Cleidocranial Dysplasia

To identify a novel mutation of Runx2 gene in Cleidocranial Dysplasia (CCD) patients and to characterize the functional consequences of this mutation. The subjects consisted of 12 Korean CCD patients. After oral epithelial cells were collected using a mouthwash technique, genomic DNA was extracted. Screening for Runx2 mutation was performed using direct sequencing of polymerase chain reaction (PCR) products for exons 1‐8. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transacting activity, cyclohexamide chase assay for Runx2 protein stability, real‐time PCR for mRNA level of Runx2 downstream bone marker genes, and alkaline phosphatase (ALP) staining assay in mesenchymal stem cells for osteoblast differentiation. Of the 12 patients, seven showed Runx2 mutations reported previously and four showed no mutation. A novel mutation, G462X in exon 8, which was located in the C‐terminus of proline/serine/threonine‐rich (PST) domain, was found in one patient. In the luciferase assay, Runx2 transacting activity was decreased in Runx2‐G462X transfected cells. In the cyclohexamide chase assay, Runx2‐G462X mutation reduced the stability of Runx2 protein. Expression of the bone marker genes (osteocalcin, ALP, Type I collagen αI, matrix metalloproteinase‐13, bone sialoprotein, and osteopontin) decreased in G462X‐transfected cells. In the ALP staining assay, osteoblast differentiation was reduced in Runx2‐G462X overexpressed cell. The G462X mutation might reduce the Runx2 transacting activity, lower the protein stability, downgrade the expression of bone marker genes, and eventually diminish osteoblast differentiation in CCD patients.     

Four novel RUNX2 mutations including a splice donor site result in the cleidocranial dysplasia phenotype

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder caused by haploinsufficiency of the RUNX2 gene. In this study, we analyzed by direct sequencing RUNX2 mutations from eleven CCD patients. Four of seven mutations were novel: two nonsense mutations resulted in a translational stop at codon 50 (Q50X) and 112 (E112X); a missense mutation converted arginine to glycine at codon 131 (R131G); and an exon 1 splice donor site mutation (donor splice site GT/AT, IVS1 + 1G > A) at exon 1-intron junction resulted in the deletion of QA stretch contained in exon 1 of RUNX2. We focused on the functional analysis of the IVS1 + 1G > A mutation. A full-length cDNA of this mutation was cloned (RUNX2Deltae1) and expressed in Chinese hamster ovary (CHO) and HeLa cells. Functional analysis of RUNX2Deltae1 was performed with respect to protein stability, nuclear localization, DNA binding, and transactivation activity of a downstream RUNX2 target gene. Protein stability of RUNX2Deltae1 is similar to wild-type RUNX2 as determined by Western blot analysis. Subcellular localization of RUNX2Deltae1, assessed by in situ immunofluorescent staining, was observed with partial retention in both the nucleus and cytoplasm. This finding is in contrast to RUNX2 wild-type, which is detected exclusively in the nucleus. DNA binding activity was also compromised by the RUNX2Deltae1 in gel shift assay. Finally, RUNX2Deltae1 blocked transactivation of the osteocalcin gene determined by transient transfection assay. Our findings demonstrate for the first time that the CCD phenotype can be caused by a splice site mutation, which results in the deletion of N-terminus amino acids containing the QA stretch in RUNX2 that contains a previously unidentified second nuclear localization signal (NLS). We postulate that the QA sequence unique to RUNX2 contributes to a competent structure of RUNX2 that is required for nuclear localization, DNA binding, and transactivation function.