Novel Mutation of the RUNX2 Gene in Patients with Cleidocranial Dysplasia.
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ABSTRACT:
SUBMITTER: Hordyjewska E
PROVIDER: S-EPMC5582518 | biostudies-literature | 2017 Aug
REPOSITORIES: biostudies-literature
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Novel Mutation of the <i>RUNX2</i> Gene in Patients with Cleidocranial Dysplasia.
Hordyjewska Ewa E Jaruga Anna A Kandzierski Grzegorz G Tylzanowski Przemko P
Molecular syndromology 20170615 5
Cleidocranial dysplasia (CCD) is an autosomal dominant disorder linked to mutations in the Runt-related transcription factor 2, encoded by the <i>RUNX2</i> gene, which is essential for osteoblast differentiation and skeletal development. Here, we describe a novel nonsense mutation (c.532C>T; p.Q178X) in <i>RUNX2</i> identified in 3 affected members of a Polish family with CCD. The localization and transcriptional transactivation studies show that the mutated form of the protein has altered the s ...[more]
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Project description:Haploinsufficiency of the runt-related transcription factor 2 (RUNX2) gene is known to cause cleidocranial dysplasia (CCD). Here, we investigated a complex, heterozygous RUNX2 gene mutation in a Chinese family with CCD and the pathogenesis associated with the variations.Genomic DNA extracted from peripheral venous blood was taken from the proband, her parents and 3 siblings, and 150 normal controls. Analysis of their respective RUNX2 gene sequences was performed by PCR amplification and Sanger sequencing. Pathogenesis associated with RUNX2 mutations was investigated by performing bioinformatics, real-time PCR, western blot analysis, and subcellular localization studies.We identified 2 complex heterozygous mutations involving a c.398-399 insACAGCAGCAGCAGCA insertion and a c.411-412 insG frameshift mutation in exon 3 of the RUNX2 gene. The frameshift mutation changed the structure of the RUNX2 protein while did not affect its expression at the mRNA level. Transfection of HEK293T cells with a plasmid expressing the RUNX2 variant decreased the molecular weight of the variant RUNX2 protein, compared with that of the wild-type protein. Subcellular localization assays showed both nuclear and cytoplasmic localization for the mutant protein, while the wild-type protein localized to the nucleus.Our findings demonstrated that the novel c.398-399insACAGCAGCAGCAGCA mutation occurred alongside the c.411-412insG frameshift mutation, which resulted in RUNX2 truncation. RUNX2 haploinsufficiency was associated with CCD pathogenesis. These results extend the known mutational spectrum of the RUNX2 gene and suggest a functional role of the novel mutation in CCD pathogenesis.
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Project description:BackgroundCleidocranial dysplasia is a rare hereditary skeletal disorder due to heterozygous loss of function mutations in the RUNX2 gene that encodes runt-related transcription factor 2 (RUNX2). Here we report a 52 year-old woman with cleidocranial dysplasia due to a novel RUNX2 mutation.Case descriptionA 52 year-old Han Chinese woman presented with short stature and skeletal dysplasia that was first noted during early childhood. She was 153 cm in height and 40 kg in weight. Her skull was deformed with hypertelorism, midface hypoplasia, protrusion of chin, and dental abnormalities. Radiological examination revealed shortened clavicles and depressed skull bone and that were consistent with the clinical diagnosis of cleidocranial dysplasia. There was no family history of a similar skeletal disorder. We sequenced the RUNX2 gene and discovered a novel heterozygous mutation in exon 3 (c.476 del G, p.G159fs175X) that is predicted to cause a frameshift and premature termination that leads to the loss of the final 347 amino acid residues. This severely truncated protein is expected to be inactive.Literature reviewRUNX2 gene controls osteoblast differentiation and chondrocyte maturation. Around 90 RUNX2 mutations have been discovered in patients with cleidocranial dysplasia.Clinical relevanceWe identified a case of cleidocranial dysplasia due to a novel mutation of RUNX2 gene at exon 3 (c.476 del G).
| S-EPMC4348862 | biostudies-literature
Project description:Cleidocranial dysplasia (CCD) is a skeletal dysplasia with autosomal-dominant inheritance. The runt related transcription factor 2 (RUNX2) gene is the only gene in which mutations are known to cause CCD. We report identification of a novel small deletions mutation in the RUNX2 gene in a Chinese family with CCD. A 29-year-old female was diagnosed as proband of CCD based on the clinical findings, which show delayed closure of the fontanels, hypoplastic or aplastic clavicles and dental anomalies. Similar dental and skeletal symptoms were also observed in the other three affected individuals. We prepared genomic DNA from all four affected individuals, unaffected individual from her family members, as well as 100 unrelated healthy controls. PCR was conducted using the above genomic DNA as template and the RUNX2 gene-specific primers. The PCR product was subjected to direct sequencing and the sequence was compared to that of RUNX2 gene within the NCBI database. We detected a small deletion CCTA from nucleotide 635 to nucleotide 638 in exon 3 of RUNX2 gene of the proband. This will lead to the introduction of a translational stop codon at codon 220, resulting in a truncated RUNX2 protein, and therefore within the runt domain of the RUNX2 protein. We detected the same mutation in the the other three affected individuals, and did not detect any mutation in the unaffected family members or the 100 unrelated healthy controls, demonstrating that this is a novel missense mutation in RUNX2 gene and therefore, contributes to the molecular diagnosis of CCD.
| S-EPMC4069909 | biostudies-literature
Project description:BackgroundCleidocranial dysplasia (CCD) is a rare genetic disorder affecting bone and cartilage development. Clinical features of CCD comprise short stature, delayed ossification of craniofacial structures with numerous Wormian bones, underdeveloped or aplastic clavicles and multiple dental anomalies. Several studies have revealed that CCD development is strongly linked with different mutations in runt-related transcription factor 2 (RUNX2) gene.ObjectiveIdentification and functional characterization of RUNX2 mutation associated with CCD.MethodsWe performed genetic testing of a patient with CCD using whole exome sequencing and found a novel RUNX2 frameshift mutation: c.1550delT in a sporadic case. We also compared the functional activity of the mutant and wild-type RUNX2 through immunofluorescence microscopy and osteocalcin promoter luciferase assay.ResultsWe found a novel RUNX2 frameshift mutation, c.1550delT (p.Trp518Glyfs*60). Both mutant RUNX2 and wild-type RUNX2 protein were similarly confined in the nuclei. The novel mutation caused abrogative transactivation activity of RUNX2 on osteocalcin promoter.ConclusionsWe explored a novel RUNX2 deletion/frameshift mutation in a sporadic CCD patient. This finding suggests that the VWRPY domain may play a key role in RUNX2 transactivation ability.
| S-EPMC9120113 | biostudies-literature
Project description:Cleidocranial dysplasia (CCD) is an autosomal dominant inheritable skeletal disorder characterized by cranial dysplasia, clavicle hypoplasia and dental abnormalities. This disease is mainly caused by heterozygous mutations in RUNX2, a gene that encodes an osteoblast-specific transcription factor. In the present study, mutational analyses of RUNX2 gene were performed on four unrelated Chinese patients with CCD. Four different RUNX2 mutations were detected in these patients, including one nonsense mutation (c.199C>T p.Q67X) and three missense mutations (c.338T>G p.L113R, c.557G>C p.R186T and c.673C>T p.R225W). Among them, two mutations (c.199C>T p.Q67X and c.557G>C p.R186T) were novel and the other two had been reported in previous literatures. Except for Q67X mutation located in the Q/A domain, other three mutations were clustered within the highly conserved Runt domain. Green fluorescent protein (GFP) and RUNX2 fusion protein analyses in vitro showed that nuclear accumulation of RUNX2 protein was disturbed by Q67X mutation, while the other two mutations (c.338T>G p.L113R and c.557G>C p.R186T) had no effects on the subcellular distribution of RUNX2. Luciferase reporter assay demonstrated that all the three novel RUNX2 mutations significantly reduced the transactivation activity of RUNX2 on osteocalcin promoter. Our findings enrich the evidence of molecular genetics that the mutations of RUNX2 gene are responsible for CCD.
| S-EPMC5524338 | biostudies-literature
Project description:Cleidocranial dysplasia (CCD) is an autosomal dominant bone disease in humans caused by haploinsufficiency of the RUNX2 gene. The RUNX2 has two major isoforms derived from P1 and P2 promoters. Over 90 mutations of RUNX2 have been reported associated with CCD. In our study, DNA samples of nine individuals from three unrelated CCD families were collected and screened for all exons of RUNX2 and 2 kb of P1 and P2 promoters. We identified two point mutations in the RUNX2 gene in Case 1, including a nonsense mutation (c.577C>T) that has been reported previously and a silent substitution (c.240G>A). In vitro studies demonstrated that c.577C>T mutation led to truncated RUNX2 protein production and diminished stimulating effects on mouse osteocalcin promoter activity when compared with full-length Runx2-II and Runx2-I isoforms. These results confirm that loss of function RUNX2 mutation (c.577C>T) in Case 1 family is responsible for its CCD phenotype.
| S-EPMC2734498 | biostudies-literature
Project description:Cleidocranial dysplasia (CCD) is an autosomal dominant human skeletal disorder comprising hypoplastic clavicles, wide cranial sutures, supernumerary teeth, short stature, and other skeletal abnormalities. It is known that mutations in the human RUNX2 gene mapped at 6p21 are responsible for CCD. We analyzed the mutation patterns of the RUNX2 gene by direct sequencing in six Taiwanese index cases with typical CCD. One of the patients was a familial case and the others were sporadic cases. Sequencing identified four mutations. Three were caused by single nucleotide substitutions, which created a nonsense (p.R391X), two were missense mutations (p.R190W, p.R225Q), and the forth was a novel mutation (c.1119delC), a one-base deletion. Real time quantitative PCR adapted to determine copy numbers of the promoter, all exons and the 3'UTR region of the RUNX2 gene detected the deletion of a single allele in a sporadic case. The results extend the spectrum of RUNX2 mutations in CCD patients and indicate that complete deletions of the RUNX2 gene should be considered in those CCD patients lacking a point mutation detected by direct sequencing.
| S-EPMC3115309 | biostudies-literature
Project description:BackgroundPathogenic variants of RUNX2, a gene that encodes an osteoblast-specific transcription factor, have been shown as the cause of Cleidocranial dysplasia (CCD), which is a rare hereditary skeletal and dental disorder with dominant mode of inheritance and a broad range of clinical variability. Due to the relative lack of clinical complications resulting in CCD, the medical diagnosis of this disorder is challenging, which leaves it underdiagnosed.Methods: In this study, nine healthy and affected members of an Iranian family were investigated. PCR and sequencing of all exons and exon-intron boundaries of runt-related transcription factor 2 (RUNX2; NM_001024630) gene was performed on proband. Co-segregation analysis was conducted in the other family members for the identified variant. Additionally, a cohort of 100 Iranian ethnicity-matched healthy controls was screened by Amplification Refractory Mutation System PCR method.ResultsThe novel splice site variant (c.860-2A>G), which was identified in the intron 6 of RUNX2 gene, co-segregated with the disease in the family, and it was absent in healthy controls. Pathogenicity of this variant was determined by several software, including , human splicing finder, which predicts the formation or disruption of splice donor sites, splice acceptor sites, exonic splicing silencer sites, and exonic splicing enhancer sites. In silico analysis predicted this novel variant to be disease causing.ConclusionThe identified variant is predicted to have an effect on splicing, which leads to exon skipping and producing a truncated protein via introducing a premature stop codon.
| S-EPMC8334394 | biostudies-literature
Project description:Cleidocranial dysplasia (CCD; MIM 119600) is a rare autosomal dominant disorder characterized by facial, dental, and skeletal malformations. To date, rearrangement and mutations involving RUNX2, which encodes a transcription factor required for osteoblast differentiation on 6p21, has been the only known molecular etiology for CCD. However, only 70% patients were found to have point mutations, 13% large/contiguous deletion but the rest of 17% remains unknown. We ascertained a family consisted of eight affected individuals with CCD phenotypes. Direct sequencing analysis revealed no mutations in the RUNX2. Real time quantitative PCR were performed which revealed an exon 2 to exon 6 intragenic deletion in RUNX2. Our patients not only demonstrated a unique gene change as a novel mechanism for CCD, but also highlight the importance of considering "deletion" and "duplication" in suspected familial cases before extensive effort of gene hunting be carried.
| S-EPMC2518658 | biostudies-literature
Project description:ObjectivesCleidocranial dysplasia (CCD) is a skeletal disorder affecting cranial sutures, teeth, and clavicles, and is associated with the RUNX2 mutations. Although numerous patients have been described, a direct genotype-phenotype correlation for RUNX2 has been difficult to establish. Further cases must be studied to understand the clinical and genetic spectra of CCD. To characterize detailed phenotypes and identify variants causing CCD in five unrelated patients and their family members.MethodologyClinical and radiographic examinations were performed. Genetic variants were identified by exome and Sanger sequencing, data were analyzed by bioinformatics tools.ResultsThree cases were sporadic and two were familial. Exome sequencing successfully detected the heterozygous pathogenic RUNX2 variants in all affected individuals. Three were novel, comprising a frameshift c.739delA (p.(Ser247Valfs*)) in exon 6 (Patient-1), a nonsense c.901C>T (p.(Gln301*)) in exon 7 (Patient-2 and affected mother), and a nonsense c.1081C>T (p.(Gln361*)) in exon 8 (Patient-3). Two previously reported variants were missense: the c.673C>T (p.(Arg225Trp)) (Patient-4) and c.674G>A (p.(Arg225Gln)) (Patient-5) in exon 5 within the Runt homology domain. Patient-1, Patient-2, and Patient-4 with permanent dentition had thirty, nineteen, and twenty unerupted teeth, respectively; whereas Patient-3 and Patient-5, with deciduous dentition, had normally developed teeth. All patients exhibited typical CCD features, but the following uncommon/unreported phenotypes were observed: left fourth ray brachymetatarsia (Patient-1), normal clavicles (Patient-2 and affected mother), phalangeal malformations (Patient-3), and normal primary dentition (Patient-3, Patient-5).ConclusionsThe study shows that exome sequencing is effective to detect mutation across ethnics. The two p.Arg225 variants confirm that the Runt homology domain is vital for RUNX2 function. Here, we report a new CCD feature, unilateral brachymetatarsia, and three novel truncating variants, expanding the phenotypic and genotypic spectra of RUNX2 , as well as show that the CCD patients can have normal deciduous teeth, but must be monitored for permanent teeth anomalies.
| S-EPMC9239300 | biostudies-literature