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1.
We identified a disease-causing mutation of the RUNX2 gene in a four-generation Chinese family affected with cleidocranial dysplasia (CCD). For mutation analysis, the coding region of RUNX2 was sequenced with DNA from two patients and three unaffected family members. The RUNX2 mutation was investigated in 50 normal controls by denaturing high pressure liquid chromatography. A heterozygous single-base deletion (c.549delC) of RUNX2, which predicts a termination site at the 185th codon and leads to a stop in the runt domain of RUNX2 protein, was detected in both patients but not in the three unaffected members of the family. This mutation was also not found in 50 controls and has not been reported previously. We demonstrated that a novel mutation (c.549delC) of RUNX2 is associated with CCD in a Chinese family, adding to the repertoire of RUNX2 mutations related to CCD.  相似文献   

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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.  相似文献   

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Zhang YW  Yasui N  Kakazu N  Abe T  Takada K  Imai S  Sato M  Nomura S  Ochi T  Okuzumi S  Nogami H  Nagai T  Ohashi H  Ito Y 《Gene》2000,244(1-2):21-28
Cleidocranial dysplasia (CCD) is an autosomal dominant human bone disease whose genetic locus has been located on chromosome 6p21, where the PEBP2alphaA/CBFA1 gene essential for osteogenesis also maps. Previously, several heterozygous mutations in PEBP2alphaA/CBFA1 were found in CCD patients. In this study, we identified six different types of mutations in PEBP2alphaA/CBFA1 in Japanese CCD patients. Four cases were similar to those reported previously: two were nonsense mutations in the Runt domain, one was a hemizygous deletion, and the other was a missense mutation in the Runt domain which abolished the DNA-binding activity of Runx2/PEBP2alphaA/CBFA1. The remaining two mutations were novel: one had a heterozygous gt-to-tt mutation at the splice donor site (gt) between the exon3-intron junction, which resulted in abnormal exon3 skipping, and the other had a mutation in exon7, which led to the introduction of a translational stop codon in the middle of the transactivation domain. Thus, defects in either the DNA-binding domain or transactivation domain of Runx2/PEBP2alphaA/CBFA1 can cause CCD. The results not only provide a strong genetic evidence that mutations involving in PEBP2alphaA/CBFA1 contribute to CCD, but also provide a useful tool to study how Runx2/PEBP2alphaA/CBFA1 plays its pivotal role during osteoblastic differentiation.  相似文献   

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Metaphyseal dysplasia with maxillary hypoplasia and brachydactyly (MDMHB) is an autosomal-dominant bone dysplasia characterized by metaphyseal flaring of long bones, enlargement of the medial halves of the clavicles, maxillary hypoplasia, variable brachydactyly, and dystrophic teeth. We performed genome-wide SNP genotyping in five affected and four unaffected members of an extended family with MDMHB. Analysis for copy-number variations revealed that a 105 kb duplication within RUNX2 segregated with the MDMHB phenotype in a region with maximum linkage. Real-time PCR for copy-number variation in genomic DNA in eight samples, as well as sequence analysis of fibroblast cDNA from one subject with MDMHB confirmed that affected family members were heterozygous for the presence of an intragenic duplication encompassing exons 3 to 5 of RUNX2. These three exons code for the Q/A domain and the functionally essential DNA-binding runt domain of RUNX2. Transfection studies with murine Runx2 cDNA showed that cellular levels of mutated RUNX2 were markedly higher than those of wild-type RUNX2, suggesting that the RUNX2 duplication found in individuals with MDMHB leads to a gain of function. Until now, only loss-of-function mutations have been detected in RUNX2; the present report associates an apparent gain-of-function alteration of RUNX2 function with a distinct rare disease.  相似文献   

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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.  相似文献   

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Cleidocranial dysplasia (CCD; OMIM: 119600) is a rare autosomal dominant skeletal dysplasia caused by RUNX2 gene mutations. The present study described a sporadic case with CCD. The clinical data of the proband with CCD was reported and genetic analysis was performed. The proband presented with typical CCD features including supernumerary impacted teeth, bilateral clavicle dysplasia, delayed closure of cranial sutures, and short stature; while his hands were normal. Sequencing analysis of the entire coding region of the RUNX2 gene revealed no pathogenic changes; however, copy-number analysis with the Affymetrix HD array found \(\sim \)500 kb genomic microdeletion. Real-time quantitative PCR validated this microdeletion in the 1–4 exons of the RUNX2 gene. The junction point of the breaking DNA was located in the directly oriented AluSz6 and AluSx repetitive elements, indicating that this microdeletion might be generated through an Alu–Alu mediated mechanism. In addition, this microdeletion existed in 21.8% of the asymptomatic mother’s peripheral blood cells, demonstrating that the mosaicism was not associated with CCD phenotypes. In summary, a pathogenic microdeletion in the RUNX2 gene located on chromosome 6 was responsible for CCD.  相似文献   

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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.  相似文献   

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Xuan D  Li S  Zhang X  Lin L  Wang C  Zhang J 《Biochemical genetics》2008,46(11-12):702-707
Cleidocranial dysplasia (CCD) is an autosomal-dominant heritable skeletal disease caused by heterozygous mutations in the RUNX2 gene. Here, the RUNX2 gene was analyzed within a CCD family from China, and a novel missense mutation (c. 475G --> C [p.G159R]) was identified. Normal and mutant RUNX2 expression vectors were then constructed and expressed transiently in NIH3T3 cells. Immunofluorescent staining and Western blotting showed that wild-type RUNX2 protein was localized exclusively in the nucleus; however, the mutant protein was found in both the nucleus and the cytoplasm, which demonstrated that transport of the RUNX2 mutant into the nucleus was disturbed by the G159R mutation. Therefore, we suggest that G159 is very important to promote RUNX2 nuclear localization. According to clinical analysis, the patient displays severe dysplasia of bones and relatively low-grade craniofacial abnormality, and we infer that G159 may be vital for normal skeletal development, other than control of tooth number. These findings confirm that mutations in the RUNX2 gene are associated with the pathogenesis of CCD across different ethnic backgrounds.  相似文献   

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Cleidocranial dysplasia (CCD) is caused by haploinsufficiency in RUNX2 function. We have previously identified a series of RUNX2 mutations in Korean CCD patients, including a novel R131G missense mutation in the Runt‐homology domain. Here, we examine the functional consequences of the RUNX2R131G mutation, which could potentially affect DNA binding, nuclear localization signal, and/or heterodimerization with core‐binding factor‐β (CBF‐β). Immunofluorescence microscopy and western blot analysis with subcellular fractions show that RUNX2R131G is localized in the nucleus. Immunoprecipitation analysis reveals that heterodimerization with CBF‐β is retained. However, precipitation assays with biotinylated oligonucleotides and reporter gene assays with RUNX2 responsive promoters together reveal that DNA‐binding activity and consequently the transactivation of potential of RUNX2R131G is abrogated. We conclude that loss of DNA binding, but not nuclear localization or CBF‐β heterodimerization, causes RUNX2 haploinsufficiency in patients with the RUNX2R131G mutation. Retention of specific functions including nuclear localization and binding to CBF‐β of the RUNX2R131G mutation may render the mutant protein an effective competitor that interferes with wild‐type function. J. Cell. Biochem. 110: 97–103, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

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The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.  相似文献   

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Mutations in the gene encoding cartilage oligomeric matrix protein ( COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). More than 40 mutations have been identified; however, genotype-phenotype relationships are not well delineated. Further, mutations other than in-frame insertion/deletions and substitutions have not been found, and currently known mutations are clustered within relatively small regions. Here we report the identification of nine novel and three recurrent COMP mutations in PSACH and MED patients. These include two novel types of mutations; the first, a gross deletion spanning an exon-intron junction, causes an exon deletion. The second, a frameshift mutation that results in a truncation of the C-terminal domain, is the first known truncating mutation in the COMP gene. The remaining mutations, other than a novel exon 18 mutation, affected highly conserved aspartate or cysteine residues in the calmodulin-like repeat (CLR) region. Genotype-phenotype analysis revealed a correlation between the position and type of mutations and the severity of short stature. Mutations in the seventh CLR produced more severe short stature compared with mutations elsewhere in the CLRs ( P=0.0003) and elsewhere in the COMP gene ( P=0.0007). Patients carrying mutations within the five-aspartates repeat (aa 469-473) in the seventh CLR were extremely short (below -6 SD). Patients with deletion mutations were significantly shorter than those with substitution mutations ( P=0.0024). These findings expand the mutation spectrum of the COMP gene and highlight genotype-phenotype relationships, facilitating improved genetic diagnosis and analysis of COMP function in humans.  相似文献   

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The fibroblast growth factor-receptor 3 (FGFR3) Lys650 codon is located within a critical region of the tyrosine kinase-domain activation loop. Two missense mutations in this codon are known to result in strong constitutive activation of the FGFR3 tyrosine kinase and cause three different skeletal dysplasia syndromes-thanatophoric dysplasia type II (TD2) (A1948G [Lys650Glu]) and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) syndrome and thanatophoric dysplasia type I (TD1) (both due to A1949T [Lys650Met]). Other mutations within the FGFR3 tyrosine kinase domain (e.g., C1620A or C1620G [both resulting in Asn540Lys]) are known to cause hypochondroplasia, a relatively common but milder skeletal dysplasia. In 90 individuals with suspected clinical diagnoses of hypochondroplasia who do not have Asn540Lys mutations, we screened for mutations, in FGFR3 exon 15, that would disrupt a unique BbsI restriction site that includes the Lys650 codon. We report here the discovery of three novel mutations (G1950T and G1950C [both resulting in Lys650Asn] and A1948C [Lys650Gln]) occurring in six individuals from five families. Several physical and radiological features of these individuals were significantly milder than those in individuals with the Asn540Lys mutations. The Lys650Asn/Gln mutations result in constitutive activation of the FGFR3 tyrosine kinase but to a lesser degree than that observed with the Lys540Glu and Lys650Met mutations. These results demonstrate that different amino acid substitutions at the FGFR3 Lys650 codon can result in several different skeletal dysplasia phenotypes.  相似文献   

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