Novel mutations in the TBX5 gene in patients with Holt-Oram Syndrome

The Holt-Oram syndrome (HOS) is an autosomal dominant condition characterized by upper limb and cardiac malformations. Mutations in the TBX5 gene cause HOS and have also been associated with isolated heart and arm defects. Interactions between the TBX5, GATA4 and NKX2.5 proteins have been reported in humans. We screened the TBX5, GATA4, and NKX2.5 genes for mutations, by direct sequencing, in 32 unrelated patients presenting classical (8) or atypical HOS (1), isolated congenital heart defects (16) or isolated upper-limb malformations (7). Pathogenic mutations in the TBX5 gene were found in four HOS patients, including two new mutations (c.374delG; c.678G > T) in typical patients, and the hotspot mutation c.835C > T in two patients, one of them with an atypical HOS phenotype involving lower-limb malformations. Two new mutations in the GATA4 gene were found in association with isolated upper-limb malformations, but their clinical significance remains to be established. A previously described possibly pathogenic mutation in the NKX2.5 gene (c.73C > 7) was detected in a patient with isolated heart malformations and also in his clinically normal father.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.     

tRNA抑制子对转录因子NKX2.5提前终止密码子的通读

人类NKX2.5基因(NK2 homeobox 5,NKX2.5)提前终止密码子(Premature termination codon,PTC)突变会引起房间隔缺损、房室传导阻滞等先天性心脏病。目前,已报道的NKX2.5 PTC突变有8个(E109X、Q149X、Q170X、Q187X、Q198X、Y256X、Y259X和C264X)。为了检测tRNA抑制子是否对PTC突变诱导通读产生有功能的全长蛋白,文章将8个NKX2.5 PTC突变克隆到pcDNA3.1(-)载体,将NKX2.5全长和E109X、Q149X及C264X克隆到pEGFP-N1载体,形成NKX2.5-EGFP融合质粒。将NKX2.5-EGFP与对应的tRNA抑制子质粒分别或共转染后观察绿色荧光数量定性判断tRNA抑制子是否诱导通读。Western blotting检测通读后全长蛋白和截短蛋白表达并计算通读效率。Real-time PCR检测NKX2.5下游重要调控基因Cx43 mRNA的表达判断通读后蛋白功能。结果表明,文章成功构建了8个基于pcDNA3.1(-)的NKX2.5表达质粒、4个基于pEGFP-N1的质粒;tRNA抑制子tRNA am能有效通读Q149X、Q170X、Q187X和Q198X,且对后三者的通读效率均在50%以上;tRNA op能有效通读C264X,通读效率约50%左右;tRNA oc不能通读NKX2.5 PTC突变;各通读后样本Cx43 mRNA相对表达量增加7%~41.7%;tRNA am和tRNA op能有效通读NKX2.5 PTC突变,产生具有功能的全长蛋白,但tRNA抑制子对细胞的其他影响还不明确,有待于进一步观察。