单纯性先天性心脏病中TBX5基因表达异常的机制

为探讨人类单纯性先天性心脏病患者中TBX5基因表达下调的可能原因, 应用变性高效液相色谱(DHPLC)方法检测100例单纯性先天性心脏病患者中TBX5基因上游1 200 bp调控区的突变情况; 应用甲基化敏感性限制性内切酶(MS-RE)法检测50例单纯性先天性心脏病患者和5例非先天性心脏病患者心肌组织TBX5基因启动子区两个CpG岛(转录起始点上游-49~-188 bp和-247~-464 bp处)的甲基化情况; 应用P-match软件预测小鼠Tbx5基因上游转录因子Nkx2-5的结合位点, 构建Nkx2-5表达载体转染小鼠H9C2(2-1)心肌细胞, RT-PCR及Western blotting检测Tbx5基因表达, 凝胶阻滞实验(EMSA)验证Nkx2-5和Tbx5基因的作用。结果在100例单纯性先天性心脏病患者中, 未检测到TBX5基因上游1 200 bp调控区突变; 非先天性心脏病患者和单纯性先天性心脏病患者在两个CpG岛存在相同的甲基化; 小鼠Tbx5基因转录起始点上游-312~-315 bp可能存在Nkx2-5的结合位点, 转染Nkx2-5表达载体后Tbx5基因在mRNA及蛋白质水平均有表达增高趋势, Nkx2-5在体外可以与Tbx5基因上游-312~-315 bp序列相结合。以上结果提示TBX5基因调控区突变和两个CpG岛的甲基化不是单纯性先天性心脏病患者心肌组织中TBX5基因表达下调的原因, TBX5基因表达下调可能由于NKX2-5的表达异常引起。     

Genetic analysis of the TBX1 gene promoter in indirect inguinal hernia

Inguinal hernia is a common disease, most cases of which are indirect inguinal hernia (IIH). Genetic factors play an important role for inguinal hernia. Increased incidences of inguinal hernia have been reported in patients with 22q11.2 microdeletion syndrome, which is mainly caused by TBX1 gene mutations. Thus, we hypothesized that altered TBX1 gene expression may contribute to IIH development. In this study, the human TBX1 gene promoter was genetically analyzed in children with IIH (n = 100) and ethnic-matched controls (n = 167). Functions of DNA sequence variants (DSVs) within the TBX1 gene promoter were examined in cultured human fibroblast cells. The results showed that two heterozygous DSVs were found, both of which were single nucleotide polymorphisms. One DSV, g.4248 C>T (rs41298629), was identified in a 2-year-old boy with right-sided IIH, but not in all controls, which significantly decreased TBX1 gene promoter activity. Another DSV, g.4199 C>T (rs41260844), was found in both IIH patients and controls with similar frequencies (P > 0.05), which did not affect TBX1 gene promoter activity. Collectively, our data suggested that the DSV within the TBX1 gene promoter may change TBX1 level, contributing to IIH development as a rare risk factor. Underlying molecular mechanisms need to be established.     

Identification of a fat cell enhancer: analysis of requirements for adipose tissue-specific gene expression.

The molecular basis for adipose-specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5'-flanking region of the adipocyte P2 (aP2) gene to direct cell-type specific gene expression. Although the proximal promoter containing AP-1 and C/EBP binding sites is capable of directing differentiation-dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that -5.4 kb of the 5'-flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at -5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation-dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis- and trans- acting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.     

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.