Prioritizing cardiovascular disease-associated variants altering NKX2-5 and TBX5 binding through an integrative computational approach.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies have mapped >90% of CVD-associated variants within the noncoding genome, which can alter the function of regulatory proteins, such as transcription factors (TFs). However, due to the overwhelming number of single-nucleotide polymorphisms (SNPs) (>500,000) in genome-wide association studies, prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1535 CVD-associated SNPs within TF footprints and putative cardiac enhancers plus 14,218 variants in linkage disequilibrium with genotype-dependent gene expression in cardiac tissues. Using ChIP-seq data from two cardiac TFs (NKX2-5 and TBX5) in human-induced pluripotent stem cell-derived cardiomyocytes, we trained a large-scale gapped k-mer SVM model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay. Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and are in cardiac tissue eQTLs. All five variants altered NKX2-5 and TBX5 DNA binding. We present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro analysis.

Disease-associated non-coding variants alter NKX2-5 DNA-binding affinity.

Genome-wide association studies (GWAS) have mapped over 90 % of disease- or trait-associated variants within the non-coding genome, like cis-regulatory elements (CREs). Non-coding single nucleotide polymorphisms (SNPs) are genomic variants that can change how DNA-binding regulatory proteins, like transcription factors (TFs), interact with the genome and regulate gene expression. NKX2-5 is a TF essential for proper heart development, and mutations affecting its function have been associated with congenital heart diseases (CHDs). However, establishing a causal mechanism between non-coding genomic variants and human disease remains challenging. To address this challenge, we identified 8475 SNPs predicted to alter NKX2-5 DNA-binding using a position weight matrix (PWM)-based predictive model. Five variants were prioritized for in vitro validation; four of them are associated with traits and diseases that impact cardiovascular health. The impact of these variants on NKX2-5 binding was evaluated with electrophoretic mobility shift assay (EMSA) using purified recombinant NKX2-5 homeodomain. Binding curves were constructed to determine changes in binding between variant and reference alleles. Variants rs7350789, rs7719885, rs747334, and rs3892630 increased binding affinity, whereas rs61216514 decreased binding by NKX2-5 when compared to the reference genome. Our findings suggest that differential TF-DNA binding affinity can be key in establishing a causal mechanism of pathogenic variants.

Association between congenital heart disease and NKX2.5 gene polymorphisms: systematic review and meta-analysis.

Aim: To analyze the association of NKX2.5 gene with congenital heart disease (CHD), and to determine if the variants rs703752, rs3729753 and rs2277923 increase the risk for developing CHD. Materials & methods: PubMed, EBSCO and Web of Science databases were screened to identify eligible studies. Through a comprehensive meta-analysis software, the association between NKX2.5 gene variants and susceptibility of CHD was calculated by pooled odd ratio (ORs) and 95% CI. Results: We observed that the allelic model of rs703752 and rs2277923 increased the risk in the overall population: OR = 1.24; 95% CI: 1.00-1.55; Z p-value = 0.049; OR = 1.18; 95% CI: 0.01-1.37; Z p-value = 0.036; respectively. Conclusion: Our results suggested that the rs703752 and rs2277923 polymorphisms of the NKX2.5 gene are associated with CHD.

Electrical stimulation applied during differentiation drives the hiPSC-CMs towards a mature cardiac conduction-like cells.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and ß-catenin). We detected ES-induced CM maturation as indicated by increased Tnni1 and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca2+ transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias.

An update on genetic variants of the NKX2-5.

NKX2-5 is a homeodomain transcription factor that plays a crucial role in heart development. It is the first gene where a single genetic variant (GV) was found to be associated with congenital heart diseases in humans. In this study, we carried out a comprehensive survey of NKX2-5 GVs to build a unified, curated, and updated compilation of all available GVs. We retrieved a total of 1,380 unique GVs. From these, 970 had information on their frequency in the general population and 143 have been linked to pathogenic phenotypes in humans. In vitro effect was ascertained for 38 GVs. The homeodomain had the biggest cluster of pathogenic variants in the protein: 49 GVs in 60 residues, 23 in its third α-helix, where 11 missense variants may affect protein-DNA interaction or the hydrophobic core. We also pinpointed the likely location of pathogenic GVs in four linear motifs. These analyses allowed us to assign a putative explanation for the effect of 90 GVs. This study pointed to reliable pathogenicity for GVs in helix 3 of the homeodomain and may broaden the scope of functional and structural studies that can be done to better understand the effect of GVs in NKX2-5 function.

Mutation screening in the genes PAX-8, NKX2-5, TSH-R, HES-1 in cohort of 63 Brazilian children with thyroid dysgenesis.

OBJECTIVE: To evaluate the candidate genes PAX-8, NKX2-5, TSH-R and HES-1 in 63 confirmed cases of thyroid dysgenesis. SUBJECTS AND METHODS: Characterization of patients with congenital hypothyroidism into specific subtypes of thyroid dysgenesis with hormone levels (TT4 and TSH), thyroid ultrasound and scintigraphy. DNA was extracted from peripheral blood leukocytes and the genetic analysis was realized by investigating the presence of mutations in the transcription factor genes involved in thyroid development. RESULTS: No mutations were detected in any of the candidate genes. In situ thyroid gland represented 71.1% of all cases of permanent primary congenital hypothyroidism, followed by hypoplasia (9.6%), ectopia (78%), hemiagenesis (6.0%) and agenesis (5.5%). The highest neonatal screening TSH levels were in the agenesis group (p < 0.001). CONCLUSIONS: Thyroid dysgenesis is possibly a polygenic disorder and epigenetic factors could to be implicated in these pathogeneses.

Mutation screening in the genes PAX-8, NKX2-5, TSH-R, HES-1 in cohort of 63 Brazilian children with thyroid dysgenesis

ABSTRACT Objective: To evaluate the candidate genes PAX-8, NKX2-5, TSH-R and HES-1 in 63 confirmed cases of thyroid dysgenesis. Subjects and methods: Characterization of patients with congenital hypothyroidism into specific subtypes of thyroid dysgenesis with hormone levels (TT4 and TSH), thyroid ultrasound and scintigraphy. DNA was extracted from peripheral blood leukocytes and the genetic analysis was realized by investigating the presence of mutations in the transcription factor genes involved in thyroid development. Results: No mutations were detected in any of the candidate genes. In situ thyroid gland represented 71.1% of all cases of permanent primary congenital hypothyroidism, followed by hypoplasia (9.6%), ectopia (78%), hemiagenesis (6.0%) and agenesis (5.5%). The highest neonatal screening TSH levels were in the agenesis group (p < 0.001). Conclusions: Thyroid dysgenesis is possibly a polygenic disorder and epigenetic factors could to be implicated in these pathogeneses.

NKX2.5 is expressed in papillary thyroid carcinomas and regulates differentiation in thyroid cells.

BACKGROUND: NKX2.5 is a transcription factor transiently expressed during thyroid organogenesis. Recently, several works have pointed out the oncogenic role of NKX2.5 in a variety of tumors. We therefore hypothesized that NKX2.5 could also play a role in thyroid cancer. METHODS: The validation of NKX2.5 expression was assessed by immunohistochemistry analysis in a Brazilian case series of 10 papillary thyroid carcinoma (PTC) patients. Then, the long-term prognostic value of NKX2.5 and its correlation with clinicopathologic features of 51 PTC patients was evaluated in a cohort with 10-years follow-up (1990-1999). Besides, the effect of NKX2.5 overexpression on thyroid differentiation markers and function was also investigated in a non-tumor thyroid cell line (PCCL3). RESULTS: NKX2.5 was shown to be expressed in most PTC samples (8/10, case series; 27/51, cohort). Patients who had tumors expressing NKX2.5 showed lower rates of persistence/recurrence (p = 0.013). Overexpression of NKX2.5 in PCCL3 cells led to: 1) downregulation of thyroid differentiation markers (thyrotropin receptor, thyroperoxidase and sodium-iodide symporter); 2) reduced iodide uptake; 3) increased extracellular H2O2 generation, dual oxidase 1 mRNA levels and activity of DuOx1 promoter. CONCLUSIONS: In summary, NKX2.5 is expressed in most PTC samples analyzed and its presence correlates to better prognosis of PTC. In vitro, NKX2.5 overexpression reduces the expression of thyroid differentiation markers and increases ROS production. Thus, our data suggests that NKX2.5 could play a role in thyroid carcinogenesis.

Intramyocardial implantation of differentiated rat bone marrow mesenchymal stem cells enhanced by TGF-ß1 improves cardiac function in heart failure rats.

The present study tested the hypotheses that i) transforming growth factor beta 1 (TGF-ß1) enhances differentiation of rat bone marrow mesenchymal stem cells (MSCs) towards the cardiomyogenic phenotype and ii) intramyocardial implantation of the TGF-ß1-treated MSCs improves cardiac function in heart failure rats. MSCs were treated with different concentrations of TGF-ß1 for 72 h, and then morphological characteristics, surface antigens and mRNA expression of several transcription factors were assessed. Intramyocardial implantation of these TGF-ß1-treated MSCs to infarcted heart was also investigated. MSCs were initially spindle-shaped with irregular processes. On day 28 after TGF-ß1 treatment, MSCs showed fusiform shape, orientating parallel with one another, and were connected with adjoining cells forming myotube-like structures. Immunofluorescence revealed the expression of cardiomyocyte-specific proteins, α-sarcomeric actin and troponin T, in these cells. The mRNA expression of GATA4 and Nkx2.5 genes was slightly increased on day 7, enhanced on day 14 and decreased on day 28 while α-MHC gene was not expressed on day 7, but expressed slightly on day 14 and enhanced on day 28. Transmission electron microscopy showed that the induced cells had myofilaments, z line-like substances, desmosomes, and gap junctions, in contrast with control cells. Furthermore, intramyocardial implantation of TGF-ß1-treated MSCs to infarcted heart reduced scar area and increased the number of muscle cells. This structure regeneration was concomitant with the improvement of cardiac function, evidenced by decreased left ventricular end-diastolic pressure, increased left ventricular systolic pressure and increased maximal positive pressure development rate. Taken together, these results indicate that intramyocardial implantation of differentiated MSCs enhanced by TGF-ß1 improved cardiac function in heart failure rats.

Intramyocardial implantation of differentiated rat bone marrow mesenchymal stem cells enhanced by TGF-ß1 improves cardiac function in heart failure rats

The present study tested the hypotheses that i) transforming growth factor beta 1 (TGF-β1) enhances differentiation of rat bone marrow mesenchymal stem cells (MSCs) towards the cardiomyogenic phenotype and ii) intramyocardial implantation of the TGF-β1-treated MSCs improves cardiac function in heart failure rats. MSCs were treated with different concentrations of TGF-β1 for 72 h, and then morphological characteristics, surface antigens and mRNA expression of several transcription factors were assessed. Intramyocardial implantation of these TGF-β1-treated MSCs to infarcted heart was also investigated. MSCs were initially spindle-shaped with irregular processes. On day 28 after TGF-β1 treatment, MSCs showed fusiform shape, orientating parallel with one another, and were connected with adjoining cells forming myotube-like structures. Immunofluorescence revealed the expression of cardiomyocyte-specific proteins, α-sarcomeric actin and troponin T, in these cells. The mRNA expression of GATA4 and Nkx2.5 genes was slightly increased on day 7, enhanced on day 14 and decreased on day 28 while α-MHC gene was not expressed on day 7, but expressed slightly on day 14 and enhanced on day 28. Transmission electron microscopy showed that the induced cells had myofilaments, z line-like substances, desmosomes, and gap junctions, in contrast with control cells. Furthermore, intramyocardial implantation of TGF-β1-treated MSCs to infarcted heart reduced scar area and increased the number of muscle cells. This structure regeneration was concomitant with the improvement of cardiac function, evidenced by decreased left ventricular end-diastolic pressure, increased left ventricular systolic pressure and increased maximal positive pressure development rate. Taken together, these results indicate that intramyocardial implantation of differentiated MSCs enhanced by TGF-β1 improved cardiac function in heart failure rats.

The c.63A>G polymorphism in the NKX2.5 gene is associated with thyroid hypoplasia in children with thyroid dysgenesis.

OBJECTIVE: To search for genetic alteration in NKX2.5 gene in patients presenting both congenital heart disease (CHD) and TD. SUBJECTS AND METHODS: Individual phenotypes were carefully analyzed in 86 children with thyroid dysgenesis (TD) using thyroid function tests, scintigraphy, ultrasound and echocardiography. DNA was extracted and NKX2.5 gene coding region was amplified by polymerase chain reaction (PCR) and sequenced. RESULTS: CHD were found in 8.1% of patients with TD. The mutation screening revealed two known polymorphisms in patients with isolated TD or TD associated with CHD. None of them are predicted to result in codon change in conserved domain. The c.63A>G polymorphism was detected in 54/86 patients (49 with isolated TD and 5 with TD combined with CHD). There was a significant association of c.63A>G polymorphism with hypoplasia (p < 0.036). The c.541G>A polymorphism was observed in only one patient with isolated thyroid hypoplasia. CONCLUSION: NKX2.5 mutations were not found. The c.63A>G polymorphism might be associated with thyroid hypoplasia.

Germline mutations in NKX2-5, GATA4, and CRELD1 are rare in a Mexican sample of Down syndrome patients with endocardial cushion and septal heart defects.

Congenital heart defects (CHD) are found in ~50 % of Down syndrome (DS) patients. Genetic variants have been implicated, including CRELD1 mutations, but no previous study has examined the candidate genes, NKX2-5 and GATA4, in DS patients with secundum atrial defects (ASDII) and ventricular septal defects (VSD). Furthermore, CRELD1 mutations have not yet been studied in Mexican DS patients with atrioventricular septal defects (AVSD). Mexican DS patients (n = 148) with standard trisomy 21 were classified as follows: group I, normal heart; group II, VSD, ASDII, or both; and group III, AVSD. Mexican healthy controls (n = 113) were also included. Sequence analysis was performed on NKX2-5 and GATA4 in all three groups, and on CRELD1 in only group III. Statistical differences in the percentages of functional variants were analyzed by Fisher's exact test. Three non-synonymous variants in NKX2-5 were identified in the heterozygous state: a novel p.Pro5Ser was found in one DS patient without CHD; the p.Glu21Gln was found in one ASDII patient; and the p.Arg25Cys (R25C) was found in three patients (one from each DS study group). The p.Glu21Gln and R25C were also documented in 0.88 % of the controls. No significant difference was observed between the DS groups and healthy controls. Germline mutations in the NKX2-5, GATA4, and CRELD1 genes do not appear to be associated with CHD in Mexican DS patients. Our findings also support the notion that the R25C variant of NKX2-5 is a polymorphism, as it was not significantly different between our DS patients and controls.

Prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation.

OBJECTIVE: The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF). METHODS: A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart. CONCLUSION: These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia.

Absence of mutations in PAX8, NKX2.5, and TSH receptor genes in patients with thyroid dysgenesis.

OBJECTIVES: To precisely classify the various forms of TD, and then to screen for mutations in transcription factor genes active in thyroid development. SUBJECTS AND METHODS: Patients underwent ultrasound, thyroid scan, and serum thyroglobulin measurement to accurately diagnose the form of TD. DNA was extracted from peripheral leukocytes. The PAX8, and NKX2.5 genes were evaluated in all patients, and TSH receptor (TSHR) gene in those with hypoplasia. RESULTS: In 27 nonconsanguineous patients with TD, 13 were diagnosed with ectopia, 11 with hypoplasia, and 3 with athyreosis. No mutations were detected in any of the genes studied. CONCLUSION: Sporadic cases of TD are likely to be caused by epigenetic factors, rather than mutations in thyroid transcription factors or genes involved in thyroid development.

Novel NKX2-5 mutations responsible for congenital heart disease.

Congenital heart disease (CHD) is the most common birth defect and is the leading cause of infant morbidity and mortality resulting from birth defects. Increasing evidence demonstrates that genetic variation in the NKX2-5 gene, which encodes a homeobox-containing transcription factor crucial to cardiogenesis, is an important molecular determinant for CHD. Nevertheless, the genetic components underlying CHD remain largely unknown. We screened NKX2-5 for potential molecular defects in patients with CHD. The entire coding region of NKX2-5 was initially sequenced in a cohort of 268 unrelated patients with CHD. The relatives of the patients carrying identified mutations and 200 unrelated control individuals were subsequently genotyped. Three novel heterozygous missense NKX2-5 mutations, p.Q22K, p.R36S, and p.E54K, were identified in three families with autosomal dominantly inherited atrial septal defect, ventricular septal defect, and tetralogy of Fallot, respectively. These mutations, absent in 200 control individuals, appear to be highly conserved evolutionarily and co-segregated with CHD in the families, with complete penetrance. These findings expand the spectrum of mutations in NKX2-5 associated with CHD and provide new insight into the molecular etiology involved in the pathogenesis of CHD, which signifies potential implications for genetic diagnosis and gene-specific therapy for this common disease in newborns.

Search of somatic GATA4 and NKX2.5 gene mutations in sporadic septal heart defects.

High prevalence of somatic mutations in the cardiac transcription factor genes NKX2.5 and GATA4 have been reported in the affected cardiovascular tissue of patients with isolated cardiac septal defects, suggesting a role of somatic mutations in the pathogenesis of these congenital heart defects (CHDs). However, all somatic mutations have been identified in DNA extracted from an archive of formalin-fixed cardiac tissues. In the present study, to address the hypothesis that somatic mutations are important in isolated CHDs, we analyzed the GATA4 and NKX2.5 genes in the fresh-frozen pathologic cardiac tissue specimen and corresponding non-diseased tissue obtained from a series of 62 CHD patients, including 35 patients with cardiac septal defects and 27 with other cardiac anomalies. We identified one variant and two common polymorphisms in the NKX2.5 gene, and six variants and two common polymorphisms in the GATA4 gene. All identified variants were seen in both the fresh-frozen pathologic cardiac tissue and the corresponding non-diseased tissue, which indicates that they all were constitutional variants. The present study has identified NKX2.5 and GATA4 constitutional variants in our CHD cohort, but was unable to replicate the previously published findings of high prevalence of somatically derived sequence mutations in patients with cardiac septal defects using fresh-frozen cardiac tissues rather than formalin-fixed tissues.

NKX2.5 mutations in patients with non-syndromic congenital heart disease.

BACKGROUND: Cardiac development is a complex and multifactorial biological process. Heterozygous mutations in the transcription factor NKX2.5 are between the first evidence of a genetic cause for congenital heart defects in human beings. In this study, we evaluated the presence and frequency of mutations in the NKX2.5 gene on 159 unrelated patients with a diverse range of non-syndromic congenital heart defects (conotruncal anomalies, septal defects, left-sided lesions, right-sided lesions, patent ductus arteriosus and Ebstein's anomaly). METHODS: The coding region of the NKX2.5 locus was amplified by polymerase chain reaction and mutational analysis was performed using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. RESULTS: We identified two distinct mutations in the NKX2.5 coding region among the 159 (1.26%) individuals evaluated. An Arg25Cys mutation was identified in a patient with Tetralogy of Fallot. The second mutation found was an Ala42Pro in a patient with Ebstein's anomaly. CONCLUSIONS: The association of NKX2.5 mutations is present in a small percentage of patients with non-syndromic congenital heart defects and may explain only a few cases of the disease. Screening strategies considering the identification of germ-line molecular defects in congenital heart disease are still unwarranted and should consider other genes besides NKX2.5.

Clinical and molecular analysis of thyroid hypoplasia: a population-based approach in southern Brazil.

BACKGROUND: Congenital hypothyroidism (CH) is mainly due to developmental abnormalities leading to thyroid dysgenesis (TD). TD encompasses very distinct morphologic subtypes of disease. This study examined and compared the phenotype in TD variants and searched for genetic alterations in sporadic thyroid hypoplasia (TH), the most misdiagnosed form of CH. This was a longitudinal study over a 14-year period (1990-2004). METHODS: A continuous series of 353 children with TD was identified using thyroid function tests [thyroxine (T4) and TSH], scintigraphy, and ultrasound as diagnostic tools. Individual phenotypes were analyzed in 253 children with TD. Mutations in the most likely candidate genes were studied in 35 cases of TH. RESULTS: The overall birth prevalence of permanent CH was 1:4795. Ectopy represented 37% of all cases of permanent primary CH, dyshormonogenesis 28%, agenesis 24%, hypoplasia 10%, and hemiagenesis 1%. The lowest screening T4 level and the highest TSH level were in the agenetic group, followed by TH. The TH group had an improvement in the thyroid function showing less-severe phenotype with aging. In the molecular analysis, one patient was identified with a mutation in the PAX8 gene (155G>C; R52P); four patients had a heterozygous G>C substitution in position -569; two patients showed a (234C>A; P52T) or (2181C>G; D727E) polymorphic variants of the TSH-R gene; and one patient presented a novel heterozygous nonsynonymous substitution, 293G>A; S98N, in the NKX2.5 gene. CONCLUSIONS: The prevalence of CH was within the previously reported range of 1:3000-4000. Ectopy was the most common etiology. Clinical analysis revealed distinct hormonal patterns in TH subgroup when compared with other variants of TD, with genetic abnormalities identified only in few cases in the TSH-R, PAX8, and NKX2.5 genes.

Transcriptional regulation of the murine Connexin40 promoter by cardiac factors Nkx2-5, GATA4 and Tbx5.

OBJECTIVE: Connexin40 (Cx40) is a gap junction protein expressed specifically in developing and mature atrial myocytes and cells of the conduction system. In this report, we identify cis-acting elements within the mouse Cx40 promoter and unravel part of the complex pathways involved in the cardiac expression of this gene. METHODS: To identify the factors involved in the cardiac expression of Cx40, we used transient transfections in mammalian cells coupled with electrophoretic mobility shift assays (EMSA) and RT-PCR. RESULTS: Within the promoter region, we identified the minimal elements required for transcriptional activity within 150 base pairs (bp) upstream of the transcriptional start site. Several putative regulatory sites for transcription factors were predicted within this region by computer analysis, and we demonstrated that the nuclear factors Sp1, Nkx2-5, GATA4 and Tbx5 could interact specifically with elements present in the minimal promoter region of the Cx40. Furthermore, co-transfection experiments showed the ability of Nkx2-5 and GATA4 to transactivate the minimal Cx40 promoter while Tbx5 repressed Nkx2-5/GATA4-mediated activation. Mutagenesis of the Nkx2-5 core site in the Cx40 promoter led to significantly decreased activity in rat smooth muscle cell line A7r5. Consistent with this, mouse embryos lacking Nkx2-5 showed a marked decrease in Cx40 expression. CONCLUSION: In this work, we cloned the promoter region of the Cx40 and demonstrated that the core promoter was modulated by cardiac transcriptional factors Nkx2-5, Tbx5 and GATA4 acting together with ubiquitous Sp1.