van Engelen K, etal., PLoS One. 2012;7(12):e52685. doi: 10.1371/journal.pone.0052685. Epub 2012 Dec 28.
NKX2-5 is a homeodomain-containing transcription factor implied in both heart and thyroid development. Numerous mutations in NKX2-5 have been reported in individuals with congenital heart disease (CHD), but recently a select
few have been associated with thyroid dysgenesis, among which the p.A119S variation. We sequenced NKX2-5 in 303 sporadic CHD patients and 38 families with at least two individuals with CHD. The p.A119S variation was identified in two unrelated patients: one was found in the proband of a family with four affected individuals with CHD and the other in a sporadic CHD patient. Clinical evaluation of heart and thyroid showed that the mutation did not segregate with CHD in the familial case, nor did any of the seven mutation carriers have thyroid abnormalities. We tested the functional consequences of the p.A119S variation in a cellular context by performing transactivation assays with promoters relevant for both heart and thyroid development in rat heart derived H10 cells and HELA cells. There was no difference between wildtype NKX2-5 and p.A119S NKX2-5 in activation of the investigated promoters in both cell lines. Additionally, we reviewed the current literature on the topic, showing that there is no clear evidence for a major pathogenic role of NKX2-5 mutations in thyroid dysgenesis. In conclusion, our study demonstrates that p.A119S does not cause CHD or TD and that it is a rare variation that behaves equal to wildtype NKX2-5. Furthermore, given the wealth of published evidence, we suggest that NKX2-5 mutations do not play a major pathogenic role in thyroid dysgenesis, and that genetic testing of NKX2-5 in TD is not warranted.
Wang J, etal., Int J Mol Med. 2011 Mar;27(3):369-75. doi: 10.3892/ijmm.2010.585. Epub 2010 Dec 16.
Ventricular septal defect (VSD) is the most common cardiovascular malformation and an important contributor to the substantial morbidity and mortality in infancy. Growing evidence suggests that genetic defects play important roles in the pathogenesis of congenital VSD. However, VSD is of great genet
ic heterogeneity and the genetic basis for VSD in the majority of the patients remains largely unhnown. In this study, the entire coding region of the NKX2-5 gene, which encodes a homeodomain-containing transcription factor crucial to cardiogenesis, was initially sequenced in 136 unrelated patients with VSD. The relatives of a proband harboring the identified mutation and 200 unrelated control individuals were genotyped. The functional characteristic of the mutant transcription factor was analyzed in contrast to its wild-type counterpart using a luciferase reporter assay system. A novel heterozygous NKX2-5 mutation, p.P59A, was identified in a family with autosomal dominant inherited VSD. Absent in the 200 control individuals, the mutation was highly conserved evolutionarily and co-segregated with VSD in the family with complete penetrance. Functional analysis revealed that the p.P59A mutation of NKX2-5 was associated with a decreased transcriptional activity. These findings expand the spectrum of the mutations in NKX2-5 linked to VSD and provide new insight into the molecular mechanisms involved in VSD. The resuls of the present study may have potential implications in the genetic diagnosis and gene-specific therapy of this common childhood disease.
Wang J, etal., Genet Mol Res. 2011 Nov 29;10(4):2905-15. doi: 10.4238/2011.November.29.1.
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-containin
g 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.
Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggeste
d that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a "shifting" site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.
Liu XY, etal., Pediatr Cardiol. 2011 Feb;32(2):193-201. doi: 10.1007/s00246-010-9859-6. Epub 2010 Dec 25.
Atrial septal defect (ASD) is a common cardiovascular malformation and an important contributor to substantial morbidity and mortality. Increasing evidence demonstrates that mutated NKX2-5, a gene encoding a homeobox transcription factor crucial to cardiogenesis
, is a significant genetic determinant for congenital ASD. Nevertheless, the genetic basis for ASD in a majority of ASD patients remains largely unknown. In the current study, the entire coding region of NKX2-5 was sequenced initially for 58 unrelated probands with familial ASD. The relatives of the probands harboring identified mutations and 200 unrelated control individuals were subsequently genotyped. Three novel heterozygous NKX2-5 mutations (p.P43GfsX59, p.C46 W, and p.S179F) were identified respectively in three families with autosomal dominantly inherited ASD. These mutations, absent in 200 control individuals, cosegregated with ASD in the families that had complete penetrance. The findings expand the spectrum of mutations in NKX2-5 linked to ASD and contribute to genetic counseling, clinical interventions, and prenatal prevention of ASD for individuals with genetic susceptibility.
OBJECTIVE: Patent foramen ovale (PFO) has been related to stroke but its existence has not been explained to date. NKX2-5 is the most implicated gene in fetal atrial septation. We studied NKX2-5 with respect to the presence
or absence of PFO in stroke patients. METHODS: A prospective analysis of NKX2-5 regarding age, gender, PFO, right-to-left shunt (RLS) size and atrial septal aneurysm (ASA) was performed in consecutive stroke patients and in 50 controls. The entire coding region and intron-exon boundaries of NKX2-5 gene were analyzed by PCR and sequencing of DNA from peripheral lymphocytes. RESULTS: One hundred patients participated in the study (mean age 56.5+/-12.4 years, 58% males) and PFO was diagnosed in 34% of them by transesophageal echocardiography. RLS was small (12%), moderate (2%) and large (20%). ASA was present in four patients. DNA revealed a novel c.2357G>A change in one PFO patient with cryptogenic stroke. Furthermore, c.182C>T, a mutation previously described in patients with cardiac defects, was detected in two non-PFO women with cryptogenic stroke. None of these changes were detected in our controls. The c.172A>G polymorphism was found in 21% of controls. It appeared more frequently in ASA patients (p=0.084), in cryptogenic PFO stroke patients (p=0.097) and in patients with known causes of stroke (p=0.037). The c.2850C>A polymorphism was also detected in our series with no differences in PFO, RLS size or ASA. CONCLUSION: Despite the fact that the NKX2-5 could account for the persistence of PFO, mutations of this gene in peripheral blood DNA were barely detected in our study.
Mutations in the gene encoding the homeobox transcription factor NKX2-5 were found to cause nonsyndromic, human congenital heart disease. A dominant disease locus associated with cardiac malformations and atrioventricular conduction abnormalities was mapped to c
hromosome 5q35, where NKX2-5, a Drosophila tinman homolog, is located. Three different NKX2-5 mutations were identified. Two are predicted to impair binding of NKX2-5 to target DNA, resulting in haploinsufficiency, and a third potentially augments target-DNA binding. These data indicate that NKX2-5 is important for regulation of septation during cardiac morphogenesis and for maturation and maintenance of atrioventricular node function throughout life.
Lu XL, etal., Eur Rev Med Pharmacol Sci. 2016;20(4):715-20.
OBJECTIVE: Rheumatic heart disease (RHD) results due to the cross reaction of the host immune system when it develops immunity against group A streptococcal infection. This autoimmune disease progress with different pathological conditions and the genes associated with it are still less understood.
MATERIALS AND METHODS: To understand the role of NKX2-5 and Smad-6 in developing an RHD, we successfully developed RHD model using BALB/c mice and we evaluate the expression of NKX2-5 and Smad-6 in different conditions. RESULTS: The disease conditions are confirmed through histological sectioning of RHD heart tissue with its associated Aschoff bodies. The histological of control heart tissue in the absence of NKX2-5 looks abnormal with an enlarged nucleus and in the absence of Smad-6 the solid nature of heart tissue loosens. The mice developed a complex form of acute RHD with tissue hardening in the absence of either NKX2-5 or Smad-6 which are confirmed in NKX2-5 or Smad-6 null mice. Immunohistochemical studies reveal that the NKX2-5 and Smad-6 expression get down regulated on developing with RHD. Through experiments, we detected that both Nkx2-5 and Smad-6 are both inter-dependable and it negatively regulated each other by inhibiting them. In the absence of NKX2-5 or Smad-6, a severe form of RHD is observed together with down-regulation of either NKX2-5 or Smad-6. CONCLUSIONS: The present investigation of NKX2-5 and Smad-6 in RHD provides a new insight of data that helps to understand the disease pathogenesis.
The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases. Using the yeast two-hybrid system with Nkx2
00;'>Nkx2-5 as the 'bait', we isolated the T-box-containing transcription factor Tbx5; mutations in TBX5 cause heart and limb malformations in Holt-Oram syndrome (HOS). Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells showed that they also associate with each other in mammalian cells. Glutathione S-transferase (GST) 'pull-down' assays indicated that the N-terminal domain and N-terminal part of the T-box of Tbx5 and the homeodomain of Nkx2-5 were necessary for their interaction. Tbx5 and Nkx2-5 directly bound to the promoter of the gene for cardiac-specific natriuretic peptide precursor type A (Nppa) in tandem, and both transcription factors showed synergistic activation. Deletion analysis showed that both the N-terminal domain and T-box of Tbx5 were important for this transactivation. A G80R mutation of Tbx5, which causes substantial cardiac defects with minor skeletal abnormalities in HOS, did not activate Nppa or show synergistic activation, whereas R237Q, which causes upper-limb malformations without cardiac abnormalities, activated the Nppa promoter to a similar extent to that of wildtype Tbx5. P19CL6 cell lines overexpressing wildtype Tbx5 started to beat earlier and expressed cardiac-specific genes more abundantly than did parental P19CL6 cells, whereas cell lines expressing the G80R mutant did not differentiate into beating cardiomyocytes. These results indicate that two different types of cardiac transcription factors synergistically induce cardiac development.
Zou Y, etal., Development. 1997 Feb;124(4):793-804.
To identify the molecular pathways that guide cardiac ventricular chamber specification, maturation and morphogenesis, we have sought to characterize factors that regulate the expression of the ventricular myosin light chain-2 gene, one of the earliest markers of ventricular regionalization during m
ammalian cardiogenesis. Previously, our laboratory identified a 28 bp HF-la/MEF-2 element in the MLC-2v promoter region, which confers cardiac ventricular chamber-specific gene expression during murine cardiogenesis, and showed that the ubiquitous transcription factor YB-1 binds to the HF-la site in conjunction with a co-factor. In a search for interacting co-factors, a nuclear ankyrin-like repeat protein CARP (cardiac ankyrin repeat protein) was isolated from a rat neonatal heart cDNA library by yeast two-hybrid screening, using YB-1 as the bait. Co-immunoprecipitation and GST-CARP pulldown studies reveal that CARP forms a physical complex with YB-1 in cardiac myocytes and immunostaining shows that endogenous CARP is localized in the cardiac myocyte nucleus. Co-transfection assays indicate that CARP can negatively regulate an HF-1-TK minimal promoter in an HF-1 sequence-dependent manner in cardiac myocytes, and CARP displays a transcriptional inhibitory activity when fused to a GAL4 DNA-binding domain in both cardiac and noncardiac cell context. Northern analysis revealed that carp mRNA is highly enriched in the adult heart, with only trace levels in skeletal muscle. During murine embryogenesis, endogenous carp expression was first clearly detected as early as E8.5 specifically in heart and is regulated temporally and spatially in the myocardium. Nkx2-5, the murine homologue of Drosophila gene tinman was previously shown to be required for heart tube looping morphogenesis and ventricular chamber-specific myosin light chain-2 expression during mammalian heart development. In Nkx2-5(-/-)embryos, carp expression was found to be significantly and selectively reduced as assessed by both whole-mount in situ hybridizations and RNase protection assays, suggesting that carp is downstream of the homeobox gene Nkx2-5 in the cardiac regulatory network. Co-transfection assays using a dominant negative mutant Nkx2-5 construct with CARP promoter-luciferase reporter constructs in cardiac myocytes confirms that Nkx2-5 either directly or indirectly regulates carp at the transcriptional level. Finally, a carp promoter-lacZ transgene, which displays cardiac-specific expression in wild-type and Nkx2-5(+/-) background, was also significantly reduced in Nkx2-5(-/-) embryos, indicating that Nkx2-5 either directly or indirectly regulates carp promoter activity during in vivo cardiogenesis as well as in cultured cardiac myocytes. Thus, CARP is a YB-1 associated factor and represents the first identified cardiac-restricted downstream regulatory gene in the homeobox gene Nkx2-5 pathway and may serve as a negative regulator of HF-1-dependent pathways for ventricular muscle gene expression.
NKX2-5 mutations are associated with different forms of congenital heart disease. Despite the knowledge gained from molecular and animal studies, genotype-phenotype correlations in humans are limited by the lack of large cohorts and the incomplete assessment of
family members. We hypothesized that studying the role of NKX2-5 in inbred populations with homogeneous genetic backgrounds and high consanguinity rates such as Lebanon could help closing this gap. We sequenced NKX2-5 in 188 index CHD cases (25 with ASD). Five variants (three segregated in families) were detected in eleven families including the previously documented p.R25C variant, which was found in seven patients from different families, and in one healthy individual. In 3/5 familial dominant ASD cases, we identified an NKX2-5 mutation. In addition to the heterogeneity of NKX2-5 mutations, a diversity of phenotypes occurred within the families with predominant ASD and AV block. We did in fact identify a large prevalence of Sudden Cardiac Death (SCD) in families with truncating mutations, and two patients with coronary sinus disease. NKX2-5 is thus responsible for dominant familial ASD even in consanguineous populations, and a wide genetic and phenotypic diversity is characteristic of NKX2-5 mutations in the Lebanese population.
Liu Z, etal., Pediatr Int. 2015 Apr;57(2):234-8. doi: 10.1111/ped.12489. Epub 2014 Dec 11.
BACKGROUND: Congenital heart disease (CHD) is one of the most common birth defects in newborns. The etiology of CHD has remained largely unknown, but it is assumed to result from the combined effects of genetic and environmental factors. Recent investigations have detected potentially pat
hogenic copy number variations (CNV) in a proportion of patients with CHD. The present case-control study evaluated whether CNV in the GATA4 and NKX2-5 genes contribute to the pathogenesis of CHD in Chinese fetuses (n = 117), by comparing them with non-CHD control subjects (n = 100). METHODS: Multiplex ligation-dependent probe amplification with the P311A probe mixture was used to detect CNV. RESULTS: The normalized signals were within the normal range for all exons in all CHD patients and non-CHD control subjects. Of the 117 CHD patients, three had a deletion of 22q11, and two had a duplication of 22q11. CONCLUSIONS: There was no evidence of a role for NKX2-5 and GATA4 CNV in fetal CHD; therefore, these CNV may not be common in fetal CHD in China.
Zheng J, etal., Int J Med Sci. 2015 Jun 12;12(7):538-43. doi: 10.7150/ijms.11700. eCollection 2015.
The purposes of this study are to investigate somatic NKX2-5 mutations in Chinese children with congenital heart disease (CHD) and assess the reliability of somatic mutation detection in formalin-fixed, paraffin-embedded (FFPE) tissues. The study cohort included
frozen and FFPE cardiac tissues as well as blood samples from 85 Chinese children with CHD who had the cardiac operations. The right atrial appendage far from the diseased heart was used as normal control. Genomic DNA was isolated from cardiac tissues and blood samples using TIANamp Blood DNA kit. Two exons and exon-intron boundaries of NKX2-5 were amplified by polymerase chain reaction (PCR) and sequenced by dideoxynucleotide chain termination approach. The acquired sequences were aligned with GenBank sequences to identify the sequence variations. No somatic mutation in the NKX2-5 gene was observed in both frozen and FFPE cardiac tissues in 85 Chinese children with CHD. Nonetheless, a common single nucleotide polymorphism (SNP), c.63 A > G (E21E), was identified in all the three kinds of DNA samples with the same allele frequency 82.3%. Moreover, another common SNP c.606 G > C (L202L) was found in 2.3% of our patients. There were no significant differences in the allele frequencies of two SNPs between the cardiac diseased tissues and right atrial appendage (P > 0.05). PCR artefact as mutations was not found in the FFPE tissues stored for one year. Our findings demonstrate that somatic NKX2-5 mutations do not represent an important aetiologic pathway in Chinese children with congenital heart disease.
Reamon-Buettner SM and Borlak J, J Med Genet. 2004 Sep;41(9):684-90.
NKX2-5 is a pivotal transcription factor in heart development. Previous studies on lymphocytic DNA provided evidence of familial NKX2-5 gene mutations in cardiac malformations. Common mutations are rare in unrelated families
. We analysed, by direct sequencing, the gene encoding NKX2-5 in the diseased heart tissues of 68 patients with complex congenital heart disease, focussing particularly on atrial, ventricular, and atrioventricular septal defects. We identified 35 non-synonymous NKX2-5 mutations in the diseased heart tissues of patients. These mutations were mainly absent in normal, for example, unaffected, heart tissue of the same patient, indicating the somatic nature and mosaicism of mutations. We also observed multiple mutations and multiple haplotypes, as well as mutations in Down's syndrome patients with cardiac malformations. Taken collectively, the above results suggest the somatic nature of NKX2-5 mutations associated with complex cardiac malformations. Somatic mutations in transcription factor genes of cardiac progenitor cells provide a novel mechanism of disease.
Boldt LH, etal., Int J Cardiol. 2010 Nov 19;145(2):316-7. doi: 10.1016/j.ijcard.2009.11.023.
Atrial fibrillation (AF) is the most frequently encountered arrhythmia in clinical practice. In a subgroup of patients, AF is regarded as idiopathic when no signs of structural heart disease or other causes of the arrhythmia can be identified during conventional clinical work-up. Recent studies have
demonstrated that AF has a substantial genetic basis in a number of cases. The entire coding sequences, including intron-exon boundaries, of the genes PITX2 and NKX2-5 were screened for genetic variants by means of initial polymerase chain reaction followed by DNA sequencing in 96 patients with idiopathic AF. Although we detected a number of variants, our candidate gene approach did not result in identification of mutations associated with AF in the coding regions of PITX2 or NKX2-5 in our well characterized AF cohort.
BACKGROUND: Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac
malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. METHODS AND RESULTS: We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg->Gly (R52G). All the heterozygous neonatal Nkx2-5(+/R52G) mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5(+/R52G) mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5(+/+) or Nkx2-5(+/-) mice. CONCLUSIONS: The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.
Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutati
ons in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies.
We report on a familial screen of five female members in three generations affected by an autosomal-dominant inherited atrioventricular (AV) conduction block associated with atrial septal defects (ASD) and other congenital cardiovascular diseases (CCVD), such as pulmonary artery stenosis (PAS), pate
nt foramen ovale (PFO) and ventricular septal defect (VSD). We tested the cardiac transcription factor NKX2-5 which is known to cause CCVD with variable phenotype and penetrance by direct sequencing of the two NKX2-5 coding exons in the index patient and identified a novel heterozygous c.325G> T mutation in exon 1 of the gene. This mutation co-segregated with the disease in the family and was present in all five affected family members, but not in 100 control chromosomes. The c.325G > T mutation is predicted to introduce a stop codon at amino-acid position 109 (p.E109X). The truncated protein lacks all of the functionally important domains of the cardiac transcription factor. Therefore, it is very likely that this novel mutation causes a complete loss of NKX2-5 function and haploinsufficiency is the pathophysiological mechanism underlying the disease in the family.
Biben C, etal., Circ Res. 2000 Nov 10;87(10):888-95.
Heterozygous mutations in the cardiac homeobox gene, NKX2-5, underlie familial cases of atrial septal defect (ASD) with severe atrioventricular conduction block. In this study, mice heterozygous for Nkx2-5-null alleles were
assessed for analogous defects. Although ASD occurred only rarely, atrial septal dysmorphogenesis was evident as increased frequencies of patent foramen ovale and septal aneurysm, and decreased length of the septum primum flap valve. These parameters were compounded by genetic background effects, and in the 129/Sv strain, septal dysmorphogenesis bordered on ASD in 17% of Nkx2-5 heterozygotes. In a proportion of neonatal heterozygotes, as well as in adults with ASD, we found that the size of the foramen ovale was significantly enlarged and altered in shape, potentially exposing the normally thin septum primum to excessive hemodynamic forces. Therefore, defective morphogenesis of the septum secundum may be one contributing factor in the generation of patent foramen ovale, septal aneurysm, and certain ASDs. Mild prolongation of P-R interval in females and an increased frequency of stenotic bicuspid aortic valves were also features of the Nkx2-5 heterozygous phenotype. Our data demonstrate that the complex effects of Nkx2-5 haploinsufficiency in mice are weaker but convergent with those in humans. As in the mouse, the phenotype of human NKX2-5 mutations may be modulated by interacting alleles.
Background. Variants of several genes encoding transcription modulators, signal transduction, and structural proteins are known to cause Mendelian congenital heart disease (CHD). NKX2-5 and GATA4 were the first CHD-causing genes identified by linkage analysis in
large affected families. Mutations of TBX5 cause Holt-Oram syndrome, which includes CHD as a clinical feature. All three genes have a well-established role in cardiac development. Design. In order to investigate the possible role of multiple mutations in CHD, a combined mutation screening was performed in NKX2-5, GATA4, and TBX5 in the same patient cohort. Samples from a cohort of 331 CHD patients were analyzed by polymerase chain reaction, double high-performance liquid chromatography and sequencing in order to identify changes in the NKX2-5, GATA4, and TBX5 genes. Results. Two cases of multiple heterozygosity of putative disease-causing mutations were identified. One patient was found with a novel L122P NKX2-5 mutation in combination with the private A1443D mutation of MYH6. A patient heterozygote for a D425N GATA4 mutation carries also a private mutation of the MYH6 gene (V700M). Conclusions. In addition to reporting two novel mutations of NKX2-5 in CHD, we describe families where multiple individual mutations seem to have an additive effect over the pathogenesis of CHD. Our findings highlight the usefulness of multiple gene mutational analysis of large CHD cohorts.
The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the de
velopment of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.
The homeobox transcription factor Nkx2-5 and the zinc metalloprotease endothelin-converting enzyme-1 (ECE-1) are essential for cardiac development. Here, we demonstrate for the first time a functional link between Nkx2-5 and
ECE-1. In transiently transfected rat H9c2 cardiomyoblasts, the alternative promoters specific for ECE-1a, ECE-1b, and ECE-1c are activated by Nkx2-5 coexpression. Lack of a consensus sequence for Nkx2-5 binding within the ECE-1c promoter and mutational analyses of Nkx2-5 consensus sequences identified in the ECE-1a and ECE-1b promoters, respectively, reveal an indirect mechanism of activation that is supported by gel shift assays. Furthermore, we have evidence of an additional direct activation mechanism of the ECE-1b promoter by Nkx2-5. With the use of RNase protection assay, Northern blot, and real-time PCR, the activating effect of Nkx2-5 on mRNA expression of ECE-1 isoforms was confirmed in the chromatin context of H9c2 and endothelial EA.hy926 cells, respectively, by stable Nkx2-5 overexpression. The interaction presented in this work provides a possible explanation for distinct phenotypic aspects of patients carrying mutations in the Nkx2-5 gene and may also be of significance for the pathophysiology of heart failure.
OBJECTIVE: Atrial septal defect (ASD) is the second most common congenital heart defect (CHD) and is observed in families as an autosomal dominant trait as well as in nonfamilial CHD. Mutations in the NKX2-5 gene, located on chromosome 5, are associat
ed with ASD, often combined with conduction disturbances, cardiomyopathies, complex CHD, and sudden cardiac death as well. Here, we show that NKX2-5 mutations primarily occur in ASD patients with conduction disturbances and heritable ASD. Furthermore, these families are at increased risk of sudden cardiac death. RESULTS: We screened 39 probands with familial CHD for mutations in NKX2-5 and discovered a novel mutation in one family (2.5%) with ASD and atrioventricular block. A review of the literature revealed 59 different NKX2-5 mutations in 202 patients. Mutations were significantly more common in familial cases compared to nonfamilial cases (P¿=¿7.1 × 10(-9) ). The majority of patients (74%) had ASD with conduction disturbance. Nineteen patients (15%) of 120 with familial ASD and conduction disturbance died from sudden cardiac death of which nine (8%) were confirmed mutation carriers, and 10 were possible carriers. CONCLUSIONS: NKX2-5 mutations mainly occur in familial CHD, the signature phenotype is ASD with conduction disturbances and mutation carriers are at increased risk of sudden cardiac death. We suggest that familial ASD patients should be screened for NKX2-5 mutations and, if they are mutation carriers, implantation of an implantable cardioverter-defibrillator should be considered in these patients.
Inga A, etal., Hum Mol Genet. 2005 Jul 15;14(14):1965-75. Epub 2005 May 25.
Human heart development requires an orderly coordination of transcriptional programs, with the homeodomain protein NKX2-5 being one of the key transcription factors required for the differentiation of mesodermal progenitor cells. Indeed, lack of Nkx2
t-weight:700;'>Nkx2-5 in mice arrests heart development prior to looping, resulting in embryonic lethality. There are 28 germline NKX2-5 mutations identified in humans that are associated with congenital heart disease, and we recently reported multiple somatic mutations in patients with complex cardiac malformations. To address the functional consequences of single and multiple mutations of NKX2-5, we developed a functional assay in the budding yeast Saccharomyces cerevisiae, which could determine transactivation capacity and specificity of expressed NKX2-5 alleles towards targeted response element (RE) sequences. We focused on mutants of the third helix, which provides DNA binding specificity, and characterized mutations that were highly associated with either ventricular (VSD) or atrioventricular (AVSD) septal defects. Individual mutants exhibited partial to complete loss of function and differences in transactivation capacity between the various REs. The mutants also exhibited gene dosage rather than dominant effects on transcription. Surprisingly, all AVSD patients (22/23) had a single K183E mutation in the DNA binding domain, which resulted in transcriptional inactivation. None of the VSD patients had this mutation; yet 14/29 had at least one mutation in the third helix leading to either inactivation or reduction of NKX2-5 transactivation. Therefore, mutations of somatic origin in the binding domains of NKX2-5 were associated specifically with AVSD or VSD and resulted in loss of protein function.
Dentice M, etal., J Clin Endocrinol Metab. 2006 Apr;91(4):1428-33. doi: 10.1210/jc.2005-1350. Epub 2006 Jan 17.
CONTEXT: Congenital hypothyroidism (CH) is a common endocrine disorder with an incidence of 1:3000-4000 at birth. In 80-85% of cases, CH is caused by defects in thyroid organogenesis, resulting in absent, ectopically located, and/or severely reduced gland [thyroid dysgenesis (TD)]. Mutati
ons in genes controlling thyroid development have demonstrated that in a few cases, TD is a Mendelian trait. However, accumulating evidence supports the view that the genetics of TD are complex, possibly with a polygenic/multifactorial basis. A higher prevalence of congenital heart disease has been documented in children with CH than in the general population. Such an association suggests a possible pathogenic role of genes involved in both heart and thyroid development. NKX2-5 encodes a homeodomain-containing transcription factor with a major role in heart development, and mutations affecting this gene have been reported in individuals with congenital heart disease. OBJECTIVE: In the present work we investigated the possible involvement of NKX2-5 mutations in TD. RESULTS: Our results indicate that Nkx2-5(-/-) embryos exhibit thyroid bud hypoplasia, providing evidence that NKX2-5 plays a role in thyroid organogenesis and that NKX2-5 mutations contribute to TD. NKX2-5 mutational screening in 241 patients with TD allowed the identification of three heterozygous missense changes (R25C, A119S, and R161P) in four patients with TD. Functional characterization of the three mutations demonstrated reduced DNA binding and/or transactivation properties, with a dominant-negative effect on wild-type NKX2-5. CONCLUSION: Our results suggest a previously unknown role of NKX2-5 in the pathogenesis of TD.
Moussa H and Sidhom I, Asia Pac J Clin Oncol. 2016 Mar;12(1):e1-10. doi: 10.1111/ajco.12119. Epub 2013 Nov 8.
AIM: Cohorts of T-cell acute lymphoblastic leukemia (T-ALL) patients show regional geographic differences in incidence, biological features and clinical outcome, implying that in different populations, cases may harbor different genetic lesions than those reported elsewhere. In this study, we prospe
ctively evaluated the frequency and the clinical relevance of NKX2-5, TLX3/HOX11L2 and SIL/TAL expression in Egyptian childhood T-ALL. METHODS: NKX2-5, TLX3/HOX11L2 and SIL/TAL expression were tested in peripheral blood and/or bone marrow of 83 newly diagnosed Egyptian childhood T-ALL patients. RESULTS: NKX2-5 expression was detected in 11/83 cases (13%), TLX3/HOX11L2 (5/83, 6%) and SIL/TAL (4/83, 5%). Initial central nervous system involvement was significantly higher in the NKX2-5 positive versus negative patients (P = 0.009). The follow-up period was a median of 65.5 months. The 5-year leukemia-free and event-free survival rates of the whole T-ALL population were 70 +/- 6% and 58 +/- 6%, respectively. The 5-year leukemia-free and event-free survival rates of NKX2-5 were 86 +/- 13% and 60 +/- 16%, respectively. There were no statistically significant differences in clinical presentation, biological features, initial response to chemotherapy, or subsequent treatments between the subgroups and the total population. CONCLUSION: Egyptian T-ALL cases seemed to have a different genetic pattern compared to other populations, with a lower incidence of TLX3/HOX11L2 and SIL/TAL but a higher incidence of NKX2-5 expression than recorded in Western countries.
AIMS: Transcription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofacto
rs such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood. METHODS AND RESULTS: A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors. CONCLUSIONS: The results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors.
The homeobox transcription factors NKX2-5 and MEIS1 are essential for vertebrate heart development and normal physiology of the adult heart. We show that, during cardiac differentiation, the two transcription factors have partially overlapping expression patte
rns, with the result that as cardiac progenitors from the anterior heart field differentiate and migrate into the cardiac outflow tract, they sequentially experience high levels of MEIS1 and then increasing levels of NKX2-5. Using the Popdc2 gene as an example, we also show that a significant proportion of target genes for NKX2-5 contain a binding motif recognized by NKX2-5, which overlaps with a binding site for MEIS1. Binding of the two factors to such overlapping sites is mutually exclusive, and this provides a simple regulatory mechanism for spatial and temporal synchronization of a common pool of targets between NKX2-5 and MEIS1.
Cao Y, etal., Int J Clin Exp Pathol. 2015 Nov 1;8(11):14917-24. eCollection 2015.
BACKGROUND: Congenital heart disease (CHD) is the most common birth abnormality, especially for sporadic CHD. However, the etiology of sporadic CHD is largely unknown. NKX2-5, the earliest sign of cardiac progenitor cell differentiation, plays a key role in car
diac morphogenesis, and the mutation of this gene can cause sporadic CHD. PURPOSE: To investigate the association of genetic variations of NKX2-5 with sporadic CHD in Chinese Bai people. METHODS: The whole 2 coding exons and flanking intron sequences of NKX2-5 gene were screened using DNA sequencing in 70 Chinese Bai patients with sporadic CHD and 136 healthy controls. RESULTS: A novel heterozygous DNA sequence variant (DSV), 1433A>G, was identified in one tetralogy of Fallot (TOF) patient and one persistent left superior vena cava (PLSVC) patient, but none in controls. The frequency of single nucleotide polymorphism (SNP) rs2277923 in CHD group was significantly higher than that in control group. The allele and genotype were associated with the occurrence of CHD. CONCLUSION: The novel DSV (1433A>G) may be relevant with TOF and PLSVC, and the SNP rs2277923 of NKX2-5 gene contributes to the risk of sporadic CHD in Chinese Bai people.
Heterozygous mutations in the NKX2-5 gene of patients with various congenital heart defects have been reported. Most of the congenital heart defects associated with the mutations in the NKX2-5 gene are conotruncal heart anom
alies, primarily the tetralogy of Fallot. In this study, the authors screened 72 Turkish children with conotruncal heart anomalies and 185 healthy control subjects to find the NKX2-5 alterations. They found one previously documented NKX2-5 missense alteration, heterozygous c.73C>T (p.Arg25Cys), in a 10-year-old boy with tetralogy of Fallot. The same heterozygous alteration was found also in the patient's healthy father and in two unrelated persons in the healthy control group. The current study shows for the first time the presence of p.Arg25Cys in healthy control subjects other than African Americans. These results show that no genetic support exists for the pathogenecity of this alteration, although a previous in vitro study and theoretical predictions suggest a structural/functional difference in the altered protein region.
