Disease relevance of NKX2-5

• Congenital heart disease caused by mutations in the transcription factor NKX2-5 [1].
• The cardiac homeobox gene NKX2-5 is deregulated by juxtaposition with BCL11B in pediatric T-ALL cell lines via a novel t(5;14)(q35.1;q32.2) [2].
• BACKGROUND: Mutations in NKX2-5 have been found in families showing secundum ASD and atrioventricular (AV) conduction block and in some individuals with tetralogy of Fallot [3].
• OBJECTIVES: We sought to examine the importance of mutations in the cardiac transcription factor gene NKX2-5 in patients with an atrial septal defect (ASD), patent foramen ovale (PFO), or hypoplastic left heart syndrome (HLHS) [3].
• The coinheritance of NKX2.5 mutations with various congenital heart defects suggests that this transcription factor contributes to diverse cardiac developmental pathways [4].

High impact information on NKX2-5

• Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation [5].
• The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases [5].
• Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells showed that they also associate with each other in mammalian cells [5].
• A dominant disease locus associated with cardiac malformations and atrioventricular conduction abnormalities was mapped to chromosome 5q35, where NKX2-5, a Drosophila tinman homolog, is located [1].
• 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 [1].

Biological context of NKX2-5

• 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 [6].
• 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 [6].
• We studied the pathology and molecular genetics of NKX2-5 in diseased heart tissues of 68 patients with complex congenital heart disease (CHD), particularly atrial (ASD), ventricular (VSD), and atrioventricular septal defects (AVSD) [7].
• We found one previously documented NKX2-5 missense mutation, T178M, in members of a family with ASD without AV conduction block [3].
• 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 [8].

Anatomical context of NKX2-5

• 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 [6].
• Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis [8].
• Furthermore, overexpression of NKX2.5 in C2C12 cells and primary cultures of human fetal myoblasts led to differentiation of myoblasts into neuron-like cells and expression of neuronal markers [9].
• The expression of NKX2.5 in terminally differentiated C2C12 myotubes resulted in a change in morphology and breakdown into smaller myotubes [9].
• High NKX2-5 expression was also found in the T-cell lines PEER and CCRF-CEM, which harbor an NKX2-5-BCL11B rearrangement, and in the embryonic kidney cell line 293 [10].

Associations of NKX2-5 with chemical compounds

• Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain [11].
• Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively [12].
• We have genetically defined a novel conserved tyrosine-rich domain (YRD) within Nkx2-5 that has co-evolved with its homeodomain [13].
• Furthermore, heterozygous mutation of all nine tyrosines to alanine created an allele with a strong dominant-negative-like activity in vivo: ES cell<-->embryo chimaeras bearing the heterozygous mutation died before term with cardiac malformations similar to the more severe anomalies seen in NKX2.5 mutant families [13].
• The detection of beta amyloid by CSX was nullified by formic acid pretreatment of the tissue section [14].
• Knockdown of PU.1, known to be trichostatin A responsive and which potentially binds TLX3/NKX2-5 promoters, effected down-regulation of both homeobox genes [15].

Enzymatic interactions of NKX2-5

• SUMO-2 linkage to Nkx2.5 was catalyzed only by PIASx and not by other PIAS proteins [16].

Regulatory relationships of NKX2-5

• In this t(5;14) variant, NKX2-5 is expressed instead of TLX3 at both RNA and protein levels [2].
• As a result of this recombination NKX2-5 was placed under influence of the TRD enhancer, resulting in strong ectopic NKX2-5 expression [10].
• Humans with heterozygous mutations in the NKX2.5 gene generally have a disorder involving progressive atrio-ventricular conduction block and atrial septal defect, although sometimes other abnormalities including tetralogy of Fallot [17].

Other interactions of NKX2-5

• Based on these findings and recent studies showing that haploinsufficiency for other cardiac transcription factor genes (e.g., TBX5, NKX2-5) causes congenital heart disease, we postulate that GATA-4 deficiency may contribute to the phenotype of patients with monosomy of 8p23.1 [18].
• Mutations in the gene encoding the homeobox transcription factor NKX2-5 were found to cause nonsyndromic, human congenital heart disease [1].
• The coding region of the NKX2-5 locus was amplified by polymerase chain reaction and sequenced [3].
• GATA-6 cannot substitute for GATA-4 for interaction with Nkx2-5 [19].
• Adipose tissue-derived stem cells (ADSCs) were transduced with two different lentiviral vectors simultaneously: (1) a lentiviral vector expressing eGFP controlled by the Nkx2.5 promoter and (2) a lentiviral vector expressing DsRed2 controlled by the myosin light chain-2v promoter (MLC-2v) [20].

Analytical, diagnostic and therapeutic context of NKX2-5

• Functional dissection of sequence-specific NKX2-5 DNA binding domain mutations associated with human heart septation defects using a yeast-based system [6].
• Expression of NKX2-5 was analyzed by real-time quantitative PCR in the T-ALL case with the NKX2-5-TRD rearrangement, 18 other cases of T-ALL, three T-ALL derived cell lines, two non-hematopoietic cell lines, peripheral blood mononuclear cells from six healthy individuals and sorted thymocyte subsets [10].
• We identified 7 novel mutations by sequence analysis of the NKX2.5-coding region in 26 individuals [4].
• In the present study, we have determined the chromosomal localization of CSX by fluorescence in situ hybridization techniques and systemic screening of a yeast artificial chromosome library using polymerase chain reaction [21].
• Immunostaining and RT-PCR analysis of the dual positive cells revealed that these cells are positive for Nkx2.5, cardiac troponin I, and L-type calcium channel alpha-1c subunit [20].

References