Mutational Spectrum of the NKX2-5 Gene in Patients with Lone Atrial Fibrillation

Abstract

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia in humans and is responsible for substantial morbidity and mortality worldwide. Emerging evidence indicates that abnormal cardiovascular development is involved in the pathogenesis of AF. In this study, the coding exons and splice sites of the NKX2-5 gene, which encodes a homeodomain-containing transcription factor essential for cardiovascular genesis, were sequenced in 146 unrelated patients with lone AF as well as the available relatives of the mutation carriers. A total of 700 unrelated ethnically matched healthy individuals used as controls were genotyped. The disease-causing potential of the identified NKX2-5 variations was predicted by MutationTaster and PolyPhen-2. The functional characteristics of the mutant NKX2-5 proteins were analyzed using a dual-luciferase reporter assay system. As a result, two heterozygous NKX2-5 mutations, including a previously reported p.E21Q and a novel p.T180A mutation, were identified in two families with AF transmitted in an autosomal dominant pattern. The mutations co-segregated with AF in the families with complete penetrance. The detected substitutions, which altered the amino acids highly conserved evolutionarily across species, were absent in 700 control individuals and were both predicted to be causative. Functional analyses demonstrated that the NKX2-5 mutants were associated with significantly decreased transcriptional activity compared with their wild-type counterpart. The findings expand the spectrum of NKX2-5 mutations linked to AF and provide additional evidence that dysfunctional NKX2-5 may confer vulnerability to AF, suggesting the potential benefit for the early prophylaxis and personalized treatment of AF.

Prevalence and spectrum of NKX2.5 mutations in patients with congenital atrial septal defect and atrioventricular block

“…** P<0.01 and * P<0.005, compared with the same quantity of wild-type NKX2.5. ANF, atrial natriuretic factor; NKX2.5, NK2 homeobox 5. homeodomain 2 (11,19,26,29,31,48,49). The NKX2.5 mutation of p.Q181X identified in the present study was located in the HD, and functional analyses revealed that the mutant protein showed loss of the ability to transcriptionally activate ANF, alone or together with GATA4.…”

“…In humans, the NKX2.5 gene is mapped to chromosome 5q34, coding for a protein of 324 amino acids, which is expressed at a high level in the human heart (11). The NKX2.5 protein contains an evolutionarily conserved homeodomain (HD), which is centrally located at amino acid positions 138-197, and functions to specifically recognize and bind to a consensus DNA motif, AAGTG, which is key in the transcriptional regulation of target genes, including ANF, brain natriuretic peptide and α-actin, either alone or in synergy with other transcription factors, including GATA4, TBX5, TBX20, heart and neural crest derivatives expressed 2 and paired-like .…”

“…By contrast, mice homozygous for a ventricle-restricted knockout of Nkx2.5 showed no cardiovascular structural defects, but exhibited marked overgrowth of trabecular muscle and progressive complete heart block owing to hypoplastic atrioventricular node at birth (38). In humans, mutations in NKX2.5 have been associated with a wide spectrum of cardiovascular diseases, including congenital heart defects, such as atrial septal defect (ASD), ventricular septal defect, tetralogy of Fallot, bicuspid aortic valve, mitral valve deformations, subvalvular aortic stenosis, abnormal systemic venous return, hypoplasia of left heart and visceral situs inversus (2,(5)(6)(7)(8)(9)39), dilated cardiomyopathy (10), arrhythmias, such as atrioventricular block (AVB), atrial fibrillation and ventricular tachycardia (10)(11)(12)34,(40)(41)(42), and sudden cardiac death (12,43,44), of which ASD and AVB are the two most frequent phenotypes in patients carrying NKX2.5 mutations. Of note, in a murine knock-in model generated by knocking in a comparable NKX2.5 missense mutation (p.R52G) previously identified in patients, pleiotropic cardiac anomalies, including ASD, ventricular septal defect, atrioventricular septal defect, Ebstein malformation of the tricuspid valve and ventricular noncompaction were observed, in addition to progressive AVB, and this was similar or more marked, compared with the cardiac anomalies found in humans harboring a heterozygous mutation of p.R52 G in NKX2.5 (45,46).…”

“…Although the genetic basis underpinning a number of these defects remains to be elucidated, the core cardiac transcription factors, which are expressed predominantly in the heart and mediate the expression of genes encoding cardiac structural proteins or regulatory proteins, are increasingly recognized as being important in the normal development of the heart (6). Mutations in certain genes encoding core cardiac transcription factors, including homeodomain-containing NK2 homeobox 5 (NKX2.5) and NKX2.6 (7)(8)(9)(10)(11)(12)(13)(14)(15)(16), GATA-binding protein (GATA) 4, GATA5 and GATA6 (17)(18)(19)(20)(21)(22)(23)(24), T-box protein (TBX) 5 and TBX20 (25)(26)(27)(28)(29), and a paired-like homeobox transcription factor, PITX2 (30-33), have emerged as major contributors to several types of congenital heart defects (2,5,6,34).…”

Genomics of Atrial Fibrillation

“…Implicated genes include those encoding connexin 40, GJA5 [7, 67–69], the transcription factor gene NKX2.5 [70, 71], and the LMNA gene[72]. Genes involved in the renin-angiotensin-aldosterone pathway, including the angiotensin converting enzyme, angiotensin gene promoter and angiotensinogen[73–76], have also been associated with AF.…”