Bicuspid aortic valve (BAV) is the most common form of congenital cardiovascular defect in humans and is associated with substantial morbidity and mortality. Emerging evidence demonstrates that genetic risk factors play an important role in the pathogenesis of BAV. However, BAV is a genetically heterogenous disorder, and the genetic defects underpinning BAV in most patients remain to be identified. In the present study, the coding exons and flanking introns of the NKX2.5 gene, which encodes a homeodomain-containing transcription factor essential for the normal development of the aortic valve, were sequenced in 142 unrelated patients with BAV. The available relatives of the mutation carrier and 200 unrelated healthy subjects used as controls were also genotyped for NKX2.5. The functional characteristics of the mutation were delineated by using a dual-luciferase reporter assay system. As a result, a novel heterozygous NKX2.5 mutation, p.K192X, was identified in a family with BAV transmitted in an autosomal dominant pattern. The nonsense mutation was absent in 400 control chromosomes. Functional analyses revealed that the mutant NKX2.5 had no transcriptional activity compared with its wild-type counterpart. Furthermore, the mutation abolished the synergistic transcriptional activation between NKX2.5 and GATA5, another transcription factor crucial for the aortic valvular morphogenesis. In conclusion, this study is the first to link an NKX2.5 loss-of-function mutation to enhanced susceptibility to human BAV, providing novel insight into the molecular mechanism of BAV and suggesting potential implications for genetic counseling and clinical care of families presenting with BAV.
In vertebrates, the heart is the first functional organ that forms during the development of embryos. Cardiac valve morphogenesis, which occurs early in fetal development, is a complex morphogenetic process that requires the temporal and spatial cooperation of cardiac cell commitment, differentiation, proliferation, and migration, and environmental and genetic pathogenic factors may interrupt this biologic process, leading to abnormal valvulogenesis and the formation of a bicuspid aortic valve (BAV). Recently, emerging evidence highlights the important roles of genetic defects in the pathogenesis of BAV, and mutations in multiple genes, including GATA5, have been implicated in the pathogenesis of BAV. As a synergistic transactivational partner of GATA5, NKX2.5 is essential for normal cardiovascular development and valvulogenesis in vertebrate species. The temporal and spatial expression profiles and functional characteristics of NKX2.5 partially overlap with those of the BAV-associated gene GATA5 during cardiogenesis, and in mice, targeted deletion of Nkx2.5 resulted in partially penetrant BAV, similar to what has been observed in Gata5-null mice. These findings warranted screening NKX2.5 as a preferred candidate gene for human BAV.
Methods
A total of 142 unrelated patients with BAV were prospectively recruited from the Chinese Han population. The available relatives of the index patient carrying an identified NKX2.5 mutation were also included. The control population comprised 200 unrelated, ethnically matched healthy subjects. Participants underwent clinical evaluation that included detailed individual and familial histories, complete physical examinations, standard 12-lead electrocardiography, and 2-dimensional echocardiography with color flow Doppler. BAV was confirmed by imaging and/or direct view during aortic valve replacement surgery. Familial BAV was defined if ≥2 affected relatives had proved BAV. A peripheral venous blood sample was taken from each participant. The study protocol conformed to the principles outlined in the Declaration of Helsinki and was approved by the local institutional ethics committee. Written informed consent was obtained from all participants or their guardians before the study.
Genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood leukocytes with the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin). The coding regions and splice junction sites of the NKX2.5 gene were amplified and sequenced in all participants, as described previously. For an identified sequence variation, the single-nucleotide polymorphism database ( http://www.ncbi.nlm.nih.gov ), the Human Gene Mutation Database ( http://www.hgmd.org ), and the 1000 Genomes Project database ( http://www.1000genomes.org ) were queried to confirm its novelty.
The plasmid NKX2.5-pEFSA and the atrial natriuretic factor (ANF)–luciferase (ANF-luc) reporter, which contains the 2,600-bp 5′-flanking region of the ANF gene, were kindly provided by Dr. Ichiro Shiojima of Chiba University School of Medicine in Japan. The plasmid GATA5-pcDNA3.1 and the eNOS-luc reporter, which contains the 1,600-bp 5′-flanking region of the endothelial nitric oxide synthase gene, were prepared as described previously. The identified mutation was introduced into the wild-type NKX2.5 using a QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, California) with a complementary pair of primers. The mutant was sequenced to confirm the desired mutation and to exclude any other sequence variations.
COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and seeded in 12-well plates before transfection. The reporter ANF-luc and an internal control plasmid pGL4.75 (hRluc/CMV; Promega) were used in transient transfection assay to assess the transcriptional activity of the NKX2.5 mutant. Twenty-four hours after plating, the COS-7 cells were transfected with 0.4 μg of wild-type or mutant NKX2.5-pEFSA, 1.0 μg of ANF-luc, and 0.04 μg of pGL4.75 using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, California). For cotransfection experiments, 0.2 μg of wild-type NKX2.5-pEFSA in combination with 0.2 μg of mutant NKX2.5-pEFSA or 0.2 μg of empty vector were used in the presence of 1.0 μg of ANF-luc and 0.04 μg of pGL4.75. Firefly luciferase and Renilla luciferase activities were measured with the Dual-Glo luciferase assay system (Promega) 48 hours after transfection. The activity of the ANF promoter was presented as fold activation of Firefly luciferase relative to Renilla luciferase. Three independent experiments were conducted at minimum for wild-type and mutant NKX2.5.
For the analysis of the synergistic transcriptional activation between NKX2.5 and GATA5, NIH 3T3 cells were transfected with 0.2 μg of wild-type or mutant NKX2-5-pEFSA, alone or together with 0.2 μg of wild-type GATA5-pcDNA3.1, in the presence of 1.0 μg of eNOS-luc and 0.04 μg of pGL4.75, using Lipofectamine 2000 (Invitrogen).
Data for promoter activity are expressed as mean ± SD, which were analyzed by Student’s unpaired t test, with p values <0.05 considered statistically significant.
Results
A cohort of 142 unrelated patients with BAV was clinically investigated in contrast to a total of 200 unrelated healthy control subjects. In the BAV group, 61% (n = 87), 37% (n = 52), and 2% (n = 3) had fusion of the right and left coronary, right coronary and noncoronary, and left coronary and noncoronary cusps, respectively. None of the study participants had established environmental risk factors for congenital heart disease, such as maternal illness and drug use in the first trimester of pregnancy, parental smoking, or long-term exposure to toxicants and ionizing radiation. There was no difference in gender or race between patient and control groups. The baseline clinical characteristics of the 142 unrelated patients with BAV are listed in Table 1 .
| Variables | Statistics |
|---|---|
| Age (years) | 47 ± 11 |
| Male gender | 82 (58%) |
| Positive family history | 45 (32%) |
| Abnormal valve function ∗ | 88 (62%) |
| Concomitant aortopathy † | 75 (53%) |
| Concomitant other cardiovascular structural defects ‡ | 51 (36%) |
| Atrial fibrillation | 12 (9%) |
| Atrioventricular block | 3 (2%) |
| Surgical repair | 109 (77%) |
∗ Aortic regurgitation and/or aortic stenosis with at least moderate severity.
† Aortic dilation ≥40 mm, affecting any part of the aorta from sinus of Valsalva to proximal descending aorta.
‡ Thirteen patients with coarctation of the aorta; 8 with ventricular septal defect; 6 with atrial septal defect; 4 with patent ductus arteriosus; 3 with patent formamen ovale; 2 with anomalous origin of the left coronary artery; 5 with coarctation of the aorta and ventricular septal defect; 3 with coarctation of the aorta and atrial septal defect; 7 with other complex cardiac defects.
By sequence analysis of the exons and exon-intron boundaries of NKX2.5, a novel heterozygous sequence variation was detected in this cohort. Specifically, a substitution of thymine for adenine in the first nucleotide of codon 192 (c.574A>T), predicted to produce a truncated protein with only amino-terminal 191 amino acids (p.K192X), was found in an index patient from family 1. The sequence electropherograms showing the identified heterozygous NKX2.5 variation in contrast to its corresponding control sequence are shown in Figure 1 . The schematic diagrams of NKX2.5 proteins showing the structural domains and location of the mutation identified in this study are presented in Figure 1 . The nonsense mutation was neither observed in 400 control chromosomes nor discovered in the single-nucleotide polymorphism, Human Gene Mutation Project, and 1000 Genomes Project databases, which were consulted again on July 16, 2014, indicating a novel mutation.
A genetic scan of the family members of the proband showed that the mutation was present in all the affected family members available but absent in the unaffected relatives examined. Analysis of the pedigree showed that in the family, BAV was transmitted in an autosomal-dominant pattern with complete penetrance. The pedigree structure of the family is shown in Figure 2 . In addition, the proband’s father (I-1) and first sister (II-4) also had atrial septal defect, aortic valve stenosis, paroxysmal atrial fibrillation, and atrioventricular conduction block. The phenotypic characteristics and results of genetic screening of the affected pedigree members are listed in Table 2 .
| Subject information | Phenotype | Genotype | ||
|---|---|---|---|---|
| Identity | Gender | Age at first diagnosis/ present study (years) | Cardiac structural defects and arrhythmias | Nkx2.5 mutation |
| Family 1 | K192X | |||
| I-1 | M | 33/64 ∗ | BAV ( R-L), secundum ASD, severe AS † , third-degree AVB, PAF | NA |
| II-1 | M | 47/55 | BAV ( R-L) | +/– |
| II-4 | F | 30/52 | BAV (R-L), secundum ASD, moderate AS, second-degree AVB, PAF | +/– |
| II-8 | F | 45/45 | BAV ( R-L) | +/– |
| III-1 | M | 28/28 | BAV ( R-L) | +/– |
| III-3 | M | 25/25 | BAV ( R-L), PFO | +/– |
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
