An enhancer polymorphism at the cardiomyocyte intercalated disc protein NOS1AP locus is a major regulator of the QT interval

Abstract

QT interval variation is assumed to arise from variation in repolarization as evidenced from rare Na- and K-channel mutations in Mendelian QT prolongation syndromes. However, in the general population, common noncoding variants at a chromosome 1q locus are the most common genetic regulators of QT interval variation. In this study, we use multiple human genetic, molecular genetic, and cellular assays to identify a functional variant underlying trait association: a noncoding polymorphism (rs7539120) that maps within an enhancer of NOS1AP and affects cardiac function by increasing NOS1AP transcript expression. We further localized NOS1AP to cardiomyocyte intercalated discs (IDs) and demonstrate that overexpression of NOS1AP in cardiomyocytes leads to altered cellular electrophysiology. We advance the hypothesis that NOS1AP affects cardiac electrical conductance and coupling and thereby regulates the QT interval through propagation defects. As further evidence of an important role for propagation variation affecting QT interval in humans, we show that common polymorphisms mapping near a specific set of 170 genes encoding ID proteins are significantly enriched for association with the QT interval, as compared to genome-wide markers. These results suggest that focused studies of proteins within the cardiomyocyte ID are likely to provide insights into QT prolongation and its associated disorders.

Figure 1

Regional Association between Variants at the NOS1AP Locus and QT Interval in ARIC The x axis shows the genomic interval annotated with NOS1AP and OLFML2B transcripts, the left y axis shows the statistical significance of association as negative log₁₀ of p values, and the right y axis shows the human recombination map based on HapMap samples. The most significant SNP, rs12143842, is shown as a purple diamond, the functional SNP, rs7539120 (see Figure 2, below), is shown as a red diamond, and the remainder are shown as gray circles. The genomic interval corresponding to the first major LD region (see Figure S2B) is highlighted in light gray on the x axis. The plot was generated with LocusZoom.

Figure 2

rs7539120 Is a Functional Variant Underlying QT Interval Association at the NOS1AP Locus (A) Firefly luciferase reporter enhancer/silencer assays in HL1 cells using alternate alleles/haplotypes for selected QT-interval-associated NOS1AP variants. Firefly luciferase expression is plotted relative to Renilla luciferase expression, normalized to the expression from empty vector (Promoter). Mean luciferase expression between alternate alleles/haplotypes constructs was compared with the t test and found to be significantly different between rs2010491 haplotypes, which also include rs7539120 alleles (p = 3 × 10⁻⁵). Error bars indicate SEM (n = 8). (B) Enhancer activity in rs7539120_rs2010491 haplotype is driven by rs7539120 and is dependent on the flanking sequence. Firefly luciferase reporter enhancer/silencer assays in HL1 cells using a deletion series derived from the 959 bp rs7539120_rs2010491 construct. In the construct design on left, small vertical lines represent the rs7539120 and rs2010491 SNPs, Δ represents the 11-base deletion encompassing rs7539120, 5′ deletions are represented by shorter lengths of the horizontal line, and an internal 250 bp deletion is represented by sloping lines. Error bars indicate SEM (n = 8). (C) rs7539120 acts as an in vivo enhancer. The rs7539120 risk haplotype construct injected (rs7539120_rs2010491_742_H2) at the 1–2 cell stage in developing zebrafish embryos drives transient reporter expression (enhanced-GFP; eGFP) in forebrain 24 hr postfertilization in ∼33% of injected embryos. Representative images from four different embryos with forebrain eGFP expression (white spots, strong expression indicated by yellow arrows) are shown. No eGFP expression was observed from the rs7539120 deletion (±5 bases) constructs (Δrs7539120_rs2010491_742_H2) (see Figure S4). Abbreviations are as follows: FB, forebrain; YS, yolk sac. (D) rs7539120 acts as a cardiac expression quantitative trait locus (eQTL). Box-whisker plots of NOS1AP mRNA expression in human left ventricle tissue from 131 heart failure subjects genotyped at rs7539120. The y axis shows –ΔCT used as a measure of expression. The risk allele T is associated with higher expression of NOS1AP (p = 4.72 × 10⁻⁵). (E) rs7539120 is bound by an uncharacterized protein(s) from HL1 cell nuclear extract. EMSAs using radiolabeled probes (P) containing nonrisk allele (A), risk allele (T), and deletion (Δ) of rs7539120 in presence or absence of HL1 cells nuclear extract (NE) and excess unlabeled competing probe (C). The black arrow indicates DNA-protein complex formed with both the A and T alleles containing probes, but lacking with the deletion probe. + indicates addition; - indicates absence.

Figure 3

Expression Levels of NOS1AP Influences Cardiac Electrophysiology (A) Left: Representative APD (in milliseconds, ms) trace at 80% repolarization (APD₈₀) in NRVMs nontransduced (top) and transduced with human NOS1AP short (middle) and long (bottom) isoforms, respectively. Overexpression of both isoforms of NOS1AP in NRVMs lead to significantly reduced APD₈₀, as compared to nontransduced cells. Right: Bar plots showing mean APD₈₀ from four replicates. Error bars indicate standard deviation and asterisks indicate p < 0.05. (B) Representative isochronal maps for CV (in cm/s) in monolayer of NRVMs nontransduced (top left) and transduced with human NOS1AP short (top right) and long (bottom left) isoform, respectively. Overexpression of both isoforms of NOS1AP in NRVMs leads to significantly increased CV, as compared to nontransduced cells. Bottom right: Bar plots showing mean CV from four replicates. Error bars indicate standard deviation and asterisks indicate p < 0.05.

Figure 4

NOS1AP Localizes to ID in Cardiac Muscle (A) Immunohistochemical staining of FFPE section of human heart (left ventricle free wall) with NOS1AP antibody. Intense staining (dark brown) was observed at ID joining adjacent cardiomyocytes. (B) NOS1AP colocalizes with proteins marking the three well-recognized structural zones within the ID: desmosomes, gap junctions, and fascia adherens. Immunohistochemical staining of FFPE section of human heart with an antibody against an ID marker protein (Plakoglobin at desmosomes, top; Connexin-43 at gap junctions, middle; and N-cadherin at fascia adherens, bottom) alone (left column) and in combination with NOS1AP antibody (right column).

Figure 5

Genes Encoding Proteins Localized at Cardiomyocytes ID Play a Significant Role in Interindividual QT Interval Variation QQ plots of QT interval GWAS, and association studies of QT interval using a specific set 170 ID genes and NOS1AP, and a control set of 166 heart-expressed genes not localized at ID. The black, blue, green, and red curves show the QQ plots for all GWASs, ID genes including NOS1AP, ID genes minus NOS1AP, and the control heart-expressed genes. The gray line shows the expected values from a theoretical χ²-distribution. For each gene we selected variants in and around (±10 kb) the gene from the GWAS. The y axis has been truncated at 35.