Transethnic Genome-Wide Association Study Provides Insights in the Genetic Architecture and Heritability of Long QT Syndrome

BACKGROUND: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. METHODS: We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. RESULTS: Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance (P&lt;5×10-8) near NOS1AP, KCNQ1, and KLF12, and 1 missense variant in KCNE1(p.Asp85Asn) at the suggestive threshold (P&lt;10-6). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (rg=0.40; P=3.2×10-3). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls (P&lt;10-13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive (P&lt;0.005). CONCLUSIONS: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.</p

Antiviral Protection via RdRP-Mediated Stable Activation of Innate Immunity

<div><p>For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or <i>a priori</i> knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (<i>Rag1</i>, <i>Mda5</i>, <i>Mavs</i>, <i>Ifnar1</i>, <i>Ifngr1</i>, and <i>Tlr3)</i> established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.</p></div

RdRP mice appear indistinguishable from WT controls.

<p>(A) Histological examination of WT and RdRP transgenic mice. Tissues were harvested from uninfected age-matched, sex-matched adult FVB mice (n = 2 per genotype). Histological examination was performed on formalin-fixed paraffin-embedded tissue after hematoxylin and eosin staining. (B) Gross tissue morphology. Tissues were harvested from uninfected age-matched, sex-matched adult FVB mice (n = 2 mice per genotype). (C) Kidney tissues were closely examined (40X) for signs of glomerulonephritis. (D) Sex-matched WT (n = 3) and RdRP (n = 3) mice were randomly chosen and photographed at different time points throughout their development. Mice are age-matched at postnatal (p) day 4, p15, 4 weeks (wk), 7 wk, and 12 wk. (E) Survival curve of female WT (n = 4) and RdRP (n = 4) mice in the absence of infection. (F and G) WT (F, n = 2) and RdRP mice (G, n = 4) from (E) were photographed at 20 months of age.</p

RdRP-induced antiviral phenotype is mediated in a MDA5-MAVS, IFNαβR-dependent manner.

<p>(A-E) Knockout mice were bred to RdRP mice and the antiviral phenotype of each cross was analyzed by immunoblotting and survival. Left, cerebrum (top panel), spinal cord (middle), and cerebellum (bottom) tissue homogenates (n = 2 uninfected mice per genotype, 1 per lane) were analyzed by immunoblotting. Right, survival curve following EMCV infection (n = 8 mice per genotype), data are representative of two independent experiments. (A) <i>IfnγR</i>^<i>-/-</i> cross. (B) <i>IfnαβR</i>^<i>-/-</i> cross. (C) <i>Tlr3</i>^<i>-/-</i> cross. (D) <i>Mda5</i>^<i>-/-</i> cross. (E) <i>Mavs</i>^<i>-/-</i> cross.</p

Rapid induction of antiviral ISG mRNAs following RdRP expression in human lung epithelial cells.

<p>(A) DOX-inducible RdRP A549 cell lines were generated using lentiviral vectors that inducibly co-express RdRP or RdRPΔcat and resistance to puromycin. Vector terminology: Tsin-TetON-RdRP-IRESpuro (TetON-RdRP) and Tsin-TetON-RdRPΔcat-IRESpuro (TetON-RdRPΔcat) lentiviral vectors. (B) TetON-A549 cell lysates were analyzed by immunoblotting to determine relative abundance of RdRP-HA protein (anti-HA-tag) following DOX treatment (hr). (C) mRNA levels of the prominent antiviral genes, <i>IFI27</i>, <i>OASL</i>, <i>ISG15</i>, <i>IFIT1</i>,and <i>IFNβ</i> in TetON-A549 cell lines prior to (0 hr) or after (24 and 48 hr) DOX treatment (data are mean ± SEM of three technical replicates, relative to <i>GAPDH</i> mRNA). * <i>P<0</i>.<i>05; ** P<0</i>.<i>01; *** P<0</i>.<i>001; **** P<0</i>.<i>0001</i>.</p

Human THP-1 cells expressing a catalytically-active RdRP display highly augmented antiviral defenses.

<p>(A) Stable RdRP THP-1 cell lines were generated using lentiviral vectors that co-express GFP-puro with RdRP, RdRPΔcat, or no RdRP (null). (B) Heat map of differentially expressed genes in THP-1 monocytes, presented as fold change (blue = 1-fold induction; purple = 8-fold upregulation; red = ≥15-fold upregulation). RdRP: subset of antiviral genes significantly upregulated >4-fold in RdRP THP-1 cells (n = 2) compared with null empty vector control THP-1 cells (n = 2). RdRPΔcat: THP-1 cells expressing the RdRPΔcat transgene (n = 2) compared with null empty vector control THP-1 cells (n = 2). For a complete list of upregulated genes see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005311#ppat.1005311.s009" target="_blank">S4</a> & <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005311#ppat.1005311.s012" target="_blank">S7</a> Tables. (C) THP-1 cell lysates were run in duplicate and analyzed by immunoblotting using antibodies to OAS2, MX1, and IFIT1. Antibody to HA-tag was used to determine relative abundance of RdRP-HA protein in each cell line. Antibody to β-ACTIN served as control, data are representative of two independent experiments. (D) Infection by an HIV-1 reporter virus (HIV-1_luc) in RdRP, RdRPΔcat, and parental THP-1 cell lines. Data are mean ± SEM of three technical replicates, and are representative of two independent experiments.</p

RdRP mice display highly augmented antiviral defenses.

<p>(A) Heat map of differentially expressed genes in CNS tissue. Microarray data are presented as fold change (blue = 1-fold induction; purple = 8-fold upregulation; red = ≥15-fold upregulation). WT+EMCV: expression profile of genes significantly upregulated >4-fold in spinal cords of WT FVB mice infected with EMCV (n = 2) compared with uninfected WT FVB mice (n = 2). RdRP: uninfected transgenic RdRP FVB mice (n = 3) compared with uninfected WT FVB mice (n = 3), data are representative of two independent experiments. RdRP+EMCV: RdRP FVB mice infected with EMCV (n = 2) compared with uninfected WT FVB mice (n = 2). For a complete list of upregulated genes see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005311#ppat.1005311.s006" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005311#ppat.1005311.s008" target="_blank">S3</a> Tables. (B) mRNA levels of the prominent antiviral genes, <i>Oasl2</i>, <i>Isg15</i>, <i>Ifit1</i>, and <i>Rig-I</i>, determined by RT-PCR in cerebrum, spinal cord, and cerebellum tissues of uninfected (WT FVB or RdRP FVB) or virally-infected FVB mice (WT+EMCV or RdRP+EMCV) (n = 5 mice per group, relative to <i>Gapdh</i> mRNA, mean ± SEM). (C and D) Cerebrum, spinal cord, and cerebellum tissue homogenates (C, n = 2 mice per group, 1 per lane; D, n = 1 uninfected mouse of specified age in months per lane) were analyzed by immunoblotting using antibodies for RIG-I and ISG15. Antibody for β-ACTIN served as control. (E) RT-PCR analysis of viral titers in murine tissues two days post infection with EMCV (n = 8 mice per genotype, relative to endogenous <i>Gapdh</i> mRNA, mean ± SEM). (F) Survival curve following EMCV infection (n = 8 mice per genotype). Results are presented as a Kaplan-Meier plot and are representative of two independent experiments. * <i>P<0</i>.<i>05; ** P<0</i>.<i>01; *** P<0</i>.<i>001; **** P<0</i>.<i>0001</i>.</p

Transethnic Genome-Wide Association Study Provides Insights in the Genetic Architecture and Heritability of Long QT Syndrome

Supplemental Digital Content is available in the text. We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance (P <5×10 −8) near NOS1AP, KCNQ1, and KLF12, and 1 missense variant in KCNE1 (p.Asp85Asn) at the suggestive threshold (P <10 −6). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (r=0.40; P =3.2×10 −3). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls (P <10−13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive (P <0.005). This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS