Genome-wide imputation study identifies novel HLA locus for pulmonary fibrosis and potential role for auto-immunity in fibrotic idiopathic interstitial pneumonia.

BACKGROUND: Fibrotic idiopathic interstitial pneumonias (fIIP) are a group of fatal lung diseases with largely unknown etiology and without definitive treatment other than lung transplant to prolong life. There is strong evidence for the importance of both rare and common genetic risk alleles in familial and sporadic disease. We have previously used genome-wide single nucleotide polymorphism data to identify 10 risk loci for fIIP. Here we extend that work to imputed genome-wide genotypes and conduct new RNA sequencing studies of lung tissue to identify and characterize new fIIP risk loci. RESULTS: We performed genome-wide genotype imputation association analyses in 1616 non-Hispanic white (NHW) cases and 4683 NHW controls followed by validation and replication (878 cases, 2017 controls) genotyping and targeted gene expression in lung tissue. Following meta-analysis of the discovery and replication populations, we identified a novel fIIP locus in the HLA region of chromosome 6 (rs7887 P meta = 3.7 × 10(-09)). Imputation of classic HLA alleles identified two in high linkage disequilibrium that are associated with fIIP (DRB1*15:01 P = 1.3 × 10(-7) and DQB1*06:02 P = 6.1 × 10(-8)). Targeted RNA-sequencing of the HLA locus identified 21 genes differentially expressed between fibrotic and control lung tissue (Q < 0.001), many of which are involved in immune and inflammatory response regulation. In addition, the putative risk alleles, DRB1*15:01 and DQB1*06:02, are associated with expression of the DQB1 gene among fIIP cases (Q < 1 × 10(-16)). CONCLUSIONS: We have identified a genome-wide significant association between the HLA region and fIIP. Two HLA alleles are associated with fIIP and affect expression of HLA genes in lung tissue, indicating that the potential genetic risk due to HLA alleles may involve gene regulation in addition to altered protein structure. These studies reveal the importance of the HLA region for risk of fIIP and a basis for the potential etiologic role of auto-immunity in fIIP.

Association between the MUC5B promoter polymorphism and survival in patients with idiopathic pulmonary fibrosis.

IMPORTANCE: Current prediction models of mortality in idiopathic pulmonary fibrosis (IPF), which are based on clinical and physiological parameters, have modest value in predicting which patients will progress. In addition to the potential for improving prognostic models, identifying genetic and molecular features that are associated with IPF mortality may provide insight into the underlying mechanisms of disease and inform clinical trials. OBJECTIVE: To determine whether the MUC5B promoter polymorphism (rs35705950), previously reported to be associated with the development of pulmonary fibrosis, is associated with survival in IPF. DESIGN, SETTING, AND PARTICIPANTS: Retrospective study of survival in 2 independent cohorts of patients with IPF: the INSPIRE cohort, consisting of patients enrolled in the interferon-γ1b trial (n = 438; December 15, 2003-May 2, 2009; 81 centers in 7 European countries, the United States, and Canada), and the Chicago cohort, consisting of IPF participants recruited from the Interstitial Lung Disease Clinic at the University of Chicago (n = 148; 2007-2010). The INSPIRE cohort was used to model the association of the MUC5B genotype with survival, accounting for the effect of matrix metalloproteinase 7 (MMP-7) blood concentration and other demographic and clinical covariates. The Chicago cohort was used for replication of findings. MAIN OUTCOMES AND MEASURES: The primary end point was all-cause mortality. RESULTS: The numbers of patients in the GG, GT, and TT genotype groups were 148 (34%), 259 (59%), and 31 (7%), respectively, in the INSPIRE cohort and 41 (28%), 98 (66%), and 9 (6%), respectively, in the Chicago cohort. The median follow-up period was 1.6 years for INSPIRE and 2.1 years for Chicago. During follow-up, there were 73 deaths (36 GG, 35 GT, and 2 TT) among INSPIRE patients and 64 deaths (26 GG, 36 GT, and 2 TT) among Chicago patients. The unadjusted 2-year cumulative incidence of death was lower among patients carrying 1 or more copies of the IPF risk allele (T) in both the INSPIRE cohort (0.25 [95% CI, 0.17-0.32] for GG, 0.17 [95% CI, 0.11-0.23] for GT, and 0.03 [95% CI, 0.00-0.09] for TT) and the Chicago cohort (0.50 [95% CI, 0.31-0.63] for GG, 0.22 [95% CI, 0.13-0.31] for GT, and 0.11 [95% CI, 0.00-0.28] for TT). In the INSPIRE cohort, the TT and GT genotypes (risk for IPF) were associated with improved survival compared with GG (hazard ratios, 0.23 [95% CI, 0.10-0.52] and 0.48 [95% CI, 0.31-0.72], respectively; P < .001). This finding was replicated in the Chicago cohort (hazard ratios, 0.15 [95% CI, 0.05-0.49] and 0.39 [95% CI, 0.21-0.70], respectively; P < .002). The observed association of MUC5B with survival was independent of age, sex, forced vital capacity, diffusing capacity of carbon monoxide, MMP-7, and treatment status. The addition of the MUC5B genotype to the survival models significantly improved the predictive accuracy of the model in both the INSPIRE cohort (C = 0.71 [95% CI, 0.64-0.75] vs C = 0.68 [95% CI, 0.61-0.73]; P < .001) and the Chicago cohort (C = 0.73 [95% CI, 0.62-0.78] vs C = 0.69 [95% CI, 0.59-0.75]; P = .01). CONCLUSIONS AND RELEVANCE: Among patients with IPF, a common risk polymorphism in MUC5B was significantly associated with improved survival. Further research is necessary to refine the risk estimates and to determine the clinical implications of these findings.

Virologic escape during danoprevir (ITMN-191/RG7227) monotherapy is hepatitis C virus subtype dependent and associated with R155K substitution.

Danoprevir is a hepatitis C virus (HCV) NS3/4A protease inhibitor that promotes multi-log(10) reductions in HCV RNA when administered as a 14-day monotherapy to patients with genotype 1 chronic HCV. Of these patients, 14/37 experienced a continuous decline in HCV RNA, 13/37 a plateau, and 10/37 a rebound. The rebound and continuous-decline groups experienced similar median declines in HCV RNA through day 7, but their results diverged notably at day 14. Plateau group patients experienced a lesser, but sustained, median HCV RNA decline. Baseline danoprevir susceptibility was similar across response groups but was reduced significantly at day 14 in the rebound group. Viral rebound in genotype 1b was uncommon (found in 2/23 patients). Population-based sequence analysis of NS3 and NS4A identified treatment-emergent substitutions at four amino acid positions in the protease domain of NS3 (positions 71, 155, 168, and 170), but only two (155 and 168) were in close proximity to the danoprevir binding site and carried substitutions that impacted danoprevir potency. R155K was the predominant route to reduced danoprevir susceptibility and was observed in virus isolated from all 10 rebound, 2/13 plateau, and 1/14 continuous-decline patients. Virus in one rebound patient additionally carried partial R155Q and D168E substitutions. Treatment-emergent substitutions in plateau patients were less frequently observed and more variable. Single-rebound patients carried virus with R155Q, D168V, or D168T. Clonal sequence analysis and drug susceptibility testing indicated that only a single patient displayed multiple resistance pathways. These data indicate the ascendant importance of R155K for viral escape during danoprevir treatment and may have implications for the clinical use of this agent.

Antiviral activity of danoprevir (ITMN-191/RG7227) in combination with pegylated interferon α-2a and ribavirin in patients with hepatitis C.

BACKGROUND: Current therapy options for patients with chronic hepatitis C virus (HCV) infection genotype 1 are effective in <50%. Danoprevir (ITMN-191/RG7227) is a potent, selective, and orally active inhibitor of the HCV NS3/4A serine protease. METHODS: The safety and antiviral efficacy of danoprevir was examined over 14 days in combination with pegylated interferon α-2a (180 µg once weekly) and ribavirin (1000-1200 mg/day) in a double-blind, placebo-controlled, phase 1b, multiple ascending dose study consisting of 6 dose cohorts (400 mg, 600 mg, and 900 mg twice daily and 100 mg, 200 mg, and 300 mg 3 times daily). RESULTS: Danoprevir in combination with pegylated interferon α-2a and ribavirin was safe and generally well tolerated. The median change in HCV RNA level from baseline to the end of treatment with danoprevir at 400 mg, 600 mg, and 900 mg twice daily was -4.7 log(10) IU/mL, -5.4 log(10) IU/mL, and -5.3 log(10) IU/mL, respectively, and at 100 mg, 200 mg, and 300 mg 3 times daily was -5.5 log(10) IU/mL, -5.7 log(10) IU/mL, and -5.6 log(10) IU/mL, respectively. Placebo administered in combination with standard of care resulted in median decrease in HCV RNA level of -2.6 log(10) IU/mL (with twice daily regimen) and -2.0 log(10) IU/mL (with 3 times daily regimen). CONCLUSIONS: Our study showed substantial antiviral efficacy of danoprevir in combination with pegylated interferon α-2a and ribavirin. Exploration of the safety and antiviral efficacy of danoprevir in longer clinical studies is warranted.

Treatment of chronic hepatitis C patients with the NS3/4A protease inhibitor danoprevir (ITMN-191/RG7227) leads to robust reductions in viral RNA: a phase 1b multiple ascending dose study.

BACKGROUND & AIMS: Danoprevir is a potent and selective inhibitor of the hepatitis C virus (HCV) NS3/4A serine protease. The present study assessed the safety, pharmacokinetics, and antiviral activity of danoprevir in a randomized, placebo-controlled, 14-day multiple ascending dose study in patients with chronic HCV genotype 1 infection. METHODS: Four cohorts of treatment-naïve (TN) patients (100 mg q12 h, 100 mg q8 h, 200 mg q12 h, 200 mg q8 h) and one cohort of non-responders (NR) to prior pegylated interferon alfa-ribavirin treatment (300 mg q12 h) were investigated. RESULTS: Danoprevir was safe and well tolerated; adverse events were generally mild, transient and were not associated with treatment group or dose level. Danoprevir displayed a slightly more than proportional increase in exposure with increasing daily dose and was rapidly eliminated from the plasma compartment. Maximal decreases in HCV RNA were: -3.9 log(10)IU/ml and -3.2 log(10)IU/ml in TN receiving 200 mg q8 h and 200 mg q12 h, respectively. End of treatment viral decline in these two cohorts was within 0.1 log(10)IU/ml of the viral load nadir. HCV RNA reduction in NR was more modest than that observed in upper dose TN cohorts. The overall incidence of viral rebound was low (10/37) and was associated with the R155K substitution in NS3 regardless of the HCV subtype. CONCLUSIONS: Danoprevir was safe and well tolerated when administered for 14 days in patients with chronic HCV genotype 1 infection. Treatment resulted in sustained, multi-log(10) IU/ml reductions in HCV RNA in upper dose cohorts. These results support further clinical evaluation of danoprevir in patients with chronic HCV.

Danoprevir monotherapy decreases inflammatory markers in patients with chronic hepatitis C virus infection.

Danoprevir is a potent and selective direct-acting antiviral agent that targets the protease activity of hepatitis C virus (HCV) NS3/4A. This agent results in a significant rapid decline in HCV RNA levels when it is used in monotherapy. The present study evaluated whether plasma concentrations of the inflammatory markers gamma interferon-inducible protein 10 (IP-10) and neopterin or the interferon-stimulated gene product 2'-5'-oligoadenylate synthetase (OAS-1) were correlated with the plasma HCV RNA concentration before or during 14-day danoprevir monotherapy. In contrast to pegylated interferon and ribavirin treatment, a higher baseline IP-10 concentration was positively correlated with a greater first-phase HCV RNA decline upon danoprevir administration. Changes in the IP-10 plasma concentration during danoprevir administration were also associated with categorical changes in HCV RNA concentration at days 7 and 14. The neopterin concentration appeared to be moderately decreased during danoprevir administration, although these changes were not statistically significant. However, changes in neopterin concentration showed a statistically significant correlation with changes in IP-10 concentration. Considerable variation in the OAS-1 concentration was observed before and during treatment, including in patients treated with placebo and/or patients with minimal virologic response. Overall, these results suggest that effective treatment with a direct-acting antiviral agent may reduce hepatic inflammation and that first-phase HCV RNA decline during treatment with an NS3/4A protease inhibitor is more robust in patients with high baseline IP-10 concentrations.

Recent advances in drug discovery of benzothiadiazine and related analogs as HCV NS5B polymerase inhibitors.

Hepatitis C virus (HCV) is a major health burden, with an estimated 170 million chronically infected individuals worldwide, and a leading cause of liver transplantation. Patients are at increased risk of developing liver cirrhosis, hepatocellular carcinoma and even liver failure. In the past two decades, several approaches have been adopted to inhibit non-structural viral proteins. The RNA-dependent RNA polymerase (NS5B) of HCV is one of the attractive validated targets for development of new drugs to block HCV infection. In this review, we report the recent progress made towards identifying and developing benzothiadiazines as HCV NS5B polymerase inhibitors. The substituted benzothiadiazine class was identified by HTS in 2002 as an NS5B inhibitor. Further optimization and modification of the core has improved the potency and pharmacokinetic properties of substituted benzothiadiazines. Research on palm site-binding benzothiadiazine analogs and related derivatives and analogs is discussed in this article.

Inhibition and binding kinetics of the hepatitis C virus NS3 protease inhibitor ITMN-191 reveals tight binding and slow dissociative behavior.

The protease activity of hepatitis C virus nonstructural protein 3 (NS3) is essential for viral replication. ITMN-191, a macrocyclic inhibitor of the NS3 protease active site, promotes rapid, multilog viral load reductions in chronic HCV patients. Here, ITMN-191 is shown to be a potent inhibitor of NS3 with a two-step binding mechanism. Progress curves are consistent with the formation of an initial collision complex (EI) that isomerizes to a highly stable complex (EI*) from which ITMN-191 dissociates very slowly. K(i), the dissociation constant of EI, is 100 nM, and the rate constant for conversion of EI to EI* is 6.2 x 10(-2) s(-1). Binding experiments using protein fluorescence confirm this isomerization rate. From progress curve analysis, the rate constant for dissociation of ITMN-191 from the EI* complex is 3.8 x 10(-5) s(-1) with a calculated complex half-life of approximately 5 h and a true biochemical potency (K(i)*) of approximately 62 pM. Surface plasmon resonance studies and assessment of enzyme reactivation following dilution of the EI* complex confirm slow dissociation and suggest that the half-life may be considerably longer. Abrogation of the tight binding and slow dissociative properties of ITMN-191 is observed with proteases that carry the R155K or D168A substitution, each of which is likely in drug resistant mutants. Slow dissociation is not observed with closely related macrocyclic inhibitors of NS3, suggesting that members of this class may display distinct binding kinetics.

Optimization of the multiple enzymatic activities of the hepatitis C virus NS3 protein.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is known to possess multiple enzymatic activities. In addition to its well-characterized protease activity, HCV NS3 also has ATP hydrolase (ATPase) and nucleic acid unwinding (helicase) activities. We systematically studied the effect of common reagents on all three enzymatic activities with a view to improving assay sensitivity for compound screening and profiling. Inclusion of the detergent lauryl dimethylamine oxide (LDAO) improves protease and helicase activities significantly, allowing robust assays at much lower NS3 concentrations. These conditions enable a particularly sensitive protease assay that uses picomolar concentrations of NS3.

HCV NS5B polymerase inhibitors 3: Synthesis and in vitro activity of 3-(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives.

3-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

HCV NS5B polymerase inhibitors 1: Synthesis and in vitro activity of 2-(1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-1-hydroxynaphthalene derivatives.

2-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-1-hydroxynaphthalene derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition and replicon activity are discussed.

HCV NS5B polymerase inhibitors 2: Synthesis and in vitro activity of (1,1-dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives.

(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

Preclinical characteristics of the hepatitis C virus NS3/4A protease inhibitor ITMN-191 (R7227).

Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C.

Monitoring post-translational modification of proteins with allosteric ribozymes.

An allosteric hammerhead ribozyme activated specifically by the unphosphorylated form of the protein kinase ERK2 was created through a rational design strategy that relies on molecular recognition of ERK2 to decrease the formation of an alternate, inactive ribozyme conformer. Neither closely related mitogen-activated protein kinases (MAPKs) nor the phosphorylated form of ERK2 induced ribozyme activity. The ribozyme quantitatively detected ERK2 added to mammalian cell lysates and also functioned quantitatively in a multiplexed solution-phase assay. This same strategy was used to construct a second ribozyme selectively activated by the phosphorylated (active) form of ERK2. This approach is generally applicable to the development of ribozymes capable of monitoring post-translational modification of specific proteins.

Immunomodulatory activities of IFN-gamma1b in combination with type I IFN: implications for the use of IFN-gamma1b in the treatment of chronic HCV infections.

The standard of care for chronic hepatitis C, pegylated interferon-alpha (IFN-alpha) and ribavirin (RBV), causes a sustained virologic response (SVR) in approximately 50% of patients. SVR is correlated with innate and adaptive immune system responses, such as natural killer (NK) cell activation, production of IFN-alpha from immature plasmocytoid dendritic cells (pDC), and polarization of CD4(+) cells to a T helper 1 (Th1) cell phenotype. To examine how these immunologic responses vary with currently available regimens for chronic hepatitis C, cell populations purified from human peripheral blood mononuclear cells (PBMC) were treated with the clinically available combinations of pegylated IFN-alpha2b (PEG-IFN-alpha2b) + RBV, IFN-alphacon1 + RBV, or IFN- alphacon1 + IFN-gamma1b, and activation of cellular immune system components was monitored. The magnitude of NK cell activation depended on regimen, with IFN-alphacon1 + IFN-gamma1b > IFN-alphacon1 + RBV > PEG-IFN- alphaa2b + RBV. The maximum human serum concentrations of IFN-alphacon1 + IFN-gamma1b saturated NK cell activation, whereas the maximum human serum concentrations of IFN-alphacon1 + RBV or PEG-IFN-alpha2b + RBV did not. IFN-gamma1b also enhanced the production of IFN-alpha from immature pDCs, which are the dominant source of IFN-alpha upon viral infection. The rank order for induction of Th1 cell phenotype and repression of Th2 cell phenotype by the cocktails described was identical to that observed for NK cell activation. Additionally, IFN- gamma1b suppressed the ability of the hepatitis C virus (HCV) NS4 protein to enhance monocyte secretion of interleukin- 10 (IL-10), a cytokine whose expression level is correlated with viral persistence. These results suggest that addition of IFN-gamma1b to HCV treatment regimens may provide unique benefits.

High-throughput ribozyme-based assays for detection of viral nucleic acids.

Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.

Discovery of danoprevir (ITMN-191/R7227), a highly selective and potent inhibitor of hepatitis C virus (HCV) NS3/4A protease.

HCV serine protease NS3 represents an attractive drug target because it is not only essential for viral replication but also implicated in the viral evasion of the host immune response pathway through direct cleavage of key proteins in the human innate immune system. Through structure-based drug design and optimization, macrocyclic peptidomimetic molecules bearing both a lipophilic P2 isoindoline carbamate and a P1/P1' acylsulfonamide/acylsulfamide carboxylic acid bioisostere were prepared that possessed subnanomolar potency against the NS3 protease in a subgenomic replicon-based cellular assay (Huh-7). Danoprevir (compound 49) was selected as the clinical development candidate for its favorable potency profile across multiple HCV genotypes and key mutant strains and for its good in vitro ADME profiles and in vivo target tissue (liver) exposures across multiple animal species. X-ray crystallographic studies elucidated several key features in the binding of danoprevir to HCV NS3 protease and proved invaluable to our iterative structure-based design strategy.

Genetic variants associated with idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide association study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF. METHODS: First, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p<5 × 10(-8)) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes. FINDINGS: In stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 [r(2)=0·07], rs5743894 [r(2)=0·16], and rs5743890 [r(2)=0·01]) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1·72 [95% CI 1·24-2·38]; p=0·0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0·097), 40% in those with the minor allele of rs111521887 (p=3·0 × 10(-4)), and 50% in those with the minor allele of rs5743894 (p=2·93 × 10(-5)) compared with homozygous carriers of common alleles for these SNPs. INTERPRETATION: Novel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP, rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease. FUNDING: National Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.

Genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis.

We performed a genome-wide association study of non-Hispanic, white individuals with fibrotic idiopathic interstitial pneumonias (IIPs; n = 1,616) and controls (n = 4,683), with follow-up replication analyses in 876 cases and 1,890 controls. We confirmed association with TERT at 5p15, MUC5B at 11p15 and the 3q26 region near TERC, and we identified seven newly associated loci (Pmeta = 2.4 × 10(-8) to 1.1 × 10(-19)), including FAM13A (4q22), DSP (6p24), OBFC1 (10q24), ATP11A (13q34), DPP9 (19p13) and chromosomal regions 7q22 and 15q14-15. Our results suggest that genes involved in host defense, cell-cell adhesion and DNA repair contribute to risk of fibrotic IIPs.

Monitoring protein modification with allosteric ribozymes.

An allosteric ribozyme is an RNA-based enzyme (ribozyme) whose catalytic activity is modulated by molecular recognition of a protein. The direct coupling of a detectable catalytic event to molecular recognition by an allosteric ribozyme enables simple assays for quantitative protein detection. Most significantly, the mode of development and molecular recognition characteristics of allosteric ribozymes are fundamentally different from antibodies, providing them with functional characteristics that complement those of antibodies. Allosteric ribozymes can be developed using native proteins and, therefore, are often sensitive to protein conformation. In contrast, antibodies tend to recognize a series of adjacent amino acids as a consequence of antigen presentation and typically are not sensitive to protein conformation. Unlike antibody development, the development of allosteric ribozymes is a completely in vitro process that allows the specificity of an allosteric ribozyme to be tightly controlled. These significant differences from antibodies allow the pre-programmed development of conformation-state-specific protein detection reagents that can be used to investigate the activation-state of signal transduction components.