Association study of human leukocyte antigen variants and idiopathic pulmonary fibrosis.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia marked by progressive lung fibrosis and a poor prognosis. Recent studies have highlighted the potential role of infection in the pathogenesis of IPF, and a prior association of the HLA-DQB1 gene with idiopathic fibrotic interstitial pneumonia (including IPF) has been reported. Owing to the important role that the human leukocyte antigen (HLA) region plays in the immune response, here we evaluated if HLA genetic variation was associated specifically with IPF risk. Methods: We performed a meta-analysis of associations of the HLA region with IPF risk in individuals of European ancestry from seven independent case-control studies of IPF (comprising 5159 cases and 27 459 controls, including a prior study of fibrotic interstitial pneumonia). Single nucleotide polymorphisms, classical HLA alleles and amino acids were analysed and signals meeting a region-wide association threshold of p<4.5×10-4 and a posterior probability of replication >90% were considered significant. We sought to replicate the previously reported HLA-DQB1 association in the subset of studies independent of the original report. Results: The meta-analysis of all seven studies identified four significant independent single nucleotide polymorphisms associated with IPF risk. However, none met the posterior probability for replication criterion. The HLA-DQB1 association was not replicated in the independent IPF studies. Conclusion: Variation in the HLA region was not consistently associated with risk in studies of IPF. However, this does not preclude the possibility that other genomic regions linked to the immune response may be involved in the aetiology of IPF.

Apolipoprotein L1 (APOL1) renal risk variant-mediated podocyte cytotoxicity depends on African haplotype and surface expression.

Homozygous Apolipoprotein L1 (APOL1) variants G1 and G2 cause APOL1-mediated kidney disease, purportedly acting as surface cation channels in podocytes. APOL1-G0 exhibits various single nucleotide polymorphisms, most commonly haplotype E150K, M228I and R255K ("KIK"; the Reference Sequence is "EMR"), whereas variants G1 and G2 are mostly found in a single "African" haplotype background ("EIK"). Several labs reported cytotoxicity with risk variants G1 and G2 in KIK or EIK background haplotypes, but used HEK-293 cells and did not verify equal surface expression. To see if haplotype matters in a more relevant cell type, we induced APOL1-G0, G1 and G2 EIK, KIK and EMR at comparable surface levels in immortalized podocytes. G1 and G2 risk variants (but not G0) caused dose-dependent podocyte death within 48h only in their native African EIK haplotype and correlated with K+ conductance (thallium FLIPR). We ruled out differences in localization and trafficking, except for possibly greater surface clustering of cytotoxic haplotypes. APOL1 surface expression was required, since Brefeldin A rescued cytotoxicity; and cytoplasmic isoforms vB3 and vC were not cytotoxic. Thus, APOL1-EIK risk variants kill podocytes in a dose and haplotype-dependent manner (as in HEK-293 cells), whereas unlike in HEK-293 cells the KIK risk variants did not.

Genome-wide SNP-sex interaction analysis of susceptibility to idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition that is more prevalent in males than females. The reasons for this are not fully understood, with differing environmental exposures due to historically sex-biased occupations, or diagnostic bias, being possible explanations. To date, over 20 independent genetic variants have been identified to be associated with IPF susceptibility, but these have been discovered when combining males and females. Our aim was to test for the presence of sex-specific associations with IPF susceptibility and assess whether there is a need to consider sex-specific effects when evaluating genetic risk in clinical prediction models for IPF. Methods: We performed genome-wide single nucleotide polymorphism (SNP)-by-sex interaction studies of IPF risk in six independent IPF case-control studies and combined them using inverse-variance weighted fixed effect meta-analysis. In total, 4,561 cases (1,280 females and 2,281 males) and 23,500 controls (8,360 females and 14,528 males) of European genetic ancestry were analysed. We used polygenic risk scores (PRS) to assess differences in genetic risk prediction between males and females. Findings: Three independent genetic association signals were identified. All showed a consistent direction of effect across all individual IPF studies and an opposite direction of effect in IPF susceptibility between females and males. None had been previously identified in IPF susceptibility genome-wide association studies (GWAS). The predictive accuracy of the PRSs were similar between males and females, regardless of whether using combined or sex-specific GWAS results. Interpretation: We prioritised three genetic variants whose effect on IPF risk may be modified by sex, however these require further study. We found no evidence that the predictive accuracy of common SNP-based PRSs varies significantly between males and females.

Multi-ancestry GWAS analysis identifies two novel loci associated with diabetic eye disease and highlights APOL1 as a high risk locus in patients with diabetic macular edema.

Diabetic retinopathy (DR) is a common complication of diabetes. Approximately 20% of DR patients have diabetic macular edema (DME) characterized by fluid leakage into the retina. There is a genetic component to DR and DME risk, but few replicable loci. Because not all DR cases have DME, we focused on DME to increase power, and conducted a multi-ancestry GWAS to assess DME risk in a total of 1,502 DME patients and 5,603 non-DME controls in discovery and replication datasets. Two loci reached GWAS significance (p<5x10-8). The strongest association was rs2239785, (K150E) in APOL1. The second finding was rs10402468, which co-localized to PLVAP and ANKLE1 in vascular / endothelium tissues. We conducted multiple sensitivity analyses to establish that the associations were specific to DME status and did not reflect diabetes status or other diabetic complications. Here we report two novel loci for risk of DME which replicated in multiple clinical trial and biobank derived datasets. One of these loci, containing the gene APOL1, is a risk factor in African American DME and DKD patients, indicating that this locus plays a broader role in diabetic complications for multiple ancestries. Trial Registration: NCT00473330, NCT00473382, NCT03622580, NCT03622593, NCT04108156.

Association study of human leukocyte antigen (HLA) variants and idiopathic pulmonary fibrosis.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia marked by progressive lung fibrosis and a poor prognosis. Recent studies have highlighted the potential role of infection in the pathogenesis of IPF and a prior association of the HLA-DQB1 gene with idiopathic fibrotic interstitial pneumonia (including IPF) has been reported. Due to the important role that the Human Leukocyte Antigen (HLA) region plays in the immune response, here we evaluated if HLA genetic variation was associated specifically with IPF risk. Methods: We performed a meta-analysis of associations of the HLA region with IPF risk in individuals of European ancestry from seven independent case-control studies of IPF (comprising a total of 5,159 cases and 27,459 controls, including the prior study of fibrotic interstitial pneumonia). Single nucleotide polymorphisms, classical HLA alleles and amino acids were analysed and signals meeting a region-wide association threshold p<4.5×10-4 and a posterior probability of replication >90% were considered significant. We sought to replicate the previously reported HLA-DQB1 association in the subset of studies independent of the original report. Results: The meta-analysis of all seven studies identified four significant independent single nucleotide polymorphisms associated with IPF risk. However, none met the posterior probability for replication criterion. The HLA-DQB1 association was not replicated in the independent IPF studies. Conclusion: Variation in the HLA region was not consistently associated with risk in studies of IPF. However, this does not preclude the possibility that other genomic regions linked to the immune response may be involved in the aetiology of IPF.

A systems biology approach uncovers novel disease mechanisms in age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness, affecting 200 million people worldwide. To identify genes that could be targeted for treatment, we created a molecular atlas at different stages of AMD. Our resource is comprised of RNA sequencing (RNA-seq) and DNA methylation microarrays from bulk macular retinal pigment epithelium (RPE)/choroid of clinically phenotyped normal and AMD donor eyes (n = 85), single-nucleus RNA-seq (164,399 cells), and single-nucleus assay for transposase-accessible chromatin (ATAC)-seq (125,822 cells) from the retina, RPE, and choroid of 6 AMD and 7 control donors. We identified 23 genome-wide significant loci differentially methylated in AMD, over 1,000 differentially expressed genes across different disease stages, and an AMD Müller state distinct from normal or gliosis. Chromatin accessibility peaks in genome-wide association study (GWAS) loci revealed putative causal genes for AMD, including HTRA1 and C6orf223. Our systems biology approach uncovered molecular mechanisms underlying AMD, including regulators of WNT signaling, FRZB and TLE2, as mechanistic players in disease.

Rare CIDEC coding variants enriched in age-related macular degeneration patients with small low-luminance deficit cause lipid droplet and fat storage defects.

BACKGROUND: The basis of Age-related macular degeneration (AMD) genetic risk has been well documented; however, few studies have looked at genetic biomarkers of disease progression or treatment response within advanced AMD patients. Here we report the first genome-wide analysis of genetic determinants of low-luminance vision deficit (LLD), which is seen as predictive of visual acuity loss and anti-VEGF treatment response in neovascular AMD patients. METHODS: AMD patients were separated into small- and large-LLD groups for comparison and whole genome sequencing was performed. Genetic determinants of LLD were assessed by common and rare variant genetic analysis. Follow-up functional analysis of rare coding variants identified by the burden test was then performed in vitro. RESULTS: We identified four coding variants in the CIDEC gene. These rare variants were only present in patients with a small LLD, which has been previously shown to indicate better prognosis and better anti-VEGF treatment response. Our in vitro functional characterization of these CIDEC alleles revealed that all decrease the binding affinity between CIDEC and the lipid droplet fusion effectors PLIN1, RAB8A and AS160. The rare CIDEC alleles all cause a hypomorphic defect in lipid droplet fusion and enlargement, resulting in a decreased fat storage capability in adipocytes. CONCLUSIONS: As we did not detect CIDEC expression in the ocular tissue affected by AMD, our results suggest that the CIDEC variants do not play a direct role in the eye and influence low-luminance vision deficit via an indirect and systemic effect related to fat storage capacity.

PCSK6 and Survival in Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. Objectives: To identify and validate molecular determinants of IPF survival. Methods: A staged genome-wide association study was performed using paired genomic and survival data. Stage I cases were drawn from centers across the United States and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplantation-free survival (TFS). Stage I variants with nominal significance (P < 5 × 10-5) were advanced for stage II testing and meta-analyzed to identify those reaching genome-wide significance (P < 5 × 10-8). Downstream analyses were performed for genes and proteins associated with variants reaching genome-wide significance. Measurements and Main Results: After quality controls, 1,481 stage I cases and 397 stage II cases were included in the analysis. After filtering, 9,075,629 variants were tested in stage I, with 158 meeting advancement criteria. Four variants associated with TFS with consistent effect direction were identified in stage II, including one in an intron of PCSK6 (proprotein convertase subtilisin/kexin type 6) reaching genome-wide significance (hazard ratio, 4.11 [95% confidence interval, 2.54-6.67]; P = 9.45 × 10-9). PCSK6 protein was highly expressed in IPF lung parenchyma. PCSK6 lung staining intensity, peripheral blood gene expression, and plasma concentration were associated with reduced TFS. Conclusions: We identified four novel variants associated with IPF survival, including one in PCSK6 that reached genome-wide significance. Downstream analyses suggested that PCSK6 protein plays a potentially important role in IPF progression.

A genome-wide association study of chronic spontaneous urticaria risk and heterogeneity.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a dermatologic condition characterized by spontaneous, pruritic hives and/or angioedema that persists for 6 weeks or longer with no identifiable trigger. Antihistamines and second-line therapies such as omalizumab are effective for some CSU patients, but others remain symptomatic, with significant impact on quality of life. This variable response to treatment and autoantibody levels across patients highlight clinically heterogeneous subgroups. OBJECTIVE: We aimed to highlight pathways involved in CSU by investigating the genetics of CSU risk and subgroups. METHODS: We performed a genome-wide association study (GWAS) of 679 CSU patients and 4446 controls and a GWAS of chronic urticaria (CU)-index, which measures IgG autoantibodies levels, by comparing 447 CU index-low to 183 CU index-high patients. We also tested whether polygenic scores for autoimmune-related disorders were associated with CSU risk and CU index. RESULTS: We identified 2 loci significantly associated with disease risk. The strongest association mapped to position 56 of HLA-DQA1 (P = 1.69 × 10-9), where the arginine residue was associated with increased risk (odds ratio = 1.64). The second association signal colocalized with expression-quantitative trait loci for ITPKB in whole blood (Pcolocalization = .997). The arginine residue at position 56 of HLA-DQA1 was also associated with increased risk of CU index-high (P = 6.15 × 10-5, odds ratio = 1.86), while the ITKPB association was not (P = .64). Polygenic scores for 3 autoimmune-related disorders (hypothyroidism, type 1 diabetes, and vitiligo) were associated with CSU risk and CU index (P < 2.34 × 10-3, odds ratio > 1.72). CONCLUSION: A GWAS of CSU identified 2 genome-wide significant loci, highlighting the shared genetics between CU index and autoimmune disorders.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Genome-wide association study across five cohorts identifies five novel loci associated with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition with poor survival times. We previously published a genome-wide meta-analysis of IPF risk across three studies with independent replication of associated variants in two additional studies. To maximise power and to generate more accurate effect size estimates, we performed a genome-wide meta-analysis across all five studies included in the previous IPF risk genome-wide association studies. We used the distribution of effect sizes across the five studies to assess the replicability of the results and identified five robust novel genetic association signals implicating mTOR (mammalian target of rapamycin) signalling, telomere maintenance and spindle assembly genes in IPF risk.

Genome-wide association study identifies kallikrein 5 in type 2 inflammation-low asthma.

BACKGROUND: Clinical studies of type 2 (T2) cytokine-related neutralizing antibodies in asthma have identified a substantial subset of patients with low levels of T2 inflammation who do not benefit from T2 cytokine neutralizing antibody treatment. Non-T2 mechanisms are poorly understood in asthma but represent a redefined unmet medical need. OBJECTIVE: We sought to gain a better understanding of genetic contributions to T2-low asthma. METHODS: We utilized an unbiased genome-wide association study of patients with moderate to severe asthma stratified by T2 serum biomarker periostin. We also performed additional expression and biological analysis for the top genetic hits. RESULTS: We identified a novel protective single nucleotide polymorphism at chr19q13.41, which is selectively associated with T2-low asthma and establishes Kallikrein-related peptidase 5 (KLK5) as the causal gene mediating this association. Heterozygous carriers of the single nucleotide polymorphisms have reduced KLK5 expression. KLK5 is secreted by human bronchial epithelial cells and elevated in asthma bronchial alveolar lavage. T2 cytokines IL-4 and IL-13 downregulate KLK5 in human bronchial epithelial cells. KLK5, dependent on its catalytic function, induces epithelial chemokine/cytokine expression. Finally, overexpression of KLK5 in airway or lack of an endogenous KLK5 inhibitor, SPINK5, leads to spontaneous airway neutrophilic inflammation. CONCLUSION: Our data identify KLK5 to be the causal gene at a novel locus at chr19q13.41 associated with T2-low asthma.

A whole genome sequencing study of moderate to severe asthma identifies a lung function locus associated with asthma risk.

Genome-wide association studies (GWAS) have identified many common variant loci associated with asthma susceptibility, but few studies investigate the genetics underlying moderate-to-severe asthma risk. Here, we present a whole-genome sequencing study comparing 3181 moderate-to-severe asthma patients to 3590 non-asthma controls. We demonstrate that asthma risk is genetically correlated with lung function measures and that this component of asthma risk is orthogonal to the eosinophil genetics that also contribute to disease susceptibility. We find that polygenic scores for reduced lung function are associated with younger asthma age of onset. Genome-wide, seven previously reported common asthma variant loci and one previously reported lung function locus, near THSD4, reach significance. We replicate association of the lung function locus in a recently published GWAS of moderate-to-severe asthma patients. We additionally replicate the association of a previously reported rare (minor allele frequency < 1%) coding variant in IL33 and show significant enrichment of rare variant burden in genes from common variant allergic disease loci. Our findings highlight the contribution of lung function genetics to moderate-to-severe asthma risk, and provide initial rare variant support for associations with moderate-to-severe asthma risk at several candidate genes from common variant loci.

Leveraging global multi-ancestry meta-analysis in the study of idiopathic pulmonary fibrosis genetics.

The research of rare and devastating orphan diseases, such as idiopathic pulmonary fibrosis (IPF) has been limited by the rarity of the disease itself. The prognosis is poor-the prevalence of IPF is only approximately four times the incidence, limiting the recruitment of patients to trials and studies of the underlying biology. Global biobanking efforts can dramatically alter the future of IPF research. We describe a large-scale meta-analysis of IPF, with 8,492 patients and 1,355,819 population controls from 13 biobanks around the globe. Finally, we combine this meta-analysis with the largest available meta-analysis of IPF, reaching 11,160 patients and 1,364,410 population controls. We identify seven novel genome-wide significant loci, only one of which would have been identified if the analysis had been limited to European ancestry individuals. We observe notable pleiotropy across IPF susceptibility and severe COVID-19 infection and note an unexplained sex-heterogeneity effect at the strongest IPF locus MUC5B.

An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

Rare Protein-Altering Telomere-related Gene Variants in Patients with Chronic Hypersensitivity Pneumonitis.

Rationale: Rare genetic variants in telomere-related genes have been identified in familial, idiopathic, and rheumatoid arthritis-associated pulmonary fibrosis. Short peripheral blood leukocyte (PBL) telomere length predicts poor outcomes in chronic hypersensitivity pneumonitis (CHP).Objectives: Determine the prevalence and clinical relevance of rare protein-altering variants in telomere-related genes in patients with CHP.Methods: Next-generation sequences from two CHP cohorts were analyzed to identify variants in TERT (telomerase reverse transcriptase), TERC (telomerase RNA component), DKC1 (dyskerin pseudouridine synthase 1), RTEL1 (regulator of telomere elongation helicase 1), PARN (poly[A]-specific RNase), and TINF2 (TERF1-interacting nuclear factor 2). To qualify, variants were required to have a minor allele frequency less than 0.005 and be predicted to be damaging to protein function. Variant status (binary variable) was used in statistical association tests, including Cox proportional hazard models for transplant-free survival. PBL telomere length was measured using quantitative PCR.Measurements and Main Results: Qualifying variants were identified in 16 of 144 patients (11.1%; 95% confidence interval [CI], 6.5-17.4) in the discovery cohort and 17 of 209 patients (8.1%; 95% CI, 4.8-12.7) in the replication cohort. Age- and ancestry-adjusted PBL telomere length was significantly shorter in the presence of a variant in both cohorts (discovery: -561 bp; 95% CI, -933 to -190; P = 0.003; replication: -612 bp; 95% CI, -870 to -354; P = 5.30 × 10-6). Variant status was significantly associated with transplant-free survival in both cohorts (discovery: age-, sex-, and ancestry-adjusted hazard ratio, 3.73; 95% CI, 1.92-7.28; P = 0.0001; replication: hazard ratio, 2.72; 95% CI, 1.26-5.88; P = 0.011).Conclusions: A substantial proportion of patients diagnosed with CHP have rare, protein-altering variants in telomere-related genes, which are associated with short peripheral blood telomere length and significantly reduced transplant-free survival.

Strategies for Pathway Analysis Using GWAS and WGS Data.

Single-allele study designs, commonly used in genome-wide association studies (GWAS) as well as the more recently developed whole genome sequencing (WGS) studies, are a standard approach for investigating the relationship of common variation within the human genome to a given phenotype of interest. However, single-allele association results published for many GWAS studies represent only the tip of the iceberg for the information that can be extracted from these datasets. The primary analysis strategy for GWAS entails association analysis in which only the single nucleotide polymorphisms (SNPs) with the strongest p-values are declared statistically significant due to issues arising from multiple testing and type I errors. Factors such as locus heterogeneity, epistasis, and multiple genes conferring small effects contribute to the complexity of the genetic models underlying phenotype expression. Thus, many biologically meaningful associations having lower effect sizes at individual genes are overlooked, making it difficult to separate true associations from a sea of false-positive associations. Organizing these individual SNPs into biologically meaningful groups to look at the overall effects of minor perturbations to genes and pathways is desirable. This pathway-based approach provides researchers with insight into the functional foundations of the phenotype being studied and allows testing of various genetic scenarios. © 2018 by John Wiley & Sons, Inc.

Previously reported placebo-response-associated variants do not predict patient outcomes in inflammatory disease Phase III trial placebo arms.

In clinical trials, a placebo response refers to improvement in disease symptoms arising from the psychological effect of receiving a treatment rather than the actual treatment under investigation. Previous research has reported genomic variation associated with the likelihood of observing a placebo response, but these studies have been limited in scope and have not been validated. Here, we analyzed whole-genome sequencing data from 784 patients undergoing placebo treatment in Phase III Asthma or Rheumatoid Arthritis trials to assess the impact of previously reported variation on patient outcomes in the placebo arms and to identify novel variants associated with the placebo response. Contrary to expectations based on previous reports, we did not observe any statistically significant associations between genomic variants and placebo treatment outcome. Our findings suggest that the biological origin of the placebo response is complex and likely to be variable between disease areas.