Pharmacologic and Genetic Downregulation of Proprotein Convertase Subtilisin/Kexin Type 9 and Survival From Sepsis.

OBJECTIVES: Treatments that prevent sepsis complications are needed. Circulating lipid and protein assemblies-lipoproteins play critical roles in clearing pathogens from the bloodstream. We investigated whether early inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) may accelerate bloodstream clearance of immunogenic bacterial lipids and improve sepsis outcomes. DESIGN: Genetic and clinical epidemiology, and experimental models. SETTING: Human genetics cohorts, secondary analysis of a phase 3 randomized clinical trial enrolling patients with cardiovascular disease (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab [ODYSSEY OUTCOMES]; NCT01663402), and experimental murine models of sepsis. PATIENTS OR SUBJECTS: Nine human cohorts with sepsis (total n = 12,514) were assessed for an association between sepsis mortality and PCSK9 loss-of-function (LOF) variants. Incident or fatal sepsis rates were evaluated among 18,884 participants in a post hoc analysis of ODYSSEY OUTCOMES. C57BI/6J mice were used in Pseudomonas aeruginosa and Staphylococcus aureus bacteremia sepsis models, and in lipopolysaccharide-induced animal models. INTERVENTIONS: Observational human cohort studies used genetic PCSK9 LOF variants as instrumental variables. ODYSSEY OUTCOMES participants were randomized to alirocumab or placebo. Mice were administered alirocumab, a PCSK9 inhibitor, at 5 mg/kg or 25 mg/kg subcutaneously, or isotype-matched control, 48 hours prior to the induction of bacterial sepsis. Mice did not receive other treatments for sepsis. MEASUREMENTS AND MAIN RESULTS: Across human cohort studies, the effect estimate for 28-day mortality after sepsis diagnosis associated with genetic PCSK9 LOF was odds ratio = 0.86 (95% CI, 0.67-1.10; p = 0.24). A significant association was present in antibiotic-treated patients. In ODYSSEY OUTCOMES, sepsis frequency and mortality were infrequent and did not significantly differ by group, although both were numerically lower with alirocumab vs. placebo (relative risk of death from sepsis for alirocumab vs. placebo, 0.62; 95% CI, 0.32-1.20; p = 0.15). Mice treated with alirocumab had lower endotoxin levels and improved survival. CONCLUSIONS: PCSK9 inhibition may improve clinical outcomes in sepsis in preventive, pretreatment settings.

Genotyping, sequencing and analysis of 140,000 adults from Mexico City.

The Mexico City Prospective Study is a prospective cohort of more than 150,000 adults recruited two decades ago from the urban districts of Coyoacán and Iztapalapa in Mexico City1. Here we generated genotype and exome-sequencing data for all individuals and whole-genome sequencing data for 9,950 selected individuals. We describe high levels of relatedness and substantial heterogeneity in ancestry composition across individuals. Most sequenced individuals had admixed Indigenous American, European and African ancestry, with extensive admixture from Indigenous populations in central, southern and southeastern Mexico. Indigenous Mexican segments of the genome had lower levels of coding variation but an excess of homozygous loss-of-function variants compared with segments of African and European origin. We estimated ancestry-specific allele frequencies at 142 million genomic variants, with an effective sample size of 91,856 for Indigenous Mexican ancestry at exome variants, all available through a public browser. Using whole-genome sequencing, we developed an imputation reference panel that outperforms existing panels at common variants in individuals with high proportions of central, southern and southeastern Indigenous Mexican ancestry. Our work illustrates the value of genetic studies in diverse populations and provides foundational imputation and allele frequency resources for future genetic studies in Mexico and in the United States, where the Hispanic/Latino population is predominantly of Mexican descent.

Common and rare variant associations with clonal haematopoiesis phenotypes.

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.

Pharmacogenomic Study of Statin-Associated Muscle Symptoms in the ODYSSEY OUTCOMES Trial.

BACKGROUND: Statin-associated muscle symptoms (SAMS) are the most frequently reported adverse events for statin therapies. Previous studies have reported an association between the p.Val174Ala missense variant in SLCO1B1 and SAMS in simvastatin-treated subjects; however, evidence for genetic predictors of SAMS in atorvastatin- or rosuvastatin-treated subjects is currently lacking. METHODS: ODYSSEY OUTCOMES (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab; n=18 924) was a double-blind, randomized, placebo-controlled study evaluating the efficacy and safety of alirocumab (a PCSK9 [proprotein convertase subtilisin/kexin type 9] inhibitor) in acute coronary syndrome patients receiving high-intensity statin therapy. The goal of this pharmacogenomic analysis was to identify genetic variants associated with atorvastatin- and rosuvastatin-mediated SAMS among ODYSSEY OUTCOMES subjects who consented to participate in the genetic study (n=11 880). We performed multi-ancestry exome-wide and genome-wide association studies and gene burden analysis across 2 phenotypes (clinical SAMS [n=10 617] and creatine kinase levels [n=9630]). RESULTS: A novel genome-wide significant association for an intronic variant (rs6667912) located within TMEM9 (odds ratio [95% CI], 1.39 [1.24-1.55]; P=3.71×10-8) for patients with clinical SAMS (cases=894, controls=9723) was identified. This variant is located ≈30 kb upstream of CACNA1S, a locus associated with severe SAMS. We replicated 2 loci, at LINC0093 and LILRB5, previously associated with creatine kinase levels during statin treatment. No association was observed between p.Val174Ala (rs4149056) in SLCO1B1 and SAMS (odds ratio [95% CI], 1.03 [0.90-1.18]; P=0.69). CONCLUSIONS: This study comprises the largest discovery exome-wide and genome-wide association study for atorvastatin- or rosuvastatin-mediated SAMS to date. These novel genetic findings may provide biological/mechanistic insight into this drug-induced toxicity, and help identify at-risk patients before selection of lipid-lowering therapies.

UGT1A1 genetic variants are associated with increases in bilirubin levels in rheumatoid arthritis patients treated with sarilumab.

Sarilumab is a human monoclonal antibody against interleukin (IL)-6Rα that has been approved for the treatment of adult patients with moderately to severely active rheumatoid arthritis (RA) and an inadequate response or intolerance to one or more disease-modifying antirheumatic drugs (DMARDs). Mild liver function test abnormalities have been observed in patients treated with sarilumab. We describe a genome-wide association study of bilirubin elevations in RA patients treated with sarilumab. Array genotyping and exome sequencing were performed on DNA samples from 1075 patients. Variants in the UGT1A1 gene were strongly associated with maximum bilirubin elevations in sarilumab-treated patients (rs4148325; p = 2.88 × 10-41) but were not associated with aminotransferase elevations. No other independent loci showed evidence of association with bilirubin elevations after sarilumab treatment. These findings suggest that most bilirubin increases during sarilumab treatment are related to genetic variation in UGT1A1 rather than underlying liver injury.

Current challenges and opportunities for pharmacogenomics: perspective of the Industry Pharmacogenomics Working Group (I-PWG).

Pharmaceutical companies have increasingly utilized genomic data for the selection of drug targets and the development of precision medicine approaches. Most major pharmaceutical companies routinely collect DNA from clinical trial participants and conduct pharmacogenomic (PGx) studies. However, the implementation of PGx studies during clinical development presents a number of challenges. These challenges include adapting to a constantly changing global regulatory environment, challenges in study design and clinical implementation, and the increasing concerns over patient privacy. Advances in the field of genomics are also providing new opportunities for pharmaceutical companies, including the availability of large genomic databases linked to patient health information, the growing use of polygenic risk scores, and the direct sequencing of clinical trial participants. The Industry Pharmacogenomics Working Group (I-PWG) is an association of pharmaceutical companies actively working in the field of pharmacogenomics. This I-PWG perspective will provide an overview of the steps pharmaceutical companies are taking to address each of these challenges, and the approaches being taken to capitalize on emerging scientific opportunities.

Sequencing of 640,000 exomes identifies GPR75 variants associated with protection from obesity.

Large-scale human exome sequencing can identify rare protein-coding variants with a large impact on complex traits such as body adiposity. We sequenced the exomes of 645,626 individuals from the United Kingdom, the United States, and Mexico and estimated associations of rare coding variants with body mass index (BMI). We identified 16 genes with an exome-wide significant association with BMI, including those encoding five brain-expressed G protein-coupled receptors (CALCR, MC4R, GIPR, GPR151, and GPR75). Protein-truncating variants in GPR75 were observed in ~4/10,000 sequenced individuals and were associated with 1.8 kilograms per square meter lower BMI and 54% lower odds of obesity in the heterozygous state. Knock out of Gpr75 in mice resulted in resistance to weight gain and improved glycemic control in a high-fat diet model. Inhibition of GPR75 may provide a therapeutic strategy for obesity.

Patients With High Genome-Wide Polygenic Risk Scores for Coronary Artery Disease May Receive Greater Clinical Benefit From Alirocumab Treatment in the ODYSSEY OUTCOMES Trial.

BACKGROUND: Alirocumab, an antibody that blocks PCSK9 (proprotein convertase subtilisin/kexin type 9), was associated with reduced major adverse cardiovascular events (MACE) and death in the ODYSSEY OUTCOMES trial (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab). In this study, higher baseline levels of low-density lipoprotein cholesterol (LDL-C) predicted greater benefit from alirocumab treatment. Recent studies indicate high polygenic risk scores (PRS) for coronary artery disease (CAD) identify individuals at higher risk who derive increased benefit from statins. We performed post hoc analyses to determine whether high PRS for CAD identifies higher-risk individuals, independent of baseline LDL-C and other known risk factors, who might derive greater benefit from alirocumab treatment. METHODS: ODYSSEY OUTCOMES was a randomized, double-blind, placebo-controlled trial comparing alirocumab or placebo in 18 924 patients with acute coronary syndrome and elevated atherogenic lipoproteins despite optimized statin treatment. The primary endpoint (MACE) comprised death of CAD, nonfatal myocardial infarction, ischemic stroke, or unstable angina requiring hospitalization. A genome-wide PRS for CAD comprising 6 579 025 genetic variants was evaluated in 11 953 patients with available DNA samples. Analysis of MACE risk was performed in placebo-treated patients, whereas treatment benefit analysis was performed in all patients. RESULTS: The incidence of MACE in the placebo group was related to PRS for CAD: 17.0% for high PRS patients (>90th percentile) and 11.4% for lower PRS patients (≤90th percentile; P<0.001); this PRS relationship was not explained by baseline LDL-C or other established risk factors. Both the absolute and relative reduction of MACE by alirocumab compared with placebo was greater in high versus low PRS patients. There was an absolute reduction by alirocumab in high versus low PRS groups of 6.0% and 1.5%, respectively, and a relative risk reduction by alirocumab of 37% in the high PRS group (hazard ratio, 0.63 [95% CI, 0.46-0.86]; P=0.004) versus a 13% reduction in the low PRS group (hazard ratio, 0.87 [95% CI, 0.78-0.98]; P=0.022; interaction P=0.04). CONCLUSIONS: A high PRS for CAD is associated with elevated risk for recurrent MACE after acute coronary syndrome and a larger absolute and relative risk reduction with alirocumab treatment, providing an independent tool for risk stratification and precision medicine.

Epigenetic modification of the FMR1 gene in fragile X syndrome is associated with differential response to the mGluR5 antagonist AFQ056.

Fragile X syndrome (FXS) is an X-linked condition associated with intellectual disability and behavioral problems. It is caused by expansion of a CGG repeat in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. This mutation is associated with hypermethylation at the FMR1 promoter and resultant transcriptional silencing. FMR1 silencing has many consequences, including up-regulation of metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. mGluR5 receptor antagonists have shown promise in preclinical FXS models and in one small open-label study of FXS. We examined whether a receptor subtype-selective inhibitor of mGluR5, AFQ056, improves the behavioral symptoms of FXS in a randomized, double-blind, two-treatment, two-period, crossover study of 30 male FXS patients aged 18 to 35 years. We detected no significant effects of treatment on the primary outcome measure, the Aberrant Behavior Checklist-Community Edition (ABC-C) score, at day 19 or 20 of treatment. In an exploratory analysis, however, seven patients with full FMR1 promoter methylation and no detectable FMR1 messenger RNA improved, as measured with the ABC-C, significantly more after AFQ056 treatment than with placebo (P < 0.001). We detected no response in 18 patients with partial promoter methylation. Twenty-four patients experienced an adverse event, which was mostly mild to moderately severe fatigue or headache. If confirmed in larger and longer-term studies, these results suggest that blockade of the mGluR5 receptor in patients with full methylation at the FMR1 promoter may show improvement in the behavioral attributes of FXS.

A genome-wide study identifies HLA alleles associated with lumiracoxib-related liver injury.

Lumiracoxib is a selective cyclooxygenase-2 inhibitor developed for the symptomatic treatment of osteoarthritis and acute pain. Concerns over hepatotoxicity have contributed to the withdrawal or non-approval of lumiracoxib in most major drug markets worldwide. We performed a case-control genome-wide association study on 41 lumiracoxib-treated patients with liver injury (cases) and 176 matched lumiracoxib-treated patients without liver injury (controls). Several SNPs from the MHC class II region showed strong evidence of association (the top SNP was rs9270986 with P = 2.8 x 10(-10)). These findings were replicated in an independent set of 98 lumiracoxib-treated cases and 405 matched lumiracoxib-treated controls (top SNP rs3129900, P = 4.4 x 10(-12)). Fine mapping identified a strong association to a common HLA haplotype (HLA-DRB1*1501-HLA-DQB1*0602-HLA-DRB5*0101-HLA-DQA1*0102, most significant allele P = 6.8 x 10(-25), allelic odds ratio = 5.0, 95% CI 3.6-7.0). These results offer the potential to improve the safety profile of lumiracoxib by identifying individuals at elevated risk for liver injury and excluding them from lumiracoxib treatment.

DOCK4, a GTPase activator, is disrupted during tumorigenesis.

We used representational difference analysis to identify homozygous genomic deletions selected during tumor progression in the mouse NF2 and TP53 tumor model. We describe a deletion targeting DOCK4, a member of the CDM gene family encoding regulators of small GTPases. DOCK4 specifically activates Rap GTPase, enhancing the formation of adherens junctions. DOCK4 mutations are present in a subset of human cancer cell lines; a recurrent missense mutant identified in human prostate and ovarian cancers encodes a protein that is defective in Rap1 activation. The engulfment defect of C. elegans mutants lacking the CDM gene ced-5 is rescued by wild-type DOCK4, but not by the mutant allele. Expression of wild-type, but not mutant, DOCK4 in mouse osteosarcoma cells with a deletion of the endogenous gene suppresses growth in soft agar and tumor invasion in vivo. DOCK4 therefore encodes a CDM family member that regulates intercellular junctions and is disrupted during tumorigenesis.

The Tre2 (USP6) oncogene is a hominoid-specific gene.

Gene duplication and domain accretion are thought to be the major mechanisms for the emergence of novel genes during evolution. Such events are thought to have occurred at early stages in the vertebrate lineage, but genomic sequencing has recently revealed extensive amplification events during the evolution of higher primates. We report here that the Tre2 (USP6) oncogene is derived from the chimeric fusion of two genes, USP32 (NY-REN-60), and TBC1D3. USP32 is an ancient, highly conserved gene, whereas TBC1D3 is derived from a recent segmental duplication, which is absent in most other mammals and shows rapid amplification and dispersal through the primate lineage. Remarkably, the chimeric gene Tre2 exists only in the hominoid lineage of primates. This hominoid-specific oncogene arose as recently as 21-33 million years ago, after proliferation of the TBC1D3 segmental duplication in the primate lineage. In contrast to the broad expression pattern of USP32 and TBC1D3, expression of Tre2 is testis-specific, a pattern proposed for novel genes implicated in the emergence of reproductive barriers. The sudden emergence of chimeric proteins, such as that encoded by Tre2, may have contributed to hominoid speciation.