Brumpton, Ben

Davey Smith, George

Fritsche, Lars G

Zheng, Jie

Åsvold, Bjørn Olav

PCSK9 inhibitors have strong effects on lowering low-density lipoprotein cholesterol (LDL-C) and subsequent risk of cardiovascular disease. While a number of trials have evaluated the safety of such inhibitors in long-term clinical trials, a broad investigation of potential outcomes over the life-time, leveraging genetic variation in PCSK9 levels, has not been conducted. We aimed to investigate genetic variants associated with serum PCSK9 and the effect of LDL-C lowering variants on a range of potential outcomes.To achive this, we used data from the Nord-Trøndelag Health Study (HUNT)(n=69,424), a large population-based health study of the inhabitants of Nord-Trøndelag county, Norway. Firstly, we preformed a genome-wide association study of serum PCSK9 measured in 3697adults. Secondly we created a genetic risk score for PCSK9 as well as utilized existing scores for PCSK9 and HMGCR, and regressed these on a borad range of outcomes in the total sample. Thirdly, we perfomed two-sample medelian randomization on 50 outcomes using publically available datasets. Finally, we assessed the genetic correlation between PCSK9 and 229 diseases or traits using LD score regression.Three independent variants were GWAS significant (rs11591147, rs499883 and rs192265866). Using both existing genetic risk scores and scores defined from this discovery GWAS; we confirmed a strong association between serum PCSK9 and lower LDL-C and reducing risk of cardiovascular disease. We did not observe any consistently strong potentially adverse or beneficial associations. We observed some strong but not statistically significant genetic correlations between serum PCSK9 and fasting insulin, urate, uric acid and caudate volume.The lack of association between the genetic risk scores for PCSK9 and HMGCR is reassuring for the use of these treatments. However, further studies are warranted to confirm our findings, follow-up on the functional influences of the variants identified and extend our investigations to other outcomes.<br/

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                          Brumpton, B., Fritsche, L. G., Zheng, J., Davey Smith, G., & Åsvold, B. O.(2019). Variation in Serum PCSK9 (Proprotein Convertase Subtilisin/KexinType 9), Cardiovascular Disease Risk, and an Investigation of PotentialUnanticipated Effects of PCSK9 Inhibition. Circulation: Genomic andPrecision Medicine, 12(1), 44-47.https://doi.org/10.1161/CIRCGEN.118.002335Peer reviewed versionLicense (if available):OtherLink to published version (if available):10.1161/CIRCGEN.118.002335Link to publication record in Explore Bristol ResearchPDF-documentThis is the accepted author manuscript (AAM). The final published version (version of record) is available onlinevia the American Heart Association at https://doi.org/10.1161/CIRCGEN.118.002335 . Please refer to anyapplicable terms of use of the publisher.University of Bristol - Explore Bristol ResearchGeneral rightsThis document is made available in accordance with publisher policies. Please cite only the publishedversion using the reference above. Full terms of use are available:http://www.bristol.ac.uk/pure/about/ebr-terms 1  Variation in serum PCSK9, cardiovascular disease risk and an investigation of potential unanticipated effects of PCSK9 inhibition: A GWAS and Mendelian randomization study in the Nord-Trøndelag Health Study, Norway  Short title: Brumpton et al. Effects of variation in serum PCSK9 Ben M. Brumpton, PhD 1,2,3, Lars G. Fritsche, PhD 1,4, Jie Zheng, PhD 2, Jonas Bille Nielsen, MD, PhD 5, Maria Mannila, MD, PhD 6, 7, Ida Surakka, PhD 5, Humaira Rasheed, PhD 1,2, Gunnhild Åberge Vie, MD, PhD 1, 8, Sarah E. Graham 5, Maiken Elvestad Gabrielsen, PhD 1, Lars Erik Laugsand, MD, PhD 8, 9, 10, Pål Aukrust, MD, PhD 11, 12, 13, Lars Johan Vatten, MD, PhD 8, Jan Kristian Damås, MD, PhD 14, 15, Thor Ueland, PhD 11, 16, 17, Imre Janszky, MD, PhD 8, John-Anker Zwart, MD, PhD 18, INVENT Consortium, Ferdinand M. van ´t Hooft, MD, PhD 7, Nabil Georges Seidah, PhD 19, Kristian Hveem, MD, PhD 1, Cristen Willer*, PhD 4, 5, 20, George Davey Smith, MD, DSc 2*, Bjørn Olav Åsvold, MD, PhD 1, 21*  *contributed equally Affiliations 1 K.G. Jebsen Center for Genetic Epidemiology, Department of Public Health and Nursing, NTNU, Norwegian University of Science and Technology, Norway.  2 MRC Integrative Epidemiology Unit, University of Bristol, UK.  3 Clinic of Thoracic and Occupational Medicine, St. Olavs Hospital, Trondheim University Hospital.   2  4 Department of Biostatistics and Center for Statistical Genetics, University of Michigan, Ann Arbor, USA.  5 Department of Internal Medicine, University of Michigan, Ann Arbor, MI, US.  6 Cardiology Unit, Department of Medicine Solna and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.  7 Cardiovascular Medicine Unit, Department of Medicine Solna and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.  8 Department of Public Health and Nursing, NTNU, Norwegian University of Science and Technology, Norway.  9 Department of Cardiology, St. Olavs Hospital, Trondheim, Norway.  10 Department of Circulation and Medical Imaging, Faculty of Medicine and Health Sciences, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.  11 Research Institute of Internal Medicine, Oslo, Norway.  12 Section Of Clinical Immunology and Infectious Diseases, Oslo University Hospital Rikshospitalet, Oslo, Norway.  13 Institute of Clinical Medicine, University of Oslo, Oslo, Norway.  14 Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, NTNU, Norwegian University of Science and Technology.  15 Department of Infectious Diseases, St Olavs hospital, Trondheim.  16 Faculty of Medicine, University of Oslo, Oslo .  17 K.G. Jebsen TREC, University of Tromsø, Tromsø, Norway.  18 Division of Clinical Neursoscience, Oslo University Hospital and University of Oslo, Norway.  19 Laboratory of Biochemical Neruroendocrinology, IRCM, Montreal, QC, Canada.  20 Department of Human Genetics, University of Michigan, Ann Arbor, USA.  21 Department of Endocrinology, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway.   3  Corresponding author: Ben Michael Brumpton, K.G. Jebsen Center for Genetic Epidemiology, Department of Public Health and Nursing, NTNU, Norwegian University of Science and Technology, Norway. Postbox 8905, MTFS, NO-7491 Trondheim, Norway. E-mail: ben.brumpton@ntnu.no Phone: +4746229495  Journal Subject Terms: Lipids and Cholesterol, Cardiovascular Disease, Epidemiology, Genetic, Association Studies Key Words: PCSK9, PCSK2, LDL-C, Cardiovascular Diseases, GWAS, Mendelian randomization    Word count: 800   4  Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors reduce serum low-density lipoprotein cholesterol (LDL-C) by increasing up-take in the liver1. While some long-term trials have evaluated their safety, broad investigations of outcomes over the life-time, leveraging genetic variation in serum PCSK9, have seldomly been conducted. We investigated effects of these variants on a range of outcomes to explore unanticipated effects of long-term PCSK9 inhibition.  We linked genotype and phenotype data from 69,422 participants in the population-based Nord-Trøndelag Health Study (HUNT) with hospital records. Coronary heart disease, myocardial infarction, heart failure, venous thrombosis, diabetes, asthma, chronic obstructive pulmonary disease, allergic rhinitis and mood disorders were indicated by ICD-9 or ICD-10 codes recorded between September 1987 and March 2016 or self-report in HUNT2 (1995-1997) or HUNT3 (2006-2008). Using self-report in HUNT, we assessed anxiety, depression, musculoskeletal pain, headaches, pain or stiffness in muscles or joints in at least 3 consecutive months during the last year, gastrointestinal pain, pain in either leg while walking and pain in the legs while at rest.  Blood pressure, height, weight and lung function were measured in HUNT by trained nurses or technicians. Non-fasting serum levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides, glucose and C-reactive protein were measured, and LDL-C was calculated. All-cause mortality was updated from the National Population Registry.  Serum concentrations of PCSK9 were measured in a sub-sample by enzyme immune-assay with antibodies obtained from R&D Systems (Minneapolis, Minnesota) as described elsewhere2.  5  Because of the sensitive nature of the data, requests to access the dataset from qualified researchers may be sent to HUNT (www.ntnu.edu/hunt). The Regional Committee for Ethics in Medical Research approved the study and participants gave informed consent.  We performed a genome-wide association study (GWAS) of serum PCSK9 in 3,697 adults using BOLT-LMM adjusted for sex, age, genotyping batch and four principal components. Serum PCSK9 measurements were rank-transformed to normality. The replication cohort included 5,304 adults from the TwinGene Study recruited between 2004 and 20083. We defined a new genetic risk score for serum PCSK9, and regressed this and an existing score (rs11206510, rs2479409, rs2149041, rs2479394, rs10888897, rs7552841, rs562556) on a range of outcomes1. For comparision, we used LDL-C-associated variants on HMGCR (rs12916, rs17238484, rs5909, rs2303152, rs10066707, rs2006760), the target of statins1. The associations of PCSK9 and HMGCR genetic risk scores with outcomes in HUNT were assessed using linear regression for continuous and logistic regression for binary outcomes, adjusted for age and sex. We perfomed two-sample Mendelian randomization on 48 outcomes using large biobanks available in MR-Base4.   We assessed 11.67 million single nucleotide and indel variants imputed from a combined reference panel including 2,202 low-pass sequenced HUNT genomes and the Haplotype Reference Consortium panel. We identified genome-wide significant associations at two loci – PCSK9 and PCSK2. Three independent genome-wide significant variants were identified, the most strongly associated variant was rs11591147 in PCSK9 (p.R46L), which was associated with a 1.30 unit decrease in rank-transformed serum PCSK9 (P=2.9x10-31) per effect allele (standard  6  deviation of original PCSK9 measurements=47.5 ng/ml, interquartile range=100.6-154.9), followed by the intronic rs499883 in PCSK9 (β=0.19, P=2.3x10-15) and rs192265866 in PCSK2 (β=-1.05, P=2.8x10-8) of which the two variants in PCSK9 (but not the variant in PCSK2) replicated in TwinGene and were used to construct a new genetic risk score for PCSK9.  Our results demonstrated probable causal associations between lower serum PCSK9 and both lower serum LDL-C (P=2.8x10-43) and lower risk of coronary heart disease (P=8.9x10-4) (Figure A). As analyses within HUNT suggested associations between PCSK9 and HMGCR genetic risk scores and risk of venous thrombosis and asthma, respectively, we investigated associations using summary level data from the INVENT Consortium and GABRIEL4, 5. Neither associations replicated (venous thrombosis, inverse variance weighted, OR=1.38, 95% CI=0.88, 2.20; asthma, inverse variance weighted, OR=1.13, 95% CI=0.64, 1.98). We did not observe any other convincing associations across a range of cardiovascular, respiratory, mental, diabetes, or pain related outcomes.  Utilising GWAS summary statistics and two-sample MR, the PCSK9 genetic risk score was associated with LDL-C, HDL-C and total cholesterol as expected (Figure B). Moreover, genetically decreased serum PCSK9 was associated with higher father’s age at death (β=0.04, 95% CI=0.01, 0.08), parents age at death (β=0.05, 95% CI=0.01, 0.10) and waist-to-hip ratio (β=0.03, 95% CI=0.01, 0.06) (Figure B). Additionally, genetically decreased serum PCSK9 was associated with decreased risk of coronary heart disease (odds ratio [OR]=0.84, 95% CI=0.78, 0.89) and myocardial infarction (OR=0.81, 95% CI=0.70, 0.93), and an increased risk of Alzheimer’s disease (OR=1.27, 95% CI=1.04, 1.55) (Figure B).  7   In summary, we confirmed two independent genetic variants associated with serum PCSK9, and causal associations of lower serum PCSK9 with lower LDL-C and reduced risk of coronary heart disease. Additioanally, we did not observe any other consistent unexpected associations when investigating a range of outcomes. The lack of association with a range of potential adverse outcomes is reassuring for long-term use of PCSK9 inhibitors.    8  Acknowledgments The Nord-Trøndelag Health Study (The HUNT Study) is a collaboration between HUNT Research Center (Faculty of Medicine and Health Sciences, NTNU, Norwegian University of Science and Technology), Nord-Trøndelag County Council, Central Norway Regional Health Authority, and the Norwegian Institute of Public Health. Sources of Funding BMB and BOA work in a research unit funded by Stiftelsen Kristian Gerhard Jebsen; Faculty of Medicine and Health Sciences, NTNU; The Liaison Committee for education, research and innovation in Central Norway; and the Joint Research Committee between St. Olavs Hospital and the Faculty of Medicine and Health Sciences, NTNU. The genotyping in HUNT was financed by the National Institute of Health (NIH); University of Michigan; The Research Council of Norway; The Liaison Committee for education, research and innovation in Central Norway; and the Joint Research Committee between St. Olavs Hospital and the Faculty of Medicine and Health Sciences, NTNU. NGS was supported by a Leducq Foundation Grant # 13 CVD 03, a CIHR Foundation Grant # 148363 and a Canada Research Chair # 950-231335. JBN was supported by grants from the Danish Heart Foundation and the Lundbeck Foundation. GÅV was supported by a research grant from the Norwegian Research Council (Grant # 250335). GDS and BMB work in the Medical Research Council Integrative Epidemiology Unit at the University of Bristol which is supported by the Medical Research Council and the University of Bristol [MC_UU_12013/1]. MM and FvH were supported by the Swedish Heart-Lung Foundation and the Leducq Foundation. CJW was supported by HL135824, HL109946, HL127564.   9  Disclosure  None  10  References  1. Ference BA, et al. Variation in PCSK9 and HMGCR and Risk of Cardiovascular Disease and Diabetes. N Engl J Med. 2016;375:2144-2153. 2. Laugsand LE, et al. Circulating PCSK9 and risk of myocardial infarction: The HUNT Study in Norway. JACC Basic Transl Sci. 2016;1:568-575. 3. Magnusson PK, et al. The Swedish Twin Registry: establishment of a biobank and other recent developments. Twin Res Hum Genet. 2013;16:317-329. 4. Hemani G, et al. The MR-Base platform supports systematic causal inference across the human phenome. Elife. 2018;7:e34408. 5. Germain M, et al. Meta-analysis of 65,734 individuals identifies TSPAN15 and SLC44A2 as two susceptibility loci for venous thromboembolism. Am J Hum Genet. 2015;96:532-542.   11    Figure. A, The association between PCSK9 and HMGCR genetic risk scores and a range of continuous traits (left) and diseases or traits (right) in the Nord-Trøndelag Health Study, scaled to a genetic risk score-determined 0.259 mmol/L (10 mg/dL) decrease in LDL cholesterol. The PCSK9 genetic risk score is defined from the discovery GWAS in this study. The existing PCSK9 score and HMGCR score are defined and weighted using results for LDL cholesterol associated variants from the Global Lipids Genetics Consortium. B, The association between genetically determined serum PCSK9 and a range of continuous traits (Amygdala volume, Birth weight, Body fat, Body mass index, Caudate volume, Chronotype, Depressive symptoms, Father’s age at death, HDL cholesterol, Hip circumference, Hippocampus volume, Intracranial volume, LDL cholesterol, Mean platelet volume, Mother’s age at death, Neuroticism, Nucleus accumbens volume, Pallidum volume, Parents' age at death, Platelet count, Putamen volume, Sitting height ratio, Sleep duration, Thalamus volume, Top 1 % survival, Total cholesterol, Triglycerides, Waist circumference, Waist-to-hip ratio, Years of schooling) (left) and diseases or traits (Alcohol dependence, Alzheimer's disease, Amyotrophic lateral sclerosis, Autism, Bulimia nervosa, Coronary heart disease, Crohn's disease, Eczema, Inflammatory bowel disease, Lung adenocarcinoma, Lung cancer, Multiple sclerosis, Myocardial infarction, Rheumatoid arthritis, Schizophrenia, Squamous cell lung cancer, Type 2 diabetes, Ulcerative colitis) (right) in MR-Base. The new PCSK9 genetic risk score had a r2 and F-statistics of 2.1% and 80.55 for serum PCSK9.  CI, confidence interval; COPD, chronic obstructive pulmonary disease; BMI, body mass index; FEV1, forced expiratory volume in one second; FVC, forced vital capacity; HADS, Hospital Anxiety and Depression Scale; HDL, high-density lipoprotein; HMGCR, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase; LDL, low-density lipoprotein; OR, odds ratio; PCSK9: Proprotein convertase subtilisin/kexin type 9. 

Variation in Serum PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9), Cardiovascular Disease Risk, and an Investigation of Potential Unanticipated Effects of PCSK9 Inhibition.

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Variation in Serum PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9), Cardiovascular Disease Risk, and an Investigation of Potential Unanticipated Effects of PCSK9 Inhibition.

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