A microarray minisequencing system for pharmacogenetic profiling of antihypertensive drug response Ulrika Liljedahl a , Julia Karlsson a , Ha ˚ kan Melhus a , Lisa Kurland a , Marie Lindersson a , Thomas Kahan c , Fredrik Nystro ¨m d , Lars Lind a,b and Ann-Christine Syva ¨ nen a We aimed to develop a microarray genotyping system for multiplex analysis of a panel of single nucleotide polymorphisms (SNPs) in genes encoding proteins involved in blood pressure regulation, and to apply this system in a pilot study demonstrating its feasibility in the pharmacogenetics of hypertension. A panel of 74 SNPs in 25 genes involved in blood pressure regulation was selected from the SNP databases, and genotyped in DNA samples of 97 hypertensive patients. The patients had been randomized to double-blind treatment with either the angiotensin II type 1 receptor blocker irbesartan or the â 1 -adrenergic receptor blocker atenolol. Genotyping was performed using a microarray based DNA polymerase assisted ‘minisequencing’ single nucleotide primer extension assay with fluorescence detection. The observed genotypes were related to the blood pressure reduction using stepwise multiple regression analysis. The allele frequencies of the selected SNPs were determined in the Swedish population. The established microarray-based genotyping system was validated and allowed unequivocal multiplex genotyping of the panel of 74 SNPs in every patient. Almost 7200 SNP genotypes were generated in the study. Profiles of four or five SNP-genotypes that may be useful as predictors of blood pressure reduction after antihypertensive treatment were identified. Our results highlight the potential of microarray-based technology for SNP genotyping in pharmacogenetics. Pharmacogenetics 13:7–17 & 2003 Lippincott Williams & Wilkins Pharmacogenetics 2003, 13:7–17 Keywords: minisequencing, pharmacogenetics, systolic blood pressure, diastolic blood pressure, haplotype, single nucleotide polymorphism, genotyping, microarrays a Department of Medical Sciences, Uppsala University, Uppsala, b Astra Zeneca R&D, Mo ¨ lndal, c Division of Internal Medicine, Karolinska Institute, Danderyd Hospital, Stockholm and d Department of Medicosurgical Gastroenterology, Endocrinology and Metabolism, University Hospital of Linko ¨ ping, Linko ¨ ping, Sweden. Correspondence to Ann-Christine Syva ¨ nen, Department of Medical Sciences, Molecular Medicine, Uppsala University Hospital, Entrance 70, Third Floor, Research Department 2, S-751 85 Uppsala, Sweden. Tel: +46 18 611 29 59; fax: +46 18 611 25 19; e-mail: ann-christine.syvanen@medsci.uu.se Received 1 July 2002 Accepted 24 October 2002 Introduction The most abundant form of genetic variation are the single nucleotide polymorphisms (SNPs) that occur on the average at one out of every thousand bases in the human genome [1,2]. As a result of the efforts of the SNP consortium, which comprises a collaboration be- tween 14 major pharmaceutical companies, and the Human Genome Project [1], there are over four million SNPs in public databases. Depending on where in the genome a SNP occurs, it may have different conse- quences on the phenotypic level. SNPs in the coding regions of genes may alter the function or the structure of the encoded proteins. While most of the SNPs are located in non-coding regions of the genome and have no direct known impact on the phenotype of an individual, they are useful as genetic markers because they may be inherited linked to the functional variants of physically closely located genes. Hypertension is a quantitative trait caused by multiple factors that interact through pathways involving cardiac function, blood volume, salt regulation, peripheral vascular tone and endothelial function [3]. Many of the components of the blood pressure regulating pathways are proteins that vary in structure and activity among individuals owing to SNPs in the genes encoding them. Twin studies show that heritable components explain approximately one-half of the variance in blood pres- sure [4,5]. Because genetic factors contribute to hyper- tension, there is good reason to hypothesize that genetic factors also contribute to individual responses to treatment with antihypertensive drugs. A large inter- individual variation in the response to antihypertensive treatment is well documented [6,7]. Despite attempts to use biochemical indicators, such as plasma renin activity, or metabolic characteristics as predictors of individual drug response, no such predictor has yet been identified to be of clinical importance [8,9]. We have recently found that the insertion/deletion polymorphism in the angiotensin-converting enzyme (ACE) gene predicted the reduction in blood pressure Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Original article 7 0960-314X & 2003 Lippincott Williams & Wilkins