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The Pakistan Risk of Myocardial Infarction Study: a resource for the study of genetic, lifestyle and other determinants of myocardial infarction in South Asia

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The burden of coronary heart disease (CHD) is increasing at a greater rate in South Asia than in any other region globally, but there is little direct evidence about its determinants. The Pakistan Risk of Myocardial Infarction Study (PROMIS) is an
  NEW STUDY The Pakistan Risk of Myocardial Infarction Study: a resourcefor the study of genetic, lifestyle and other determinantsof myocardial infarction in South Asia Danish Saleheen   Moazzam Zaidi   Asif Rasheed   Usman Ahmad   Abdul Hakeem   Muhammed Murtaza   Waleed Kayani   Azhar Faruqui   Assadullah Kundi   Khan Shah Zaman   Zia Yaqoob   Liaquat Ali Cheema   Abdus Samad   Syed Zahed Rasheed   Nadeem Hayat Mallick   Muhammad Azhar   Rashid Jooma   Ali Raza Gardezi   Nazir Memon   Abdul Ghaffar   Fazal-ur-Rehman   Nadir Khan   Nabi Shah   Asad Ali Shah   Maria Samuel   Farina Hanif    Madiha Yameen   Sobia Naz   Aisha Sultana   Aisha Nazir   Shehzad Raza   Muhammad Shazad   Sana Nasim   Muhammad Ahsan Javed   Syed Saadat Ali   Mehmood Jafree   Muhammad Imran Nisar   Muhammad Salman Daood   Altaf Hussain   Nadeem Sarwar   Ayeesha Kamal   Panos Deloukas   Muhammad Ishaq   Philippe Frossard   John Danesh Received: 22 October 2008/Accepted: 11 March 2009   The Author(s) 2009. This article is published with open access at Abstract  The burden of coronary heart disease (CHD) isincreasing at a greater rate in South Asia than in any otherregion globally, but there is little direct evidence about itsdeterminants. The Pakistan Risk of Myocardial InfarctionStudy (PROMIS) is an epidemiological resource to enablereliable study of genetic, lifestyle and other determinants of CHD in South Asia. By March 2009, PROMIS hadrecruited over 5,000 cases of first-ever confirmed acutemyocardial infarction (MI) and over 5,000 matched con-trols aged 30–80 years. For each participant, informationhas been recorded on demographic factors, lifestyle, med-ical and family history, anthropometry, and a 12-lead D. Saleheen    A. Rasheed    U. Ahmad    M. Ishaq    P. FrossardCenter for Non-Communicable Diseases (CNCD), Karachi,PakistanD. Saleheen ( & )    N. Sarwar    J. DaneshDepartment of Public Health and Primary Care, StrangewaysResearch Laboratory, University of Cambridge, Cambridge CB18RN, UK e-mail: D. Saleheen    M. Zaidi    A. Rasheed    U. Ahmad    A. Hakeem   M. Murtaza    W. Kayani    N. Khan    N. Shah   A. A. Shah    M. Samuel    F. Hanif     M. Yameen    S. Naz   A. Sultana    A. Nazir    S. Raza    M. Shazad    S. Nasim   M. A. Javed    S. S. Ali    M. Jafree    M. I. Nisar    A. Hussain   P. FrossardDepartment of Biological and Biomedical Sciences, Aga KhanUniversity, Stadium road, Karachi, PakistanA. Faruqui    A. Kundi    K. S. Zaman    Z. Yaqoob   L. A. CheemaNational Institute of Cardiovascular Diseases, Karachi, PakistanA. Samad    S. Z. Rasheed    M. IshaqKarachi Institute of Heart Diseases, Karachi, PakistanN. H. Mallick     M. Azhar    M. S. DaoodPunjab Institute of Cardiology, Lahore, PakistanR. JoomaJinnah Post-graduate Medical Centre, Karachi, PakistanA. R. GardeziMultan Institute of Cardiology, Multan, PakistanN. Memon    A. GhaffarCivil Hospital, Hyderabad, PakistanFazal-ur-RehmanRed Crescent Institute of Cardiology, Hyderabad, PakistanA. KamalSection of Neurology, Department of Medicine, Aga KhanUniversity, Karachi, PakistanP. DeloukasWellcome Trust Sanger Institute, Hinxton, Cambridge, UK   1 3 Eur J EpidemiolDOI 10.1007/s10654-009-9334-y  electrocardiogram. A range of biological samples has beencollected and stored, including DNA, plasma, serum andwhole blood. During its next stage, the study aims toexpand recruitment to achieve a total of about 20,000 casesand about 20,000 controls, and, in subsets of participants,to enrich the resource by collection of monocytes, estab-lishment of lymphoblastoid cell lines, and by resurveyingparticipants. Measurements in progress include profiling of candidate biochemical factors, assay of 45,000 variants in2,100 candidate genes, and a genomewide association scanof over 650,000 genetic markers. We have established alarge epidemiological resource for CHD in South Asia. Inparallel with its further expansion and enrichment, thePROMIS resource will be systematically harvested to helpidentify and evaluate genetic and other determinants of MIin South Asia. Findings from this study should advancescientific understanding and inform regionally appropriatedisease prevention and control strategies. Keywords  Myocardial Infarction    Case-control study   South Asia    Pakistan    MI    Risk factors Introduction Coronary heart disease (CHD) is the single leading causeof death worldwide and a major cause of disability [1]. Theprojected increase in CHD is expected to be much greaterin South Asia than in any other region worldwide [2],where it is expected to more than double over the next20 years [3]. The burden of CHD in South Asians extends beyond regional concerns, as CHD mortality and morbidityremain higher in immigrant South Asians living in westernregions compared with native western populations [4]. There is limited evidence, however, about the determinantsof CHD in South Asia, although such evidence shouldcontribute importantly to scientific understanding and tothe development of regionally appropriate strategies toprevent and control CHD. In particular, it has been pro-posed that populations in South Asia are predisposed tocardiometabolic conditions owing to a combination of hereditary and lifestyle factors that promote metabolicdysfunction [5, 6], but direct evidence is sparse. Until the initiation of the Pakistan Risk of MyocardialInfarction Study (PROMIS), fewer than 1,000 patients anda similar number of controls had been assessed in allavailable epidemiological studies of CHD in Pakistan, acountry with a population of 175 million. The value of large case-control studies of CHD in relation to genetic andlifestyle factors has been demonstrated in non-Pakistanipopulations by ISIS (14,000 cases of acute myocardialinfarction (MI), 16,000 controls: [ 95% white British) [7] and INTERHEART (15,000 acute MI cases, 15,000controls: \ 5% Pakistani) [8]. An important challenge is, therefore, to establish epidemiological resources of con-siderable scale in a rigorous yet cost-effective manner inSouth Asian populations where there is unmet scientificand public health need.The objectives of this report are to describe the methodsused in the establishment of a epidemiological resource forthe study of CHD in Pakistan and to outline the rationalefor the study’s plan of further expansion and sub-studies toenable scientific enrichment. Methods Study participantsPROMIS is a retrospective case-control study of acute MI.Following screening by medically-qualified research offi-cers, patients aged 30–80 years admitted to the emergencyrooms of collaborating hospitals (Fig. 1 lists recruitmentcentres active as of March 2009) have been eligible forinclusion as cases if they fulfil all of the following criteria:(1) sustained clinical symptoms suggestive of MI lastinglonger than 20 min within the previous 24 h; (2) ECGchanges of MI (i.e., new pathologic Q waves, at least 1 mmST elevation in any 2 or more contiguous limb leads or anew left bundle branch block, or new persistent ST–T wavechanges diagnostic of a non-Q wave MI); (3) confirmatorytroponin-T measurements; and (4) no previous cardiovas-cular diseases, defined as self-reported history of angina,MI, coronary revascularisation, transient ischaemic attack,stroke or evidence of CHD on prior ECG or in othermedical records.Controls have been individuals without a self-reportedhistory of cardiovascular disease (who had no ECG chan-ges consistent with a previous MI) drawn from individualsconcurrently identified in the same hospitals as index cases.Controls have been recruited in the following order of priority: (1) visitors of patients attending the out-patientdepartment; (2) patients attending the out-patient depart-ment for routine non-cardiac complaints, or (3) non-bloodrelated visitors of index MI cases. Controls were fre-quency-matched to cases on sex and age (in 5-year bands).Participants were not recruited into PROMIS if any of thefollowing features were evident: (1) a previous history of cardiovascular disease (including self-reported MI, angina,coronary revascularization, stroke, transient ischaemicattack, or peripheral vascular disease, and, in cases, pres-ence of cardiogenic shock); (2) a history of a viral orbacterial infection in the previous 2 weeks; (3) documentedchronic conditions, such as malignancy, any chronicinfection, leprosy, malaria or other bacterial/parasiticinfections, chronic inflammatory disorders, hepatitis or D. Saleheen et al.  1 3  renal failure on past medical history; (4) pregnancy; or (5)refusal to give consent.Prior to adoption of the approach described above forthe selection of controls, we carefully assessed severalother options, such as choosing reference groups who had:an unrelated disease; population based community con-trols; controls from occupational settings or health-check clinics. Our chosen approach was considered desirablebecause it achieved a balance between feasibility and sci-entific rigor and because it was scalable in Pakistan. (Bycontrast, whereas use of population based communitycontrols may be desirable in principle, it is considerablymore labour-intensive and expensive and cannot guaranteethat such controls will necessarily represent the catchmentareas from which hospital-based cases are derived, partic-ularly since referral patterns to hospitals are complex.)Furthermore, by analogy with the Wellcome Trust Case-Control Consortium (WTCCC) [9], PROMIS controls canbe efficiently and validly used for patients with other car-diometabolic conditions owing to the broad geographicaland ethnic scope in PROMIS.PROMIS has received approval by the relevant researchethics committee of each of the institutions involved inparticipant recruitment. Informed consent has beenobtained from each participant recruited into the study,including for use of samples in genetic, biochemical andother analyses.Questionnaire administration and anthropometryTo maximise consistency and quality-control acrossrecruitment centres, staff in each recruitment centre havebeen trained in the use of standard operating procedures.Research medical officers have administered pre-pilotedepidemiological questionnaires to participants that seek atotal of  [ 200 items of information in relation to: ethnicity(e.g, personal and paternal ethnicity, spoken language,place of birth and any known consanguinity); demo-graphic characteristics; lifestyle factors (e.g., tobacco andalcohol consumption, dietary intake and physical activ-ity); and personal and family history of cardiovasculardisease; and medication usage (Table 1). As dietary habitsand types of tobacco consumption, in particular, can besubstantially different in South Asia than in westernpopulations, it has been necessary to develop a locallyrelevant questionnaire that adequately records suchinformation (Table 2). Development of the dietary com-ponent of this questionnaire involved completion of a24 h dietary recall questionnaire and the completion of a7-day food diary by 200 healthy adults, which enabled theidentification of 160 different foods consumed that werecategorised into 54 groups by a local nutritionist. Aprovisional food-frequency questionnaire was then furtherrefined after its piloting among 100 additional healthyadults (e.g., the final version added further questionsabout foods typically consumed only during Ramadan, themonth of fasting). For MI cases, questionnaires have beenadministered only after medical stabilization and relate tohabits and characteristics during the time before thediagnosis of acute MI. Using standardized procedures andequipment, research officers have obtained measurementsof height, weight, waist and hip circumference, systolicand diastolic blood pressure, and heart rate. Waist cir-cumference has been assessed over the abdomen at thewidest diameter between the costal margin and the iliaccrest, and hip circumference has been assessed at the levelof the greater trochanters. Research officers have alsorecorded a standard 12-lead ECG. Information extractedfrom questionnaires, physical measurements and ECGrecordings has been entered by two different operators Recruitment centre CityPredominant ethnicity Punjab Institute of Cardiology LahorePunjabiKarachiUrduKarachiUrdu Multan Multan Institute of CardiologyMultanPunjabiCivil HospitalRed Crescent HospitalNational Institute of Cardiovascular DiseasesKarachi Institute of Heart DiseasesHyderabad HyderabadSindhiUrdu Fig. 1  Location and predominant ethnicities of PROMIS recruitment centresThe Pakistan Risk of Myocardial Infarction Study  1 3  onto the central database, which is securely held both inKarachi, Pakistan, and Cambridge, UK.Collection of biological samples and initialbiochemistryNon-fasting blood samples (with the time since last mealrecorded) have been drawn by phlebotomists from eachparticipant and centrifuged within 45 min of venepuncture.In MI cases, blood sampling has been due to occur within24 h of the onset of MI symptoms and prior to theadministration of any thrombolytics, with the time sinceonset of symptoms recorded to the nearest hour. Becauseblood is collected from MI cases while they are in arecumbent position (e.g, at about 45  ), blood samples havebeen drawn in the same manner from controls to limit thepossibility of systematic differences (e.g, plasma volumesmay be higher in the supine position than when sitting [10, 11]). A total of 29 ml of blood has been drawn from eachparticipant in 2  9  6 ml serum tubes and 3  9  5 ml EDTA Table 1  Summary of questionnaire-based information collectedType of information Detail recordedSymptoms, arrival and managementat the hospitalDate of MI, time of onset, time since last meal, review of symptoms, diagnosis and management at thehospital and any investigations ordered by the attending physicianMedication and medical history Medication class and duration of use for each of anti-coagulant, blood pressure- and lipid-lowering,diabetes related, hormonal and, for women, contraceptive and HRT medicationsFemale reproductive history Age at first menstrual period, age periods stopped, hormones for menopause treatment, oral contraceptiveuseFamily history Approximate age of diagnosis/occurrence of hypertension, diabetes, angina, MI, stroke, cancer or suddendeath, for each of mother, father, sister, brother, son and daughterEthnicity and other genetic relatedinformationPlace of birth, personal and parental ethnicity (e.g, Urdu, Punjabi, Pathan, Balooch, Sindhi, Memon,Gujrati), parental co-sanguinitySocio-economic status Occupation(s), monthly income, level and duration of education, marital status, no. of dependants,ownership/wealthPhysical activity Type, frequency, duration and intensity of activity for each of occupational, work related commuting andleisure timeTobacco consumption See Table 2 for detailsPsychosocial factors Experience of traumatic events in the past year (e.g., loss of crop, family bereavement), perceived level of occupational, domestic and financial stress, perceived level of mental and physical healthAnthropometry Height, weight, waist and hip circumferenceHRT, hormone replacement therapy Table 2  Information collected on dietary intake and tobacco consumptionType of food Routinely recorded information Additional information recorded to reflect local habitsCooking medium Oil (recording type), butter, margarine Ghee (type of fat)Breads N/A Naan, chapatti (recording type of flour used), paratha (butter-coated chapatti)Fruits and vegetables Separately for cooked and raw, green leafyvegetables, yellow vegetables, cruciferousvegetables, salad items, fruitsMethod of cooking (grilled, curried and fried)Meats Chicken, beef, mutton, lamb and fish Method of cooking (grilled, curried and fried)Legumes Nuts and seeds Pulses (including different daals)Sweets Bakery items Kheer (dairy based dessert), halwa, mithai, jalabe (unrefinedsugars), nimko (fried desserts)Brewed beverages Tea, coffee, herbal tea Qahwa (local form of herbal tea)Other beverages Milk, carbonated and non-carbonated soft drinks,fruit juicesAlcohol (including local varieties), lassi (yoghurt based drink,recorded separately for sweet and salty)Tobacco consumption Usage status, quantity, frequency and lifetime.Information on sources of passive smoking alsorecordedFor each of cigarettes, beedies (rolled tobacco), huqqa (waterpipe), paan (chewing tobacco), naswar (snuff), gutka andsupari (preparations of crushed betel nut, tobacco, and sweetor savory flavourings).D. Saleheen et al.  1 3  tubes (a further citrate tube has been obtained only insuspected MI cases for troponin assay). Hence, a total of five blood tubes have been collected per participant,including serum, EDTA plasma and whole blood whichhave been stored in cryogenic vials. All samples have beenstored temporarily at each recruitment centre at  - 20  C. Asecond sample (2 ml) has been drawn in cases 10 h afterthe onset of clinical symptoms for measurement of tropo-nin. Serum, plasma and whole blood samples are trans-ported daily to the local laboratory where they are stored at - 80  C. The long-term - 80  C sample repository is kept inboth Karachi and Cambridge. Measurement of total cho-lesterol (using an enzymatic colorimetric method, RocheDiagnostics GmbH, USA) and high-density lipoprotein-cholesterol (using a homogeneous enzymatic colorimetricmethod, Roche Diagnostics GmbH, USA) has been com-pleted in over 5,000 MI cases and over 5,000 controls thusfar.DNA quality, quantity and initial genotypingResearch technicians trained in accordance with standardoperating procedures in laboratories in Karachi haveextracted DNA from leucocytes using a reference phenol-chloroform protocol [12]. PCR-RFLP has been performed in all samples on the day of extraction to provide an initialquality check. DNA concentrations have been determinedby fluorescence measurement using PicoGreen molecularprobes. The yield of DNA per participant has typicallybeen between 600 and 800 ng/  l l in a total volume of about 750  l l. To minimise any systematic biases arisingfrom plate- or batch-specific genotyping error and/ornonrandom missingness, stock plates have been used togenerate genotyping plates which contain a mixture of cases and controls along with negative and positive con-trols designed to address genotyping quality control (QC),plate identification and orientation. As part of the QC,samples have been subject to molecular bar-coding andchecked for degradation by running samples on agarosegels. Samples passing QC have been diluted in tris borateEDTA buffer to 50 ng/  l l and arrayed into 96-well masterplates.Further expansion and measurementsBy March 2009, PROMIS had recruited over 5,000 con-firmed first-ever MI cases and over 5,000 controls. Themain objectives of the study’s next stages are: to expandthe study to 20,000 MI cases and 20,000 controls; to enrichthe bioresource in various ways in order to increase itsscientific value; and to accelerate harvesting of its bio-logical resources. Assays have been completed in the first2,000 MI cases and in the first 2,000 controls of 45,000genetic markers in 2,100 candidate genes in the Illumina Table 3  Biological measurements in progress in PROMISApproach Analytical strategy Potential value Genomics Genomewide association study Illumina 610 quad array  ?  60 K forCNV analysis(1) discovery of genetic variants associated with MI; (2) investigationof genetic architecture in Pakistani populationIBC Illumina cardiochip 45,000 variants selected from 2,100candidate genesComprehensive evaluation of candidate genes in MISequencing In-depth sequencing to investigate LDstructureInvestigation of LD and haplotypes to identify causal genetic variants Transcriptomics Genomewide expression study Illumina high throughput beadchipplatform (46,000 gene targets)(1) assessment of transcript levels associated with cis-acting sequencevariants; (2) comparison of resting and activated transcript levels incases and controls; (3) functional consequences of established andnovel variantsLmyphoblastoid cell lines Transfection with Epstein-Barr virus Functional genomics Metabonomics Spectroscopy 1H NMR and GC-MS Metabolic determinants of MI Biomarkers Circulating candidate analytes Total cholesterol, HDL-C, LDL-C,triglycerides, apo-AI, apo-B, Lp(a),CRP, Lp-PLA 2 , adiponectin(1) associations of biomarkers with MI; (2) genetic determinants of the measured biomarkers in controlsCNV, copy number variants; LD, linkage disequilibrium;  1 H NMR, nuclear magnetic resonance; GC-MS, gas chromatography mass spec-troscopy; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; apo-AI, apolipoprotein-A1; apo-B, apoli-poprotein B100; Lp(a), lipoprotein (a); CRP, C-reactive protein; Lp-PLA 2 , Lipoprotein-associated phospholipase A 2 The Pakistan Risk of Myocardial Infarction Study  1 3
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