Methodology

Cross Sectional Study.

Study population:

Patients who are dyslipidemic (LDL-C 100 mg/dl diabetics and /or CAD patients, LDL-C

130 mg/dl others)will be recruited for the study. Patients will be recruited from Out Patient

Department (OPD) of Medicine, and Cardiology JIPMER.

Intervention:

Atorvastatin 10 and 40 mg for 6 weeks.

Methodology

Patients will be recruited from Out Patient Department (OPD) Department of Medicine, and

Cardiology JIPMER. Patients who are dyslipidemic will be recruited for the study and they will

be initiated on atorvastatin treatment based on their lipid levels for 6 weeks. At the end of 6

weeks their lipid profile will be assessed for lipid response and plasma will be collected for

assessment of drug and metabolite levels. The effect of genetic polymorphisms on the percentage

reduction in LDL-C level will be evaluated.

List of candidate genes to be studied:

1. HMGCR: Gene encoding for HMGCoA Reductase enzyme -a rate limiting enzyme in the

synthesis of cholesterol

2. CP7A1- Gene encoding for Cholesterol 7 alpha hydroxylase-a rate limiting enzyme in the

synthesis of bile acids. Bile acid is formed by the metabolism of cholesterol.

3. LDLR- Gene encoding for LDL- receptor on the surface of the hepatocytes.

4. MDR-1- Gene encoding for multi-drug resistance protein involved in the efflux of drugs.

5. APOE- Gene encoding for Apolipoprotein E which acts as a ligand for LDL receptor as well

as for transport of cholesterol.

6. CYP3A5- Gene encoding for CYP3A5 enzyme which metabolises Atorvastatin.

List of SNP’S to be studied.

Gene rs id SNP Chromosomallocation Allele frequency Significance

HMGCR 17238540

5908

12916

29G/T

A/G

C/T

5q.13.3 G- 0.06 T- 0.9421

A- 0.85, G-0.1521

C-0.55, T-0.4521

Variants associatedwith altered LDL-C reduction withatorvastatin

CYP7A1 3808607 -204 A/C 8q.11.q.12 A-0.63, C-0.3721 Variants associatedwith diminishedLDL-C reductionwith atorvastatin

LDLR 688 C1773/THin C II

19p.13.3 H+ 0.56 H- 0.4424 Variants associatedwith diminishedLDL-C reductionwith atorvastatin

ABCB1 2032582

1045642

G2677T/A

C3435T

7q.21.1

7q.21.1

G-0.44, T- 0.52,A-0.0425

C-0.46, T-0.5425

Variants associatedwith diminishedLDL-C reductionwith atorvastatin

APOEE2

E4

7412

429358

Cys176Arg

Cys130Arg

19q.13.2

19q.13.2

E2 -0.0526

E3- 0.8826

E4- 0.0726

VariableLDL-C reductionwithAtorvastatin

CYP3A5 776746 6986 A/G 7q.21.1 A-0.37 G-0.6422

Associated withvariable LDL-Creduction withatorvastatin

Sample size

1000 patients (including adjustment for 15 % drop out)

Minor allele Frequency- 0.0425

Alpha error-0.05

Power 80%

Effect size – 5%

Inclusion criteria

1. Newly diagnosed dyslipidemic patients

2. Patients of both gender aged 30- 65 yrs

3. Patients of Tamilian origin.(Those who are living in Tamilnadu/Puducherry for the past 3

generations and those whose mother tongue is Tamil)

Exclusion criteria

1. Patients with impaired liver or kidney function .

2. Patients receiving CYP3A4 inducers (Barbiturates, carbamazepine, glucocorticoids,

pioglitazone, phenytoin, rifampin).

3. Patients receiving CYP3A4 inhibitors (erythromycin, fluconazole, itraconazole,

ketoconazole, ritonavir, troleandomycin).

4. Pregnant and lactating women.

5. Patients on other lipid lowering agents

6. Patients with hypothyroidism.

7. Patients with LDL-C >250 mg/dl.

8. Triglycerides >400 mg/dl.

9. Patients who had/have a change in anti-diabetic drug/dose one month prior to the study and

during the study period.

Study Procedure

Clinical Evaluations:

Patients fulfilling the inclusion and exclusion criteria and volunteering to give informed consent

will be enrolled for the study. Informed written consent will be obtained from all subjects.

Patient characteristics namely age, sex, BMI, waist circumference, blood pressure and relevant

history will be obtained.

Blood collection and storage:

Initial visit- 7ml of fasting blood sample will be collected in an anticoagulant(EDTA) container

after an overnight fasting for 12-14 hrs and plasma and blood components will be separated and

stored at -80C until analysis of lipid profiles and genotyping. At the end of the study 5 ml of

venous blood will be collected after overnight fasting and 5ml after 2 hours of drug intake to

estimate the drug and metabolite levels.

Genotyping:

The genomic DNA will be isolated from whole blood using phenol-chloroform method.

Genotyping will be done by allelic discrimination assay in Real Time PCR using Taqman

Technology.

Chromatography:

The plasma levels of atorvastatin and its metabolites will be determined using Liquid

Chromatography/ Mass Spectroscopy. 27

Statistical analysis:

All the continuous study variables will be given as Mean± SD and paired t test will be used to

analyze pre & post lipid parameters and one way ANOVA will be carried out to analyze the data

between groups. Pearson’s chi square test was used for statistical comparisons between the

groups wild-type versus variants, CAD versus CAD-free, and comparison of percentage

reduction across genotypes. Chi square test will be used to see if the genotypes are in Hardy

Weinberg equilibrium.

Multivariate analysis using logistic regression analysis will be done for the data obtained to find

the association between the genotype and lipid levels and between other variables

Plasma levels of atorvastatin will be compared by C/D values (Concentration/Dose). Spearman

Rank Correlation will be used to analyze the relationship between genotype & atorvastatin

plasma level. P value less than 0.05 was considered to be significant.

Patient Data Sheet

Title of the study: Pharmacogenetics of lipid lowering response ofAtorvastatin among dyslipidemic Tamilian Population.

Date: Serial no:

Patient is enrolled from...Medicine/ Cardiology Out Patient Department, JIPMER, Pondicherry.

Name: Age: Sex:

Hospital no:

Address:

Phone no:

Religion: Caste: Sub-division

Baseline characteristics:

Height: Weight: BMI: kg/m2 Waist circumference: W/H ratio

Alcohol consumption : Never

Occasional

Regular

Smoking : Never 1-10 /day 11-20 /day > 20cig/day

Beedi/Cigarette

Duration of Smoking: _________ months/years

Tobacco Chewers : Yes / No

Dietary habits : Vegetarian / non-vegetarian (egg/fish/meat predominantly)

Occupation:

Educational status: Ill-literate/ literate

Monthly income:

Physical activity.

Level 1 Level 2 Level 3 Level 4Level 1 -Inactive lifestyle(Sitting most of the day and drive or ride wherever possible)

Level 2 –Moderately active lifestyle(sit most of the day ,but take occasional ,low intensity exercise)

Level 3- Active lifestyle(job requires physical labour and/or some amount of regular exercise 4- 5 times a week

Level 4- Very active lifestyle/athlete(job requires heavy physical labour or involved in a programme of intense

aerobic exercise of over 10 hours per week with a resting heartbeat of below 60 bpm)

Associated medical illness of the patient:

H/O Diabetes Mellitus - Yes No Duration

H/O Hypertension - Yes No Duration

H/O Angina/M.I Yes NoFamily history of Angina/M.I Yes No

History of chronic illness/other disease.

Investigations

Blood pressure: SBP DBPHeart rate

Lipid profileTime interval TC TG HDL-C LDL-C

Baseline

6wks

ECG Report

Coronary angiogram report

Month & Year of diagnosis of CAD:

Atorvastatin dose:

Concomitant medication

s.no Name of the drug Dose duration

Information about patient genotype status

Gene Rs id Genotype status

HMGCR 17238540

5908

12916

CYP7A1 3808607

LDLR 688

MDR1 2032582

1045642

APOE 7412

429358

CYP3A5 776746

Baseline diagnostic information:

LFT Baseline Post treatment

ALT

AST

RFT

BUN

Creatinine

FBS

Hb1ac

Creatine phosphokinase

TSH

Sample Code:

Adverse Reactions associated with Atorvastatin use:

Gastrointestinal disturbances

Headaches

Central nervous system disturbances

Sleep disorders

Myalgia,

Hepatotoxicity

Myopathy.

Rhabdomyolysis

1

Reference

1. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on

Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment

Panel III) Final Report. Circulation 2002;106:3143–21.

2. Keefe JHO, Carter MD, Lavie CJ. Primary and Secondary Prevention of Cardiovascular

Diseases: A Practical Evidence-Based Approach. Mayo Clinic Proceedings. 2009

Aug;84(8):741–57.

3. Grundy SM, Cleeman JI, Merz CN, Brewer HB , Clark LT, Hun-ninghake DB, Pasternak

RC, Smith SC Jr, Stone NJ. Implications of recent clinical trials for the National Cholesterol

Education Program Adult Treatment Panel III guidelines. Circulation.2004;110:227–39.

4. Waters DD, Brotons C, Chiang C-W, Ferrières J, Foody J, Jukema JW, et al. Lipid

Treatment Assessment Project 2. Circulation. 2009 ;120(1):28–34.

5. Kathiresan S, Melander O, Anevski D, Guiducci C, Burtt NP, Roos C, et al. Polymorphisms

Associated with Cholesterol and Risk of Cardiovascular Events. N Engl J Med.

2008;358(12):1240–9.

6. Shah SH. Genetics of LDL Cholesterol. emedicine.medscape.com/article/1783664-overview

7. Thompson JF, Man M, Johnson KJ, Wood LS, Lira ME, Lloyd DB,Banerjee P, Milos PM,

Myrand SP, Paulauskis J, Milad MA, Sasiela WJ. An association study of 43 SNPs in 16

candidate genes with atorvastatin response. Pharmacogenomics J. 2005;5: 352–8.

8. Lin Y, Mousa SS, Elshourbagy N, Mousa SA. Current status and future directions in lipid

management: emphasizing low-density lipoproteins, high-density lipoproteins, and

triglycerides as targets for therapy. Vasc Health Risk Manag. 2010;6:73–85.

9. Sanmukhani J, Shah V. Statins: Cost analysis in Indian scenario from eight major clinical

trials J Postgrad Med 2010;56:196-200.

10. Voora D, Shah SH, Reed CR, Zhai,J , Crosslin DR, Messer C, Salisbury BA , Ginsburg

GS. Pharmacogenetic Predictors of Statin-Mediated Low-Density Lipoprotein Cholesterol

Reduction and Dose Response. Circ Cardiovasc Genet 2008;1:100-106.

11. Mega JL, Morrow DA, Brown A, Christopher P. Cannon , Sabatine MJ. Identification of

Genetic Variants Associated With Response to Statin Therapy. Arterioscler Thromb Vasc

Biol 2009; 29;1310-15.

12. Thompson JF, Hyde CL, Wood LS, Paciga SA, Hinds DA, Cox DR, et al. Comprehensive

Whole-Genome and Candidate Gene Analysis for Response to Statin Therapy in the

Treating to New Targets (TNT) Cohort / Clinical Perspective. Circulation: Cardiovascular

Genetics. 2009 Apr 1;2(2):173 -81.

2

13. Hoenig MR, Kostner KM, Read SJ, Walker PJ, Atherton JJ. Implications of the obesity

epidemic for statin therapy: shifting cholesterol metabolism to a high synthesis and low

dietary absorption state. Endocr Metab Immune Disord Drug Targets. 2007 Sep;7(3):153-

66.

14. Chasman DI, Posada D, Subrahmanyan L, Cook NR, Stanton VP, Ridker PM.

Pharmacogenetic Study of Statin Therapy and Cholesterol Reduction. JAMA: The Journal

of the American Medical Association. 2004 Jun 16;291(23):2821 -27.

15. Chien K-L, Wang K-C, Chen Y-C, Chao C-L, Hsu H-C, Chen M-F, et al. Common

sequence variants in pharmacodynamic and pharmacokinetic pathway-related genes

conferring LDL cholesterol response to statins. Pharmacogenomics. 2010 Mar;11(3):309-17.

16. Poduri A, Khullar M, Bahl A, Sehrawat BS, Sharma Y, Talwar KK. Common variants of

HMGCR, CETP, APOAI, ABCB1, CYP3A4, and CYP7A1 genes as predictors of lipid-

lowering response to atorvastatin therapy. DNA Cell Biol. 2010 Oct;29(10):629-37.

17. Hutz MH, Fiegenbaum M. Impact of genetic polymorphisms on the efficacy of HMG-CoA

reductase inhibitors. Am J Cardiovasc Drugs. 2008;8(3):161-70.

18. Kivisto KT, Niemi M, Schaeffeler E, Pitkala K, Tilvis R, Fromm MF, et.al.,Lipid-lowering

response to statins is affected by CYP3A5 polymorphism. Pharmacogenetics 2004;14:523–

25.

19. Kajinami K, Takekoshi N, Brousseau ME, Ordovas JM, SchaeferEJ. Additive gene-gene

interaction between CYP7A1 and APOE as genetic determinants of LDL cholesterol-

lowering response to atorvastatin (abstract). J Am Coll Cardiol 2004; 43(Suppl A):505A.

20. Zhu H, Tucker HM, Grear KE, Simpson JF, Manning AK, Cupples LA, et al. A Common

Polymorphism Decreases Low-Density Lipoprotein Receptor Exon 12 Splicing Efficiency

and Associates with Increased Cholesterol. Hum Mol Genet. 2007 Jul 15;16(14):1765–72.

21. Poduri A, Khullar M, Bahl A, Sharma YP, Talwar KK. A combination of proatherogenic

single-nucleotide polymorphisms is associated with increased risk of coronary artery disease

and myocardial infarction in Asian Indians. DNA Cell Biol. 2009 Sep;28(9):451-60.

22. Krishnakumar D, Gurusamy U, Dhandapani K, Surendiran A, Baghel R, Kukreti R,

Gangadhar R, Prayaga U, Manjunath S, Adithan C. Genetic polymorphisms of drug-

metabolizing phase I enzymes CYP2E1, CYP2A6 and CYP3A5 in South Indian

population.Fundam Clin Pharmacol. 2011 Jan 25. [Epub ahead of print].

23. Harikrishnan S, Rajeev E , Tharakan JA, Titus T, Ajithkumar VK, Sivasankaran S,

Krishnamoorthy KM, NairK. Do we Achieve target lipid levels with statins in patients with

Coronary Artery Disease? KMJ August 2008; 2:5-9.

3

24. Salazar LA, Hirata MH, Giannini SD, Forti N, DiamenteJ, Issa JS, and Hirata RDC. Effects

of Ava II and Hinc II Polymorphisms at the LDL Receptor Gene on Serum Lipid Levels of

Brazilian Individuals With High Risk for Coronary Heart Disease. Journal of Clinical

Laboratory Analysis 1999; 13:251–8.

25. Umamaheswaran G, Krishnakumar D, Kayathiri D, Rajan D, Shewade DG, Steven AD et

al. Inter and intra ethnic differences in the distribution of the molecular variants of TPMT,

UGT1A1 and MDR1 genes in south Indian population. Mol Biol Rep. DOI:

10.1007/s11033-012-1456-8 [Epub ahead of print].

26. Padmaja N. Pharmacogenomics of Coronary Heart Disease in a South Indian

Population.Ph.D Thesis. Pondicherry University 2009.

27. Guillén D, Cofán F, Ros E, Millán O, Cofán M, Rimola A, et al. Determination of

atorvastatin and its metabolite ortho-hydroxyatorvastatin in human plasma by on-line anion-

exchange solid-phase extraction and liquid chromatography tandem mass spectrometry.

Analytical and Bioanalytical Chemistry. 2009 Jun 9;394(6):1687–96.