Online Results Section

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The V433M variant of the CYP4F2 is associated with ischemic stroke in male SwedES beyond its effect on blood pressure.

Short title: CYP4F2 V433M polymorphism and ischemic stroke

Cristiano Favaa,b, MD, PhD; Martina Montagnanaa,c, MD; Peter Almgrena, BS; Lena Rosberga, BS; Giuseppe Lippic, MD; Bo Hedblada, MD, PhD; Gunnar Engströma, MD, PhD; Göran Berglunda, MD; Pietro Minuz b, MD; Olle Melandera, MD, PhD

aDepartment of Clinical Sciences, Lund University, University Hospital of Malmö, Sweden.

bDepartment of Biomedical and Surgical Sciences and

cDepartment of Morphological-Biomedical Sciences, University Hospital of Verona, Italy.

Correspondence and requests for reprints to Cristiano Fava, MD, Department of Biomedical and Surgical Sciences, Division of Internal Medicine C, Piazza LA Scuro 10, 37134 Verona, Italy.

Tel: +39 45 8124414; fax: +39 45 508815;

e-mail:

ONLINE METHODS SECTION

Between 1991 and 1996, all women aged 45 to 73 years and all men aged 46 to 73 years, with residency in Malmo¨ (approximately 250 000 habitants), Sweden, were invited by mail or by newspaper advertisement to participate in the Malmo¨ Diet and Cancer (MDC) Study, a population-based prospective study. 1 In all, 28 449 participated out of an eligible population of 74 000. The participants were asked to complete a self-administered questionnaire at home, which included items on lifestyle factors, medication, previous and current diseases. The participants also underwent a health examination at the university hospital, performed by project nurses, including blood pressure, height, and weight. During the visit, the questionnaire was checked for completeness. 1 From this cohort, 6103 subjects were randomly selected to participate in a cardiovascular cohort (MD-CVA), which seeks to investigate risk factors for cardiovascular disease.

Bolod pressure adjustment

Fixed addition

Based on the known average treatment effects, fixed increments of 10 mmHg systolic BP and 5 mmHg diastolic BP were added to treated pressures.

Stepped addition

To account for the number of drugs, stepped increments of 8/4, 14/10, and 20/16 mmHg were added to the measured systolic BP/diastolic BP of treated individuals taking one, two, and three drug classes, respectively.

Laboratory parameters

Blood glucose was determined by a routine hexokinase method. Triglycerides and total cholesterol were determined on a DAX 48 automatic analyzer with use of reagents and calibrators from the supplier of the instrument (Bayer AB, Göteborg, Sweden). High-density lipoprotein (HDL) cholesterol was determined by the same procedure as used for total cholesterol but after precipitation of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) with dextran–sulphate.2 LDL-cholesterol was calculated from the values for triglycerides, total cholesterol and HDL-cholesterol according to the Friedewald formula: LDL = total cholesterol—HDL—(triglycerides/2.2). When serum triglyceride levels were above 4.00 mmol/l, LDL cholesterol was not determined.

Genotyping

DNA was extracted from frozen granulocyte or buffy-coat samples using QIAamp-96 spin blood kits (QIAGEN, Stockholm, Sweden) at the DNA extraction facility supported by SWEGENE. The CYP4A11 F434S polymorphism (dbSNP accession number, rs4233507) and the CYP4F2 V433M polymorphism (dbSNP accession number, rs2108622) were analyzed using respectively:

CYP4A11 forward primer 5¢-GTGGCTGTGTTGAGCAGAAC-3¢,

CYP4A11 reverse primer 5¢-GTGCTCTCTCTGCAGGTGTTT-3¢

CYP4F2 forward primer 5¢-AGCAGACAGAGAGAGGAG-3¢,

CYP4F2 reverse primer 5¢-TGTTTCCTAAGATGATTTAATGT-3¢,

synthesized by Applied Biosystems (Foster City, California, USA).

TaqMan MGB probes were custom synthesized by Applied Biosystems:

for the CYP4A11 F434S [wild-type (A), VIC-AAAACGG[A]AAGGGTC;

mutant (G), FAM-AACGG[G]AAGGGTC] and

for the CYP4F2 V433M [wild-type (G), VIC-CAACCCAGCT[G]TGTGGCCGGA;

mutant (A), FAM-CAACCCAGCT[A]TGTGGCCGGA]

ONLINE RESULTS SECTION

The genotyping success rate was 98.7% for the CYP4A11 F434S polymorphism (n=5975) and 94.9% for the CYP4F2 V433M (n=5797). For the CYP4A11 F434S polymorphism we had 4589 (76.8%) F434F homozygotes, 1275 (21.3%) F434S heterozygotes and 111 (1.9%) S434S homozygotes. For the CYP4F2 V433M polymorphism we had 3159 (54.5%) V433V homozygotes, 2248 (38.8%) V433M heterozygotes and 390 (6.7%) M433M homozygotes.

These genotype frequencies for the CYP4A11 F434S polymorphism are similar to those previously reported in Caucasians,3-5 whereas the minor allele frequency for the CYP4F2 V433M polymorphism was higher respect to that previously reported in African American and European American subjects.6 Genotype distributions did not deviate from Hardy–Weinberg equilibrium for the CYP4A11 F434S polymorphism (c2=1.89, P=0.39 in the whole sample; c2=0.38, P=0.83 in MDC-CVA in participants without antihypertensive treatment) and for the CYP4F2 V433M polymorphism (c2=0.06, P=0.97 in the whole sample; c2=0.01, P=0.99 in MDC-CVA in participants without antihypertensive treatment).

This study had 80% power to detect an odds ratio for CYP4A11 S434S risk genotype for hypertension greater than 1.77, according to a recessive mode of inheritance (CYP4A11 S434S vs. F434S+F434F) and to detect an odds ratio for CYP4F2 V433M for hypertension greater than 1.17, according to a dominant model of inheritance (CYP4F2 V433V vs. M433M+V433M). Regarding continuous BP variables the study had 80% power to detect a BP difference of >5.1 mmHg in systolic BP and >2.6 mmHg in diastolic BP, between CYP4A11 S434S homozygotes and F-carriers and to detect a BP difference of >1.4 mmHg in systolic BP and >0.7 mmHg in diastolic BP, between CYP4F2 V433V homozygotes and CYP4F2 M-carriers.

Regarding the survival analysis this study had 80% power to detect a hazard ratio for CYP4A11 S434S risk genotype for ischemic stroke (n=198)/coronary event (n=274) greater than 1.49/1.49, according to a recessive mode of inheritance (CYP4A11 S434S vs. F434S+F434F) and to detect a hazard ratio for CYP4F2 V433M for ischemic stroke (n=194)/coronary event (n=270) greater than 1.11/1.11, according to a dominant mode of inheritance (CYP4F2 V433V vs. M433M+V433M).

In males, this study had 80% power to detect an odds ratio for CYP4A11 S434S risk genotype for hypertension greater than 2.67, according to a recessive mode of inheritance (CYP4A11 S434S vs. F434S+F434F) and to detect an odds ratio for CYP4F2 V433M for hypertension greater than 1.28, according to a dominant mode of inheritance (CYP4F2 V433V vs. M433M+V433M). Regarding continuous BP variables the study had 80% power to detect a BP difference of >7.9 mmHg in systolic BP and >3.7 mmHg in diastolic BP, between CYP4A11 S434S homozygotes and F-carriers and to detect a BP difference of >2.14 mmHg in systolic BP and >1.1 mmHg in diastolic BP, between CYP4F2 V433V homozygotes and CYP4F2 M-carriers.

Regarding the survival analysis, this study had 80% power to detect a hazard ratio for CYP4A11 S434S risk genotype for ischemic stroke (n=111)/coronary event (n=174) greater than 1.91/1.90, according to a recessive mode of inheritance (CYP4A11 S434S vs. F434S+F434F) and to detect a hazard ratio for CYP4F2 V433M for ischemic stroke (n=108)/coronary events (n=173) greater than 1.18/1.18, according to a dominant mode of inheritance (CYP4F2 V433V vs. M433M+V433M).

In females, this study had 80% power to detect an odds ratio for CYP4A11 S434S risk genotype for hypertension greater than 2.37, according to a recessive mode of inheritance (CYP4A11 S434S vs. F434S+F434F) and to detect an odds ratio for CYP4F2 V433M for hypertension greater than 1.22, according to a dominant mode of inheritance (CYP4F2 V433V vs. M433M+V433M). Regarding continuous BP variables the study had 80% power to detect a BP difference of >6.9 mmHg in systolic BP and >3.2 mmHg in diastolic BP, between CYP4A11 S434S homozygotes and F-carriers and to detect a BP difference of >1.88 mmHg in systolic BP and >0.88 mmHg in diastolic BP, between CYP4F2 V433V homozygotes and CYP4F2 M-carriers.

Regarding the survival analysis this study had 80% power to detect a hazard ratio for CYP4A11 S434S risk genotype for ischemic stroke (n=87)/coronary event (n=100) greater than 1.74/1.74, according to a recessive model of inheritance (CYP4A11 S434S vs. F434S+F434F) and to detect a hazard ratio for CYP4F2 V433M for ischemic stroke (n=86)/coronary event (n=97) greater than 1.15/1.15, according to a dominant mode of inheritance (CYP4F2 V433V vs. M433M+V433M).

REFERENCES

1. Berglund G, Elmstahl S, Janzon L, Larsson SA. The Malmo Diet and Cancer Study. Design and feasibility. J Intern Med. 1993;233:45-51.

2. Nilsson PM, Engstrom G, Hedblad B. The metabolic syndrome and incidence of cardiovascular disease in non-diabetic subjects--a population-based study comparing three different definitions. Diabet Med. 2007;24:464-472.

3. Gainer JV, Bellamine A, Dawson EP, Womble KE, Grant SW, Wang Y, Cupples LA, Guo CY, Demissie S, O'Donnell CJ, Brown NJ, Waterman MR, Capdevila JH. Functional variant of CYP4A11 20-hydroxyeicosatetraenoic acid synthase is associated with essential hypertension. Circulation. 2005;111:63-69.

4. Mayer B, Lieb W, Gotz A, Konig IR, Aherrahrou Z, Thiemig A, Holmer S, Hengstenberg C, Doering A, Loewel H, Hense HW, Schunkert H, Erdmann J. Association of the T8590C polymorphism of CYP4A11 with hypertension in the MONICA Augsburg echocardiographic substudy. Hypertension. 2005;46:766-771.

5. Mayer B, Lieb W, Gotz A, Konig IR, Kauschen LF, Linsel-Nitschke P, Pomarino A, Holmer S, Hengstenberg C, Doering A, Loewel H, Hense HW, Ziegler A, Erdmann J, Schunkert H. Association of a functional polymorphism in the CYP4A11 gene with systolic blood pressure in survivors of myocardial infarction. J Hypertens. 2006;24:1965-1970.

6. Stec DE, Roman RJ, Flasch A, Rieder MJ. Functional polymorphism in human CYP4F2 decreases 20-HETE production. Physiol Genomics. 2007;30:74–81.

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Online table S1. Linear and logistic regression analysis of systolic, diastolic blood pressure and hypertension prevalence

Variables / Non adjusted Systolic BP / Non adjusted Diastolic BP / Hypertension prevalence
Regression
Coefficient
(95%C.I) / P / Regression
Coefficient
(95%C.I) / P / O.R.
(95%C.I) / P
Gender † / -2.76 (-3.67/-1.85) / <0.001 / -3.06 (-3.52/-2.59) / <0.001 / 0.54 (0.46/0.64) / <0.001
Age, year / 0.99 (0.91/1.06) / <0.001 / 0.16 (0.12/0.2) / <0.001 / 1.12 (1.10/1.14) / <0.001
BMI, Kg/m2 / 0.94 (0.82/1.05) / <0.001 / 0.57 (0.52/0.63) / <0.001 / 1.20 (1.17/1.23) / <0.001
CYP4A11 F434S‡ / 3.47 (0.16/6.78) / 0.04 / 1.49 (-0.2/3.17) / 0.084 / 2.015 (1.07/3.78) / 0.029
Model / <0.001 / <0.001 / <0.001

BMI, body mass index; C.I., confidence interval.

* N.B. Seven subjects were not included in the analysis due to missing BMI.

† male sex is coded as 1 and female sex as 2.

‡ For the CYP4A11 F434 S polymorphism subjects carrying at least one F-allele are coded as 0 and S434S homozygotes as 1.

§ The statistical variables used for the interaction terms (CYP4A11 F434S‡x SEX§, CYP4A11 F434S‡x BMI, CYP4A11 F434S‡x AGE) have been computed by multiplying the CYP4A11 F434S genotype respectively with sex, BMI and age.

The Interaction terms CYP4A11 F434S‡ x SEX§, CYP4A11 F434S‡ x BMI§ and CYP4A11 F434S‡ x AGE§ were discarded from the regression model because not significant.

Online table S2. Linear and logistic regression analysis of systolic, diastolic blood pressure and hypertension prevalence

Variables / Non adjusted Systolic BP / Non adjusted Diastolic BP / Hypertension prevalence
Regression
Coefficient
(95%C.I) / P / Regression
Coefficient
(95%C.I) / P / O.R.
(95%C.I) / P
Gender † / -1.66 (-2.9/-0.41) / 0.009 / -7.8 (-3.2/-1.9) / 0.009 / 0.80 (0.68/0.93) / 0.006
Age, year / 0.99 (0.92/1.07) / <0.001 / 0.16 (0.12/0.2) / <0.001 / 1.087 (1.08/1.10) / <0.001
BMI, Kg/m2 / 0.94 (0.82/1.05) / <0.001 / 0.56 (0.50/0.62) / <0.001 / 1.13 (1.11/1.15) / <0.001
CYP4F2 V433M ‡ / 4.0 (0.96/7.04) / 0.01 / 1.85 (0.31/3.4) / 0.019 / 0.86 (0.74/0.99) / 0.039
CYP4F2 V433M ‡x SEX§ / -2.24 (-4.08/-0.39) / 0.017 / -1.08 (-2.02/-0.14) / 0.024 / 1.33 (1.06/1.68) / 0.016
Model / <0.001 / <0.001 / <0.001

BMI, body mass index; C.I., confidence interval.

* N.B. Seven subjects were not included in the analysis due to missing BMI.

† male sex is coded as 1 and female sex as 2.

‡ For the CYP4F2 V433M polymorphism “wild type” subjects carrying two V-alleles are coded as 0 and M-carriers as 1.

§ The statistical variables used for the interaction terms (CYP4F2 V433M ‡x SEX§, CYP4F2 V433M ‡x BMI, CYP4F2 V433M ‡x AGE) have been computed by multiplying the CYP4F2 V433M genotype respectively with sex, BMI and age.

The Interaction terms CYP4F2 V433M ‡x BMI§ AND CYP4F2 V433M ‡x AGE§ were discarded from the regression model because not significant.