Use of Genomic Profiling to Assess Risk for Cardiovascular Disease

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Appendix 1:

Detailed information for each gene/polymorphism/SNP included in this review and its relationship with Coronary Heart Disease (CHD) and Stroke

Gene abbreviation (aka)

APOE

CBS (HIP4)

CETP (HDLCQ10 )

CYBA (p22-PHOX)

Gene Name Appendix Page

ACE (ACE D , ACE-1) Angiotensin-converting enzyme D ……………………. 2

AGT (serpin peptidase inhibitor) Ang iotensinogen………………………………………….. 5

AGTR1

APOB

APOC3 ( APOC-III )

Angiotensin II receptor, type 1 …………………………. 7

Apolipoprotein B …………………………………………… 9

Apolipoprotein C-III ………………………………………. 11

Apolipoprotein E ....................................................... 16

Cystathionine-beta-synthase ………………………….. 19

Cholesteryl ester transfer protein ……………………… 20

Cytochrome b-245, alpha polypeptide …………………. 23

CYP11B2

F2

F5

( FII, prothrombin)

(FV, FVL, PCCF)

GNB3 (beta polypeptide 3)

GPX1

IL1B (IL-1, IL1F2, IL1-BETA)

Cytochrome P450, family 11, subfamily B, polypeptide 2 .. 24

Coagulation factor II ……………………………………….. 25

Coagulation factor V …………………………………… 28

Guanine nucleotide binding protein ……………………… 31

Glutathione peroxidase 1 ………………………………. 34

Interleukin 1, beta ………………………………………... 36

Interleukin 6 ………………………………………………… 38 IL6 ( HGF, IL-6, IFNB2 )

ITGB3 (

MTHFR

GP3a, CD61

LPL (KID, HDLCQ11

)

)

Integrin, beta 3 ……………………………………………. 41

Lipoprotein lipase ……………………………………….. 44

5,10-methylenetetrahydrofolate reductase ……………... 48

5-methyltetrahydrofolate-homocysteine methyltransferase

… 53 MTR (MS )

MTRR (MSR ) 5-MTR reductase ……………………………………….. 56

NOS3 (eNOS, ECNOS, NOS III ) Nitric oxide synthase 3 (endothelial cell) ……………… 58

PAI-1 (SERPINE, SERBP1, PAI) Plasminogen activator inhibitor type 1 …………………. 61

PON1 (ESA, PON) Paraoxonase 1 ……………………………………………. 65

SELE (ELAM, ESEL, CD62E)

SOD2 (MNSOD, Mn-SOD

SOD3(EC-SOD)

9p21 SNPs

References

)

TNF (DIF, TNFA, TNF-alpha )

Selectin E ………………………………………………… 68

Superoxide dismutase 2

………………………………. 70

Superoxide dismutase 3 ………………………………. 71

Tumor necrosis factor

…………………………………… 72

……………………………………………………………….. 75

……………………………………………………..………… 77

Appendix 1: Genomic Panels and CVD EGAPP - Confidential 1

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ACE (angiotensin-converting enzyme D)

The ACE gene (aka ACE D , ACE-1 ) is located on chromosome 17 (17q23.3). According to

NCBI Entrez Gene, “This gene encodes an enzyme involved in catalyzing the conversion of angiotensin I into a physiologically active peptide angiotensin II. Angiotensin II is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluidelectrolyte balance. This enzyme plays a key role in the renin-angiotensin system. Studies have associated the presence [Insertion] or absence [Deletion] of a 287 bp Alu repeat element in this gene with the levels of circulating enzyme or cardiovascular pathophysiologies. The two most abundant alternatively spliced variants of this gene encode two isozymes - the somatic form and the testicular form that are equally active .”

Literature search

A HuGE Navigator (V1.1) search for the ACE gene identified 1,161 articles on 343 disease terms. A search for meta-analyses ( ACE and Meta-analysis[Text+MeSH)>>Brain Ischemia,

Cardiovascular disease, unspecified, Apoplexy, Coronary heart disease, Congestive heart failure, Myocardial Infarction, Myocardial ischemia, Subarachnoid Hemorrhage[MeSH]) identified eight articles. Four studies were excluded from analysis due to a non-Caucasian population (Asian, non-European), or because they focused on diseases that were outside of our categorization areas (premature CHD, cardiac function).

1-4 The remaining four articles are summarized below.

Genotype frequencies

For the ACE gene insertion/deletion polymorphism, I represents the presence of the sequence (insertion) while D indicates the deletion. The at-risk genotype is homozygous for the deletion (DD). In a general Caucasian population, genotype frequencies for the wild (II) and heterozygous (ID) combined is 73%, while homozygotes (DD) represent the remaining

27%. Under Hardy-Weinberg, the three genotypes separately would be approximately 23%,

50% and 27%, respectively.

5

Coronary Heart Disease (Primary Myocardial Infarction)

A meta-analysis from 2003 5 provided a systematic review of published studies, abstracts and letters regarding the relationship between ACE (and two other markers) and primary myocardial infarction (MI). Studies that were family based, reported on recurrent disease or focused on subgroups ( e.g., pregnant women, diabetics) were also excluded. Analytic methods included random effects modeling, formal tests for heterogeneity, and identification of possible publication bias. Overall, 43 studies were included in the analysis. The summary odds ratio for the recessive model (DD vs. II+ID) was 1.22 (95% CI 1.11 to 1.34), p<0.001. The total number of subjects was 53,942. However, there was high heterogeneity

(Q = 107, p<0.001, I 2 = 61%) and an obvious trend for smaller studies to show a larger effect.

This indicates a possible publication bias. The summary ORs for small, median and large studies are 1.49, 1.15 and 1.07, respectively.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘ A ’ (size: over 11,000 cases/controls with a DD genotype), ‘ C ’ (replication: I 2 of 61% indicating considerable he terogeneity), and ‘ ‘ (this indicates that the bias component of the Venice criteria was not evaluated because at least


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120 one previous component had already been assigned a grade of C). There is, however, clear indication of bias related to sample size.

Using a large study analysis, as described in methods, a revised OR estimate was generated by reanalyzing only the seven studies with 500 study subjects (Figure B.1). The revised summary OR (DD vs. II+ID is 1.04, (95% CI 0.95 to 1.15), p=0.4. There was moderate heterogeneity (Q = 9.7, I 2 = 38%).

CVD and 9p21 (homozygotes vs heterozygotes)

Model Study name

Random

Fujimura 1997

Pfohl 1998

Lindpaintner 1995

Kee 2000

Agerholm-Larsen 1997

Gardemann (A) 1995

Keavney 2000

Statistics for each study

Odds Lower Upper ratio limit

0.73

1.15

1.09

0.92

1.00

1.19

1.09

1.04

0.53

0.85

0.86

0.74

0.81

0.99

1.00

0.95

limit

1.00

1.55

1.38

1.14

1.22

1.43

1.19

1.15

p-Value MI n / Total

Not

MI

0.05

75 / 635 99 / 635

0.36

157 / 511 96 / 345

0.49

126 / 387 453 / 1475

0.45

217 / 861 215 / 801

0.98

129 / 494 2409 / 9203

0.06 328 / 1066 326 / 1201

0.05 1359 / 4629 1637 / 5934

0.39

Odds ratio and 95% CI

0.5

1 2

Favours A Favours B

Figure B.1 Summary analysis of the ACE I/D polymorphism and myocardial infarction

(MI), after restriction to seven large studies

Meta Analysis

Coronary Heart Disease (Ischemic Heart Failure)

A meta-analysis from 2007 7 provided a systematic review of published studies regarding the relationship between ACE and ischemic heart failure (IHF). Inclusion criteria included a clinical diagnosis of IHF and controls free of heart failure as well as validated molecular methods. Studies of progress, response to treatment or survival were excluded along with family based studies, case reports and previous reviews. Analytic methods included random effects modeling and formal tests for heterogeneity. Overall, five studies were included in the analysis including three in Caucasians and two in East Asians. A total of 372 cases and 719 controls were included. The summary odds ratio for the recessive model

(DD vs. II+ID) was 0.95 (95% CI 0.60 to 1.52), p = NS. There was moderate to high heterogeneity (I 2 = 51%, p = 0.09).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘C’ (size: under 500 cases/controls with a DD genotype); ‘C’ (replication: I 2 of 51% indicates the possibility of considerable heterogeneity even though it is not statistically significant); and ‘-‘ (bias: not evaluated due to at least one already assigned grade of C). Given the consistency of these findings with the previous one for MI (Figure B.1), we have chosen to report only the 2003 5 meta-analysis for MI and ACE in the evidence manuscript.


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Stroke (Ischaemic Stroke)

A meta-analysis from 2004 8 provides a systematic review of published studies, letters and abstracts, focusing on ischemic stroke. Exclusion criteria included those studies focused on children, intermediate genotype, no frequencies, duplicate publications. Analytic methods included random effects modeling, formal tests for heterogeneity, and identification of possible publication bias. Overall, 11 studies were included in the analysis with 2,990 cases and 11,305 controls. The summary odds ratio for the recessive model (DD vs. II+ID) was

1.21 (95% CI 1.08 to 1.35, p < 0.001). There was low heterogeneity (Q = 11, I 2 = 10%, p =

0.9).

Applying the Venice criteria results in a credibility grade of ‘A’ (size: over 11,000 cases/controls with a DD genotype); ‘A’ (replication: I 2 of 10% indicating homogeneity), and

‘B‘ (bias: none obvious, but considerable missing information).

Large study analysis -- The five studies with 500 or more cases resulted in a summary OR

(for DD vs. II+ID) of 1.16, (95% CI 1.03 to 1.32); a significant finding (p = 0.02). These results were also homogeneous (Q = 2.1, I 2 = 0%, p = 0.7). Figure B.2 shows this analysis.

Stroke and ACE I/D

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Model Study name Statistics for each study

Random

Odds Lower Upper ratio limit limit Z-Value p-Value

Agerholm-Larsen 1997 1.150

0.934

1.417

Catto 1996

Szolonoki 2002

Ueda 1995

Zee 1999

0.942

1.287

0.654

1.004

1.357

1.650

1.261

0.845

1.883

1.123

0.815

1.546

1.162

1.025

1.319

1.315

-0.321

1.994

1.135

0.709

2.337

0.189

0.748

0.046

0.256

0.478

0.019


0.5

1 2

DD is protective DD is at risk

Meta Analysis

to large studies

Stroke (Subarachnoid Hemorrhage)

A 2004 article 9 reported results for 90 case and 128 control individuals. The odds ratio (DD vs. II+ID) was 0.88 (95% CI 0.46 to 1.68), p = NS. This article was published after the metaanalysis for ischemic stroke 8 and would have slightly reduced their odds ratio of 1.21.

However, it is a small study and would have no impact on our summary estimate from large studies of 1.16.


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AGT (angiotensinogen)

The AGT gene (aka serpin peptidase inhibitor, serpine-1) is located on chromosome 1q42-q43.

The most widely studied polymorphism is M235T. According to NCBI Entrez Gene, “the protein encoded by this gene, pre-angiotensinogen or angiotensinogen precursor, is expressed in the liver and is cleaved by the enzyme renin in response to lowered blood pressure. The resulting product, angiotensin I is then cleaved by angiotensin converting enzyme (ACE) to generate the physiologically active enzyme angiotensin II. The protein is involved in maintaining blood pressure and in the pathogenesis of essential hypertension and preeclampsia.

”

Literature search

A HuGE Navigator (V1.1) search ( AGT and Meta-analysis[Text+MeSH)>> Coronary heart disease, Congestive heart failure, Myocardial Infarction, Myocardial ischemia[MeSH]) identified four articles.

7,10-12 A 2008 meta-analysis 10 was selected as being the most recent, and the most complete.


The at-risk genotype is TT. In a general Caucasian population, genotype frequencies for the wild (MM) and heterozygous (MT) combined is 57%, while homozygotes (TT) represent the remaining 43%. Under Hardy-Weinberg, the three genotypes separately would be approximately 32%, 50% and 18%, respectively.

5

Coronary Heart Disease (Myocardial Infarction)

A 2007 meta-analysis 11 provided a review of the AGT polymorphism M235T (T allele) and

CHD. The authors excluded studies of recurrent events, case-only studies, insufficient data to create confidence intervals, and studies containing duplicate data. Analytic methods include fixed effects modeling, formal tests for heterogeneity, stratified analyses and identification of possible publication bias. Overall, 38 studies were included in the analysis and for a total of 32,005 subjects. The summary odds ratio for the model MT vs. MM was

1.02 (95% CI 0.91 to 1.14), p=0.7 and for the model TT vs. MM was 1.15 (95% CI 1.00 to

1.32), p = 0.05. However, there was significant heterogeneity (Q = 76, p < 0.001, I 2 = 51% for the former and Q = 79, p < 0.001, I 2 = 53% for the latter). The majority of heterogeneity was explained by study size, with small studies showing larger effects.

Venice criteria 6 for evaluating the credibility of genetic association meta-analysis were applied and resulted in the three grades of ‘ A ’ (size: over 8,000 cases/controls with a TT genotype), ‘ C ’ (replication: I 2 of 56 % indicating considerable heterogeneity), and ‘ ‘ (bias: not evaluated due to at least one already assigned grade of C).

A 2008 meta-analysis examined the M235T polymorphism of AGT and MI.

10 Analytic methods included random effects modeling, formal tests for heterogeneity and the evaluation of potential publication bias. There were 38 studies included in this metaanalysis; however, only 25 studies were conducted in Caucasian populations for a total of

26,489 study participants. The summary odds ratio for the model MT vs. MM was 1.03

(95% CI 0.94 to 1.14), p = 0.5 and for the model TT vs. MM was 1.19 (95% CI 1.02 to 1.38), p = 0.02. However, there was significant heterogeneity (Q = 32, p = 0.1, I 2 = 25% for the former and Q = 60, p < 0.001, I 2 = 60% for the latter). Some of the heterogeneity was


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The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘A’ (size: over 4,700 cases/controls with a TT genotype in the Caucasian studies), ‘C’ (replication: I 2 of 25% - 60% indicating considerable heterogeneity), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis -- When the analysis was restricted to the seven studies with 500 or more cases of MI 10 , the summary OR for the MT vs. MM comparison (Figure B.3) was reduced to 1.00 (95% CI 0.92 to 1.08), p = NS. The heterogeneity was low (Q = 3.3, I 2 =

0%, p = 0.8). The summary OR for the TT vs. MM comparison (Figure B.4) was reduced to

AGT Failsafe N Analysis: MT vs MM for MI

Odds ratio and 95% CI Model

Random

Study name Statistics for each study

Odds Lower Upper ratio limit limit

Katsuya 1996

Tiret 1995

1.055

0.912

Gardemann 1999 1.167

Olivieri 2001 0.899

Sethi 2001

Tobin 2004

Renner 2005

0.982

1.036

0.951

0.996

0.779

0.721

0.934

0.627

0.846

0.795

0.789

0.917

1.429

1.152

1.459

1.290

1.140

1.350

1.146

1.082

p-Value

0.730

0.438

0.174

0.564

0.811

0.792

0.599

0.927

0.1 0.2

0.5

1 2

MT is protectiv e

5

Mt is at-risk

10

restriction to the seven large studies.

AGT Failsafe N Analysis: TT vs MM for MI

Model

Random


Odds Lower Upper ratio limit limit p-Value

Katsuya 1996 1.689

Tiret 1995 1.015


Olivieri 2001

Sethi 2001

Tobin 2004

Renner 2005

0.886

0.937

0.941

1.095

1.025

1.135

0.734

0.692

0.577

0.764

0.655

0.864

0.897

2.514

1.403

1.234

1.359

1.150

1.350

1.386

1.171

0.010

0.928

0.592

0.579

0.534

0.740

0.453

0.717


0.1 0.2

0.5

1 2 5 10

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Meta Analysis


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AGTR1 (angiotensin II receptor, type 1)

The AGTR1 gene (aka AT1R ) is located on chromosome 1 (3q21-q25). The most widely studied polymorphism is A1166C. According to NCBI Entrez Gene, “Angiotensin II is a potent vasopressor hormone and a primary regulator of aldosterone secretion. It is an important effector controlling blood pressure and volume in the cardiovascular system. It acts through at least two types of receptors. This gene encodes the type 1 receptor which is thought to mediate the major cardiovascular effects of angiotensin II. This gene may play role in the generation of reperfusion arrhythmias following restoration of blood flow to ischemic or infarcted myocardium. It was previously thought that a related gene, denoted as

AGTR1B , existed; however, it is now believed that there is only one type 1 receptor gene in humans. At least five transcript variants have been described for this gene. Additional variants have been described but their full-length nature has not been determined.

”

Literature search

A HuGE Navigator (V1.1) search ( AGTR1 and Clinical Trial, HuGE Review, Metaanalysis[StudyType)>>Myocardial Infarction) identified one article 13 which is summarized below.


The at-risk genotype is CC. In a general Caucasian population, genotype frequencies for the wild (AA) and heterozygous (AC) combined is 72%, while homozygotes (CC) represent the remaining 28%. Under Hardy-Weinberg, the three genotypes separately would be approximately 52%, 40% and 8%, respectively.

5


A 2007 HuGE review 13 systematically reviewed the literature relating the AGTR1 A1166C polymorphism (C is the risk allele) to MI. Analytic methods include random effects modeling, formal tests for heterogeneity, stratified analyses and identification of possible publication bias. 20 studies reported effect measures for the recessive model in European Caucasians.

A total of 24,331 study participants were included. The summary OR for the recessive model (CC vs. AC+AA) was 1.32 (95% CI 1.10 to 1.59), p = NR. Heterogeneity was high (Q

= 46, I 2 = 56%, p=NR). 21 studies reported effect measures for the dominant model in

European Caucasians. A total of 25,388 study participants were included. The summary

OR for the dominant model was 1.11 (95% CI 1.01 to 1.21), p = NR. Heterogeneity was moderate (Q = 37, I 2 = 44%, p = NR).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘A’ (size: estimated at least 6,750 cases/controls with a CC genotype using 28% genotype frequency), ‘C’ (replication: I 2 of

44% and 56% indicating considerable heterogeneity), and ‘-‘ (bias: not evaluated due to at least one already assigned grade of C). According to the original analysis 13 there is a clear indication that bias related to sample size and other sources of bias (such as publication bias) contribute to the between-study heterogeneity in this meta-analysis.


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Large study analysis (Figure B.5) – Raw numbers were not available from the metaanalysis, so the point estimate and 95% confidence intervals were used for the three studies with the smallest confidence intervals, corresponding to studies having approximately 500 cases, the allele-specific OR (*C allele) was 0.98, (95% CI 0.93 to 1.01), p = NS.


Random

Gardemann 1998

Kee 2000

Keavney 2000


Point Lower Upper estimate limit limit

1.040

0.905

1.175

1.030

0.875

1.185

0.960

0.910

1.010

0.975

0.930

1.020

Point estimate and 95% CI

-2.00

-1.00

0.00

1.00

2.00

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three large studies.

Meta Analysis


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APOB (apolipoprotein B)

The APOB gene is located on chromosome 2 (2p24-p23). Polymorphisms are Xbal, signal peptide, and EcoR1. According to NCBI Entrez Gene, “this gene product is the main apolipoprotein of chylomicrons and low density lipoproteins. It occurs in plasma as two main isoforms, apoB-48 and apoB-100: the former is synthesized exclusively in the gut and the latter in the liver. The intestinal and the hepatic forms of apoB are encoded by a single gene from a single, very long mRNA. The two isoforms share a common N-terminal sequence.

The shorter apoB-48 protein is produced after RNA editing of the apoB-100 transcript at residue 2180 (CAA->UAA), resulting in the creation of a stop codon, and early translation termination.

”

Literature search

A HuGE Navigator (V1.1) query ( APOB [Text+MeSH]>>Meta-analysis, HuGE

Review[StudyType] identified two summary analyses, both performed in 2003.

14,15 We chose the Chiodini et al., analysis 15 , as it included heterozygotes in their odds ratios.

Boekholdt et al 14 , computed ORs for only the wild and homozygous groups (heterozygotes were ignored).




For the polymorphism Xbal (C2488T), the at-risk genotype is TT. In a European

Caucasian population in the US, the C allele frequency is about 0.51.

16 Under Hardy-

Weinberg, the three genotypes separately would be approximately 42%, 46% and 12%, respectively.



For the polymorphism EcoRI (G4154A), the at-risk genotype is AA. In a European

Caucasian population in the US, the G allele frequency is about 0.82.

16 Under Hardy-

Weinberg, the three genotypes separately would be approximately 70%, 27% and 3%, respectively.



For the signal peptide insertion/deletion (Sp Ins/Del), the at-risk genotype is DD. In a

European Caucasian population in the US, the I allele frequency is about 0.67.

16 Under

Hardy-Weinberg, the three genotypes separately would be approximately 47%, 43% and

10%, respectively.


A 2003 HuGE review 15 systematically reviewed the literature relating APOB to MI/CAD.

Analytic methods include random effects modeling, formal tests for heterogeneity, stratified analyses and identification of possible publication bias.



For the Xbal polymorphism, 19 studies (including various races) were included for the recessive model and 20 studies were included for the dominant model. The summary odds ratio for the recessive model was 1.19 (95% CI 1.01 to 1.39), p = 0.03, and for the dominant model was 1.14 (95% CI 0.88 to1.48), p = 0.3. However, there was moderate heterogeneity (I 2 = 29%) for the former and significant heterogeneity for the latter (I 2 =

49%). Only one included study reported on more than 500 cases.



For the EcoRI polymorphism, 14 studies (including various races) were included. The summary odds ratio for the recessive model was 1.73 (95% CI 1.19 to 2.50), p=0.004 and for the dominant model was 1.32 (95% CI 1.14 to 1.54), p < 0.001 with low heterogeneity (I 2 = 0% and I 2 = 13%, respectively). None of the included studies reported on 500 or more cases.


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

For the polymorphism Sp Ins/Del, 22 studies (including various races) were included.

The summary odds ratio for the recessive model was 1.19 (95% CI 1.05 to 1.35), p =

0.006 and for the dominant model was 1.15 (95% CI 1.06 to 1.24), p < 0.001 with modest evidence of heterogeneity (I 2 = 23% and I 2 = 12%, respectively). Only one included study reported on 500 or more cases.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for



Xbal: ‘A’ (size: approximately 1,104 cases with a TT genotype (2,628 cases * 0.42 reported frequency of TT in the analysis), ‘B’ (replication: I 2 of between 29% and 49%, depending on model, indicating considerable heterogeneity), and ‘C‘ (bias: the authors reported clear indication of bias related to sample size.



EcoRI: ‘C’ (size: approximately 46 cases with an AA genotype (1,721 * 0.027), ‘A’

(replication: I 2 of 0% indicating a low level of heterogeneity) and ‘-‘ (no value assigned given a grade of C in a previous category).



Ins/Del: ‘B’ (size: approximately 595 cases with a DD genotype (6,007 * 9.9%), ‘A’

(replication: I 2 of 0% indicating a low level of heterogeneity), ‘B‘ (bias: none obvious, but considerable missing information).


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APOC3 (apolipoprotein C-III)

The APOC3 gene (aka APOCIII, APOC-III ) is located on chromosome 11 (11q23.1-q23.2). The most widely studied polymorphism is C3238G, but several others have also been studied.

According to NCBI Entrez Gene, “Apolipoprotein C-III is a very low density lipoprotein

(VLDL) protein. APOC3 inhibits lipoprotein lipase and hepatic lipase; it is thought to delay catabolism of triglyceride-rich particles. The APOA1 , APOC3 and APOA4 genes are closely linked in both rat and human genomes. The A-I and A-IV genes are transcribed from the same strand, while the A-1 and C-III genes are convergently transcribed. An increase in

APOC3 levels induces the development of hypertriglyceridemia.

”

Literature search

A HuGE Navigator (V1.1) search for APOC3 gene identified 135 articles on 54 disease terms. No meta-analyses were found in the area of CVD. A search

( APOC3 [Text+MeSH]>>Arterial Sclerosis, Atherosclerosis, Cardiovascular disease, unspecified, coronary artery disease, coronary artery disease, Coronary heart disease,

Vascular Diseases[Mesh]) identified 40 articles; none of which were summary articles. Of these, the majority looked at intermediate outcomes (e.g., lipid measurements) or studied subpopulations (e.g., diabetics). Ten studies remained.

17-26 Four additional studies were examined after searching references.

27-30 After limiting studies to the Caucasian population with a primary CVD outcome, nine studies remained and are summarized below.




For the polymorphism Sst-1 (also referred to as the C3238G variant), the at-risk allele is

S2. In a US population of 371 healthy predominately Caucasian controls, the S1 (wild) allele frequency is 0.90 and the S2 (at-risk) allele is 0.10.

27 The reported genotype frequencies were 80%, 19% and 1%, for S1/S1, S1/S2 and S2/S2, respectively. Under

Hardy-Weinberg, the three genotypes would be approximately 81%, 18% and 1%, respectively. Because of the low prevalence of the S2/S2 genotype, the usual grouping is S1/S2 + S2/S2 vs. S1/S1. In some publications, these genotypes would be reported as CC, CG and GG, respectively.



For the T455C polymorphism, the at-risk allele is C. In a cohort study of 505 European

Caucasians (English), the T allele frequency is 0.65 and the C allele is 0.35.

28 The reported genotype frequencies were 42%, 46% and 12%, for TT, TC and CC, respectively. Under Hardy-Weinberg, the three genotypes are expected to be 42%, 45% and 12%, respectively.



For the C482T polymorphism, the at-risk allele is T. In a cohort study of 505 European

Caucasians (English), the C allele frequency is 0.75 and the T allele is 0.25.

28 The reported genotype frequencies were 56%, 38% and 6%, for CC, CT and TT, respectively. Under Hardy-Weinberg, the three genotypes are expected to be 56%, 38% and 6%, respectively.

Coronary Heart Disease



S1S2: Seven studies 24-30 were available for analysis using the recessive model (S1S2 +

S2S2 vs. S1S1). Total numbers included are 2,541 cases and 6,316 controls. None of the individual studies found a significant effect. Figure B.6 shows that the summary OR is 1.00 (95% CI 0.89 to 1.13), p = NS. The heterogeneity is low (Q = 5.8, I 2 = 0%, p =

0.4. Similar results were found for the S1S1 + S1S2 vs. S2S2 model (data not shown).


392




Random

Russo 2001 0.756

0.497

1.149

0.190

Olivieri 2002 1.241

0.822

1.874

0.304

Izar 2003

Liu 2004

0.691

1.133

0.377

0.796

1.264

1.613

0.230

0.488

Tobin 2004 0.986

0.712

1.365

0.930

Relvas 2005 0.572

0.191

1.717

0.319

Ruiz 2005 1.034

0.874

1.224

0.695

1.004

0.889

1.133

0.951


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0.5

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Meta Analysis

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the APOC3 Sst1 polymorphism results in three grades: ‘B’

(size: approximately 500 cases with a TT genotype (2,541 cases * 20% reported genotype frequency for S2+ in the analysis), ‘A’ (replication: I 2 of between 29% and

49%, depending on model, indicating considerable heterogeneity), and ‘B ‘ (bias: none obvious, but considerable missing information).

Large study analysis (Figure B.7) – Five of the studies included 500 or more subjects.

The large study OR is 1.03 (95% CI 0.91 to 1.16), p = NS. Heterogeneity was low (Q =

3.3, I 2


Model Study name Subgroup within study

Random

Russo 2001 CHD

Olivieri 2002 CHD

Liu 2004 MI

Tobin 2004 MI

Ruiz 2005 MI



0.756

0.497

1.149

0.190

1.241

0.822

1.874

0.304

1.133

0.796

1.613

0.488

0.975

0.704

1.349

0.878

1.034

0.874

1.224

0.695

1.026

0.905

1.162

0.690


0.5

1 2

Favours B restriction to the five large studies.

Meta Analysis


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

T455C: Four studies 21,25,28,29 were available for the TC vs. TT (Figure B.8) and CC vs.

TT (Figure B.9) comparisons.

The summary OR for the heterozygous comparison (TC vs. TT) was 1.07 (95% CI 0.91 to 1.25), p= 0.4. The results showed modest heterogeneity (Q = 4.3, I 2 = 30%, p = 0.3).

Model Study name Statistics for each study TC / Total

Odds Lower Upper ratio limit limit p-Value CAD

Not

CAD

Random

Olivieri 2002 1.216

0.883

1.674

Olivieri 2003 0.889

0.623

1.267

Tobin 2004 1.258

0.972

1.628

Ruiz 2005 0.964

0.796

1.166

1.067

0.909

1.253

0.232 253 / 447 118 / 228

0.515 158 / 303 114 / 207

0.081 284 / 495 229 / 443

0.704 524 / 881 527 / 873

0.430


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1 2


Figure B.8. Analysis of four APOC3 studies of the T455C polymorphism (TC vs. TT) for CHD/MI

Meta Analysis

The summary OR for the homozygous comparison (CC vs. TT) was 1.32 (95% CI 0.82 to 2.12), p = 0.3. The results showed high heterogeneity (Q = 16.3, I 2 = 82%, p = 0.001).

Model Study name Statistics for each study CC / Total


Not

CAD

Random

Olivieri 2002 2.515

1.511

4.185

Olivieri 2003 1.745

1.030

2.956

Tobin 2004 0.851

0.562

1.288

Ruiz 2005 0.930

0.727

1.191

1.319

0.821

2.119

0.000 102 / 296 23 / 133

0.039 68 / 213 25 / 118

0.445 52 / 263 62 / 276

0.567 192 / 549 200 / 546

0.253


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0.2

0.5

1 2 5 10


Figure B.9. Analysis of four APOC3 studies of the T455C polymorphism (CC vs. TT) for CHD/MI

Meta Analysis

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the APOC3 T455C polymorphism results in three grades: ‘B’

(size: approximately 300 cases with a TT genotype (2,541 cases * 12% reported genotype frequency for TT in the analysis), ‘C’ (replication: I 2 values of 30% and 82%, depending on model, indicating considerable heterogeneity), and ‘-‘ (bias: no value assigned given a grade of C in a previous category).


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Large study analysis – For both comparisons, all four studies are considered large, so the same summary OR’s and measures of heterogeneity apply.



C482T: Three studies 19,28,29 were available for the TC vs. TT (Figure B.8) and CC vs. TT

(Figure B.10) comparisons.

The summary OR for the heterozygous comparison (CT vs. CC) was 0.99 (95% CI 0.85 to 1.15), p = 0.9. The results showed modest heterogeneity (Q = 2.5, I 2 = 21%, p = 0.3).

For this analysis, 1,814 cases and 1,800 controls were availble for study.


Odds ratio and 95% CI Model Study name Statistics for each study CT / Total


Not

CAD

Random

Tobin 2004 1.174

0.913

1.510

0.211 233 / 524 193 / 476

Ruiz 2005 0.937

0.776

1.131

0.497 516 / 898 522 / 884

Dallongeville 2006 0.901

0.683

1.189

0.463 155 / 392 185 / 440

0.992

0.852

1.154

0.912

0.5

1 2

Figure B.10

Analysis of three APOC3 studies of the C482T polymorphism (CT vs.

CC) for CHD/MI

The summary OR for the homozygous comparison (TT vs. CC) was 0.91 (95% CI 0.74 to 1.12), p = 0.4. The results showed low heterogeneity (Q = 1.6, I 2 = 0%, p = 0.4). For this analysis, 1,142 cases and 1,148 controls were availble for study.


Study name Statistics for each study TT / Total


Not

CAD

Tobin 2004

Ruiz 2005

0.771

0.436

1.365

0.373 23 / 314 29 / 312

0.877

0.682

1.128

0.306 174 / 556 188 / 550

Dallongeville 2006 1.215

0.726

2.033

0.459 35 / 272 31 / 286

0.910

0.738

1.123

0.379


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1 2

Figure B.11

Analysis of three APOC3 studies of the C482T polymorphism (TT vs.

CC) for CHD/MI

Meta Analysis

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the APOC3 C482T polymorphism results in three grades: ‘B’

(size: approximately 120 cases with a TT genotype (2,046 cases * 6% reported


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478 genotype frequency for TT in the analysis), ‘A’ (replication: I 2 values of 0% and 21%, depending on comparison, indicating low heterogeneity), and ‘B‘ (bias: none obvious, but considerable missing information).

Large study analysis – For both comparisons, all three studies are considered large, so the same summary OR’s and measures of heterogeneity apply.

Stroke

A HuGE Navigator (V1.1) search for APOC3 gene (articlesAPOC3[Text+MeSH]>>Arterial

Sclerosis, Atherosclerosis, Brain Ischemia, Apoplexy[Mesh]) identified 5 articles, none of which were relevant. No analyses were performed.


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APOE (apolipoprotein E)

The APOE gene is located on chromosome 19 (19q13.2). The most widely studied polymorphisms are ε2 (C112/C158), ε3 (C112/A158) and ε4 (A112/A158). According to

NCBI Entrez Gene, “[c]hylomicron remnants and very low density lipoprotein (VLDL) remnants are rapidly removed from the circulation by receptor-mediated endocytosis in the liver. Apolipoprotein E, a main apoprotein of the chylomicron, binds to a specific receptor on liver cells and peripheral cells. APOE is essential for the normal catabolism of triglyceriderich lipoprotein constituents. The APOE gene is mapped to chromosome 19 in a cluster with

APOC1 and APOC2 . Defects in apolipoprotein ε results in familial dysbetalipoproteinemia, or type III hyperlipoproteinemia (HLP III), in which increased plasma cholesterol and triglycerides are the consequence of impaired clearance of chylomicron and VLDL remnants.”

Literature search

A HuGE Navigator (V1.1) search ( APOE [Text+MeSH]>>Meta-analysis, Clinical trial, HuGE review[StudyType]>>Brain Ischemia, Cardiovascular disease, unspecified, Cerebral

Hemorrhages, Apoplexy, Coronary heart disease, Intracranial Arteriosclerosis, Intracranial

Hemorrhages, Ischemia, Myocardial Infarction, Apoplexy, Subarachnoid

Hemorrhage[Mesh]). Articles were excluded if they evaluated only intermediate outcomes, examined non-Caucasian populations, were not being published in English or have a more recent study published on the same topic. Five meta-analyses remained.

8,31-34 Two of these 31,34 related to CHD. We chose the analysis by Bennet and colleagues 31 because it was more recent (2007 vs. 2004 for Song and colleagues) and it was restricted to only large studies. Among the three meta-analyses of stroke 8,32,33 , we chose the analysis by Sudlow and colleagues 33 as it was larger and more recent. The Lanterna report 32 was a summary of case-only studies.


Allele frequencies in a mainly Caucasian population of controls are 7% for ε2, 82% for ε3, and 11% for ε4.

31 The at-risk genotype includes at least one ε4 allele. In a general

Caucasian population, the wild genotype is ε3/ε3. Under Hardy-Weinberg, the six genotypes (to the nearest 1%) would be approximately 1%, 11%, 2%, 67%, 18% and 1%, for ε2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4, ε4/ε4, respectively. The ε2/* group (ε2/ε2 and ε2/ε3) is about 12%, while the ε4/* group (ε2/ε4, ε3/ε4 and ε4/ε4 is about 21%). The reference group is usually the remaining ε3/ε3.


A meta-analysis from 2007 31 provided a systematic review of published studies of APOE genotypes and MI. Studies with fewer than 1,000 participants cases were excluded, coronary stenosis defined as one or more vessels > 50% occlusion and collected some grey data from the original authors. Analytic methods included random effects modeling, formal tests for heterogeneity and tests for publication bias. Overall 17 studies were included in the analysis. The odds ratio for ε2/* vs. ε3/ε3 is 0.80 (95% CI 0.70 to 0.90). There was significant heterogeneity (I 2 = 72%). The odds ratio for ε4* vs. ε3/ε3 is 1.06 (95% CI 0.99 to

1.13). There was modest heterogeneity (I 2 = 44%). Because of the large sample size the authors were able to show a dose-response relationship from lower to higher odds of


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532 disease. Genotypes ε2/ε2, ε2/ε3, ε2/ε4, had reduced ORs, ε3/ε3 was the referent category with OR=1, and the genotypes ε3/ε4, ε4/ε4 had gradually higher ORs.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘A’ (size: estimated at least 2500 cases/controls with an ε2/* genotype using a 12% genotype frequency), ‘C’ (replication: I 2 of 72% and 44% indicating considerable heterogeneity), and ‘-‘ (bias: not evaluated due to at least one already assigned grade of C).

Although all studies were relatively large, the authors compared smaller versus larger studies and found a strong protective effect of ε2 in large studies that was absent from smaller studies, and v irtually no effect for ε4* in large studies, but a strong and significant effect in small studies (OR = 1.66, 95% CI 1.50 to 1.84). These findings reinforce the replication grade of C.

Stroke (Ischemic Stroke)

A meta-analysis from 2006 33 provided a systematic review of published studies of APOE genotypes and various forms of stroke. Studies were excluded if the disorder was not explicitly stated or if the study focused on recurrent events. Ethnicity/race was not restricted and varied widely; 50% were Caucasian. A total of 4,096 cases and 16,117 controls were included. Analytic methods included the Mantel-Haenszel method of pooling

ORs and examination of results for publication bias. Overall 24 studies were included in the analysis of ischemic stroke.



The OR for the ε4+ vs. ε4- was 1.11 (95% CI 1.01 to 1.22), p = 0.03. There was significant heterogeneity (I 2 = 68%).



The OR for the ε2+ vs. ε2- was 0.99 (0.87 to 1.13), p = NS. There was no report for the heterogeneity.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘A’ (size: over 1,000 ε4+ cases/controls with an

ε4+ genotype), ‘C’ (replication: I 2 of 72% and 44% indicating considerable heterogeneity), and ‘-‘ (bias: not evaluated due to at least one already assigned grade of C). The authors reported smaller and non-significant associations when only studies of 200 or more subjects were used, or when studies with selection bias of controls were removed.

Large study analysis - Seven of the included studies reported results for 500 or more cases/controls for the ε4+/ε4- comparison (Figure B.12). The summary OR is 0.91 (95% CI

0.79 to 1.03), p = NS. The heterogeneity was low (Q = 5.3, I 2 = 0%, p = 0.6).


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Failsafe N Analysis: ApoE (e4+ v other) for Stroke

Model Study name Statistics for each study Odds ratio and 95% CI


Random

Ferrucci

Aalto

Catto

Kokubo

Frikke

Morrison

Um

0.900

0.610

1.328

0.596

0.840

0.594

1.188

0.324

0.820

0.592

1.135

0.232

0.880

0.598

1.295

0.517

1.000

0.745

1.342

1.000

1.200

0.849

1.697

0.303

0.690

0.462

1.031

0.070

0.906

0.794

1.034

0.142

0.5

1

Figure B.12 Summary analysis of the APOE gene (



Meta Analysis

2


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CBS (cystathionine-beta-synthase)

The CBS gene (aka HIP4) is located on chromosome 21 (21q22.3). The most widely studied polymorphisms is c.844ins68. According to NCBI Entrez Gene, “the protein encoded by this gene is involved in the transsulfuration pathway. The first step of this pathway, from homocysteine to cystathionine, is catalyzed by this protein. CBS deficiency can cause homocystinuria which affects many organs and tissues, including the eyes and the skeletal, vascular and central nervous systems.

”

Literature search

A HuGE Navigator (V1.1) search for the CBS gene identified 81 articles on 57 disease terms. Four meta-analyses/HuGE Reviews were found, but none were in the area of CVD

(three on various cancers, and on congenital anomalies). A search

( CBS [Text+MeSH]>>Arterial Sclerosis, Brain Ischemia, Cardiovascular disease, unspecified, Apoplexy, Cerebrovascular Disorders, coronary artery disease, Coronary heart disease, Myocardial Infarction, Myocardial ischemia, Vertebral Artery Dissection[Mesh]) identified 20 articles. Of these, 16 were considered non-relevant, mostly because they focused exclusively on intermediate outcomes. The two remaining studies are summarized below.

35,36


W represents the wild allele, while M represents the presence of the insertion. The at-risk genotype is MM with a prevalence of 2.5%. In a general Caucasian population, allele frequencies are .85 and .15, respectively (based on 591 control individuals.

35 Under Hardy-

Weinberg, the three genotypes separately would be approximately 72%, 25.5% and 2.5%, respectively.

Coronary Heart Disease (Coronary Artery Disease)

A 2003 study 35 reported on 869 Caucasians from the Czech Republic; 278 cases (confirmed

WHO criteria) and 591 control individuals. The OR for the recessive model (MM vs.

WW+WM) was 0.55 (95% 0.36 to 0.88), p = 0.015.


A 2001 study 36 from Atlanta reported on 295 African Americans; 110 cases (65 and younger patients for follow-up of clinically defined MI) and 185 matched controls (outpatients at the same hospital with no history of MI, matched for age sex and race). The odds ratio for recessive model (WW+WM) vs. MM) comparison was 1.10 (95% 0.69 to 1.86), p = 0.6.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘C’ (size: less than 100 in the smallest category), ‘ C ’ (replication: failed replication is subsequent study), and ‘ ‘ (bias: not evaluated because both earlier grades were C).

Large study analysis -- No studies were sufficiently large to perform this analysis.


No studies were found that reported CBS genotypes and stroke outcomes.


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CETP (cholesteryl ester transfer protein)

The CETP gene (aka HDLCQ10) is located on chromosome 16 (16q21). The most widely studied polymorphisms is TaqIB (C629A) .

According to NCBI Entrez Gene, “Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters between lipoproteins. CETP may affect susceptibility to atherosclerosis.

“

Literature search

A HuGE Navigator (V1.1) search for the CETP gene identified 177 articles on 57 disease terms. Two ( CETP [Text+MeSH]>>Clinical trial, Meta-analysis[StudyType]>>Cardiovascular disease, unspecified[Mesh]) identified two articles. One dealt with natural genetic variation and the role of CETP in lipid levels and disease.

14 The remaining meta-analysis is summarized below. To investigate stroke the search ( CETP [Text+MeSH]>>Arterial

Sclerosis, Atherosclerosis, Apoplexy, Apoplexy[Mesh]) identified seven studies, two of which are summarized below.


B1 represents the wild allele, while B2 represents the presence of the variant. The at-risk genotype is B2/B2 with a 17.6% prevalence. In a general Caucasian population, allele frequencies are 0.57 and 0.42, respectively (based on over 10,000 controls individuals.

35

Under Hardy-Weinberg, the three genotypes separately would be approximately 33.6%,

48.7% and 17.6%, respectively.

Coronary Heart Disease (Coronary Artery Disease - CAD)

The 2005 meta-analysis 37 was restricted to studies reporting on 500 or more Caucasian individuals. Patient specific data were available for analysis from the original authors.

Analytic methods included random effects modeling, adjusting for confounding variables, formal tests for heterogeneity and identification of possible publication bias. Overall, seven studies included in the analysis reported results in 2,857 cases and 8,815 controls. It appears that the authors mislabeled Figure 2. The three columns should be B2/B2, B1/B2 and B1/B1 (the first and last column labels seem reversed). The odds ratios were adjusted for age, sex, smoking, diabetes, BMI, blood pressure, LDL and use of alcohol (the odds ratios without adjustment are nearly identical). Among large studies, the adjusted summary

OR for the B1/B2 vs. B1/B1 comparison was 0.96 (95% CI 0.92 to 0.99), p = 0.03.

Unadjusted OR was 0.97 (95% CI 0.88 to 1.06), p = 0.5. The adjusted summary odds ratio for the B2/B2 vs. B1/B1 comparison was 0.82 (95% CI 0.75 to 0.91), p < 0.001. Unadjusted

OR was 0.97 (0.76 to 0.99), p = 0.3. There was significant heterogeneity reported for both summaries, but no statistical results were provided.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of

‘A’

(size: approximately 18% of the 2,857 cases would be B1/B1 of 515 with more than that number of controls). ‘ B ’ (reliability: we reanalyzed the raw data 37 to obtain a reliable estimate of heterogeneity. For both the B1/B2

(Q = 1.5, I 2 = 0%, p = 0.9) and the B2/B2 (Q = 3.7, I 2 = 0%, p = 0.7) comparison to B1/B1, heterogeneity was low. ‘B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis

– The seven studies summarized above were already ‘large’.


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Stroke (Ischemic Stroke )

A 2007 study 38 involved 451 Caucasians in Sardinia. Of these, 215 had an ischemic stroke

(clear clinical parameters persisting for 24+ hours confirmed by CT scan). Controls were unrelated individuals from the same hospital who had no known history of CVD. The study included both males and females. The OR for the recessive model [(B1/B1 + B1/B2) vs.

B2/B2] was 0.53 (95% CI 0.32 to 0.88), p = 0.01. The effect was larger for females than for males. A second study of 98 case and 100 control individuals 39 did not report specific numbers, but concluded that “neither polymorphism examined in this study appears to be significantly associated with ischemic stroke”.

Overall, we conclude that there is low quality evidence that no significant association exists between this CETP polymorphism and stroke.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘C’ (size: less than 100 in the smallest category), ‘ C ’ (replication: failed replication is subsequent study), and ‘ ‘ (bias: not evaluated because both earlier grades were C.



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CYBA (cytochrome b-245, alpha polypeptide)

The CYBA gene (aka p22-PHOX, CYBA8 ) is located on chromosome 16 (16q24). The most widely studied polymorphism is C242T. According to NCBI Entrez Gene, “Cytochrome b is comprised of a light chain (alpha) and a heavy chain (beta). This gene encodes the light, alpha subunit which has been proposed as a primary component of the microbicidal oxidase system of phagocytes. Mutations in this gene are associated with autosomal recessive chronic granulomatous disease (CGD), which is characterized by the failure of activated phagocytes to generate superoxide, which is important for the microbicidal activity of these cells.

”

Literature search

A HuGE Navigator (V1.1) search ( CYBA [Text+MeSH]>>Meta-analysis[StudyType]>>) identified one article that is summarized below.


For the C242T polymorphism, C represents the wild allele, T represents the at-risk variant.

The at-risk genotypes are TT and CT. In the US population, the T allele frequency is 0.34.

Under Hardy-Weinberg, the three genotypes separately would be approximately 12% (TT),

44% (TC) and 44% (CC), respectively.


A 2008 meta-analysis reported the association of CYBA with CHD, with eight reports having the outcome of coronary artery disease (CAD). Three additional publications found by that group that focused on other vascular risk, including diabetes, were not included.

40 Overall,

6,253 cases and 3,984 controls were included. Statistical analyses included verification of

Hardy-Weinberg, fixed and random effects modeling, and formal analysis of heterogeneity.



For the CT+TT vs. CC (dominant) model the random effects OR is 0.94 (95% CI 0.77 to

1.14). Heterogeneity was high using a fixed effects model (I 2 = 65%). Significant heterogeneity was also reported for the random effects model, but no I 2 value was reported.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘A’ (size: 16% of 6,253 cases), ‘ C ’

(replication: high heterogeneity

), and ‘

-

‘ (bias: not evaluated because of a replication grade of C).

Large study analysis -- Figure B.13 shows the analysis of the four large studies. The summary OR for the dominant model is 0.92 (95% CI 0.77 to 1.10), p = 0.4.

Heterogeneity is high (Q = 7.5, I 2 = 60%, p = 0.06).


730



Statistics for each study CT&TT / Total Odds ratio and 95% CI


Not

CAD

Random


0.861

1.286

0.616 955 / 1706 273 / 499

Cai 1999 1.144

0.787

1.662

0.481 315 / 550 75 / 139

Yamada 2002 0.755

0.629

0.906

0.003 344 / 1784 258 / 1073

Morgan 2007 0.887

0.718

1.095

0.264 392 / 739 367 / 655

0.921

0.770

1.103

0.373

0.5

1 2

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

restriction to the four large studies.



For the CC+CT vs. TT (recessive) model, the random effects OR is 1.17 (95% CI 0.97 to

1.41), p = NS. No evidence of heterogeneity (I 2 = 4%).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘A’ (size: 16% of 6,253 cases), ‘ A ’ (replication: high heterogeneity), and

‘B‘

(bias: large amount of data relating to bias that was not reported).

Large study analysis -- Figure B.14 shows the analysis of the four large studies. The summary OR for the recessive model is 1.16 (95% CI 0.86 to 1.57), p = NS. Heterogeneity is moderate to high (Q = 6.0, I 2




TT / Total

CAD

Not

CAD

Random


0.684

1.243

0.594 207 / 1706 65 / 499

Cai 1999 1.299

0.694

2.431

0.413

65 / 550 13 / 139

Yamada 2002 0.939

0.499

1.767

0.845 25 / 1784 16 / 1073

Morgan 2007 1.537

1.127

2.097

0.007 121 / 739 74 / 655

1.160

0.859

1.566

0.333


0.5

1 2

747

748

749

750

751


Meta Analysis


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759

CYP11B2 (cytochrome P450, family 11, subfamily B, polypeptide 2)

The CYP11B2 gene is located on chromosome 8 (8q21-q22). The polymorphism of interest is

C344T. According to NCBI Entrez Gene, “[t]his gene encodes a member of the cytochrome

P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the mitochondrial inner membrane. The enzyme has steroid 18-hydroxylase activity to synthesize aldosterone and 18-oxocortisol as well as steroid 11 beta-hydroxylase activity. Mutations in this gene cause corticosterone methyl oxidase deficiency.

”

Literature search

A HuGE Navigator (V1.0) search ( CYP11B2 [Text+MeSH]>>Cardiovascular disease, unspecified, Coronary heart disease, Coronary restenosis, Myocardial Infarction[Mesh]) identified 10 articles. Only a single paper from 2004 41 was found, and it is summarized below.


For the C344T polymorphism, T represents the wild allele, C represents the at-risk variant.

The at-risk genotypes are TC and CC. In the US population, the CC genotype is approximately 20% (Payne 2004). Under Hardy-Weinberg, the three genotypes separately would be approximately 20% (CC), 49% (TC) and 31% (TT, the referent category), respectively.


A 2004 study reported the association of CYP11B2 with CAD in a cohort of 187 cases and

2,303 controls.

41 The study group was all Caucasian males after exclusion of people with diabetes and those with unstable angina. CAD was defined as sudden cardiac death or a symptomatic MI, silent MI or coronary revascularization. Controls were middle-aged

Caucasian males screened for a negative history of CHD. The summary OR for the TC vs.

TT genotypes was 1.25 (no CI, but termed ‘not significant’). The OR for the CC vs. TT genotype was 0.80 (no CI but term ed ‘not significant’).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades for both models of ‘C’ (size: 1<100 cases with the

CC genotype), ‘ C ’ (replication: no studies replicating this finding), and ‘ ‘ (bias: not evaluated because of previous grades of C).



819

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797

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799

F2 (coagulation factor II, prothrombin)

The F2 gene (aka prothrombin, Factor II ) is located on chromosome 11 (11p11). The most widely studied polymorphism is G20210A (alleles G and A; ‘A’ is the at-risk allele).

According to NCBI Entrez Gene, “ F2 is proteolytically cleaved to form thrombin in the first step of the coagulation cascade which ultimately results in the stemming of blood loss. F2 also plays a role in maintaining vascular integrity during development and postnatal life.

Mutations in F2 lead to various forms of thrombosis and dysprothrombinemia.

”

Literature search

A HuGE Navigator (V1.1) search was performed ( Factor II [Text+Mesh]>>Metaanalysis[StudyType]) which returned 11 meta-analyses. Four of these related to CVD.

42-45

We chose to use Burzotta and colleagues for CHD as it is more recent and larger.

42 We used the meta-analysis by Kim for the stroke analysis 43 as it was the most recent one for this outcome.


For the C20210A polymorphism, G represents the wild allele and A represents the presence of the variant. The allele frequency for the ‘A’ variant in a general Caucasian population is only 0 .011; the frequency for ‘G’ is then .989.

46 Based on Hardy-Weinberg, the expected genotype frequencies are 97.8% GG and 2.2% GA or AA. Because of the very low frequency for the A allele and therefore, the AA genotype, the homozygous variant (AA) is usually combined with the heterozygote (AG) and the evaluation comparisons are reported as (AG + AA) vs. GG (dominant model).

Coronary Heart Disease (Ischemic Heart Disease)

A 2004 meta-analysis included 19 studies that evaluated the 20210 variant and its association to ischemic heart disease.

42 A total of 4,944 cases and 7,090 controls were included in the meta-analysis. Because of the rare homozygosity for the A allele, the homozygous (AA) and heterozygous (AG) variants were combined and compared to the wild genotype of GG. Analytic methods included X² analysis and pooling according to Mantel-

Haenszel. The summary odds ratio was 1.21 (95% CI 0.99 – 1.58). Heterogeneity was moderate (Q = 26, i 2 = 36% p = 0.1).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grade s of ‘ B ’ (size: about 150 cases/control with the atrisk genotypes), ‘ B ’ (replication: I 2 of 36% indicating moderate heterogeneity), and ‘B ‘ (bias: large amount of data relating to bias that was not reported). There is, however, the possibility of bias related to sample size.

Large study analysis -- Eight of the individual studies included more than 500 study subjects. The results of the analysis are shown in Figure B.15. The summary OR is 1.10

(95% CI 0.80 to 1.51), p = 0.6. There was moderate heterogeneity (Q = 8.2, I 2 = 32%, p =

0.3).


845

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858


Model Study name Statistics for each study GA & AA / Total

Odds Lower Upper ratio limit limit p-Value IHD

Ridker 1999

Arruda 1998

Doggen 1998

0.756

0.406

1.410

4.782

0.984 23.247

1.450

0.568

3.700

Franco 1999

Croft 1999

2.707

0.720

0.785

0.324

9.340

1.602

Prohaska 1999 1.027

0.341

3.091

Random

Coulet 2000 1.110

0.582

2.118

Eikelboom 1998 1.056

0.554

2.013

1.097

0.797

1.509

not

IHD

0.380 12 / 404 69 / 1774

0.052 7 / 220 2 / 293

0.437 10 / 560

0.751 19 / 599

0.869 19 / 644

0.571

8 / 646

0.115 7 / 263 4 / 400

0.421 11 / 539 14 / 498

0.963 6 / 284 7 / 340

19 / 663

19 / 679


0.1

0.2

0.5

1 2 5 10


Figure B.15 Summary analysis of the F2 gene and IHD, after restriction to the eight large

studies.

Meta Analysis


A 2003 meta-analysis 43 evaluated the relationship between the F2 G20210A variant and ischemic stroke from 10 published studies. Overall, 1,625 cases and 5,050 controls were included. Four of these studies involved patients of all ages, while the remaining six only included patients diagnosed prior to age 55. The reported summary OR for all studies is

1.30 (95% CI 0.91 to 1.87), p = NS. Heterogeneity was low (Q = 11.5, I 2 = 22%, p = 0.2). In order to determine whether the restriction to age at diagnosis was important, we stratified the 10 trials (Figure B.16) by age of diagnosis. The summary OR for the general population was 0.93 (95% CI 0.60 to 1.47), p = 0.7. The summary OR for the six studies with early age of diagnosis was 1.67 (95% CI 1.05 to 2.67), p = 0.02. The test for between group differences was nearly significant (p = 0.08), indicating that the association may be very week in the general population of stroke victims, but much stronger for the less common early stroke (under age 55).


Model Group by

Subgroup within study


All

All

All

All

Random All

Early

Early

Early

Early

Early

Early

Random Early

Random Overall

Study name

Ridker 1999

Smiles 2002

Ferraresi 1997

Hankey 2001

All

All

All

All

Lopaciuk 2001 Early

Longstreth 1998 Early

De Stefano 1998 Early

Margaglione 1999 Early

Madonna 2002 Early

Voetsch 2000 Early

Subgroup within study Statistics for each study


1.567

5.514

1.203

1.362

2.110

1.672

1.239

1.096

0.624

0.573

1.659

0.935

0.951

0.572

0.287

0.058

0.491

0.595

0.181

0.594

0.669

0.657

1.049

0.895

2.100

1.355

5.697

5.601

1.469

4.985

0.184

13.342

1.782

17.064

2.435

2.773

6.774

2.667

1.714

0.681

0.003

0.608

0.395

0.210

0.031

0.196

0.782

0.233

0.634

0.415

0.770

0.953

0.1

0.2

0.5

1 2 5 10


Figure B.16 Reanalysis of F2 versus stroke, stratified by age of stroke diagnosis.

Meta Analysis


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The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grade s of ‘ B ’ (size: about 50 cases and 150 controls have the at-risk genotype ), ‘ B ’ (replication: I 2 of 22% indicating moderate heterogeneity), and ‘B‘

(bias: large amount of data relating to bias that was not reported). Given the issue of age at diagnosis, we have chosen to report the OR of 0.93 (95% CI 0.60 to 1.47), based on the four general population studies.

Large study analysis -- Only two of the four general population stroke studies had 500 or more subjects, and only one of the six early diagnosis studies were large. No large study analysis was done.


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F5 (coagulation factor V)

The F5 gene (aka Factor V , Factor V Leiden , FVL ) is located on chromosome 1 (1q23). The polymorphism of interest is G1691A (alleles G and A; ‘A’ is the at-risk allele). This polymorphism is also referred to as the ‘Leiden’ mutation, Arg534Gln, R506Q or rs6025.

According to NCBI Entrez Gene, “[t]his gene encodes coagulation factor V which is an essential factor of the blood coagulation cascade. This factor circulates in plasma, and is converted to the active form by the release of the activation peptide by thrombin during coagulation. This generates a heavy chain and a light chain which are held together by calcium ions. The active factor V is a cofactor that participates with activated coagulation factor X to activate prothrombin to thrombin. Defects in this gene result in either an autosomal recessive hemorrhagic diathesis or an autosomal dominant form of thrombophilia, which is known as activated protein C resistance.

”

Literature search

A HuGE Navigator (V1.1) search was performed ( Factor V [Text+MeSH]>>Metaanalysis[StudyType]>>Cardiovascular disease, unspecified, Coronary heart disease,

Coronary Restenosis, Myocardial Infarction, Myocardial ischemia[Mesh]) and 14 articles were identified. Four of these were relevant.

43-45,47 We chose to use the most recent 43 and included more studies and more study subjects. These data are summarized below.


For the G1691A polymorphism, G represents the wild allele and A represents the presence of the variant. The allele frequency for the ‘A’ variant in a general Caucasian population is only 0 .026; the frequency for ‘G’ is then 0.974.

46 Because of the very low frequency for the

A allele and therefore, for the AA genotype, the homozygous variant (AA) is often combined with the heterozygote (AG) and the evaluation comparisons are reported as AG+AA vs. GG.

Under Hardy-Weinberg, the expected genotype frequencies for GG and AG+AA are 90% and 10%, respectively.


A 2003 meta-analysis 43 reported on the association between the F5 polymorphism G1691A and several outcomes. We focused on MI as the outcome of interest. Patient specific data were available for analysis from the original authors. Analytic methods included random effects modeling, but did not perform formal tests for heterogeneity or examine possible publication bias. A total of 20 studies were included for MI, representing 5,313 cases and

14,047 controls. There was no restriction by race noted by the authors. Using the dominant model (AG+AA vs. GG), the summary OR was 1.10, (95% CI 0.88 to 1.36), p = 0.4. The corresponding Forest plot appeared to be symmetric (no obvious publication bias). We reentered the data and performed a formal test of heterogeneity. There was moderate heterogeneity (Q = 29, I 2 = 35%, p = 0.06).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grade s of ‘ A ’ (size: about 1900 with the least common genotype studied - 10% of about 19,000 study subjects with the AG or AA genotypes) , ‘ B ’

(replication: I 2 of 35% indicating moderate heterogeneity), and ‘B ‘ (bias: large amount of data relating to bias that was not reported).


918

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922

Large study analysis – There were six studies of 500 or more study subjects (Figure B.17).

The summary OR for these large studies was 0.94 (95% CI 0.75 to 1.19), p = 0.6. There was moderate heterogeneity (Q = 7.1, I 2 = 30%, p = 0.2).


923

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Model Study name Statistics for each study GA & AA / Total

Odds Lower Upper ratio limit limit p-Value MI not

MI

Random

Doggen 1998

Juul 2002

Psaty 2001

1.397

0.860

2.268

0.176 38 / 560 32 / 646

Ridker 1995

Cushman 1998

1.033

0.448

0.611

0.172

1.745

1.167

0.904 23 / 374

0.100

5 / 152

42 / 704

34 / 482


0.502

1.140

0.182 40 / 1038 59 / 1172

1.035

0.703

0.811

0.335

1.321

1.473

0.781 79 / 962 629 / 7907

0.350

0.944

0.749

1.190

0.624

9 / 232 39 / 718


0.5

1


Figure B.17 Summary analysis of the F5 gene (AG+AA vs. GG) and MI, after

restriction to the six large studies included in a published meta-analysis

Meta Analysis

2


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A 2004 meta-analysis 8 reported on the association between the F5 R506Q (Leiden) polymorphism and ischemic stroke. Overall, 26 individual studies and 4,588 cases and

13,798 controls were included in the analysis. Analytic methods included random effects modeling, formal tests for heterogeneity and examination for possible publication bias. The summary OR for the dominant model (AG+AA vs. GG) was 1.31 (95% CI 1.10 to 1.56), p =

0.001. There was modest heterogeneity (I 2 = 35%). The authors identified one report that was responsible for much of the heterogeneity and recomputed the OR to be 1.18 (95% CI

0.98 to 1.42), p = 0.08. The heterogeneity was then considered low (I 2 =0%).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grade s of ‘ B ’ (size: about 1,800 with the at-risk genotypes

– about 10% of nearly 18,000 cases/controls with a AG or AA genotype), ‘ B ’ (replication: I 2 of 0% after removal of one outlying study), and ‘B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis -- The summary OR (AG + AA vs. GG) for the six studies having 500 or more combined cases and controls was 1.22, (95% CI 0.69 to 2.16); p = 0.5. There was high heterogeneity (Q=28, I² = 82%, p = <0.001). Figure B.18 summarizes these results.

Again, one study is a significant outlier.

48 If this was removed, the summary OR would be

1.07 (95% CI 0.82 to 1.38), p = 0.6. Moderate heterogeneity remains (Q = 6.3, I 2 = 36, p =

0.2).

FV Failsafe N Analysis: AG + AA vs GG for Stroke



Catti 1995

Juul 2002

0.750

0.919

Margaglione 1999 4.028

Ridker 1995 0.627

Szolonoki 2002

Zunker 2001

1.539

0.949

1.219

0.355

0.557

2.459

0.290

1.029

0.378

0.687

1.583

1.516

6.598

1.358

2.301

2.379

2.163

0.450

0.741

0.000

0.237

0.036

0.910

0.498


0.1 0.2

0.5

1 2 5 10

952

953

954

restriction to the six large studies.

Meta Analysis


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GNB3 (guanine nucleotide binding protein (G protein))

The GNB3 gene is located on chromosome 12 (12p13). The most widely studied polymorphisms is C825T (alleles C and T; T is the at-risk allele). According to NCBI Entrez

Gene, “ GNB3 encodes a beta subunit of a heterotrimeric guanine nucleotide-binding protein

(G protein). Beta subunits are important regulators of alpha subunits, as well as of certain signal transduction receptors and effectors. A single-nucleotide polymorphism (C825T) in this gene is associated with essential hypertension and obesity. This polymorphism is also associated with the occurrence of the splice variant GNB3-s, which appears to have increased activity. GNB3-s is an example of alternative splicing caused by a nucleotide change outside of the splice donor and acceptor sites. Additional splice variants may exist for this gene, but they have not been fully described.

”

Literature search

A HuGE Navigator (V1.2) search was performed (GNB3 [Text+MeSH]>>Arterial Sclerosis,

Atherosclerosis, Cardiovascular disease, unspecified, coronary artery disease, Coronary heart disease, Myocardial Infarction, Myocardial ischemia, Vascular Diseases[Mesh]) and 21 articles were identified. None were meta-analyses. All but five studies 49-53 were excluded.

These studies are summarized below.


For the C825T polymorphism in the GNB3 gene, the reported genotype frequencies in 1,332 control non-Hispanic Caucasians from the US were 50%, 41% and 9% for the CC, CT and

TT genotype, respectively.

53 The at-risk genotype is TT. The corresponding allele frequencies were 0.704 and 0.296 for C and T, respectively. Under Hardy-Weinberg, the expected genotype frequencies would be 8.8%, 41.6%, and 49.6%, respectively. Usually, the comparisons reported are for TT vs. (CT + CC), but often both TT and CT are compared separately against the wild genotype CC.

Coronary Heart Disease (Coronary Artery Disease, Myocardial Infarction, Peripheral Arterial

Disease).

Among the five included studies, three studied MI alone 50-52 , one studied CAD 49 and one studied both CAD and PAD.

53 All examined the same polymorphism (C825T) and provided raw data so that both heterozygotes (CT) and homozygotes (TT) could separately be compared with the wild genotype of CC. Figures B.19 and B.20 show these comparisons.

Overall, there were 2,865 cases and 4,568 controls included. The OR for the heterozygotes was 1.05 (95% CI 0.94 to 1.19), p = 0.4. The heterogeneity was low (Q = 5.7, I 2 = 13%), p =

0.3. The corresponding OR for the homozygotes was 1.13 (95% CI 0.95 to 1.35), p = 0.2.

The heterogeneity was low (Q = 4.4, I 2 = 0%, p = 0.5).



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Model Study name Statistics for each study CT / Total


Not

CAD

Random

Renner 2007

Klintschar 2005

Morrison 2002 (CHD) 0.94

Morrison 2002 (PAD) 1.14

Hengstenbert 2001 1.01

Naber 2000

1.05

0.84

1.49

1.05

0.87

1.27

0.50

1.44

0.72

1.23

0.76

1.69

0.83

1.22

1.06

2.09

0.94

1.19

0.61 574 / 1229 301 / 662

0.53

47 / 96 67 / 126

0.64 149 / 342 269 / 596

0.53 56 / 117 274 / 613

0.94 263 / 540 893 / 1841

0.02 135 / 247 131 / 293

0.38


0.1

0.2

0.5

1 2 5 10


Figure B.19

Analysis of five GNB3 studies of the C825T polymorphism (CT vs. CC) for

CHD

Meta Analysis

Model


Study name Statistics for each study TT / Total


Not

CAD

Renner 2007 1.102

0.805

1.509

0.544 140 / 795 70 / 431

Klintschar 2005 2.080

0.904

4.787

0.085 19 / 68 11 / 70

Morrison 2002 (CHD) 0.875

0.548

1.399

0.578 31 / 224 60 / 387

Morrison 2002 (PAD) 1.323

0.708

2.474

0.380 15 / 76 63 / 402

Random

Hengstenbert 2001 1.071

0.787

1.455

0.664 66 / 343 211 / 1159

Naber 2000 1.512

0.804

2.845

0.200 23 / 135 22 / 184

1.131

0.947

1.350

0.174


0.1

0.2

0.5

1 2 5 10


Figure B.20 Analysis of five GNB3 studies of the C825T polymorphism (TT vs. CC) for

CHD

Meta Analysis

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grade s of ‘ B ’ (size: about 273 with TT genotype (7,433 study subjects with 8.8%), ‘ A ’ (replication: both estimates of heterogeneity <10%), and ‘B ‘


Large study analysis – Three of the studies included 500 or more combined cases and controls.

49,51,53 The OR for the heterozygous comparison was 1.02, (95% CI 0.91 to 1.15), p

= 0.7. The heterogeneity was low (Q = 0.8, I 2 = 0%, p = 0.9). For the homozygous comparison, the OR was 1.07 (95% CI 0.88 to 1.29), p = 0.5. There was low heterogeneity

(Q = 1.2, I 2 = 0%, p = 0.8). No figures were made as only two entries were removed (see

Figures B.19 and B.20 with the removal of Klintschar 2005 and Naber 2000).


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Stroke

Only one study reported the association of GNB3 and stroke, in a Caucasian population.

54

This study involved 231 incident clinical ischemic stroke cases and 984 control individuals representing a stratified random sample of the ARIC (Atherosclerosis Risk in Communities) cohort. After adjustment for age and sex, the hazard ratio for the CT+TT vs. CC comparison was 1.45 (95% CI 1.04 to 2.00), p = 0.02.

One other study 55 reported no association between the GNB3 T allele and stroke (cases were restricted to cerebral thrombosis or lacunar infarctions) in a Chinese population of 990 cases and 1,124 controls. The genotype frequency for the at-risk TT allele was higher in this population than in a Caucasian population 53 (21% vs. 8.8%). The observed OR for the recessive model (CC+CT vs. TT) was computed from their raw data and found to be 0.98

(95% CI 0.80 to 1.21), p = 0.9.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grade s of ‘ C ’ (size: about 41 with TT genotype (231 cases

*2 study subjects with 8.8%), ‘ C ’ (replication: no study replicating this finding), and ‘B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis – Not performed because of limited relevant studies.


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GPX1 (glutathione peroxidase 1)

The GPX1 gene (glutathione peroxidase) is located on chromosome 3 (3p21.3). The relevant polymorphisms found in the literature are ALA n

(or ALA5, ALA6 and ALA7) where ALA6 is the at-risk allele for CAD. Both the ALA5 and ALA7 polymorphisms code for Pro, while the

ALA6 codes for Leu. Thus, this is sometimes referred to as the Pro/Leu polymorphism where Leu/Leu is the at-risk genotype for stroke). According to NCBI Entrez Gene “This gene encodes a member of the glutathione peroxidase family. Glutathione peroxidase functions in the detoxification of hydrogen peroxide, and is one of the most important antioxidant enzymes in humans. This protein is one of only a few proteins known in higher vertebrates to contain selenocysteine, which occurs at the active site of glutathione peroxidase and is coded by UGA, that normally functions as a translation termination codon.

In addition, this protein is characterized in a polyalanine sequence polymorphism in the Nterminal region, which includes three alleles with five, six, or seven alanine (ALA) repeats in this sequence. The allele with five ALA repeats is significantly associated with breast cancer risk. Two alternatively spliced transcript variants encoding distinct isoforms have been found for this gene.

”

Literature search A HuGE Navigator (V1.1) search for the GPX1 gene identified 52 articles on

47 disease terms. A search for meta-analyses yielded no results. However searching

( GPX1 [Text+Mesh]>>coronary artery disease, Coronary heart disease, Coronary

Restenosis, Myocardial Infarction[Mesh]) identified 3 articles. Two of these 56,57 were excluded because they only measured intermediate outcomes (restenosis after coronary stenting or artery calcification). The remaining study 58 , is summarized in the Coronary Heart

Disease section below. Searching ( GPX1 [Text+Mesh]>>Apoplexy[Mesh]) yielded 1 individual study 59 which is summarized in the Stroke section below.

Genotype Frequencies

For the ALA n

polymorphism, ALA6 represents the at-risk allele and ALA5 or ALA7 represents the wild allele. The allele frequencies in a Northern European population of 146 are 0.54, 0.21 and 0.25 for ALA5 through ALA7, respectively.

58 The reported genotype frequencies for ALA6 present and ALA6 absent are 40% and 60%, respectively.

58

Coronary Heart Disease (Coronary Artery Disease)

A single paper from 2003 58 described an association between the GPX1 ALA6 allele being present or absent and CAD. A total of 88 men with CAD and 146 control men were studied.

All cases had >50 stenosis of one or more major arteries. The OR for ALA6 allele-carriers vs. non-carriers (ALA6+ vs. ALA6-) was 1.30 (95% CI 0.85 to 2.0), p = 0.2.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘C’ (size: 1<100 cases with the CC genotype),

‘ C ’ (replication: no studies replicating this finding), and ‘ ‘ (bias: not evaluated because of previous grades of C).



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Stroke

A single paper from 2000 59 evaluated an association between the Pro/Leu variants in the

GPX1 gene and stroke in a Swedish population. The reported OR for (Leu/Leu vs. Pro/Pro

+ Pro/Leu) is 0.92 (95% CI 0.37 to 2.3), p = 0.8. In terms of the ALA n

nomenclature, this comparison is (ALA6/ALA6 vs. other). Others include all ALA6 heterozygotes and combinations of ALA5 and ALA7.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results in the three grades of ‘C’ (size: 1<100 cases with the Leu/Leu genotype), ‘ C ’ (replication: no studies replicating this finding), and ‘ ‘ (bias: not evaluated because of previous grades of C).



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IL1B (interleukin 1, beta)

The IL1B gene (aka IL-1; IL1F2; IL1-BETA ) is located on chromosome 2 (2q14). The most widely studied polymorphism is C511T (T is the at-risk allele). According to NCBI Entrez

Gene, ”[t]he protein encoded by this gene is a member of the interleukin 1 cytokine family.

This cytokine is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1 (CASP1/ICE). This cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis.

”

Literature search

A HuGE Navigator (V1.1) search for the IL1B gene identified 802 articles on 376 disease terms. A search for relevant outcomes ( IL-1 [Text+Mesh]>>Arterial Occlusive Diseases,

Arterial Sclerosis, Atherosclerosis, Brain Ischemia, coronary artery disease, Coronary heart disease, Coronary Restenosis, Coronary Stenosis, Myocardial Infarction, Apoplexy,

Subarachnoid Hemorrhage[Mesh]) identified 29 articles. Eleven were excluded from analysis because they examined either intermediate or irrelevant outcomes. Two articles were excluded because they were published in a language other than English. One article did not investigate the variant of interest. Two more articles were excluded because the studies were conducted in solely non-Caucasian populations. Of the remaining nine studies, only one was suitable for CHD 60 and three were suitable for stroke.

61-63 None of the four articles were meta-analyses.


For the C511T polymorphism, C represents the wild allele and T represents the presence of the variant. Allele frequencies were calculated from the 122 genotyped individuals in a general Northern Italian Caucasian population. The allele frequency for the C variant is

0.642; the frequency for T allele is 0.357.

60 The reported genotype frequencies from that same report the genotype frequencies for IL1B C511T are CC = 37%, CT = 54% and TT =

9%. Using Hardy-Weinberg, the expected genotype frequencies are 41%, 46% and 13%, respectively.


Licastro and colleagues 60 reported data from Northern Italy. A total of 139 elderly males with MI and randomly selected males in good health (somewhat younger than cases).

Patients with MI who were included did not have any associated neoplastic, autoimmune diseases, coagulation disorders or chronic renal failure. The authors did not report an OR, but it could be computed from the raw data. The summary OR for the TT vs. CC comparison is 0.90 (95% CI, 0.38 to 2.2), p = 0.8. The summary OR for the CT vs. CC comparison is 0.65 (95% CI 0.39 to 1.1), p = 0.1. Using the dominant model, the OR is 0.69

(95% CI 0.42 to 1.1), p = 0.1.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘C’ (size: less than 100 in the smallest category), ‘ C ’ (replication: no other studies), and ‘ ‘ (bias: not evaluated because both earlier grades were C. Both OR estimates received this grade.



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Three articles 61-63 reported the IL1B C511T polymorphism and risk of ischemic stroke.

Overall, 586 cases and 533 controls were examined. Using a random effects model, we were able to pool the results of these three studies (Figures B.21 and B.22). The summary

OR for the CT vs. CC comparison is 1.07 (95% CI 0.83 to 1.38), p = 0.6, with little heterogeneity ( I² = 0%, p = 0.6). The summary OR for the TT vs. CC comparison is 1.52

(95% CI 0.65 to 3.59), p = 0.3, with high heterogeneit y (I² = 75%).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘C’ (size: less than 100 in the smallest category), ‘ C ’ (replication: no other studies), and ‘ ‘ (bias: not evaluated because both earlier grades were C). Both OR estimates receive this score.

Large study analysis -- No studies were sufficiently large to perform this analysis for either comparison.

IL1B: CT vs CC for Stroke

Odds ratio and 95% CI Model

Random

Study name Subgroup within study Statistics for each study


Dziedic 2005

Vohnout 2003

CT

CT

Iacoviello 2005 CT

0.96

1.02

1.33

1.07

0.59

0.70

0.80

0.83

1.56

1.47

2.22

1.38

0.8564

0.9249

0.2675

0.5986

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0.2

0.5

1 2 5 10

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1171 stroke less likely stroke more likely

Figure B.21 Analysis of the three IL1B studies of the C511T polymorphism (CT vs. CC)

Meta Analysis for Stroke

IL1B: TT vs CC for Stroke

Odds ratio and 95% CI Model Study name

Random

Dziedic 2005 TT

Vohnout 2003 TT

Iacoviello 2005 TT

Subgroup within study Statistics for each study

Odds ratio

1.87

0.73

2.96

1.52

Lower Upper limit limit

0.84

0.41

1.25

0.65

p-Value

4.16

0.1252

1.31

0.2948

7.01

0.0135

3.59

0.3370

0.1

0.2

0.5

1 2 5 10 stroke less likely stroke more likely

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1175 for Stroke


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IL6 (interleukin 6)

The IL6 gene (aka HGF, IL-6, IFNB2 ) is located on chromosome 7 (7p21). The polymorphism of interest is G174C.

According to NCBI Entrez Gene, “ IL6 is an immunoregulatory cytokine that activates a cell surface signaling assembly composed of IL6, IL6RA (IL6R; MIM

147880), and the shared signaling receptor gp130 ( IL6ST; MIM 600694).”

Literature search

A HuGE Navigator (V1.1) search on the IL6 gene identified 675 articles on 361 disease terms. A search for meta-analyses ( IL6 [Text+MeSH]>>Meta-analysis, HuGE Review,

Clinical trial[StudyType]) identified 11 such studies; only one of which was relevant to

CVD.

64 That study is summarized below. A separate search was performed for stroke

( IL6 [Text+MeSH]>>Atherosclerosis, Intracranial Hemorrhages, Apoplexy, Subarachnoid

Hemorrhage[Mesh]). No meta-analyses were identified, but 18 articles were retrieved. Of these articles, 13 were excluded for various reasons. Two only looked at those with a history of stroke, three studied stroke in patient subpopulations (those with type 2 diabetes, pediatric patients, or post operative stroke victims), six measured non-stroke or intermediate outcomes only, and three were conducted exclusively in non-Caucasian populations. The remaining five articles 65-69 are described in the stroke section below.


For the G174C polymorphism, G represents the wild allele, while C represents the presence of the variant. The at-risk genotype is CC with a prevalence of 17%. In a general

Caucasian population, allele frequencies are 0.59 and 0.41, respectively (based on 5,674 control individuals.

64 The observed genotype frequencies were similar to observed (GG =

36%, GC= 47% and CC=17%).

Coronary Heart Disease (CHD and MI)

A 2006 meta-analysis 64 identified 7 studies reporting the G174C polymorphism and the risk of CHD and/or MI in European individuals. The authors then included their own data as well. Analytic methods included random effects modeling, formal tests for heterogeneity and identification of possible publication bias. Data from eight studies (6,927 cases and 12,871 controls) were included in the analysis. The summary odds ratio for the model (GC+CC vs.

GG) was 1.12 (95% CI 0.97 to 1.29), p = 0.12. There was evidence for heterogeneity (

I² =

67%), but much of that was due to one small study 60 with a high OR. If this one study were to be removed, the OR is 1.06 (95% CI 0.95 to 1.18), p = 0.3. Heterogeneity is reduced to

(Q = 10.7, I 2 = 44%, p = 0.1).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘A’ (size: more than 1000 in the smallest genotype category ), ‘ C ’ (replication: I 2 of 67% ), and ‘ ‘ (bias: not evaluated because of an earlier grade of C).

Large study analysis -- When restricted to the six studies with 500 or more cases (Figure

B.23), the summary odds ratio was 1.04 (95% CI 0.93 to 1.15), p = 0.5. There was still moderate heterogeneity (I²=41%).


Failsafe N: IL6 G174C genotype and CAD

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Model

Random

Study name

Georges 2001

Basso 2002

Nauck 2002

Bennet 2003

Lieb 2004

Sie 2005

Odds ratio

1.37

1.07

0.96

0.97

0.92

1.09

1.04


Lower limit

Upper limit p-Value

1.08

0.85

0.80

0.81

0.77

0.89

0.93

1.74

1.34

1.15

1.16

1.09

1.33

1.15

0.0097

0.5601

0.6514

0.7395

0.3417

0.3960

0.5095


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0.2

0.5

1 2 5 10

CAD less likely CAD more likely

Meta Analysis to the six large studies

Stroke (Ischemic Stroke, Aneurysmal Subarachnoid Haemorrhage ).

Five studies reported the association between various forms of stroke and the IL6 G174C polymorphism.

65-69 One of these studies was a case-only 68 and another 69 did not give sufficient information to compute an OR, but did state that “no relation was observed for genotype of the IL6 G174G polymorphisms and stroke (data not shown)”. Chamorro and colleagues studied four types of stroke (89 lacunar, 82 large vessel disease, 53 embolic and

49 undetermined cause) in 273 patients and 105 control individuals.

65 The OR for CC vs.

(CG + GG) was 1.57 (95% CI 0.69 to 3.66), p = 0.2. They also reported that the strongest association was with lacunar stroke with an OR of 3.22 (95% CI 1.1 to 9.1), p = 0.3. This finding has not been confirmed (or refuted) in subsequent studies. Lalouschek and colleagues reported on 404 cases of ischemic stroke or transient ischemic attack and 415 control individuals.

66 The OR for CC vs. (CG + GG) was 1.10 (95% CI 0.82 to 1.48), p = 0.5.

The group also reported that “the exclusion of patients with transient ischemic attack (N=81) also had no notable effect”. Fontanella and colleagues studied 179 cases (aneurysmal subarachnoid hemorrhage) and 156 controls.

67 The OR for CC vs. (CG + GG) was 0.66

(95% CI 0.30 to 1.42), p = 0.2.

For the three studies, 856 cases and 676 control subjects were included (Figure B.24), and the combined OR is 1.07 (95% CI 0.77 to 1.48), p = 0.7. The heterogeneity was low (I² =

11%).


IL6 G174C genotype and Stroke

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Model

Random

Study name

Chamorro 2005

Fontanella 2008

Lalouscheck 2006

Odds ratio

1.57

0.66

1.10

1.07


Lower limit


0.68

0.30

0.82

0.77

3.62

1.44

1.48

1.48

0.2893

0.2948

0.5269

0.6997


0.1

0.2

0.5

1 2 5 10

Stroke less likely Stroke more likely

Figure B.24 Summary analysis of the IL6 gene (GC+CC vs. GG) and stroke

Meta Analysis

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘B’ (size: about 260 in the smallest genotype category: 1,532 * 17% ), ‘ A ’ (replication: ), and ‘ B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis -- Only one study included more than 500 subjects, so no analysis was performed.


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ITGB3 (integrin, GP3A)

The ITGB3 gene (aka GP3A, CD61, integrin ) is located on chromosome 17 (17q21). The polymorphism of interest is C1565T (aka Leu/Pro).

According to NCBI Entrez Gene, “The

ITGB3 protein product is the integrin beta chain beta 3. Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain. A given chain may combine with multiple partners resulting in different integrins. Integrin beta 3 is found along with the alpha

IIb chain in platelets. Integrins are known to participate in cell adhesion as well as cellsurface mediated signaling.

”

Literature search

A HuGE Navigator (V1.1) search on the ITGB3 gene ( ITGB3 [Text+MeSH]>>Metaanalysis[StudyType]) identified seven meta-analyses. Of these, five were potentially useful.

5,45,70-72 We chose to use the review by Morgan and colleagues, as it includes far more publications and study subjects, and applied more refined statistical analyses than the others. The selected meta-analysis is described in more detail below.

5


For the C1565T polymorphism, C represents the wild allele, while T represents the presence of the variant. The at-risk genotype is TT. In a meta-analysis based mainly on Caucasians, the allele frequencies in 5,674 controls were 0.85 and 0.15 for the C and T allele, respectively.

70 Under Hardy-Weinberg, the expected genotype frequencies would be about

72%, 26% and 2% for the CC, CT and TT genotypes, respectively. Because of the low allele frequency of T, studies usually grouped CT and TT genotype together.


A 2003 meta-analysis 5 identified 30 studies reporting the ITGB3 C1565T polymorphism and the risk of MI in mainly Caucasian individuals. Analytic methods included random effects modeling, formal tests for heterogeneity and identification of possible publication bias.

There were 6,173 cases and 6,994 controls included in the analysis. The summary OR for the dominant model (CT+TT vs. CC) was 1.13 (95% CI 1.02 to 1.26) p = 0.2. There was evidence for heterogeneity (Q = 42.9, I² = 42%, p = 0.047). They explored the possibility of publication bias using a funnel plot (p = 0.04 for the existence of bias).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘A’ (size: over 3,000 in the smallest genotype category: 13,000 * 28%), ‘ B ’ (replication: I 2 value of 42%), and ‘ C ‘ (bias: significant evidence of publication bias).

Large study analysis -- When restricted to the eight studies with 500 or more cases (Figure

B.25), the summary odds ratio was 1.08 (95% CI 0.97 to 1.21), p = 0.2. There was low heterogeneity (Q = 8.5,

I² = 18%, p = 0.3).


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A2A2 & A2A1 / Total

MI not

MI

Random

Anderson 1999

Joven 1998

1.392

0.949

2.044

0.091

76 / 225 74 / 276

1.092

0.756

1.579

0.638

89 / 250 84 / 250

Mikkelsson 2990 1.080

0.764

1.527

0.661

78 / 281 101 / 385

Mamotte 1998 1.024

0.760

1.380

0.874

97 / 367 148 / 570

Ridker 1997 0.935

0.701

1.246

0.647

94 / 374 186 / 704

Herrrmann 1997 1.090

0.826

1.440

0.543 133 / 428 153 / 523

Bottiger 2000 1.386

1.066

1.803

0.015 232 / 689 119 / 444


0.775

1.126

0.475 278 / 1061 328 / 1191

1.079

0.965

1.206

0.184


0.5

1 2


Figure B.25 Summary analysis of the ITGB3 gene and the C1565T polymorphism

(CT+TT vs. CC) and MI, after restriction to the eight large studies

Meta Analysis

Stroke (Cerebral Vascular Accident)

A 2001 meta-analysis 45 provided information about the ITGB3 C1565T polymorphism and the risk of stroke in mainly Caucasian individuals. They identified three studies that reported information on 479 cases and 1,376 controls. Analytic methods did not include any of the standard methodology (random effects modeling, formal tests for heterogeneity and identification of possible publication bias). The summary OR for the dominant model

(CT+TT vs. CC) was 0.80 (95% CI 0.62 to 1.04). Unfortunately, the summary OR is smaller than that reported by any of the three individual studies bringing this result into question.

We retrieved the three original articles 73-75 and reanalyzed the data (Figure B.26). We found the overall OR for the (CT+TT vs. CC) comparison to be 0.99 (95% CI 0.74 to 1.34), p = 0.9.

2 CVD and 9p21 (homozygotes vs heterozygotes)

Model Study name Statistics for each study Pro+ / Total

Odds Lower Upper ratio limit limit p-Value Stroke Controls

Random

Ridker 1997 0.946

0.664

1.348

Kekomaki 1998 1.010

0.479

2.128

Reiner 2000 1.252

0.568

2.762

0.994

0.739

1.337

0.759 53 / 209 186 / 704

0.979 11 / 36 105 / 346

0.577 57 / 234 9 / 44

0.970


0.1

0.2

0.5

1 2 5 10


Figure B.26 Our re-analysis of the data from a 2001 meta-analysis of stroke and ITGB3 gene (CT+TT vs. CC) and stroke

Meta Analysis


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The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘B’ (size: 500 in the smallest genotype category: about 1,800 * 28%), ‘ A ’ (replication: I 2 value of 42%), and ‘B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis -- Only one study included more than 500 subjects, so no analysis was performed.


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LPL (lipoprotein lipase)

The LPL gene (aka KID, HDLCQ11 ) is located on chromosome 8 (8p22). The most common polymorphisms are S447X, Ans291Ser and PvuII. According to NCBI Entrez Gene, “ LPL encodes lipoprotein lipase, which is expressed in heart, muscle, and adipose tissue. LPL functions as a homodimer, and has the dual functions of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake. Severe mutations that cause

LPL deficiency result in type I hyperlipoproteinemia, while less extreme mutations in LPL are linked to many disorders of lipoprotein metabolism.

”

Literature search

A HuGE Navigator (V1.1) search on the LPL gene identified 207 articles on 77 disease terms. A search for meta-analyses ( LPL [Text+MeSH]>>Meta-analysis, Clinical

Trial[StudyType] ) identified four such studies; all were relevant to CVD.

76-79 For the S447X polymorphism, we also included one additional study 80 found in one of the reference lists.

Results from these studies are summarized below. A separate search (v1.3) was performed for stroke ( LPL [Text+MeSH]>> Brain Ischemia, Cerebral Infarction, Apoplexy, Transient

Ischemic Attack, Apoplexy[Mesh]). No meta-analyses were identified, but 11 articles were retrieved. Of these, nine were removed from consideration because of language, exclusively non-Caucasian population or studied an intermediate outcome. The two included studies 39,53 are examined below.




S447X: S represents the wild allele, while X represents the presence of the variant. The atrisk ‘protective’ genotype (SX + XX) is found in 19% of the Caucasian population.

Allele frequencies are 0.9 and 0.1, respectively (based on about 8,000 control individuals.

79 The expected genotype frequencies were 81%, 18% and 1%, respectively.



Ans291Ser: A represents the wild allele, while S represents the presence of the variant.

The at-risk genotype (AS + SS) is found in 5% of the Caucasian population. Allele frequencies are 0.975 and 0.025, respectively.

80 The expected genotype frequencies were 95%, 5% and < 0.1%, respectively.



PvuII: P- represents the wild allele and P+ represents the presence of the variant. The at-risk genotype (P+/P+) with an 18% prevalence. In a general Caucasian population, allele frequencies are 0.58 and 0.42, respectively (based on 800 control individuals.

76

The expected genotype frequencies were 35%, 47% and 18%, respectively.

Coronary Heart Disease (Ischemic Heart Disease - IHD)



S447X: The 2002 meta-analysis 79 reported S447X and the risk of IHD from six studies in men and two studies in women. o Data in the six studies of men (unknown model) showed an overall OR of 0.82

(95% CI 0.71 to 0.95

), p = 0.01. Heterogeneity was low (I² = 0%). Overall 2,509 cases and 7,007 controls were included in the analysis. Using the dominant model, the OR is 0.85 (95% CI 0.74 to 0.98), p=0.025. Heterogeneity was low

(I 2 =0%).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of

‘A’

(size: about 1800 in


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Large study analysis – Three studies included 500 or more subjects (cases and controls). The revised OR using these large studies was 0.86 (95% CI 0.73 to

1.0), p = 0.06. There was limited heteroge neity (I² = 0%). Figure B.27 shows this analysis.

Failsafe N: LPL S447X (SX & XX v SS) for IHD

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Model Statistics for each study IHD / Total

Odds Lower Upper ratio limit limit Z-Value p-Value Carriers Non-carriers

Jemma 1995 0.854

0.657

1.111

-1.175

Galton 1996 0.729

0.532

0.999

-1.966

Random

Wittrup 2002 0.955

0.746

1.224

-0.362

0.859

0.735

1.004

-1.904

0.240 118 / 761

0.049 61 / 479

0.717 84 / 578

0.057

154 / 871

169 / 1013

584 / 3865


0.5

1 2

Figure B.27 Summary analysis of the LPL gene (SX+XX vs. SS) and IHD, after

Favours A Favours B restriction to the three large studies

Meta Analysis o Data in two studies of women (413 cases and 6,101 controls) showed an overall

OR of 0.97 (95% CI 0.68 to 1.38), p = 0.9.

79 Heterogeneity was low ( I² = 0%).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘B’ (size: about 140 in the smallest genotype category: 413*2 NB: used twice the number of cases as the Venice criteria assume an approximate 1:1 case/control ratio), ‘ A ’

(replication: I 2 of 0%), and ‘B‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis -- Only two studies, so no analysis was performed.



Ans291Ser: The 2006 meta-analysis 77 published on an unknown number of studies reporting the relationship between the Ans291Ser polymorphism and CHD. A total of

1,203 carriers and 6,192 non-carriers were included. Using a random effects model the summary OR for the AS+SS vs AA comparison was 1.48 (95% CI 1.09 to 2.00), p =

0.01. There was significant unexplained heterogeneity ( χ 2 = 13.8, p = 0.003). It was not possible to verify or reanalyze this data as only this summary information was provided.

By examining the reference list, we estimated that the author included eight studies.

This would result in an I 2 value of 49%.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘C’ (size: about 60 in the smallest genotype category: 1,203*2 NB: used twice the number of cases as the Venice criteria assume an approximate 1:1 case/control ratio), ‘ B ’ (replication: I 2 estimated to be 49% ), and ‘ ‘ (bias: not evaluated because of an earlier grade of C).


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Large study analysis – Without raw data, the analysis could not be performed.



PvuII: A 2007 meta-analysis 76 examined the polymorphism for CAD in seven published studies (six in Caucasians). Only four studies provided sufficient information to compare

PvuII genotype and CAD. No formal analysis was performed and no summary statistic was reported. We analyzed the data from these four studies1,092 cases and 800 controls and found the OR for heterozygotes vs. the wild genotype (P+/P- vs. P-/P-) was

1.17 (95% CI 0.94 to 1.46), p = 0.2 (Figure B.28). Heterogeneity was low (Q = 3.1, I² =

4%, p = 0.4). The corresponding OR for homozygous vs. wild (P-/P- vs. P+/P+) is 0.93

(95% CI 0.60 to 1.46), p = 0.8 (Figure B.29). Heterogeneity was high (Q=6.1, I² = 51%, p = 0.1).

LPL: Pvull +/- vs -- for CAD



Random

Abu-Amero 2003a 1.197

0.899

1.592

1.232

0.218

Isbir 2004a 0.662

0.334

1.315

-1.178

0.239

Anderson 1999a

Duman 2004a

1.312

1.365

0.882

0.569

1.952

3.276

1.340

0.697

0.180

0.486

1.167

0.936

1.455

1.372

0.170

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Figure B.28 Analysis of four LPL studies of the Pvull polymorphism (P+/P- vs. P-/P-) for CAD

Meta Analysis

LPL: Pvull +/+ vs -- for CAD

Model Study name Odds ratio and 95% CI

Random

Abu-Amero 2003

Isbir 2004

Anderson 1999

Duman 2004



1.107

0.631

1.386

0.413

0.934

0.749

0.245

0.843

0.159

0.597

1.637

1.620

2.281

1.068

1.461

0.510

-0.958

1.287

-1.824

-0.301

0.610

0.338

0.198

0.068

0.764

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0.5

1 2 5 10

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Figure B.29 Analysis of four LPL studies of the Pvull polymorphism (P+/P+ vs. P-/P-)

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘B’ (size: about 340 in the smallest


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1451 genotype category of P+/P+: 18% of about 1900 subjects ), ‘ C ’ (replication: I 2 estimated to be 51% and 4% ), and ‘ ‘ (bias: not evaluated because of an earlier grade of C).

Large study analysis – Only two of these studies 81,82 have 500 or more study subjects, so no analysis could be performed.

Stroke (Incident Ischemic Stroke)

Two studies were relevant.

39,53 One study from the US 53 reported on the LPL S447X polymorphism in stroke patients (incident ischemic stroke confirmed by hospitalizations, hospital records and death records for cerebrovascular events – subclinical stroke was defined by MRI). We restricted the analysis to incident ischemic stroke (removing the subclinical stroke patients). There were 113 male cases and 540 controls; 100 females cases and 397 controls. The population was racially diverse and ranged in age from 45 to

64. They reported ORs separately for men and women. Among males, the corresponding crude OR was 0.69. Among females, the crude OR for the (SX + XX) vs. SS comparison was 1.58. After adjusting for age and race, the estimates changed dramatically. The adjusted rates were 1.94 and 0.95 for males and females, respectively. We chose to use these latter rates. The second study 39 reported on a cohort of Greek stroke patients aged

65 – 92 years; control group was age and sex matched. The population was approximately half male and half female. The results were not separated by gender.

Figure B.30 shows the summary analysis for these two studies with a total of 311 cases and

1,030 controls. Overall, the OR is 1.33 (95% CI 0.88 to 2.02), p = 0.2. There was low heterogeneity (Q = 2.2, I² = 11%, p = 0.3).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘B ’ (size: about 120 in the smallest genotype category: 311*2 * 20%

), ‘

B

’ (replication: I 2 estimated to be 11%), and

‘B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis -- Only one study had more than 500 subjects, so no analysis could be performed.

LPL S447X (SX & XX v SS) for Stroke

Model Study name Statistics for each study SS / Total

Odds Lower Upper ratio limit limit p-Value Stroke non-stroke

70 / 93 Fidani 2005

Morrison (males) 2002

1.205

1.940

Random

Morrison (females) 2002 0.950

1.335

0.614

2.365

1.020

3.690

0.464

1.946

0.882

2.020

0.588 77 / 98

0.043

0.888

0.172


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0.2

0.5

1 2 5 10

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Meta Analysis

Favours B


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MTHFR (5,10-methylenetetrahydrofolate reductase)

The MTHFR gene is located on chromosome 1 (1p36.3). According to Entrez Gene,

“Methylenetetrahydrofolate reductase (EC 1.5.1.20) catalyzes the conversion of 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine.

”

Literature search

A HuGE Navigator (V1.1) search on the MTHFR gene identified 1,363 articles on 376 disease terms. Sixty-five meta-analyses or HuGE Reviews were identified. Among these articles, the search ( MTHFR [Text+MeSH]>>Meta-analysis, HuGE

Review[StudyType]>>Arterial Sclerosis, Brain Ischemia, Cardiovascular disease, unspecified, Apoplexy, coronary artery disease, Coronary heart disease, Myocardial

Infarction, Peripheral Vascular Diseases[Mesh]) identified 12 meta-analyses/HuGE Reviews.

We chose to use original meta-analyses that were most recent and most methodologically advanced.

Genotype frequency

For the C677T polymorphism, C represents the wild allele while T represents the presence of the variant. The at-risk genotype (TT) has a 12.2% prevalence in North American

Caucasians.

83 In a general Caucasian population, this translates in approximate allele frequencies of 0.65 and 0.35, respectively (based on more than 2,500 control individuals.

83

The expected genotype frequencies for CC, CT and TT are about 42%, 45% and 12%, respectively.

Coronary Heart Disease (Cardiovascular Disease, Ischemic Heart Disease, Coronary, Heart

Disease, Myocardial Infarction and Angina )

Lewis and colleagues is the most recent meta-analysis in which they explored the association between the MTHFR C677T polymorphism and CHD (using the search terms listed above).

84 Analytic methods included random effects modeling, adjusting for confounding variables, formal tests for heterogeneity and identification of possible publication bias. Overall, 80 studies were included in the analysis with 26,000 cases and

31,183 controls. With all studies included, the summary OR for the TT vs. CC genotypes was 1.14 (95% CI 1.05 to 1.24), p = 0.01. There was moderate hetero geneity (I² = 38%) with much of the variability explained by geographical region (Europe, Australia and North

America showing smaller effects than the Middle East and Asia).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘A’ (size: over 1000 in the smallest genotype category: over 50,000*12% ), ‘ B ’ (replication: I 2 estimated to be 38%), and ‘B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis -- When restricted to the 12 European studies (mainly Caucasians) having 500 or more cases (Figure B.31), the summary OR for the TT vs. CC comparison was 1.03 (95% CI 0.94 to 1.13), p = NS. There was low heterogeneity (Q = 6.4, I 2 = 0%, p =

0.8). Fewer large studies were available from the US, but the summary OR for three large studies 85-87 with >500 cases) was 1.07, (95% CI 0.71 to 1.24), p = 0.6, consistent with the larger experience in Europe (data not shown).


Failsafe N: MTHFR (TT v CC) for CHD


Random

Meisel 2001

Girelli 1998/2003

Rothenbacher 2002

Frederiksen (cohort) 2004

Tobin 2004

Frederiksen C/C 2004

Italian Study Group 2003

Kolling 2004

Kluijtmans 1997

Fernandez-Arcas 1999

Meleady 2003

Kozich (unpub from Klerk 2002



0.820

0.593

1.135

0.860

0.535

1.383

0.880

0.541

1.432

0.960

0.758

1.216

0.970

0.654

1.439

1.040

0.880

1.230

1.060

0.839

1.339

1.070

0.790

1.450

1.210

0.871

1.681

1.220

0.742

2.006

1.240

0.824

1.866

1.280

0.798

2.054

1.032

0.946

1.126

0.231

0.534

0.607

0.735

0.880

0.646

0.625

0.662

0.256

0.433

0.302

0.306

0.478


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1 2 5 10


Figure B.31 Summary analysis of the MTHFR gene (TT vs. CC) and CHD, after

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The meta-analysis by Lewis and colleagues 84 did not contain data for the heterozygote comparison (CT vs. CC genotypes) with CHD. However, an earlier 2002 meta-analysis did.

83 Among 11,162 cases and 12,758 controls, the OR for this comparison was 1.04 (95%

CI 0.98 to 1.10). p = NS . There was significant heterogeneity (I² = 60%). We applied the

Venice criteria to the data and assigned ‘ A ’ for size (more than 1000 subjects with least common genotype), ‘ C ’ for replication (I 2 >50%), and ‘ ‘ for bias (previous grade of C).

Large study analysis - Insufficient data were available to conduct a large study analysis.


A meta-analysis 88 included all published studies (1996 to 2004) using the terms

“cerebrovascular accident and cerebrovascular disorders” and limited studies to those with

“a clinical syndrome consistent with recent ischemic stroke (TIA excluded), with neuroimaging confirmation”. A total of 31 studies were included with 6,110 cases and 8,760 controls. Analytic methods included random effects modeling, formal tests for heterogeneity and identification of possible publication bias. Twelve of these studies were in Asians and one in Blacks; these were removed and the remaining 19 studies reanalyzed by us. There were 3,527 cases and 4,141 controls remaining. The summary OR for the CT vs. CC comparison was 1.03 (95% CI 0.89 to 1.18), p = 0.7. Figure B.32 shows the results ordered by study size, from small to large. Moderate between study heterogeneity was identified (Q

= 31, I 2 = 41%, p = 0.03).


MTHFR Stroke TT vs CC (Cronin)

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Random

Gallai 2001

Pazzini 2003

McLiroy 2002

Topic 2001

Lalousckek 1999

Press 1999

Voetsch 2000

Reuner 1998

Kostulas 1998

Lopacluk 2001

Harmon 1999

Duca 1997

Salooja 1998

Elkelboom 2000

Markus 1997

Kelly 2003

Margaglione 1999

Gaustadnes 1999

Szolnoki 2003

0.893

1.088

0.890

1.000

0.712

1.087

1.051

1.185

1.101

1.173

1.027

3.250

2.170

0.241

1.041

0.846

1.543

0.896

1.459

1.000

0.903

11.696

0.854

5.515

0.116

0.501

0.537

2.020

0.439

1.629

0.803

2.967

0.515

1.558

0.857

2.483

0.592

1.690

0.541

1.477

0.697

1.698

0.573

1.384

0.664

1.506

0.474

1.068

0.727

1.625

0.705

1.565

0.825

1.700

0.804

1.508

0.949

1.450

0.892

1.183

-0.439

0.370

-0.516

0.002

-1.643

0.408

0.244

0.919

0.601

1.476

0.373

1.804

1.627

-3.813

0.120

-0.501

1.302

-0.390

1.393

0.000

0.660

0.712

0.606

0.999

0.100

0.683

0.808

0.358

0.548

0.140

0.709

0.071

0.104

0.000

0.905

0.616

0.193

0.697

0.164

1.000

0.1

0.2

0.5

1 2 5 10


Figure B.32

Re-analysis of 19 MTHFR studies of the C677T polymorphism (CT vs. CC) for stroke


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The corresponding summary OR for the TT vs. CC comparison (Figure B.33) was 1.19 (95%

MTHFR Stroke TT vs CC (Cronin)



Random

McLiroy 2002

Topic 2001

Press 1999

Gallai 2001

Pazzini 2003

Lalousckek 1999

Reuner 1998

Kostulas 1998

Voetsch 2000

Lopacluk 2001

Salooja 1998

Harmon 1999

Elkelboom 2000

Kelly 2003

Markus 1997

Duca 1997

Gaustadnes 1999

Margaglione 1999

Szolnoki 2003

0.256

0.022

3.002

-1.084

17.920

2.096

153.177

2.636

4.500

0.543

37.311

7.000

1.293

37.909

1.394

2.258

3.656

0.989

13.516

0.914

0.323

2.589

1.943

-0.169

0.699

0.276

1.000

0.430

1.772

2.326

-0.754

0.000

1.240

0.550

1.059

0.496

0.933

0.458

1.651

0.850

0.861

0.457

1.487

0.808

0.789

0.436

0.649

0.373

1.373

0.822

2.798

2.262

1.897

3.208

0.518

0.148

-0.193

1.481

1.625

-0.461

2.738

1.274

1.429

-0.782

1.128

-1.533

2.296

1.211

1.569

1.026

1.191

0.874

1.190

0.959

2.400

1.624

1.476

2.077

1.107

1.583

0.604

0.883

0.847

0.139

0.645

0.203

0.434

0.125

0.226

0.278

0.008

0.163

0.024

0.052

0.866

0.451

1.000

0.038

0.268

0.113

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0.2

0.5

1 2 5 10


Figure B.33

Our re-analysis of 19 MTHFR studies of the C677T polymorphism (TT vs.

CC) for stroke

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the MTHFR C677T polymorphism and stroke results in three grades: ‘B ’ (size: approximately 423 cases with a TT genotype (3,527 cases * 12% TT), ‘C ’

(replication: I 2 of 50% or higher for both models, indicating considerable heterogeneity), and

‘-‘ (bias: not evaluated due to an earlier grade of C).


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Large study analysis -- Four studies performed in a Caucasian population reported at least

500 participants.

48,89-91 The summary OR for the CT vs. CC comparison was 1.15 (95% CI

0.99 to 1.33), p = 0.07. There was low heterogeneity (Q = 0.2, I² = 0%, p = 0.9). For the TT vs. CC comparison, the OR was 1.24 (95% CI 0.98 to 1.57), p = 0.08. There was low heterogeneity (Q = 3.6, I² = 17%, p = 0.3). The corresponding data are shown in Figures

B.34 and B.35.

MTHFR Failsafe N: C677T CT vs CC for Stroke



Random

Markus 1997

Margaglione 1999

1.087

0.727

1.625

0.408

0.683

1.185

0.825

1.700

0.919

0.358

Gaustadnes 1999 1.101

0.804

1.508

0.601

0.548

Szolnoki 2003 1.173

0.949

1.450

1.476

0.140

1.147

0.990

1.329

1.829

0.067


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0.2

0.5

1 2 5 10

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MTHFR Failsafe N: C677T TT vs CC for Stroke



Random

Markus 1997

Margaglione 1999

0.789

0.436

1.429

-0.782

0.434

1.569

1.026

2.400

2.077

0.038

Gaustadnes 1999 1.373

0.822

2.296

1.211

0.226

Szolnoki 2003 1.191

0.874

1.624

1.107

0.268

1.237

0.975

1.569

1.754

0.079


0.1

0.2

0.5

1 2 5 10


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MTR (5-methyltetrahydrofolate-homocysteine methyltransferase)

The MTR gene (aka MS – methionine synthase) is located on chromosome 1 (1q43). According to Entrez Gene, “MTR encodes the enzyme 5-methyltetrahydrofolate-homocysteine methyltransferase. This enzyme, also known as cobalamin-dependent methionine synthase, catalyzes the final step in methionine biosynthesis. Mutations in MTR have been identified as the underlying cause of methylcobalamin deficiency complementation group G.

”

Literature search

A HuGE Navigator (V1.1) search on the MTR gene identified 124 articles on 83 disease terms. Five meta-analyses or HuGE Reviews were identified; none were relevant to CVD.

The search ( MTR [Text+MeSH]>>Cardiovascular disease, unspecified, Apoplexy,

Cerebrovascular Disorders, coronary artery disease, Coronary heart disease, Ischemia,

Myocardial Infarction, Myocardial ischemia[Mesh]) identified nine studies. Of these, five contained sufficient data for either CHD or stroke. One of these studies 92 was removed because controls were patients undergoing clinically indicated angiography that had negative findings. The remaining three studies are summarized below.


The polymorphism of interest in the MTR gene is A2756G, where A is the wild allele and G is the at-risk allele. The at-risk genotype is GG with a frequency of about 3%. The allele frequencies for A and G are 0.84 and 0.16, respectively (based on 361 control individuals.

93,94 The expected genotype frequencies of AA, AG and GG would be about

71%, 26% and 3%, respectively.

Coronary Heart Disease (Myocardial Infarction-MI, Coronary Heart Disease – CHD, Coronary

Artery Disease - CAD)

One study 95 studied the A2756G polymorphism in a Caucasian population of 381 cases

(confirmed MI) and 767 control individuals. The OR for the AG vs. AA comparison (without adjustments) was 1.03 (95% CI 0.78 to 1.35), p = 0.8. The OR for the GG vs. AA comparison (with adjustments) was 0.56 (95% CI 0.23 to 1.31), p = 0.1. The authors adjusted these ORs for age and smoking status as well as for age, smoking status, MTHFR genotype, diabetes, angina, hypertension, BMI, aspirin use, alcohol intake and family history

(adjusted OR 0.97 and 0.51). None of the adjusted ORs were statistically significant.

Another study 96 studied 123 individuals with CHD (patients undergoing angiography with at least 90% occlusion in one major coronary vessel and at least 40% occlusion in another major coronary vessel) and 540 control individuals. The OR for the AG vs. AA comparison

(without adjustments) was 1.01 (95% CI 0.65 to 1.58), p = NS. The OR for the GG vs. AA comparison (with adjustments) was 3.48 (95% CI 1.48 to 8.54), p <0.001. The authors adjusted these ORs for age and sex (adjusted OR 0.83 and 3.34) as well as for age, sex, total cholesterol, systolic and diastolic blood pressure, current smoking and alcohol use

(adjusted OR 0.71 and 4.00). A third study 93 did not find any association between this polymorphism (allelic odds ratio 1.00) and CAD in 530 patients (angiographically defined, with >50% occlusion in at least one major coronary or peripheral vessel) and 248 age and sex matched control individuals. We estimated the numbers of cases and controls with each genotype under Hardy-Weinberg in order to combine this information with the other two studies (365, 150 and 15 in cases and 171, 70 and 7 in controls for AA, AG and GG genotype, respectively). A fourth study 94 also reported no association between MTR


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1665 genotype and CAD in 140 patients (before age 55, at least one episode of MI, angina pectoris and/or coronary artery bypass surgery) and 113 control individuals matched for age, sex and geographic area. The allele frequencies were again provided and we estimated the genotypes (101, 36, 3 in cases and 82, 29 and 3 in controls).

We combined results from these four studies using a random effects model (Figures B.36 and B.37). Overall, there were 1,175 cases and 1,668 controls. The OR for the AG to AA comparison was 1.02 (95% CI 0.85 to 1.22) p = 0.8. There was low heterogeneity (Q = 1.9,

I 2 = 0%, p = 0.7). The OR for the GG to AA comparison was 1.25 (95% CI 0.52 to 3.0), p =

0.6. There was low heterogeneity (Q = 4.5, I 2 = 10%, p = 0.3).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the MTR A2756G polymorphism and CHD results in three grades:

‘C’ (size: approximately 23 cases with a TT genotype (1,176 cases * 3% TT), ‘A’

(replication: I 2 of 10% or lower for both models, indicating considerable heterogeneity), and

‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study Analysis – Because three of these four studies already included 500 or more

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1671 summary analysis shown in Figure B.36 and B.37.


Random

Chen 2001

Klerk 2003

Urreitzi 2007

Gueant-Rodriguez 2005

Odds ratio


Lower limit


1.03

1.01

1.01

1.00

1.02

0.78

0.65

0.57

0.72

0.85

1.36

1.57

1.78

1.41

1.22

0.8327

0.9650

0.9785

0.9819

0.8581


0.1

0.2

0.5

1 2 5 10


Figure B.36 Analysis of four MTR studies of the A2745G polymorphism (AG vs. AA) for CAD

Meta Analysis

MTR: A2756G GG vs AA for CAD

Model

Random

Study name

Chen 2001

Klerk 2003

Urreitzi 2007

Gueant-Rodriguez 2005

Odds ratio

0.56

3.48

1.22

1.00

1.25


Lower limit

0.23

1.45

0.20

0.40

0.52

Upper limit

1.34

8.36

7.46

2.51

3.02

p-Value

0.1914

0.0053

0.8313

0.9933

0.6251


0.1

0.2

0.5

1 2 5 10


Figure B.37 Analysis of four MTR studies of the A2745G polymorphism (GG vs. AA) for CAD


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Stroke (Ischemic Cerebral Vascular Disease)

One study 97 examined 131 cases (with cortical lesions documented by CAT scan or MRI) and 121 control individuals. All were European Caucasians (Sicily). The OR for the AG to

AA comparison is 1.18 (95% CI 0.67 to 2.07), p = 0.5. The corresponding OR for the GG to

AA comparison is 4.00 (95% CI 0.41 to 96), p = 0.2. Only one homozygote control and four cases were included in the study, leading to the wide confidence interval. No other studies were identified. Based on this single study, we conclude there is low confidence in a nonsignificant relationship between stroke and the MTR A2756G polymorphism. There is not enough evidence to report a summary measure.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the MTR A2756G polymorphism and stroke results in three grades:

‘C’ (size: fewer than 100 with a TT genotype, ‘C’ (replication: single study), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis – Not performed as no studies had 500 or more study subjects.


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MTRR (5-methyltetrahydrofolate-homocysteine methyltransferase reductase)

The MTRR gene (aka MSR - methionine synthase reductase) is located on chromosome 5

(5p15.3-p15.2). The most common polymorphism studied is A66G. According to Entrez

Gene, “[m]ethionine is an essential amino acid required for protein synthesis and onecarbon metabolism. Its synthesis is catalyzed by the enzyme methionine synthase.

Methionine synthase eventually becomes inactive due to the oxidation of its cob(I)alamin cofactor. The protein encoded by this gene regenerates a functional methionine synthase via reductive methylation. It is a member of the ferredoxin-NADP(+) reductase (FNR) family of electron transferases. Patients of the cbl-E complementation group of disorders of folate/cobalamin metabolism are defective in reductive activation of methionine synthase.

Alternative splicing of this gene results in multiple transcript variants encoding distinct isoforms.

”

Literature search

A HuGE Navigator (V1.1) search on the MTRR gene identified 97 articles on 66 disease terms. Four meta-analyses were identified; none were relevant. The search

( MTRR [Text+MeSH]>>Cardiovascular disease, unspecified, Cerebrovascular Disorders, coronary artery disease, Coronary heart disease, Myocardial ischemia[Mesh]) identified seven studies. Three of these seven were excluded because one reported on pediatric stroke, another on DNA damage and a third was conducted in a patient sub-population.

One of these studies 92 was removed because controls were patients undergoing clinically indicated angiography that had negative findings. The remaining three studies provided sufficient information for analysis; three for CAD and one for stroke. These are summarized below.


For the A66G polymorphism, A represents the wild allele while G represents the presence of the variant. The at-risk genotype (GG) with a prevalence of 22%. In a general Caucasian population, allele frequencies are 0.53 and 0.47, respectively (based on about 234 control individuals.

94,97 The expected genotype frequencies were 28%, 50% and 22%, respectively.

Coronary Heart Disease (Coronary Artery Disease – CAD, Cardiovascular Disease – CVD)

Three articles 93,94,98 provided information about MTRR genotype and CAD risk (usually defined as one or more major arteries with 50% or more occlusion). The studies included a total of 1,052 cases and 483 controls. Two of the studies 93,94 provided only allele frequencies and we estimated genotype frequencies under the assumption of Hardy-

Weinberg (both studies reported agreement with HWE). We combined all three studies together in a random effects model (Figure B.38) and found a summary OR of 1.06 (95% CI

0.53 to 2.13), p = 0.9. Heterogeneity was high (Q = 12, I ² = 83%, p <0.003). Of the three studies, two agreed that the GG genotype was associated with ORs of 1.3 to 1.7, with the third 93 reporting an OR roughly equivalent to the reciprocal (reported OR of 0.57, 1/0.57 =

1.8).


MTRR (AG + GG) vs. AA for CAD

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Model

Random

Study name

Brilakis

Gueant-Rodriguez

Urreitzi

Odds ratio

1.312

0.568

1.732

1.063


Lower limit

0.795

0.378

0.960

0.532

Upper limit

2.167

0.853

3.127

2.125

p-Value

0.288

0.006

0.068

0.862


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Figure B.38 Analysis of three MTRR studies of the A66G polymorphism (AG+GG vs.

AA) for CAD

Meta Analysis

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the MTRR A66G polymorphism and CAD results in three grades:

‘B’ (size: 330 subjects with the GG genotype (about 0.22 * 1500)), ‘C’ (replication: high heterogeneity with I 2 >50%), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis – Only two studies had 500 or more subjects 98 so no analysis is possible.

Stroke (Ischemic Cerebrovascular Disease - ICVD)

One article 97 reported on 131 Italians (Sicily) with ischemic stroke and corticoid lesions documented by imaging at least one year earlier. Controls consisted of 121 individuals with a normal clinical report (exclusion criteria also included any family history of hypertension,

CVD, diabetes or cerebral occlusions). The A66G polymorphism was measured in all. The

OR (AG+GG v AA) was 0.79 (95% CI 0.45 to 1.40), p = NS.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the MTRR A66G polymorphism and stroke results in three grades:

‘C’ (size: fewer than 100 subjects with an AA genotype), ‘C’ (replication: single study), and ‘-

‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis – Not performed as there was only one large study.


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NOS3 (nitric oxide synthase 3 (endothelial cell))

The NOS3 gene (aka eNOS; ECNOS; NOS III ) is located on chromosome 7 (7q36). The most common polymorphisms are G894T (aka Gly298Asp), intron4, T786C. According to Entrez

Gene, “Nitric oxide is a reactive free radical which acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. Nitric oxide is synthesized from L-arginine by nitric oxide synthases. Variations in this gene are associated with susceptibility to coronary spasm. Multiple transcript variants encoding different isoforms have been found for this gene.

”

Literature search

A HuGE Navigator (V1.1) search on the NOS gene identified 1359 articles on 455 disease terms. A search for meta-analyses ( NOS [Text+MeSH]>>Meta-analysis, HuGE Review,

Clinical trial[StudyType]) identified 11 such studies. The most important one 99 is summarized below.




G894T (or Glu298Asp) G represents the wild allele, while T represents the presence of the at-risk polymorphism. The at-risk genotype is TT with a 25% prevalence. In a general Caucasian population, allele frequencies are 0.50 and 0.50, respectively (based on over 13,042 controls individuals.

99 Under Hardy-Weinberg, the three genotypes separately would be approximately 25%, 50% and 25%, respectively.



Intron4 B (4B) represents the wild allele, while A represents the presence of the at-risk polymorphism. The at-risk genotype is AA with a 2% prevalence. In a general

Caucasian population, allele frequencies are 0.86 and 0.14, respectively (based on

3,222 control individuals).




T786C C represents the wild allele, while T represents the presence of the at-risk polymorphism. The at-risk genotype is TT with a 18% prevalence. In a general

Caucasian population, allele frequencies are 0.58 and 0.42, respectively (based on

13,562 control individuals.


Coronary Heart Disease

The 2006 meta-analysis 99 summarized the published literature in Caucasians for the association between CHD and the three NOS3 polymorphisms referred to above. Analytic methods included random effects modeling, formal tests for heterogeneity and identification of possible publication bias.



G894T Overall, 40 studies were included in the analysis with 13,876 cases and 13,042 controls were included in the analysis. The OR for an allele model was 1.17 (95% CI

1.07 to 1.28), p = 0.001. There was significant heterogeneity (I 2 =68%, p <0.001). In this model, the OR of 1.17 would be for the wild vs. heterozygote comparison while the corresponding OR for the wild vs. homozygotes comparison would be 1.37 (1.17*1.17) would be. Larger studies provided lower OR estimates (1.42, 1.18, 0.97 for study sized of <200, 200 to 499 and >500 subjects, respectively).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the NOS3 G298A polymorphism and CHD results in three


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Large study analysis – The authors also examined the nine studies of 500 or more cases

(7,321 cases, 3,447 controls), and found the OR was 0.97 (95% CI 0.87 to 1.09), p =

NS. However, there was still significant heterogeneity (I 2 = 61%).



Intron 4 Overall, 31 studies were included in the analysis with. Overall 9,925 cases and

9,407 controls were included in the analysis. The OR for an allele model was 1.12 (95%

CI 1.01 to 1.24), p = 0.02. There was high heterogeneity (I 2 = 55%, p <0.001). Larger studies again provided lower estimates (1.16, 1.14, 1.02, for the same three size categories).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the NOS3 Intron 4 polymorphism and CHD results in three grades: ‘A’ (size: more than 1,000 subjects in with the at-risk allele, ‘C’ (replication: I 2 over 50% indicating high heterogeneity), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis – The authors also examined the six studies of 500 or more cases

(5,128 cases, 2,413 controls), and found the OR was 1.02 (95% CI 0.84 to 1.23), p =

NS. There was still significant heterogeneity remaining (I 2 = 69%).



T786C Overall, 22 studies were included in the analysis with. Overall, 11,236 cases and 24,798 controls were included in the analysis. The OR for an allele model was 1.17

(95% CI 1.07 to 1.28), p=0.001. Heterogeneity was high (I 2 =63%, p <0.001). Larger studies provided lower estimates (1.05, 1.32 and 1.03 for the same three size categories).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the NOS3 T786C polymorphism and CHD results in three grades: ‘A’ (size: more than 1,000 subjects in with the at-risk allele), ‘C’ (replication: I 2 over 50% indicating high heterogeneity), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis – The authors also examined the seven studies of 500 or more cases (7,158 cases, 4,502 controls), and found the OR was 1.03 (95% CI 0.96 to 1.11), p = NS. The remaining heterogeneity was low (I 2 = 1%).


The same 2006 meta-analysis 99 also summarized the published literature in Caucasians for the association between ischemic stroke and the G894T polymorphism. Overall, three studies were included with 1,086 cases and 1,089 controls. The OR for the recessive model was 0.98 (95% CI 0.76 – 1.26), p = NS. The author noted that since that analysis had been performed, two additional studies 101,102 have also reported no significant associations between G894T and stroke. Figure B.39 contains our summary of all five studies, for both


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Model Group by

Subgroup within study hetero hetero hetero hetero hetero

Random hetero homo homo homo homo homo

Random homo

Random Overall the heterozygous and homozygous combinations. Overall, there were 1,791 cases and

1,984 controls. For the first comparison (CT vs. CC), the OR is 0.91, (95% CI 0.71 to 1.17), p=0.5. There was high heterogeneity (Q = 12.7, I 2 = 69%, p = 0.01). For the second comparison (TT vs. CC), the OR is 1.05 (95% CI 0.81 to 1.36), p = 0.7. There was moderate heterogeneity (Q = 5.2, I 2 = 24, p = 0.3).


Odds ratio and 95% CI Study name

Markus 1998

Elbaz 2000

Hassan 2004

Szolnoki 2005

Subgroup within study

MacLeod M 1999 hetero hetero hetero hetero hetero

MacLeod M 1999 homo

Markus 1998

Elbaz 2000

Hassan 2004

Szolnoki 2005 homo homo homo homo

Statistics for each study T allele / Total

Odds Lower Upper ratio limit limit p-Value Stroke Controls

0.870

0.625

1.212

1.387

0.972

1.981

0.620

0.468

0.821

0.971

0.720

1.310

0.918

0.674

1.249

0.912

0.710

1.171

1.217

0.709

2.086

1.007

0.606

1.674

0.722

0.481

1.081

1.401

0.894

2.197

1.154

0.574

2.319

1.045

0.808

1.351

0.974

0.814

1.165

0.410 125 / 234 203 / 357

0.072 187 / 314 104 / 202

0.001 187 / 399 232 / 395

0.847 138 / 255 294 / 536

0.585 179 / 384 137 / 281

0.468

0.476 31 / 140 36 / 190

0.977 47 / 174 36 / 134

0.114 61 / 273 65 / 228

0.141 42 / 159 62 / 304

0.687 23 / 228 14 / 158

0.739

0.774

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Figure B.39 Analysis of five NOS3 studies of the G894T polymorphism (GT vs. GG and

TT vs. GG) and stroke

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the NOS3 G894T polymorphism and stroke results in three grades:

‘B’ (size: about 950 subjects with the TT genotype - 25% of about 3,800), ‘C’ (replication: high heterogeneity, especially in the heterozygote comparison), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis

– all five studies enrolled 500 or more subjects, so the Large study OR is the same as that shown in Figure B.39.


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PAI-1 (plasminogen activator inhibitor type 1)

The PAI1 gene (aka serpin peptidase inhibitor, serpine1, serpine, PAI, PLAT) is located on chromosome 7 7q21.3-q22). The most widely studied polymorphism is 4G/5G. There was no relevant information found on NCBI Entrez Gene.

Literature search

A HuGE Navigator (V1.1) search for the PAI-1 gene identified 344 articles on 187 disease terms. A search for meta-analyses ( PAI-1 [Text+MeSH]>>Meta-analysis[StudyType]) identified 6 articles. Three were excluded from analysis due to a non-Caucasian population or because they studied diseases outside of our categorization (venous thrombosis, preeclampsia). Of the remaining three meta-analyses, one studied coronary heart disease 44 and two studied ischemic stroke.

103,104 These articles are summarized below.


For the 4G/5G polymorphism, 4G is the at-risk allele with an allele frequency of 52.5% in a non-Hispanic Caucasian population in the Unites States.

46 Based on the allele frequency previously reported, and assuming Hardy-Weinberg equilibrium, the homozygous wild

(5G5G) would have an expected genotype frequency 22.6% while the homozygous at-risk variant (4G4G) would have an expected genotype frequency of 27.6%. The heterozygote genotype frequency would be 49.8%.


The Boekholdt 2001 meta-analysis 44 included seven studies (eight populations) with a total of 2,813 cases and 3,358 controls. That study reported a fixed effects result for the 4G4G vs. 5G5G comparisons of 1.20 (95% CI 1.04 to 1.39), p = 0.04. We re-analyzed and confirmed the OR (after adjusting one reported case number from 106 to 160) using a random effects model and found the heterogeneity to be high (Q = 27.6, I 2 = 75%, p <0.001).

The authors did not mention the possibility of publication bias, but among the four smallest studies (under 250 subjects), three of the four had ORs above 2, while the ORs of the three largest studies were close to 1.0. We reanalyzed the data u sing the ‘trim and fill’ method of

Duval and Tweedie 105 to account for publication bias and the revised OR was 1.16 (95% CI

0.90 to 1.51), p = NS. The originally published OR of 1.20 will be used as the literature’s estimate.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the PAI-1 4G/5G polymorphism and MI results in three grades: ‘ B ’

(size: about 500 with the 4G4G genotype - 25% of 2000 subjects), ‘C’ (replication: high heterogeneity with I 2 >50%), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis: -- When restricted to the three studies of 500 subjects or more (Figure

B.40), the OR for this comparison was 1.06 (95% CI .89 to 1.27), p = 0.5. Heterogeneity was low (Q = 0, I² = 0%, p = 0.9).



Model Study name Statistics for each study CAD / Total


4G4G p-Value and 4G5G 5G5G

Ye 1995 (France) 1.04

Ridker 1997 1.07

Random

Gardemann 1999 1.07

1.06

0.70

1.56

0.8379

92 / 235 66 / 173

0.73

1.56

0.7476 101 / 234 82 / 197

0.85

1.34

0.5791 382 / 791 226 / 484

0.89

1.27

0.5051


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restriction to the three large studies.

Meta Analysis


The Attia 2007 meta-analysis 104 included 12 individual case-control studies that assessed the relationship of PAI-1 polymorphisms and ischemic stroke. Another meta-analysis 103 was excluded even thought it was published the same year and included many of the same studies. That study also included several studies with study populations that are considered beyond the scope of this review. Using a random effects model, the OR reported for the heterozygote comparison (4G5G vs. 5G5G) was 0.99 (95%CI 0.85 to 1.15), p = NS.

Heterogeneity was low (Q=11, I² = 5%, p=0.4) and no publication bias was found (p = 0.3).

The OR for the homozygote comparison (4G4G vs. 5G5G) was 0.89 (95% CI 0.55 to 1.20), p = NS. Heterogeneity was high (Q = 34, I 2 = 68% p <0.001) and no publication bias was found (p = 0.3).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the PAI-1 G455A polymorphism and results in three grades: ‘A’

(size: over 1,000 with the 4G4G genotype - 25% of 6,000 subjects), ‘C’ (replication: high heterogeneity with I 2 >50% in the homozygous comparison), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis – Although the Attia study 104 did not report the number of subjects in each study, only the weights and totals, we used the contemporary meta-analysis by

Tsantes published in the same year 103 to identify large studies (500 or more subjects).

When the analysis was restricted to the four larger studies the OR for the 4G5G vs. 5G5G comparison (Figure B.41) becomes 1.07 (95%CI 0.84 – 1.36), p = 0.6. Heterogeneity is moderate (Q = 5.2, I 2 = 43%, p = 0.2). For the 4G4G vs. 5G5G comparison (Figure B.42), the OR becomes 1.12 (95%CI 0.78 to 1.60), p = 0.6. Heterogeneity is high (Q = 11, I 2 =

72%, p = 0.01).




Random

Catto 1997 4G5G

Crainich 2003 4G5G

Jood 2005 4G5G

Wiklund 2005 4G5G



0.92

1.50

1.12

0.83

1.07

0.59

1.43

0.7120

1.01

2.22

0.0424

0.84

1.50

0.4436

0.57

1.20

0.3206

0.84

1.36

0.5871


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Figure B.41 Summary analysis of the PAI-1 gene (4G5G vs. 5G5G) and stroke, after


Meta Analysis


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1973


Random

Catto 1997 4G4G

Crainich 2003 4G4G

Jood 2005 4G4G

Wiklund 2005 4G4G



0.72

1.64

0.89

1.48

1.12

0.45

1.16

0.1739

1.07

2.52

0.0236

0.65

1.22

0.4739

1.02

2.14

0.0381

0.78

1.62

0.5430


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1 2 5 10


Figure B.42 Summary analysis of the PAI-1 gene (4G4G vs. 5G5G) and stroke, after


Meta Analysis


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PON1 (paraoxonase 1)

The PON1 gene (aka ESA, PON ) is located on chromosome 7 (7q21.3). The most widely studied polymorphisms are Q192R (with the R allele being the at-risk allele) and L55M (with the M allele being the at-risk allele). According to NCBI Entrez Gene , “The enzyme encoded by this gene is arylesterase that mainly hydrolyzes paroxon to produce pnitrophenol. Paroxon is an organophosphorus anticholinesterase compound that is produced in vivo by oxidation of the insecticide parathion. Polymorphisms in this gene aer a risk factor in coronary artery disease. The gene is found in a cluster of three related paraoxonase genes at 7z21.3.

”

A literature search

A HuGE Navigator (V1.3) search on the PON1 gene identified 277 articles on 116 disease terms. Three meta-analyses or HuGE Reviews were identified; two were relevant to CVD; none were relevant to stroke. We excluded one of these meta-analyses because it only reported on one of the polymorphisms of interest.

106 The other meta-analysis 107 reported on two polymorphisms and is summarized in the Coronary Heart Disease sections below. The search for studies that evaluated stroke: ( PON1 [Text+MeSH]>>Cerebral Infarction,

Apoplexy, Apoplexy[Mesh] identified 14 studies. Of these, eight were excluded because they were conducted in patient subpopulations, their outcome of interest was an intermediate outcome or there were no Caucasians in the study population. The remaining

6 studies contained sufficient data for stroke and one or both of the polymorphisms of interest. These are also summarized below.




Q192R polymorphism - Q represents the wild allele and R represents the presence of the variant. In a general Caucasian population, the expected genotype frequencies are

47%, 42% and 11% for the QQ, QR and RR genotypes, respectively.



L55M polymorphism - L represents the wild allele and M represents the presence of the variant. In a general Caucasian population, the expected genotype frequencies are

50%, 40% and 10% for the LL, LM and MM genotypes, respectively.




The Wheeler 2004 meta-analysis 107 included 19 studies that evaluated the association between the Q192R polymorphism and MI. A total of 5,723 cases and 8,063 controls were included. Using a random effects model, the OR reported for the QR vs. QQ comparison was 1.32 (95%CI 1.04 – 1.67), p = 0.03. Heterogeneity was high (I² = 85%).

The OR for the RR vs. QQ comparison was 1.38 (95% CI 1.04 to 1.80), p = 0.02.

Heterogeneity was again high (I²=71%).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the PON1 Q192R polymorphism and MI results in three grades:

‘A’ (size: over 1,000 subjects with the RR genotype - 11% of nearly 14,000 subjects), ‘C’

(replication: high heterogeneity with I 2 >50% in the both comparisons), and

‘-‘

(bias: not evaluated due to an earlier grade of C).

Large study analysis – When restricted to the six large studies these estimates from publication bias (as was also done in the original analysis 107 to avoid possible publication


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2033 bias), the OR for QR vs. QQ became 1.52 (95%CI 1.08-2.15), p = 0.02. The heterogeneity was still high (I² = 91%). The OR for RR vs. QQ comparison became 1.58

(95%CI 1.07-2.33), p = 0.02. The heterogene ity was still high (I² = 81%).



The same 2004 meta-analysis 107 included 9 studies that evaluated the association between the L55M polymorphism and myocardial infarction (only eight studies reported on the MM vs. LL comparison). A total of 3,189 cases and 3,650 controls were included.

Using a random effects model, the OR reported for the LM vs. LL comparison was 0.99

(95%CI 0.86 to1.15), p = 0.9. Heterogeneity was moderate ( I² = 33%). The OR reported for the MM vs. LL comparison was 0.99 (95% CI 0.83 to 1.19), p = 0.9. There was low heterogeneity (Q = 6.4, I² = 0%, p = 0.5).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the PON1 L55M polymorphism and MI results in three grades:

‘ B ’ (size: about 700 subjects with the MM genotype - 10% of nearly 7,000 subjects), ‘ B ’

(replication: moderate heterogeneity with I 2 = 33% in the heterozygous comparison), and

‘B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis – When restricted to the five large studies, the OR for the LM vs. LL comparison became 0.95 (95%CI 0.84 -1.08), p = 0.5. Heterogeneity was low ( I² = 0%).

The OR for the MM vs. LL comparison became 0.95 (95%CI 0.76 – 1.17), p = 0.7. The heterogeneity was low ( I² = 0%).

Stroke (Small vessel disease, large vessel disease, cardio-embolic stroke, cerebrovascular disease, ischemic stroke, arterial ischemic stroke)



Q192R Six articles 108-113 reported the Q192R polymorphism and risk of stroke. Overall,

1,300 cases and 1,468 controls were examined. One study 110 took place in a Polish population and reported three effect measures, each evaluating a different type of stroke; small vessel disease, large vessel disease and cardioembolic stroke. Topic et al.

111 reported on 56 Croatian stroke patients and 124 healthy volunteers. Can

Demird öğen’s study population comprised 108 unrelated, Caucasian patients with acute ischemic stroke from Anatolia, Turkey.

112 A total of 78 controls were selected from the same geographic region and were described as “symptom-free”. Another study,

Schiavon 108 , enrolled 126 survivors that had been referred to the stroke unit of Legnago

Hospital in Legnago, Italy. 92 volunteer controls were matched for sex and age and had no history of stroke or other cardiovascular conditions. In the UK, 397 Caucasian ischaemic stroke patients and 405 controls were evaluated by Pasdar.

109 In the final study, Aydin et al.

113 , 65 patients with acute ischemic cerebrovascular disease from a research hospital in Istanbul, Turkey were evaluated along with 84 healthy volunteers with no history of stroke as the control group.

Using a random effects model, we pooled the results of these six studies (Figure B.43) and found that the summary OR for the QR vs. QQ comparison was 1.00 (95% CI 0.80 to 1.24), p=0.9. There was moderate heterogeneity (Q=11.4, I² = 39%, p = 0.1). The summary OR for the RR vs. QQ comparison (Figure B.44) was 1.35 (95% CI 1.01 to

1.81), p = 0.04) with v ery little heterogeneity (I² = 3%).


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Random

Slowik1 2007

Slowik2 2007

Slowik3 2007

QR

QR

QR

Topic 2001 QR

Demirdogen 2007 QR

Schiavon 2007 QR

Pasdar 2006

Aydin 2006

QR

QR

Subgroup within study Statistics for each study Stroke / Total


QR or RR QQ

0.856

0.545

1.345

0.941

0.597

1.483

0.951

0.656

1.380

0.781

0.404

1.509

0.678

0.363

1.268

1.180

0.665

2.094

1.002

0.749

1.340

3.102

1.447

6.649

0.998

0.803

1.241

0.499 48 / 140 75 / 198

0.793 46 / 134 75 / 210

0.792 105 / 192 151 / 270

0.462

23 / 84 28 / 86

0.224

42 / 82 48 / 79

0.571

56 / 92 58 / 102

0.990 164 / 331 199 / 402

0.004

40 / 75 14 / 52

0.986

0.1

0.2

0.5

1 2 5 10

Fav ours A Fav ours B

Figure B.43 Analysis of six PON1 studies of the Q192R polymorphism (QR vs. QQ) for stroke

Meta Analysis



Random

Slowik1 2007

Slowik2 2007

Slowik3 2007

Topic 2001

Demirdogen 2007 RR

Schiavon 2007 RR

Pasdar 2006

Aydin 2006

RR

RR

RR

RR

RR

RR



QR p-Value or RR QQ

1.991

0.928

4.273

1.800

0.834

3.885

1.051

0.479

2.306

2.071

0.554

7.747

1.661

0.621

4.438

0.759

0.311

1.849

0.990

0.590

1.661

2.714

0.963

7.651

1.354

1.014

1.806

0.077 17 / 31 75 / 198

0.134 15 / 30 75 / 210

0.902 16 / 28 151 / 270

0.279 5 / 10 28 / 86

0.312 18 / 25 48 / 79

0.543 12 / 24 58 / 102

0.970 33 / 67 199 / 402

0.059 11 / 22 14 / 52

0.040


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0.2

0.5

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2086 stroke

Meta Analysis

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the PON1 Q192 polymorphism and stroke and results in three grades: ‘ B ’ (size: about 300 subjects with the MM genotype - 11% of nearly 3000 subjects), ‘ B ’ (replication: moderate heterogeneity with I 2 = 39% in the heterozygous comparison), and ‘B ‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis – Only one study included more than 500 study subjects so no analysis was possible.



L55M All but one of these studies 111 also reported results for the L55M polymorphism and stroke. A total of 1,244 cases and 1,341 controls were included in the analysis.

Using a random effects model, we pooled the results of these studies to obtain a summary OR for the LM vs. LL comparison (Figure B.45) of 1.04 (95% CI 0.88 to 1.23), p = 0.6. There was low heterogeneity (Q = 4.6, I² = 0%, p = 0.6). The summary OR for the MM vs. LL comparison (Figure B.46) was 0.92 (95% CI .72 to 1.19), p = 0.52. There was low heterogeneity (Q = 3.8, I 2 = 0%, p = 0.7).


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2111



Random

Slowik1 2007

Slowik2 2007

LM

LM

Slowik3 2007 LM

Demirdogen 2007 LM

Schiavon 2007

Pasdar 2006

Aydin 2006

LM

LM

LM



LM or MM LL

1.038

0.660

1.631

1.011

0.645

1.584

0.997

0.681

1.460

0.860

0.455

1.627

1.223

0.694

2.154

0.978

0.726

1.318

2.414

1.023

5.693

1.041

0.879

1.233

0.873 67 / 175 55 / 147

0.964 66 / 180 55 / 151

0.987 127 / 232 108 / 197

0.644

41 / 71 54 / 88

0.486 61 / 100 55 / 98

0.883 183 / 369 163 / 325

0.044

42 / 71 12 / 32

0.639

0.1

0.2

0.5

1 2 5 10


Figure B.45 Analysis of five PON1 studies of the L55M polymorphism (LM vs. LL) for stroke

Meta Analysis



Random

Slowik1 2007

Slowik2 2007

MM

MM

Slowik3 2007 MM

Demirdogen 2007 MM

Schiavon 2007

Pasdar 2006

Aydin 2006

MM

MM

MM



LM or MM LL

1.075

0.545

2.122

1.007

0.492

2.062

1.270

0.708

2.281

0.585

0.245

1.393

0.782

0.298

2.048

0.907

0.587

1.400

0.524

0.196

1.403

0.921

0.716

1.185

0.834 18 / 46 55 / 147

0.985 15 / 41 55 / 151

0.423 37 / 61 108 / 197

0.226 13 / 27 54 / 88

0.616 10 / 20 55 / 98

0.658 52 / 109 163 / 325

0.198 11 / 46 12 / 32

0.523


0.1

0.2

0.5

1 2 5 10


Figure B.46 Analysis of five PON1 studies of the L55M polymorphism (MM vs. LL) for stroke

Meta Analysis

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the PON1 L55M polymorphism and stroke and results in three grades: ‘ B ’ (size: about 250 subjects with the MM genotype - 10% of about 2,500 subjects), ‘A’ (replication: low heterogeneity with I 2 <25% for both comparisons), and ‘B‘


Large study analysis – There were too few large studies for analysis.


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SELE (selectin E, E selectin)

The SELE gene (aka ELAM; ESEL; CD62E; ELAM1; LECAM2 ) is located on chromosome 1

(1q22-q25). The most widely studied polymorphism is S128R. According to NCBI Entrez

Gene, “[t]he protein encoded by this gene is found in cytokine-stimulated endothelial cells and is thought to be responsible for the accumulation of blood leukocytes at sites of inflammation by mediating the adhesion of cells to the vascular lining. It exhibits structural features such as the presence of lectin- and EGF-like domains followed by short consensus repeat (SCR) domains that contain six conserved cysteine residues. These proteins are part of the selectin family of cell adhesion molecules. Adhesion molecules participate in the interaction between leukocytes and the endothelium and appear to be involved in the pathogenesis of atherosclerosis.

”

Literature search

A HuGE Navigator (V1.3) search on the SELE gene ( SELE [Text+MeSH]>>Acute Coronary

Syndrome, Arterial Sclerosis, Atherosclerosis, Brain Ischemia, Cardiovascular disease, unspecified, Apoplexy, Cerebrovascular Disorders, coronary artery disease, Coronary heart disease, Ischemia, Myocardial Infarction, Apoplexy[Mesh]) resulted in 29 articles being identified; none were meta-analyses or HuGE reviews. Eight appeared relevant after reviewing the title and abstract and three contained sufficient data relating SELE genotypes and CVD outcomes.

114-116 None of these articles studied a European Caucasian population, but are summarized below.


For the S128R polymorphisms, S is the wild allele and R is the at-risk allele. In a European

Caucasian population of 244 control adults 117 , the S128A genotype frequencies were 81.1%,

18.4% and 0.4% for the SS, SR and RR genotypes, respectively. The corresponding allele frequencies for S and R are 0,904 and 0.096, respectively. Interestingly, the S allele frequency is even higher in South Asians (0.92) and those of African origin (0.96). Due to the low frequency of the RR genotype, SR and RR are usually combined together.

Coronary Heart Disease (Myocardial Infarction, Coronary Artery Disease)

The study by Yoshida 114 was performed in a Japanese population of 135 patients with MI and 327 control subjects. The study by Abu-Amero 116 was performed in an Arabic population of 1,112 patients with angiographically determined CAD (>70% narrowing or at least one vessel) along with 427 controls. However, this study appeared to be performed in a diabetes setting, as 67% of controls and 92% of cases were diabetic. In both of these studies, about two-thirds of cases and controls were males. In the study by Leshinsky-

Silver 115 , the 1,000 patients with CVD (part of the BIP – bezafibrate infarction prevention study) were Israeli, and the 1,480 controls were French, from the Stanislas cohort. An argument can be made that none of these studies are appropriate for use. However, in order to provide some estimate of the effect and of the reliability of existing data, we chose to combine these three studies using a random effects model. The summary OR for the

(SR+RR vs. SS) comparison (Figure B.47) was 1.51 (95% CI 1.21 to 1.90), p <0.001.

Heterogeneity was low (Q = 1.0, I 2 = 0%, p = 0.6).


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Model Study name Statistics for each study R+ / Total

Odds Lower Upper ratio limit limit p-Value CHD not

CHD

Yoshida 2003

Abu-Amero 2007

2.014

1.033

3.928

0.040

17 / 134 22 / 327

1.285

0.688

2.400

0.431

20 / 186 24 / 280

Random

Leshinsky-Silver 2006 1.487

1.149

1.923

0.003 128 / 1000 133 / 1480

1.510

1.207

1.889

0.000


0.1

0.2

0.5

1 2 5

SS) for CHD


The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the SELE S128R polymorphism and CHD and results in three grades: ‘B’ (size: about 650 subjects with the SR+RR genotype - 19% of about 3,500 subjects), ‘A’ (replication: low heterogeneity with I 2 <25% for both comparisons), and ‘C‘

(bias: likely bias due to inappropriate comparison groups and a high prevalence of diabetes in one study).

Large study analysis -- There were too few large studies for analysis.

Stroke

No studies for SELE genotyping and the occurrence of stroke were identified.

10


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SOD2 (superoxide dismutase 2)

The SOD2 gene (aka MNSOD; Mn-SOD) is located on chromosome 6 (6q25.3). The most common polymorphism is C47T. According to NCBI Entrez Gene, “[t]his gene is a member of the iron/manganese superoxide dismutase family. It encodes a mitochondrial protein that forms a homotetramer and binds one manganese ion per subunit. This protein binds to the superoxide byproducts of oxidative phosphorylation and converts them to hydrogen peroxide and diatomic oxygen. Mutations in this gene have been associated with idiopathic cardiomyopathy (IDC), premature aging, sporadic motor neuron disease, and cancer.

Alternate transcriptional splice variants, encoding different isoforms, have been characteri z ed.

”

Literature search

A HuGE Navigator (V1.1) search on the SOD2 gene identified 149 articles on 92 disease terms. No relevant meta-analyses were identified. The search

( SOD2 [Text+MeSH]>>Atherosclerosis, Cardiovascular disease, unspecified[Mesh]) identified four studies. Of these, only one provided CHD or stroke related outcomes.

118


For the C47T polymorphism, C represents the wild allele while T represents the presence of the variant. The at-risk genotype (TT) has a prevalence of 23%. In a general Caucasian population, allele frequencies are 0.52 and 0.48, respectively (based on about 5,773 control individuals.

118 Under Hardy-Weinberg, the expected genotype frequencies are 27%, 50% and 23% for CC, CT and TT, respectively.

Cardiovascular Disease

The 2006 Genkinger article 118 studied ‘deaths due to CVD’ in Maryland (racial distribution not provided). The population was followed-up for 15 years and cause of death identified.

Among the 6,151 individuals studied (2/3 women) a total of 378 deaths due to CVD were observed. Existing sample banks were used to obtain SOD2 genotypes. The OR for the heterozygote comparison (CC vs. CT) was 1.04 (95% CI 0.82 to 1.33), p = NS. The OR for the homozygote comparison (CC vs. TT) was 0.86 (95% CI 0.64 to1.16), p = NS.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the SOD2 C47T polymorphism and CVD results in three grades:

‘B’ (size: about 190 have the TT genotype NB: used 2 * cases as Venice assumes controls are about equal to cases), ‘C’ (replication: single study), and ‘-‘ (bias: not evaluated due to an earlier grade of C).

Large study analysis -- There were too few large studies for analysis.

Stroke

No studies were identified for stroke and SOD2 polymorphisms.


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SOD3 (superoxide dismutase 3)

The SOD3 gene is located on chromosome 4 (4p15.3-p15.1). The most common polymorphism is C47T. According to NCBI Entrez Gene, “[t]his gene is a member of the superoxide dismutase (SOD) protein family. SODs are antioxidant enzymes that catalyze the dismutation of two superoxide radicals into hydrogen peroxide and oxygen. The product of this gene is thought to protect the brain, lungs, and other tissues from oxidative stress. The protein is secreted into the extracellular space and forms a glycosylated homotetramer that is anchored to the extracellular matrix (ECM) and cell surfaces through an interaction with heparan sulfate proteoglycan and collagen. A fraction of the protein is cleaved near the Cterminus before secretion to generate circulating tetramers that do not interact with the

ECM.

”

Literature search

A HuGE Navigator (V1.1) search on the SOD3 gene identified 10 articles on 10 disease terms. Five meta-analyses or HuGE Reviews were identified; none were relevant to CVD.

The search ( SOD3 [Text+MeSH]>>Cardiovascular disease, unspecified[Mesh]) identified two articles.

119,120 Neither of these articles were appropriate for our analysis because they did not evaluate risk of cardiovascular disease; they focused on pulmonary function measures 119 and cigarette-induced cardiovascular disease.

120

No Venice score and no large study analyses were possible.


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TNF (tumor necrosis factor)

The TNF gene (aka DIF; TNFA; TNFSF2; TNF-alpha ) is located on chromosome 6 (6p21.3).

The most widely studied polymorphisms are G308A (A is the at-risk allele) and G238A (A is the at-risk allele). According to NCBI Entrez Gene, “[t]his gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor ( TNF ) superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer .”

Literature search

A HuGE Navigator (V1.3) search ( TNF [Text+MeSH]>>Meta-analysis[StudyType]) identified a total of 33 meta-analyses or HuGE Reviews; two were relevant to CVD. One evaluated coronary artery disease 121 and the other evaluated the risk of ischemic heart disease as well as ischemic stroke.

122




G308A Where G represents the wild allele, while A represents the at-risk allele.

According to the first meta-analysis 121 , the allele frequencies for G and A are 0.83 and

0.17, respectively in 250 controls. Under Hardy-Weinberg, the expected genotype frequencies are 68%, 30% and 2% for GG, GA and AA, respectively.



G238A Where G also represents the wild allele, while A represents the at-risk allele.

The same meta-analysis listed allele frequencies for G and A as 0.94 and 0.06 respectively. Under Hardy-Weinberg, the expected genotype frequencies are 88%, 12% and <1% for GG, GA and AA, respectively. It was estimated that 12.4% of the

Caucasian population has at least one copy of the at-risk allele (GA+AA).

Coronary Heart Disease (Ischemic Heart Disease, Coronary Artery Disease)



G308A According to the 2007 meta-analysis by Pereira and colleagues 122 of the TNF

G308A polymorphism and IHC, there were17 groupings from 14 studies identified. A total of 7,533 IHD cases and 5,678 controls were included. The OR for the dominant model (GA+AA vs. GG) was 1.07 (95% CI 0.94 to 1.21), p = 0.3. There was significant heterogeneity (I 2 = 49%, p = 0.01). Similar results were found when a few of the studies outside of Europe were removed. No clear evidence for publication bias was found, but two studies were considered outliers (one of these required that the participants have diabetes as well). After removal of these two studies, the OR was 1.00 (95% CI 0.92 to

1.09), p = 0.9.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the TNF G308A polymorphism and CHD results in three grades:

‘A’

(size: over 1,000 with the at-risk genotypes GA+AA 32% * 13,000 study subjects), ‘B’ (replication: modest heterogeneity with I 2 as high as 49%), and ‘B‘ (bias: large amount of data relating to bias that was not reported).


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Large study analysis – When restricted to the eight large studies, the OR for the GA+AA vs. GG comparison became 1.01 (95% CI 0.91 to 1.10), p = 0.9. Heterogeneity was low

(Q = 2.7, I² = 0%, p = 0.9).


Odds ratio and 95% CI Model Study name Statistics for each study


A+ / Total

CHD not

CHD

Allen 2001 0.866

0.583

1.285

0.475

53 / 180 107 / 329

Tulyakova 2005 0.868

0.612

1.231

0.426 115 / 455 69 / 246

Georges 2003 0.929

0.698

1.236

0.613 236 / 849 92 / 314

Tobin 2004 1.000

0.774

1.293

0.999 182 / 547 168 / 505

Random

Bennet 2006 1.038

0.880

1.225

0.656 368 / 1167 460 / 1497

Szalai 2002 1.050

0.724

1.523

0.798

89 / 318 67 / 248

Koch 2001 1.072

0.816

1.407

0.618 296 / 998 96 / 340

Antonicelli 2005 1.184

0.805

1.741

0.390

69 / 293 64 / 310

1.008

0.914

1.110

0.877

0.1

0.2

0.5

1 2 5 10


Figure B.48 Summary analysis of the TNF gene (GA+AA vs. GG) and CHD, after restriction to the eight large studies.

Meta Analysis



G238A The 2007 meta-analysis by Pererira 122 did not address this polymorphism, but according to the 2001 meta-analysis by Allen and colleagues 121 , there were two relevant studies. Overall, 1,126 cases and 1,181 controls were included in the analysis. The pooled odds ratio for the dominant model (GA+AA vs. GG) reported in their paper was

0.95 (95% CI 0.71 to 1.27), p = NS. Although no formal test of heterogeneity was performed, the wide confidence intervals for the two studies broadly overlap.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analysis results for the TNF G238A polymorphism and CVD results in three grades: ‘B’ (size: about 156 have the GA+AA genotype – 12% of about 1,300), ‘B’

(replication: not formally computed, but likely to be moderate), and ‘B‘ (bias: large amount of data relating to bias that was not reported).

Large study analysis – There were too few large studies for analysis.


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A 2007 meta-analysis 122 examined the association between TNF polymorphism G308A and risk of Ischemic stroke. The study utilized random effects modeling, formal tests of heterogeneity and checked for publication bias. Five of the eight identified studies were conducted in primarily Caucasian populations and were, therefore, most appropriate for our analysis. The summary OR for the dominant model (GA+AA vs. GG) was 1.23 (95% CI

0.80 to 1.88), p = 0.4. There was high heterogeneity (I² = 77%, p = 0.001).

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘B’ (size: about 400 in the GA+AA group), ‘ C ’

(replication: I 2 over 50% ), and ‘ ‘ (bias: not calculated due to a previous grade of C).

Large study analysis – When the analysis is restricted to the three studies with 500 or more subjects, the OR for the dominant model (GA+AA vs. GG) becomes 0.97 (95% CI 0.51 to

1.55), p = 0.9. The heterogeneity was still high (Q = 10, I 2 Failsafe N Analysis: TNFa (AA+AG v GG) for Stroke

Odds ratio and 95% CI Model Study name Statistics for each study AA & GA / Total

Odds Lower Upper not ratio limit limit p-Value Stroke Stroke

Random

Harcos 2006 0.622

0.440

0.880

0.007 74 / 336 104 / 333

Balding 2004 1.169

0.757

1.808

0.481 46 / 105 156 / 390

Lalouscheck 2006 1.277

0.940

1.736

0.118 122 / 404 105 / 415

0.974

0.611

1.553

0.911

0.1

0.2

0.5

1 2 5 10

restriction to the three large studies.

Meta Analysis


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9p21 SNPs (near CDKN2A and CDKN2B)

The 9p21 SNPs (aka near CDKN2A ; near CDKN2B ) is located on chromosome 9 (9p21). The most widely studied SNPs are rs10757274, rs2981168, rs1333049, and rs2383207. These

SNPs are located near the tumor suppressor genes CDKN2A and CDKN2B, but are in a non-coding region. The SNPs are in strong linkage disequilibrium; it is therefore reasonable to combine them to obtain summary estimates. The mechanism by which these SNPs influence CVD risk is not known.

Literature search

A meta-analysis examining the association between 9p21 SNPs and cardiovascular disease has been previously published.

123 (9p21[Text+MeSH]>>Brain Ischemia, Apoplexy[MeSH]) identified one study.

124 Reviewing the publications from the earlier search identified an additional reference.

125 We identified an electronically published letter via an internet search 126 that was eventually published in 2009. This communication summarized the literature (including known abstracts) regarding stroke and 9p21 SNPs and is summarized below


Although the exact genotype frequencies for each of the 9p21 SNPs vary slightly, the allele frequencies of the at-risk genotype is slightly above (or below) 0.5. We have to assume that, on average, genotype frequencies of 25%, 50%, and 25% for the wild (low risk), heterozygotes (referent group) and homozygotes (higher risk), respectively. The choice of heterozygotes rather than wild type individuals to be the referent group is reasonable, because they are the most common genotype.

CHD/MI

The summary analysis by Palomaki 123 identified 16 articles that met inclusion criteria.

There were 37 distinct datasets comprising four of the SNPs in the 9p21 region to be investigated with respect to coronary heart disease (rs10757274, rs1333049, rs2383207 and rs2891168). The summary OR using a random effects model for individuals with two at-risk alleles compared with one at-risk allele was 1.27 (95% CI 1.23 to 1.32, P <0.001).

Heterogeneity was low (Q=38 , I²=7%, p=0.35). A meta-regression show a significant linear trend with larger ORs being associated with datasets that enrolled cases with younger age-of-onset. The regressed OR was 1.46 for an age cut-off of 50 years and younger, and 1.16 for 80 years and younger. Remaining heterogeneity was low (Q=23,

I²= 0%, p= 0.74).

The application of the Venice criteria for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘ A ’ (size: more than 1,000 cases and controls in the least common genotype ), ‘ A ’ (replication: based on the low heterogeneity for the meta-regression (Q=23

, I²= 0%, p= 0.74) and for the 12 later age-of-onset analyses (Q=9,

I²= 0%, p= 0.58) and ‘ A ’ (the potential for bias was deemed low/not likely).

Stroke

The summary analysis by Di Castelnuovo 126 identified five sources of data for the association of 9p21 SNPs and stroke, including new data they reported from populations in

Italy and Germany. The studies included 1,682 cases and 16,474 controls. Overall, the


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2405 rs10757278 SNP (or another SNP in absolute linkage disequilibrium) was reported for seven populations from four separate studies. The summary OR (using the additive model) was

1.02 (95% CI 0.94-1.11), p = NS. There was no significant heterogeneity reported (p = 0.3).

For the rs10757274 SNP, two studies reported on three populations. For the heterozygous vs. wild comparison the OR was 0.98 (95% CI 0.73 to 1.31), p = NS. For the homozygous at-risk vs. wild comparison, the OR was 1.04 (95% CI 0.74 to 1.47). The heterogeneity was not significant for either comparison (p = 0.9, p = 0.4, respectively). Although I 2 was not reported, all the OR estimates were close to 1.0 and I 2 would be expected to be low.

The application of the Venice criteria 6 for evaluating the credibility of genetic association meta-analyses, results in the three grades of ‘B’ (size: about 400 - 25% * 1,628 in either the low risk or high risk groups ), ‘ A ’ (replication: tests for heterogeneity were not significant).and

‘ B ‘ (bias: large amount of data relating to bias that was not reported as abstracts were included).

Large study analysis – Insufficient data were presented to exclude the two estimates from the rs10757278 analysis of seven studies. One reported an OR slightly below one and the other slightly above. For this reason, we will use the point estimate provided, with five studies of 500 or more subjects. Heterogeneity would be considered low.


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Use of Genomic Profiling to Assess Risk for Cardiovascular Disease

Stroke and ACE I/D

AGT Failsafe N Analysis: MT vs MM for MI

AGT Failsafe N Analysis: TT vs MM for MI

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

FV Failsafe N Analysis: AG + AA vs GG for Stroke

IL1B: CT vs CC for Stroke

IL1B: TT vs CC for Stroke

Failsafe N: IL6 G174C genotype and CAD

IL6 G174C genotype and Stroke

Failsafe N: LPL S447X (SX & XX v SS) for IHD

LPL: Pvull +/- vs -- for CAD

LPL: Pvull +/+ vs -- for CAD

LPL S447X (SX & XX v SS) for Stroke

Failsafe N: MTHFR (TT v CC) for CHD

MTHFR Stroke TT vs CC (Cronin)

MTHFR Stroke TT vs CC (Cronin)

MTHFR Failsafe N: C677T CT vs CC for Stroke

MTHFR Failsafe N: C677T TT vs CC for Stroke

MTR: A2756G GG vs AA for CAD

MTRR (AG + GG) vs. AA for CAD

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

Related documents

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Support

Use of Genomic Profiling to Assess Risk for Cardiovascular Disease

Stroke and ACE I/D

AGT Failsafe N Analysis: MT vs MM for MI

AGT Failsafe N Analysis: TT vs MM for MI

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

FV Failsafe N Analysis: AG + AA vs GG for Stroke

IL1B: CT vs CC for Stroke

IL1B: TT vs CC for Stroke

Failsafe N: IL6 G174C genotype and CAD

IL6 G174C genotype and Stroke

Failsafe N: LPL S447X (SX & XX v SS) for IHD

LPL: Pvull +/- vs -- for CAD

LPL: Pvull +/+ vs -- for CAD

LPL S447X (SX & XX v SS) for Stroke

Failsafe N: MTHFR (TT v CC) for CHD

MTHFR Stroke TT vs CC (Cronin)

MTHFR Stroke TT vs CC (Cronin)

MTHFR Failsafe N: C677T CT vs CC for Stroke

MTHFR Failsafe N: C677T TT vs CC for Stroke

MTR: A2756G GG vs AA for CAD

MTRR (AG + GG) vs. AA for CAD

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

CVD and 9p21 (homozygotes vs heterozygotes)

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