Reference
Complete the bibliographic reference for the article according to AJE format.

Levesque, J., et al. Implication of an AGT Haplotype in a Multigene Association Study With Pregnancy Hypertension. Hypertension. 2004; 43: 1-8.

Category of HuGE information
Specify the types of information (from the list below) available in the article:

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

1. Prevalence of a gene variant
2. Gene-disease association

1. To validate 10 previously published associations by examining 10 polymorphisms in 9 genes.
   
2. To determine if polymorphisms previously found to be associated with preeclampsia are associated with essential hypertension and also to determine if polymorphisms found to be associated with essential hypertension are also associated with preeclampsia.

Gene(s)
Identification of the following:

1. Gene name
2. Chromosome location
3. Gene product/function
4. Alleles
5. OMIM #

1. Gene name: Angiotensinogen (AGT)
2. Chromosome location: 1q42-q43
3. Gene product/function: A ngiotensinogen, which is produced in the liver and also found in the alpha-globulin fraction of plasma produces angiotensin.
4. Alleles:Met235Thr, Thr174Met
5. OMIM #: 106150

1. Gene name: Angiotensin I-Converting Enzyme (ACE)
2. Chromosome location: 17q23
3. Gene product/function: Believed to play an important role for blood pressure regulation and electrolyte balance by hydrolyzing angiotensin I into angiotensin II.
4. Alleles: I intron 16
5. OMIM#: 106181

1. Gene name:5-10 methylenetetrahydrofolate reductase (MTHFR)
2. Chromosome location: 1p36.3
3. Gene product/function: C atalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.
4. Alleles: Ala222Val
   OMIM#:607093

1. Gene name: factor V (F5)
2. Chromosome location: 1q23
3. Gene product/function: Contributes to the activation of prothrombin to the clotting enzyme thrombin.
4. Alleles: Gln506Arg
5. OMIM#: 227400

1. Gene name:Angiotensin Receptor 1 (AGTR1)
2. Chromosome location: 3q21-q25
3. Gene product/function: Receptor for angiotensin II.
4. Alleles: A1166C
5. OMIM#: 106165

1. Gene name: Glutathione S-Transferase, PI (GSTP1)
2. Chromosome location: 11q13
3. Gene product/function: Catalyzes the conjugation of reduced glutathione to hydrophobic and electrophilic compounds.
4. Alleles: Ile105Val
5. OMIM#:134660

1. Gene name:Apolipoprotein B (APOB)
2. Chromosome location: 2p24
3. Gene product/function: Recognition signal for cellular binding and internalization of LDL particles by the APOB/E receptor.
4. Alleles: Thr2488Thr
5. OMIM#: 107730

1. Gene name: Transforming Growth Factor, Beta 1 (TGFB1)
2. Chromosome location: 19q13.1
3. Gene product/function: Peptide that controls proliferation, differentiation, and other functions in many cell types such as transformation and acting as a negative autocrine growth factor.
4. Alleles: Arg25Pro
5. OMIM#: 190180

1. Gene name:tumor necrosis factor- (TNF)
2. Chromosome Location: 6p21.3
3. Gene product/function: Proinflammatory cytokine affecting lipid metabolism, coagulation, insulin resistance, and endothelial function.
4. Alleles: G-308A
5. OMIM#: 191160

1. Age
2. Gestational age at delivery
3. Birth weight
4. Body Mass Index
5. Maximal systolic blood pressure during pregnancy
6. Maximal diastolic blood pressure during pregnancy

Health outcome(s)
Identification of the major health outcome(s) studied

1. Case-control
2. Cohort 
3. Cross-sectional
4. Descriptive or case series
5. Clinical trial
6. Population screening

1. Case-control Study

1. Disease case definition
2. Exclusion criteria
3. Gender
4. Race/ethnicity
5. Age
6. Time period
7. Geographic location
8. Number of participants

1. Disease case definition: Preexisting Chromic Essential Hypertension: used criteria established by the Canadian Hypertension Society. This includes, 2 measurements, at least four hours apart, of diastolic blood pressure ≥ 90mmHg. This must have been reported before pregnancy or < 20 weeks gestational age and persisted 6 weeks without proteinuria.
2. Exclusion criteria: Multiple pregnancies, preexisting secondary hypertension, and preexisting hypertension with superimposed preeclampsia.
3. Gender: Female
4. Race/ethnicity: White, French Canadian descent
5. Age: 28±5 (mean ± standard deviation)
6. Time period: 1996-2000
7. Geographic location: St-François d’Assise hospital in Quebec City , Canada
8. Number of participants: 203 (5.9% of women in original prospective study of development of hypertensive disorders in pregnancy, n=3391)

1. Disease case definition: Preeclampsia: used criteria established by the Canadian Hypertension Society. This is the occurrence of a diastolic blood pressure ≥ 90 mmHg after 20 weeks gestation with proteinuria, followed by the return to normal blood pressure 6 weeks after delivery, without the presence of proteinuria.
2. Exclusion criteria: Gestational hypertension without proteinuria, multiple pregnancies, preexisting secondary hypertension, and preexisting hypertension with superimposed preeclampsia
3. Gender: Female
4. Race/ethnicity: White, French Canadian decent
5. Age: 26±5
6. Time period: 1996-2000
7. Geographic location: St-François d’Assise hospital in Quebec City , Canada
8. Number of participants: 180 (5.3% of women in original prospective study of development of hyptensive disorders in pregnancy, n=3391)

1. Control selection criteria
2. Matching variables
3. Exclusion criteria
4. Gender
5. Race/ethnicity
6. Age
7. Time period
8. Geographic location
9. Number of participants

1. Control selection criteria: Taken from prospective sample of 3391 nulliparous French Canadian pregnant women, investigated for the development of hypertensive disorder at St-François d’Assise hospital.
2. Matching variables: maternal age (±5 years), gestational age at delivery (±1 week), body mass index (kg/m2, <20, 20-24, 25-27, and >27), and month of the year of delivery. Attempted to match 2 control subjects for every case. See Table 1.
3. Exclusion criteria: Multiple pregnancies, preexisting secondary hyptertension, and preexisting hypertension with superimposed preeclampsia.
4. Gender: Female
5. Race/ethnicity: White, French Canadian descent
   Age: 27±5 (for controls matched for Chronic Essential hypertension), 26±5 (for controls matched for preeclampsia)
6. Time period: 1996-2000
7. Geographic Location: St-François d’Assise hospital in Quebec City , Canada
8. Number of participants: 357 controls for Chronic Essential Hypertension case group (10.5% of original prospective study) and 310 controls for Preeclampsia case group (9.76% of original prospective study).

1. Environmental factor
2. Exposure assessment
3. Exposure definition
4. Number of participants with exposure data (% of total eligible)

1. Environmental factor:Not applicable, no environmental factors examined in this study.
2. Exposure assessment:
3. Exposure definition:
4. Number of participants with exposure data:

1. Gene
2. DNA source
3. Methodology
4. Number of participants genotyped (% of total eligible) 

1. Gene(s): ACE, AGT, AGTR1, TGFB1, APOB, F5, GSTP1, MTHFR, TNF
2. DNA source: DNA from whole blood samples
3. Methodology: Purified DNA was stored at -20ºC until time of analysis. All polymorphisms, except for the ACE insertion/deletion polymorphism, were analyzed by means of allele specific PCR. The ACE insertion/deletion was analyzed by PCR. The products of PCR were examined in 1.5% agarose gel using ethium bromide staining. SYBR Green I (molecular probes) was added to each reaction tube to quantify the products by detecting fluorescence.
4. Number of participants genotyped: 100%, All cases and controls for both Preeclampsia and Chronic Essential Hypertension were genotyped for all listed genes and their associated polymorphisms.

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

1. (a.)Prevalence of gene variant for chronic essential hypertension for both present study and published comparison study. Table 2.
   
   (b.) Prevalence of gene variant for preeclampsia for both present study and published comparison study. Table 3.
   
2. (a.)Gene-disease association for chronic essential hypertension for both present study and published comparison study. Table 2.
   
   (b.) Gene-disease association for preeclampsia for both present study and published comparison study. Table 3.

A strong association between a specific AGT haplotype and preeclampsia was observed in the study population, without replicating previous published associations with either preeclampsia or essential hypertension. Results from this study and other studies consistently support the role for AGT in the genetic susceptibility to preeclampsia.

Study Strengths:
This study’s main strength is that it attempts to replicate and verify previous associations that have been published. This study did not replicate any of the previous study results, which demonstrates the importance of performing repeated studies when looking at gene-disease associations.

The use of a nested case-control study provided an homogenous sample population, which added strength to the study.

Study Limitations:
One possible limitation to this study is that no potential confounders or gene environment interactions for preeclampsia and chronic essential hypertension were addressed other than the six factors that were used for matching.

The study attempts to match for several factors, but does not address the matching in the statistical analysis, only using simple chi square analysis.

Recommendations:
The newly reported association between AGT and Thr174Met for preeclampsia should be replicated before any conclusions are made.

Table 1
Table 2
Table 3