Allelic variations in angiogenic pathway genes are associated with preeclampsia

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Allelic variations in angiogenic pathway genes are associated with preeclampsia Sindhu K. Srinivas, MD, MSCE; Alanna C. Morrison, PhD; Christina M. Andrela, MS; Michal A. Elovitz, MD OBJECTIVE: This study investigates the association of allelic variation

in angiogenic pathway genes and preeclampsia. STUDY DESIGN: Data for cases with preeclampsia and term control

subjects were collected prospectively. Maternal DNA was extracted, and 124 tagging single nucleotide polymorphisms in 6 genes (vascular endothelial growth factor A, B, and C; fms-like tyrosine kinase 1 and 4; endoglin) were genotyped. Multivariable logistic regression was used to evaluate the association between tagging single nucleotide polymorphisms and preeclampsia; data were controlled for age. All models were evaluated in black women and white women separately. Haplotype analyses were performed. RESULTS: We analyzed data from 606 women (489 black women [184

cases] and 117 white women [32 cases]). In black women, the fms-like tyrosine kinase 1 rs12584067 (odds ratio [OR], 1.55; 95% confidence

interval [CI], 1.01–2.36; P ⫽ .05) and rs7335588 (OR, 1.61; 95% CI, 1.06 –2.43; P ⫽ .01) and the vascular endothelial growth factor C rs1485766 (OR, 1.56; 95% CI, 1.05–2.30; P ⫽ .03) and rs6838834 (OR, 1.60; 95% CI, 1.05–2.45; P ⫽ .03) single nucleotide polymorphisms were associated with preeclampsia. In white women, the fmslike tyrosine kinase 1 rs722503 (OR, 2.12; 95% CI, 1.07– 4.19; P ⫽ .03), fms-like tyrosine kinase 4 rs307826 (OR, 3.06; 95% CI, 1.18 – 7.91; P ⫽ .01), and vascular endothelial growth factor C rs7664413 (OR, 2.04; 95% CI, 0.99 – 4.17; P ⫽ .04) single nucleotide polymorphisms were associated with preeclampsia. CONCLUSION: Allelic variations in the fms-like tyrosine kinase 1 and

vascular endothelial growth factor C genes are associated with preeclampsia in both ethnic groups. Key words: angiogenesis, gene, preeclampsia, sFlt

Cite this article as: Srinivas SK, Morrison AC, Andrela CM, et al. Allelic variations in angiogenic pathway genes are associated with preeclampsia. Am J Obstet Gynecol 2010;202:445.e1-11.

P

reeclampsia is a pregnancy-specific syndrome that is defined broadly as the presence of hypertension and proteinuria. It affects approximately 5-7% of all pregnancies.1 However, it encompasses a wide spectrum of diseases that range from simple hypertension to the development of seizures and multiorgan system involvement. It is a major contributor to maternal and perinatal morbidity worldwide and accounts for approximately 20% of maternal deaths in the United States.2

The inability to develop satisfactory prevention and prediction strategies for preeclampsia is not due to lack of concerted research effort. A variety of theories have been proposed to explain the development of preeclampsia.1,3-7 Epidemiologic studies have demonstrated consistent risk factors for the development of preeclampsia that have included chronic hypertension, obesity, pregestational diabetes mellitus, collagen vascular disorders, renal insufficiency, and maternal race.8-10 Because observable

From the Maternal and Child Health Research Program, Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women’s Health, University of Pennsylvania School of Medicine, Philadelphia, PA (Drs Srinivas and Elovitz and Ms Andrela), and the Human Genetics Center and Division of Epidemiology and Disease Control, University of Texas Health Science Center at Houston, Houston, TX (Dr Morrison). Presented orally at the 30th Annual Meeting of the Society for Maternal-Fetal Medicine, Chicago, IL, Feb. 1-6, 2010. The racing flag logo above indicates that this article was rushed to press for the benefit of the scientific community. Received Nov. 23, 2009; revised Dec. 29, 2009; accepted Jan. 15, 2010. Reprints not available from the authors. Supported in part by Grant no. K12HD001265 (S.K.S.) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, and by the University Research Foundation, University of Pennsylvania. 0002-9378/$36.00 • © 2010 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2010.01.040

maternal phenotypes alone have not been able to explain most cases of preeclampsia,8 there has been a shift in focus to the evaluation of genetic predispositions to this disease, as has occurred in research of other complex disorders. Suggesting a genetic component to preeclampsia are (1) studies that demonstrate an increased risk of recurrence of preeclampsia in subsequent pregnancies,11,12 (2) studies that observe an increased risk in first-degree relatives of women with preeclampsia,13,14 and (3) familial studies that demonstrate that a family history that is positive for cardiovascular disease also predicts the occurrence of preeclampsia.15 Investigation into the genetic cause of preeclampsia has focused largely on candidate genes that are related to thrombophilias, folate metabolism, lipid metabolism, oxidative stress, and the renin-angiotensin system.3,16 To date, these genetic studies have not yielded consistent results, and investigation of additional candidate gene pathways is warranted. Soluble fms-like tyrosine kinase (sFlt1) is a soluble deactivating re-

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ceptor for vascular endothelial growth factor (VEGF) and placental growth factor. Both VEGF and placental growth factor are potent stimulators of angiogenesis and regulators of endothelial function. It has been demonstrated that increased levels of sFlt1 are associated with preeclampsia and are present in the serum of patients who are destined to have preeclampsia as early as 5 weeks before the onset of clinical symptoms. This suggests a role for this biomarker in the cause of preeclampsia rather than this elevated level being a consequence of the disease.17-21 Levels of endoglin, which is a glycoprotein that is involved in vascular remodeling, have also been elevated in the serum of preeclamptic women compared with control subjects, with significant elevations detectable before the onset of clinical symptoms of preeclampsia.22 Given these findings, genes that are involved in the angiogenesis pathway are suited ideally for exploration in understanding the cause of preeclampsia. This study aimed to investigate whether haplotype-tagging single-nucleotide polymorphisms (tagSNPs) in 6 genes in the angiogenic pathway (VEGFA, -B, -C; FLT1 and -4, and endoglin) are associated with preeclampsia in white and black women. To the best of our knowledge, no other study has investigated the role of tagSNPs in these biologically plausible genes in this number of women with preeclampsia.

M ATERIALS AND M ETHODS Study population We performed a prospective case-control study (Preeclampsia: Mechanisms and Consequences) between March 2005 and August 2007 at the Hospital of the University of Pennsylvania. Institutional Review Board approval was obtained before enrollment began. Cases were women with preeclampsia; control subjects were women who delivered at term (ⱖ37 weeks of gestation) without preeclampsia. All women who were admitted to Labor and Delivery with preeclampsia were eligible for enrollment and were invited to participate in the overall study. Cases were identified on 445.e2

www.AJOG.org the basis of prespecified maternal criteria for preeclampsia, according to standard criteria of the American College of Obstetricians and Gynecologists. Preeclampsia was defined as elevated blood pressure (ⱖ140/90 mm Hg on 2 measurements ⱖ6 hours apart) with ⱖ1⫹ proteinuria. Based on these prespecified criteria, case eligibility was determined at the time of enrollment and not by the treating physician. Control subjects were enrolled prospectively from women who underwent delivery at term (ⱖ37 weeks of gestation) for scheduled induction of labor, scheduled cesarean section delivery, spontaneous rupture of membranes, or term labor. Race and ethnicity were categorized according to the standard National Institutes of Health definitions and were obtained by direct patient selfreport at the time of enrollment. Cases and control subjects in the overall study were frequency matched for self-reported race (black vs other). However, only patients who identified as black or white are included in this analysis. Additional descriptions of this study are provided elsewhere.23-26 Trained research nurses collected information on height, race, ethnicity, and history of hypertension by patient interview at the time of enrollment. Additional medical history was collected from prenatal and hospital chart abstraction by trained research nurses. The primary investigator (S.K.S.) reviewed all data sheets for completeness, consistency, and accuracy. Maternal whole blood was collected from a subset of subjects who were enrolled from 2006-2007 within 24 hours of enrollment. After centrifugation, serum samples were aliquoted and placed at – 80°C. Commercially available enzyme-linked immunosorbent assays were used to measure levels of sFlt1 (nanograms per milliliter) in the maternal serum (R &D Systems, Minneapolis, MN) by personnel who were neither aware of the case-control status nor genotyping results.

Genotyping methods Maternal DNA was derived from whole blood samples that were collected from all women within 24 hours of enroll-

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ment. Specimens were stored and then processed within approximately 6 hours of collection. Samples were genotyped for the IBCv2 array (Illumina Inc, San Diego, CA) that contained 50,000 genecentric SNPs that were selected for their relevance to cardiovascular, metabolic, and inflammatory syndromes.27 From the panel of genotyped SNPs, this study focused on 124 tagSNPs in 6 genes that were involved in angiogenesis. Thirtythree SNPs were monomorphic in white women, which resulted in a total of 91 SNPs that were available for analysis in this ethnic group. Three SNPs were monomorphic in black women, which resulted in a total of 121 SNPs that were available in this ethnic group.

Statistical analyses Within each ethnic group, demographic characteristics were compared between preeclampsia cases and control subjects. For continuous variables, the means were calculated and compared with the use of a Student t test. ␹2 analyses or Fisher’s exact tests were used to compare categoric variables, as appropriate. Agreement of genotype frequencies with Hardy-Weinberg equilibrium expectations was evaluated by a ␹2 goodness-offit test. SNPs that deviated from HardyWeinberg equilibrium (P ⬍ .001) in control subjects were not analyzed. Logistic regression was used to evaluate the association between the angiogenic factor gene SNPs and preeclampsia; all models were adjusted for maternal age. Odds ratios were calculated with an assumption of an additive effect of the SNP alleles. All models were evaluated separately in white and black women. To account for the number of statistical tests that were performed in each ethnic group, a permutation-based procedure that involved 1000 null permutations was performed to generate empiric probability values.28 For genes that demonstrated a significant association with preeclampsia for at least 1 SNP, haplotypes were estimated in the program PLINK28 with the use of the expectation-maximization algorithm.29 Logistic regression was used for the haplotype-based association analyses. For H haplotypes, a single H-1

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TABLE 1

Demographics Variable

Black cases (n ⴝ 184)

Black control subjects (n ⴝ 305)

Mean maternal age, y

26.2 ⫾ 6.2

25.5 ⫾ 6

Mean body mass index at screening, kg/m

30.9 ⫾ 9.8

29.9 ⫾ 8

Chronic hypertension, n (%)

27 (14.7)

16 (5.3)

White cases (n ⴝ 32)

White control subjects (n ⴝ 85)

.22

33.4 ⫾ 5.7

32.3 ⫾ 5.2

.35

.26

26.8 ⫾ 6

25.4 ⫾ 5.8

.25

P value

P value

................................................................................................................................................................................................................................................................................................................................................................................ 2 ................................................................................................................................................................................................................................................................................................................................................................................

⬍ .001

5 (15.6)

2 (2.4)

.007

................................................................................................................................................................................................................................................................................................................................................................................

Pregestational diabetes mellitus, n (%)

6 (3.3)

8 (2.6)

1 (3.1)

2 (2.4)

1.00

Primiparous, n (%)

87 (47.3)

96 (31.5)

⬍ .001

.78

21 (65.6)

39 (45.9)

.06

History of preeclampsia, n (%)

41 (22.3)

27 (8.9)

⬍ .001

5 (15.6)

2 (2.4)

.002

Mean gestational age at delivery, wk

35.8 ⫾ 3.8

39.1 ⫾ 1.3

⬍ .001

35.6 ⫾ 3.4

39 ⫾ 1.2

⬍ .001

Intrauterine grown restriction ⬍10%, n (%)

31 (57)

53 (17)

⬍ .001

4 (4.7)

⬍ .001

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

9 (28)

................................................................................................................................................................................................................................................................................................................................................................................

Srinivas. Allelic variations in angiogenic pathway genes. Am J Obstet Gynecol 2010.

degree of freedom omnibus test was performed that jointly evaluated all haplotype effects. The omnibus test was applied to haplotypes estimated within a sliding window of fixed haplotype size of 2-4 SNPs with the use of the program PLINK.28 The program Graphical Assessment of Sliding P values (GrASP) was used to visualize probability values from the haplotype analyses and to help identify gene regions of interest.30

R ESULTS We analyzed data from 606 women; 489 black women (305 cases) and 117 white women (32 cases) were evaluated. The demographic characteristics of our population are shown in Table 1. Probability values that are reported represent within-race group comparison (black cases vs control subjects and white cases vs control subjects). Of the 91 SNPs that were available for analysis in white women, all were in accordance with Hardy-Weinberg expectations. Of the 121 SNPs in black women, 120 were in accordance with Hardy-Weinberg expectations. The risk allele is defined as the allele that conferred increased risk of preeclampsia in a particular ethnic group. The frequency of the risk allele is reported based on the frequency in control subjects. Results are reported with the use of empiric probability values. In black women, the FLT1 rs12584067 (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.01–2.36; P ⫽ .05) and

rs7335588 (OR, 1.61; 95% CI, 1.06 –2.43; P ⫽ .01) SNPs are associated with preeclampsia and are not in linkage disequilibrium (r2 ⫽ 0.05). Two VEGFC SNPs, rs1485766 (OR, 1.56; 95% CI, 1.05–2.30; P ⫽ .03) and rs6838834 (OR, 1.60; 95% CI, 1.05–2.45; P ⫽ .03) SNPs, were also associated significantly with preeclampsia and not in linkage disequilibrium (r2 ⫽ 0.02). A single VEGFA SNP rs833068 approached significance (OR, 1.28; 95% CI, 0.99 –1.67; P ⫽ .05). No VEGFB, endoglin, or FLT4 SNPs were associated with preeclampsia in black women. In white women, FLT1 rs722503 (OR, 2.12; 95% CI, 1.07– 4.19; P ⫽ .03), FLT4 rs307826 (OR, 3.06; 95% CI, 1.18 –7.91; P ⫽ .01), and VEGFC rs7664413 (OR, 2.04; 95% CI, 0.99 – 4.17; P ⫽ .04) SNPs were associated significantly with preeclamspia. No VEGFA, VEGFB, or endoglin SNPs were associated with preeclampsia in white women (Table 2). Given the significant univariate association with SNPs in FLT1 and VEGFC in black and white women, these genes were selected for haplotype estimation and association with preeclampsia. Although a single SNP in FLT4 was associated significantly with preeclampsia in white women, this was the only tagSNP to be evaluated in white women; therefore, haplotypes were not estimated for FLT4 in white women. The results from an omnibus test that was evaluated for sliding windows of haplotypes comprised of 2-4 SNPs in each window

is shown for FLT1 in Figure 1 and VEGFC in Figure 2. This novel haplotyping approach identified 2 regions of significantly associated SNPs in the FLT1 gene in black women that were not identified by univariate analyses alone. In black women, an omnibus test of haplotypes in FLT1 comprised of 3 SNPs (rs7999615, rs7992940, and rs10507386) was associated significantly with preeclampsia (P ⫽ .033). This region encompassed 27919011 base pairs (bp) to 27926554 bp and corresponds to an intronic gene region. An additional intronic region between 27817330 bp and 27822832 bp was identified in black women by analysis of 3 FLT1 SNPs (rs3936415, rs2296192, and rs9513085) that demonstrated a significant haplotype association with preeclampsia (P ⫽ .047). Haplotype analyses of FLT1 in white women and VEGFC in both ethnic groups did not yield significant additional associations with preeclampsia. We next compared mean serum sFlt levels between cases and control subjects, by risk alleles, given the significant associations between tagSNPs in the FLT1 and VEGFC genes in both ethnic groups. SFlt1 levels were available in 278 black women (74 cases), and 69 white women (18 cases) were evaluated (Table 3). Among black and white cases, there was no significant difference in mean sFlt levels, compared with control subjects by presence of risk alleles in significant tagSNPs.

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TABLE 2

Association between tagging single nucleotide polymorphisms and preeclampsia Single nucleotide polymorphism identification no.

Gene

Risk allele

Risk allele frequency

Odds ratio (95% CI)

P value

Empiric P value

Black women

.......................................................................................................................................................................................................................................................................................................................................................................

Endoglin

rs17557600

T

0.90

1.46 (0.98-2.17)

.06

.057

FLT1

rs12584067

G

0.14

1.55 (1.01–2.36)

.04

.047

FLT1

rs670084

T

0.38

1.29 (0.97–1.71)

.08

.069

FLT1

rs7335588

G

0.93

1.61 (1.06–2.43)

.02

.013

FLT1

rs880704

G

0.92

1.47 (0.94–2.31)

.09

.087

FLT1

rs9319425

C

0.45

1.26 (0.98–1.62)

.07

.07

VEGFA

rs833068

A

0.57

1.28 (0.99–1.67)

.06

.053

VEGFC

rs1485766

A

0.90

1.56 (1.05–2.30)

.03

.026

VEGFC

rs6838834

C

0.91

1.60 (1.05–2.45)

.03

.032

....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................

White women

.......................................................................................................................................................................................................................................................................................................................................................................

FLT1

rs722503

A

0.79

2.12 (1.07–4.19)

.03

.034

FLT4

rs307826

A

0.94

3.06 (1.18–7.91)

.02

.007

VEGFC

rs7664413

C

0.85

2.04 (0.99–4.17)

.05

.036

....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................

CI, confidence interval; FLT, fms-like tyrosine kinase; VEGF, vascular endothelial growth factor. Srinivas. Allelic variations in angiogenic pathway genes. Am J Obstet Gynecol 2010.

C OMMENT The association between genetic variation in 6 angiogenesis pathway genes and preeclampsia was investigated in white and black women from our large case control study. Three SNPs in black women and 3 in white women were statistically associated significantly with preeclampsia, which suggests that genetic variation in FLT1, FLT4, and VEGFC genes may play a role in this disease. The most significant finding was that allelic variants in both the FLT1 and VEGFC genes were associated with preeclampsia in both ethnic groups. Further, a novel haplotyping method revealed additional genetic regions that were not identified by univariate analyses alone. However, interestingly, the allelic variants that were associated significantly with preeclampsia in each ethnic group were not associated with differences in serum sFlt levels within cases and control subjects in those ethnic groups. Evaluation of the role of allelic variants in the angiogenesis pathway is biologically plausible, given the previous literature that 445.e4

suggested that angiogenic and antiangiogenic factors (particularly sFlt1) are associated with preeclampsia.17-21,31-33 However, despite this plausibility, few studies have truly evaluated genetic variations in this pathway in association with adverse pregnancy outcomes that include preeclampsia.34 Papazoglou et al35 evaluated 3 VEGF gene polymorphisms, found no association with preeclampsia in 42 women with preeclampsia, and compared the findings with 73 postmenopausal women. However, they did find increased allele frequency of 1 VEGF 936C/T polymorphism in women with severe preeclampsia, compared with control subjects. Another study that evaluated global placental gene expression in 16 women found an up-regulation of FLT1, FLT4, and endoglin genes in placentas from women with severe preeclampsia.36 One additional study from Korea did not find an association between a functional FLT1 gene polymorphism and preeclampsia.37 To our knowledge, our study is the first to evaluate genes comprehensively in the angiogenic pathway with the use of tagSNPs and a novel haplotype approach in a large number of women with preeclampsia.

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Strengths of our study include the prospective identification of cases and a large number of patients. The diagnosis of preeclampsia was made using an a priori definition, thereby removing the potential bias that could arise from relying on the caring physician’s assessment or ICD-9 coding. Additionally, the size of our study allowed for stratified analyses in 2 ethnic groups, which is an important consideration when genetic studies are performed. Our study is not without limitations. Given the difference in sample sizes between the white and black preeclampsia cases and control subjects, it is not surprising that a particular SNP was not associated consistently with preeclampsia within each ethnic group. It is important to note that, for many loci, there was a clear difference in the minor allele frequency between ethnic groups (data not shown). This difference in minor allele frequency coupled with variable patterns of linkage disequilibrium between the populations does not allow for comparison between white and black women. However, it is also important to note that both VEGFC and FLT1 genes had SNPs that were associated with preeclampsia in both ethnic groups, which

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FIGURE 1

Graphic Assessment of Sliding P values plot of haplotype results for fms-like tyrosine kinase 1 in black and white women

White Women

P

P

PP

PP

Black Women

P

The asterisk denotes uncorrected probability values reported. SNP, single-nucleotide polymorphism. Srinivas. Allelic variations in angiogenic pathway genes. Am J Obstet Gynecol 2010.

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FIGURE 2

Graphic Assessment of Sliding P values plot of haplotype results for vascular endothelial growth factor C in white and black women

White Women

P

P

PP

PP

Black Women

P

The asterisk denotes uncorrected probability values reported. SNP, single-nucleotide polymorphism. Srinivas. Allelic variations in angiogenic pathway genes. Am J Obstet Gynecol 2010.

TABLE 3

Mean (ⴞSD) sFlt serum levels by FLT1 or 4 genotypes Single nucleotide polymorphisms identification no. GENE AA

AB

BB

P value AA

AB

BB

P value

Black control Black cases subjects (n ⫽ 74) (n ⫽ 204) ................................................................................................................................................................................................................................................................................................................................................................................ a rs12584067 FLT1 6.90 ⫾ 4.97 10.79 ⫾ 9.61 11.62 ⫾ 12.36 .73 10.60 ⫾ 0 8.53 ⫾ 6.92 8.41 ⫾ 6.12 .94 ................................................................................................................................................................................................................................................................................................................................................................................ b rs7335588 FLT1 7.85 ⫾ 3.73 11.14 ⫾ 12.61 11.73 ⫾ 11.88 .82 8.11 ⫾ 5.47 8.87 ⫾ 8.88 3.39 ⫾ 5.86 .93 ................................................................................................................................................................................................................................................................................................................................................................................

White control White cases subjects (n ⫽ 18) (n ⫽ 51) ................................................................................................................................................................................................................................................................................................................................................................................ c rs722503 FLT1 15.29 ⫾ 17.83 7.08 ⫾ 8.58 3.4 ⫾ 3.91 .36 11.06 ⫾ 10.77 7.91 ⫾ 4.14 3.50 ⫾ 0 .45 ................................................................................................................................................................................................................................................................................................................................................................................

rs307826d

FLT4

10.43 ⫾ 16.07

11.20 ⫾ 9.42

9.90 ⫾ 0

.99

9.52 ⫾ 8.62

14.86 ⫾ 15.41 —

.23

................................................................................................................................................................................................................................................................................................................................................................................

sFlt, soluble fms-like tyrosine kinase. sFlt levels are in nanograms per milliliter. a

Rs12584067 was associated significantly with preeclampsia in black women (P ⫽ .04); b rs7335588 was associated significantly with preeclampsia in black women (P ⫽ .02); c rs722503 was associated significantly with preeclampsia in white women (P ⫽ .03); d rs307826 was associated significantly with preeclampsia in white women (P ⫽ .02).

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www.AJOG.org implicates these genes in the development of preeclampsia. Additionally, when a case-control study is performed, the control population is subject to scrutiny. Our control population did have women with a history of preeclampsia and conditions such as chronic hypertension and diabetes mellitus. It is possible that this biased our findings toward the null. However, despite this, we found tagSNPs that are associated significantly with preeclampsia in both ethnic groups in 2 genes. Further, there are several possible reasons for the lack of association between the significant FLT1 tagSNPs in our study and serum sFlt levels within each ethnic group: (1) The tagSNPs that are associated with preeclampsia do not represent functional variation; (2) allelic variants in the FLT1 gene are not responsible for the elevated sFlt levels that were seen in women with preeclampsia, and (3) the sample size of the our subset of women is not large enough to see statistically significant differences in serum levels. In summary, this study demonstrated that angiogenic factor genes are associated with preeclampsia. In-depth analysis of functional SNPs in these pathways is now warranted. Further characterization of whether FLT1 and VEGFC, in particular, contribute to increased preeclampsia risk may inform our understanding of the cause of preeclampsia, and the gene products may prove to be targets for future therapeutic or preventive strategies. f REFERENCES 1. Sibai B, Dekker G, Kupferminc M. Preeclampsia. Lancet 2005;365:785-99. 2. Centers for Disease Control. Morbidity and mortality weekly report: pregnancy-related mortality surveillance—United States, 1991–1999. CDC MMWR 2003;52:SS-2. 3. Roberts JM, Cooper DW. Pathogenesis and genetics of pre-eclampsia. Lancet 2001;357:53-6. 4. Wang Y, Gu Y, Zhang Y, Lewis DF. Evidence of endothelial dysfunction in preeclampsia: decreased endothelial nitric oxide synthase expression is associated with increased cell permeability in endothelial cells from preeclampsia. Am J Obstet Gynecol 2004;190:817-24. 5. Redman CW. Current topic: pre-eclampsia and the placenta. Placenta 1991;12:301-8. 6. Robertson SA, Ingman WV, O’Leary S, Sharkey DJ, Tremellen KP. Transforming growth factor beta: a mediator of immune deviation in seminal plasma. J Reprod Immunol 2002;57:109-28. 7. Redman CW, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory

response to pregnancy. Am J Obstet Gynecol 1999;180:499-506. 8. Catov JM, Ness RB, Kip KE, Olsen J. Risk of early or severe pre-eclampsia related to pre-existing conditions. Int J Epidemiol 2007;36:412-9. 9. Bodnar LM, Ness RB, Markovic N, Roberts JM. The risk of preeclampsia rises with increasing prepregnancy body mass index. Ann Epidemiol 2005;15:475-82. 10. Diagnosis and management of preeclampsia and eclampsia. In: American College of Obstetricians and Gynecologists Compendium 2008: practice bulletin, no. 33;2002;2002. 11. Campbell DM, MacGillivray I, Carr-Hill R. Pre-eclampsia in second pregnancy. BJOG 1985;92:131-40. 12. Dildy GA 3rd, Belfort MA, Smulian JC. Preeclampsia recurrence and prevention. Semin Perinatol 2007;31:135-41. 13. Cnattingius S, Reilly M, Pawitan Y, Lichtenstein P. Maternal and fetal genetic factors account for most of familial aggregation of preeclampsia: a population-based Swedish cohort study. Am J Med Genet A 2004;130A:365-71. 14. Dawson LM, Parfrey PS, Hefferton D, et al. Familial risk of preeclampsia in Newfoundland: a population-based study. J Am Soc Nephrol 2002;13:1901-6. 15. Ness RB, Markovic N, Bass D, Harger G, Roberts JM. Family history of hypertension, heart disease, and stroke among women who develop hypertension in pregnancy. Obstet Gynecol 2003;102:1366-71. 16. Lachmeijer A, Dekker G, Pals G, Aarnoudse J, Kate L, Arngrimsson R. Searching for preeclampsia genes: the current position. Eur J Obstet Gynecol Reprod Biol 2002;105:94-113. 17. Levine RJ, Maynard SE, Qian C, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672-83. 18. Salahuddin S, Lee Y, Vadnais M, Sachs BP, Karumanchi SA, Lim KH. Diagnostic utility of soluble fms-like tyrosine kinase 1 and soluble endoglin in hypertensive diseases of pregnancy. Am J Obstet Gynecol 2007;197:28.e1-6. 19. Rana S, Karumanchi SA, Levine RJ, et al. Sequential changes in antiangiogenic factors in early pregnancy and risk of developing preeclampsia. Hypertension 2007;50:137-42. 20. Moore Simas TA, Crawford SL, Solitro MJ, Frost SC, Meyer BA, Maynard SE. Angiogenic factors for the prediction of preeclampsia in high-risk women. Am J Obstet Gynecol 2007; 197:244.e1-8. 21. Romero R, Nien JK, Espinoza J, et al. A longitudinal study of angiogenic (placental growth factor) and anti-angiogenic (soluble endoglin and soluble vascular endothelial growth factor receptor-1) factors in normal pregnancy and patients destined to develop preeclampsia and deliver a small for gestational age neonate. J Matern Fetal Neonatal Med 2008;21:9-23. 22. Levine RJ, Lam C, Qian C, et al. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med 2006; 355:992-1005.

Research

23. Edlow A, Srinivas S, Elovitz M. Investigating the risk of hypertension shortly after pregnancies complicated by preeclampsia. Am J Obstet Gynecol 2009;200:e60-2. 24. Mazar R, Srinivas S, Sammel M, Andrela C, Elovitz M. Metabolic score as a novel approach to assessing preeclampsia risk. Am J Obstet Gynecol 2007;197:411.e1-5. 25. Srinivas SK, Edlow AG, Neff PM, Sammel MD, Andrela CM, Elovitz MA. Rethinking IUGR in preeclampsia: dependent or independent of maternal hypertension? J Perinatol 2009;29:680-4. 26. Srinivas SK, Sammel MD, Bastek J, et al. Evaluating the association between all components of the metabolic syndrome and preeclampsia. J Matern Fetal Neonatal Med 2009; 22:501-9. 27. Keating B, Tischfield S, Murray S, et al. Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies. PLoS One 2008;3:e3583. 28. Purcell S, Neale B, Todd-Brown K, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007;81:559-75. 29. Excoffier L, Slatkin M. Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population. Mol Biol Evol 1995;12:921-7. 30. Mathias RA, Gao P, Goldstein JL, et al. A graphical assessment of p-values from sliding window haplotype tests of association to identify asthma susceptibility loci on chromosome 11q. BMC Genet 2006;7:38. 31. Shibata E, Rajakumar A, Powers RW, et al. Soluble fms-like tyrosine kinase 1 is increased in preeclampsia but not in normotensive pregnancies with small-for-gestational-age neonates: relationship to circulating placental growth factor. J Clin Endocrinol Metab 2005;90:4895-903. 32. McKeeman GC, Ardill JE, Caldwell CM, Hunter AJ, McClure N. Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is increased throughout gestation in patients who have preeclampsia develop. Am J Obstet Gynecol 2004;191:1240-6. 33. Chedraui P, Lockwood CJ, Schatz F, et al. Increased plasma soluble fms-like tyrosine kinase 1 and endoglin levels in pregnancies complicated with preeclampsia. J Matern Fetal Neonatal Med 2009;565-70. 34. Rogers MS, D’Amato RJ. The effect of genetic diversity on angiogenesis. Exp Cell Res 2006;312:561-74. 35. Papazoglou D, Galazios G, Koukourakis MI, et al. Vascular endothelial growth factor gene polymorphisms and pre-eclampsia. Mol Hum Reprod 2004;10:321-4. 36. Sitras V, Paulssen RH, Gronaas H, et al. Differential placental gene expression in severe preeclampsia. Placenta 2009;30:424-33. 37. Kim SY, Lim JH, Yang JH, et al. Dinucleotide repeat polymorphism in Fms-like tyrosine kinase-1 (Flt-1) gene is not associated with preeclampsia. BMC Med Genet 2008;9:68.

MAY 2010 American Journal of Obstetrics & Gynecology

445.e7

Research

Obstetrics

www.AJOG.org

APPENDIX

Summary of all SNPs analyzeda Blacks (n ⴝ 489)

Variable

b

Whites (n ⴝ 117)

Marker

SNP ID

Gene name

Odds ratio

P value

Odds ratiob

P value

M341

rs10121110

ENG

1.08

.59

0.94

.84

M342

rs10760503

ENG

1.04

.79

1.27

.38

M343

rs10819309

ENG

0.97

.87

1.19

.56

M344

rs11792480

ENG

0.77

.37

0.87

M345

rs12001427

ENG

M346

rs17557600

ENG

M347

rs1800956

ENG

M348

rs2900256

ENG

1.40

.50

M349

rs3739817

ENG

1.23

.57

1.73

.36

M350

rs4836585

ENG

0.90

.45

1.17

.70

M351

rs4837192

ENG

0.95

.71

1.30

.51

M352

rs7034078

ENG

1.01

.96

M353

rs10507386

FLT1

0.76

.12

1.03

.95

M355

rs11149520

FLT1

0.83

.23

1.42

.30

M356

rs11838619

FLT1

0.75

.12

M357

rs12428494

FLT1

1.22

.62

M358

rs12429309

FLT1

1.05

.75

M359

rs12584067

FLT1

1.55

.04

M360

rs12858139

FLT1

0.75

.10

0.81

.52

M362

rs1408243

FLT1

1.04

.86

0.62

.42

M363

rs1408245

FLT1

0.80

.20

1.34

.51

M364

rs1555641

FLT1

1.19

.25

1.09

.81

M365

rs17086565

FLT1

1.16

.66

mono

M366

rs17086574

FLT1

1.32

.38

mono

M367

rs17086586

FLT1

0.73

.31

mono

M368

rs17086602

FLT1

0.93

.72

mono

M369

rs17086609

FLT1

1.30

.26

0.96

M370

rs17086620

FLT1

1.33

.11

0.28

M371

rs17086636

FLT1

0.65

.20

mono

M372

rs17086729

FLT1

0.76

.25

mono

M373

rs17086741

FLT1

0.85

.54

mono

M374

rs17625898

FLT1

1.03

.87

M375

rs2147188

FLT1

1.03

.93

M376

rs2296187

FLT1

1.14

.64

0.32

.29

M377

rs2296188

FLT1

1.19

.19

2.07

.13

M378

rs2296190

FLT1

0.80

.42

1.07

.88

M379

rs2296192

FLT1

0.98

.91

0.82

.78

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

.66

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

1.46

.06

1.11

.85

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

1.28

.54

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

.89

................................................................................................................................................................................................................................................................................................................................................................................

.10

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

0.66

.48

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................

Srinivas. Allelic variations in angiogenic pathway genes. Am J Obstet Gynecol 2010.

445.e8

American Journal of Obstetrics & Gynecology MAY 2010

(continued )

Obstetrics

www.AJOG.org

Research

APPENDIX

Summary of all SNPs analyzeda

(continued)

Blacks (n ⴝ 489)

Variable

b

Whites (n ⴝ 117) Odds ratiob

P value

0.66

.72

.42

1.15

.65

1.24

.21

1.08

.79

FLT1

1.64

.12

FLT1

1.20

.40

1.13

.83

rs3794402

FLT1

1.18

.23

1.53

.57

M389

rs3936415

FLT1

0.83

.20

0.92

.82

M391

rs6491283

FLT1

1.20

.39

1.43

.77

M392

rs670084

FLT1

1.29

.08

0.53

.20

M393

rs674834

FLT1

1.02

.90

M394

rs678714

FLT1

0.70

.37

0.89

M395

rs679791

FLT1

0.84

.22

0.84

M396

rs7139483

FLT1

0.90

.63

M397

rs718273

FLT1

1.33

.26

M398

rs720661

FLT1

1.11

.47

M399

rs722503

FLT1

1.08

.64

M400

rs7319123

FLT1

0.89

.59

mono

M401

rs7319213

FLT1

0.78

.24

mono

M402

rs7323184

FLT1

0.95

.71

0.35

M403

rs7326277

FLT1

0.94

.63

2.18

M405

rs7335588

FLT1

1.61

.02

M406

rs7337610

FLT1

0.94

.67

M407

rs7337866

FLT1

0.91

.55

M408

rs7982957

FLT1

0.89

.40

1.16

.75

M409

rs7987291

FLT1

0.96

.78

0.92

.82

M410

rs7987649

FLT1

1.10

.46

0.88

.70

M411

rs7992940

FLT1

0.97

.80

1.04

.90

M412

rs7995813

FLT1

0.85

.37

M413

rs7995976

FLT1

0.92

.56

0.90

.75

M414

rs7999615

FLT1

0.77

.15

0.86

.81

M415

rs880703

FLT1

1.21

.40

M416

rs880704

FLT1

1.47

.09

M417

rs9319425

FLT1

1.26

.07

1.12

.70

M418

rs9319427

FLT1

0.79

.21

1.35

.40

M419

rs9508016

FLT1

0.87

.34

1.09

.86

M420

rs950860

FLT1

1.05

.81

0.83

.87

M421

rs9513070

FLT1

1.02

.88

0.78

.40

Marker

SNP ID

Gene name

Odds ratio

M382

rs3088111

FLT1

M383

rs3751395

FLT1

1.12

M384

rs3751397

FLT1

M385

rs3794396

M386

rs3794399

M387

P value

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

.81

................................................................................................................................................................................................................................................................................................................................................................................

.61

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

1.02

.94

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

2.12

.03

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

.18

................................................................................................................................................................................................................................................................................................................................................................................

.16

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

1.04

.91

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................

Srinivas. Allelic variations in angiogenic pathway genes. Am J Obstet Gynecol 2010.

(continued )

MAY 2010 American Journal of Obstetrics & Gynecology

445.e9

Research

Obstetrics

www.AJOG.org

APPENDIX

Summary of all SNPs analyzeda

(continued)

Blacks (n ⴝ 489)

Variable

b

Whites (n ⴝ 117)

Marker

SNP ID

Gene name

Odds ratio

P value

Odds ratiob

P value

M424

rs9513085

FLT1

0.92

.57

1.73

.18

M425

rs9513089

FLT1

0.89

.43

0.86

.60

M426

rs9513099

FLT1

0.96

.94

1.10

.82

M427

rs9554320

FLT1

0.88

.44

1.63

.12

M429

rs9582036

FLT1

1.05

.77

0.67

.27

M430

rs307826

FLT4

0.88

.78

3.06

.02

M431

rs35874891

FLT4

1.00

.99

M559

rs3024994

VEGFA

0.44

.29

M560

rs3025010

VEGFA

1.03

.80

M561

rs3025019

VEGFA

0.91

.86

mono

M562

rs3025023

VEGFA

0.93

.85

mono

M563

rs3025030

VEGFA

1.11

.62

M565

rs3025034

VEGFA

0.89

.63

M566

rs3025035

VEGFA

1.14

.41

0.94

.91

M569

rs833068

VEGFA

1.28

.06

0.90

.75

M570

rs833070

VEGFA

1.10

.48

0.85

.60

M571

rs9369425

VEGFA

M572

rs9472138

VEGFA

M573

rs2286612

VEGFB

1.11

.46

M574

rs2429458

VEGFB

0.83

.44

M575

rs3741403

VEGFB

0.94

.68

0.91

.76

M576

rs4672

VEGFB

2.72

.12

1.39

.53

M577

rs633561

VEGFB

0.89

.47

0.37

.48

M578

rs10000679

VEGFC

1.25

.14

0.55

.60

M579

rs10007002

VEGFC

0.88

.58

0.96

.97

M580

rs10012721

VEGFC

0.82

.42

1.42

.50

M581

rs1485762

VEGFC

M582

rs1485766

VEGFC

1.56

.03

1.25

.46

M583

rs1692787

VEGFC

0.81

.11

1.77

.27

M584

rs17063598

VEGFC

0.82

.15

M586

rs17697305

VEGFC

1.78

.68

1.64

.60

M588

rs3755975

VEGFC

0.99

.94

2.20

.47

M589

rs3775189

VEGFC

1.25

.20

0.53

.57

M590

rs3775194

VEGFC

1.11

.57

1.46

.22

M591

rs3775195

VEGFC

0.86

.43

1.39

.35

M592

rs3775202

VEGFC

1.08

.58

1.13

.69

M594

rs4146612

VEGFC

1.07

.66

0.65

.37

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

1.05

.87

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

0.69

.48

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

0.40

.38

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................

Srinivas. Allelic variations in angiogenic pathway genes. Am J Obstet Gynecol 2010.

445.e10

American Journal of Obstetrics & Gynecology MAY 2010

(continued )

Obstetrics

www.AJOG.org

Research

APPENDIX

Summary of all SNPs analyzeda

(continued)

Blacks (n ⴝ 489)

Variable

b

Whites (n ⴝ 117)

Marker

SNP ID

Gene name

Odds ratio

P value

Odds ratiob

P value

M595

rs4557213

VEGFC

0.81

.14

0.71

.50

M596

rs4604006

VEGFC

1.07

.63

0.77

.43

M598

rs553513

VEGFC

1.17

.32

0.60

.65

M599

rs6815174

VEGFC

0.84

.29

M600

rs6816640

VEGFC

0.94

.69

M602

rs6838834

VEGFC

1.60

.03

M603

rs6857752

VEGFC

1.22

.15

1.42

.50

M604

rs689142

VEGFC

1.03

.87

1.49

.73

M605

rs7440052

VEGFC

1.23

.15

1.42

.50

M606

rs7664413

VEGFC

1.06

.68

2.04

.05

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................

1.54

.37

................................................................................................................................................................................................................................................................................................................................................................................

mono

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

mono, monomorphic; SNP, single-nucleotide polymorphism. a

Monomorphic (these SNPs were excluded); blanks represent instances with no cases or controls with a variant allele (ie, very rare SNPs) and therefore, logistic regression could not be performed; Adjusted for age.

b

Srinivas. Allelic variations in angiogenic pathway genes. Am J Obstet Gynecol 2010.

MAY 2010 American Journal of Obstetrics & Gynecology

445.e11