Mol. Hum. Reprod. Advance Access originally published online on October 22, 2004
Molecular Human Reproduction 2005 11(1):73-77; doi:10.1093/molehr/gah116
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Haplotypic association of DDAH1 with susceptibility to pre-eclampsia
1Department of Obstetrics and Gynaecology, Kuopio University Hospital, 2Research Institute of Public Health, University of Kuopio, 3Oy Jurilab Ltd and 4Atherosclerosis Research Unit, Clinical Trial Centre, University of Kuopio, Kuopio, Finland
5 To whom correspondence should be addressed at: Research Institute of Public Health, University of Kuopio, P.O.Box 1627, 70211 Kuopio, Finland. Email: jukka.salonen{at}uku.fi or Email: jukka.salonen{at}jurilab.com
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Abstract |
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Association between pre-eclampsia (PEE1) and the dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 genes, which play a role in the regulation of nitric oxide synthesis and release, was studied. In a case–control study design single nucleotide polymorphisms (SNPs) were determined at eight sites in the DDAH1 gene and at one site (Pro231Pro) in the DDAH2 gene from 132 women with pre-eclampsia and 112 healthy controls. Three SNPs in the DDAH1 gene were associated with pre-eclampsia, showing complete linkage disequilibrium with each other, but none of the associations in the allele or genotype data reached statistical significance in either of the genes after the correction for multiple testing. Haplotype frequencies were estimated using a population based on a maximum likelihood method (EM algorithm). Four common DDAH1 haplotypes were present and a significant association of haplotypes H2 and H3 with pre-eclampsia (P=0.03) was found. The risk of pre-eclampsia was greatest in individuals (odds ratio: 3.93; 95% confidence interval: 1.54–9.99) who had two copies of the high-risk haplotypes (H2 or H3). The observed haplotypic association provides the first evidence of the importance of DDAH1 polymorphisms in pre-eclampsia susceptibility.
Key words: DDAH1/association/pre-eclampsia/relative risk/haplotype
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Introduction |
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Pre-eclampsia (PEE1; MIM 189800) is a multisystem disorder (Norris et al., 1999
), which involves dysfunction of vascular endothelium and imbalance between endothelium-derived constricting and relaxing factors (Faxen et al., 2001) with the initiating event being postulated to be reduced placental perfusion (Granger et al., 2001a). Endothelial dysfunction is considered to underlie many clinical manifestations of pre-eclampsia, including hypertension, proteinuria, and oedema (Pascoal et al., 1998; Chambers et al., 2001). Recent evidence suggests that nitric oxide (NO)—a potent vasodilator derived from endothelium—plays a role in the regulation of vascular resistance during normal pregnancy and pre-eclampsia (Faxen et al., 2001). Chronic NO synthase inhibition in pregnant rats is known to produce a hypertensive state associated with peripheral and renal vasoconstriction, proteinuria, intrauterine growth retardation, and increased fetal morbidity, a pattern that closely resembles the features of pre-eclampsia (Pascoal et al., 1998; Granger et al., 2001a,b; Nakatsuka et al., 2002), suggesting that NO deficiency might be responsible for the disease process during pre-eclampsia.
In humans, NO synthesis is inhibited by endogenous asymmetric methylarginines. Free methylarginines—that is, NG,NG asymmetric dimethylarginine (ADMA), NG,NG' symmetric dimethylarginine and NG methylarginine—have a widespread distribution in the body and are found in cell cytosol, plasma and tissues. The asymmetric form (ADMA) inhibits all three isoforms of NO synthesis. Savvidou et al. (2003) have shown that pre-eclamptic women and women with evidence of impaired placental perfusion have significantly higher levels of ADMA than women with normal pregnancies. The primary route of elimination of ADMA is by catabolism, carried out by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), to citrulline and di- or mono-methylamine. The enzyme is encoded by a gene which has two isoforms—DDAH1 (MIM 604743) and DDAH2 (MIM 604744)—located on chromosome 1p22 and 6p21.3, respectively. When DDAH is inhibited, NO synthesis is decreased because of increased intracellular concentration of ADMA (Murray-Rust et al., 2001). Low ADMA concentrations are also found in areas of low tissue blood flow mainly due to pronounced increase in expression of DDAH at the protein and mRNA level, which catabolizes ADMA (Laussmann et al., 2002).
Taken together, these findings indicate that DDAH is a biological candidate gene for pre-eclampsia. We, therefore, designed a case–control study to test the possible association between DDAH gene polymorphisms and pre-eclampsia. The effects of these polymorphisms were not tested by biochemical assays, gene expression and structure-functional analyses of the mutations and this is one area, needing further exploration.
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Materials and methods |
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Ethics
Written approval for the study was obtained from the Ethics Committee of Kuopio University Hospital, and the protocol was approved by the Institutional Review Board. All subjects gave written informed consent for participation, which was documented.
Study group
Information was collected retrospectively in connection with 132 consecutive pre-eclamptic singleton pregnancies and 112 healthy control women who delivered at Kuopio University Hospital between January 1997 and December 1998. Using the Birth Registry at Kuopio, pre-eclamptic patients were called and blood samples were drawn. During the same time, blood samples were collected from controls who had given birth in the same hospital after uncomplicated pregnancies and who had at least two normal pregnancies, including the current one. From controls, blood was drawn for DNA analysis at enrolment. To ensure homogeneity of the genetic background, controls originating from a regional population with no clinical signs of the disorder were enrolled by random selection in this study. All study and control women were Caucasian and they were derived only from women with singleton deliveries at our hospital during the study period. Pre-eclampsia was defined as the development of hypertension and new-onset proteinuria (>300 mg of urinary protein in 24 h) in the absence of urinary tract infection after the 20th week of gestation in women with no proteinuria at baseline. Hypertension was defined according to current guidelines that accept 140 and/or 90 mmHg of systolic and diastolic pressure (Korotkoff phase V), respectively, or higher, as hypertension, when measured on two consecutive occasions at least 24 h apart (Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy, 2000). Women with a previous history of chronic hypertension, renal disease or diabetes mellitus were excluded from the study.
Genotyping
Genotyping of the DDAH1 c.260C > T, Thr87Met variant (SNP1) was done with the restriction fragment length polymorphism method. The PCR amplification was conducted in a 20 µl volume: the reaction mixture contained 60 ng human genomic DNA (extracted from peripheral blood), 1x GeneAmp® Gold Buffer (Applied Biosystems), 1.25 mM of MgCl2 (Applied Biosystems), 100 µM of each of the nucleotides (dATP, dCTP, dGTP, dTTP), 0.5 µM of the PCR primers (Table I), 1 unit of DNA polymerase (AmpliTaq Gold, Applied Biosystems) and 1 M of Betaine (Sigma-Aldrich). The PCR programme conditions were as follows: first the reaction was held for 7 min at 94°C, then the following two steps were repeated for 40 cycles: 45 s at 94°C, 1 min and 30 s at 68°C, after which the reaction was kept at 72°C for 5 min, and finally held at 4°C. The PCR product was digested for 6 h with BsmAI restriction endonuclease in 1x NEBuffer 3 (New England BioLabs), mixed with 6x loading dye solution and run in 1.7% agarose gel electrophoresis. Identification of normal and mutant alleles were based on different sizes of the restriction fragments in electrophoresis, resulting in distinct bands [normal homozygote form of Thr87 (Thr/Thr): 486, 284, 112, 10 bp; heterozygote form of mutation Thr87Met (Thr/Met): 770, 486, 287, 112, 10 bp; and homozygote form of mutation Thr87Met (Met/Met): 770, 112, 10 bp].
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Other target single nucleotide polymorphisms (SNPs 2–8 and the DDAH2 gene variant, Table II) were amplified in a multiplex PCR and genotyped with a primer extension method (SNaPshot, Applied Biosystems). The reason for choosing all these SNPs was that they were found to be polymorphic in the Finnish population in a pilot study carried out on 48 control subjects prior to the present study.
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The PCR was conducted in a 20 µl volume. The reaction mixture contained 60 ng human genomic DNA (extracted from peripheral blood), 1x PCR buffer (1.25 mM of MgCl2, QIAGEN), 100 µM of each of the nucleotides (dATP, dCTP, dGTP, dTTP), 5 pmol of each of the PCR primers (Table I) and 1 unit of DNA polymerase (Hot Start Taq DNA Polymerase, QIAGEN). In the PCR reaction, the samples were incubated at 95°C for 10 min and then subject to 35 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 1 min 30 s in a PTC-220 DNA Engine Dyad PCR machine (MJ Research). The PCR products were purified with SAP (Shrimp Alkaline Phosphatase, USB Corporation) and ExoI (Exonuclease I, USB Corporation) treatment: 5 units of SAP and 2 units of ExoI were added to 15 µl of the PCR product. Reaction was mixed and incubated at 37°C for 1 h, at 75°C for 15 min and then kept at 4°C. In the subsequent primer extension reaction, 5 µl of SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems), 3 µl of purified PCR products, 1 µl of pooled extension primers (depending on the signal in the SNaPshot reaction, the primer concentrations in the mix ranged between 0.05 and 1 µM) and 1 µl water were mixed in a tube and incubated at 94°C for 2 min and subject to 25 cycles of 95°C for 5 s, 50°C for 5 s and 60°C for 5 s in a PTC-220 DNA Engine Dyad PCR machine (MJ Research). After the primer extension reaction, 1 unit of SAP was added to the reaction mix and incubated at 37°C for 1 h, at 75°C for 15 min and then kept at 4°C. Aliquots of 1 µl of pooled SNaPshot products, 9 µl of Hi-Di formamide (Applied Biosystems) and 0.25 µl GeneScan-120 LIZ size standard (Applied Biosystems) were combined in a 96-well 3100 optical microamp plate (Applied Biosystems). The reactions were denatured at 95°C for 5 min and then loaded onto an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Electrophoresis data were processed and the genotypes were visualized by using the GeneScan Analysis version 3.7 (Applied Biosystems).
Data processing and statistical analysis
Data were analysed using the Statistics Package for Social Sciences (SPSS), Version 10.0 (SPSS Inc, Chicago, IL). Deviation from Hardy–Weinberg equilibrium and differences in genotype and allele distributions between groups were evaluated by 
2-test and Fisher's exact test. Linkage disequilibrium (LD) was measured as Lewontin D' (Lewontin, 1964). Haplotypes were constructed by an EM-algorithm using Snphap software (The Cambridge Institute for Medical Research, Clayton D, http://www-gene.cimr.cam.ac.uk/clayton/software). Joint analysis of DDAH1 haplotypes and DDAH2 genotypes was done by a multiple logistic model.
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Results |
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The characteristics of the subjects and the clinical data are summarized in Table III. In comparison with controls, women with pre-eclampsia had higher systolic and diastolic blood pressure, BMI, and lower gestational age at delivery with no significant differences in age.
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A total of nine SNPs were studied in the study sample (Table II); however, the first variant DDAH1 c.260C > T (Thr87Met) (SNP1) occurred only once in both study groups and was not included in further statistical analyses. The allele and genotype distribution in case and control groups of the eight variants are given in Table IV. All variants were in Hardy–Weinberg equilibrium. To determine whether the DDAH SNPs were associated with pre-eclampsia, we initially compared the single-point genotype and allele distributions of the SNPs between pre-eclamptic and control groups (Table IV). Genotype or allele distributions of these SNPs were not statistically different between the groups after the Bonferroni correction for eight independent tests (P-value of a single test should be below 0.006 to keep the overall significance of 0.05).
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Haplotypes were constructed on the basis of maximum likelihood. Three SNPs (5, 7 and 8) that had significant allelic associations with pre-eclampsia were in complete linkage disequilibrium with each other, as estimated by Lewontin D' (D'=1.0, P<0.001). Another interesting observation was of a tight association between the SNPs 2, 4 and 6 (D'=1.0, P<0.001). Consequently, we observed only five of the theoretically possible haplotypes, and the haplotype breakdown for the 490 chromosomes is shown in Table V. These five observed haplotypes were assembled and their identification numbers were assigned according to the total sample frequencies.
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With the exception of haplotypes 4 and 5, all other haplotypes, that is, 96% of the control population were in the background of IVS3-7C (SNP3) and either IVS4-68C (SNP5), IVS5-71A (SNP7), 3'UTR + 16C (SNP8) or IVS4-68T (SNP5), IVS5-71T (SNP7), 3'UTR + 16G (SNP8). Therefore, haplotype 3 seems to be a variant of haplotype 1 and haplotypes 4 and 5 are variants of haplotype 2. Haplotype 5 was found to have a frequency of only 0.2% and we elected not to pursue it further. Thus, haplotype 5 was excluded from the joint analysis and estimated haplotype frequencies of the four common haplotypes (H1–H4) of subjects with pre-eclampsia were compared with those of controls.
The data presented in Table V suggest that haplotypes H1 and H4 are protective and haplotypes H2 and H3 are predisposing haplotypes to pre-eclampsia. Haplotypes H2, H3 and H4 were found to have the minor alleles at SNP sites 5, 7 and 8. Interestingly, the frequency of haplotype H4 containing the low frequency variant at SNP site 3 was more frequent in the control than in the study group, while haplotypes H2 and H3 containing the wild-type allele at SNP site 3 were less frequent in the pre-eclampsia group (0.40 versus 0.29). Thus, effective sites correspond to SNP3 and an LD-group based on SNPs 5, 7 and 8. Since the effective sites were identical for haplotypes H2 and H3, they were pooled in the analyses. As expected from the genotype data, the difference in haplotype frequencies between cases and controls was statistically significant (P=0.03), with carriers of haplotypes H2 or H3 having an odds ratio (OR) of 1.63 [95% confidence interval (CI): 1.12–2.38] to develop pre-eclampsia compared to haplotypes H1 and H4.
In the same line of thought, there were also more individuals having two copies of high-risk haplotypes (combinations H2/H2, H2/H3, and H3/H3) in the study group (18%) than in the controls (5%). This difference is statistically significant (P=0.003) and corresponds to an OR of 3.93 (95% CI: 1.54–9.99).
Since DDAH2 is located at a different chromosome (chromosome 6), it obviously did not have significant LD with DDAH1 variants. Genotype and allele distributions of the Pro231Pro polymorphism in the DDAH2 gene did not reveal statistically significant single-point association with pre-eclampsia. Logistic regression with DDAH1 haplotype (coding 1 if the haplotype combination was H2/H2, H2/H3, or H3/H3 and 0 otherwise) and DDAH2 genotype as independent variables and pre-eclampsia as a dependent variable (0 for controls, 1 for cases) gave a non-significant result for the DDAH2 genotype, whereas the result for the DDAH1 haplotype was significant (OR 4.07, 95% CI: 1.60–10.40, P=0.003).
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Discussion |
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In the current study, we tested the association of the polymorphisms in both the DDAH isoforms—DDAH1 and DDAH2—with pre-eclampsia and found that polymorphisms in the DDAH1 gene may modify disease susceptibility. Association tests, haplotype analyses and conditional logistic regression were used to test eight DDAH1-specific SNPs and the DDAH2 Pro231Pro polymorphism in our study sample. Genotype distributions of these eight DDAH1 SNPs did not reveal statistically significant single-point association with pre-eclampsia, whereas three of these polymorphisms gave evidence for allelic association, but evidence for nominal association disappeared after correcting for multiple tests. Analysing these polymorphisms together as a haplotype constructed on the basis of maximum likelihood, we found a significant DDAH1 haplotypic association. This finding is in agreement with the results of conditional regression analyses that gave no evidence for interactions between DDAH1 and DDAH2 polymorphism, but pointed to a significant association with pre-eclampsia and DDAH1 haplotypes. Being a carrier of haplotypes H2 and H3 appeared to result in a two-fold increase in risk, and viewed alternatively, two copies of these haplotypes actually conferred susceptibility to pre-eclampsia at an OR of 4.
To explore which part of the gene contributes most to the disease risk, we looked at the association in each set of eight consecutive DDAH1 polymorphisms. Although the LD between these polymorphisms was generally high across the entire gene, the strongest association signals were observed in the 3' portion of the gene. Furthermore, sites at the 3' end of the DDAH1 locus appeared to be more important in explaining pre-eclampsia than the sites at the 5' end. Effective sites corresponded to SNP3 and a structured linkage disequilibrium block was based on SNPs 5, 7 and 8. These were also the most promising sites based on individual site analysis. The functional impact of the polymorphisms of the DDAH1 gene is uncertain, but different haplotypes containing functional variants may lead to inter-individual variation in DDAH1 transcription or expression and to a malfunctioning enzyme by folding or maturation defects, which in turn result in altered NO metabolism and vasoconstriction seen in pre-eclampsia.
To the best of our knowledge, this is the first study to implicate the polymorphisms of DDAH as a genetic risk factor for pre-eclampsia in a sufficient number of cases and controls. Furthermore, case–control association studies of pre-eclampsia and polymorphisms in the eNOS gene have been largely negative (Lachmeijer et al., 2002). We tested for significance using single-point as well as haplotype association analyses. Genotype distributions of the SNPs did not reveal statistically significant single-point association of the DDAH1 gene with pre-eclampsia and to further explore the association we undertook haplotype estimation analysis. The validity of such an approach may be questioned, but predictions based on empirical data drawn from the literature currently support the feasibility of haplotype estimation analysis for detecting association more efficiently than single-point association analysis (Martin et al., 2000). However, association findings in case–control studies may occur due to chance or bias due to population stratification, and replication studies in other populations will be needed to argue against both these alternatives. On the other hand, any genetic variation is likely to make only a small individual contribution to the complex disease phenotype and may be population specific.
In general, the statistical power to detect a real association or linkage is limited by the background noise in the population under study. This noise consists of all possible combinations of environmental and genetic factors present in the population. Therefore, in heterogeneous populations, large sample sizes would be needed to obtain sufficient statistical power to detect genetic risk factors. More homogeneous populations, such as genetically isolated populations, have been proposed as a possible alternative for these large sample sizes, because environmental variation might be lower and the genetic make-up of these populations is expected to be less complex owing to founder effects, thus improving the signal-to-noise ratio. Use of genetic isolates has been considered especially useful in the studies of complex disorders (Peltonen et al., 2000; Heutink and Oostra, 2002). In this context, Finland offers an advantage to detect even a small contribution of genes in multifactorial diseases like pre-eclampsia because the population has a more similar way of living and eating habits that reduces environmental variation, and because population expansion mainly occurred by population growth and not by immigration.
In summary, the present study on Finnish women supports the role of DDAH1 gene polymorphisms and endothelial dysfunction in pre-eclampsia risk and we advocate further studies using larger and ethnically differing subjects to extend and confirm the present results. The effect of the polymorphisms found in our study was not tested with plasma and urinary levels of NO and ADMA and in future these aspects need to be explored in expression studies to obtain evidence of causality by gene expression and structure-functional studies and biochemical assays.
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Acknowledgements |
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This project was supported by The Centre for International Mobility, CIMO.
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References |
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Submitted on August 16, 2004; resubmitted on September 7, 2004; accepted on September 12, 2004.
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