WO2005078128A1

WO2005078128A1 - Method for detecting the risk of preeclampsia by analysing a dimethylarginine dimethylaminohydrolase gene

Info

Publication number: WO2005078128A1
Application number: PCT/FI2005/050037
Authority: WO
Inventors: Jukka T. Salonen; Mia Pirskanen; Pekka Uimari; Tomi-Pekka Tuomainen; Seppo Heinonen
Original assignee: Oy Jurilab Ltd
Priority date: 2004-02-18
Filing date: 2005-02-17
Publication date: 2005-08-25
Also published as: FI20040255A0; FI20040255A

Abstract

The invention provides a method and kit for detecting or diagnosing a risk of or predisposition to preeclampsia in a subject, the method comprising the steps of providing a biological sample taken from the subject to be tested and detecting the presence or absence of a variant genotype of a dimethylarginine dimethylaminohydrolase (DDAH) gene in the biological sample. The invention further provides a kit comprising means to carry out the method of the invention and a method for treatment and prevention of preeclampsia.

Description

Method for detecting the risk of preeclampsia by analysing a dimethylarginine dimethylaminohydrolase gene FIELD OF THE INVENTION

The present invention resides in the fields of molecular genetics and diagnostics. The invention provides a method and kit for detecting or diagnosing a risk of or predisposition to preeclampsia in a subject, the method comprising the steps of providing a biological sample taken from the subject to be tested and detecting the presence or absence of a variant genotype of a DDAH gene in the biological sample. The invention further provides a kit comprising means to carry out the method of the invention and a method for treatment and prevention of preeclampsia.

BACKGROUND OF THE INVENTION

Preeclampsia (PEEl [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. 2001). Endothelial dysfunction is considered to underlie many clinical manifestations of preeclampsia; including hypertension, proteinuria, and edema (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 preeclampsia (Faxen et al. 2001). Chronic NOS 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 preeclampsia (Pascoal et al. 1998; Granger et al. 2001a, 2001b; Nakatsuka et al. 2002), suggesting that NO deficiency might be responsible for the disease process during preeclampsia.

In humans, NO synthesis is inhibited by endogenous asymmetric methylarginines. Free methylarginines — that is, NG, NG asymmetric dimethylarginine (ADMA), NG, NG' symmetric dimethylarginine (SDMA) and NG methylarginine (L-NMMA) — have a wide spread distribution in the body and are found in cell cytosol, plasma and tissues. The asymmetric form (ADMA) inhibits all three isoforms of nitric oxide synthesis. The primary route of elimination of this potent NO inhibitor 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 lp22 and 6p21.3, respectively. When DDAH is inhibited, nitric oxide synthesis is decreased because of increased intracellular concentration of ADMA (Murray-Rust et al. 2001). High NO concentrations are also found in areas of low tissue blood flow mainly due to pronounced increased expression of DDAH at protein and mRNA level, which inhibited ADMA shown by corresponding low concentration of this compound in those areas (Laussmann et al. 2002).

Taken together, these findings were the basis of our hypothesis and invention that the DDAH is a biological candidate gene for preeclampsia. To test this hypothesis, we designed a case-control study to investigate the possible association between DDAH gene polymoφhisms and preeclampsia. In the study, we showed the presence of four SNP based haplotypes (H1-H4) in DDAH1 gene. A significant association between haplotypes H2 or H3 with preeclampsia (P = 0.03) was found. The risk of preeclampsia was greatest in individuals (OR: 3.93; 95% CI: 1.54 - 9.99) who had two copies of the high-risk haplotypes (H2 or H3). The observed haplotypic association provided the first evidence on the importance of DDAH 1 polymoφhisms in preeclampsia susceptibility.

SUMMARY OF THE INVENTION

The object of this invention is to provide a method for screening to assess if a subject is at risk to develop preeclampsia by detecting DDAH gene polymoφhisms in a biological sample from the subject. More particularly, the invention provides a method for detecting genetic variation or polymoφhism, i.e. a mutation, in a DDAH gene comprising the steps of: i) providing a biological sample taken from a subject to be tested, ii) detecting the presence or absence of a variant genotype of the DDAH gene in the biological sample, the presence of a variant DDAH genotype indicating an increased risk of preeclampsia in said subject. Said DDAH gene can be selected from DDAH1 and DDAH2 genes.

Said method can also be used diagnostically.

Furher object of the invention is to provide a kit comprising means to carry out the detection method of the invention and a method for treatment and prevention of preeclampsia.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides means for prognostic or diagnostic assays for determining if a subject is likely to develop preeclampsia, which is associated with the variation or dysfunction of a DDAH gene. Basically, such assays comprise a detection step, wherein the presence or absence of a genetic alteration or defect in DDAH is determined in a biological sample taken from the subject. Said detection step can be performed, e.g., by methods involving sequence analysis, nucleic acid hybridisation, primer extension, restriction enzyme site mapping or antibody binding. These methods are well-known in the art (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons: 1992).

In particular, the present invention is directed to a method of determining the presence or absence of a DDAH 1 SNP in a biological sample from a female for assessing the predisposition of the individual to preeclampsia. Said method comprises determining the sequence of the nucleic acid from a female at one or more of the following positions in the DDAH1 gene or mRNA: c.260 OT, Thr87Met (reference type form is set forth in SEQ ID NO:29 and variant form in SEQ ID NO:31 , variant site is at position 260);

IVS2-330T (reference type form is set forth in SEQ ID NO:21 and variant form in SEQ ID NO:22, variant site is at position 174); IVS3-70T (reference type form is set forth in SEQ ID NO:23 and variant form in SEQ ID NO:24, variant site is at position 145); c.561 A>G, Alal87Ala (reference type form is set forth in SEQ ID NO:29 and variant form in SEQ ID NO:37, variant site is at position 561);

IVS4-680T (reference type form is set forth in SEQ ID NO:25 and variant form in SEQ ID NO:26, variant site is at position 104); IVS5-88G>A (reference type form is set forth in SEQ ID NO:27 and variant form in SEQ ID NO:28, variant site is at position 152);

IVS5-71A>T (reference type form is set forth in SEQ ID NO:27 and variant form in SEQ ID NO:28, variant site is at position 163); and

3'UTR+16C>G (reference type form is set forth in SEQ ID NO:27 and variant form in SEQ ID NO:28, variant site is at position 368);

and determining the status of the female by reference to polymoφhism in DDAH1 gene. However, a person skilled in the art may carry out various SNP discovery methods to find other functional DDAH gene mutations for use in the method of the invention. Such variants are deemed to be within the scope of the present invention from the teachings herein.

Numerous genotyping methods have been described in the art for analysing nucleic acids for the presence of specific sequence variations e.g. SNPs, insertions and deletions (for review see Syvanen, 1999, Human Mutation 13:1-10). In these methods a sample containing nucleic acid (e.g. blood, tissue biopsy or buccal cells) is obtained from the patient and the sequence variations of interest are identified and assessed from the nucleic acids.

Allelic variants in genes can be discriminated by enzymatic methods (with the aid of restriction endonucleases, DNA polymerases, ligases etc.), by electrophoretic methods (e.g. single strand conformation polymoφhism (SSCP), heteroduplex analysis, fragment analysis and DNA sequencing), by solid-phase assays (dot blots, microarrays, microparticles, microtiter plates etc.) and by physical methods (e.g. hybridisation analysis, mass spectrometry and denaturing high performance liquid chromatography (DHPLC)). In most of the genotyping assays different polymerase chain reaction (PCR) applications are used both to increase the signal to noise ratio as well as spare sample nucleic acid before allele discrimination. Detectable labels (fluorochromes, radioactive labels, biotin, modified nucleotides, haptens etc) can be used to enhance visualization of allelic variants.

In a preferred embodiment of the invention a biological sample is contacted with oligonucleotide primers so that the nucleic acid region containing the potential single nucleotide polymoφhism is amplified by polymerase chain reaction prior to determining the sequence. The final results can be obtained by using a method selected from, e.g., allele specific nucleic acid amplification, allele specific nucleic acid hybridisation (e.g. with a capturing probe), oligonucleotide ligation assay or restriction fragment length polymoφhism (RFLP). These methods are well-known for a skilled person of the art (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons:1992, or Landegren et al, "Reading Bits of Genetic Information: Methods for Single-Nucleotide Polymoφhism Analysis", Genome Research 8:769-776).

The detection step of the method can also be a specific DNA-assay, such as a gene or DNA chip, microarray, strip, panel or similar combination of more than one genes, mutations or RNA expressions to be assayed..

The biological sample for the method can be, e.g., a blood sample or buccal swab sample. From said sample genomic DNA is isolated.

The subject to be tested is preferably a mammal, more preferably a primate, and most preferably a human.

The polymoφhic sites can be analyzed individually or in sets for prognostic purposes. The conclusion drawn from the analysis depends on the nature and number of polymoφhic sites analyzed. Some polymoφhic sites have variant polymoφhic forms that are causative of disease. Detection of such a polymoφhic form provides at least a strong indication of presence or susceptibility to disease. Other polymoφhic sites have variant polymoφhic forms that are not causative of disease but are in equilibrium dislinkage with a polymoφhic form that is causative. Thus, detection of noncausative polymoφhic forms may also indirectly provide an indication of the risk of presence or susceptibility to disease. Preferably, multiple variant forms at several polymoφhic sites in a DDAH gene are detected to provide an indication of increased risk of presence or susceptibility to disease. The results from analyzing the polymoφhic sites of the invention can be combined with analysis of other loci that associate with the same disease {i.e., preeclampsia). Alternatively or additionally, the risk of disease can be confirmed by performing conventional medical diagnostic tests of patient symptoms.

The detection method of the invention may further comprise a step of combining information concerning the subject's age (years), BMI (kg/m²), systolic blood pressure (mmHg), and/or diastolic blood pressure (mmHg), as well as newborn's birth weight (gm), and/or gestational age at delivery (weeks).

The results from the above step of the method render possible a step of calculating the probability of preeclampsia using a logistic regression equation.

The score that predicts the probability of preeclampsia may be calculated using a multivariate failure time model or a logistic regression equation as follows:

Probability of preeclampsia = [1 + e H-^{a + ∑}O^51**¹⁾⁾] ^_1 ₅ wherein e is Napier's constant, Xjare variables related to preeclampsia, bj are coefficients of these variables in the logistic function, and a is the constant term in the logistic function. The model may additionally include any interaction (product) or terms of any variables Xj, e.g. bjXj. Alternative statistical models are a failure-time models such as the Cox's proportional hazards' model and neural networking models.

The present invention also provides a method for treating or targeting the treatment of preeclampsia in a subject with preeclampsia by determining the pattern of alleles encoding a variant DDAH gene, i.e. by determining if said subject's genotype of the DDAH gene is of the variant type, comprising the steps presented in the above detection method, and treating a subject of the variant genotype with a drug affecting DDAH production or metabolism of the subject. The treatment may comprise a therapy which enhances DDAH availability, production or concentration in the circulation of the human subject or animal. Such treatment can be a dietary treatment, a vaccination, gene therapy or gene transfer (see e.g. US patent No: 6,627,615). Gene therapy is carried out, e.g., by transferring a non- variant DDAH gene or fragment or derivative thereof.

The invention also features prognostic kits for use in detecting the presence of DDAH SNP in a biological sample. The kit provides means for assessing the predisposition of an individual to preeclampsia mediated by variation or dysfunction of DDAH. The kit can comprise a labeled compound capable of detecting DDAH polypeptide or nucleic acid (e.g. mRNA) in a biological sample. The kit can also comprise nucleic acid primers or probes capable of hybridising specifically to at least of portion of a DDAH gene or allelic variant thereof. The kit can be packaged in a suitable container and preferably it contains instructions for using the kit and optionally software to inteφret the results of the detection.

The kit can be based on a capturing nucleic acid probe specifically binding to the variant genotype as defined in the invention, and/or on a DNA chip, microarray, DNA strip, DNA panel or real-time PCR based tests.

The publications and other materials used herein to illuminate the background of the invention, and in particular, to provide additional details with respect to its practice, are incoφorated herein by reference.

The invention will be described in more detail in the experimental section. However, it will be appreciated that the methods of the present invention can be incoφorated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive. EXPERIMENTAL SECTION

Material and Methods

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 to participation, which was documented.

Study group Information was collected retrospectively in connection with 132 consecutive preeclamptic 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, preeclamptic 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. The study and control groups were derived only from women with singleton deliveries at our hospital during the study period. Preeclampsia 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 20 week of gestation in women with no proteinuria at baseline. Hypertension was defined according to current guidelines that accept 140 and/or 90 mm Hg 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.260OT, Thr87Met variant was done with RFLP method. The PCR (polymerase chain reaction) amplification was conducted in a 20 μl volume: the reaction mixture contained 60 ng human genomic DNA (extracted from peripheral blood), IX 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 each of the PCR primers 5'- GTC CCC CGC CTC CGC ATA CTT -3' (SEQ ID NO:l) and 5'- CCA CCT GCC CGA GAC CGT ACA A -3' (SEQ ID NO:2), 1 unit of the DNA- polymerase (AmpliTaq Gold, Applied Biosystems) and 1 M of Betaine (Sigma- Aldrich). The PCR program conditions were as follows: first the reaction was hold 7 minutes at 94°C, then the following two steps were repeated for 40 cycles: 45 seconds at 94°C, 1 minute and 30 seconds at 68°C, after which the reaction was kept at 72°C for 5 minutes, and finally hold at 4°C.

The PCR-product was digested for 6 hours with BsmA I restriction endonuclease in the concentration of IX 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 was based on different size of the restriction fragments in electrophoresis, resulting in distinct bands (normal homozygote form of Thr87 (Thr/Thr); 486 bp, 284 bp, 112 bp, 10 bp, heterozygote form of mutation Thr87Met (Thr/Met); 770 bp, 486 bp, 287 bp, 112 bp, 10 bp and homozygote form of mutation Thr87Met (Met/Met); 770 bp, 112 bp, 10 bp).

Other target SNPs (Table 1) were amplified in a multiplex PCR and genotyped with a primer extension method (SNaPshot, Applied Biosystems). The PCR was conducted in a 20 μl volume. The reaction mixture contained 60 ng human genomic DNA (extracted from peripheral blood), IX 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 primers (Table 2) and 1 unit of the DNA-polymerase (Hot Start Taq DNA Polymerase, QIAGEN). In the PCR reaction the samples were incubated at 95°C for 10 minutes and then subject to 35 cycles of 94°C for 30 sec, 50°C for 30 sec and 72°C for 1 min 30 sec in a PTC-220 DNA Engine Dyad PCR machine (MJ Research).

The nucleotide sequence of the PCR primer pair for the amplification of the human DDAHl gene IVS2-330T (SNP2) variant was as follows: 5' - ATC CTG CTT TCT GCC CTT T -3' (SEQ ID NO:3) and 5'- AAG CCA GTG AAG CGT AAA CAC -3' (SEQ ID NO:4). The nucleotide sequence of the PCR primer pair for the amplification of the human DDAHl gene IVS3-70T (SNP3) variant was as follows: 5'- CAA TAT CCA AAT CTG TGG GTC -3' (SEQ ID NO:5) and 5'- AAA TAC CTG CCT GTT CTC TCC -3' (SEQ ID NO:6).

The nucleotide sequence of the PCR primer pair for the amplification of the human DDAHl gene c.561A>G, Alal87Ala (SNP4) variant was as follows: 5'- CAA TAT CCA AAT CTG TGG GTC -3' (SEQ ID NO:5) and 5'- AAA TAC CTG CCT GTT CTC TCC - 3' (SEQ ID NO:6).

The nucleotide sequence of the PCR primer pair for the amplification of the human DDAHl gene IVS4-680T variant was as follows: 5'- ATC AAG TTT TCC TTT TCT GG -3' (SEQ ID NO:7) and 5'- CTC CTA TTG GTC ACT CCT TT -3' (SEQ ID NO:8).

The nucleotide sequence of the PCR primer pair for the amplification of the human

DDAHl gene IVS5-88G>A variant was as follows: 5'- AAC CAC ATT TCT GAC ACA TCT TTG -3' (SEQ ID NO:9) and 5'- GCA CAG TGG CAC AGT AGA TTG T -3' (SEQ ID NO: 10).

DDAHl gene IVS5-71A>T variant was as follows: 5'- AAC CAC ATT TCT GAC ACA TCT TTG -3' (SEQ ID NO:9) and 5'- GCA CAG TGG CAC AGT AGA TTG T -3' (SEQ ID NO: 10).

The nucleotide sequence of the PCR primer pair for the amplification of the human DDAHl gene 3'UTR+16C>G variant was as follows: 5'- AAC CAC ATT TCT GAC ACA TCT TTG -3' (SEQ ID NO:9) and 5'- GCA CAG TGG CAC AGT AGA TTG T -3' (SEQ ID NO: 10).

The nucleotide sequence of the PCR primer pair for the amplification of the human DDAH2 gene C.6930T, Pro231Pro variant was as follows: 5'- CAG TCC GTC CCC AGC CCT TAG T -3' (SEQ ID NO:l 1) and 5'- GCA CAC CCC CTT TAT TTC CCT CAT -3' (SEQ ID NO: 12). The PCR products were purified with SAP (Shrimp Alkaline Phosphatase, USB Coφoration) and Exol (Exonuclease I, USB Coφoration) treatment. 5 units of SAP and 2 units of Exol were added to 15 μl of the PCR product. Reaction was mixed and incubated at 37°C for 1 hour, at 75°C for 15 minutes 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 of the signal in the SNaPshot reaction, the primer concentrations in the mix can range between 0.05 μM and 1 μM) and 1 μl water were mixed in a tube and incubated at 94°C for 2 minutes 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 hour, at 75°C for 15 minutes and then kept at 4°C.

Aliquots of 1 μl of pooled SNaPshot products, 9.00 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 minutes and then loaded onto an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Electrophoresis data was processed and the genotypes were visualized by using the GeneScan Analysis version 3.7 (Applied Biosystems).

The nucleotide sequence of the extension primer for the genotyping of human DDAHl IVS4-680T variant in a SNaPShot reaction was: 5' - TTT TTT TTC AAA CAG AAG GAA GGC A - 3 ' (SEQ ID NO: 13)

The nucleotide sequence of the extension primer for the genotyping of human DDAHl IVS2-330T variant in a SNaPShot reaction was: 5' - TTT TTT TTT TTT GTA CAG TCA CTG GTG CCA - 3' (SEQ ID NO: 14)

The nucleotide sequence of the extension primer for the genotyping of human DDAHl IVS3-70T variant in a SNaPShot reaction was: 5' - TTT TTT TTT TTT TTT TTG CTT GTT TTT CTA TTG TC - 3' (SEQ ID NO: 15) The nucleotide sequence of the extension primer for the genotyping of human DDAHl c.561A>G, alal87 variant in a SNaPShot reaction was: 5' - TTT TTT TTT TTT TTT TTT TTT CAG ATT CAC TAG ACC CAA T - 3 ' (SEQ ID NO: 16)

The nucleotide sequence of the extension primer for the genotyping of human DDAHl IVS5-88G>A variant in a SNaPShot reaction was: 5' - TTT TTT TTT TTT TTT TTT TTT TTT TTA TGA ATG AAA AGC CTA GAT - 3' (SEQ ID NO: 17)

The nucleotide sequence of the extension primer for the genotyping of human DDAHl IVS5-71 A>T variant in a SNaPShot reaction was: 5' - TTT TTT TTT TTT TTT TTT TTT _{τττ τττ τττ τττ TCA GAA GχG GAG AAT CAA 3} , ^Q _{ro NO:} ! )

The nucleotide sequence of the extension primer for the genotyping of human DDAHl 3'UTR+16C>G variant in a SNaPShot reaction was: 5' - TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TAG CTG CAG AGT CCC CCC - 3 ' (SEQ ID NO: 19)

The nucleotide sequence of the extension primer for the genotyping of human DDAH2 C.6980T, Pro231 variant in a SNaPShot reaction was: 5' - TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT ACG GTG CAG GAG GAA AGG - 3 ' (SEQ ID NO:20)

Data processing and statistical analysis

Data were analyzed using SPSS, Version 10.0 (SPSS Inc, Chicago, 111) statistical package. Deviation from Hardy- Weinberg equilibrium and differences in genotype and allele distributions between groups were evaluated by chi-squared test and Fisher's exact test. Linkage disequilibrium (LD) was measured as Lewontin D' (Lewontin 1964). Haplotypes were constructed by EM-algorithm using Snphap software. Joint analysis of DDAHl haplotypes and DDAH2 genotypes was done by multiple logistic model.

Empirical findings

The characteristics of the subjects and the clinical data are summarized in Table 3. In comparison with controls, women with preeclampsia had higher systolic and diastolic blood pressure, BMI, and lower gestational age at delivery with no significant differences in age.

A total of 9 single nucleotide polymoφhisms (SNPs) were studied in the study sample (Table 1), however the first variant DDAHl : C.260OT, 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 4. All variants were in Hardy-Weinberg equilibrium. To determine whether the DDAH SNPs were associated with preeclampsia, we initially compared the single-point genotype and allele distributions of the SNPs between preeclamptic and control groups

(Table 4). Genotype distributions of these SNPs were not statistically different between the groups. However, the allele frequency differences of SNPs 5, 7, and 8 were statistically significant between the studied groups (P=0.041) given odds ratio of 0.68 (95% CI: 0.47- 0.98) for subjects that carried allele IVS4-68C compared to allele T. After the Bonferroni correction for eight independent tests none of the comparisons reached the significance (P- value of a single test should be below 0.006 to keep the overall significance of 0.05).

Haplotypes were constructed on the basis of maximum likelihood. Three SNPs (5, 7 and 8) that had significant allelic associations with preeclampsia were in complete linkage disequilibrium with each other, as estimated as Lewontin D' (D'=l .0, PO.001). Another interesting observation was of a tight association between the IVS2-330T, c.561 A>G Alal87Ala, and IVS5-88G>A variations (SNPs 2,4 and 6) (D'=1.0, PO.001). Consequently, we observed only five of theoretically possible haplotypes and the haplotype breakdown for the 490 chromosomes is shown in Table 5. These five observed haplotypes were assembled and their identification numbers were assigned according to the total sample frequencies.

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 be 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 statistical analysis and estimated haplotype frequencies of the four common haplotypes (H1-H4) of subjects with preeclampsia were compared with those of controls.

The Table 5 clearly (even without statistical significance) indicates that haplotypes HI and H4 are protective and haplotypes H2 and H3 are predisposing haplotypes to preeclampsia. 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 3 site were less frequent in the preeclampsia group (0.40 vs. 0.29). Thus, effective sites correspond to SNP3 and a 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); carriers of haplotypes H2 or H3 having an odds ratio of 1.63 (95% CI: 1.12-2.38) to develop preeclampsia compared to haplotypes HI and H4.

In the same line of thought, there were also more individuals having two copies of high- risk haplotypes (combinations H2/TT2, 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 odds ratio 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 DDAHl variants. Genotype and allele distributions of the Pro231Pro polymoφhism in the DDAH2 gene (reference type form is set forth in SEQ ID NO:33 and variant form in SEQ ID NO:35) did not reveal statistically significant single point association with preeclampsia. Logistic regression with DDAHl haplotype (coding 1 if the haplotype combination was H2/TT2, H2/H3, or H3/H3 and 0 otherwise) and DDAH2 genotype as independent variables and preeclampsia as dependent variable (0 for controls, 1 for cases) gave a non-significant result for DDAH2 genotype whereas the result for the DDAHl haplotype was significant (odds ratio 4.07, 95% CI: 1.60-10.40, P=0.003). Implications of our findings

In the current study we tested the association of the polymoφhisms in both of the DDAH isoforms - DDAHl and DDAH2 with preeclampsia and found that polymoφhisms in the DDAH 1 gene may modify disease susceptibility. Association tests, haplotype analyses and conditional logistic regression were used to test eight DDAHl specific SNPs and the DDAH2 Pro231Pro polymoφhism in our study sample. Genotype distributions of these eight DDAHl SNPs did not reveal statistically significant single-point association with preeclampsia whereas three of these polymoφhism gave significant evidence for allelic association but evidence for nominal association disappeared after correcting for multiple tests. Analyzing these polymoφhisms together as a haplotype constructed on the basis of maximum likelihood, a significant DDAHl haplotypic association was found. This finding is in agreement with the results of conditional regression analyses that gave no evidence for interactions between DDAHl and DDAH2 polymoφhism, but pointed to a significant association with preeclampsia and DDAHl haplotypes. Being a carrier of haplotypes H2 and H3 appeared to result in a 2-fold increase in risk, and viewing alternatively, two copies of these haplotypes actually conferred susceptibility to preeclampsia at an odds ratio of 4.

To explore which part of the gene contributes most to the disease risk we looked at the association at each set of eight consecutive DDAHl polymoφhisms. Although the LD between these polymoφhisms 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 DDAHl locus appeared to be more important in explaining preeclampsia than the sites at 5' end. Effective sites corresponded to SNP3 and a structured linkage disequilibrium block based on SNPs 5, 7, and 8. These were also the most promising sites based on individual site analysis. The functional impact of the polymoφhisms of the DDAHl gene is uncertain, but different haplotypes containing functional variants may lead to inter individual variation in DDAHl transcription or expression and to malfunctioning enzyme by folding or maturation defects.

To our best knowledge, this is the first study to implicate the polymoφhisms of DDAH as a genetic risk factor for preeclampsia in a sufficient number of cases and controls. Furthermore, case-control association studies of preeclampsia and polymoφhisms 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 DDAHl gene with preeclampsia. To further explore the association we undertook haplotype estimation analysis, since 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).

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 preeclampsia because 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. Thus, the present study on Finnish women strongly supports the role of DDAH gene polymoφhisms and endothelial dysfunction in preeclampsia risk.

Table 1. DDAHl and DDAH2 gene sequence variations

Table 2. Nucleotide sequences of the primers used in polymerase chain reaction (PCR) for the amplification of the DDAHl exons 1, 3-6 and the DDAH2 exon 5.

Table 3. Clinical characteristics of women with preeclampsia (n=132) and healthy pregnant controls (n=112).

Table 4. Genotype and allele distributions of DDAHl and DDAH2 gene variants among women with preeclampsia (n=132) and healthy pregnant controls (n=l 12).

Table 4 (continued).

^aBased on chi-squared test ^bBased on Fisher's exact test

Table 5. Distribution of DDAHl haplotypes among women with preeclampsia (n=132) and healthy pregnant controls (n=l 12).

References

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Faxen M, Nisell H, Kublickiene KR (2001) Altered mRNA expression of ecNOS and iNOS in myometrium and placenta from women with preeclampsia. Arch Gynecol Obstet. 265:45-50.

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Claims

Claims:

1. A method for detecting genetic variation or polymoφhism, i.e. a mutation, in a DDAH gene comprising the steps of: i) providing a biological sample taken from a subject to be tested, ii) detecting the presence or absence of a variant genotype of the DDAH gene in the biological sample, the presence of a variant DDAH genotype indicating an increased risk of preeclampsia in said subject.

2. The method according to claim 1, wherein said variant genotype of the DDAH gene is a homo- or heterozygote form of the mutation.

3. The method according to claim 1, wherein the detection step is a DNA-assay.

4. The method according to claim 1, wherein the detection step is carried out using a gene or DNA chip, microarray, strip, panel or similar combination of more than one genes, mutations or RNA expressions to be assayed.

5. The method according to claim 1, wherein the allelic pattern is determined using polymerase chain reaction.

6. The method according to claim 1, wherein the biological sample is a blood sample or buccal swab sample.

7. The method according to claim 1, wherein the detection step is based on a capturing probe.

8. The method according to claim 1, wherein said preeclampsia involves dysfunction of vascular endothelium and/or imbalance between endothelium derived constricting and relaxing factors.

9. The method according to claim 8, wherein said dysfunction of vascular endothelium causes hypertension, proteinuria and/or edema.

10. The method according to any one of the previous claims, wherein said DDAH gene is DDAH 1 and/or DDAH2 gene.

11. The method according to claim 10, wherein one or more of the following variations in the DDAHl gene or mRNA is detected:

C.260OT Thr87Met (SNP1), IVS2-330T (SNP2), IVS3-70T (SNP3), c.561 A>G, Alal87Ala (SNP4), IVS4-680T (SNP5), IVS5-88G>A (SNP6), IVS5-71A>T (SNP7), and 3'UTR+16C>G (SNP8).

12. The method according to claim 10, wherein a DDAHl haplotype is detected.

13. The method according to claim 12, wherein said haplotype is HI, H2, H3, H4, or H5.

14. The method according to claim 13, wherein said haplotype is H2 or H3.

15. The method according to claim 1, wherein the detection of the presence or absence of a nucleic acid as defined in claim 10 or 11, i.e. target nucleic acid, in a biological sample comprises the steps of: a) treating said sample to obtain single stranded target nucleic acid, or if the target nucleic acid are already single stranded, directly employing step (b); b) contacting said target nucleic acid with a capturing nucleic acid probe and a detector nucleic acid probe; c) detecting the complex of capturing probe, target nucleic acid and detector probe.

16. The method according to claim 15, wherein the capturing nucleic acid probe is attached or capable of attaching to a solid phase, and comprises a cDNA corresponding to the gene coding a variant DDAH protein, and wherein a detected signal from the solid phase is an indication of the presence in the sample of a nucleic acid as defined in claim 10 or 11.

17. The method according to claim 15, wherein the capturing nucleic acid probe is attached or capable of attaching to a solid phase, and comprises a cDNA corresponding to the gene coding a wild-type DDAH protein, and wherein a detected signal from the solid phase is an indication of the absence of the nucleic acid as defined in claim 10 or 11 in the sample.

18. The method of claim 1, wherein said method is used for diagnostics of preeclampsia.

19. A kit for detecting genetic variation or polymoφhism, i.e. a mutation, in a DDAH gene for the determination of a risk of preeclampsia in a subject, comprising means for DDAH gene allele detection, and optionally software to inteφret the results of the determination.

20. The kit according to claim 19, wherein said DDAH is as defined in claim 10.

21. The kit according to claim 20, wherein the genetic variation to be detected is as defined in claim 11.

22. The kit according to claim 19 comprising a capturing nucleic acid probe specifically binding to the variant genotype as defined in claim 11.

23. The kit according to any one of claims 19-22, comprising a DNA chip, microarray, DNA strip, DNA panel or real-time PCR based tests.

24. The kit according to any one of claims 19-23, wherein said kit is used for diagnostics of preeclampsia.

25. A method for targeting the treatment of preeclampsia in a subject with preeclampsia or prevention of preeclampsia by determining the pattern of alleles encoding a variant DDAH gene, i.e. by determining if said subject's genotype of the DDAH gene is of the variant type, comprising the steps presented in claim 1, and treating a subject of the variant genotype with a drug affecting DDAH production or metabolism of the subject.

26. The method according to claim 25, wherein said DDAH gene is as defined in claim 11.

27. The method according to claim 25, wherein said method comprises a therapy enhancing DDAH availability, production or concentration in the circulation of the human subject or animal.

28. The method according to claim 27, wherein the said method of treating is a dietary treatment, a vaccination, gene therapy or gene transfer.

29. The method according to claim 28, wherein said therapy comprises the transfer of the non- variant DDAH gene or fragment or derivative thereof.