WO1999066069A1 - Genetic marker for mortality risk of an individual predisposed to hypertension or cardiovascular disease - Google Patents

Abstract

A method of testing an individual predisposed to hypertension or other cardiovascular disease to ascertain the general survival rate of the individual, the method comprising determining the genotype in the individual of a polymorphism in the inducible nitric oxide synthase (iNOS) gene (NOS2A), wherein the presence of one allele of the polymorphism is indicative of an improved survival rate over a hypertensive individual with a contrasting allele of the polymorphism.

Description

GENETIC MARKER FOR MORTALITY RISK OF AN INDIVIDUAL PREDISPOSED TO HYPERTENSION OR CARDIOVASCULAR DISEASE

Technical Field

The present invention relates to a method of predicting the general survival rate of an individual predisposed to or having hypertension or other cardiovascular disease.

Background Art

A locus for essential hypertension has been reported recently on chromosome 17 after sibpair analysis using microsatellite markers. The broad linkage region identified borders, at cen-qll.2, the inducible nitric oxide synthase (iNOS) gene (NOS2A), which spans —37 kb and contains 26 exons, exons 22-26 also being represented as 4 partially duplicated sequences in humans and great apes at this (NOS2B, -C) and other (-E) loci.

NOS2A, along with the neuronal (n)NOS and endothelial (e)NOS isoform genes, NOSl and NOS3 (chromosomes 12q24.2-q24.31 and 7q35- q36), respectively, are involved in the generation of nitric oxide (NO), a potent vasodilator. NOS2A is expressed in various tissues, including some relevant to the cardiovascular system, viz. cardiac and vascular smooth muscle, renal tubules and afferent arteriole. There is, moreover, evidence that intrarenal expression of iNOS can regulate arterial pressure. Although NO is reduced in essential hypertension, which may contribute to vascular and cardiac hypertrophy, and NO markers correlate inversely with blood pressure, there is no evidence to date that iNOS has a pathogenic role. In the spontaneously hypertensive rat (SHR), however, although iNOS expression is similar in vascular smooth muscle cells of pre hypertensive rats and Wistar-Kyoto controls, sustained NO production is lower in SHR, and NOS2 transcription differs between cells of each strain, leading to a suggestion that iNOS could be involved in the early rise in blood pressure. Others have found abnormal expression later in SHR hypertension. In Sabra DOCA-salt-hypertensive rats iNOS expression is reduced compared with their salt-resistant control strain and greater NO generation could contribute to the salt resistance of the latter. Dahl salt-sensitive hypertensive rats may have a defect in NO synthesis, seen in vivo as well as in primary cultures of aortic smooth muscle cells, and a transversion, T2140C (Ser714Pro), has been noted in the iNOS gene of Dahl rats. Moreover, various rat strains show a hypertension linkage region in the vicinity of Nos2. Two polymorphisms have been described for NOS2A. Both concern variation in repeated sequences and each is located in the 5'-flanking DNA. One is in an AAAT/AAAAT repeat at -756 to -716 relative to the major transcription start site ( + 1) and involves an insertion or deletion of one repeat unit. The other, located 2.7-2.5 kb upstream consists of 8 alleles of a

CCTTT_n pentanucleotide repeat with heterozygosity 0.80. Of possible relevance to a disease like hypertension, strand slippage in such repeats provides a rapid evolutionary mechanism for response to environmental change, with 9-16 repeat units in humans, but only 3-9 in chimpanzees. Similar sequences form a triplex structure in vivo, leading to Si nuclease- sensitive sites that may affect gene regulation. Poly-Pur/Pyr elements also occur in the 5 '-flanking DNA of the β-globin and rat ATla angiotensin receptor genes, and, as for NOS2A, correspond to DNase I hypersensitive sites in active chromatin. Moreover, variation in repeat number in the insulin promoter affects promoter activity and onset of insulin-dependent diabetes mellitus (IDDM).

Using these polymorphisms of NOS2A as markers, the present inventors have conducted the first disease association and linkage studies of this gene. As well as examining hypertension itself, whether there was any effect on mortality was determined. Disclosure of Invention

The present invention consists in a a method of testing an individual predisposed to hypertension or other cardiovascular disease to ascertain the general survival rate of the individual, the method comprising determining the genotype in the individual of a polymorphism in the inducible nitric oxide synthase (iNOS) gene (NOS2A), wherein the presence of one allele of the polymorphism is indicative of an improved survival rate over a hypertensive individual with a contrasting allele of the polymorphism. In one preferred embodiment, the polymorphism is a bi-allele polymorphism located approximately 0.7 kb upstream of the NOS2A transcription initiation site ( + 1). Preferably, the polymorphism is within a nucleotide (AAAT/AAAAT) (SEQ ID NO: 1) repeat running from -756 to -716, wherein the insertion/deletion (+/-) of one of the repeating units giving a difference in length of the overall block of repeating units of 4 base pairs (bp). Particular regions of interest for amplification and analysis are 313 bp

("-" allele) or 317 bp (" + " allele) embracing within the nucleotide repeat polymorphism located approximately 756 to 716 bp upstream from the NOS2A gene transcription initiation site.

The method is preferably performed using polymerase chain reaction (PCR) techniques to amplify from biological samples (extracted DNA) the upstream or promoter region of NOS2A in which the polymorphism of interest is likely to exist. It will be appreciated, however, that other genetic techniques such as sequencing, allele-specific hybridization and various other DNA hybridization or ligase technologies would also be applicable. The present inventors have found that the PCR primers listed below are particularly suitable to amplify the upstream region of the NOS2A transcription initiation site to locate the presence of the polymorphism of interest: sense:- 5'-TGG TGC ATG CCT GTA GTC C-3¹; (SEQ ID NO: 3); and antisense:- 5'-GAG GCC TCT GAG ATG TTG GTC-3' (SEQ ID NO: 4). The ability to screen hypertensive individuals (or other individuals having cardiovascular disease) for the polymorphism according to the present invention will allow the identification of "high-risk" individuals that may be at further risk for long term survival. The identified individuals may require additional medical monitoring or support to assist them to live longer. Further research may also be targeted towards the newly identified "high risk" population to assist or manage their hypertension. Similarly, the identification of a particular "high risk" population may allow further understanding of the role of the identified polymorphism of NOS2A, or other genetic abnormalities, in patient survival. Another polymorphism tested by the present inventors was a multi- allelic marker located 2.7-2.5 kb upstream from the NOS2A gene transcription initiation site consisting of 8 alleles of a CCTTT_n (SEQ ID NO: 2) pentanucleotide repeat with heterozygosity 0.80. It was found, however, that this polymorphism was not indicative of survival potential. For the detection of the multi-allelic marker, the present inventors have found that PCR primer sequences listed below were particularly suitable: forward:- 5'-ACC CCT GGA AGC CTA CAA CTG CAT-3' (SEQ ID NO: 5): and reverse:- 5'-GCC ACT GCA CCC TAG CCT GTC TCA-3' (SEQ ID NO: 6). In a second aspect, the present invention consists in the use of PCR primers: sense:- 5'-TGG TGC ATG CCT GTA GTC C-3^*; (SEQ ID NO: 3) and antisense:- 5'-GAG GCC TCT GAG ATG TTG GTC-3' (SEQ ID NO: 4) in a method of testing an individual predisposed to hypertension to ascertain the general survival rate of the individual, wherein the primers being directed to a polymorphism in the inducible nitric oxide synthase (iNOS) gene (NOS2A) of the individual.

The present invention is particularly suitable for potential hypertensive individuals. Analysis of subjects having or predisposed to angina, a manifestation of cardiovascular disease, also had a bearing on the results obtained for hypertensive individuals. From the results obtained by the present inventors, it will be appreciated that the method according to the present invention would be suitable for screening or testing for survival rates in individuals having, or predisposed to, cardiovascular disease including, for example, coronary artery disease, stroke, myocardial infarction, and complications stemming from cardiovascular disease including kidney related problems and disease.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

In order that the present invention may be more clearly understood, preferred forms will be described in the following examples.

Modes for Carrying Out the Invention METHODS

Subjects for Hypertension Study Association study The association study involved hypertensives with a strong genetic background (two hypertensive parents). These have a greater likelihood of showing an existing association than for only one affected first degree relative or an unselected hypertensive group. This group has, moreover, been the subject of a number of previous molecular genetic studies of hypertension, wherein ascertainment details have been described. In all there were 112 unrelated, age- and sex-matched, non-diabetic, treated Caucasian essential hypertensive patients and a control group of 164 normotensive subjects who also had parents who both had the same blood pressure status. Hypertensives with two affected parents represent —10% of all hypertensive patients. Characteristics of the groups are shown in Table 1. These studies had human ethical approval and all subjects gave informed consent.

Table 1. Demographic Parameters (meaan ±_ SD) for Normotensive (NT) and Hypertensive (HT) Groups Used for Association and Sibpair Studies of a Bi- allelic (Bi-) and Multi-allelic (Multi-) Polymorphism Upstream of NOS2A.

Association Studies Sibpair Studies

Bi- Multi- Bi- Multi-

Parameter HT NT HT NT HT HT

Male/female 50/59 81/68 52/60 91/73 78/156 76/151

Age (y) 52 47 53 47 61 60

± 12 ± 10 ± 12 ± 10 ± 10 ± 10

(n) (109) (149) (112) (164) (219) (212)

BMI (kg m²) 26 26 26 26 27 27

± 5 ±4 ±5 ±4 ±5 ±5

(n) (109) (144) (107) (164) (201) (197)

Systolic pressure pre- 175 119 176 120 171 172 treatment (mmHg) ± 25 ± 10 ± 24 ± 10 ± 25 ±25

(n) (89) (147) (88) (164) (103) (102)

Diastolic pressure pre- 112 73 111 73 103 103 treatment (mmHg) ± 19 ±8 ± 19 ±8 ± 14 ± 14

(n) (89) (147) (88) (164) (96) (95) Sibpair study- Two hundred and thirty-four individuals from 98 hypertensive sibships ( = 2 or more affected sibs) were contacted by community advertising. All were Anglo-Caucasians, mainly from eastern Australia, principally Sydney, and had to have systolic/diastolic blood pressure of > 140/90 mm Hg and not have diabetes or renal disease. After adjustment for 14 trios and 6 quartets the weighted sibpair number was 156. Table 1 shows their characteristics. Subjects for Coronary Artery Disease Study

Subjects for this association study were a high risk population of patients referred to the Eastern Heart Clinic at Prince of Wales Hospital for coronary angiogram, with a provisional diagnosis of coronary artery disease (CAD). Each patient's medical history was obtained using a questionnaire with standardized choices of answers to be ticked during the interview and DNA samples were collected for each patient as described previously. The severity of CAD was determined by the number of significantly stenosed coronary arteries. Each angiogram was classified as revealing either normal coronary arteries or having no coronary lesion with _> 50% luminal stenosis or as having one, two, or three major epicardial coronary arteries with more than 50% luminal obstructions. An angiographically normal coronary artery was defined as smooth contour and no luminal stenosis. Genotyping

This involved leukocyte DNA. Genotypes for the bi-allelic polymorphism were determined by PCR using the following primers: sense - 5'-TGG TGC ATG CCT GTA GTC C-3'; antisense - 5'-GAG GCC TCT GAG ATG TTG GTC-3'. The former was labelled with FAM during synthesis by

Bresatec (Adelaide, South Australia). The PCR mix (25 μl) consisted of 50 ng DNA, 20 nmol each primer, 0.25 mmol/L each dNTP, 1 U AmpliTaq Gold™ DNA polymerase (Perkin-Elmer, Norwalk, CT, USA), 50 mmol/L KC1, 10 mmol/L Tris-HCl, pH 8.3, 1.7 mmol/L MgCl₂ and 4 μg BSA. A "hot-start" protocol was employed in which after initial denaturation at 94°C for 5 min, there were 10 cycles of 94, 65 and 72°C for 1 min each, followed by 15 cycles of 94, 60 and 72°C for 1 min each, and finally 20 cycles of 94, 58 and 72°C for 1 min each, finishing with a step at 72°C for 30 min.

For the multi-allelic marker, PCR primer sequences were: forward, 5'- ACC CCT GGA AGC CTA CAA CTG CAT-3' (FAM-labelled) ; reverse, 5'-GCC

ACT GCA CCC TAG CCT GTC TCA-3'. The final PCR mix (8 μl) comprised 50 ng DNA, 5.4 nmol each primer, 0.24 mmol/L each dNTP, 0.4 U AmpliTaq Gold™ , 49 mM KCl, 10 mmol/L Tris-HCl, pH 8.3, and 2 mmol/L MgCl . After an initial denaturation step at 94°C for 12 min, 35 cycles of 94°C, 55°C and 72°C for 30 s each were performed, finishing with a 10 min step at 72°C. All PCR products were electrophoresed on an ABI 377 automated sequencer (Applied Biosystems, Foster City, CA, USA) and genotypes were assigned using ABI Genotyper™ software, according to " + " (plus; 317 bp) or "- " (minus; 313 bp). Statistical Analysis Genotype data was used to calculate total alleles on all chromosomes and these data were tested by χ² analysis using Excel (Microsoft, Redmond, WA, USA). The comparison of different parameters across genotypes involved one-way ANOVA. Linkage analysis was performed using programs suitable for complex traits. Since different programs have relative advantages and disadvantages, the use of several is deemed desirable. These included: SPLINK, which uses allele shared IBD estimates for all possible pairs in a sibship and computes probabilities for each marker genotype when parents are not available; ASPEX, which uses an alternate restriction to SPLINK when performing maximum likelihood calculations, although has been criticized for its unacceptably high rate of false positive results; the Affected Pedigree Member (APM) method, which uses a non-random co- segregation statistic to test distortions in alleles shared IBS at a marker locus; and MAPMAKER/SIBS. The significance thresholds that were set for acceptance of linkage were in accord with current recommendations. Determination of linkage disequilibrium between the different polymorphisms involved analysis of haplotype frequencies in the largest group as described by Thomson and co-worker in 1988.

In the study of CAD patients, all the continuous variables are presented as mean ±_ SEM. Hardy- Weinberg equilibrium was assessed by χ analysis as described previously. A contingency table χ² analysis was employed to estimate the contribution of the NOS2A polymorphism to the occurrence and severity of CAD and to evaluate relationships between the NOS2A genotypes and other medical conditions including myocardial infarction, family history of CAD, and hypertension. Logistic regression analysis (stepwise linear model) was used to assess the association between the polymorphism and CAD with other known risk factors being controlled. Transient Expression Analyses

Recombinant DNA Constructs:

NOS2A promoter constructs were made by cloning one kilo-base pairs of NOS2A 5'-flanking DNA into the pGL3 basic reporter gene vector (Promega, Madison, WI, USA) after one subcloning step. To do this, leukocyte DNA was taken from subjects previously genotyped as homozygous for either the minus or the plus allele, and used nested PCR to obtain a 1.2 kb fragment of NOS2A extending from + 105 to —1090, relative to the transcription start site ( + 1). Primers for the external PCR were: forward, 5'-GGA CGG TGA GAT CAA GGT GAC-3'; reverse, 5'-GAC AGT CAA ACC AGG AAG-3'. Primers for the internal PCR were: 5'-TGC TTC TCA ACT TCT CCC-3' and 5'-AGA CCT GTG GCC TTG AGA ACT-3'. PCR involved 30 cycles of 94°C for 30 s, 48°C for 30 s and 72°C for 1.5 min. PCR products were then digested withNsil (cutting site: -1090) / Xbal (cutting site: + 105) and ligated into Psrøbαl-cut pBSIIKS (Stratagene, La Jolla, CA, USA). These subclones were finally digested with Kpnl/Xbal and cloned into pGL3 linearized with Kpnl/Nhel. CMV-pGL2, which was constructed on a pGl-2 backbone, was used as a positive control. Transfection efficiency control, CMV-SEAP, was constructed by cutting pIRES-EGFP (Clontech, Palo Alto, CA, USA) with Notl/Bglϊl and cloned into Notl/Bglll linearized pSEAP2-Basic (Clontech). All constructs were verified by restriction enzyme analysis and DNA sequencing. Cell culture

Cell culture involved cells obtained from the American Type Culture Collection (ATCC. Rockville, MD) and media from Gibco BRL (New York, NY, USA). A549 cells (ATCC: CCL 185), a human alveolar type II epithelium-like pulmonary adenocarcinoma cell line, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 units/ml penicillin/streptomycin. Calu-6 cells (ATCC: HTB-S6) were grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum, sodium pyruvate and non-essential amino acids as described previously. Cells were kept at 37°C under 5% CO2. Transfection and luciferase assay

A549 and Calu-6 cells were transfected at 70% confluency in 60 mm petri dishes using Fugene 6 (Boehringer Mannheim, GmbH, Germany). Cells were incubated with 1 μg of DNA and 3 μl of Fugene according to the directions supplied by the manufacturer. As a control for transfection efficiency, all cells were co-transfected with pCMV-SEAP (secreted alkaline phosphatase). Luciferase assay was performed 24 h after transfection using a kit (Luciferase Assay System, Promega) and the protocol supplied by the manufacturer. Luciferase activity was measured in a Berthold model LB9501 luminometer (Germany). Luciferase activity was calculated relative to activity of basic pGL3 vector after correction for transfection efficiency. SEAP assays were performed using the Great EscAPe chemiluminescent system (Clontech), as well as by luminometry. Four independent experiments were performed for each construct in each cell line. RESULTS

Bi-allelic Marker Association Study

Genotype and derived allele frequencies are shown in Table 2(a). Hardy-Weinberg equilibrium was observed. Frequency of the minor allele ( + ) was 0.14 in the normotensive group and 0.18 in the hypertensive group.

The difference was not significant. Minor allele frequency was 0.16 in male and 0.20 in female hypertensives (P = 0.5), and 0.18 in both lean (BMI < 26 kg/m²) and obese (= 26 kg/m²) patients. Comparison by 1-way ANON A of the various parameters in Table 1 across genotypes did not reveal any significant differences. For example, systolic blood pressure (mean ± S.D., mm Hg) in the hypertensives was 174 ± 25, 179 ± 25 and 170 ± 21 for -/-, +/- and +/+ genotypes (n = 62, 22 and 5), respectively, and for diastolic pressure was 112 ± 17, 11 ± 24 and 114 ± 21. Values for plasma lipids, plasma renin, plasma angiotensinogen and plasma ACE were similar to those described previously in the subjects studied. Values did not differ between genotypes, although since these were only determined on a limited number of plasma samples n values were too low to provide adequate power to allow any conclusion to be drawn. Sibpair Study Minor allele frequency in hypertensive sibs was 0.17. The bi-allelic marker was not very informative (information content = 0.17). Linkage analysis of sibpair data by SPLINK, either weighted or unweighted, gave P - 0.50. ASPEX, allowing for slight dominance variance, gave maximum log-like value (MLOD) of 1.8, and with no dominance variance, MLOD was 1.0. The APM method gave P = 0.66. Thus each test indicated no significant excess allele sharing. Table 2. Genotype and Allele Frequencies of the Deletion/Insertion (-/+) Polymorphism of NOS2A in (a) the Hypertensive Group (HT) and (b) Different Age Groups of HTs, Compared with the Normotensive Group (NT).

Genotype Frequency Total Alleles (Proportion) (Proportion)

Group -/- -/+ +/+ +

All NTs

149 113 29 7 255 43

(0.76) (0.19) (0.05) (0.86) (0.14)

(a) All HTs

109 76 27 6 1.4 0.5 179 39 1.1 0.3

(0.70) (0.25) (0.05) (0.82) (0.18)

(b) Different Age groups of HTs

χ^" values are for comparison of data for HT, or each age-group of HTs, with data for NT group

Multi-allelic Marker

Association study Frequencies of the 8-alleles in each group are shown in Table 3.

Observed heterozygosity was 0.74 in the normotensive group, 0.81 in the hypertensive group and 0.77 in the hypertensive sibs. Comparison of allele frequencies by χ² analysis showed no significant difference between the groups. Blood pressure and other parameters were also similar for each genotype. Table 3. Frequency of Alleles of 8-allele Pentameric Repeat Upstream of NOS2A in the Hypertensive Association and Sibpair Groups, the Normotensive Group, and in Another Caucasian Population

Genotype (size Df PCR product in bp) and frequency

Group n 178 183 188 193 198 203 208 213

HT sibs 227 0.01 0.03 0.14 0.19 0.37 0.19 0.04 0.03

HT 112 0.02 0.04 0.13 0.19 0.30 0.14 0.13 0.04

NT 164 0.01 0.04 0.13 0.21 0.30 0.21 0.07 0.03

Xu et al. * 202 - 0.03 0.12 0.19 0.29 0.23 0.10 0.03

*Data for a general population in which the polymorphism was first described

Sibpair study

Linkage analysis of sibpair data by SPLINK, unweighted and weighted, gave P values of 0.56 and 0.57, respectively, indicating no excess allele sharing. APM produced P = 0.4. A log-like value of 0.00 was obtained by

MAPMAKER/SIBS, and an exclusion Lod score of -1.6 was noted assuming a relative risk to sibling (λ_s) of 1.6. Linkage disequilibrium

The two markers were in weak linkage disequilibrium (D' = 70%, P = 0.05) with alleles 193, 198 and 203 of the multi-allelic marker being associated with the + allele of the bi-allelic marker, and alleles 178, 183, 188, 208 and 213 being associated with the - allele. Analysis in Different Age-groups

Genotype frequency is fixed from conception and should remain constant throughout life. Any deviation with age could indicate an effect on longevity. To test this, data in Table 2(a) were re-analyzed after subdivision into age groups of < 50 years, 50-59 and greater than or equal to 60, as done previously to reveal a deleterious effect of the deletion allele of an ACE gene variant in the same group. As can be seen in Table 2(b), although frequencies were virtually identical with control in the younger age-groups, the older hypertensives had double the frequency of the χ² allele (0.28), with correspondingly lower - allele frequency (χ² = 7.4, P = 0.006). In contrast, the normotensives showed no difference with age. Comparison of -/- with -/+ and +/+ frequencies combined gave χ² = 7.9, P = 0.005. The hypertensive group, but not the normotensives, displayed, moreover, a significant interaction of age with genotype: -/- = 50.7 ± 11.9 S.D. y (n = 76), -/+ = 57.3 ± 11.8 (n = 27) and +/+ = 53.8 ± 13.3 (n = 6) (P = 0.05 by ANOVA). Pre-treatment blood pressures were similar for each genotype, in each age group; e.g., in the older subgroup, systolic = 183 ± 24 S.D., 182 ± 20, 180 ±

14 for -/-, -/+ and +/+ (n = 16, 20 and 2, respectively); diastolic = 112 ± 15, 112 ± 23, 113 ± 4. All parameters were similar for older vs younger patients, except that pre-treatment systolic pressure was higher in the older subgroup (182 ± 21 cf. 171 ± 26 S.D. mmHg; P = 0.03). Similar analyses in the hypertensive sibs, whose blood pressure was lower overall (mean diastolic = 103 cf. 112 mm Hg; Table 1), showed no difference across age groups (- frequency: 0.15, 0.20, 0.16 for age < 50, 50-59 and greater than or equal to 60 y, respectively). However, restriction to sibs with more severe hypertension (diastolic greater than or equal to 100 mm Hg; mean = 107 ± 10 S.D.), revealed a genotypic difference in the 20 aged greater than or equal to 60 y: viz. 0.60, 0.40 and 0.0 for -/-, -/+ and +/+ , respectively [P = 0.03 vs normotensives). For the 38 sibs aged < 60 (with diastolic greater than or equal to 100 mmHg) genotype values (0.76, 0.21 and 0.03) for -/-. -/+ and +/+ were similar to the normotensives. Age for each genotype of the more severely hypertensive sibs was 57 ± 10 S.D. y, 59 ± 10 and 57, respectively (n = 48, 20 and 1).

For the multi-allelic marker, although numbers for each allele were smaller, no age-related differences could be seen. Coronary Artery Disease Study Genotype results for 401 consecutive CAD patients are shown in Table

4 and these did not differ significantly from control. Using univariate χ analysis, no association was also found between NOS2A genotypes and past history of myocardial infarction in the CAD patients, nor hypertension, smoking status, number of diseased coronary arteries, as well as occurrence of CAD. The lack of correlation between the NOS2A genotype and CAD was confirmed using a logistic regression model in which a list of known risk factors was controlled. In particular, there was no interaction with either smoking status nor with lifetime smoking dose. Table 4. Frequency of genotypes of NOS2A promoter polymorphism in patients with coronary artery disease (CAD).

NOS2A Genotypes and ^frequencies

CAD Patients -/- +/- +/+ Total

Males 221 (74.7%) 63 (21.3%) 12 (4.1%) 296 Females 82 (78.1%) 21 (20.0%) 2 (1.9%) 105

Total 303 (75.6%) 84 (20.9%) 14 (3.5%) 401

Association of NOS2A Promoter Polymorphism with Angina Severity

A significant association was observed between with NOS2A genotype and severity of angina (Table 5), the + allele being elevated in progressing from no angina to stable and thence to unstable angina.

Table 5. Frequencies of enotypes of NOS2A promoter polymorphism in CAD patients without and with angina, either stable or unstable.

NOS2A Genotypes and Frequencies

Status -/- -/+ +/+ Total

No angina 3 (83.0%) 14 (15.9%>) 1 (1.1%) 88 Stable angina 104 (77.0%) 25 (18.5%) 6 (4.4%) 135 Unstable angina 125 (70.6%) 45 (25.4%) 7 (4.0%) 177

Total 302 84 14 400

If both variables are regarded as ordinal variables, i.e., the effect is dose dependent, the Spearman correlation is 0.113, P=0.024.

Association of NOS2A Promoter Variant with Other Risk Factors

Next NOS2A genotype was examined in relation to conventional risk factors including lipid profiles, lifetime smoking dose and demographic data. This involved 1-way analysis of variance (ANOVA). There was no association between any lipid profiles and NOS2A genotypes. However, there was an association between the NOS2A polymorphism and waist/hip ratio, as well as plasma glucose concentration (Table 6). This was more pronounced in male patients. Table 6. Waist/hip ratio and plasma glucose (mmol/L) for each genotype of the NOS2A promoter polymorphism in CAD patients.

NOS2A Genotypes

Parameter -/- -/+ +/+ Total

Waist/hip (total)* 0.945 + 0.005 0.941 + 0.01 1.013 + 0.02 0.947 + 0.004

Waist/hip (males)® 0.979 + 0.004 0.966 + 0.009 1.026 + 0.01 0.978 + 0.003

Glucose (total) ^Λ 6.06 +_0.18 7.12 ±_0.53 8.22 + 2.2 6.34 + 0.19

Glucose (males)# 5.94 + 0.20 7.53 + 0.67 8.22 + 2.2 6.36 + 0.23

*P = 0.024 (n=384) (ωP = 0.014 (n = 286) Λ P = 0.018 (n = 215) #P = 0.005 (n = 164)

Effect of Genotype on Promoter Activity The present inventors were able to detect low basal NOS2A mRNA in cultured A549 cells by RT-PCR, but none was observed in Calu-6 cells. Transfection results for 4 separate transient expression experiments with NOS2A promoter-luciferase reporter constructs are shown in Table 7. The positive control, involving a construct containing the ubiquitously expressed CMV promoter, showed high luciferase expression in the case of both the

A549 and the Calu-6 cells, this being greater in the former. For the NOS2A promoter constructs we found that luciferase activity of those made from the -/- genotype indicated 10-times higher luciferase reporter gene expression than that of +/+ constructs in both cell types. Calu-6 cells did not appear to possess low level iNOS expression, although like many cells might be capable of being induced to do so by appropriate stimuli, and the level of luciferase activity observed in Calu-6 cells was one-third that seen in the A549 cells. Table 7. Promoter activity of NOS2A constructs containing 1.1 kb of proximal promoter DNA from subjects homozygous for the deletion allele (-/-) and for the insertion allele (+/+) of tetranucleotide-repeat polymorphism 0.7 kb upstream. Shown also are results for positive control involving the CMV promoter.

Luciferase activity (relative to pGL3 negative control) -/- +/+ P CMV +ve control

A549 cells 10.2 ± 0.4 1.1 ± 0.2 < 0.0001 220 ± 24 Calu-6 cells 3.4 ± 0.7 0.2 ± 0.03 0.005 20 ± 1.2

Mean ± SE (n = 4 experiments); P values are for comparison of -/- with +/+ .

DISCUSSION The present study found no evidence for association or linkage of markers at the NOS2A locus with essential hypertension. Both bi-allelic and multi-allelic polymorphisms were tested. The former are preferred in association studies, whereas the latter can provide the necessary informativeness for linkage analyses. Association studies test whether a disease and an allele show correlated occurrence in a population, whereas linkage studies test whether they show a correlated transmission within a pedigree. Bi-allelic allele frequency in our normotensives and younger hypertensives was similar to what others have observed in unselected European Caucasians (minor allele frequency = 0.15, n = 35). Since, in the absence of exhaustive studies, it is generally believed that results of association analyses only apply to the polymorphism tested, the present findings do not rule out hypertension association for (an) other variant(s) in or near NOS2A that is not in linkage disequilibrium with the markers tested. The negative sibpair linkage result suggests either that the NOS2A locus is not linked to essential hypertension or the size of the study group was insufficient to provide sufficient power to reveal a small genetic contribution of NOS2A or a linked gene to hypertension. For strong family history and disease onset prior to age 55 y, relative risk to a sibling (λ_s) is 3.8, meaning that in a complex disease —100 sibpairs would be sufficient to provide 90% power to show significant linkage at the Lod = 3 (P = 0.0001) level. For a disease with multiple weak contributing loci (e.g., λ_s values of ~2), 156 sibs should have 90% power to provide evidence at the Lod = 2 (P = 0.001) level, as indeed was found with the number of sibs for markers on chromosome 1 and 17 and as others have reported for the angiotensinogen locus. Thus a negative result from both association and linkage analysis helps provide some assurance about the validity of the conclusion reached.

The two groups of hypertensives had similar inclusion criteria and geographic location. However, the stronger family history of hypertensives with two hypertensive parents may explain why their age of onset of hypertension (32 ± 10 S.D. y) was earlier than the hypertensive sibs (43 ± 13 y). so accounting for their lower age (53 ± 12 S.D. cf. 61 ± 10 y), and more severe hypertension (Table 1). Moreover, the fact that age of onset for the older hypertensives (33 ± 7 y) was similar to the younger hypertensives rules out a different etiology involving late-onset disease.

There has been no previous molecular genetic study of NOS2A in hypertension, so the present results add to findings ioτNOSl , where an 8- allele microsatellite (heterozygosity 0.52) in exon 29 failed to show association with hypertension in Japanese patients, and NOS3, where a 24- allele dinucleotide repeat (polymorphism information content 0.92) in intron 13 showed no linkage with hypertension in 269 Caucasian sibpairs, nor association in 88 hypertensives with two hypertensive parents. In Japanese, although no difference was observed for the whole group, hypertensives without left ventricular hypertrophy showed weak (P = 0.02) association with hypertension. Two other single base substitution polymorphisms, in introns 18 and 23, have also proved negative for hypertension in Caucasians, but a T/G variant in exon 7 that causes an amino acid substitution

(Asp298Glu) was 16% more frequent (P = 0.004). It displayed, however, no association with blood pressure. Thus results to date fov NOSl and NOS3 also provide little support for each in hypertension etiology.

Although the present inventors found no association in the group as a whole or in younger hypertensives, in the case of the bi-allelic variant, older more severely affected hypertensives of either group displayed a 2-fold elevation in +/- genotype frequency, and a 1/3 reduction in -/- genotype frequency. The -/+ hypertensives were also 7 years older than -/- hypertensives. No conclusion could be made about +/+ homozygotes, since patient number and population frequency for this genotype was low. A possible cause of altered genotype frequency with age may involve effects on survival or mortality. The present results would be consistent with a deleterious effect in high risk patients with the -/- genotype or a survival advantage in those with the -/+ genotype. What this would mean is that, at the level of the gene, the promoter variant tested could affect NOS2A expression or be in linkage disequilibrium with (an) other variant(s) that confers altered promoter activity, mRNA stability, or is a sequence variant of iNOS having different enzymatic activity. Thus alteration in iNOS-mediated NO formation could affect death rate in at- risk individuals. Depending on the tissue, cytotoxic effects of elevated iNOS activity might be either beneficial, e.g., in reducing tumour growth, or harmful, e.g., in β-cell destruction and onset of NIDDM, atherosclerosis and coronary disease. Infections result in an iNOS response, which may be beneficial or deleterious, depending on the pathogen. Survival effects could involve its immune or pro-inflammatory functions and enhanced vasodilatory actions could be cardioprotective. The effects of iNOS therefore appear dichotomous. However, the greatest cause of death in patients aged > 60 y with moderate to severe hypertension is heart attack and stroke. Atherosclerosis is an exacerbatory factor, for which iNOS has both pathogenic and protective functions. Both iNOS and ACE are elevated markedly in human coronary plaque macrophages. Higher iNOS levels in -/- patients could thus have a survival disadvantage.

However, results of the CAD study provide no support for an association of NOS2A genotype with heart disease. One possibility is that stroke and/or renal disease could be the possible cause(s). Since the type of hypertensives studied (offspring of two hypertensive parents) represent 10% of the total population of hypertensives, their spectrum of terminal events could differ from those of hypertensives as a whole. In this regard, their level of blood pressure is high and could contribute more to, for example, renal damage, than might mild hypertension. End-stage renal disease affects > 3% of patients with severe hypertension and elevates relative risk of renal failure 12 fold. Hypertension also exacerbates atherosclerosis, which leads to ischaemic nephropathy.

It is noteworthy that the present inventors only found associations with clinical manifestation of CAD but not with pathological changes in the arterial wall. It could be that whereas iNOS is the major NO-generating isoform in normal coronary arteries, iNOS may play a more important role in coronary arteries with existing atherosclerotic changes. In these arteries, iNOS is more likely to be depressed due to endothelial cell dysfunction. Activation of iNOS becomes the major source for vascular wall bioactive NO. Although iNOS is classically regarded as requiring induction before it appears, it is quite likely that it could be expressed in a continuous manner in these diseased tissues since the stimuli may be there all the time. Such "constitutive" iNOS expression would not only help in vasodilatation of these diseased vessels, it could also play a role in remodelling. This argument could also help to explain the association between the NOS2A promoter polymorphism and clinical expression of angina. Patients who have low NOS2A promoter activity would have lower NO production. Therefore these patients may have a reduced compensatory mechanism, with the result that they exhibit more severe and unstable ischaemic pain. Thus the association of the " + " allele of the NOS2A genotype with severity of angina is consistent with the functional data on NOS2A promoter activity. Higher NOS2A promoter activity will lead to higher iNOS expression and therefore higher NO synthesis in or near cardiac myocytes. This may be expected to confer a greater vasodilatory ability in subjects with the "-" allele of the NOS2A tetranucleotide promoter and lower vasodilatory ability with the " + " allele". A reduced ability to vasodilate would be expected to result in predisposition to inadequacy of blood supply to cardiac muscle. Genotyping for the NOS2A marker should therefore help predict severity or angina. Taken together, the data are consistent with the following scenario:

Subjects with the + allele, by experiencing greater severity of angina (i.e., chest pain) will be more likely to seek medical attention, get treatment and have greater compliance with treatment. Thus they are not only more likely to be treated for cardiovascular disease (severe hypertension exacerbates CAD), but any treatment will have a more effective outcome. The memory of pain will be more likely to ensure diet and lifestyle modifications needed to improve prognosis. Thus the + allele patients will be more likely to survive. In contrast, the -/- patients will not be getting as strong a warning signal in the form of chest paid, so their frequency of seeking medical attention and subsequent compliance with advice and treatment may occur at a lower relative rate. As a result will be more likely to die from a terminal event such as a heart attack.

It could also be the case that iNOS expression in vascular smooth muscle cells may be able to delay the onset of restenosis following percutaneous transluminal coronary angiplasty.

The higher waist hip ratio and plasma glucose are markers of severity of the insulin resistance syndrome. The mechanism whereby lower iNOS expression could confer increased prevalence of this is not known. However, markers of inflammation predict CAD. Tumor necrosis factor-α is a stimulatory cytokine for iNOS expression and may produce insulin resistance by phosphorylating insulin receptor substrate 1 that then inhibits insulin receptor tyrosine kinase to attenuate insulin receptor signalling, reduce GLUT-4 in muscle and raise plasma free fatty acids. However, it could also be that the association between NOS2A genotype and glucose could be an indirect effect of central obesity, as represented here by waist/hip ratio.

Furthermore, higher NOS2A expression in -/- subjects would lead to higher NO, and thus greater blood flow and consequent glucose uptake, i.e., implies that NOS2A genotype could be primary determinant of insulin resistance and could be involved in etiology and other insulin-resistant syndromes. The data point to the value of NOS2A genotype testing as an adjunct to current clinical assessment. Degree of angina needs to be evaluated in the context of NOS2A genotype, as the latter can affect the former. Thus less severe angina in patients with the -/- genotype could indicate a higher risk profile than less severe angina in a patient with a + allele. The finding of higher mortality in the -/- hypertensives may reflect higher rate of heart attack arising from reduced management of their cardiovascular condition. The transient expression results showing that genotype has a marked effect on NOS2A promoter activity could indicate an effect of the tetranucleotide-repeat polymorphism itself, by, for example, an influence on transcription factor binding, or it could be that the insertion/deletion polymorphism at -0.7 kb serves merely as a marker for (an)other genetic variant(s) in the proximal 1.1 kb of promoter DNA that is in linkage disequilibrium with it. Notwithstanding the possibility that other polymorphisms could exist, the particular variant at -0.7 kb is the only polymorphism reported so far in the literature in the proximal 1.1 kb of

NOS2A 5 '-flanking DNA of Caucasians. The NOS2A promoter is complex. An enormous number of actual or potential transcription factor binding sites have been demonstrated, with sequences of functional significance extending as far as 8.3 kb upstream. Within the region of promoter DNA tested, a NF-kB site at -115 is particularly important, with disruption reducing promoter activity by 67% in A549 cells.

In the rat, a binding site for the cyclic AMP response element binding protein (CREB), and members of the C/EBP family of transcription factors, has been found just upstream of the NF-kB site. A heterodimer of a C/EBP family member and CREB exerts a synergistic effect on the NF-kB-mediated expression of iNOS in rat cardiac myocytes in response to cytokine stimulation. A binding site for Jun/Fos (collectively referred to as AP-1) has also been noted, as well as one for STAT family members. There is also evidence to suggest that the 3'-untranslated region of human NOS2A interacts cooperatively with the proximal 0.4 kb promoter to confer inducible expression, with the 3' region able to reduce basal reporter activity.

However, no information is available to indicate whether the tetranucleotide repeat at -0.7 kb has an influence on NOS2A transcription.

The possibility of a genetic effect on iNOS expression has been seen previously only in poultry. In this work, lipopolysaccharide (LPS) -stimulated macrophages from Cornell K-Strain chickens (B¹⁵B¹⁵), which are used for meat production, and a transformed cell line (MQ-NCSU) produced significantly higher levels of iNOS mRNA and NO (measured as nitrite) than macrophages from GBl (B¹³B¹³) and GB2 (B6B6) chickens, varieties used for egg production. This was also reflected in differences in iNOS enzyme activity. Although the difference could likely involve a promoter variant, this possibility was not explored.

That mutations in the NOS2A promoter can be selected for in human populations, presumably as a disease resistance mechanism, is evident in Africa. In Gabon, a locality hyperendemic for Plasmodium falciparum malaria, a NOS2A promoter mutation (G-969C) not known in Caucasian populations has been found. In children, the frequency of heterozygotes for this mutation was 17% in severe and 30% in mild cases (P = 0.04). In the Caucasian population of industrialized nations selection pressure on the NOS2A promoter variant or polymorphisms in linkage disequilibrium with it could reflect emergence of resistance to diseases that have been prevalent in

European populations during their evolutionary history. The deletion (-) allele, that was found in the present study to be associated with elevated NOS2A promoter activity, appears to predispose to a death rate higher than that for the insertion ( + ) allele. Perhaps the rarer (+) allele is a marker for the evolution of a resistant phenotype. However, the nature of the disease(s) responsible for any such selection pressure remains to be determined.

Moreover, selection for a predominance of the deletion (-) allele as a result of an advantage it, or linked variant(s), may have conferred in previous millennia, may in more recent times have altered. This allele may now have become deleterious, as a result of alteration in environmental influences, life expectancy and profile of major diseases affecting population mortality today.

In conclusion, the present work provides no support for involvement of the iNOS gene (NOS2A) in the genetics of essential hypertension or coronary artery disease, but suggests that genetic variation involving a tetranucleotide repeat in the promoter is a marker for mortality risk in subjects having moderate to severe cardiovascular disease such as hypertension. This could be because of association with severity of angina, which then results in greater attention and thus treatment effectiveness.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. In particular it is recognised that one or other genetic variants in linkage disequilibrium with the marker described may exist and can also serve as markers for cardiovascular disease survival potential.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: The University of Sydney

(B) STREET:

(C) CITY: Sydney

(D) STATE: New South Wales

(E) COUNTRY: Australia

(F) POSTAL CODE (ZIP): 2006

(ii) TITLE OF INVENTION: Genetic Marker for Hypertension Survival Potential

(iii) NUMBER OF SEQUENCES: 6

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0. Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 base pairs

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Claims

CLAIMS:

1. A method of testing an individual predisposed to hypertension or other cardiovascular disease to ascertain the general survival rate of the individual, the method comprising determining the genotype in the individual of a polymorphism in the inducible nitric oxide synthase (iNOS) gene (NOS2A), wherein the presence of one allele of the polymorphism is indicative of an improved survival rate over a hypertensive individual with a contrasting allele of the polymorphism.

2. The method according to claim 1 wherein the polymorphism is a bi- allele polymorphism located approximately 0.7 kb upstream of the NOS2A transcription initiation site (+ 1).

3. The method according to claim 2 wherein the polymorphism is within a nucleotide (AAAT/AAAAT) (SEQ ID NO: 1) repeat running from -756 to - 716, and wherein an insertion/deletion (+/-) of one of the repeating units giving a difference in length of the overall block of repeating units of 4 base pairs (bp).

4. The method according to claim 3 wherein regions of interest for amplification and analysis are 313 bp ("-" allele) or 317 bp ("+" allele) embracing within the tetranucleotide repeat polymorphism located approximately 756 to 716 bp upstream from the NOS2A gene transcription initiation site.

5. The method according to any one of claims 1 to 4 performed using genetic techniques including polymerase chain reaction (PCR), sequencing, allele-specific hybridization and various other DNA hybridization or ligase technologies.

6. The method according to claim 5 performed using PCR techniques to amplify the upstream or promoter region of NOS2A in which the polymorphism of interest is likely to exist from extracted DNA from biological samples obtained from the individual.

7. The method according to claim 6 wherein PCR primers suitable to amplify the upstream region of the NOS2A transcription initiation site to locate the presence of the polymorphism of interest are as follows: sense:- 5'-TGG TGC ATG CCT GTA GTC C-3'; (SEQ ID NO: 3) and antisense:- 5'-GAG GCC TCT GAG ATG TTG GTC-3' (SEQ ID NO: 4).

8. Use of PCR primers sense:- 5'-TGG TGC ATG CCT GTA GTC C-3': (SEQ ID NO: 3) and antisense:- 5^*-GAG GCC TCT GAG ATG TTG GTC-3' (SEQ ID NO: 4) in a method of testing an individual predisposed to hypertension or other cardiovascular diease to ascertain the general survival rate of the individual. wherein the primers being directed to a polymorphism in the inducible nitric oxide synthase (iNOS) gene [NOS2A] of the individual.

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SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: The University of Sydney

(B) STREET:

(C) CITY: Sydney

(D) STATE: New South Wales

(E) COUNTRY: Australia

(F) POSTAL CODE (ZIP) : 2006

(ii) TITLE OF INVENTION: Genetic Marker for Hypertension Survival Potential

(iii) NUMBER OF SEQUENCES: 6

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 base pairs

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TGGTGCATGC CTGTAGTCC 19

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(C) STRANDEDNESS: single

(D) TOPOLOGY: linear 3/3

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GAGGCCTCTG AGATGTTGGT C 21

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

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(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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ACCCCTGGAA GCCTACAACT GCAT 24

(2) INFORMATION FOR SEQ ID NO: 6:

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(C) STRANDEDNESS: single

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