Essential hypertension is the most common reason for adult visits to office-based physicians and for the use of medication 123. It is estimated that 24% of the US adult population (70% in persons over 70) and at least 50% of the Irish adult population aged 50 or over are hypertensive45. Ethnic differences exist in the prevalence of hypertension between populations, for example between Africans and Northern Europeans 678. Mendelian forms of hypertension resulting from single gene defects are rare 91011. The major contribution to the etiology of this disorder is proposed to result from the combined effects of genes that modify the response of blood pressure to environmental stresses such as diet and 'environmental susceptibility genes' 121314. Essential hypertension does not follow a clear pattern of inheritance but exhibits familial aggregation of cases. This multifactorial trait increases the affected individuals' risk of myocardial infarction, stroke and end-stage renal disease, and is one of the leading causes of morbidity and mortality in adults815. Population-wide application of preventative measures and candidate gene analysis to predict modifiable risks in addition to treatment are thus very worthwhile 16171819.

Trimethylaminuria (TMAuria) is an inborn error of metabolism (MIM # 275700) resulting from diminished oxidation of the tertiary amine trimethylamine to trimethylamine N-oxide resulting in a severe body odour in affected individuals. The microsomal NADPH-dependent flavin-containing monooxygenases (FMOs) (E.C. 1.14.12.8) are a family of chemical and drug metabolising enzymes that catalyze the phase 1 oxygenation of a wide variety of nucleophilic heteroatom containing compounds including catecholamines2021. FMO3, a phase 1 drug metabolising gene, is the main liver dependent human isoform22. We and others have shown that recessivity for mutations of FMO3 cause TMAuria 2021222324.

We have previously noted that a number of patients with TMAuria have idiopathic hypertension 2122. In addition, Cashman et al 25 have reported a proposed link in African-American males between hypertension and increased excretion of trimethylamine. We have identified increased excretion of catecholamines in a proband with TMAuria who is homozygous for a deletion of the FMO3 gene 20suggesting a possible association between abnormal catecholamine metabolism and variants of the FMO3 gene. Endogenous substrates for FMO3 include tyramine and phenylethylamine 2627 and tyramine has a known pressor effect. As high levels of circulating catecholamines contribute to hypertension, it is proposed that polymorphisms of FMO3 gene could contribute to impaired catecholamine metabolism and hypertension.

We have previously described the population frequencies of a number of common polymorphisms of the FMO3 gene (E158K, V257M, E308G) in a North American population2528. Expression studies indicate that the polymorphisms E158K and V257M exhibit decreased tyramine oxidation in vitro25. We and others have noted that the E308G polymorphism shows a substantial decrease in FMO3 activity in the presence of the E158K polymorphism causing mild TMAuria2229. While these common polymorphisms exhibit some difference in expression of the functional FMO3 enzyme, this effect is minimal in comparison to that of the null mutations.

We hypothesised that common FMO3 polymorphisms might predispose to essential hypertension. While null alleles in the FMO3 gene may be too rare to cause any appreciable effect on the population burden of hypertension, prevalent polymorphisms with documented in vitro evidence of variation in catecholamine metabolism may however potentially associate with and predispose to this condition.

Herein we report our investigations into a possible association of haplotypes of three polymorphic variants of the FMO3 gene with the phenotype of blood pressure in an occupational Irish adult population and with presence of essential hypertension in an Irish cardiovascular disease (CVD) population.

The AIB Phase II population consisted of 387 subjects, 224 (57.8%) of which were male. In comparison to the females, the males had on average higher blood pressure measurements, triglycerides and total cholesterol. Slightly more males in this group were smokers (Table 1).

The CVD group consisted of 1649 subjects, 691 (41.9%) of which were classified as having a history of hypertension requiring drug treatment. A greater percentage of those classified as hypertensive also had hypercholesterolemia and diabetes mellitus in comparison to those who were classified as non-hypertensive. As might be expected from a CVD group, most of the subjects were taking medication, with almost the same frequency of hypertensives and non-hypertensives taking each group of medications (Table 2). Blood pressure measurements are only presented to characterise, rather than compare, the groups, since medication is lowering blood pressure in most of the CHD group, but not in most of the AIB Phase II study group.

The most notable difference between the AIB Phase II study group and the CVD group is that there were a much greater proportion of current or ex smokers in the CVD group than in the AIB Phase II study group (73% vs. 30.1%).

The 3 SNPs were found to be in Hardy-Weinberg Equilibrium. 4 common haplotypes were inferred in the two populations (Table 3), with similar frequencies in the AIB Phase II study group, the hypertensives and in the non-hypertensives of the CVD group. However the haplotype KVG, which is the 2^nd most frequent haplotype in the AIB Phase II study group, was the 3^rd most frequent haplotype in the CVD group. However the difference in frequency was only 4%, which is most likely due to chance.

No statistically significant associations were found between any of the four common haplotypes and mean daytime systolic blood pressure in the AIB Phase II study (overall p = 0.65, 3 d.f) (See table 3). Nor was this statistically significant when adjusted for age, sex and daily alcohol intake (overall p = 0.729, 3 d.f) (See table 3). Neither did we find any statistically significant associations between any of the haplotypes and hypertension status in all subjects with and without adjustment of sex and age (see table 3). When stratified by gender, there were still no statistically significant associations between any of the specific haplotypes and daytime SBP in the AIB Phase II study (males overall p = 0.91, 3 df and females overall p = 0.61, 3 df) or with hypertension status in the CVD patients (males overall p = 0.59, 3 df and females overall p = 0.82, 3 df). Table 4 displays the genotypic frequencies of the 3 SNPs in the AIB Phase II population, in the hypertensives of the CVD group and in the non-hypertensives of the CVD group. When genotypic frequencies were compared among 3 groups, there were no statistically significant differences (E158K p = 0.30, V257M p = 0.47, E308G p = 0.15). There were no statistically significant associations between any of the individual genotypes with daytime SBP in the AIB Phase II study group, even when adjusted for age, sex and daily alcohol consumption (E158K p = 0.91, V257M p = 0.85, E308G p = 0.68).

The mean daytime SBP and mean daily alcohol consumption are broken down by genotype in table 5 and it is clear that there is not much difference between the genotype groups. Stratifying the analysis by gender, there was still no association between genotypic frequencies in the AIB Phase II study group (females E158K p = 0.92, V257M p = 0.86, E308G p = 0.13, males E158K p = 0.37, V257M p = 0.87, E308G p = 0.38).

There was no genotypic difference between hypertensive and non-hypertensive CVD subjects, even after adjustment for age and sex (E158K p = 0.84, V257M p = 0.44, E308G p = 0.91). When this analysis was stratified by gender, still no statistically significant differences are noted (females E158K p = 0.79, V257M p = 0.54, E308G p = 0.29, males E158K p = 0.71, V257M p = 0.22, E308G p = 0.66).

Testing for any associations between haplotype and daytime diastolic blood pressure, night time systolic blood pressure and night time diastolic blood pressure in the AIB Phase II study yielded no statistically significant associations (Table 6). Likewise it was shown by logistic regression that none of the SNPs were significant predictors for cardiovascular disease when the CVD and the AIB Phase II study groups were compared (E158K z = 0.60 p = 0.55, V257M z = -0.31 p = 0.76, E308G z = 0.49 p = 0.63), though diabetes was found to be a significant predictor.

The observation that a number of patients with TMAuria are hypertensive was the basis to the hypothesis that there may be an association between variants in the FMO3 gene and hypertension. The FMO3 enzyme has a broad substrate specificity, which includes catecholamines as a minor pathway2126. Catecholamines are produced in response to stress and modulate heart rate and blood pressure. Variants of the FMO3 gene resulting in decreased enzymatic activity could result in decreased catabolism of catecholamines, which impact on blood pressure homeostasis. As catecholamines are at peak levels during the day, we elected to study the association between daytime SBP and variants in the FMO3 gene as a primary endpoint. In our analysis, we adjusted for alcohol use as tyramine is a substrate for FMO3 oxidation26. The FMO3 enzyme is also modulated by the sex hormones28. Females with moderate to severe TMAuria note exacerbations perimenstrually and it is proposed that decreases in FMO3 enzymatic activity resulting from polymorphisms could also be influenced by hormonal events31. For this reason the analysis was stratified by treating males and females as separate groups.

In this study, by constructing haplotypes based on the 3 SNPs, we discovered that none of the haplotypes were statistically significantly associated with either daytime systolic blood pressure in the AIB Phase II study group or with hypertensive status in the CVD group. There was also a lack of association between any of the genotypes of the SNPs with daytime SBP or with hypertension status. Neither did the stratified analyses show any statitically significant associations.

While the definition of hypertension in the CVD population provides a rather arbitrary estimation of hypertension, the 24-hour blood pressure measurements in the AIB Phase II study were carefully controlled. Even so, the results from both groups showed that the variants had no association with blood pressure or hypertension status.

Why are FMO3 variants maintained in population, and in particular the E158K variant? It is proposed that genes such as FMO3, a drug and toxin detoxicating gene, may have survived as balanced polymorphisms in an ancient human or primate population to neutralise the effect of harmful exposures3233. Evolutionary changes in FMO3 may have evolved in different geographic locations to buffer changes in the highly polymorphic drug metabolising genes, which show substantial geographic and ethnic variation. It is possible that 'low penetrance' polymorphisms of FMO3 alone may not independently affect blood pressure homeostasis but severe loss of function causing mutations may unmask pressor effects of variation in other drug metabolising enzymes previously buffered by FMO334. This hypothesis is supported by the observation that only some, but not all, TMAuria patients exhibit hypertensive symptoms. Therefore other common FMO genes may be potential hypertension candidates. There could be identified by typing other FMO genes in TMAuria patients with hypertensive symptoms or by directly assessing the candidate genes in a large study of hypertension.

In conclusion, these data indicate that these three polymorphisms of the FMO3 gene inherited singly or in specific haplotype combinations do not represent an independent susceptibility risk for hypertension as an absolute trait or continuous variable in a representative Irish population.

CD carried out the statistical analysis and drafted the manuscript. DCS assisted in the statistical analysis and participated in the study design. AS contributed to the interpretation of the hypertension data and participated in the study design. EOB designed the data collection for the AIB Phase II study and participated in the study design. DML helped draft the manuscript. JKOB led the genotypic analysis component. EPT conceived the study and helped to draft the manuscript. All authors read and approved the final manuscript.