Monoamino oxidase alleles correlate with the presence of essential hypertension among hypogonadic patients

Abstract Background Monoamine oxidase (MAO) activity has been traditionally implicated in blood pressure through its effects on biogenic amine levels such as catecholamines, serotonin, and dopamine. Nowadays, this role is considered relegated to side‐effects such as orthostatic hypotension and/or hypertensive crisis derived from MAO‐inhibitory treatments in patients with psychiatric disease. Methods In the present work we have found an association between a polymorphic variant of MAOB gene and arterial hypertension in obese hypogonadic patients. The study cases comprised a series of 219 nondiabetic males with a body mass index ≥30 kg/m2 and aged <45 years. Hypogonadism was defined as subnormal testosterone concentrations, when free testosterone values ranged <65 pg/ml. Results MAOB rs3027452‐A allele carriers were significantly over‐represented among hypertensive (HT) patients (25.49%) in comparison to either the non‐HT patients (10%, OR = 3.079 CI95 [1.364–6.952], p = .005, Chi‐square test) and the control population series of nonobese nor hypogonadic males (also 10%, p = .003 Chi‐square test). Upon adjusted, an independent association was shown with the hypogonadic group with hypertension when compared with nonhypertensive hypogonadics (Beta = 3.653, p = .005). When quantitative analysis was performed, hypertensive patients harboring rs3027452‐A allele showed higher systolic blood pressure values (p = .038, Mann–Whitney U‐test) as well as an increased Systolic‐Diastolic range despite following HT treatment (∆mmHg 54 vs. 48 for rs3027452‐A and rs3027452‐G respectively, p‐value .019, Mann–Whitney U‐test). Previous studies on MAOB revealed that rs3027452‐A allele has been correlated to a lower activity of the enzyme, what gives a functional evidence over our observation. Conclusion If this result could be extrapolated to other hypertensive patient groups, it would implicate a review of the markers and therapeutic targets on human hypertension.

The most suitable way to determine MAOB activity is by analyzing thrombocyte-MAO activity (Trbc-MAO). It has been shown that MAO activity in the cerebral spinal fluid is associated to the serotonin activity . Moreover, Trbc-MAO activity has been described to be higher in women than in men (Harro et al., 2001). This difference in MAOB activity is thought to be due to a possible effect of sex steroid hormones or to an inappropriate imprinting X-inactivation, since MAOB gene expression has been shown to be upregulated by a decreased DNA methylation (Good et al., 2003;Launay et al., 2009;Shih, Chen, & Ridd, 1999;Shih, Wu, & Chen, 2011). On the other hand, Trbc-MAO activity has also been associated with several psychiatric syndromes, personality traits, and mood disorders (Shih et al., 1999).
Moreover, hypogonadism has been associated to hypertension. For instance, it has been described that men presenting a history of hypertension have higher prevalence of hypogonadism (Valerie Berry, 2005). It has also been reported that testosterone supplementation can lower blood pressure. This could be through an indirect action, via the decrease in adiposity, or by a direct action, via the decrease in plasma endothelin 1 (ET1, OMIM 131240), the contractile RhoA/ Rho-kinase (ROCK, OMIM 601702 and OMIM 604002) signaling pathway, the nitric oxide synthase (NOS) and an increase in the asymmetric dimethylarginine (ADMA) (Fahed, Gholmieh, & Azar, 2012). Thus, the aim of this work was to study the distribution of MAOA and MAOB polymorphisms in a group of nondiabetic obese patients with and without hypogonadism to discern the possible relationship between this genetic factor with the blood pressure in hypogonadism. This would be of clinical interest since could give clues to a better treatment of hypogonadism and its related complications as the hypertension.

| Study population
The study cases comprised a series of 219 nondiabetic obese males with a body mass index (BMI) ≥ 30 kg/m 2 and aged <45 years, consecutively referred from six primary care centers from Malaga (Spain) recruited between 2013 to June 2015. Hypogonadism was defined as subnormal testosterone concentrations, defined when free testosterone values <65 pg/ml or total testosterone <3.5 ng/ml. Exclusion criteria for this study were previous diagnoses of hypogonadism, diabetes mellitus, use of antidiabetic medication, or being under any treatment known to affect the gonadal axis. Subjects with hepatic or renal impairment, established cardiovascular disease or cancer were also excluded. All subjects had a normal pubertal development and referred intact sense of smell. The control population consisted on 228 males recruited among healthy volunteers from the School of Medicine, University of Malaga. The study was reviewed and approved by the Ethics and Research Committee of Virgen de la Victoria Clinical University Hospital, Malaga (Spain), and was conducted according to the principles of the Declaration of Helsinki. The participants, all volunteers, provided signed consent after being fully informed of the study goal and its characteristics.

| DNA extraction and genotyping
Cases provided a blood sample while healthy controls provided buccal swap that was used to extract genomic DNA using salting out method followed by ethanol precipitation. DNA quantity was determined using nanodrop prior to genotyping. Genotyping was outsourced to Genologica SL. SNP analysis was performed using the TaqMan Open Array Genotyping System from Applied Biosystems. The results obtained were processed using TaqMan Genotyper Software. The selected SNPs were chosen from the literature among those affecting the MAO activity (Gonzalez, Polvillo, Ruiz-Galdon, Reyes-Engel, & Royo, 2019;Xu et al., 2017;Zhang, Martin, Morris, & Li, 2009). Call ratios where >95% for every SNP.

| Statistical analysis
Statistical analysis was performed using the SPSS statistical package (version 22 for Windows; SPSS). Bivariate regressions were used to adjust genetic Odds Ratios (OR). Multivariate results were expressed as standardized coefficient (beta). Significance was established for p-values < .05. Haplotypes were estimated using THESIAS software as previously described (Tregouet & Garelle, 2007).

| RESULTS
In this study, we have evaluated the distribution of MAOA and MAOB polymorphisms among a hypogonadic population. No differences were observed between the genotype distribution of rs3788862, rs979605 nor rs3027452 between control population and the hypogonadic series. However, when the hypogonadic population was stratified according to the hypertensive status we found that rs3027452-A carriers were significantly over-represented among hypertensive (HT) patients (25.49%) in comparison to either the non-HT patients (10%, p = .005, Chi-square test) and the control population series of nonobese nor hypogonadic males (also 10%, p = .003 Chi-square test; .003 Abbreviations: HT, hypertensive; OR, odds ratios. Bold values indicate results statistically significant. Table 1). When bivariate logistic regression was performed in an intracohort study taking into account BMI and age as covariates, the association persisted (Beta = 3.653, p = .005; Table 2). However, there is no difference in the genotypic distribution between the control group and the hypogonadic subjects without differentiating by their hypertensive state, nor between control normotensives and non-HT hypogonadics. Interestingly, those HT patients harboring rs3027452-A allele showed higher systolic blood pressure values despite that fact that they were under HT treatment (139 mmHg ± 17 for rs3027452-G vs. 147 mmHg ± 10 for rs3027452-A, pvalue = .038, Mann-Whitney U-test; Figure 1; Table S1). The MAOA polymorphisms rs3788862 and rs979605 were in linkage disequilibrium and not associated with hypertension in hypogonadic patients. However, MAOA and MAOB polymorphism were not found in partial linkage and therefore were analyzed in combination ( Table 3). The MAOA-MAOB joint analysis showed that the rs3027452-A allele had an increased Odds Ratio regardless the MAOA haplotype, although only with the G-G combination was found to be statistically significant after adjusted by age and BMI. This might be a result of statistical power, since the G-G haplotype is more prevalent than the A-A. Thus, taking these data altogether we propose that the effect over hypertension shall be attributed to rs3027452 on its own.

| DISCUSSION
In this study we report a strong association of the polymorphism rs3027452 of the MAOB with essential hypertension in hypogonadic patients. The effects of the activity of MAOA and MAOB on blood pressure are controversial and dual. Thus, it has been related to variations in biogenic amines levels, and in most cases induced by pharmacological agents such as serotonin reuptake inhibitors, producing, serotonergic crisis (Boyer & Shannon, 2005), treatments with Levodopa, (Murphy, 2000) and combinations of neuropeptide-enhancing drugs such as selective serotonin reuptake inhibitors (SSRIs) and MAO inhibitors (MAOIs) in which there may be seizures in both hypo and hypertensive senses (Buu, Kuchel, Manger, Hulse, & Chute, 1986;Caston, Eaton, Gheorghiu, & Ware, 2002;Cockhill & Remick, 1987;Lefebvre, Noblet, Moore, & Wolf, 1995).
To the beast of our knowledge, we could not find any recent study linking MAO activity with essential hypertension, neither related to genetic determinants (polymorphisms or mutations) nor to therapeutic alternatives (MAOIs). Nevertheless, a genome linkage study found that two HT loci within the X chromosome, the first one pinpointed between base pair 10,000,000-52,058,362 and the second between 123,825,825,787. In this sense, MAO locus is located between base pair 43,654,907-43,746,824 and therefore the MAO locus might explain the first of the two linkage results (Ciccarelli et al., 2017).
Our obese hypogonadic population is diagnosed by low levels of free testosterone (<65 pg/ml) and all of them with a BMI >30 kg/m 2 . We have not found a correlation between the studied polymorphisms and free or total testosterone levels. On the other hand, the association between sympathetic effects of obesity in the generation of HT is well known (Re, 2009). It has been shown that the main reservoir of neuropeptides are platelets and platelet MAOB activity can be determinant for neuropeptides levels, (Jansson et al., 2005) however, no correlation was found between the number of platelets and genotypes in this population of hypogonadic subjects with or without hypertension (data not shown).
Associations between blood pressure and the studied MAO alleles were only present in the hypogonadic HT group even though these subjects were treated with several antihypertensive drugs. Thus, systolic blood pressure was associated with the allele rs3027452-A independently of the MAOA genotype. A previous work quantified MAO activity in platelets for the different MAOB polymorphisms (Jansson et al., 2005). They observed that the MAOB haplotype (C-C-A-G) (rs1181252, rs2283729, rs3027452, rs1799836) was the least active in males. This haplotype is the only one that contains the MAOB rs3027452-A allele, which is the polymorphism we have found to be overrepresented among our hypogonadic HT patients. Therefore, it might happen that the higher blood pressure found in these hypogonadic HT subjects could be due to the lower activity of MAOB associated to the A-allele.
In conclusion we report an association between the Aallele of the rs3027452 polymorphism for MAOB (which in turn can be associated to a lower MAOB activity) with blood pressure in obese hypogonadic patients with HT regarding the possible HT treatment. This would be of clinical interest since the assessment of genetic background in hypertension subjects could serve as starting point for personalized treatment. However, we are aware that our sample size might compromise the robustness of our findings. For this reason, further studies are necessary to replicate our results and eventually dig on the molecular mechanism by which these genetic variations can influence HT. Therefore we propose a genetic variant as a new etiological agent for HT that in turn may serve as a key target for new therapeutic approaches.

ACKNOWLEDGMENTS
This study was supported by research grants from the Andalusian Health Public System (grant number SAS PI-0173-2013). JCF-G and FC were supported by research contracts from the Andalusian Health Public System (B-0003-2017 and Nicolas Monardes C-0032-2016, respectively) by Fondo Europeo de Desarrollo Regional -FEDER. Daniel Castellano-Castillo was supported by a grant "FPU" (FPU13/04211) from the "Ministerio de Educación, Cultura y Deporte" co-funded by the "Fondo Europeo de Desarrollo Regional-FEDER."