Genetic polymorphisms in the serotonin receptor 7 (HTR7) gene are associated with cortisol levels in African American young adults

Serotonin is a neurohormone involved in biological processes, Introduction: such as behavior and immune function. Chronic psychosocial stressors may cause serotonin release resulting in immune system dysregulation, as evidenced by increased or far decreased levels of cortisol, a blood biomarker of stress and immune function. We hypothesize that genetic polymorphisms in the gene are associated with both hypoand hyper-cortisolism. HTR7 Methods: The study population included 602 African American subjects between 18-34 years of age, living in Washington, D.C. Five single nucleotide polymorphisms (SNPs) in , rs2420367, rs12412496, rs2185706, rs7089533, and HTR7 rs7093602 were genotyped by restriction fragment length polymorphism or the TaqMan assay. Statistical analysis, using the program SNPstat, was performed to determine their associations with cortisol measured in the study population. While an increased risk of hypocortisolism was found to be associated Results: with rs2420367, rs2185706, and rs7093602 in a gender specific manner, no genotypes could be associated with hypercortisolism. Inversely, a decreased risk of hypocortisolism was found with the haplotype CGGCC (p=0.033), which remained significant in males. When adjusting for gender, females associated with the haplotype AGACC. Hypercortisolism was also associated with a decreased risk for the haplotypes AAACC (p=0.042) and AAGTT (p=0.001). Based on these results, genetic variation in the gene may Discussion: HTR7 contribute to both stress and inflammation, and will provide a new glimpse into stress-related inflammation psychophysiology. 1 2,3 4 5

Psychosocial stressors, such as exposure to interpersonal and community violence, may impact immune function (O'Connor et al., 2000), leading to the development of a number of health disparities (Black, 2003;Murali et al., 2007). During exposure to a stressor, many hormones are released in the brain, including serotonin (5-HT). 5-HT is a neurohormone that functions in the regulation of a variety of psychological and physiological processes, including behavior and inflammation (Idzko et al., 2004;Mikulski et al., 2010). Serotonin receptor 7 (HTR7) is the most recently described serotonin receptor, and it is found expressed on a number of immune cells, including macrophages and T lymphocytes (Ahern, 2011). Studies indicate that HTR7 activation results in the production of pro-inflammatory cytokines from immune cells, such as microglial cells, dendritic cells, and monocytes (Dürk et al., 2005;Mahé et al., 2005;Müller et al., 2009). Cytokines, such as interleukin-6 (IL-6), are then involved in the production of a variety of blood biomarkers, including cortisol, the major stress hormone (de Kloet et al., 2005;Howren et al., 2009;Maeda et al., 2010).
Cortisol is involved in the regulation of a number of biological processes, such as cellular metabolism and immune function (Anagnostis et al., 2009;Lundberg, 2005;Webster Marketon & Glaser, 2008). Both hypercortisolism and hypocortisolism have recently been associated with a number of health disparities, including cardiovascular disease and asthma (Buske-Kirschbaum et al., 2003;Manenschijn et al., 2013). During a stress response, cortisol concentration is increased resulting in a shift towards an inflammatory and humoral response (Murali et al., 2007;Straub et al., 2002). Under normal circumstances, this response will be limited and shut off when cortisol levels decrease back to normal levels (Ehlert et al., 2001). If a stressor persists for an extended period of time, the cortisol concentration will continue to increase until the system exhausts its supply (McEwen, 2004). Both cases, whether amplified or depressed, can lead to serious conditions, such as metabolic syndrome and irritable bowel syndrome (Anagnostis et al., 2009;Fries et al., 2005).
An increasing number of single nucleotide polymorphisms (SNPs) in genes for neurohormone receptors have also been linked to a number of health disparities. These diseases include breast cancer, depression, diabetes, and asthma (Deming et al., 2012;Kim et al., 2011;Kring et al., 2009;Lucae et al., 2010). While SNPs in these genes have been identified, there is limited data on the molecular pathways involved in these relationships. With regard to SNPs found in serotonin receptors, the mechanisms used by serotonin receptor 2A (HTR2A) in relation to disease state have begun to be identified (Beretta et al., 2008;Snir et al., 2013). It is possible that SNPs within HTR7 modulate the induction of pro-inflammatory cytokines from immune cells during times of stress, leading to a predisposition towards the development of health disparities. Previous findings prompted us to investigate whether genetic variation in HTR7 associates with the changes in function of the stress and immune system measured by cortisol levels in African American (AA) young adults.

Methods and materials
Study population All DNA and sample data were collected during a previous study done between 2010 and 2012 entitled "Gender Differences in the Experience of Violence, Discrimination, and Stress Hormone in African Americans: Implications for Public Health", which examined the genetic markers for alcohol and depression, violence exposure, and drug use in AA young adults (unpublished study; Jackson L, Shestov M, Abbas M and Saadatmand F). In total, DNA samples from 602 AA individuals living in the Washington, D. C. area were available for genotyping. All individuals, both male and female, ranged between the ages of 18 and 34 years old. In an Audio Computer-Assisted Self-Interviewing (ACASI) survey, only 590 participants provided information concerning behavioral data, including information concerning housing, income, and violence exposure ( Differences between CEU and YRI populations was of interest as African American's are likely to possess frequencies that fall between those of Caucasian and Yuroban descent. Four SNPs (rs12412496, rs2185706, rs7089533, and rs7093602) were selected for inclusion in the study based on a large difference in allele frequency between the CEU and YRI populations. The SNP rs2420367 was selected based on no frequency data being recorded with any population besides CEU ( Table 2). All of the chosen SNPs were located in the first intron region of the gene, as indicated on NCBI's SNP: Gene View. Genotyping Genotypes were determined using one of two methods. Restriction fragment length polymorphisms (RFLPs) were used for the genotyping of rs2420367, rs7089533, and rs7093602. Samples were amplified by polymerase chain reaction (PCR) in 96 well plates. The Taq polymerase and dNTP mix utilized for PCR amplification was obtained from Thermo Fisher Scientific (Waltham, MA, USA). SNP primers were supplied by Integrated DNA Technologies (Coralville, IA, USA). The concentration of magnesium chloride was 1.8 mM (rs2420367, rs7089533) and 1.5 mM (rs7093602). A total of 40 cycles were used, in which the annealing temperature was decreased by 2°C every 5 cycles until reaching the optimum annealing temperature of 57°C for each primer set. After confirming proper DNA amplification, the PCR products were digested with the appropriate restriction enzyme. The restriction enzymes, SmlI, AflII, and AvaII, were used at a concentration of 0.2 µL for the three SNPs rs2420367, rs7089533, rs7093602, respectively, (New England BioLabs, Ipswich, MA, USA). Incubation time for SmlI was increased to two hours at 55°C, while incubation for AflII and AvaII remained at the recommended 15 minutes. Visualization of the digestion was done using a 3% agarose gel.
The TaqMan SNP genotyping assay was used to genotype rs12412496 and rs2185706. A volume 1.5 µL of DNA was used, as well as the 10 µL of the prepared master mix as per manufacturer's instructions. The plates were then run using the TaqMan SNP genotyping assay protocol by Applied Biosystems.

Statistical analysis
The genotype and allele frequencies for each SNP were determined using the data generated from all 602 DNA samples. Genotype and haplotype associations were made using the software program SNPstat, which tested the data for Hardy-Weinberg equilibrium, linear and logistic regression, and linkage disequilibrium statistics (http://bioinfo.iconcologia.net/SNPstats).

Results
All 602 samples were used for the determination of allele and genotype frequencies within the AA population ( Cortisol, the major glucocorticoid in the stress response, is also an important biomarker of an immune response (Cavigelli & Chaudhry, 2012;Straub et al., 2002). Cortisol is shown to first be involved in the development of an inflammatory response, before eventually acting to depress this response (Elenkov, 2008;Kunz-Ebrecht et al., 2003). It has also been shown to shift the adaptive immune response towards the humoral response (Cavigelli & Chaudhry, 2012;Murali et al., 2007). As such, it was desired to identify potential associations between both hyper-and hypocortisolism and the five intronic SNPs in HTR7. None of the five SNPs were found to be associated with hypercortisolism.
Hypocortisolism was found to be associated with rs2420367, rs2185706, and rs7093602 when adjusting for gender (Table 3 and  Table 4). Females showed an association with the genotype A/C (rs2420367) by an 11-fold increase in the risk of hypocortisolism (OR=11.64[1.52-89.24]), when categorizing the interaction first by SNP then by gender (SNP within gender; Table 4). Similarly, males showed a 2-fold increased risk of hypocortisolism with the genotype A/A (rs2420367) when the interaction was categorized first by gender then by SNP (gender within SNP; Table 3).
Only males showed an association to hypocortisolism when categorizing in a gender within SNP manner for both rs2185706  et al., 2011). As such, it should follow that SNP genotypes and haplotypes in the HTR7 gene should also be associated with changes in the stress response, resulting in an effect on immune function. In this paper, we report the allele, genotype, and haplotype frequencies for five intronic SNPs of the HTR7 gene in an African American population.
During times of stress, cortisol secretion will initially increase, and over time may lead to hypercortisolism if the stressor remains persistent (Fries et al., 2005). In the case of chronic stress, it is possible to reach a state of adrenal exhaustion or hypocortisolism (McEwen, 2004). Both states, hyper-and hypocortisolism, have been associated with a number of stress-related diseases. While hypercortisolism has been shown to contribute to diseases such as depression, heart disease, and type 2 diabetes (Lundberg, 2005;Tse & Bond, 2004), hypocortisolism contributes to post-traumatic stress disorder, asthma, and irritable bowel syndrome (Buske-Kirschbaum et al., 2003;Ehlert et al., 2001;Fries et al., 2005;Heim et al., 1999). To test the hypothesis that there would be an association between SNPs in the HTR7 gene and both hyper-and hypocortisolism, the program SNPstat was used. While hypercortisolism was not associated to any specific SNP genotypes, associations were found to hyper-and hypercortisolism with the haplotypes AAACC and AAGTT, respectively. Both haplotypes provided a decreased risk of 77% and 98%, respectively, and may identify individuals who are less likely to develop hypercortisolism, and the potential resulting health disparities.
An increased risk of hypocortisolism was found to associated with three SNP genotypes in a gender specific manner. For rs2420367, females with the A/C genotype and males with the A/A genotype showed an 11-fold and 2-fold increased risk, respectively. Males, but not females also showed a 5-fold and 2-fold increased risk of hypocortisolism with the genotypes A/A (rs2185706) and C/C (rs7093602), respectively. As such, it is possible to conclude that individuals with these genotypes will be at an increased risk of developing hypocortisolism, and this may also increase the likelihood of developing health disparity diseases.
The haplotype CGGCC was determined to provide a 79% decreased risk of hypocortisolism in the population, and this association remained in males when adjusting for gender. Females however, showed a decreased risk of 78% with the haplotype AGACC. These haplotypes may identify the individuals in the AA population who are at a decreased risk of developing hypocortisolism and associated diseases.
In this work, we demonstrate that genetic variation in HTR7 influences the production of cortisol in response to chronic stress. While individual SNP genotypes indicate a risk towards the development of hypocortisolism, haplotypes within HTR7 seem to be protective against both hyper-and hypocortisolism. This protective effect may be a biological indicator of resilience towards maladaptive effects of chronic stress. This supports previous findings that individual genetics are involved in the degree of adaptability seen in response to stress (Feder et al., 2009;Gillespie et al., 2009). To our knowledge, this is the first evidence for a functional link between the data for hypocortisolism, only the haplotype CGGCC (p=0.033) was associated with 79% decreased risk (OR=0.19[0.04-0.87]; Table 5).
When adjusting for gender, males remained associated with hypocortisolism by the CGGCC haplotype (OR=0.01[0.00-0.19]) providing a 99% decreased risk (Table 6). This relationship held true regardless of the categorization of the interaction. While females did not remain associated with the CGGCC haplotype, a 91% decreased risk was found associated with hypocortisolism by the haplotype AGACC (OR=0.11[0.02-0.69]) and by 83% for the grouping of extremely rare haplotypes (OR=0.17[0.03-0.88]) ( Table 6). These associations held when categorized in a haplotype within gender manner. Due to the low number of individuals denoted as case for both analyses (case1=22; case2=85), the majority of haplotypes were unable to be used for analysis purposes. The results obtained indicate that certain haplotypes are associated with cortisol level in the AA population by decreasing the risk of having either hyper-or hypocortisolism.

Discussion
Despite the existing data on the impact of both genetic and environmental factors on immune function, few studies have examined relationships between these variables, especially in African Americans. We know relatively little about potentially sequential relationships between genetic risk factors, environmental stress, and immune function. In the brain, 5-HT is produced primarily by neurons in the midbrain, especially the dorsal raphe nucleus, which acts to innervate the majority of the brain, allowing 5-HT to modulate the response to stress (Holmes, 2008;Hornung, 2003). HTR7 is located throughout the brain and periphery, functioning in the regulation of circadian rhythmicity and smooth muscle tone, both in the cardiovasculature and gastrointestinal tract. (Abdouh et al., 2004;Hornung, 2003;Mahé et al., 2005) During times of stress, HTR7 expression is shown to increase and has been linked to depression (Guscott et al., 2005;Holmes, 2008). SNP genotypes and haplotypes in various genes, including serotonin receptors, have also been associated with changes in immune function or disease state (Ikeda et al., 2006;Snir et al., 2013; a genetic polymorphism in the HTR7 receptor gene and immunologically important subphenotypes related to differential levels of a blood biomarker for both stress and the immune response. Further study is required to investigate this relationship between genetic polymorphisms in HTR7, the stress response, and the immune response. Due to the low number of individuals in the case grouping, haplotype analysis was incomplete. By increasing the sample set data with regards to cortisol measurements, a clearer picture of the relationship between these five SNPs and cortisol may be formed. This will enable a more detailed picture of the relationship between HTR7 polymorphisms and cortisol production. Furthermore, the cytokine shift induced by cortisol during a response to stress suggests that a relationship may exist between these five SNPs and both the inflammatory and humoral response (Maeda et al., 2010;Murali et al., 2007;Pepys & Hirschfield, 2003), and it would be beneficial to analyze whether any association between the five intronic SNPs and biomarkers of the inflammatory and humoral response. This would aid in the understanding of how stress exposure contributes to the development of disease, and the role in which genetics plays in this pathway. Author contributions MA conceived the project and designed the experiments. GS, SM, and AA carried out the experiments. GS performed the statistical analysis and prepared the manuscript. MA, CL, GD, FS, and BW were involved in the revision of the manuscript. All authors agreed to the final content.

Competing interests
No competing interests were disclosed.

Grant information
The "Biological and Social Correlates of Drug Use in African American Adults" dataset was collected under Dr. Kathy Sanders-Phillips and was supported by the National Institute of Minority Health and Health Disparities (grant number, 5P20MD000198) and the National Institutes of Health (NIH) "Re-Engineering the Clinical Research Enterprise" (grant #UL1TR000101). The research reported in this publication was supported by the National Institute on Drug Abuse (NIDA) of the NIH (award number, R24DA021470), as well at the National Science Foundation under the Louis Stokes Alliance for Minority Participation (award numbers, HRD-1000286 and HRD-1503192). The content reported is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the National Science Foundation.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.