The Relationship of Single Nucleotide Polymorphisms in the TRPV1 Gene with Lipid Profile,Glucose,and Blood Pressure in Mexican Population

Abstract

Aim:

We analyzed the frequencies of the rs222749 G>A, rs222747 G>C, rs224534 G>A, and rs8065080 C > T polymorphisms in the TRPV1 gene and their relationships with biomarkers in a Mexican population.

Materials and Methods:

We included 195 students from two Mexican universities (72.3% female and 27.7% male, mean age, 20.8 ± 3.3 years). The biomarkers analyzed were lipid profile, glucose levels, blood pressure (BP), and body mass index. DNA was obtained from leukocytes by the dodecyltrimethylammonium bromide and cetyltrimethylammonium bromide method and polymorphisms were determined with TaqMan single nucleotide polymorphism (SNP) genotyping assays.

Results:

Alterations in lipid profile were total cholesterol ≥200 mg/dL in 9.7% of participants, triglycerides (TG) ≥150 mg/dL in 9.2%, high-density lipoprotein (HDL) <35 mg/dL in 6.7%, and low-density lipoprotein (LDL) ≥130 mg/dL in 6.2% of participants. Moreover, 8.2% of the subjects had BP values consistent with hypertension. The most frequent alleles were rs222749G (89.2%), rs222747G (69.2%), rs224534G (59.7%), and rs8065080T (62.3%). An analysis of the associations between the genotypic data and the biomarkers showed that the rs222749GA and rs224534GA genotypes were associated with higher diastolic and systolic BP values, respectively; the rs222747CC genotype was associated with lower LDL levels; the rs224534AA genotype was associated with higher HDL levels and lower triglycerides and LDL. The GGGC/GCAT and GGGT/GCAT haplotypes were associated with higher systolic BP.

Conclusions:

This study suggests a possible association between TRPV1 gene polymorphisms and BP and lipid profiles in a Mexican population.

Introduction

The TRPV1 gene encodes for the transient receptor potential vanilloid type 1 (TRPV1) protein, which belongs to a family of transient receptor potential voltage-dependent channels. TRPV1 activates at heat, capsaicin, and low pH levels; it is also involved in nociception, neurogenic inflammation, and pruritus, and functions as a transducer of painful stimuli in vivo. The agonist of this receptor is capsaicin, the active ingredient in hot chili peppers. The pungent activity of capsaicin is mediated by TRPV1, which is expressed in sensory neurons or nociceptors sensitive to noxious heat. Capsaicin also desensitizes nociceptors and is clinically useful as an analgesic (Bevan et al., 2014).

TRPV1 channels are widely distributed in the human body and are expressed in the heart, pancreas, liver, lung, kidney, adipose tissue, intestine, brain, uterus, testis, salivary glands, and the nervous system (Randhawa and Jaggi, 2017). Functional studies of the TRPV1 receptor have been conducted in several populations around the world. The TRPV1 function has been related to gastric cancer (López-Carrillo et al., 2003), metabolic syndrome, insulin resistance, obesity (Panchal et al., 2018), and hypertension (Randhawa and Jaggi, 2017). In subjects with sensitive skin, the expression of TRPV1 has been positively correlated with symptom intensity and pigmentation (Ehnis-Pérez et al., 2016).

Other authors have studied the relationship between TRPV1 and insulin secretion in beta cells; although they revealed that this receptor did not contribute to glucose-induced insulin secretion, it remains possible that TRPV1 might control insulin sensitivity in peripheral tissues (Diaz-Garcia et al., 2014). In addition, TRPV1 activation by capsaicin diminishes tension in frog fast muscle fibers (Trujillo et al., 2015).

Single nucleotide polymorphisms (SNPs) are located in the human TRPV1 gene, and affect structural domains of TRPV protein; p.P91S (rs222749) is situated at the intracellular amino terminus; p.M315I (rs222747) is found in domain 5 that contains ankyrin repeats that play a role in the mediation of protein-protein interactions and channel homotetramerization; p.T469I (rs224534) is located on the extracellular loop between helices 1 and 2; and p.I585V (rs8065080) is expressed on transmembrane domain of helix 5 affecting this domain that confers responsiveness to capsaicin. In addition to the structural changes, rs222749 and rs222747 SNPs modify the functional properties of the channel and induce an increase in TRPV1 protein expression due to a greater number of copies (Xu et al., 2007; Van Esch et al., 2009).

Some of these TRPV1 gene polymorphisms have been associated with pain perception, neuropathic pain (Binder et al., 2011), type 1 diabetes (Sadeh et al., 2013), salty taste perception (Dias et al., 2013), pancreatitis (Van Esch et al., 2009), and coughing (Smit et al., 2012). However, no study has investigated SNPs in the TRPV1 gene in the Mexican population.

The aim of this study was to determine the frequencies of rs222749 (c.271G>A), rs222747 (c.945G>C), rs224534 (c.1406G>A), and rs8065080 (c.1753C>T) SNPs and their relationships with biomarkers, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), glucose, as well as blood pressure (BP), and body mass index (BMI).

Materials and Methods

Study population

For this study, 195 participants from two western Mexican universities (Universidad de Guadalajara and Universidad de Colima) were enrolled. All subjects agreed to participate and signed an informed consent form; they were interviewed to collect complete personal and family data, their spicy food intake (yes/no), and spicy food tolerance (no/little or high); height and weight were measured. BMI was calculated with the international formula (WHO, 2020) and BP was measured according to the criteria proposed by NOM-030-SSA2-2009.

Furthermore, a sample of 10 mL of peripheral blood was collected in the fasting state for at least 9 hours to obtain the following biomarker levels: TC, TG, HDL, LDL, and glucose as well as DNA extraction. Biomarker measurements were performed using enzymatic methods with commercial kits and a semiautomatic ALS 2000 spectrophotometer. Teco diagnostics (Anaheim, CA), Trinder GOD-POD, and Spinreact (Esteve de Bas, Girona, Spain) were employed to determine the lipid profile (TC, TG, HDL, and LDL, using the Friedewald formula) and glucose, respectively. The Bioethics Committee of the University Center of Biomedical Research at Universidad de Colima approved the study.

Genotyping

DNA extraction was performed with the dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB) method (Gustincich et al., 1991). DNA integrity was verified on agarose 2% gels stained with SYBR Safe DNA gel stain (Invitrogen). The concentrations and purity of DNA were obtained using a Thermo Scientific NanoDrop 2000c Spectrophotometer.

We used the allelic discrimination method with TaqMan SNP genotyping assays to analyze the SNPs of the TRPV1 gene: rs222749 (C___1093692_30), rs222747 (C___1093688_20), rs224534 (C___1093674_1_), and rs8065080 (C__11679656_10) (Applied Biosystems, Foster City, CA). The PCR was performed in a final volume of 15 μL containing 50 ng of genomic DNA, 7.5 μL TaqMan Genotyping Master Mix, 0.375 μL TaqMan probe, and 6.125 μL water DNase/RNase free; the program was 10 min at 95°C, followed by 43 cycles of 15 s at 95°C and 1 min at 60°C. Reactions were performed on ABI Prism 7000 sequence detection system (Applied Biosystems PRISM 700 Sequence Detection System.).

Statistical analysis

Genotypic and allelic frequencies were determined by direct counting. The Hardy-Weinberg equilibrium (HWE) and the comparison between tolerance to spicy foods and the SNPs studied were calculated by the chi-square test; haplotypes were inferred with a Bayesian algorithm. Linkage disequilibrium (LD) was measured in terms of D’ and r² (values >0.33 were considered significant). The relationship of biomarkers (i.e., TC, TG, HDL, LDL, glucose, systolic BP, diastolic BP, and BMI) with SNPs was calculated by means analysis of variance, chi-square, Student's t, correlation, and linear regression. These analyses were made using alleles, genotypes, genotype combination, and the different inheritance models (dominant, recessive, and additive). The programs used were Arlequin v3.01 software (Excoffier et al., 2005) and SPSS version 20.0. p-Values <0.05 were considered statistically significant.

Results

As genotypic and allelic frequencies were statistically similar in participants from Colima and Guadalajara populations, we combined subjects into a single group for the analysis. We enrolled 195 students: 72.3% female and 27.7% male, with a mean age 20.8 ± 3.3 years and BMI of 24.0 ± 4.1 kg/m²; of these, 34.3% were either overweight or obese. Systolic BP was ≥140 mmHg in 4.1% of individuals and diastolic BP was ≥90 mmHg in 5.6% of participants; out of these, three subjects had elevations in both systolic and diastolic BP; therefore, 8.2% (16/195) had abnormal pressure the accurate diagnosis of primary arterial hypertension must be established by NOM-030-SSA2-2009 guidelines. All subjects presented glucose <100 mg/dL, and 40 individuals (20.5%) showed alteration in any lipid biomarker: TC, TG, HDL, or LDL. High tolerance to spicy food was reported by 47.2% of individuals. Table 1 shows the general characteristics of the study population.

Table 1.

Characteristics of the Study Population

Biomarker	Mean ± SD	Cutoff values	n	%
Systolic BP (mmHg)	110.6 ± 14.3	<140	187	95.9
Systolic BP (mmHg)	110.6 ± 14.3	≥140	8	4.1
Diastolic BP (mmHg)	71.9 ± 10.1	<90	184	94.4
Diastolic BP (mmHg)	71.9 ± 10.1	≥90	11	5.6
Glucose (mg/dL)	76.2 ± 7.8	<100	195	100
Glucose (mg/dL)	76.2 ± 7.8	≥100	0	0
Total cholesterol (mg/dL)	149.2 ± 34.6	<200	176	90.3
Total cholesterol (mg/dL)	149.2 ± 34.6	≥200	19	9.7
Triglycerides (mg/dL)	89.2 ± 54.9	<150	177	90.8
Triglycerides (mg/dL)	89.2 ± 54.9	≥150	18	9.2
HDL (mg/dL)	48.8 ± 11.6	≥35	182	93.3
HDL (mg/dL)	48.8 ± 11.6	<35	13	6.7
LDL (mg/dL)	82.1 ± 29.8	<130	183	93.8
LDL (mg/dL)	82.1 ± 29.8	≥130	12	6.2
Variable	n (%)
Spicy food consumption	167 (85.6)
Spicy food tolerance
No/little tolerance	103 (52.8)
High tolerance	92 (47.2)

Three participants had elevated both diastolic and systolic BP.

BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SD, standard deviation.

Analysis of genotypic, allelic, and haplotypic frequencies

The minor allele frequencies were 10.8% for rs222749A, 30.8% for rs222747C, 40.3% for rs224534A, and 62.3% for rs8065080T; the rs222749AA genotype was not observed (Table 2). All SNPs analyzed were in HWE.

Table 2.

Genotypes and Alleles Distributions of the Studied Polymorphisms in the TRPV1 Gene

TRPV1 gene
	rs222749 G > A			rs222747 G > C			rs224534 G > A			rs8065080 C > T
		n	%		n	%		n	%		n	%
Genotype frequencies	GG	153	78.5	GG	92	47.2	GG	63	32.3	CC	30	15.4
	GA	42	21.5	GC	86	44.1	GA	107	54.9	CT	87	44.6
	AA	0	0	CC	17	8.7	AA	25	12.8	TT	78	40.0
Allele frequencies	G	348	89.2	G	270	69.2	G	233	59.7	C	147	37.7
Allele frequencies	A	42	10.8	C	120	30.8	A	157	40.3	T	243	62.3

TRPV1, transient receptor potential vanilloid type 1.

We determined the haplotypes with four SNPs in the TRPV1 gene (rs222749, rs222747, rs224534, and rs8065080), where 11 different combinations were detected. The two most frequent were GGGT (43.8%) and GCAC (20.6%). The remaining nine haplotypes did not exceed 12% (GGGC, 12.0%; GCAT, 7.3%; AGAT, 4.9%; AGAC, 4.1%; GCGT, 2.5%; GGAT, 2.5%; AGGT, 1.3%; GGAC, 0.5%; and ACAC, 0.5%). In addition, LD was observed in the pair of loci rs222747/rs224534 (r² = 0.49, D’ = 0.86; p < 0.0001).

TRPV1 gene polymorphisms and their association with biomarkers

We compared biomarkers (i.e., TC, TG, HDL, LDL, BMI, glucose, systolic BP, and diastolic BP) between alleles, genotypes, genotype combination, and under the different inheritance models. The significant results obtained were, for genotypes, in diastolic BP between rs222749 GA and GG (74.7 mmHg vs. 70.9 mmHg, p = 0.040); in systolic BP, between rs224534 GA and GG (113.2 mmHg vs. 107.1 mmHg, p = 0.022). For different inheritance models: in diastolic BP between rs222749 GG and GA+AA (70.8 mmHg vs. 74.8 mmHg, p = 0.032), in LDL between rs222747 GG + GC and CC (84.0 mg/dL vs. 68.1 mg/dL, p = 0.033), for rs224534, in systolic BP between GG and GA+AA (107.2 mmHg vs. 112.2 mmHg, p = 0.021), and between GG + GA and AA in TG (90.5 mg/dL vs. 73.4 mg/dL, p = 0.005), in HDL (48.0 mg/dL vs. 54.4 mg/dL, p = 0.011), and in LDL (84.3 mg/dL vs. 71.5 mg/dL, p = 0.047). Also, we found significant differences for genotype combination in systolic BP between GGGC/GCAT and GGGT/GGGT (114.7 mmHg vs. 102.4 mmHg, p = 0.005), and between GGGT/GCAT and GGGT/GGGT (117.1 mmHg vs. 102.4 mmHg, p = 0.026).

It should be noted that systolic BPs between rs222747 GC and GG genotypes (113.2 mmHg vs. 109.0 mmHg, p = 0.050) and between rs8065080 CT and TT (113.1 mmHg vs. 109.1 mmHg, p = 0.051) were parameters with borderline significance.

In contrast, we found no significant differences between tolerance to spicy foods and the genotypes of the studied SNPs (p > 0.05).

Discussion

The TRPV1 receptor is controlled by physical and chemical stimuli; in particular, it is activated by capsaicin, the spicy “hot” component of chili. Capsaicin has been an essential tool for investigating both physiological and pathological processes, as well as the relevance of TRPV1 channels (Gregorio-Teruel et al., 2014). TRPV1 is one of the most studied receptors in obesity and diabetes mellitus (Zsombok and Derbenev, 2016).

The four polymorphisms here investigated showed high frequencies of minor alleles (range 10.8% to 62.3%). In all SNPs, the three expected genotypes were detected, except in rs222749, for which the mutated genotype (AA) was not observed (Table 2).

The minor allele frequencies of the four SNPs investigated in this study were statistically like those reported in the 1000 Genomes Database for individuals in the Los Angeles population with Mexican ancestry (rs222749 G>A: A 10.8% vs. A 15.6%, p = 0.141; rs222747 G>C: C 30.8% vs. C 24.2%, p = 0.157; rs224534 G>A: A 40.3% vs. A 41.4%, p = 0.818; and rs8065080 C>T: T 62.3% vs. T 61.7%, p = 0.905). However, other population frequencies were significantly different from those of our study (p < 0.0001). For example, minor alleles in East Asian and African populations were rs222749A 24% and 0%, rs222747C 56% and 11%, rs224534A 77% and 3%, and rs8065080T 41% and 89%, respectively (The 1000 Genomes Project Consortium, 2015). This comparison highlights the genetic variability among different populations around the world.

The activation of TRPV1 channels can protect against the development of atherosclerosis and systemic hypertension. An increase in the systemic BP activates TRPV1 channels, in consequence, the activation of endothelial TRPV1 channels enhances endothelial nitric oxide synthase phosphorylation to increase nitric oxide levels that, in turn, induce vasodilation and decrease peripheral resistance and BP (Randhawa and Jaggi, 2017). Such a process could be directly related to our results on genotypes and BP, since we found that subjects with rs222749GA and rs224534GA genotypes had higher diastolic BP and systolic BP, respectively, than those with wild genotypes. Consequently, the presence of a mutated allele might bring the receptor to act abnormally, thus increasing the BP. Therefore, it would be interesting to carry out association studies to relate these two SNPs with high BP in hypertensive patients.

It is interesting that 11 of the 16 subjects with elevated diastolic or systolic BP also had BMI values >25 kg/m². Several studies have shown that high BP is strongly correlated with overweight and obesity and that obese subjects have a 3.5-fold increased likelihood of having hypertension (Seravalle and Grassi, 2017; Chaudhary et al., 2019).

Regarding the borderline significant result of the rs222747GC and rs8065080CT genotypes and the relationship with higher systolic BP, additional studies with a larger sample size and more representative of the Mexican population are necessary to clarify the role of these SNPs with BP.

Other findings in this study were that individuals with rs222747CC genotype presented lower LDL levels, and those with rs224534AA genotype presented higher values in HDL and lower in TG and LDL; that is, these mutated genotypes are related with healthy lipid levels, since the lipid profile of patients with obesity is characterized by a decrease in HDL as well as an increase in TG and sometimes LDL known as dyslipidemia atherogenic (Mbundu Ilunga et al., 2018).

In this study, subjects were asked on their tolerance to spicy foods and their daily intake of chili peppers, nearly half of subjects being very tolerant to spicy foods (47.2%). We found no association between this characteristic and the genotypes based on the studied SNPs. Given the reliance on self-report measures, however, perceptions of and opinions on spicy food tolerance were subjective. Therefore, this finding should be investigated more formally in studies that use an objective evaluation regarding the sensitivity of spicy foods. Okamoto et al., for example, have investigated the effects of SNPs in TRPV1 on capsaicin sensitivity in Japanese adults and found the rs8065080CC genotype to be related to significantly high capsaicin sensitivity (Okamoto et al., 2018).

The four TRPV1 SNPs here analyzed have been previously related to several conditions and diseases in diverse populations around the world; our findings have shown that rs222749 and rs224534 SNPs are related to higher BP in younger subjects who apparently are healthy. This information can give rise to further research about their possible role on pathological conditions such as systemic hypertension, diabetes, and obesity in the Mexican population. Another factor that supports continuing to study these SNPs is the high frequency of rare alleles in our population.

In conclusion, we found that the genotype rs222749GA confers higher diastolic BP and genotype rs224534GA confers higher systolic BP, meanwhile for lipids, rs222747CC is related to lower LDL levels, and rs224534AA to higher values in HDL and lower in LDL and TG. GGGC/GCAT and GGGT/GCAT genotype combinations conferred relatively high systolic BP.

Footnotes

Acknowledgments

Some data were collected as part of a GMA postdoctoral CONACyT project conducted in the laboratory of Dr. Huerta M. The authors thank UAPSU-CUCS-Universidad de Guadalajara and the Medical Faculty at the Universidad de Colima for providing facilities, as well as Cobian JG PhD for his collaboration with the obtainment of the biomarkers.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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