Association of serotonin gene (5-HTTLPR &amp; 5-HTTVNTR) polymorphism with Fibromyalgia Syndrome in North Indian Women

doi:10.21203/rs.3.rs-1396711/v1

Download PDF

Research Article

Association of serotonin gene (5-HTTLPR & 5-HTTVNTR) polymorphism with Fibromyalgia Syndrome in North Indian Women

https://doi.org/10.21203/rs.3.rs-1396711/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Several psychiatric disorders have been found associated with serotonin transporter gene polymorphisms. The purpose of this study was to explore the correlation between serotonin gene polymorphism (5-HTTLPR & 5-HTTVNTR) in North Indian Women with Fibromyalgia Syndrome (FMS).

Results

105 FMS patients and 105 controls were enrolled in the study. Polymerase chain reaction (PCR) method was used to analyse the 5-HTTLPR & 5-HTTVNTR gene polymorphism. The psychopathology status of the 105 FMS patients and 105 healthy controls was assessed using the Beck Depression Inventory (BDI) and the Symptom Checklist-90-Revised questionnaires (SCL-90-R). Patients with psychiatric symptoms were excluded from the study. No significant differences were found in 5-HTTVNTR genotypes; 10/10 (OR 0.74, P = 1.000), 10/12 (OR 0.72, P = 0.4691) and 12/12 (OR 0.92, P = 0.3697) or 5-HTTLPR genotypes; L/L (OR 0.59, P = 0.7212), S/L (OR 1.10, P = 0.6700) and S/S (OR 0.92, P = 0.8890) between patients and controls.

Conclusion

Hence, we conclude that serotonin gene polymorphism (5-HTTLPR & 5-HTTVNTR) is not associated with FMS in north Indian women.

Fibromyalgia syndrome

Serotonin transporter gene Polymorphism

5-HTTLPR

5- HTTVNTR

PCR

Fibromyalgia (FMS) is a syndrome characterized by persistent widespread pain, extreme sensitivity on palp, and other problems such as insomnia, stiffness, tiredness, and psychosocial problems [1]. Disturbances in serotonin metabolism and transmission are hypothesized to be pathophysiologic mechanisms that cause FMS [2]. This hypothesis is supported by numerous studies. According to Das et al., patients with FMS have a lower level of 5-HT in their blood plasma, which can be associated with sleep disorders and pain [3]. In the cerebrospinal fluid of FMS patients, similar alterations in 5-HT metabolism have been reported [4].

The sodium-dependent serotonin transporter gene (5-HTT), encoded by SLC6A4 which transports serotonin from synaptic space into presynaptic neurons for recycling, therefore playing a crucial role in serotonergic transmission termination [5]. The location of 5-HTT gene on chromosome is 17q11.1-q12, which have been reported for two polymorphism. first, in the second intronic region, that contains 17 variable number tandem repeats (VNTR) with 9, 10, and 12 number repeats [6]. Several studies have associated 5-HTTVNTR to a various mental disease, including schizophrenia and bipolar disorder [7, 8]. But several researches have concluded that this polymorphism is not significantly associated with psychiatric disorders [9, 10].

Second, another polymorphic region is the 5-HTTLPR promoter region, a 44-base pair polymorphism that results in two different alleles, including deletion(S/short) alleles and insertion(L/long) alleles [7]. The presence of the S allele reduced the transcription activity of the 5-HTT promoter gene as compared to the L allele, resulting in lower serotonin transporter binding and uptake [11]. As a result, this genetic variation may raise the risk of developing psychiatric diseases; indeed, studies have related the 5-HTTLPR polymorphism to significant psychiatric problems [12]. Only a few studies have found that the 5-HTT gene polymorphism are related with depression and anxiety in patients with FMS [13]. These studies provide evidence supporting that the 5-HTTVNTR and the 5-HTTLPR could be candidate gene polymorphism for psychiatric disorders.

Currently, there is insufficient data supporting the role of 5-HTT gene polymorphism in FMS. However, the use of 5-hydrooxytrptamine type-3 receptor antagonist and re-uptake blockers in the treatment of FMS suggests that genes influencing the serotonergic pathway may be involved in the pathogenesis of FMS [14]. Depression, anxiety, and fatigue are common in FMS patients, and the serotonergic pathway indirectly influencing these symptoms [15]. Therefore, it is possible that 5-HTT gene polymorphism may contribute to the clinical symptoms in FMS. To our knowledge, no study has reported the association of the 5-HTTVNTR and the 5-HTTPLR gene polymorphism with clinical phenotypes or symptoms in North Indian women with FMS. Therefore, the present study investigates the association of the 5-HTTVNTR and the 5-HTTLPR gene polymorphisms in North Indian women with FMS.

Patients and controls selection

All procedures performed in this study involving human participants were done in according to the ethical standards of Era’s Lucknow Medical College and Hospital (ELMCH), Era University, Lucknow, India. This study was approved by the ethics committee of the ELMCH. 105 FMS patients and 105 healthy control participants from ELMCH were recruited in this case control study. The study includes only those FMS patients who satisfied the American College of Rheumatology 2016 inclusion criteria for FMS [16]. The patient with multiple comorbidities like diabetes, rheumatoid arthritis, systematic lupus erythematosus and multiple myeloma were excluded from the study. The patients suffering from any other endocrine disorder were also excluded. The FMS patients and controls were all female belonging from the same ethnicity and geography. The mean age of the FMS patients was 36.1 ± 11.1 years and controls were 34.6 ± 10.3 years. The duration of illness for the patients suffering from FMS was 19.5 ± 2.5 weeks. The blood sample were collected from all patients and controls after signing an informed consent form. The psychiatric test of all the patients and controls were evaluated using Beck Depression Inventory (BDI) and symptom Checklist-90-revised (SCL-90-R). Therefore, BDI and SCL-90-R were used to identify the psychological distress in all patients and controls. The patients with psychiatric disorders were excluded from the study. Demographic and clinical data of the patients and controls are given in Table 1.

Table 1

Demographic and clinical data (including data on depression and psychological problems as measured by the BDI and the SCL-90-R) in the FMS patients and controls.
Variable	Patients n = 105	Controls n = 105	P-value
Age (years)	35.9 ± 11.1	34.6 ± 10.2	0.38
Duration of symptoms (weeks)	19.5 ± 2.5	-
BDI score	21.2 ± 2	18.3 ± 11.5	0.06
SCL-90-R score include:
Somatization	1.2 ± 0.5	1.1 ± 0.6	0.33
Obsessive-Compulsive	1.1 ± 0.4	1.0 ± 0.4	0.20
Interpersonal Sensitivity	1.1 ± 0.3	1.0 ± 0.4	0.07
Depression	1.1 ± 0.3	1.0 ± 0.5	0.06
Anxiety	1.3 ± 0.5	1.1 ± 0.3	0.08
Hostility	1.0 ± 0.4	0.9 ± 0.2	0.22
Phobic Anxiety	0.9 ± 0.4	0.8 ± 0.3	0.66
Paranoid Ideation	0.7 ± 0.5	0.6 ± 0.1	0.08
Psychoticism	0.6 ± 0.5	0.5 ± 0.1	0.08
GSI (Global Severity Index)	1.0 ± 0.3	0.9 ± 0.2	0.05

Genotyping of 5-HTTVNTR and 5-HTTLPR

The peripheral blood was taken in a blood collection tube from the subjects after taking written consent. Salting out method was used for isolating DNA from the blood samples. Extracted DNA was used for the amplification of the 5-HTTVNTR and 5-HTTLPR gene polymorphism by Polymerase chain reaction (PCR) method. The primer details for the 5-HTTVNTR are given in the Table 2.

Table 2

Primer sequences used for 5-HTTVNTR and 5-HTTLPR PCR.
Primer		Sequence
5-HTTVNTR	Forward	5′-GTCAGTATCACAGGCTGCGAG-3′
5-HTTVNTR	Reverse	5′-TGTTCCTA GTCTTACGCCAGTG-3′
5-HTTLPR	Forward	5′-GGCGTTGCCGCTCTGAATGCC-3′
5-HTTLPR	Reverse	5′-CAGGGGAGATCCTGGGAGAGGT-3′

PCR was performed in a final volume of 25µl consisting of 150-200ng genomic DNA, 10pmol of each primer and 2x master mix (Takara) per tube using a gradient Master-Cycler (BIO-RAD). PCR amplification consisted of an initial denaturation at 94 ^oC for 5 min, followed by 30 cycles of denaturation were carried out at 94 ^oC for 40 sec, annealing was carried out at 61.1 ^oC for 40 sec, extension was carried out at 72 ^oC for 40 sec, followed by final extension at 72 ^oC for 5 min. PCR products were run on agarose gel (3%) containing ethidium bromide and visualized by gel documentation apparatus (EZ BIO-RAD, California), which allow the identification of 267 bp product (10 repeats) and 300 bp product (12 repeats), constituting three types of genotypes, 10/10, 10/12, and 12/12 (Fig. 1)

Figure 1. Representative agarose gel showing three types of genotypes of 5-HTTVNTR. Lane 2- 267 bp (10/10); Lane 1 & 7- 300 bp, 267 bp (10/12); Lane 3, 4, 5, 6 & 8- 300 bp (12/12); M- DNA Ladder (50 bp).

Primer details for 5-HTTLPR are given in Table 2. PCR was performed in a final volume of 25µl consisting of 150-200ng genomic DNA, 10pmol of each primer and 2x master mix (Takara) per tube using a gradient Master-Cycler (BIO-RAD). PCR amplification consisted of an initial denaturation at 94 ^oC for 5 min, followed by 30 cycles of denaturation were carried out at 94 ^oC for 40 sec, annealing was carried out at 61.1 ^oC for 40 sec, extension was carried out at 72 ^oC for 40 sec, followed by final extension at 72 ^oC for 5 min. PCR products were run on agarose gel (3%) containing ethidium bromide and visualized by gel documentation apparatus (EZ BIO-RAD, California), which allows for the identification of long (265 bp) and short (221 bp) alleles, resulting in three genotypes: S/S, S/L, and L/L (Fig. 2).

Figure 2. Representative agarose gel showing three types of genotypes of 5-HTTLPR. Lane 1 & 3- 221 bp (S/S); Lane 2 & 4- 221 bp, 265bp (S/L); Lane 5- 265 bp(L/L); M-DNA Ladder (100 bp).

Statistical Analysis

The statistical analysis was carried out using Prism, version 6. (GraphPad Inc., CA, USA). Student’s t-test was used to compare demographic and clinical data between patients and control. The results are reported as mean ± SD. Fisher exact test was used to compare the frequency distribution of genotypes between patients and control population. Association between the genotypes and FMS was analysed using 2x2 Contingency table and applying Fisher's exact test. The presence of Hardy-Weinberg equilibrium was examined using a chi-squared test. In the present study, a P < 0.05 value was considered statistically significant.

The age and sex of 105 patients and 105 controls were similar (test, P > 0.05). Neither FMS patients nor controls had psychiatric disorders, and their psychiatric test results (BDI and SCL-90-R score) were not different (test, P > 0.05). Table 1 summarized the test result.

Table 3

Genotype frequency of the 5-HTTVNTR gene polymorphism in the FMS patients and control groups.
5-HTTVNTR Genotype	Patients (n = 105)	Controls (n = 105)	Odds ratio (95% CI)	P-value (Two tailed)
10/10 10/12 12/12	4/101 21/84 80/25	3/102 16/89 86/19	0.74 (0.16–3.4) 0.72 (0.35–1.5) 0.92 (0.72–2.8)	1.0000 0.4691 0.3697
Hardy–Weinberg equilibrium test: (χ2 = 2.68, df = 1, p = 0.10 in FMS patients), (χ2 = 3.69, df = 1, p = 0.05 in control groups).

Table 3 shows the genotype frequency of the 5-HTTVNTR gene polymorphism in the FMS patients (n = 105) and healthy control groups (n = 105). In both FMS patients and controls, the 10/10, 10/12 and 12/12 genotypes were represented in 3.8% and 2.9%, 20% and 15.2%, and 76.2% and 81.90%, respectively. There was no significant difference in the 5-HTTVNTR genotypes distribution between FMS patients and controls (OR 0.74, P = 1.000), (OR 0.72, P = 0.4691) and (OR 0.92, P = 0.3697), respectively.

Table 4

Genotype frequency of 5-HTTLPR gene polymorphism in the FMS patients and controls.
5-HTTLPR Genotype	Patients (n = 105)	Controls (n = 105)	Odds ratio (95% CI)	P-value (Two tailed)
L/L S/L S/S	5/100 38/67 62/43	3/102 42/63 60/45	0.59 (0.14–2.5) 1.1 (0.65-2.0) 0.92 (0.53–1.6)	0.7212 0.6700 0.8890
Hardy–Weinberg equilibrium test: (χ2 = 0.07, df = 1, p = 0.79 in FMS patients), (χ2 = 1.9, df = 1, p = 0.17 in controls).

Table 4 shows the genotype frequency of the 5-HTTLPR gene polymorphism in the FMS patients and control groups (n = 105). In both FMS patients and healthy controls, the L/L, S/L and S/S genotypes were represented in 4.8% and 2.8%, 36.2% and 40.3%, and 59% and 57%, respectively. Again, there was no significant difference in the 5-HTTLPR genotypes distribution between FMS patients and controls (OR 0.59, P = 0.7212), (OR 1.10, P = 0.6700) and (OR 0.92, P = 0.8890), respectively.

This study was designed to investigate the association of 5-HTTNVTR and 5-HTTLPR gene polymorphisms in North Indian women with FMS. 5-HTT gene polymorphisms have been most extensively investigated in various population. To our knowledge, this is the first study to investigate the correlation between 5-HTTNVTR and 5-HTTLPR gene polymorphism and clinical symptoms in North Indian women with FMS. According to our findings, no significant difference between the psychiatric test results and 5-HTT gene polymorphism (5-HTTVNTR and 5-HTTLPR) in North Indian women with FMS. Similar result was also reported by Gursoy (2002), who showed that neither 5-HTTVNTR nor 5-HTTLPR gene polymorphism were association with FMS susceptibility in patients with normal psychiatric status [10]. Likewise, a meta-analysis found no correlation between the variations in the 5-HTT gene and FMS [17].

Cohen et al. (2002), investigated polymorphism in 5-HTTLPR (5-HTT promoter region) in FMS patients. Female patients with FMS selected from two different ethnic group of Israelis like Arab and Jewish were genotyped. Tridimensional Personality Questionnaire (TPQ) was used to assess each patient with a self-reported assessment consisting one hundred Yes/No questions. The result showed that FMS and 5-HTTLPR polymorphism were associated in both Israeli groups. Moreover, TPQ scores of FMS patients were associated with the 5-HTTLPR polymorphism [18]. Leschi et al. (1996), found that the S allele of 5-HTTLPR polymorphism was associated with anxiety [19]. The result of both studies provided support to the findings that 5-HT play important roles in the regulation of FM symptoms.

Offenbaecher et al. (1999), studied the genotypes in the 5-HTT gene in FM patients. The genotype S/S was high, comprising 31% in FM patients compared to 16% in controls. Moreover, the patients with S/S genotype had more severe depressive symptoms and psychological problem, as demonstrated by the high scores in SCL-90-R and BDI compared with group of L/L and L/S genotype patients. However, they did not analyse 5-HTTVNTR gene polymorphism [20].

We examined both 5-HTTLPR and 5-HTTVNTR gene polymorphism in North Indian women with FMS. There was no statistical difference between FMS patients and controls. The following explanations may account for the above findings. First, the sample size of this study is small; second, the genetic variations depend on the region or the population being studied; third, genetic diversity could have also affected the outcome. Also, no enough information on the influence of 5-HTT gene polymorphism on FMS susceptibility is available. Consequently, role of 5-HTT gene variants in the pathogenesis of FMS patients remained inconclusive till date. Further research with large sample size is needed to confirm our observation in FMS patients.

Our study correlated the association of the 5-HTTVNTR and the 5-HTTLPR gene polymorphisms in North Indian women with FMS. Our study found no association of 5-HTTVNTR and 5-HTTLPR gene polymorphism in FMS. Thus, we could say that 5-HTTVNTR and 5-HTTLPR gene is not important contributor for FMS in North Indian Ethic group.

5-HTT

Serotonin transporter

FMS

Fibromyalgia Syndrome

VNTR

variable number tandem repeat polymorphism

LPR

linked promoter region

BDI

Beck Depression Inventory

SCL-90-R

Symptom Checklist-90-Revised questionnaires

TPQ Tridimensional Personality Questionnaire

Short

Long.

Ethical approval and consent to participate

The ethics committee of the Era’s Lucknow Medical College and Hospital (ELMCH), Era University, Lucknow, India approved this study. All the procedures including human subjects were performed in accordance with the ethical standards of ELMCH. Written consent was taken from all the patients and controls involved in the study.

Consent for publication

The consent was taken from all the patients and controls participated in the study.

Availability of data and material

On reasonable request, the corresponding author will provide the datasets created and analysed during the current study.

Competing interests

No conflict of interest.

Funding

We thank the Indian Council of Medical Research vide Award letter no. 3/1/2(11)/CD/2021-NCD-II for providing fund for this work.

Authors’ contributions

Conceived and designed the experiment: SP, GF. Performed the experiment: SP. Analysed the data: MMK, SP, GF, FM, JF, NRH. Wrote the paper: SP, GF.

Acknowledgment

We greatly acknowledge the Indian Council of Medical Research vide Award letter no. 3/1/2(11)/CD/2021-NCD-II for providing grant for this work.

Costa IS, Gamundí A, Miranda JGV, França LGS, De Santana CN, and Montoya P (2017) Altered functional performance in patients with fibromyalgia. Front Hum Neurosci 11:14. doi: 10.3389/fnhum.2017.00014.
Wolfe F, Russell IJ, Vipraio G, Ross K, Anderson J (1997) Serotonin levels, pain threshold, and fibromyalgia symptoms in the general population. J Rheumatol 24:555–9.
Fatima G, Das SK, Khan FH, Mahdi AA, Verma NS (2013) Circadian variations of 5- hydroxytryptamine in female with fibromyalgia syndrome: A case control study. Sleep Biol Rhythms 11:261–267. https://doi.org/10.1111/sbr.12038
Russell IJ, Vaeroy H, Javors M, Nyberg F (1992) Cerebrospinal fluid biogenic amine metabolites in fibromyalgia/fibrositis syndrome and rheumatoid arthritis. Arthritis Rheum 35:550–6. doi: 10.1002/art.1780350509.
Chen FX, Chen XS, Guo JC, Zheng BA, Guo M (2019) Serotonin transporter-linked polymorphic region genotypes in relation to stress conditions among patients with papillary thyroid carcinoma. Int J Clin Exp Pathol 12:968–977.
Hranilovic D, Stefulj J, Schwab S, Borrmann-Hassenbach M, Albus M, Jernej B, et al (2004a) Serotonin transporter promoter and intron 2 polymorphisms: relationship between allelic variants and gene expression. Biol Psychiatry 55:1090–4.
Battersby S, Ogilvie AD, Smith CA, Blackwood DH, Muir WJ, Quinn JP, et al (1996) Structure of a variable number tandem repeat of the serotonin transporter gene and association with affective disorder. Psychiatr Genet 6:177–81. doi: 10.1016/j.biopsych.2004.01.029.
Ogilvie AD, Battersby S, Bubb VJ, Fink G, Harmar AJ, Goodwim GM, et al (1996) Polymorphism in serotonin transporter gene associated with susceptibility to major depression. Lancet 347:731–3. https://doi: 10.1016/s0140-6736(96)90079-3.
Stober G, Jatzke S, Heils A, Jungkunz G, Fuchs E, Knapp M, et al (1998) Susceptibility for schizophrenia is not influenced by a functional insertion/deletion variant in the promoter of the serotonin transporter gene. Eur Arch Psychiatry Clin Neurosci 248:82–6. https://doi: 10.1007/s004060050022.
Gursoy S (2002) Absence of association of the serotonin transporter gene polymorphism with the mentally healthy subset of fibromyalgia patients. Clin Rheumatol 21:194–197. https://doi: 10.1007/s10067-002-8284-5.
Ming Q, Zhang Y, Yi J, Wang X, Zhu X, Yao S (2015) Serotonin transporter gene polymorphism (5-HTTLPR) L allele interacts with stress to increase anxiety symptoms in Chinese adolescents: A multiwave longitudinal study. BMC Psychiatry 15:1–8. https://doi: 10.1186/s12888-015-0639-y.
Basu A, Chadda R, Sood M, Kaur H, Kukreti R (2019) A preliminary association study between serotonin transporter (5-HTTLPR), receptor polymorphisms (5-HTR1A, 5-HTR2A) and depression symptom-clusters in a north Indian population suffering from Major Depressive Disorder (MDD). Asian J Psychiatry 43:184–188. https://doi.org/10.1016/j.ajp.2019.05.028.
Tour J, Löfgren M, Mannerkorpi K, Gerdle B, Larsson A, Palstam A, et al (2017) Gene-to-gene interactions regulate endogenous pain modulation in fibromyalgia patients and healthy controls-antagonistic effects between opioid and serotonin-related genes. Pain 158:1194–1203. doi: 10.1097/j.pain.0000000000000896.
Hrycaj P, Stratz T, Mennet P, Muller W (1996) Pathogenetic aspects of responsiveness to ondansetron (5-hydroxytryptamine type 3 receptor antagonist) in patients with primary fibromyalgia syndrome: a preliminary study. J Rheumatol 23:1418–1423.
Murphy DL, Andrews AM, Wichems CH, Li Q, Tohda M, Greenberg B (1998) Brain serotonin neurotransmission: an overview and update with an emphasis on serotonin subsystem heterogeneity, multiple receptors, interactions with other neurotransmitter systems, and consequent implications for understanding the actions of serotonergic drugs. J Clin Psychiatry 59 Suppl. 15:4–12
Wolfe F, Clauw DJ, Fitzcharles MA, Goldenberg DL, Häuser W, Katz RL, et al. (2016) 2016 Revisions to the 2010/2011 fibromyalgia diagnostic criteria. Semin Arthritis Rheum 46(3):319–329. https://doi.org/10.1016/j.semarthrit.2016.08.012
Lee YH, Choi SJ, Ji JD, Song GG (2012) Candidate gene studies of fibromyalgia: a systematic review and meta-analysis. Rheumatol Int 32:417–426. https://doi.org/10.1007/s00296-010-1678-9
Cohen H, Buskila D, Neumann L, Ebstein RP (2002) Confirmation of an association between fibromyalgia and serotonin transporter promoter region (5-HTTLPR) polymorphism, and relationship to anxiety-related personality traits. Arthritis Rheum 46:845–847. https://doi.org/10.1002/art.10103
Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, et al (1996) Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. N Y Sci J 274:1527–1531. https://doi.org/10.1126/science.274.5292.1527.
Offenbaecher M, Bondy B, de Jonge S, Glatzeder K, Krüger M, Schoeps P, et al (1999) Possible association of fibromyalgia with a polymorphism in the serotonin transporter gene regulatory region. Arthritis Rheum 42:2482–2488. https://doi.org/10.1002/1529-0131(199911)42:11<2482::AID-ANR27>3.0.CO;2-B

Download PDF

Version 1

posted

You are reading this latest preprint version

Association of serotonin gene (5-HTTLPR & 5-HTTVNTR) polymorphism with Fibromyalgia Syndrome in North Indian Women

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Background

Methods

Patients and controls selection

Genotyping of 5-HTTVNTR and 5-HTTLPR

Statistical Analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Version 1