- Email: [email protected]
Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population
DIAB-6256; No. of Pages 5 diabetes research and clinical practice xxx (2015) xxx–xxx
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population A. Hameed *, M. Ajmal, M. Nasir, M. Ismail Institute of Biomedical and Genetic Engineering, 24-Mauve Area, G-9/1, Islamabad, Pakistan
article info
abstract
Article history:
Aims: It is well established that the serotonergic system contributes to the regulation of
Received 19 September 2014
glucose homeostasis and feeding and therefore it has been presumed to contribute to the
Received in revised form
biological susceptibility to type 2 diabetes mellitus (T2DM) and body-mass index (BMI). 5-
3 December 2014
HTTLPR is a serotonin transporter (5-HTT) gene-linked polymorphic region that regulates the
Accepted 14 January 2015
transcriptional activity of 5-HTT. Our aim was to investigate the possible association of 5-
Available online xxx
HTTLPR polymorphism (L and S alleles) in the promoter region of the serotonin transporter
Keywords:
Methods: In this study, 574 subjects diagnosed with T2DM and 402 unrelated normal
gene with T2DM and/or higher BMI in Pakistani population. 5-HTTLPR polymorphism
controls from the general Pakistani population were genotyped for 5-HTTLPR polymorphism
Type 2 diabetes mellitus
by PCR amplification and agarose gel electrophoresis. The genotyping data (S/S, S/L and L/L)
Body mass index
were recorded and analysed statistically using various software and online available tools.
Genetic association.
Results: In the total sample, patients with type 2 diabetes and controls without diabetes, genotypes were distributed according to Hardy–Weinberg equilibrium, and S allele frequency was 61.52% (0.61). There was no statistical association between 5-HTTLPR polymorphism and the development of T2DM in this Pakistani population ( p = 0.12). Conclusions: No significant statistical association of 5-HTTLPR polymorphism with type 2 diabetes and obesity in Pakistani population shows that 5-HTTLPR polymorphism is not a major factor in determining type 2 diabetes and obesity in Pakistan. # 2015 Published by Elsevier Ireland Ltd.
1.
Introduction
Type 2 Diabetes mellitus (T2DM) is a common metabolic disorder, affecting 85–90% of all people with diabetes Over the past few decades the rate of obesity and type T2DM has increased alarmingly worldwide. Obesity and T2DM are a growing epidemic in South Asian countries with a higher prevalence. Pakistan was ranked amongst the top 10 countries of the world with the highest number of people with diabetes
in 2004 and an estimated 14.5 million Pakistanis will have diabetes by the year 2025. According to a recent national survey, 35% people over the age of 45 years have diabetes in Pakistan. Similar observations of higher prevalence of T2DM amongst immigrant South Asians have also been made. Obesity and T2DM frequently co-occur, indicating that these conditions may share common pathological mechanisms, including complex interactions between genetic and environmental factors [8]. Therefore, both environmental as well as genetic factors contribute to the incidence of this disease [10].
* Corresponding author. Tel.: +92 51 9106282; fax: +92 51 9106283. E-mail address: [email protected] (A. Hameed). http://dx.doi.org/10.1016/j.diabres.2015.01.009 0168-8227/# 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009
DIAB-6256; No. of Pages 5
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diabetes research and clinical practice xxx (2015) xxx–xxx
Table 1 – Major clinical characteristics of the analyzed patients and control subjects. Clinical characteristics Age (Years)
Gender BMI (Kg/M2)
Mean SD <40 41–60 >61 Male Female Mean SD <25 (Non-Obese) >25 (Obese)
Type 2 diabetic patients (n = 574)
Non-diabetic controls (n = 402)
Total
p Value
51.4 11.3 71(12.4%) 390 (68%) 113 (19.6%) 187 (32.5%) 387 (67.5) 27.6 6.0 188 (33%) 386 (67%)
53.1 11.6 18 (4.5%) 294 (73%) 90 (22.5) 212 (53%) 190 (47%) 24.37 6.0 254 (63%) 148 (37%)
976 89 684 203 399 577 976 442 534
0.0224 <0.0001 0.0812 0.3055 <0.0001 <0.0001 0.0001 <0.0001 <0.0001
It is complex or a multi-gene disorder with a number of genes and their polymorphisms contributing to disease pathogenesis have been identified [9]. The genes of the serotonergic system have been included in the candidate gene list due to their function in the brain and gastrointestinal tract. The serotonergic system contributes substantially to the regulation of feeding and glucose homeostasis. 5-HTTLPR is serotonin transporter (5-HTT) gene-linked polymorphic region that regulates the transcriptional activity of 5-HTT gene. The human 5-HTT gene (SLC6A4) spans 37.8 kb and is located on chromosome 17q11.1-12. The gene is composed of 14 exons and encodes a protein of 630 amino acid residues. Transcriptional activity of 5-HTT gene is modulated by a repetitive element of varying length in the 50 flanking region located approximately 1.4 kb upstream of the transcription start site, termed as 5-HTT gene linked polymorphic region (5-HTTLPR). A 44 bp insertion/deletion has been identified in the promoter region, which results into long (L) and short (S) alleles. In vitro transfection studies have demonstrated that S allele is dominant over the long allele (L) and its presence suppresses the expression of long allele resulting in lower expression of 5HTT gene, thus reducing the capability to take up and release serotonin transporter. In contrast, L allele has an almost three fold transcription rate of 5-HTT gene [5,6]. This suggests that 5HTTLPR is associated with an altered response of the serotonin system. In previous studies a possible association of 5-HTTLPR with obesity and T2DM has been reported [1,7]. Keeping in view previous reports, this study was aimed to compare the frequencies of different alleles of 5-HTT gene between patients with type 2 diabetes and the normal controls and to find out association of 5-HTT gene polymorphism with T2DM or BMI in Pakistani population.
2.
Material and methods
A total of 574 unrelated subjects diagnosed with T2DM (187 males and 387 females) and 402 unrelated controls (212 males and 190 females) from Pakistani population were collected and studied. All individuals were recruited from various hospitals located in Islamabad and in its vicinity. Inclusion of subjects in the group with diabetes was based on medical diagnosis, according to the criteria recommended by the WHO for T2DM [2]. Subjects with either diagnosis or a first-degree family history of type T2DM were excluded from the control sample. BMI was calculated as the ratio between weight and the square of the height (kg/m2). Subjects were also divided into overweight/obese (BMI > 25) and non-obese (BMI < 25)
groups. The other clinical parameters of the patients recorded were age of onset of the diabetes, systolic/diastolic blood pressures, fasting/random blood glucose, HbA1C are shown in Table 1. The study protocol for human experimentation was approved by the institutional ethics committee. Informed consent was obtained from each subject before participation in the study. Genomic DNA was isolated from peripheral blood with standard organic method of DNA extraction.
2.1.
5-HTTLPR polymorphism genotyping
Polymerase Chain Reaction (PCR) for 5-HTTLPR polymorphism genotyping was performed as previously described Cook et al. (1997), with small modifications. The polymorphic region was amplified by using 5-HTT_F, 50 -TGAATGCCAGCACCTAACCC30 , and 5-HTT_R, 50 -TTCTGGTGCCACCTAGACGC-30 as forward and reverse primers respectively. This set of primers generated Amplicons of 406 bp for S allele and Amplicons of 450 bp for L allele. Each reaction was carried out in a total volume of 25 ml containing 200 mM of dNTP, 1 unit of Taq DNA polymerase (Fermentas), 1.5 mM of MgSO4, 5% DMSO and 10 mM of TrisHCl buffer. PCR amplification was performed in a GeneAmp1 PCR system 9700 System (Applied Biosystem) Thermal Cycler. PCR amplification was carried out for 40 cycles consisting of 30 s at 95 8C, 30 s at 61 8C, and 1 min at 71 8C, followed by 10 min at 72 8C. PCR products (Amplicons) were separated by electrophoresis on a 6% polyacrylamide gel at 120 V for 3 h. The gels were stained with ethidium bromide, visualised under UV-transillumination and photographed. Genotyping was performed by classifying subjects into three genotypes: individuals homozygous for the short allele SS, those heterozygous for the short and long allele LS, and those homozygous for the long allele LL.
2.2.
Statistical analysis
Hardy–Weinberg equilibrium (HWE) was analysis, genotype distribution and allele frequencies between T2DM patients and controls without diabetes were determined using online available statistical tools, such as SNPStats [12], Vasser stats, HWE calculator for two alleles and SPSS software. The clinical data is shown as means and standard deviation (SD). Deviation from Hardy-Weinberg equilibrium was confirmed by x2 test analysis of genotype distribution. The association between 5-HTTLPR and T2DM/BMI, was defined by logistic regression analysis, assuming an additive model. Considering gender, age and T2DM as the appropriate covariates, associations were adjusted accordingly. The odds ratios (ORs) with
Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009
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Discussion
The serotonergic system contributes substantially to the regulation of glucose homeostasis and feeding. 5-HTTLPR regulates the transcriptional activity of serotonin transporter
S L SS LS LL
0.5078 0.4386
0.92 (0.715-1.18)
0.4824 0.4934
1.107 (0.8331-1.472)
P (df = 1) Chi-Square
OR (95% CI)
0.9627 0.0022
0.25 450 (39) 698 (61) 0.8246 0.84 316 (41) 488(59) 0.31 766 (39) 1186 (61) non-diabetic controls. P HWE Allele n (%) (df = 2) L S 0.736 152 222 (41) (59) 0.7854 0.7596 162 262 (38) (62) 0.2265 298 476 (39) (61) 0.6122 0.9507 154 226 (41) (59)
P (df = 1) Chi-Square Allele n (%) (n = 976) HWE P (df = 2) Chi-Square 5-HTTLPR Polymorphism Genotype n (%)
Type 2 Diabetic Patients 95 (17) 260 (45) 219 (38) Non-Diabetic Controls 63 (16) 190 (47) 149 (37) 0.3856 All Subjects n (%) 158 (16) 450 (46) 368 (38) 1.07 A gender wise breakdown association test of 5HTTLPR polymorphism in type 2 diabetic patients and Gender Affection Status 5-HTTLPR Polymorphism Chi-Square Genotype n (%) LL LS SS Male Type 2 Diabetic Patients 32 88 67 (17) (47) (36) Non-Diabetic Controls 32 98 82 0.09 (15) (46) (39) Female Type 2 Diabetic Patients 63 172 152 (16) (44) (39) Non-Diabetic Controls 31 92 67 0.00 (16) (48) (35)
4.
5HTTLPR LL, LS and SS genotypes distribution and L and S allele frequencies in type 2 diabetic patients and non-diabetic controls.
As per WHO criteria for diagnosis, 574 individuals had T2DM (58.81%), whereas 402 were normal (41.18%) controls with matched ethnic background. Gender distribution among 574 T2DM patients was 67.5% females and 32.5% males. Major clinical characteristics and distribution age, gender, and BMI are shown in Table 1. The mean age in patients group was 51.4 years and in control group was 53.1 years, which is statistically significantly different ( p = <0.001). Overall T2DM patient group majority of the subjects (67%) were obese, whereas the obesity was less prevalent in control samples (37%) The statistical analysis and the frequency of the resulting genotypes i.e individuals homozygous for long allele (LL), individuals homozygous for short allele (SS) and heterozygote (LS) was carried out to find their association with type 2 diabetes. 5-HTLPR polymorphism genotype distribution (LL, LS and SS) and allele frequencies in T2DM patient and control groups are shown in Table 2. 5-HTLPR polymorphism was found to be in Hardy–Weinberg equilibrium in the total sample ( p = 0.31), T2DM patients ( p = 0.25) and normal control ( p = 0.84). The genotype distributions of 5-HTT gene polymorphism were not significantly different between the T2DM patient and control groups (Table 2). None of the three genotypes SS, LL or LS showed any statistically significant association with the disease ( p < 0.05). Similarly, neither the S nor L allele showed any association with T2DM. This study shows that there is no association between patients with T2DM and serotonin transporter gene polymorphism in the studied Pakistani population. A gender and BMI wise breakdown association test of 5HTTLPR polymorphism in T2DM and control groups was performed. Again no association was present (Tables 2 and 3). Since age is immutable risk factor for T2DM, we performed an age-stratified analysis to study whether age influences the contribution of 5-HTTLPR polymorphism to the risk of T2DM. The results are presented in Table 3 shows only a significant association in age group with age less than 40 years (genotype: x2 = 16.81; df = 2; p = 0.0002). No significant association was obtained in the groups with ages above 40 years (Table 3). Further, association under the dominant and recessive models was also unable to yield a significant association of 5-HTTLPR polymorphism with T2DM (Table 4). Interaction analysis with covariate sex was also not statistically significant (Table 4).
Affection Status
Results
Table 2 – 5HTTLPR LL, LS and SS genotypes distribution and L and S allele frequencies.
3.
OR (95% CI)
95% confidence intervals (CIs) are presented in the tables with respect to the risk allele. A p value of 0.05 was considered significant. Statistical analysis was performed using SPSS version 17.0 (SPSS, Chicago, IL, USA) and web tools, SNPStats and Vasserstats for the analysis of association studies.
0.9956 (0.8915-1.116)
diabetes research and clinical practice xxx (2015) xxx–xxx
Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009
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diabetes research and clinical practice xxx (2015) xxx–xxx
Table 3 – A BMI and age wise breakdown association test of 5HTTLPR polymorphism in type 2 diabetic patients and nondiabetic controls. A BMI and age wise breakdown association test results BMI (Kg/M2)
Affection Status
5-HTTLPR Polymorphism Genotype n (%) LL
LS
ChiSquare
P (df = 2)
HWE
SS
<25
Type 2 Diabetic 24 95 69 Patients (13) (50) (37) Non-Diabetic 44 115 95 Controls (17) (45) (38) Type 2 Diabetic 71 165 150 >25 Patients (18) (43) (39) Non-Diabetic 19 75 54 Controls (13) (51) (36) An age wise breakdown association test results Affection Status 5-HTTLPR Age (Years) Polymorphism Genotype n (%) LL LS SS Type 2 Diabetic 9 33 29 <40 Patients (13) (46) (41) Non-Diabetic 0 18 0 Controls (0) (100) (0) Type 2 Diabetic 71 175 144 41-60 Patients (18) (45) (37) Non-Diabetic 47 130 117 Controls (16) (44) (40) >60 Type 2 Diabetic 15 52 46 Patients (13) (46) (41) Non-Diabetic 16 42 32 Controls (18) (47) (36)
0.3231 2.101
0.35
0.3683 0.0343
3.614
0.1642
0.3709
ChiSquare
P (df = 2)
HWE
0.0002
0.000 0.1670
0.8570
0.6515
0.2839 0.9598
1.016
0.6017
(5-HTT). In a study carried out by Iordanidou et al. (2010) demonstrated a strong association of S allele of 5-HTTLPR with type 2 diabetes. In another study carried out by Wilhelm K and his colleagues have shown that 5-HTTLPR genotype is associated with psychological distress in patients with diabetes type 2. Similarly, in a case-control study by PeraltaLeal V demonstrated that 5-HTTLPR polymorphism is not significantly associated with T2DM in Mexican population
L
S
143 (38) 203 (40) 307 (40) 113 (38)
233 (62) 305 (60) 465 (60) 183 (62)
ChiSquare
P (df = 1)
0.3374
0.5613
0.9221 (0.7013-1.212)
0.3999
0.5271
1.093 (0.8303-1.438)
ChiSquare
P (df = 1)
OR (95% CI)
2.400
0.1213
0.5604 (0.2679-1.172)
0.9089
0.3404
1.113 (0.8934-1.386)
0.9871
0.3205
0.8157 (0.5456-1.220)158450
Allele n (%)
0.9349 16.81
Allele n (%)
0.7312
L 51 (36) 18 (50) 317 (41) 224 (38) 82 (36) 74 (41)
S 91 (64) 18 (50) 463 (59) 364 (62)) 144 (64) 106 (59)
OR (95% CI)
but logistic regression analysis of the L allele and increased BMI association was reported. In a longitudinal study of Japanese women, association analysis of 5-HTTLPR polymorphism with fasting blood glucose (FBG) was carried out. They identified the protective role of S allele on FBG levels and improvement in FBG values [13, 14]. A study on large cohorts of 1584 unrelated individuals in US found that SS and SL 5-HTTLPR genotypes are significantly associated
Table 4 – Association of 5-HTTLPR Polymorphism with type 2 diabetes under he dominant and recessive models and interaction analysis results with covariate as gender. Association under the dominant and recessive models adjusted by age, sex and BMI Affection Status
Dominant N (%) LL + LS
ChiSquare
P (df = 1)
OR (95% CI)
Recessive
SS
Type 2 Diabetic 355 (61.9) 219 (38.1) 0.1193 0.7298 Patients Non-Diabetic 253 (62.9) 149 (37.1) Controls 5-HTTLPR Polymorphism and Sex cross-classification interaction Female Male Genotype OR (95% CI) NonNonType 2 Diabetic Diabetic Diabetic Controls Controls Patients S/S-S/L 159 324 1.00 180 31 63 1.02 (0.63-1.65) 32 L/L Interaction p-value: 0.82
LL 0.9547 95 (0.8903-1.242) 63
ChiP Square (df = 1)
OR (95% CI)
LS + SS 479
0.1346
0.7137
1.067 (0.7539-1.511)
339
table (n = 976, adjusted by Age + BMI) Type 2 Diabetic Patients 155 32
OR (95% CI)
0.52 (0.38-0.70) 0.57 (0.33-0.98)
Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009
DIAB-6256; No. of Pages 5 diabetes research and clinical practice xxx (2015) xxx–xxx
with a higher BMI [3]. This association was further confirmed in South American population samples of adolescents and adults of European ancestry, in which the SS genotype was found to be a risk factor [11,13]. To date only one study has reported the S allele as a risk factor for T2DM, in a Caucasian population from Greece, interestingly, the presence of the S allele was not found to be associated with increased BMI values [8]. Previously reported T2DM risk allele S was higher. High S allele frequencies have already been reported in various world populations, including Native Americans, Japanese, Chinese and Korean populations [4]. The reports from various word populations differ significantly differ from each other. Nonetheless, all these studies are in agreement by pointing out that SLC6A4 polymorphism is associated with BMI, and ethnicity may be a contributing factor towards the differences between our study and previous reports. Association studies of 5-HTTLPR with glucose metabolism, changes in the body-mass index (BMI) and T2DM are relatively recent. Therefore, based on the reported findings, this case-control association study was aimed to explore the possible contribution of 5-HTTLPR polymorphism to the risk of T2DM in Pakistani population. As already mentioned no significant association of 5-HTTLPR and T2DM or obesity was observed. In conclusion, our findings indicate that 5-HTTLPR polymorphism is not linked with T2DM or BMI in Pakistani population. Genetic polymorphisms or variants of a number of genes have been associated to T2DM among other world populations, mainly in Europe. However, the contributions of these genetic variants in other populations or ethnic groups are unclear. Since the susceptibility of different ethnic groups differ due to different environmental factors and genetic background, it is important to replicate the findings to unravel the genetics of T2DM. Pakistan is one of the leading countries of the world with large number of patients with diabetes type 2. Several recent studies in Asian populations have replicated the association of a few genes with larger effect size compared to those reported for European populations. However, reports from several Asian populations give a heterogeneous picture due to its diverse ethnicity. The major issue to address in diabetes biology is to identify the genetic changes in the disease and their occurrence in different populations. Uncovering these genetic changes in diabetes may be important in defining the functional role of specific genetic alterations and developing potential biomarkers. Keeping in view this, further investigations and functional genomic studies are required to be carried to define the possible role of 5-HTTLPR polymorphism in T2DM, BMI, and obesity. It is important to study the role of the serotonin transporter as a risk factor for the obesity phenotype and suggesting potential new avenues for its pharmacological intervention.
Conflict of interest statement None to declare
5
Acknowledgement We acknowledge institutional administration for providing financial support to conduct this study. We hereby acknowledge, Mr. Husnain Haider for providing the technical assistance. We also acknowledge our colleagues who have contributed in clinical data, sample collection and in DNA extraction
references
[1] Cook Jr EH, Courchesne R, Lord C, Cox NJ, Yan S, Lincoln A, et al. Evidence of linkage between the serotonin transporter and autistic disorder. Mol Psychiatry 1997;2:247–50. [2] Deckers JG, Schellevis FG, Fleming DM. WHO diagnostic criteria as a validation tool for the diagnosis of diabetes mellitus: a study in five European countries. Eur J Gen Pract 2006;12:108–13. [3] Fuemmeler BF, Agurs-Collins TD, McClernon FJ, Kollins SH, Kail ME, Bergen AW, et al. Genes implicated in serotonergic and dopaminergic functioning predict BMI categories. Obesity 2008;16:348–55. [4] Goldman N, Glei DA, Lin YH, Weinstein M. The serotonin transporter polymorphism (5-HTTLPR): allelic variation and links with depressive symptoms. Depress Anxiety 2010;27: 260–9. [5] Greenberg BD, Tolliver TJ, Huang SJ, Li Q, Bengel D, Murphy DL. Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets. Am J Med Genet 1999;88(83-87):592. [6] Peralta-Leal V, Leal-Ugarte E, Meza-Espinoza JP, Da´valosRodrı´guez IP, Bocanegra-Alonso A, Acosta-Gonza´lez RI, et al. Association of a serotonin transporter gene (SLC6A4) 5-HTTLPR polymorphism with body mass index categories but not type 2 diabetes mellitus in Mexicans. Genet Mol Biol 2012;35(3):589–93. [7] Heils A, Teufel A, Petri S, Stober G, Riederer P, Bengel D, et al. Allelic variation of human serotonin transporter gene expression. J Neurochem 1996;66:2621–4. [8] Iordanidou M, Tavridou A, Petridis I, Arvanitidis KI, Christakidis D, Vargemezis V, et al. The serotonin transporter promoter polymorphism (5-HTTLPR) is associated with type 2 diabetes. Clin Chim Acta 2010;411:167–71. [9] Knowler WC, Saad MF, Pettitt DJ, Nelson RG, Bennett PH. Determinants of diabetes mellitus in the Pima Indians. Diabetes Care 1993;16:216–27. [10] Prokopenko I, McCarthy MI, Lindgren CM. Type 2 diabetes: new genes, new understanding. Trends Genet 1993;24:613–21. [11] Sookoian S, Gemma C, Garcia SI, Gianotti TF, Dieuzeide G, Roussos A, et al. Short allele of serotonin transporter gene promoter is a risk factor for obesity in adolescents. Obesity 2007;15:271–6. [12] Sole´ X, Guino´ E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics 2006;22(15 (Aug 1)):1928–9 [Epub 2006 May 23]. [13] Sookoian S, Gianotti TF, Gemma C, Burgueno A, Pirola CJ. Contribution of the functional 5-HTTLPR variant of the SLC6A4 gene to obesity risk in male adults. Obesity 2008;16:488–91. [14] Yamakawa M, Fukushima A, Sakuma K, Yanagisawa Y, Kagawa Y. Serotonin transporter polymorphisms affect human blood glucose control. Biochem Biophys Res Commun 2005;334:1165–71.
Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population A. Hameed *, M. Ajmal, M. Nasir, M. Ismail Institute of Biomedical and Genetic Engineering, 24-Mauve Area, G-9/1, Islamabad, Pakistan
article info
abstract
Article history:
Aims: It is well established that the serotonergic system contributes to the regulation of
Received 19 September 2014
glucose homeostasis and feeding and therefore it has been presumed to contribute to the
Received in revised form
biological susceptibility to type 2 diabetes mellitus (T2DM) and body-mass index (BMI). 5-
3 December 2014
HTTLPR is a serotonin transporter (5-HTT) gene-linked polymorphic region that regulates the
Accepted 14 January 2015
transcriptional activity of 5-HTT. Our aim was to investigate the possible association of 5-
Available online xxx
HTTLPR polymorphism (L and S alleles) in the promoter region of the serotonin transporter
Keywords:
Methods: In this study, 574 subjects diagnosed with T2DM and 402 unrelated normal
gene with T2DM and/or higher BMI in Pakistani population. 5-HTTLPR polymorphism
controls from the general Pakistani population were genotyped for 5-HTTLPR polymorphism
Type 2 diabetes mellitus
by PCR amplification and agarose gel electrophoresis. The genotyping data (S/S, S/L and L/L)
Body mass index
were recorded and analysed statistically using various software and online available tools.
Genetic association.
Results: In the total sample, patients with type 2 diabetes and controls without diabetes, genotypes were distributed according to Hardy–Weinberg equilibrium, and S allele frequency was 61.52% (0.61). There was no statistical association between 5-HTTLPR polymorphism and the development of T2DM in this Pakistani population ( p = 0.12). Conclusions: No significant statistical association of 5-HTTLPR polymorphism with type 2 diabetes and obesity in Pakistani population shows that 5-HTTLPR polymorphism is not a major factor in determining type 2 diabetes and obesity in Pakistan. # 2015 Published by Elsevier Ireland Ltd.
1.
Introduction
Type 2 Diabetes mellitus (T2DM) is a common metabolic disorder, affecting 85–90% of all people with diabetes Over the past few decades the rate of obesity and type T2DM has increased alarmingly worldwide. Obesity and T2DM are a growing epidemic in South Asian countries with a higher prevalence. Pakistan was ranked amongst the top 10 countries of the world with the highest number of people with diabetes
in 2004 and an estimated 14.5 million Pakistanis will have diabetes by the year 2025. According to a recent national survey, 35% people over the age of 45 years have diabetes in Pakistan. Similar observations of higher prevalence of T2DM amongst immigrant South Asians have also been made. Obesity and T2DM frequently co-occur, indicating that these conditions may share common pathological mechanisms, including complex interactions between genetic and environmental factors [8]. Therefore, both environmental as well as genetic factors contribute to the incidence of this disease [10].
* Corresponding author. Tel.: +92 51 9106282; fax: +92 51 9106283. E-mail address: [email protected] (A. Hameed). http://dx.doi.org/10.1016/j.diabres.2015.01.009 0168-8227/# 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009
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2
diabetes research and clinical practice xxx (2015) xxx–xxx
Table 1 – Major clinical characteristics of the analyzed patients and control subjects. Clinical characteristics Age (Years)
Gender BMI (Kg/M2)
Mean SD <40 41–60 >61 Male Female Mean SD <25 (Non-Obese) >25 (Obese)
Type 2 diabetic patients (n = 574)
Non-diabetic controls (n = 402)
Total
p Value
51.4 11.3 71(12.4%) 390 (68%) 113 (19.6%) 187 (32.5%) 387 (67.5) 27.6 6.0 188 (33%) 386 (67%)
53.1 11.6 18 (4.5%) 294 (73%) 90 (22.5) 212 (53%) 190 (47%) 24.37 6.0 254 (63%) 148 (37%)
976 89 684 203 399 577 976 442 534
0.0224 <0.0001 0.0812 0.3055 <0.0001 <0.0001 0.0001 <0.0001 <0.0001
It is complex or a multi-gene disorder with a number of genes and their polymorphisms contributing to disease pathogenesis have been identified [9]. The genes of the serotonergic system have been included in the candidate gene list due to their function in the brain and gastrointestinal tract. The serotonergic system contributes substantially to the regulation of feeding and glucose homeostasis. 5-HTTLPR is serotonin transporter (5-HTT) gene-linked polymorphic region that regulates the transcriptional activity of 5-HTT gene. The human 5-HTT gene (SLC6A4) spans 37.8 kb and is located on chromosome 17q11.1-12. The gene is composed of 14 exons and encodes a protein of 630 amino acid residues. Transcriptional activity of 5-HTT gene is modulated by a repetitive element of varying length in the 50 flanking region located approximately 1.4 kb upstream of the transcription start site, termed as 5-HTT gene linked polymorphic region (5-HTTLPR). A 44 bp insertion/deletion has been identified in the promoter region, which results into long (L) and short (S) alleles. In vitro transfection studies have demonstrated that S allele is dominant over the long allele (L) and its presence suppresses the expression of long allele resulting in lower expression of 5HTT gene, thus reducing the capability to take up and release serotonin transporter. In contrast, L allele has an almost three fold transcription rate of 5-HTT gene [5,6]. This suggests that 5HTTLPR is associated with an altered response of the serotonin system. In previous studies a possible association of 5-HTTLPR with obesity and T2DM has been reported [1,7]. Keeping in view previous reports, this study was aimed to compare the frequencies of different alleles of 5-HTT gene between patients with type 2 diabetes and the normal controls and to find out association of 5-HTT gene polymorphism with T2DM or BMI in Pakistani population.
2.
Material and methods
A total of 574 unrelated subjects diagnosed with T2DM (187 males and 387 females) and 402 unrelated controls (212 males and 190 females) from Pakistani population were collected and studied. All individuals were recruited from various hospitals located in Islamabad and in its vicinity. Inclusion of subjects in the group with diabetes was based on medical diagnosis, according to the criteria recommended by the WHO for T2DM [2]. Subjects with either diagnosis or a first-degree family history of type T2DM were excluded from the control sample. BMI was calculated as the ratio between weight and the square of the height (kg/m2). Subjects were also divided into overweight/obese (BMI > 25) and non-obese (BMI < 25)
groups. The other clinical parameters of the patients recorded were age of onset of the diabetes, systolic/diastolic blood pressures, fasting/random blood glucose, HbA1C are shown in Table 1. The study protocol for human experimentation was approved by the institutional ethics committee. Informed consent was obtained from each subject before participation in the study. Genomic DNA was isolated from peripheral blood with standard organic method of DNA extraction.
2.1.
5-HTTLPR polymorphism genotyping
Polymerase Chain Reaction (PCR) for 5-HTTLPR polymorphism genotyping was performed as previously described Cook et al. (1997), with small modifications. The polymorphic region was amplified by using 5-HTT_F, 50 -TGAATGCCAGCACCTAACCC30 , and 5-HTT_R, 50 -TTCTGGTGCCACCTAGACGC-30 as forward and reverse primers respectively. This set of primers generated Amplicons of 406 bp for S allele and Amplicons of 450 bp for L allele. Each reaction was carried out in a total volume of 25 ml containing 200 mM of dNTP, 1 unit of Taq DNA polymerase (Fermentas), 1.5 mM of MgSO4, 5% DMSO and 10 mM of TrisHCl buffer. PCR amplification was performed in a GeneAmp1 PCR system 9700 System (Applied Biosystem) Thermal Cycler. PCR amplification was carried out for 40 cycles consisting of 30 s at 95 8C, 30 s at 61 8C, and 1 min at 71 8C, followed by 10 min at 72 8C. PCR products (Amplicons) were separated by electrophoresis on a 6% polyacrylamide gel at 120 V for 3 h. The gels were stained with ethidium bromide, visualised under UV-transillumination and photographed. Genotyping was performed by classifying subjects into three genotypes: individuals homozygous for the short allele SS, those heterozygous for the short and long allele LS, and those homozygous for the long allele LL.
2.2.
Statistical analysis
Hardy–Weinberg equilibrium (HWE) was analysis, genotype distribution and allele frequencies between T2DM patients and controls without diabetes were determined using online available statistical tools, such as SNPStats [12], Vasser stats, HWE calculator for two alleles and SPSS software. The clinical data is shown as means and standard deviation (SD). Deviation from Hardy-Weinberg equilibrium was confirmed by x2 test analysis of genotype distribution. The association between 5-HTTLPR and T2DM/BMI, was defined by logistic regression analysis, assuming an additive model. Considering gender, age and T2DM as the appropriate covariates, associations were adjusted accordingly. The odds ratios (ORs) with
Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009
DIAB-6256; No. of Pages 5
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Discussion
The serotonergic system contributes substantially to the regulation of glucose homeostasis and feeding. 5-HTTLPR regulates the transcriptional activity of serotonin transporter
S L SS LS LL
0.5078 0.4386
0.92 (0.715-1.18)
0.4824 0.4934
1.107 (0.8331-1.472)
P (df = 1) Chi-Square
OR (95% CI)
0.9627 0.0022
0.25 450 (39) 698 (61) 0.8246 0.84 316 (41) 488(59) 0.31 766 (39) 1186 (61) non-diabetic controls. P HWE Allele n (%) (df = 2) L S 0.736 152 222 (41) (59) 0.7854 0.7596 162 262 (38) (62) 0.2265 298 476 (39) (61) 0.6122 0.9507 154 226 (41) (59)
P (df = 1) Chi-Square Allele n (%) (n = 976) HWE P (df = 2) Chi-Square 5-HTTLPR Polymorphism Genotype n (%)
Type 2 Diabetic Patients 95 (17) 260 (45) 219 (38) Non-Diabetic Controls 63 (16) 190 (47) 149 (37) 0.3856 All Subjects n (%) 158 (16) 450 (46) 368 (38) 1.07 A gender wise breakdown association test of 5HTTLPR polymorphism in type 2 diabetic patients and Gender Affection Status 5-HTTLPR Polymorphism Chi-Square Genotype n (%) LL LS SS Male Type 2 Diabetic Patients 32 88 67 (17) (47) (36) Non-Diabetic Controls 32 98 82 0.09 (15) (46) (39) Female Type 2 Diabetic Patients 63 172 152 (16) (44) (39) Non-Diabetic Controls 31 92 67 0.00 (16) (48) (35)
4.
5HTTLPR LL, LS and SS genotypes distribution and L and S allele frequencies in type 2 diabetic patients and non-diabetic controls.
As per WHO criteria for diagnosis, 574 individuals had T2DM (58.81%), whereas 402 were normal (41.18%) controls with matched ethnic background. Gender distribution among 574 T2DM patients was 67.5% females and 32.5% males. Major clinical characteristics and distribution age, gender, and BMI are shown in Table 1. The mean age in patients group was 51.4 years and in control group was 53.1 years, which is statistically significantly different ( p = <0.001). Overall T2DM patient group majority of the subjects (67%) were obese, whereas the obesity was less prevalent in control samples (37%) The statistical analysis and the frequency of the resulting genotypes i.e individuals homozygous for long allele (LL), individuals homozygous for short allele (SS) and heterozygote (LS) was carried out to find their association with type 2 diabetes. 5-HTLPR polymorphism genotype distribution (LL, LS and SS) and allele frequencies in T2DM patient and control groups are shown in Table 2. 5-HTLPR polymorphism was found to be in Hardy–Weinberg equilibrium in the total sample ( p = 0.31), T2DM patients ( p = 0.25) and normal control ( p = 0.84). The genotype distributions of 5-HTT gene polymorphism were not significantly different between the T2DM patient and control groups (Table 2). None of the three genotypes SS, LL or LS showed any statistically significant association with the disease ( p < 0.05). Similarly, neither the S nor L allele showed any association with T2DM. This study shows that there is no association between patients with T2DM and serotonin transporter gene polymorphism in the studied Pakistani population. A gender and BMI wise breakdown association test of 5HTTLPR polymorphism in T2DM and control groups was performed. Again no association was present (Tables 2 and 3). Since age is immutable risk factor for T2DM, we performed an age-stratified analysis to study whether age influences the contribution of 5-HTTLPR polymorphism to the risk of T2DM. The results are presented in Table 3 shows only a significant association in age group with age less than 40 years (genotype: x2 = 16.81; df = 2; p = 0.0002). No significant association was obtained in the groups with ages above 40 years (Table 3). Further, association under the dominant and recessive models was also unable to yield a significant association of 5-HTTLPR polymorphism with T2DM (Table 4). Interaction analysis with covariate sex was also not statistically significant (Table 4).
Affection Status
Results
Table 2 – 5HTTLPR LL, LS and SS genotypes distribution and L and S allele frequencies.
3.
OR (95% CI)
95% confidence intervals (CIs) are presented in the tables with respect to the risk allele. A p value of 0.05 was considered significant. Statistical analysis was performed using SPSS version 17.0 (SPSS, Chicago, IL, USA) and web tools, SNPStats and Vasserstats for the analysis of association studies.
0.9956 (0.8915-1.116)
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Table 3 – A BMI and age wise breakdown association test of 5HTTLPR polymorphism in type 2 diabetic patients and nondiabetic controls. A BMI and age wise breakdown association test results BMI (Kg/M2)
Affection Status
5-HTTLPR Polymorphism Genotype n (%) LL
LS
ChiSquare
P (df = 2)
HWE
SS
<25
Type 2 Diabetic 24 95 69 Patients (13) (50) (37) Non-Diabetic 44 115 95 Controls (17) (45) (38) Type 2 Diabetic 71 165 150 >25 Patients (18) (43) (39) Non-Diabetic 19 75 54 Controls (13) (51) (36) An age wise breakdown association test results Affection Status 5-HTTLPR Age (Years) Polymorphism Genotype n (%) LL LS SS Type 2 Diabetic 9 33 29 <40 Patients (13) (46) (41) Non-Diabetic 0 18 0 Controls (0) (100) (0) Type 2 Diabetic 71 175 144 41-60 Patients (18) (45) (37) Non-Diabetic 47 130 117 Controls (16) (44) (40) >60 Type 2 Diabetic 15 52 46 Patients (13) (46) (41) Non-Diabetic 16 42 32 Controls (18) (47) (36)
0.3231 2.101
0.35
0.3683 0.0343
3.614
0.1642
0.3709
ChiSquare
P (df = 2)
HWE
0.0002
0.000 0.1670
0.8570
0.6515
0.2839 0.9598
1.016
0.6017
(5-HTT). In a study carried out by Iordanidou et al. (2010) demonstrated a strong association of S allele of 5-HTTLPR with type 2 diabetes. In another study carried out by Wilhelm K and his colleagues have shown that 5-HTTLPR genotype is associated with psychological distress in patients with diabetes type 2. Similarly, in a case-control study by PeraltaLeal V demonstrated that 5-HTTLPR polymorphism is not significantly associated with T2DM in Mexican population
L
S
143 (38) 203 (40) 307 (40) 113 (38)
233 (62) 305 (60) 465 (60) 183 (62)
ChiSquare
P (df = 1)
0.3374
0.5613
0.9221 (0.7013-1.212)
0.3999
0.5271
1.093 (0.8303-1.438)
ChiSquare
P (df = 1)
OR (95% CI)
2.400
0.1213
0.5604 (0.2679-1.172)
0.9089
0.3404
1.113 (0.8934-1.386)
0.9871
0.3205
0.8157 (0.5456-1.220)158450
Allele n (%)
0.9349 16.81
Allele n (%)
0.7312
L 51 (36) 18 (50) 317 (41) 224 (38) 82 (36) 74 (41)
S 91 (64) 18 (50) 463 (59) 364 (62)) 144 (64) 106 (59)
OR (95% CI)
but logistic regression analysis of the L allele and increased BMI association was reported. In a longitudinal study of Japanese women, association analysis of 5-HTTLPR polymorphism with fasting blood glucose (FBG) was carried out. They identified the protective role of S allele on FBG levels and improvement in FBG values [13, 14]. A study on large cohorts of 1584 unrelated individuals in US found that SS and SL 5-HTTLPR genotypes are significantly associated
Table 4 – Association of 5-HTTLPR Polymorphism with type 2 diabetes under he dominant and recessive models and interaction analysis results with covariate as gender. Association under the dominant and recessive models adjusted by age, sex and BMI Affection Status
Dominant N (%) LL + LS
ChiSquare
P (df = 1)
OR (95% CI)
Recessive
SS
Type 2 Diabetic 355 (61.9) 219 (38.1) 0.1193 0.7298 Patients Non-Diabetic 253 (62.9) 149 (37.1) Controls 5-HTTLPR Polymorphism and Sex cross-classification interaction Female Male Genotype OR (95% CI) NonNonType 2 Diabetic Diabetic Diabetic Controls Controls Patients S/S-S/L 159 324 1.00 180 31 63 1.02 (0.63-1.65) 32 L/L Interaction p-value: 0.82
LL 0.9547 95 (0.8903-1.242) 63
ChiP Square (df = 1)
OR (95% CI)
LS + SS 479
0.1346
0.7137
1.067 (0.7539-1.511)
339
table (n = 976, adjusted by Age + BMI) Type 2 Diabetic Patients 155 32
OR (95% CI)
0.52 (0.38-0.70) 0.57 (0.33-0.98)
Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009
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with a higher BMI [3]. This association was further confirmed in South American population samples of adolescents and adults of European ancestry, in which the SS genotype was found to be a risk factor [11,13]. To date only one study has reported the S allele as a risk factor for T2DM, in a Caucasian population from Greece, interestingly, the presence of the S allele was not found to be associated with increased BMI values [8]. Previously reported T2DM risk allele S was higher. High S allele frequencies have already been reported in various world populations, including Native Americans, Japanese, Chinese and Korean populations [4]. The reports from various word populations differ significantly differ from each other. Nonetheless, all these studies are in agreement by pointing out that SLC6A4 polymorphism is associated with BMI, and ethnicity may be a contributing factor towards the differences between our study and previous reports. Association studies of 5-HTTLPR with glucose metabolism, changes in the body-mass index (BMI) and T2DM are relatively recent. Therefore, based on the reported findings, this case-control association study was aimed to explore the possible contribution of 5-HTTLPR polymorphism to the risk of T2DM in Pakistani population. As already mentioned no significant association of 5-HTTLPR and T2DM or obesity was observed. In conclusion, our findings indicate that 5-HTTLPR polymorphism is not linked with T2DM or BMI in Pakistani population. Genetic polymorphisms or variants of a number of genes have been associated to T2DM among other world populations, mainly in Europe. However, the contributions of these genetic variants in other populations or ethnic groups are unclear. Since the susceptibility of different ethnic groups differ due to different environmental factors and genetic background, it is important to replicate the findings to unravel the genetics of T2DM. Pakistan is one of the leading countries of the world with large number of patients with diabetes type 2. Several recent studies in Asian populations have replicated the association of a few genes with larger effect size compared to those reported for European populations. However, reports from several Asian populations give a heterogeneous picture due to its diverse ethnicity. The major issue to address in diabetes biology is to identify the genetic changes in the disease and their occurrence in different populations. Uncovering these genetic changes in diabetes may be important in defining the functional role of specific genetic alterations and developing potential biomarkers. Keeping in view this, further investigations and functional genomic studies are required to be carried to define the possible role of 5-HTTLPR polymorphism in T2DM, BMI, and obesity. It is important to study the role of the serotonin transporter as a risk factor for the obesity phenotype and suggesting potential new avenues for its pharmacological intervention.
Conflict of interest statement None to declare
5
Acknowledgement We acknowledge institutional administration for providing financial support to conduct this study. We hereby acknowledge, Mr. Husnain Haider for providing the technical assistance. We also acknowledge our colleagues who have contributed in clinical data, sample collection and in DNA extraction
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Please cite this article in press as: Hameed A, et al. Genetic association analysis of serotonin transporter polymorphism (5-HTTLPR) with type 2 diabetes patients of Pakistani population. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.01.009