Functional Variants in TLR4 and The Risk of Morbid Obesity
2Department of Biostatistics and Medical Informatics, Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey
3Department of Endocrinology and Metabolism, Umraniye Training and Research Hospital, Istanbul, Turkey
4Division of Endocrinology and Metabolism, Department of Internal Medicine, Bezmialem University, Bezmialem Medical Faculty,Istanbul, Turkey
5Acibadem Healthcare Services, Acibadem University Kerem Aydinlar Campus, Labgen, Istanbul, Turkey
6Department of Medical Biology and Genetics, Istanbul Aydin University, Medical Faculty, Istanbul, Turkey
Morbid obesity (MO) is an inflmmatory disorder with multifactorial etiology, caused by a combination of genetic, autoimmune and environmental factors.Toll-like receptor 4 (TLR4) has been linked with several autoimmune and inflmmatorydisorders. We, therefore, investigated the genetic variations between TLR4, and MOsusceptibility. TLR4 asp299Gln rs4986790, Thr399Ile rs4986791 were genotyped in213 female patients with MO and 204 female healthy controls using the PCR-RFLPmethod. The frequency of AA genotype and A allele of rs4986790 were signifiantlyhigher in MO patients compared to control group (p=0.0378, p=0.0423; respectively), whereas differences in rs4986791 polymorphism were not statistically signifiant.TLR4 rs4986790 and rs4986791 SNPs demonstrated statistical signifiance amongthe combined genotypes AA+CC genotype (p=0.001) in the patient and control subjects. Our fidings prove that both single and combined genotype analysis of rs986790and rs4986791 polymorphisms indicate that the wild genotypes of AA genotype ofrs4986790 provide a risk factor against MO disease and AA+CC combined genotypeare strongly associated with enhanced risk of MO in a group of women in a Turkishpopulation.
Morbid obesity (MO), the most severe form of obesity, is defined as body mass index (BMI) >40 kg/m2and mainly characterized by low-grade inflammation in both blood and white adipose tissue (WAT). Part of the systemic inflammation associated withobesity in which many inflammatory cells tend toaccumulate originates from WAT itself. ThereforeMorbid obesity (MO), is characterized by excess adipose tissue mass which is considered as an important participant in the regulation of many pathological processes including inflammation (1).Toll-like receptors (TLRs), originally characterizedin the innate immune system, are pattern recognition receptors that are activated by pathogen-associated molecular patterns, such as bacterial cellwall components and induce inflammatory responses (2). TLRs activate the nuclear factor-kappa B (NF-KB) pathway, which results in the synthesis and release of pro-inflammatory cytokines,thereby augmenting the local inflammatory responses (3,4).Among the 10 human TLRs identified so far, TLR2and TLR4 are the most defined members (5). Thepotential clinical and biological significance ofTLR4 receptor has been reported in several infectious, autoimmune and inflammatory disorders(6,7). Two common gene polymorphisms in theextracellular domain of the TLR4 receptor havealso been defined. An A/G transition at aminoacid299 resulting in an asparagine to glycine change(Asp299Gly) and a C/T transition at amino acid399 resulting in a threonine to isoleucine change(Thr399Ile) which is reported to change receptors’ligand binding site, leading to a reduced capacity toelicit inflammation (8). Asp299Gly and Thr399Ilegene polymorphisms of human TLR4 have beenshown to be associated with functional changesthat predispose people to be less responsive to lipopolysaccharide (LPS) and have an increasedrisk of severe infection susceptibility to pathogenicbacterial infections (9-11). It has been suggestedthat the carriage of these gene polymorphisms isrelated to a risk of atherosclerosis and other chronic inflammatory diseases (6,7,12) through the activation of the inflammatory cells via the NF-KBpathway (13).All in all, polymorphisms in TLR4 gene could beattractive candidates for searching morbid obesity risk, opening the question whether individualvariations in the expression of these genes might account for different susceptibilities to MO. Therefore, we analyzed the association of two SNPs(TLR4 Asp299Gly rs4986790 and TLR4 Thr399IIers4986791) alone or combined with susceptibilityto MO in a group of women in a Turkish populationas a case-control study.
The study included 213 female morbid obese patients and 204 female healthy control subjects atthe Department of Endocrinology and Metabolism,Bezmialem University Hospital, Istanbul, Turkey.The age distribution was 38.0±5.7 years for patients and 39.0±7.3 for control subjects. The inclusion criteria were BMI > 35 kg/m2 for morbid obesity and BMI <25 kg/m2 for healthy control subjects.The exclusion criteria were alcohol abuse, havingany systemic disease including chronic kidney,liver or cardiovascular diseases, or malignancies,rheumatologic disorders or any inflammatory diseases. All study subjects have Turkish origin andprovided signed informed consent prior to the sample and data collection, and the study protocol wasapproved by the Institutional Ethical Committeeof Bezmialem University (03/06/2015 - 11/10).
DNA Isolation and Polymorphism Analysis
DNA isolation was performed from the collectedperipheral blood of samples using a commercialkit (Roche Diagnostics GmbH, Mannheim, Germany; Product No. 11796828001) and the DNAsamples with OD ratio 1.8±0.1 were included inthe study.For all PCRs described here, a total of 100 to 200ng of DNA was amplified in a 25 μl PCR reaction mixture. All PCR and digestion procedureswere carried out within Veriti DX Thermal Cycler(Applied Biosystems, USA) and the PCR anddigestion products were analyzed in ethidium bromide-stained 3.5% metaphor agarose gels. For TLR4 Asp299Gly, genomic DNA was amplifiedto have a 468 bp length product. The PCR product was then digested with the restriction enzymeNcoI (New England Biolabs). During digestion, theproducts were incubated at 37 °C for 15 min andrun on an agarose gel for 60 minutes at 75 V, anddirectly screened under UV light. The heterozygous genotype (AG) gives three bands of 468 bp,446 bp, and 22 bp; as only one strand of the amplicon is cut. The homozygous mutant genotype (GG)is observed as two bands of 446 and 22 bps whereas homozygote wild genotype (AA) is observed as asingle band with 468 bp (Figure 1).For TLR4 Thr399Ile genomic DNA was amplifiedto have a 385 bp length product. The PCR product was then digested with the restriction enzymeHinfI (New England Biolabs). RFLP incubation condition was the same as NcoI enzyme used forTLR4 Asp299Gly polymorphism. The heterozygous genotype (CT) gives three bands of 385 bp,365 bp, and 20 bp, as only one strand of the amplicon is cut. The homozygous mutant genotype (TT)is observed as two bands of 365 and 20 bps, whereas homozygote wild genotype (CC) is observed as asingle band with 385 bp (Figure 2).Genotyping verification for randomly opted PCRproducts was performed with Sanger sequencing.The reaction mix was prepared with DTCS Mix(Beckman Coulter, USA) and run on GenomeLabGeXP Genetic Analysis System (Beckman Coulter,USA).
Data Processing and Analysis
Sanger sequencing data analysis was carried outwith DNAStar Software (Version 22.214.171.124). Forsingle gene only genotype analysis GraphPadPrism (Version 5), for logistic regression analysisSPSS software version 18, for haplotype analysisHaploview (Version 4.2) were used for the patientand control samples’ values. Hardy-Weinbergequilibrium (HWE) test and genotype-allele frequencies’ comparison between the cases and thecontrols were performed using chi-square analysis.Odds ratio (OR) and respective 95% confidence intervals (CIs) were reported to evaluate the effectsof any difference between allelic and genotype distribution. A two-sided p-value <0.05 was considered statistically significant.
The distribution of the genotype and allele frequencies of all polymorphisms studied is shown Table1. 213 patients and 204 control subjects were successfully genotyped for the TLR4 rs4986790 andrs4986791. The distribution of TLR4 rs4986790but not TLR4 rs4986791 were consistent withHWE (p=0.371, p=0.0803 respectively). The frequency of rs4986790 AA genotype was statisticallyhigher in the patients compared with control group(p=0.0378, OR: 2.128, 95% CI: 1.029-4.401). Bycomparing the allele frequencies, we found that,having the A allele accelerated the risk for MO by2.061 times (p=0.0423, OR: 2.061, 95% CI:1.011 -4.200). According to our results, when we compared MO patients and healthy control group, genotypesand alleles frequencies of rs4986791 polymorhismof TLR4 were not found any significant differences(Table 1).
Combined Effects of TLR4 rs4986790 andrs4986791 Polymorphisms
In addition, the associations of TLR4rs4986790and rs4986791 polymorphisms were calculatedby performing logistic regression analysis. TLR4rs4986790 and rs4986791 SNPs demonstratedstatistical significance among the combined genotypes in the patient and control subjects (Table2). We observed a significantly increased risk forMO in the case of the combined AA+CC genotype(p=0.001).
The linkage disequilibrium (LD) was assessedusing Haploview with block graphically identified from the LD intensity expressed in D’. Haplotype associations are shown in Table 3. The ACand AT haplotypes were found to be associatedwith MO (χ2=15.474, p=8.3658E-5 and χ2=15.874,p=6.7687E-5; respectively). The haplotypes ofrs4986790 and rs4986791 have the frequenciesof 94.0% AC, 1.8 % AT, 0.1 % GT, and 4.1 % GC(D’=0.021, LOD=0.03 and r2=0.0).
Chronic low-grade inflammation represents a bridge in the explanation of the mechanism between obesity and obesity-related disorders (14). The studies in obese patients focused on the inflammation-related genetic structural alterations are very rare, therefore, there is still no certain marker being identified. TLRs is one of the NF-KB stimulators, that initiates NF-KB cascade (15). TLR4, for example, has been implicated in several immune and inflammatory disorders (16). Many studies have reported that the process of inflammation could potentially be influenced by the variations within the TLR4genes. In this study, we raised the question whether polymorphisms ofTLR4 gene are associated with the risk of developing MO. TLR4 rs4986790 but not TLR4 rs4986791 are found to be highly associated with MO which correlates with the major data previously published by other groups. Tlr4 loss of function mutation was described as preventing diet-induced obesity and insulin resistance in an animal model study (17). Jermendy has reported that serum cytokine levels change by TLR4 polymorphisms in obese children. Therefore, changes in the TLR4 probably lead to alterations in protein functioning that may contribute to the development of MO (18). Recently, two SNPs in the coding region of TLR4 gene have been identified that encode aspartic acid-to-glycine and threonine-to-isoleucine amino acid substitutions at positions 299 and 399 within the ectodomain; respectively, D299G and T399I (9,19). Asp299Glyn and Thr399Ile polymorphisms can lead to abnormal signaling by altering the ligand binding affinity of TLR4 and create an imbalance between pro and anti-inflammatory cytokine secretions, resulting in the risk of chronic inflammation (20). Asp- 299Gly is reported to be a risk factor for Crohn’s disease, ulcerative colitis (UC) (21), infection of patients with HIV infection (22), infection of patients with Streptococcus pyogenes (23) but has a protective role in atherosclerosis (6), recurrent cystitis (24), obesity-associated hypertension (25) and nonallergic asthma (26). In contrast, some published studies in autoimmune diseases in general (27) and atherothrombosis (28) represent no association of TLRs. Thr399Ile is reported to be more frequent in Europe whereas Asp299Gly is to be in Africa (29). The Asp299Gly polymorphism but not Thr399Ile, blunts TLR4 function, as assessed by cytokine production and NF-KB stimulation in response to LPS (30), whereas the T399I polymorphism has either a significantly reduced or no inhibitory impact on TLR4 functions (8). By this point of view, it is understandable that Asp299Gly but not Thr399Ile is more common in disease association. We found no associations of TLR4 rs4986791 with MO in the presence of HWE inconsistency, which is probably due to control subjects being sampled from different ethnic groups or excess of heterozygotes as a result of an unlikely genotyping error. Interestingly, Thr399Ile is reported as a risk factor in UC (31) and end-stage renal disease patients on peritoneal dialysis (32). Studies mainly deal either with the Asp299Gly or the Thr399Ile polymorphism but neglect the fact that these polymorphisms also exist in a haplotype (Asp299Gly/Thr399Ile) way (33). Individuals having co-segregating polymorphisms of TLR4 have more susceptible to gram-negative infections as they are less responsive to LPS (9, 34). Ferwerda, in 2007, has reported that Asp299Gly/Thr399Ile haplotype shows selective neutrality (29). Moreover, AC and AT haplotypes are both found to be risk factors for MO. According to our combined genotype analysis of TLR4 polymorphisms, Asp299Gly/ Thr399Ile is highly associated with increased MO risk. 1.949As TLR4 was determined as a molecular bridge between inflammation and the innate immune system (35), this association may explain a potential pathophysiological link between obesity and inflammation. All in all, this study shows that TLR4 rs4986790 is associated with susceptibility to MO, implying that these polymorphisms are possible genetic risk factors for the progression of MO. Moreover, the AC and AT haplotypes were found to be associated with MO. TLR4 seems to modulate the immune response in MO although how exactly pathway is in the pathological process including inflammation is to be researched. Major limitations of our study are the small sample size which may have influenced the statistical power of our analysis, HWE inconsistency in one of the investigated polymorphisms, the single-gender preference of both patient and control samples. Different population studies are necessary to understand whether these variants play considerable roles in MO.
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