ORIGINAL ARTICLE Single-nucleotide Polymorphisms in Genes Encoding Toll-like Receptor -2, -3, -4, and -9 in a CaseeControl Study with Bladder Cancer Susceptibility in a North Indian Population Vibha Singh, a Neena Srivastava, b Rakesh Kapoor, a and Rama Devi Mittal a a Department of Urology and Renal Transplantation, Sanjay Gandhi Post Graduate Institute of Medical Science, Lucknow, Uttar Pradesh, India b Department of Physiology, CSM Medical University, Lucknow, Uttar Pradesh, India Received for publication May 8, 2012; accepted October 12, 2012 (ARCMED-D-12-00242). Background and Aims. Impairment of the immune system may contribute to the risk for having cancer as Toll-like receptors are important for innate immunity. We examined the association between candidate disease-susceptibility polymorphisms in the single nucle- otide polymorphism (SNPs) like TLR2 (196 to174del), TLR3 (C1377T), TLR4 (Thr399Ile) and TLR9 (G2848A) genes in patients with bladder cancer in a North Indian population. Methods. SNPs were comprised of TLR2 (196 to 174 Del), TLR3(C1377T), TLR4 (Thr399Ile) and TLR9 (G2848A) genes. Allelic and genotypic frequencies of these TLRs SNP from histopathologically confirmed patients of bladder cancer (n 5 200) and unre- lated healthy controls of similar ethnicity (n 5 200) were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Results. In TLR2 I/D gene polymorphism, the combination of IDþDD showed a signifi- cant 3-fold increased risk ( p 5 0.001). TLR2 with heterozygous genotype ID showed a 3- fold risk and combination of heterozygous and variant genotype (IDþDD) also showed a 5-fold risk with tumor stage/grade of patients with bladder cancer. The other genotypes of TLR3, 4 and 9 did not exhibit any significant association with bladder cancer risk. Conclusions. Our results suggested the involvement of TLR2 (196 to174 del) in bladder cancer susceptibility; however, TLR3, 4 and 9 genes were not associated with risk of bladder cancer, implicating that polymorphisms in these tested TLRs genes are not likely to be associated with increased risk for developing bladder cancer. Functional studies in ethnically diverse populations may provide a more comprehensive involvement of innate immunity in identifying the disease-associated variants for the etiology of bladder cancer. Ó 2013 IMSS. Published by Elsevier Inc. Key Words: TLR gene polymorphism, Bladder cancer, bacillus Calmette-Guerin, PCR-RFLP. Introduction Bladder cancer is an increasingly important international public health problem. As a multifactorial disease, both environmental and genetic factors are involved in its devel- opment and progression in bladder cancer is nearly three to four times more common in males than in females (1,2). The incidence in India is comparatively low (3.2 in males and 0.7 in females/100,000 persons) (3). Risk factors for the development of bladder cancer can be classified into three subsets: genetic and molecular abnormalities, chemi- cal or environmental exposures, and chronic irritation (4). TLR genes are critical in linking innate and acquired immu- nity and in serving as detectors of infectious pathogens and cancer debris (5e7). TLRs belong to the family of pattern-recognition recep- tors (PRRs), which are expressed, among others, on antigen-presenting cells like dendritic cells or T-cells. After induction through pathogen-associated molecules, TLRs Address reprint requests to: Dr. Rama Devi Mittal, Professor (Biochemistry), Department of Urology, SGPGIMS, Raebareli Road, Lucknow 226014, India; Phone: 091-522-2668004-8, ext. 4323; FAX: 091-522-2668017; E-mail: ramamittal@gmail.com 0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2012.10.008 Archives of Medical Research 44 (2013) 54e61