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Abdelhabib Semlali,1 Mikhlid Almutairi,2 Narasimha Reddy Parine,1 Abdullah Al Amri,1 Jilani P Shaik,1 Abdulrahman Al Naeem,3 Sana Abdulla Ajaj,4 Mahmoud Rouabhia,5 Mohammad Saud Alanazi1 1Department of Biochemistry, 2Zoology Department, College of Science, King Saud University, 3Department of Women’s Imaging, King Fahad Medical City, 4Family Medicine Department, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia; 5Groupe de Recherche en Écologie Buccale, Département de Stomatologie, Faculté de Médecine Dentaire, Université Laval, Québec, QC, Canada Abstract: Breast cancer (BC) is the most common cause of cancer-related deaths among women in the Kingdom of Saudi Arabia. An association between the dysregulation of innate immunity, primarily the deregulation of Toll-like receptors (TLRs), and BC development was described a long time ago. Several studies have reported that BC risk factors appear to be related to the interaction between certain genes and exposure to various environmental factors. Here, we investigated the potential correlation of three TLR2 single-nucleotide polymorphisms (SNPs; rs3804100, rs4696480, and rs3804099) with the development of BC in female patients from Saudi Arabia. We collected 126 blood samples from women with BC and 146 blood samples from healthy women without any clinical signs of BC. The genotypic frequencies of TLR2 polymorphisms were assayed. Our results showed that the genotypic and allelic frequencies of TLR2 did not differ significantly between BC patients and healthy controls. However, the distributions of rs3804100 (1350 T/C) genotypes in BC groups were 1%, 19%, and 80% for CC, CT, and TT, respectively. In the control group, the rs3804100 (1350 T/C) genotype distributions were 3%, 18%, and 79% for CC, CT, and TT, respectively. The SNP rs3804100 homozygous “TT” genotype was not associated with the risk of developing BC in the BC patients compared with controls (odds ratio [OR], 4.5; confidence interval [CI], 0.49–41.02; P=0.145). The TLR2 rs4696480 AA genotype was observed in 23% of BC patients compared to 18% of control individuals, the AT genotype was seen in 40% of BC patients and 46% of control individuals, and the TT genotype was observed in 37% of BC patients and 36% of normal controls. Our results did not show any difference in genotypic frequency between BC patients and normal controls for the TLR2 rs3804099 SNP; however, the (C) phenotypic frequency was 49% in BC patients and 53% in controls. The (T) phenotypic frequency was 51% and 47% in BC patients and normal patients, respectively. These findings indicate that there is no association between the TLR2 polymorphisms tested and BC susceptibility in the female population from the Kingdom of Saudi Arabia. We suggest using other TLR2 SNPs to investigate the possible relationship between innate immunity deregulation by disruption of TLR2 and potential BC development. Keywords: breast cancer, genotyping, polymorphism, Toll-like receptors

Muhammad Kohailan,1 Mohammad Alanazi,1 Mahmoud Rouabhia,2 Abdullah Al Amri,1 Narasimha Reddy Parine,1 Abdelhabib Semlali1 1Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 2Groupe de Recherche en Écologie Buccale, Département de Stomatologie, Faculté de Médecine Dentaire, Université Laval, Québec, QC, Canada Abstract: Defects in the innate immune system, particularly in Toll-like receptors (TLRs), have been reported in several cigarette smoke-promoted diseases. The aim of this study was to examine the impact of tobacco smoke on allelic frequencies of TLR4 single-nucleotide polymorphisms (SNPs) and to compare the genotypic distribution of these SNPs in a Saudi Arabian population with that in previously studied populations. DNA was extracted from 303 saliva samples collected from smokers and nonsmokers. Two transitional SNPs in the promoter region of TLR4 were selected, rs2770150 (T/C) and rs10759931 (G/A). Genotype frequencies were determined using quantitative polymerase chain reaction. Our results showed a slight effect of smoking on the distribution of rs2770150 and rs10759931. However, the differences were not significant. Thus, we conclude that the SNPs selected for this study were independent of smoking and may not be related to smoking-induced diseases. Keywords: Toll-like receptor 4, polymorphism, genetic variation, smoking

Abdelhabib Semlali,1 Maroua Jalouli,2 Narasimha Reddy Parine,1 Abdullah Al Amri,1 Maha Arafah,3 Abdulrahman Al Naeem,4 Sanaa Abdullah Ajaj,5 Mahmoud Rouabhia,6 Mohammad Saud Alanazi1 1Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 2Centre de recherche du CHU de Québec, L’Hôtel-Dieu de Québec, Department of Molecular Biology, Medical Biochemistry and Pathology, Université Laval, Quebec, QC, Canada; 3College of Medicine, King Saud University, 4Department of Women’s Imaging, King Fahad Medical City, 5Department of Family Medicine, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 6Groupe de Recherche en Écologie Buccale, Department of Stomatology, Faculty of Dentistry, Université Laval, Quebec, QC, Canada Abstract: The aim of this study was to investigate the association of the common polymorphisms of Toll-like receptor 4 (TLR-4) with breast cancer development in the Saudi Arabian population. Four TLR-4 polymorphisms (rs2770150, rs10759931, rs10759932, and rs4986790) were studied using 127 breast cancer patients and 117 controls. Relative expression of TLR-4 protein in the breast tumor and the matched normal breast tissues was determined in a large cohort of 70 clinical breast samples in a tissue micro-array format by immunohistochemistry using a specific anti-TLR-4 antibody. Our results demonstrated an increase in TLR-4 expression in estrogen receptor (ER)-, postmenopausal breast cancer patients compared to normal. We also demonstrated that the G allele of single-nucleotide polymorphism rs10759931 was found to be significantly higher in frequency among patients (36.3%) compared to the control group (26.7%), suggesting that this polymorphism is strongly associated with the development of breast cancer in this ethnic population. In addition, the TLR-4 polymorphism rs2770150 was shown to be highly correlated with breast cancer in patients over 48 years of age. The TLR-4 polymorphism rs4986790 was also found to be associated with this malignancy in the ER- patient groups. Our results suggested firstly that the variation in TLR-4 gene expression may influence breast cancer development and secondly a closely linked association between TLR-4 gene polymorphism and ER status. Our study provides support for a better understanding of the implication of TLR-4 polymorphism in breast tumorigenesis and for its eventual use as a cancer biomarker. Keywords: Toll-like receptor 4, single-nucleotide polymorphisms, breast cancer, Saudi population, innate immunity, estrogen receptor

Muhammad Kohailan,1 Mohammad Alanazi,1 Mahmoud Rouabhia,2 Abdullah Alamri,1 Narasimha Reddy Parine,1 Abdullah Alhadheq,1 Santhosh Basavarajappa,3 Abdul Aziz Abdullah Al-Kheraif,3 Abdelhabib Semlali1 1Genome Research Chair, Department of Biochemistry, Collegeof Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 2Département de Stomatologie, Faculté de Médecine Dentaire, Groupe de Recherche en Écologie Buccale, Université Laval, Québec City, QC, Canada; 3Dental Biomaterial Research Chair, Department of Dental Health, College of Applied Medical Sciences, King Saud University, Riyadh, Kingdomof SaudiArabia Background: Cigarette smoking is a major risk factor for lung cancer, asthma, and oral cancer, and is central to the altered innate immune responsiveness to infection. Many hypotheses have provided evidence that cigarette smoking induces more genetic changes in genes involved in the development of many cigarette-related diseases. This alteration may be from single-nucleotide polymorphisms (SNPs) in innate immunity genes, especially the toll-like receptors (TLRs).Objective: In this study, the genotype frequencies of TLR2 and TLR6 in smoking and nonsmoking population were examined.Methods: Saliva samples were collected from 177 smokers and 126 nonsmokers. The SNPs used were rs3804100 (1350 T/C, Ser450Ser) and rs3804099 (597 T/C, Asn199Asn) for TLR2 and rs3796508 (979 G/A, Val327Met) and rs5743810 (745 T/C, Ser249Pro) for TLR6.Results: Results showed that TLR2 rs3804100 has a significant effect in short-term smokers (OR =2.63; P=0.04), and this effect is not observed in long-term smokers (>5 years of smoking). Therefore, this early mutation may be repaired by the DNA repair system. For TLR2 rs3804099, the variation in genotype frequencies between the smokers and control patients was due to a late mutation, and its protective role appears only in long-term smokers (OR =0.40, P=0.018). In TLR6 rs5743810, the TT genotype is significantly higher in smokers than in nonsmokers (OR =6.90). The effect of this SNP is observed in long-term smokers, regardless of the smoking regime per day.Conclusion: TLR2 (rs3804100 and rs3804099) and TLR6 (rs5743810) can be used as a potential index in the diagnosis and prevention of more diseases caused by smoking. Keywords: polymorphism, toll-like receptor, genotyping, smoking, TLR2, TLR6