rs401502 and rs11575934 Polymorphisms of the IL-12 Receptor Beta 1 Gene are Protective Against Colorectal Carcinogenesis.

Background: Colorectal cancer (CRC) is a major public health problem worldwide and in Tunisia. It ranks among the main cancers in terms of incidence and cancer-related cause of death. Its pathogenesis is currently considered to be multifactorial involving genetic and environmental factors. Recent studies have suggested that the gene encoding the ß1 subunit of the IL-12 receptor, an important pro-inflammatory cytokine of the anti-tumor response, could be involved in the susceptibility to inherited CRC. Hence, it would be interesting to study the role of single nucleotide polymorphisms (SNPs) within the IL-12RB1 gene (rs401502 and rs11575934) in CRC susceptibility. Aim: Our purpose was to assess whether genetic variants IL-12RB1 +1196G/C (rs401502) and IL-12RB1 +705A/G (rs11575934) within the IL-12RB1 gene are associated with the sporadic CRC risk. Methods: A total of 110 Tunisian patients with sporadic CRC and 141 healthy control subjects were included in this study. Genotyping was performed by high-resolution melting (HRM) analysis. All results were confirmed by direct DNA sequencing or PCR-RFLP methods. Later, the allele frequencies and genotype distribution were established and compared between the control group and CRC patients. Results: The obtained results showed that the two target SNPs were in Hardy-Weinberg equilibrium (HWE) in both patients and controls. Minor allele frequencies of rs401502 SNP were 16.4% in CRC cases and 23.8% in controls. Mutant allele of rs11575934 SNP was present with 21.4% in CRC patients and 29.8% in control group. An association study showed a significant association of two target polymorphisms with CRC, according to the dominant genetic model with OR = 0.577, 95% CI = [0.343 to 0.972], p = 0.038 and OR = 0.547, 95% CI = [0.328 to 0.911], p = 0.02, respectively. Conclusion: In this study, we found, for the first time, a potential protective effect of two SNPs in the IL-12RB1 gene, namely rs401502 and rs11575934, in sporadic colorectal cancer in Tunisians.

Association of HLA-G 3'UTR polymorphisms and haplotypes with colorectal cancer susceptibility and prognosis.

Human leukocyte antigen (HLA)-G has been considered as an immune modulator in several types of cancers. Its genetic polymorphisms may potentially affect the risk of developing colorectal cancer (CRC). The overall purpose of this study was to analyze the implication of HLA-G 3'untranslated region (3'UTR) polymorphisms particularly 14 pb insertion/deletion (Ins/Del; rs371194629) and + 3142C/G (rs1063320) in CRC susceptibility and progression. A comparative analysis between patients (N = 233) and controls (N = 241) demonstrated that Del allele (Odds Ratios (OR) = 1.41, 95% CI = 1.091-1.819, p = 0.008), the homozygous Del/Del genotype (OR = 1.80, 95% CI = 1.205-2.664, p = 0.003) and the codominant C/G genotype (OR = 1.59, 95% CI = 1.106-2.272, p = 0.013) were associated to CRC risk. As expected, the DelG haplotype was associated with CRC susceptibility (OR = 1.47, 95% CI = 1.068-2.012, p = 0.018). Assessment of patients' survival by Kaplan-Meier analysis indicated that the Del allele and the homozygous Del/Del genotype were associated with reduced event free survival (EFS) (Respectively, p = 0.009 and p = 0.05). Interestingly, the Del allele and the homozygous Del/Del genotype have been revealed as independent prognostic factors for poor EFS in patients with CRC. Additionally, haplotypes analysis revealed that DelG haplotype was linked with significant increase in CRC risk (log-rank; EFS: p = 0.02). Inversely, the InsC haplotype was associated with a significant reduced CRC risk (log-rank; Overall survival (OS): p < 10-6; EFS: p = 0.01). Multivariate Cox regression analysis revealed that the InsC haplotype was independently associated with significantly longer EFS (p = 0.021, HR = 0.636, 95% CI = 0.433-0.935). These findings support the implication of HLA-G polymorphisms in the CRC susceptibility suggesting HLA-G as a potent prognostic and predictive indicator for CRC. Insight into mechanisms underlying HLA-G polymorphisms could allow for the development of targeted care for CRC patients according to their genetic profile.

Rapid high-resolution melting method to identify human leukocyte antigen-G (HLA-G) 3' untranslated region polymorphism +3142C/G (rs1063320).

BACKGROUND: HLA-G is a non-classical class I gene of the human Major Histocompatibility encoding molecules with immune-modulatory properties. Expression of HLA-G is being largely studied in pathological conditions, such as tumors, viral infections, inflammation, and autoimmune diseases, grafted tissues, among others. HLA-G +3142C/G (rs1063320: dbSNP database) polymorphism is located in 3' UTR of HAL-G and plays a key role in determining the magnitude of gene and protein expression. The detection of HLA-G +3142C/G polymorphism in the most published report is done through polymerase chain reaction followed by enzymatic digestion. Therefore, it is so interesting to develop a rapid and sensitive assay to genotype HLA-G +3142C/G polymorphism. High-resolution melt analysis (HRM) is a technology that is based on the analysis of the melting profile of PCR products through gradual temperature increase. The aim of this work is to apply high-resolution melt method for genotyping the HLA-G +3142C/G polymorphism. METHODS: DNA from 118 individuals was extracted from whole blood with QIAamp® DNA blood mini kit (Qiagen, Germany). Primer couple was designed using Primer 3 online tools so as to have only one SNP in the target sequence for high HRM efficiency. Positive Controls were identified using DNA sequencing and used as reference when assigning genotypes for trial samples. RESULTS: We were able to recognize the three genotypes with similar accuracy than DNA sequencing using high resolution melting method. Hardy-Weinberg equilibrium test shows that our population is in equilibrium for the studied SNP. Genotypes frequencies of +3142C/G polymorphism in Tunisian general population are 0.475 for heterozygote G/C, 0.186 for homozygote G/G and 0.339 for homozygote C/C. CONCLUSION: HRM is a cost-effective method suitable for SNP genotyping.

Association of CCR5Δ32 Deletion and Human Cytomegalovirus Infection With Colorectal Cancer in Tunisia.

Background and objectives: Human cytomegalovirus (HCMV) and genetic polymorphisms of the chemokine receptor 5 have been suggested as factors associated with the progression of colorectal cancer (CRC). The aim of the study was to evaluate the associations of both CCR5Δ32 genetic deletion and/or HCMV virus infection with CRC in Tunisia. MATERIALS AND METHODS: The association between HCMV and CRC was validated by Nested PCR technology performed for HCMV and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. Experiments were carried out on 40 tumor and 35 peri-tumor tissues, 100 blood from CRC patients and on 140 blood samples from healthy subjects and finaly serum samples of 80 patients with CRC and 100 healthy individuals. A conventional PCR has been optimized for the detection of CCR5Δ32 in100 CRC patients and 100 healthy subjects. RESULTS: Our results show that HCMV is significantly active in 93% of patients compared to 60% in controls (p < 0.0001, OR = 8.85, 95% CI: 3.82 -20.50). Compared to the healthy controls, the titers of IgG and IgM antiCMV antibodies in CRC patients were significantly higher than in healthy subjects (p value < 0,0001 for IgG and IgM). Statistical analysis revealed a lack of association between CCR5Δ32 mutation and colorectal cancer (p = 0.788, OR = 1.265, 95% CI: 0.228-7.011). CONCLUSION: our data confirmed that the HCMV infection was related to the development of CRC and that CRC cells may be infected more favorably by HCMV. Given the importance of the CCR5 in inflammation and therefore CRC progression, further studies still needed to evaluate CCR5 role as a potential candidate gene for CRC susceptibility under other polymorphisms.