Tetra-primer Amplification Refractory Mutation System-polymerase Chain Reaction Research Articles

Association between Interleukin-6 Gene Polymorphism (rs1800795 and rs1800796) and Type 2 Diabetes Mellitus in a Ghanaian Population: A Case-Control Study in the Ho Municipality.

There is no conclusive evidence on the association between interleukin- (IL-) 6 gene polymorphism and type 2 diabetes mellitus (type 2 DM). Thus, this study is aimed at evaluating the role of rs1800795 and rs1800796 polymorphisms in the pathogenesis of type 2 DM among Ghanaians in the Ho Municipality. We recruited into this hospital-based case-control study 174 patients with type 2 DM (75 DM alone and 99 with DM+HTN) and 149 healthy individuals between 2018 and 2020. Demographic, lifestyle, clinical, anthropometric, and haemodynamic variables were obtained. Fasting blood samples were collected for haematological, biochemical, and molecular analyses. Genomic DNA was extracted, amplified using Tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) technique, and genotyped for IL-6 gene polymorphism. Logistic regression analyses were performed to assess the association between IL-6 gene polymorphism and type 2 DM. The minor allele frequency (MAF) of the rs1800795 and rs1800796 polymorphisms was higher in DM alone (57.5%, 62.0%) and DM with HTN groups (58.3%, 65.3%) than controls (33.1%, 20.0%). Carriers of the rs1800795GC genotype (aOR = 2.35, 95% CI: 1.13-4.90, p = 0.022) and mutant C allele (aOR = 2.41, 95% CI: 1.16-5.00, p = 0.019) as well as those who carried the rs1800796GC (aOR = 8.67, 95% CI: 4.00-18.90, p < 0.001) and mutant C allele (aOR = 8.84, 95% CI: 4.06-19.26, p = 0.001) had increased odds of type 2 DM. For both polymorphisms, carriers of the GC genotype had comparable levels of insulin, HOMA-IR, and fasting blood glucose (FBG) with those who carried the GG genotype. IL-6 levels were higher among carriers of the rs1800796GC variant compared to carriers of the rs1800796GG variant (p = 0.023). The rs1800796 polymorphism, dietary sugar intake, and exercise status, respectively, explained approximately 3% (p = 0.046), 3.2% (p = 0.038, coefficient = 1.456), and 6.2% (p = 0.004, coefficient = -2.754) of the variability in IL-6 levels, suggesting weak effect sizes. The GC genotype and mutant C allele are risk genetic variants associated with type 2 DM in the Ghanaian population. The rs1800796 GC variant, dietary sugar intake, and exercise status appear to contribute significantly to the variations in circulating IL-6 levels but with weak effect sizes.

Rapid Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Protocol for Detection of Y132F Mutation in Fluconazole Resistant Candida parapsilosis.

There is an emerging fluconazole resistance in Candida parapsilosis in recent years. The leading mechanism causing azole resistance in C. parapsilosis is the Y132F codon alteration in the ERG11 gene which encodes the target enzyme of azole drugs. In this study, we evaluated the sensitivity, compatibility, and specificity of a novel tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method for rapid detection of the Y132F mutation in fluconazole nonsusceptible C. parapsilosis. Antifungal susceptibility tests for detection of fluconazole resistance were performed by broth microdilution according to the CLSI guidelines. All susceptible and nonsusceptible C. parapsilosis isolates were analyzed for ERG11 mutations with Sanger sequencing. T-ARMS-PCR was fully concordant with the Sanger sequencing (100% of sensitivity and specificity) for detection of Y132F mutations. T-ARMS-PCR method could be a rapid, simple, accurate, and economical assay in the early detection of the most common cause of fluconazole resistance in C. parapsilosis isolates. In routine laboratories with high C. parapsilosis isolation rates, performing the T-ARMS-PCR for early detection of the most common reason of fluconazole resistance in C. parapsilosis, could be a life-saving approach for directing antifungal therapy before obtaining the definitive antifungal susceptibility tests results.

The association between MMP-13 rs478927 gene polymorphism and dental caries susceptibility in children with mixed dentition from Birjand, Iran: A case-control study.

Gene polymorphisms are responsible for at least part of the variation in caries susceptibility despite similar environmental factors. Genes involved in enamel formation like matrix metalloproteinase-13 (MMP-13) may participate in caries process. The aim was to investigate the association between MMP-13 rs478927 polymorphism and caries susceptibility in 6-years-old children from Birjand, Iran. Six-years old children from Birjand, Iran, participated in this study. The total decayed, missing, and filled teeth were calculated and defined as caries index (CI). Based on this CI, two groups of high-caries (case) and low-caries (control) were taken into account. Saliva samples were collected and DNA was extracted. The allele and genotypes of MMP-13 rs478927 polymorphism were determined by tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. p Value was significant if p < 0.05. Three hundred sixty-seven children consisted of 186 low-caries children and 181 high-caries children were included in this study. The mean CI was 6.02 ± 0.81. There was no significant association between high and low-caries groups based on socioeconomic status, eating sweet snacks, parents' susceptibility to dental caries, duration of breastfeeding, and the brushing habit (p > 0.05). There wasn't any significant association between genotype distribution of MMP-13 rs478927 polymorphism and CI groups (p = 0.924). This polymorphism was associated with increased caries susceptibility under all genetic models but this effect was not significant (p > 0.05). The MMP-13 rs478927 gene polymorphism was not significantly associated with dental caries susceptibility in Birjandi children with mixed dentition. It is recommended to conduct studies on children of different dentitions to better understand the role of this polymorphism on caries susceptibility in primary and permanent teeth of children.

The obesity associated FTO gene polymorphism and the risk of preeclampsia in Iranian women: A case–control study

ABSTRACT Background Preeclampsia (PE) is one of the leading disorders in pregnant women with maternal and fetal complications. Obesity is considered an important risk factor for the development of PE. Genetic variations in fat mass and obesity associated (FTO) gene may play a role in the development of PE. This study aimed to investigate the possible association between FTO gene rs9939609 and PE risk in a sample of Iranian pregnant women. Material and methods In this case–control study, 312 pregnant women were included, including 128 with PE and 184 without PE. Demographic data and blood samples were obtained from all individuals. The genotyping of rs9939609 polymorphisms was performed by the tetra‐primer amplification refractory mutation system‐polymerase chain reaction (TP‐ARMS‐PCR) method, and the results of TP‐ARMS‐PCR were confirmed using DNA sequencing. Results The genotype frequency was 50%, 47.7%, and 2.3% in pregnant patients and 37%, 47.8%, and 15.2% in healthy controls for TT, AT, and AA, respectively. The risk of PE was significantly reduced in the pregnant women having the AA genotype. Conclusion Based on the results of the present study, rs9939609 polymorphism in the FTO gene may play a protective role against PE. However, further studies are warranted.

Prognostic significance of the genetic variant of lymphotoxin alpha (p.Thr60Asn) in egyptian patients with advanced hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide in terms of mortality, and susceptibility is attributed to genetic, lifestyle, and environmental factors. Lymphotoxin alpha (LTA) has a crucial role in communicating the lymphocytes with stromal cells and provoking cytotoxic effects on the cancer cells. There are no reports on the contribution of the LTA (c.179 C>A; p.Thr60Asn; rs1041981) gene polymorphism to HCC susceptibility. The main aim of this study is to investigate the association of LTA (c.179 C>A; p.Thr60Asn; rs1041981) variant with the HCC risk in the Egyptian population.MethodsThis case-control study included 317 participants (111 HCC patients, and 206 healthy controls). The LTA (c.179 C>A; p.Thr60Asn; rs1041981) polymorphism was assessed by tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) technique.ResultsThe frequencies of the dominant and recessive models (CA + AA; AA) of the LTA (c.179 C>A; p.Thr60Asn; rs1041981) variant were statistically significant among HCC patients in comparison to controls (p = 0.01; p = 0.007; respectively). The A-allele of LTA (c.179 C>A; p.Thr60Asn; rs1041981) variant was statistically significant in HCC patients in comparison to controls (p ˂ 0.001).ConclusionThe LTA (c.179 C>A; p.Thr60Asn; rs1041981) polymorphism was independently associated with an increased risk for hepatocellular carcinoma in the Egyptian population.

Association of GSTTI, M1 and Polymorphism in GSTPI with Chronic Periodontal Disease in a Pakistani Population.

Chronic periodontal disease (CP) is a multifactorial infectious and inflammatory disease that occurs due to the challenge between the immune response of the host and specific periodontal bacteria, and that can lead to tooth loss due to damage inflicted to the supporting tissue. The current study investigates the genotypes of the GSTM1 and GSTT1 genes, along with the allelic frequency of the single nucleotide polymorphism [SNP; rs1695] in the GSTP1 gene and correlates them individually or in various combinations with the incidence of CP. A total of 203 clinically confirmed CP patients and 201 control subjects were enrolled from Multan and Dera Ghazi Khan Districts in Pakistan from April to July 2022. Multiplex Polymerase Chain Reaction (PCR) and tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) approaches were applied to determine the genotypes of the studied GSTs. The association of rs1695 in GSTP1 with CP was studied both individually and in various combinations with GSTM1 and T1. The absence of GSTM1, the presence of GSTT1 and the presence of the mutant allele (G) at rs1695 in GSTP1 were found to be significantly associated with CP. Patients aged between 10 and 30 years were more affected by CP. Our results indicate that the genotypes of the analyzed GSTs affect the levels of protection from oxidative stress and may therefore influence the disease progression in CP.

Src homology 2-B adapter protein 3 C784T and Methylenetetrahydrofolate reductase C677T Polymorphisms and Inflammation Markers in ST-segment Elevation Myocardial Infarction Patients.

Neutrophil-lymphocyte (NLR), platelet-lymphocyte (PLR), eosinophil-lymphocyte (ELR), and monocyte-lymphocyte (MLR) ratios are systemic inflammatory markers related to myocardial infarction. The aim of this study is to investigate the association of Src homology 2-B adapter protein 3 (SH2B3) C784T and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms (SNP) with systemic inflammatory markers and the severity of coronary artery disease (CAD) in 150 ST-elevation myocardial infarction (STEMI) patients. Single nucleotide polymorphisms were genotyped using the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. The inflammatory markers were calculated. An interventional cardiologist blinded to other data assessed the SYNTAX (SX) Score. Eosinophil and platelet counts were significantly higher in SH2B3 variants than in the wild type. Additionally, SH2B3 variants had significantly higher ELR than the wild type (.12 ± .19 vs .25 ± .34, p = .018). NLR, PLR, ELR, and MLR were considerably higher in MTHFR variants than in the wild type (p < .05). The SX score was significantly higher in both SH2B3 C784T (21.24 ± 8.90 vs 15.29 ± 9.40, p = .00) and MTHFR C677T (20.34 ± 10.21 vs 16.08 ± 8.39, p = .00) variants when compared with wild type. In conclusion, these polymorphisms are associated with several markers of systemic inflammation as well as the severity of CAD.

Association of ANRIL Gene Single-Nucleotide Polymorphisms With Allergic Rhinitis in Kurdish Population From Kermanshah, Iran.

Allergic rhinitis (AR) is the most common inflammatory disorder of the upper airway caused by aberrant immune responses to allergens in genetically predisposed individuals. Recently, the long noncoding RNA (lncRNA) antisense noncoding RNA in the INK4 locus (ANRIL) has been identified as a novel genetic factor associated with increased AR risk. This study aimed to evaluate the potential correlation of ANRIL gene single nucleotide polymorphisms (SNPs) with AR risk in the Kurdish population of Kermanshah, Iran. In this case-control study, 130 AR patients and 130 healthy controls were recruited to genotype for two SNPs of the ANRIL gene (rs1333048 and rs10757278) using the Tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) method. Our results showed no significant difference for the alleles and genotypes frequency distribution of lncRNA ANRIL SNPs (rs1333048 and rs10757278) between AR patients and healthy controls (p > 0.05). Additionally, the dominant, additive and recessive genetic models of both SNPs were not associated with altered susceptibility to AR risk (p > 0.05). The results demonstrated that the ANRIL gene rs1333048 and rs10757278 polymorphisms might not be associated with susceptibility to AR in the Kurdish population of Kermanshah, Iran.

Analysis of bone morphogenetic protein-15 gene polymorphism in Konkan Kanyal Goats

Konkan Kanyal goats are highly prolific and can withstand high rainfall, hot and humid conditions. Increasing prolificacy offers excellent potential for improving reproduction rate and production efficiency. The fecundity gene BMP-15 is responsible for increasing the ovulation rate in small ruminants. Therefore, the study was aimed to investigate the BMP-15 gene polymorphism and its association with litter size in Konkan Kanyal goats. The genomic DNA was isolated from 50 blood samples of Konkan Kanyal goats. Tetra primer amplification refractory mutation system - polymerase chain reaction (TARMS-PCR) was used to detect the mutation of BMP-15 loci (FecXG, FecXH, FecXI, FecXL, FecXB). The TARMS-PCR successfully amplified wild and control type amplicons at FecXH (203 bp and 395 bp), FecXI (261bp and 418bp), FecXL (252bp and 398bp) loci while, only control type amplicon at FecXG (158bp) and FecXB (308bp) loci of BMP-15 gene. The study revealed monomorphic nature of BMP-15 gene at FecXH, FecXI, FecXL loci while, FecXB and FecXG loci could not be identified.

The risk of relapse in breast cancer patients is associated with MMP-9 gene polymorphism: a prospective study in a sample of the Iranian population

We investigated the association of MMP-9 rs1056628 and rs17576 polymorphisms with breast cancer (BC) relapse in a cohort of prospectively observed BC patients. The polymorphisms were genotyped in 200 BC subjects with(case) and without(control) relapse by Tetra-primer Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) method. A significant association was found between the rs1056628C allele and increased risk of BC relapse (OR = 1.8, P = 0.006). Also, rs17576 allele and genotypes were shown protective effects against BC relapse. Increased risk of relapse was observed in haplotype CG (OR = 2.1, P = 0.001). Our results suggest for the first time that rs1056628 and rs17576 polymorphisms may influence BC relapse.

Visual Detection of ACE I/D Polymorphism Using T-ARMS-PCR Combined with a Lateral Flow Assay and Its Clinical Application.

Insertions/deletions (indels) variations have been recognized as a promising marker for the development of various diseases. However, methods used for the genotyping of indels in studies were tedious, complicated, and required sophisticated or expensive instruments, as well as complex data analysis, which makes it difficult to meet the demand of point of care testing. Herein, we presented a fast and accurate biosensor (T-ARMS-PCR-LFA) by the combination of tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) and GoldMag lateral flow assay (LFA) for visual genotyping of ACE I/D polymorphism. ACE I/D can be distinguished by employing four primers in one PCR reaction, and genotyping results were presented by the visual inspection of colors on the nitrocellulose membrane of LFA strips within 5 min. And 50 of the human genomic DNA samples were used for the detection of ACE I/D to further validate the accuracy of the T-ARMS-PCR-LFA system. As a demonstration, we showed that ACE I/D could be genotyped using a low amount of DNA sample (25 ng) with an accuracy of 100%, without complicated operation steps and data analysis, which is better than that of the conventional method (agarose gel electrophoresis analysis after common PCR). In conclusion, the biosensor is highly applicable for genotyping specific large indel variants in clinical practices, which enables rapid clinical decision-making, improves the management of disease diagnosis, and facilitates personalized medicine.

Detection of Tri-allelic Single Nucleotide Polymorphisms of ABCB1 and CRP Genes by Penta-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction.

Objective: The standard methods for tri-allelic single nucleotide polymorphism (SNP) genotyping require special equipment and are costly to perform. The aim of this study was to establish a fast, simple, and low-cost method to differentiate among tri-allelic SNPs in general laboratories. Methods: Based on the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) typing of bi-allelic SNPs, we developed a penta-primer amplification refractory mutation system-polymerase chain reaction (P-ARMS-PCR) method characterize tri-allelic SNPs. The two most studied tri-allelic SNPs, rs2032582 and rs3091244, were genotyped using P-ARMS-PCR in 110 volunteers, and the results were verified by direct DNA sequencing. Results: For rs2032582, there were 20 samples (18.18%) with the GG genotype, 3 (2.73%) with the AA genotype, 24 (21.82%) with the TT genotype, 43 (39.09%) with the GT genotype, 11 (10.00%) with the AG genotype, and 9 (8.18%) with the AT genotype. For rs3091244, there were 67 samples (60.91%) with the CC genotype, 1 (0.91%) with the AA genotype, 8 (7.27%) with the CT genotype, 31 (28.18%) with the CA genotype, and 3 (2.73%) with the AT genotype. The genotypic distributions of rs2032582 (p = 0.482) and rs3091244 (p = 0.492) were in Hardy-Weinberg equilibrium. The DNA sequencing results were entirely consistent with the results of P-ARMS-PCR. Conclusion: P-ARMS-PCR is an accurate, rapid, simple, and low-cost characterization method for tri-allelic SNP genotyping of rs2032582 and rs3091244.

Autophagy ATG16L1rs2241880 impacts the colorectal cancer risk: A case-control study.

BackgroundDespite many efforts to discover the important role of the autophagy process in the pathogenesis of colorectal cancer (CRC), the exact involved molecular mechanism still remains to be elucidated. Recently, a limited number of studies have been employed to discover the impact of autophagy genes’ variants on the development and progression of CRC. Here, we evaluated the association between two single‐nucleotide polymorphisms (SNPs) in the main components of the autophagy genes, ATG16L1 rs2241880, and ATG5 rs1475270, and the CRC risk in an Iranian population.MethodsDuring this investigation, a total of 369 subjects, including 179 CRC patients and 190 non‐cancer controls have been genotyped using Tetra‐primer amplification refractory mutation system‐polymerase chain reaction (TP‐ARMS‐PCR) method.ResultThe results demonstrated that the T allele of the ATG16L1 rs2241880 was significantly associated with the increased risk of CRC in the studied population (OR 1.64, 95% CI: 1.21–2.22, p = 0.0015). Moreover, ATG16L1 rs2241880 TT genotype increased the susceptibility to CRC (OR 3.31, 95% CI: 1.64–6.69, p = 0.0008). Furthermore, a significant association was observed under the recessive and dominant inheritance models (p = 0.0015 and p = 0.017, respectively). No statistically significant differences were found in the ATG5 rs1475270 alleles and genotypes between the cases and controls.ConclusionThe results of the present study may be helpful concerning the risk stratification in CRC patients based on the genotyping approach of autophagy pathways and emphasize the need for further investigations among different populations and ethnicities to refine our conclusions.

Association between TBXT rs2305089 polymorphism and chordoma in Iranian patients identified by a developed T-ARMS-PCR assay.

BackgroundChordoma is a locally aggressive bone tumor with a high capability of recurrence. Because chordoma often occurs at critical locations next to neurovascular structures, there is an urgent need to introduce validated biomarkers. T‐box transcription factor T (TBXT; OMIM: 601397) plays an important role in the pathogenesis and survival of chordoma cells.MethodsHerein, we aimed to show whether rs2305089 polymorphism is correlated with chordoma in the Iranian population. In order to detect rs2305089, tetra‐primer amplification refractory mutation system‐polymerase chain reaction (T‐ARMS‐PCR) was used. In total, 19 chordoma patients and 108 normal healthy individuals were recruited and screened using T‐ARMS‐PCR. The results were subsequently validated by Sanger sequencing.ResultsThe genotype distributions and allele frequencies were significantly different among the patient and healthy groups (p‐value <0.05). The A allele of rs2305089 showed a significant positive association with chordoma risk (p‐value <0.05). DNA sequencing verified the T‐ARMS‐PCR results as well. This study demonstrated the association between TBXT rs2305089 and chordoma in an Iranian population using a simple, accurate, and cost‐effective T‐ARMS‐PCR assay.ConclusionsOur results were in line with those of previous studies showing that TBXT rs2305089 is associated with chordoma development. We also developed an efficient T‐ARMS‐PCR assay to determine the genotype of rs2305089.

Association between risk of brucellosis and genetic variations in MicroRNA-146a

BackgroundSingle nucleotide polymorphisms (SNPs) are the most common types of DNA changes in the human genome that leading to phenotypic differences in humans. MicroRNAs (miRNAs) are usually affected by various bacterial infections, and they are involved in controlling the immune responses. MicroRNA-146a (miR-146a) plays an essential role in the development of infectious and inflammatory diseases. The aim of the present study was to investigate the association between risk of brucellosis and genetic variations in miR-146a.MethodsThis case–control study was conducted on 108 Brucellosis patients and 108 healthy controls. We genotyped two SNPs (rs2910164 and rs57095329) of the miR-146a using tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) and restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) methods.ResultsThe rs2910164 SNP was significantly associated with brucellosis in co-dominant [OR = 4.27, 95% CI = (2.35–7.79, P = 0.001] and dominant [OR = 3.52, 95% CI = (1.97–6.30, P = 0.001] models. Co-dominant (P = 0.047) and recessive (P = 0.018) models were significant at position rs57095329 between the two groups of patient and healthy. The A C haplotype (rs2910164 and rs57095329) was associated with brucellosis in the assessed population [OR (95% CI) = 1.98 (1.22–3.20), P = 0.0059].ConclusionsConsequently, our study demonstrated significant differences in genotype and haplotype frequencies of miR-146a variants between brucellosis patients and controls. Further studies on the larger sample sizes are required to verify the observed associations.

HSP70 functional gene polymorphism and haplotype as a genetic factor for type 2 diabetes mellitus

Oxidative stress is a major cause underlying the development of Type 2 Diabetes Mellitus (T2DM). The heat shock protein 70 (HSP70) is a redox sensor for intracellular oxidative stress. The present study was aimed to understand the association of HSPA1L (HSP70-hom) rs2075800 and HSPA1B (HSP70-2) rs6457452 gene polymorphisms and haplotype with susceptibility to T2DM. In this case-control study, 200 T2DM patients and 200 healthy people were genotyped for HSPA1L rs2075800 and HSPA1B rs6457452 polymorphisms by Tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. The HSPA1B rs6457452 CT (OR = 0.52, 95%CI: 0.33–0.81, P = 0.004) and TT (OR = 0.05, 95%CI: 0.07–0.43, P = 0.005) genotypes were associated with decreased risk of T2DM. Moreover, the T allele was detected as a protective genetic factor against T2DM (OR = 0.43, 95%CI: 0.29–0.63, P < 0.001). We didn't find any significant differences in genotype and allele distributions between T2DM patients and controls for HSPA1L rs2075800 polymorphism. Haplotype analysis showed significant results for all haplotype windows except AC (rs6457452/rs2075800) haplotype. Haplotype GT (OR = 0.554; 95%CI = 0.332–0.924; p = 0.022) and AT (OR = 0.388; 95%CI = 0.230–0.654; p = 0.0002) showed highly significant association with decreased risk of T2DM. Increased risk was observed in haplotype GC (OR = 1.505; 95%CI = 1.132–1.999; p = 0.004). In conclusion, we conducted for the first time, that these polymorphism and haplotype are associated with T2DM in an Iranian population.

The Association of Fat-Mass-and Obesity-Associated Gene Polymorphism (rs9939609) With Colorectal Cancer: A Case-Control Study.

Background and AimThe association between the rs9939609 polymorphism of fat mass and obesity-associated gene (FTO) and risk of colorectal cancer is controversial. This study aims to evaluate the relationship between FTO rs9939609 polymorphism and colorectal cancer (CRC) in Iranian people.MethodsA case-control study was conducted on 125 patients with CRC and 250 healthy subjects in Tehran, Iran. Demographic data and blood samples were collected from all participants. Genotyping of rs9939609 polymorphism was performed by the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method.ResultsThe occurrence of AA genotype of FTO rs9939609 polymorphism in the colorectal cancer patients was significantly higher compared to that of healthy subjects (16.4 vs. 2.9%, respectively, P=0.02). The association between the frequency of risk allele of the FTO polymorphism and CRC (B=1.67, P=0.042) remained significant after adjustment for age. Further adjustment for gender (model 2) and marital status (model 3) did not change this result (B=1.67, P= 0.042 and B=1.67, P=0.043, respectively). The results remained significant after additional adjustment for ethnicity (B=1.57, P= 0.047).ConclusionWe found a positive association between the A allele of the rs9939609 polymorphism and CRC. Future studies are required to identify the underlying mechanisms.

Association of novel stop-gained leukaemia inhibitory factor receptor gene (rs121912501) variant, leukaemia inhibitory factor and ovarian steroids with unexplained infertility among Pakistani women.

Embryo implantation is a complex process that requires sequential steps at the interface of embryo interaction with decidual endometrium. Many women after experiencing multiple attempts of assisted reproductive techniques fail to get implantation because of instability of leukaemia inhibitory factor and leukaemia inhibitory factor receptor-signal transducer and activator of transcription factor 3 (LIF-LIFR STAT3) signalling cascade. Therefore, this study explores the association of ovarian steroids, LIF and LIFR stop-gained variant using the tetra primer amplification refractory mutation system-polymerase chain reaction (TARMS-PCR) with unexplained infertility (UEX-IF) among Pakistani women. This is a case-control study, a total of 81 unexplained infertile women and 162 fertile controls (with age and BMI matched) were inducted. Serum estradiol, progesterone and LIF were determined using enzyme-linked immunosorbent assay (ELISA). T-ARMS-PCR was designed using Primer 1 software. Genomic DNA was extracted from peripheral blood and amplified using T-ARMS-PCR followed by sequencing for validation and comprehensive concordance. This study established differences in LIF levels (χ2 =9.857, P<.05) between patients and controls as well as explored the decreased LIF significantly raised the risk of UEX-IF (OR=2.316; 95% CI=1.214, 4.416). Progesterone (P) was significantly associated with UEX-IF between fertile and infertile counterparts (χ2 =20.347, P<.05). It was also observed that increased Progesterone reduced the risk of UEX-IF (OR=0.306; 95% CI=0.166, 0.567). A rapid and inexpensive method for genotyping novel LIFR gene polymorphism through T- ARMS-PCR was successfully developed. LIFR gene SNP (rs121912501) had significant association (χ2 =200.681, P<.05) with UEX-IF. LIFR rs121912501 "TT" genotype (OR=5.417; 95% CI=1.868, 15.709) and "CT" genotype (OR=3.104, 95% CI=1.586,6.076) were at increased risk of infertility. UEX-IF can be caused by LIFR gene variation irrespective of increased P. It may open the doors for the discovery of new management plans for infertile women.

Interactions of anthropometric indices, rs9939609 FTO gene polymorphism and breast cancer: A case-control study.

Contradictory results were reported on the effect of fat mass‐ and obesity‐associated (FTO) gene and anthropometric measurements on breast cancer (BC). This study aimed to assess the interactions between rs9939609 polymorphism of FTO gene, anthropometric indices and BC risk in Iranian women. This case‐control study was performed on 540 women including 180 women with BC and 360 healthy women in Tehran, Iran. Physical activity and dietary intakes were assessed by validated questionnaires. Data on sociodemographic and pathologic factors of the participants as well as their blood samples were collected. The rs9939609 FTO gene polymorphism was genotyped using the tetra‐primer amplification refractory mutation system‐polymerase chain reaction (T‐ARMS‐PCR). No significant association was found between BC and risk allele of FTO rs9939609 polymorphism after adjustments for the confounders. However, there was a significant association between rs9939609 polymorphism risk allele and BC risk in females with overweight, even after adjusting for age, family history of BC, abortion, BMI and the number of pregnancies (P < .05). The association was disappeared after further adjustments for lifestyle factors including smoking, alcohol consumption, calorie and macronutrients intake, and physical activity. The FTO gene polymorphism was associated with the risk of BC in overweight individuals. This association was influenced by environmental factors including diet, alcohol consumption and smoking. Future studies are required to confirm the association between the FTO gene and BC in overweight females and to identify the underlying mechanisms.

Polymorphic variants INSIG2 rs6726538, HLA‐DRB1 rs9272143, and GCNT1P5 rs7780883 contribute to the susceptibility of cervical cancer in the Bangladeshi women

ObjectiveCervical cancer is a gynecological health problem, affecting nearly 500,000 women each year worldwide. Genome‐wide association studies have revealed multiple susceptible genes and their polymorphisms for cervical carcinoma risk. We have carried out this case‐control study to investigate the association of INSIG2 rs6726538 (A; T), HLA‐DRB1 rs9272143 (T; C), and GCNT1P5 rs7780883 (G; A) with cervical cancer.MethodsThe present study recruited 234 cervical cancer patients as cases and 212 healthy females as controls. We have applied the tetra‐primer amplification refractory mutation system polymerase chain reaction (T‐ARMS‐PCR) method for genotyping.ResultsThe SNP rs6726538 was significantly associated with increased risk of cervical cancer in all genetic models (AT vs. AA: OR = 3.30, 95% CI = 2.19–4.97, p < 0.0001; TT vs. AA: OR = 8.72, 95% CI = 3.87–19.7, p < 0.0001; AT+TT vs. AA: OR = 3.87, 95% CI = 2.61–5.73, p < 0.0001; T vs. A: OR = 2.97, 95% CI = 2.20–4.01, p < 0.0001) except the recessive model which showed a significantly reduced risk (TT vs. AA+AT: OR = 0.20, 95% CI = 0.09–0.44, p = 0.0001). rs9272143 showed significantly reduced risk for the additive model 1, dominant model, and allelic model (TC vs. TT: OR = 0.46, 95% CI = 0.31–0.70, p = 0.0004; TC+CC vs. TT: OR = 0.47 95% CI = 0.32–0.70, p = 0.0002; C vs. T: OR = 0.56, 95% CI = 0.40–0.78, p = 0.0006, respectively). The third variant, rs7780883, was significantly associated with increased risk in additive model 2, dominant, and allelic models (AA vs. GG: OR = 5.08, 95% CI = 2.45–10.5, p < 0.0001; GA+AA vs. GG: OR = 1.54, 95% CI = 1.06–2.24, p = 0.0237; A vs. G: OR = 1.88, 95% CI = 1.34–2.52, p < 0.0001, consecutively), whereas recessive model reduced the risk of cervical cancer (AA vs. GG+GA: OR = 0.20, 95% CI = 0.09–0.41, p < 0.0001). Other models of these SNPs were not associated with cervical cancer. All significant associations for three SNPs withstand after Bonferroni correction except the additive model 2 of rs7780883.ConclusionOur study concludes that INSIG2 rs6726538, HLA‐DRB1 rs9272143, and GCNT1P5 rs7780883 polymorphisms may contribute to the development of cervical cancer in the Bangladeshi population.