TaqMan SNP Genotyping Assay Research Articles

SNP Diversity in CD14 Gene Promoter Suggests Adaptation Footprints in Trypanosome Tolerant N'Dama (Bos taurus) but not in Susceptible White Fulani (Bos indicus) Cattle.

Immune response to infections has been shown to be mediated by genetic diversity in pattern recognition receptors, leading to disease tolerance or susceptibility. We elucidated naturally occurring variations within the bovine CD14 gene promoter in trypanosome-tolerant (N’Dama) and susceptible (White Fulani) cattle, with genomic and computational approaches. Blood samples were collected from White Fulani and N’Dama cattle, genomic DNA extracted and the entire promoter region of the CD14 gene amplified by PCR. We sequenced this region and performed in silico computation to identify SNP variants, transcription factor binding sites, as well as micro RNAs in the region. CD14 promoter sequences were compared with the reference bovine genome from the Ensembl database to identify various SNPs. Furthermore, we validated three selected N’Dama specific SNPs using custom Taqman SNP genotyping assay for genetic diversity. In all, we identified a total of 54 and 41 SNPs at the CD14 promoter for N’Dama and White Fulani respectively, including 13 unique SNPs present in N’Dama only. The significantly higher SNP density at the CD14 gene promoter region in N’Dama may be responsible for disease tolerance, possibly an evolutionary adaptation. Our genotype analysis of the three loci selected for validation show that mutant alleles (A/A, C/C, and A/A) were adaptation profiles within disease tolerant N’Dama. A similar observation was made for our haplotype analysis revealing that haplotypes H1 (ACA) and H2 (ACG) were significant combinations within the population. The SNP effect prediction revealed 101 and 89 new transcription factor binding sites in N’Dama and White Fulani, respectively. We conclude that disease tolerant N’Dama possessing higher SNP density at the CD14 gene promoter and the preponderance of mutant alleles potentially confirms the significance of this promoter in immune response, which is lacking in susceptible White Fulani. We, therefore, recommend further in vitro and in vivo study of this observation in infected animals, as the next step for understanding genetic diversity relating to varying disease phenotypes in both breeds.

Exploring new genetic variants within COL5A1 intron 4-exon 5 region and TGF-β family with risk of anterior cruciate ligament ruptures.

Variants within genes encoding structural and regulatory elements of ligaments have been associated with musculoskeletal soft tissue injury risk. The role of intron 4-exon 5 variants within the α1 chain of type V collagen (COL5A1) gene and genes of the transforming growth factor-β (TGF-β) family, TGFBR3 and TGFBI, was investigated on the risk of anterior cruciate ligament (ACL) ruptures. A case-control genetic association study was performed on 210 control (CON) and 249 participants with surgically diagnosed ruptures (ACL), of which 147 reported a noncontact mechanism of injury (NON). Whole-exome sequencing data were used to prioritize variants of potential functional relevance. Genotyping for COL5A1 (rs3922912 G>A, rs4841926 C>T, and rs3124299 C>T), TGFBR3 (rs1805113 G>A and rs1805117 T>C), and TGFBI (rs1442 G>C) was performed using Taqman SNP genotyping assays. Significant overrepresentation of the G allele of TGFBR3 rs1805113 was observed in CON vs ACL (P = .014) and NON groups (P = .021). Similar results were obtained in a female with the G allele (CON vs ACL: P = .029; CON vs NON: P = .016). The TGFBI rs1442 CC genotype was overrepresented in the female ACL vs CON (P = .013). Associations of inferred allele combinations were observed in line with the above results. COL5A1 intron 4-exon 5 genomic interval was not associated with the risk of ACL ruptures. Instead, this novel study is the first to use this approach to identify variants within the TGF-β signaling pathway to be implicated in the risk of ACL ruptures. A genetic susceptibility interval was identified to be explored in the context of extracellular matrix remodeling.

Modification effect of sex and obesity on the correlation of LEP polymorphisms with leptin levels in Taiwanese obese women.

BackgroundObesity has become the main health issue in developed countries as it impacts life expectancy and increases mortality of cerebrovascular or cardiovascular diseases. The leptin is one of the adipokines which presents in the serum in proportion to the amount of adipose tissue and is translated from LEP gene. It involves in energy homeostasis, lipid and glucose metabolisms, modulation of immune systems, and thermogenesis. Many previous studies have revealed controversial results between LEP polymorphisms and leptin levels in different ages and ethnicities. Herein, we investigated the impacts of LEP polymorphism against leptin levels in Taiwanese subjects.MethodsIn 599 Taiwanese subjects, excluding clinically overt systemic disease, age below 18 years old, and C‐reactive protein (CRP) level of above 10 mg/L, few of LEP polymorphisms were genotyped with TaqMan SNP genotyping assays, were further analyzed for association with leptin level in univariate and multivariate linear regression analyses with Bonferroni correction for multiple tests in stratified groups. The univariate and stepwise multivariate linear regression analyses were performed to determine the coefficient of determinant of LEP polymorphisms over leptin level.ResultsSignificant associations were found between LEP polymorphisms and leptin levels in obese women. Circulating leptin level was positively correlated with inflammatory, insulin resistance markers, and visceral obesity markers in all subjects. Furthermore, stratified and interaction analyses revealed that LEP polymorphisms, rs7799039 and rs2167270, were significantly associated with leptin levels in obese women—8%–10% of which could be explained by LEP polymorphisms.ConclusionThe LEP polymorphisms are independently associated with leptin levels in Taiwanese obese women. Further, the genetic determinants for leptin levels may be different between obese and nonobese, and in different sex individuals. The obesity status and female sex may exert modification effect on transcription of LEP, particularly in obese women.

Investigation of Oxidative Stress-Related Candidate Genes as Risk Factors for Drug-Induced Liver Injury due to Co-Amoxiclav.

The liver is susceptible to drug toxicity due to its vital role in xenobiotic metabolism and elimination. In addition to human leukocyte antigen (HLA) variants, which were previously determined as risk factors for drug-induced liver injury (DILI) due to co-amoxiclav, other non-HLA genes may contribute to hepatotoxicity risk. In this study, the association between DILI due to co-amoxiclav and several non-HLA genes was investigated. Association of variants in candidate genes (SOD2, GPX1, GSTM1, and GSTT1) with DILI due to various drugs was reported previously in other DILI cohorts. This study examined relevance in a co-amoxiclav-DILI cohort. One hundred sixty-five co-amoxiclav DILI cases were recruited from several European countries by two different studies (DILIGEN and iDILIC). A North-East England population group (n = 334) was used as the control group. PCR assays were used to genotype for the GSTM1 and GSTT1 null alleles with TaqMan SNP genotyping assays used for SOD2 (rs4880) and GPX1 (rs1050450). Fisher's exact test was used to assess differences in significance between cases and controls. None of the studied variants (SOD2 rs4880, GPX1 rs1050450, GSTM1 null allele, and GSTT1 null allele) was significantly associated with co-amoxiclav DILI compared with the control group. No significant differences between cases and controls were seen when combined SOD2/GPX1 genotypes and GST genotypes were considered. Despite the possible functional relevance and the previously reported contribution of the selected genes to DILI, our study failed to confirm associations between the selected genes and liver injury induced by co-amoxiclav.

An Effective and Viable DNA Extraction Protocol for FFPE Tissues and its Effect on Downstream Molecular Application

Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues are a readily available source of DNA for retrospective studies including genetic predisposition. FFPE tissues present significant challenges for extraction of pure genomic DNA in adequate amounts due to the harsh fixation conditions and long-term storage associated with samples. The aim of the study is to propose a simple, in-expensive, viable protocol for the extraction of DNA from FFPE tissues and to optimize the same for the detection of 3’UTR miRNA binding site polymorphism in ADAMTS1 gene in colorectal cancer tissues and normal colonic mucosa archival tissues using Sanger sequencing and TaqMan SNP genotyping assay. Five different DNA extraction methods were compared and analyzed. NanoDrop quantification demonstrated that out of the various methods tested, highest quantity and pure DNA was obtained from Modified non heating extraction. When we further applied this method for colorectal cancer tissues and normal colonic mucosa archival tissues the NanoDrop readout indicated that the DNA was intact and pure. Intra samples comparison over a time period of 6 months revealed that the DNA was intact. The samples isolated with Modified non heating extraction were found suitable for downstream processes such as Sanger sequencing and TaqMan SNP genotyping assay. This cost effective and non-arduous protocol can successfully extract even minute quantities of DNA from FFPE tissues and facilitate the downstream molecular analysis of a large number of archival specimens for retrospective studies.

The PLCE1 rs2274223 variant is associated with the risk of laryngeal squamous cell carcinoma.

Background: Laryngeal squamous cell carcinoma (LSCC) ranks second in the mortality rate in respiratory malignant tumors and has potential similarity in genomic alterations with the esophageal squamous cell carcinoma (ESCC). The PLCE1 rs2274223 variant is the most significant susceptibility loci identified in ESCC. Whether it is also associated with LSCC susceptibility is still unclear.Materials and Methods: A total of 331 LSCC patients and 349 healthy controls were recruited in this study. The PLCE1 rs2274223 variant was genotyped by using the Taqman SNP Genotyping Assay. Association between PLCE1 rs2274223 variant and LSCC risk was estimated by logistic regression analysis, which was performed using SAS software.Results: The PLCE1 rs2274223 variant was identified to be significantly associated with the susceptibility of LSCC in the additive model (OR = 1.40, 95% CI: 1.06-1.86, P=0.019). Compared with the wild-type (AA) carriers, the risk genotype (GG) carriers had a 2.8-fold risk of LSCC (95% CI: 1.13-7.06, P=0.026). Stratified analysis showed that the association between rs2274223 and LSCC risk was with higher significance in individuals above 60 (P = 0.027) males (P = 0.030) or non-smokers (P = 0.026).Conclusion: The PLCE1 rs2274223 variant was significantly associated with risk of LSCC, which may be a potential biomarker and therapeutic target for the LSCC.

Associations between B vitamin and amino acid intake, MTHFR genotype, atherogenic indices, and homocysteine levels in postmenopausal women

AbstractIt has been hypothesized that insufficiency of one-carbon metabolism may aggravate lipid metabolism disturbances. Lipid metabolism abnormalities and metabolic syndrome (MetS)—which is associated with abnormally high levels of triglycerides (TG) and low levels of high-density lipoproteins (HDL)—often occur in postmenopausal women. The purpose of this study was thus to determine the relationships between amino acid and B vitamin intake, MTHFR genotype, lipid profile, and atherogenic indices in postmenopausal women.131 postmenopausal women were enrolled for the study. The mean macronutrient, amino acid, and B vitamin intake was evaluated using dietary records. Lipid profile and concentrations of apolipoprotein A1 (ApoA1) and B (ApoB) in serum were measured using the colorimetric method. The total homocysteine (tHcy) and glutathione (GSH) level in plasma were measured using high-performance liquid chromatography. MTHFR genotype (rs1801133) was determined using a single tube TaqMan SNP Genotyping Assay. We calculated atherogenic indices, such as Castelli's risk index I and II (CRI-I and CRI-II), the atherogenic coefficient (AC), and the TG/HDL ratio.No association was found between MetS and tHcy or GSH concentrations. MTHFR genotype and folate intake also did not affect tHcy, GSH, lipid profile, or atherogenic indices. Subjects with tHcy levels over the median had lower total protein intake, lower animal protein/plant protein ratio, lower protein/carbohydrates ratio, and higher amino acid intake (methionine, lysine, arginine, glycine, and tryptophan) than did subjects with lower tHcy levels. Women who failed to meet their vitamin B12 intake needs had almost 40% higher levels of tHcy than women who did met those needs.MTHFR genotype, folate intake, and tHcy levels in plasma are not associated with lipid metabolism in postmenopausal women. tHcy levels may depend on the intake of vitamin B12, and of protein or particular amino acids.The project was financed by the National Science Centre, Poland (grant number 2015/17/N/NZ9/04133).

Detection of FAS and FASLG polymorphisms with TaqMan SNP genotyping assays in recurrent miscarriage

Amaç: Bu çalışmada apoptotik uyarıcı Fas hücre yüzey ölüm reseptörü (FAS) ve FAS ligandı genleri (FASLG) polimorfizmlerinin tekrarlayan gebelik kaybı (TGK) etiyolojisindeki yeri araştırıldı.Gereç ve Yöntem: Bu vaka-kontrol çalışmasında, TGK tanısı almış 70 kadın ve kontrol grubu olarak 70 kadından genomik DNA izole edildi. Her iki gruptaki FAS -670 A>G, FAS -1377 G>A ile FASLG -124 A>G polimorfizmlerinin genomik dağılımı ve allel frekansları real-time PCR yöntemi ile belirlenerek karşılaştırıldı.Bulgular: Çalışmamızda, FASLG -124 A>G polimorfizmi için TGK ve kontrol gruplarının genotip frekans farkının istatistiksel olarak anlamlı olduğu saptandı. Ancak FAS −670 A>G ve FAS-1377 G>A genotipleri ile TGK arasında istatistiksel düzeyde anlamlı bir ilişki saptanmadı. FASLG −124 A>G polimorfizmi için A allelini taşıyanlarda hastalığın görülme olasılığı A allelini taşımayanlara göre 3,94 kat daha yüksek bulundu.Sonuç: Bulgularımız, apoptozla ilişkili FASLG -124 A>G polimorfizminin TGK oluşumuna katkısı olabileceğini düşündürmektedir.

EVALUATION OF INTERLEUKIN 6 POLYMORPHISMS AND ITS ASSOCIATION WITH RHEUMATOID ARTHRITIS IN BRAZILIAN WOMEN

Objective To verify the association between serum interleukin 6 (IL-6) levels and IL-6 genetic polymorphisms in patients with rheumatoid arthritis (RA), as well as its association with disease activity and exocrine gland function. Methods. The study included 295 women, 173 had a diagnosis of RA and 122 were healthy. Clinical data and blood samples were collected. Serum IL-6 levels were measured by flow cytometry, and IL-6 genotypes were performed by real-time polymerase chain reaction (PCR) using TaqMan SNP genotyping assay. Results Patients in the RA group had higher levels of IL-6 (P Conclusions The presence of the C allele of IL-6 rs1800796 plays an important role in the pathogenesis of RA and, if confirmed in other populations, may be used as a genetic marker of the disease.

The Role of Phosphatidylethanolamine-N-methyltransferase (PEMT) Gene rs12449964 Polymorphism in the Development of Acute Pancreatitis and its Complications

Background. Acute pancreatitis is considered to be an important issue in modern medicine. The phosphatidylethanolamine-N-methyltransferase enzyme plays a significant role in the regulation of lipid metabolism by catalyzing the process of methylation of phosphatidylethanolamine to phosphatidylcholine. These lipids are key components of mitochondrial and cell membranes, providing their fluid and plastic properties and participating in the transport of fats, fatty acids and cholesterol. Along with its function in the synthesis of phosphatidylcholine, the methylation of phosphatidylethanolamine promotes the turnover of S-adenosylmethionine for the synthesis of cysteine and glutathione through transulphurisation. PEMT is a gene encoding the phosphatidylethanolamine-N-methyltransferase enzyme.Aim. To determine the role of PEMT C/T rs12449964 polymorphism in the risk of developing acute pancreatitis and its complications among Russian residents in Central Russia.Materials and methods. Whole blood samples were collected from 502 unrelated patients with acute non-biliary pancreatitis (97 women and 405 men) of Russian nationality who had been admitted to the surgical departments of the city of Kursk from 2015 to 2018, as well as from 513 unrelated individuals of Russian nationality without gastrointestinal diseases (101 women and 412 men). The average age of patients and healthy individuals was 48.9 ± 13.1 and 47.89 ± 12.1 years, respectively. Genomic DNA was isolated by a standard phenol-chloroform extraction method. Genotyping of rs12449964 polymorphism was performed using real-time PCR by allelic discrimination using a CFX96 Bio-Rad Laboratories amplifier (USA) with TaqMan probes and commercial TaqMan SNP Genotyping Assays reagents purchased from Applied Biosystems (USA).Results. The study has shown that the frequency of the C allele and the C/C PEMT C/T rs12449964 genotype was higher in the group of patients with acute pancreatitis, while the C/T genotype was predominant in the control group. C/T — T/T genotypes demonstrated a protective effect on the development of infected pancreatic necrosis, purulent necrotic peripancreatitis and severe acute pancreatitis.Conclusions. The disruption of phosphatidylethanolamine methylation processes increases the sensitivity of cells to oxidative stress, which can lead to the development of acute pancreatitis.

Abstract LB-B19: Investigating the effect of pharmacogenetic variations on voriconazole levels in pediatric cancer patients

Abstract Background: Voriconazole is a second-generation triazole antifungal with broad-spectrum activity against Aspergillosis, metabolized mainly by CYP2C19 in the liver. Despite the use of weight-based dosing, plasma voriconazole blood levels show inter-individual variability, which is attributed to many factors such as weight, age, food, drug interactions, and CYP2C19 polymorphism. Voriconazole metabolism in pediatrics has been reported to be profoundly impacted by CYP2C19 genotypes. Aim: To evaluate the frequencies of CYP2C19 variant alleles (*2, *3, and *17), genotypes, and phenotypes in Egyptian pediatric cancer patients and to investigate the correlation between CYP2C19 genetic variability and other factors with voriconazole plasma levels. Methods: This study was conducted on 100 pediatric cancer patients with probable fungal infection treated with voriconazole. DNA was extracted from blood samples and analyzed by the TaqMan SNP genotyping assay. Identified genotypes were classified accordingly into various predicted phenotypes. Results: Voriconazole plasma levels widely varied, with only 49.5% reaching therapeutic levels. Frequencies of CYP2C19*2 and *17 allelic variants were 10.6 and 18.3%, respectively, while allele *3 was not detected in the study population. A significant correlation was found between plasma levels and CYP2C19 phenotypes: ultra-rapid metabolizers had significantly lower plasma levels (P=0.003) relative to the extensive metabolizer genotype. Conclusion: Determination of CYP2C19 genotype at initiation of therapy, followed by drug monitoring, would serve as a valuable tool for the optimization of voriconazole therapy for pediatric cancer patients. It would also be an essential step in the advancement of precision medicine to optimize therapeutic efficacy and minimize toxicity. Citation Format: Dina Saeed, Lobna Shalaby, Mohamed Nagy, Ahmed ElZeiny, Tarek M. Mostafa, Marwa T. ElRakaiby, Mohammed M. Nooh, Alaa El-Haddad, Maggie Abbassi, Ramy K. Aziz. Investigating the effect of pharmacogenetic variations on voriconazole levels in pediatric cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B19. doi:10.1158/1535-7163.TARG-19-LB-B19

Carcinogen Metabolism Pathway and Tumor Suppressor Gene Polymorphisms and Gallbladder Cancer Risk in North Indians: A Hospital-Based Case-Control Study.

Background:Carcinogen metabolism pathway and tumor suppressor gene polymorphisms have been reported to be associated with increased gallbladder cancer risk. However, the association of genetic variants and gallbladder cancer risk in Indians are not well studied. We examined whether genetic polymorphisms of metabolic enzymes cytochrome P450 1A1 and glutathione S-transferase and tumor suppressor gene p53 (TP53) are associated with an increased risk of gallbladder cancer in North Indians. Methods:This hospital-based case-control study was conducted in 96 gallbladder cancer patients with gallstones (cases) and 93 cholelithiasis patients (controls) at the Sanjay Gandhi Postgraduate Institute of Medical Sciences in Lucknow, India from July 2014 through May 2017. Genomic DNA was extracted from white blood cells of each patient using a simple salting-out procedure. The genotypic frequencies of CYP1A1 rs4646903, CYP1A1 rs1048943, and TP53 rs1042522 polymorphisms were investigated using TaqMan SNP Genotyping Assay and GSTM1 and GSTT1 polymorphisms were analyzed using the multiplex PCR assay. Results:The frequency of CC genotype of TP53 rs1042522 polymorphism was 27.1% (26/96) in cases and 12.9% (12/93) in controls. The CC genotype was associated with an increased risk of gallbladder cancer in North Indians (age- and sex-adjusted odds ratio, 2.81; 95% confidence interval, 1.19–6.61; P = 0.02). No significant differences in genotypic and allelic frequencies of the metabolic pathway gene polymorphisms were found between cases and controls. Conclusions:Our data provide preliminary evidence that the CC genotype of the TP53 rs1042522 polymorphism may be associated with an increased risk of gallbladder cancer in North Indians.

Polymorphisms of FDPS, LRP5, SOST and VKORC1 genes and their relation with osteoporosis in postmenopausal Romanian women.

This study aimed to assess the relationship between bone mineral density and genotypes of four polymorphisms in previously detected osteoporosis-candidate genes (FDPS rs2297480, LRP5 rs3736228, SOST rs1234612, VKORC1 rs9934438) in postmenopausal Romanian women with primary osteoporosis. An analytical, prospective, transversal, observational, case-control study on 364 postmenopausal Romanian women was carried out between June 2016 and August 2017 in Cluj Napoca, Romania. Clinical data and blood samples were collected from all study participants. Four polymorphisms were genotyped using TaqMan SNP Genotyping assays, run on a QuantStudio 3 real-time PCR machine. Women with TT genotype in FDPS rs2297480 had significantly lower bone mineral density values in the lumbar spine and total hip, and the presence of the T allele was significantly associated with the osteoporosis. Women carrying the CC genotype in LRP5 rs3736228 tend to have lower bone mineral density values in the femoral neck and total hip. No significant association was found for the genotypes of SOST rs1234612 or VKORC1 rs9934438. Our study showed a strong association between bone mineral density and polymorphisms in the FDPS gene, and a borderline association with LRP5 and SOST polymorphisms in postmenopausal Romanian women with osteoporosis. No association was found for VKORC1.

Interferons (IFN-A/-B/-G) Genetic Variants in Patients with Mixed Connective Tissue Disease (MCTD).

Mixed connective tissue disease (MCTD) is a rare complex autoimmune disease in which autoantigens are recognized by endosomal TLRs. Their activation induces a higher secretion of the type I interferons, IFN-γ and the up-regulation of the INF-inducible genes. The present study aimed to investigate whether SNPs that are located in the IFN-A, IFN-B, and IFN-G genes are associated with MCTD. 145 MCTD patients and 281 healthy subjects were examined for IFN-A, IFN-B, and IFN-G genetic variants by TaqMan SNP genotyping assay. ELISA determined IFN-α/-β/-γ serum levels. Among the seven tested SNPs, four polymorphisms: IFN-A rs10757212, IFN-A rs3758236, IFN-G rs2069705, IFN-G rs2069718, as well as INF-G rs1861493A/rs2069705A/rs2069718G haplotype were significantly associated with a predisposition for MCTD. Raynaud’s phenomenon, erosive arthritis, swollen hands and fingers, and sclerodactyly were significantly more frequently observed in MCTD patients with IFN-G rs2069718 G allele than in patients with IFN-G rs2069718 A allele. We also found that anti-U1-A autoantibodies most frequently occurred in MCTD patients with rs2069718 GA genotype, while the IFN-G rs2069705 AG and rs2069718 GA genotypes might be a marker of anti-Ro60 presence in MCTD patients. Our results indicate that IFN-G genetic variants may be potential genetic biomarkers for MCTD susceptibility and severity.

98P - Influence of DPYD*9, DPYD*6 and GSTP1 ile105val genetic polymorphisms on capecitabine and oxaliplatin (CAPOX) associated toxicities in colorectal cancer patients

BackgroundCapecitabine and oxaliplatin combination regimen (CAPOX) is the standard chemotherapeutic care for the treatment of colorectal cancer (CRC). CAPOX treatment is found to be equivalent to FOLFOX and FOLFRI in efficacy and preferred over them due to its easy management and convenience in administration. However, CAPOX treatment was associated with several dose-limiting toxicities and high inter-individual variation in toxicity profile. These toxicities may limit treatment effectiveness as they impose treatment interruption or even discontinuation. Therefore, there is a critical need for identifying the predictive biomarkers for CAPOX related toxicities. MethodsPatients and methods: In an intent treat analysis, 145 CRC patients were recruited. Patients received a standard treatment schedule of oxaliplatin 130 mg/m2 infusion over 2 hours on day 1 and oral capecitabine 1000mg/m2 in divided doses twice daily for the next 14 days of a 21-day cycle. 5 ml of the venous blood was collected from each patient and genomic DNA was extracted by the phenol-chloroform method. The genotyping analysis of the selected SNP’s was carried out by real-time PCR using TaqMan SNP genotyping assays from applied biosystems. ResultsThe major dose-limiting toxicities observed with CAPOX treatment were thrombocytopenia, HFS and PN. DPYD*9 carries were found to be at higher risk for HFS, diarrhoea and thrombocytopenia when compared to patients with wild allele. No significant association was found between DPYD*6, GSTP1 ile105val polymorphisms and CAPOX related toxicities except for thrombocytopenia. ConclusionsA significant association was observed between DPYD*9A A>G polymorphism and CAPOX induced dose-limiting toxicities like HFS, diarrhoea and thrombocytopenia strengthening its role as a predictive biomarker. Legal entity responsible for the studyAshok Varma. FundingJawaharlal Institute of Post-Graduate Medical Education and Research (JIPMER). DisclosureThe author has declared no conflicts of interest.

359. TLR7 Gene Polymorphisms Influence Development of Hepatic Fibrosis in HCV/HIV Coinfection

BackgroundHepatic fibrosis in individuals with HCV/HIV coinfection or HCV mono-infection may be modulated by a variety of host factors. In this study, we investigated the role of gene polymorphisms of putative genes that might influence fibrosis progression including MICA (rs2596542), interferon-stimulated gene OAS2 (rs1293762) and the pathogen recognition receptors TLR7 (rs179009) and TLR9 (rs187084). Effect on a cytokine panel was evaluated.MethodsLongitudinal samples were obtained from subjects enrolled in the NCI Multicenter Hemophilia Cohort Study. Within the cohort, a subset of subjects were included based upon presence or absence of the CCR5 delta-32 mutation which was previously shown to influence the rate of fibrosis progression. Hepatic fibrosis change was determined using the serum-derived Enhanced Liver Fibrosis (ELF) Index. Four putative genes with polymorphisms that have been previously associated with the development or progression of hepatic fibrosis were evaluated using Taqman SNP genotyping assays. Cytokine assays were performed using Luminex chipsets. Samples were analyzed using Statistix 10.0 using ANOVA and least square regression models.Results58 unique subjects were evaluated. The mean age was 38 years, and all were male. 74% were HIV infected and 97% were HCV infected (76.8% coinfection). Controlling for the effect of CCR5, only the TLR7 A -> G polymorphism was predictive of change in the ELF Index. There was no statistically significant predictive difference between genotypes in the other three polymorphisms. Subjects with the TLR7 A allele (n = 47) had an average increase in ELF of 0.79 units, while the G allele (n = 11) had an increase in ELF of 2.1 units (P = 0.008). A regression model identified TLR7 as a key factor in ELF change, as well as HCV/HIV coinfection. Interferon alfa-2 levels were highly associated (increased, P = 0.0007) with the TLR7 A -> G polymorphism, while RANTES levels were inversely associated (decreased, P = 0.0443) with it.ConclusionOf the gene polymorphisms investigated, only TLR7 (rs179009) is an independent predictor of development of hepatic fibrosis in HCV/HIV coinfected subjects. The mechanism may involve modulation of inflammatory response pathways.Disclosures All authors: No reported disclosures.

Galectin-9 gene (LGALS9) polymorphisms are associated with rheumatoid arthritis in Brazilian patients.

IntroductionRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and hyperplasia, as well as cartilage and bone destruction. Several proteins are associated with the pathogenesis of the disease. Galectin-9 belongs to the family of lectins that are involved in various biological processes and have anti-inflammatory activity.ObjectiveTo investigate associations between the SNPs of the GAL-9 gene (LGALS9) and serum levels in rheumatoid arthritis patients. We extracted DNA from 356 subjects, 156 RA patients and 200 healthy controls from northeastern Brazil. Three polymorphisms (rs4795835, rs3763959, and rs4239242) in the LGALS9 gene were selected and genotyped using TaqMan SNP genotyping assay. Serum concentrations of galectin-9 were analyzed by ELISA.ResultsThe rs4239242 TT genotype showed a positive association with RA (p = 0.0032, odds ratio = 0.28), and heterozygous TC were prevalent in the control group compared to RA patients (p = 0.0001, odds ratio = 7.99). Galectin-9 serum levels were significantly increased in RA patients compared to the control group (p<0.0001). Patients in remission had high levels of galectin compared to the moderate activity group (p<0.0001). Regarding the Clinical Disease Activity Index (CDAI), patients in remission or low activity presented high levels of galectin when compared to patients in severity (p<0.0001). Patients performing moderate activity had a significant value compared to patients who were in high disease severity (p = 0.0064). Interestingly, the AG genotype (rs3763959) has been associated with a higher presence of bone erosion in RA patients (p = 0.0436). The SNP rs4239242 TT genotype showed a positive association with RA in comparison to the control group. The AG genotype (rs3763959) has been associated with a higher presence of bone erosion in RA patients.

Evaluation Of HHIP Polymorphisms And Their Relationship With Chronic Obstructive Pulmonary Disease Phenotypes.

PurposeWe aimed to correlate three polymorphisms of the Hedgehog Interacting Protein (HHIP) gene with the three main phenotypes of the chronic obstructive pulmonary disease (frequent exacerbator (FE), asthma/COPD overlap (ACO), and emphysema with hyperinflation).Patients and methodsA cross-sectional study was carried out in the Department of Pulmonology at the Rio de Janeiro State University from February 2015 to July 2018. A total of 81 patients diagnosed with COPD according to the criteria of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) were enrolled. The subjects were divided into three distinct groups according to their phenotypes (FE, ACO and emphysema-hyperinflation). Three polymorphisms of the HHIP gene that are often reported as allegedly involved in the pathogenesis of COPD were analysed: rs1828591, rs13118928, and rs6537296. Real-time PCR - TAQMAN SNP Genotyping Assay was performed. The statistical analysis was carried out with the SPSS program with a multivariate analysis with a 95% confidence interval.ResultsAn increase in the frequency of the A allele of the rs13118928 HHIP gene polymorphism was observed in the group of subjects with COPD and emphysema-hyperinflation phenotype when compared with those in the FE phenotype (p=0.019) and subjects with ACO (p=0.04). However, the subjects with emphysema-hyperinflation phenotype presented more often the A allele (p=0.04). The genotypic analysis confirmed the difference between the emphysema-hyperinflation and ACO phenotypes, with a higher prevalence of the AA genotype in the emphysema-hyperinflation group (p=0.04). The ACO and FE phenotype subjects showed no difference in these polymorphisms. No difference was found in the frequency of the polymorphisms rs1828591 (p= 0.552) and rs6537296 (p=0.296) in the three phenotypes evaluated.ConclusionThe presence of the A allele in the rs13118928 polymorphism of the HHIP gene may be related to the emphysema-hyperinflation phenotype.

Influence of DPYD*9A, DPYD*6 and GSTP1 ile105val Genetic Polymorphisms on Capecitabine and Oxaliplatin (CAPOX) Associated Toxicities in Colorectal Cancer (CRC) Patients

Aim:CAPOX treatment in CRC patients was reported to cause several dose-limiting toxicities, and are found responsible for treatment interruption or even discontinuation. Therefore there is a critical need for identifying the predictive biomarkers for such toxicities to prevent them. The aim of our present study is to find the influence of DPYD*9A, DPYD*6 and GSTP1 ile105val gene polymorphisms on CAPOX treatment-associated toxicities in south Indian patients with CRC. Patients and Methods:We have recruited 145 newly diagnosed and treatment naive CRC patients in the study. Each Patient received a standard treatment schedule of oxaliplatin 130 mg/m2 infusion over 2 hours on day 1 and oral capecitabine 1000mg/m2 in divided doses twice daily for the next 14 days of a 21-day cycle. 5 ml of the venous blood was collected from each patient and genomic DNA extraction and genotyping. The genotyping analysis of the selected genetic polymorphisms was carried out by real-time PCR using TaqMan SNP genotyping assays obtained from applied biosystems. Results:The major dose-limiting toxicities observed with CAPOX treatment were thrombocytopenia, HFS and PN. DPYD*9A carries were found to be at higher risk for HFS, diarrhoea and thrombocytopenia when compared to patients with wild allele. No significant association was found between DPYD*6, GSTP1 ile105val polymorphisms and CAPOX related toxicities except for thrombocytopenia. Conclusion:A significant association was observed between DPYD*9A polymorphism and CAPOX induced dose-limiting toxicities strengthening its role as a predictive biomarker.

Association between polymorphisms in GGPS1 and RANKL genes and postmenopausal osteoporosis in Romanian women

Objectives: This study aimed to assess the relationship between bone mineral density, fragility fractures, fracture risk and polymorphisms of two osteoporosis-candidate genes (GGPS1 and RANKL) in Romanian women with postmenopausal osteoporosis. Methods: An analytical, prospective, transversal, observational, case-control study on 364 postmenopausal women, of which 228 were previously diagnosed with osteoporosis, was carried out between June 2016 and August 2017 in Cluj Napoca, Romania. Clinical data and blood samples were collected from all study participants. Polymorphisms in GGPS1 and RANKL genes were genotyped using TaqMan SNP Genotyping assays, run on a QuantStudio 3 real-time PCR machine. Results: The CT genotype in GGPS1 rs10925503 was associated with significant lower bone mineral density values at lumbar spine and femoral neck sites and a higher fracture risk compared to controls. No significant association was found between genotypes of RANKL rs2277439 with bone mineral density or fracture risk compared to the healthy controls. Conclusions: Our study showed a strong association between low bone mineral density and genotype CT of GGPS1 rs10925503 polymorphisms. No association was found for RANKL rs2277439 polymorphism.