Can Pharmacogenetic Variants in TPMT, MTHFR and SLCO1B1 Genes Be Used as Potential Markers of Outcome Prediction in Systemic Sclerosis Patients?

Systemic sclerosis (SSc) is a rare connective tissue disorder with highest morbidity and mortality among rheumatologic diseases. Disease progression is highly heterogeneous between patients, implying a strong need for individualization of therapy. Four pharmacogenetic variants, namely TPMT rs1800460, TPMT rs1142345, MTHFR rs1801133 and SLCO1B1 rs4149056 were tested for association with severe disease outcomes in 102 patients with SSc from Serbia treated either with immunosuppressants azathioprine (AZA) and methotrexate (MTX) or with other types of medications. Genotyping was performed using PCR-RFLP and direct Sanger sequencing. R software was used for statistical analysis and development of polygenic risk score (PRS) model. Association was found between MTHFR rs1801133 and higher risk for elevated systolic pressure in all patients except those prescribed with MTX, and higher risk for kidney insufficiency in patients prescribed with other types of drugs. In patients treated with MTX, variant SLCO1B1 rs4149056 was protective against kidney insufficiency. For patients receiving MTX a trend was shown for having a higher PRS rank and elevated systolic pressure. Our results open a door wide for more extensive research on pharmacogenomics markers in patients with SSc. Altogether, pharmacogenomics markers could predict the outcome of patients with SSc and help in prevention of adverse drug reactions.

Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection.

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.

Pharmacogenomic Markers of Methotrexate Response in the Consolidation Phase of Pediatric Acute Lymphoblastic Leukemia Treatment.

Methotrexate (MTX) is one of the staples of pediatric acute lymphoblastic leukemia (ALL) treatment. MTX targets the folate metabolic pathway (FMP). Abnormal function of the enzymes in FMP, due to genetic aberrations, leads to adverse drug reactions. The aim of this study was to investigate variants in pharmacogenes involved in FMP and their association with MTX pharmacokinetics (MTX elimination profile) and toxicity in the consolidation therapy phase of pediatric ALL patients. Eleven variants in the thymidylate synthetase (TYMS), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), SLC19A1 and SLCO1B genes were analyzed in 148 patients, using PCR- and sequencing-based methodology. For the Serbian and European control groups, data on allele frequency distribution were extracted from in-house and public databases. Our results show that the A allele of SLC19A1 c.80 variant contributes to slow MTX elimination. Additionally, the AA genotype of the same variant is a predictor of MTX-related hepatotoxicity. Patients homozygous for TYMS 6bp deletion were more likely to experience gastrointestinal toxicity. No allele frequency dissimilarity was found for the analyzed variants between Serbian and European populations. Statistical modelling did not show a joint effect of analyzed variants. Our results indicate that SLC19A1 c.80 variant and TYMS 6bp deletion are the most promising pharmacogenomic markers of MTX response in pediatric ALL patients.

Exploring inflammatory and apoptotic signatures in distinct Crohn's disease phenotypes: Way towards molecular stratification of patients and targeted therapy.

BACKGROUND: Crohn's disease (CD) is chronic inflammatory bowel disease with different phenotypic characteristics influencing disease prognosis and therapeutic strategies. The aim of this pilot study was to analyze selected inflammatory and apoptotic markers in non-inflamed and inflamed samples of ileal mucosa of non-stricturing/non-penetrating (NS/NP) and stricturing (S) CD mucosal phenotypes in order to characterize their distinct profiles. METHODS: From twenty CD patients (9 NS/NP, 11 S) paired non-inflamed and inflamed ileal biopsies were collected and used for analysis of cytokine (TNF and IL6) and apoptotic (Bcl2, Bax, Fas and FasL) genes' expression levels by real-time PCR, while NFκB transcriptional potency was assessed by electromobility gel shift assay. RESULTS: Our results demonstrated significant upregulation of TNF and IL6 in inflamed area of both NS/NP (p = 0.03, p = 0.01) and S phenotypes (p = 0.04, p = 0.04), respectively. However, TNF increase was more prominent in NS/NP compared to S inflamed mucosa (p = 0.02). Also, level of proapoptotic Bax was significantly higher in NS/NP compared to S inflamed mucosa (p = 0.01). Opposing transcription potency of NFκB has been detected between two phenotypes: being decreased in NS/NP (p = 0.07) and increased in S (p = 0.1) inflamed compared to non-inflamed mucosa, demonstrating trend towards statistical significance. CONCLUSIONS: We found that two distinct CD phenotypes have specific molecular signatures. Obtained results could direct improvement of current and development of new therapeutic strategies based on more specific molecular stratification of CD patients.

Clinical Application of Thiopurine Pharmacogenomics in Pediatrics.

BACKGROUND: Thiopurine drugs are used for the treatment of pediatric diseases. Inter-individual differences in the metabolism of these drugs greatly influence the risk of thiopurine induced toxicity and therapy failure. These differences are the consequence of genomic, epigenomic and transcriptomic variability among patients. Pharmacogenomics aims to individualize therapy according to the specific genetic signature of a patient. Treatment protocols based on thiopurine drugs have already been improved by applying pharmacogenomics in pediatric clinical practice. OBJECTIVE: The aim of this review was to summarize the application of thiopurine pharmacogenomics in pediatric patients suffering from acute leukemias, different types of autoimmune and inflammatory diseases, as well as in posttransplant care. METHODS: We searched PubMed/Medline database to identify thiopurine pharmacogenomic markers clinically relevant in pediatric diseases. RESULTS: TPMT and NUDT15 pharmacogenomic testing is done in pediatric care, contributing to the reduction of thiopurine induced toxicity. Data on numerous novel potential pharmacogenomic markers relevant for optimization of thiopurine treatment are still controversial (ITPA, ABCC4, NT5C2, PRPS1, GSTM1, FTO gene variants). Majority of evidences regarding thiopurine pharmacogenomics in pediatrics have been acquired by studying acute lymphoblastic leukemia and inflammatory bowel disease. For other pediatric diseases, namely acute myeloid leukemia, non-Hodgkin lymphoma, juvenile idiopathic arthritis, atopic dermatitis, juvenile autoimmune hepatitis and renal allograft transplantation, data are still scarce. CONCLUSION: Thiopurine pharmacogenomics has shown to be one of the best examples of successful application of pharmacogenomics in pediatrics.

Metabolic Syndrome in Inflammatory Bowel Disease: Association with Genetic Markers of Obesity and Inflammation.

Background: This study analyzed poorly understood relationship of two overlapping conditions: metabolic syndrome (MeS) and inflammatory bowel disease (IBD), both associated with inflammation in the visceral adipose tissue. Methods: Newly diagnosed 104 IBD patients, of which 50 Crohn's disease (CD) and 54 ulcerative colitis (UC), and 45 non-IBD controls were examined for MeS-related obesity and lipid markers. Th-17 immune genes IL17A, IL17F, IL23A, and TLR9 mRNAs were measured in intestinal mucosa by qRT-PCR. Subjects were genotyped for obesity-associated FTO variant rs9939609 by polymerase chain reaction-amplification refractory mutation system. Results: CD was associated with MeS (P = 0.01), while both CD and UC were associated with central obesity (P = 10-5, P = 0.002, respectively) and low levels of high-density lipoprotein (HDL) cholesterol (P = 5 × 10-6, P = 6 × 10-6, respectively). IBD lipid profile was characterized by decreased total and HDL cholesterol, while low-density lipoprotein cholesterol was reduced only in CD. Negative correlations were found between total cholesterol and CD activity index (P = 0.005), waist circumference and IL17A as well as IL17F mRNA levels in inflamed CD colon (P = 0.003, P = 0.001, respectively). Carriers of FTO rs9939609 AA genotype showed increased risk of CD (OR 2.6, P = 0.01). Conclusions: MeS, central obesity, and dyslipidemia could be important for IBD pathogenesis. This could influence therapeutic approaches and prevention strategies in high-risk groups.

Influence of variants in folate metabolism genes on 6-mercaptopurine induced toxicity during treatment for childhood acute lymphocytic leukemia.

PURPOSE: To analyze influence of variants in TYMS, MTHFR, SLC19A1 and DHFR genes on 6-mercaptopurine (MP) induced toxicity during maintenance phase of treatment for childhood acute lymphocytic leukemia (ALL). METHODS: One-hundred twenty-seven children with ALL that received maintenance therapy were involved in this study. All patients were treated according to Berlin-Frankfurt-Muenster (BFM) based protocols. Myelotoxicity and hepatotoxicity were evaluated using surrogate markers (median 6-MP dose, number of leukopenic episodes and levels of bilirubin and transaminases on each visit). RESULTS: Higher number of leukopenic episodes, as a surrogate marker of 6-MP myelotoxicity, was found in carriers of TYMS 3R3R and 3R4R genotypes (p=0.067) as well as in TYMS 3R6bp+ (28bp VNTR, 6bp indel) haplotype carriers (p=0.015). Carriers of DHFR CATAG (-680, -675, -556, -464, -317) haplotype were also found to have higher number of leukopenic episodes (p=0.070). SLC19A1 c.80A allele (p=0.079) and TYMS 2R6bp+ (5'UTR VNTR, 6bp indel) haplotype carriers (p=0.078) had fewer leukopenic episodes. No difference in genotype frequencies between the control group of volunteered blood donors and childhood ALL patients was found. CONCLUSIONS: Variants in TYMS, SLC19A1 and DHFR genes are potential biomarkers of myelotoxicity and could be used for 6-MP therapy individualization in maintenance phase of childhood ALL treatment, alongside with well-established TPMT variants.

Expression Pattern of Long Non-coding RNA Growth Arrest-specific 5 in the Remission Induction Therapy in Childhood Acute Lymphoblastic Leukemia.

BACKGROUND: Long non-coding RNA growth arrest-specific 5 (GAS5) is deregulated in many cancers because of its role in cell growth arrest and apoptosis. Additionally, GAS5 interacts with glucocorticoid receptor, making it a potential pharmacotranscription marker of glucocorticoid (GC) therapy. In this study, we aimed at analysing GAS5 expression in the remission induction therapy phase of childhood acute lymphoblastic leukemia (ALL), in which GCs are mandatorily used, and to correlate it with therapy response. METHODS: GAS5 expression was measured in peripheral blood mononuclear cells taken from 29 childhood ALL patients at diagnosis, on day 15 and day 33 of remission induction therapy using RT-qPCR methodology. RESULTS: Our results have shown interindividual differences in GAS5 expression at all time points. For each ALL patient, GAS5 expression was higher on day 15 in comparison to its level at diagnosis (p<0.0005). On day 33, the level of GAS5 expression decreased in comparison with day 15 (p<0.0005), but it was still significantly higher than at diagnosis for the majority of patients (p=0.001). Patients whose number of blasts on day 8 was below 100 per µL of peripheral blood had a higher GAS5 expression at diagnosis (p=0.016), and lower ratio day 15/diagnosis (p=0.009). CONCLUSIONS: Our results suggest that the expression level of GAS5 could be a potential marker of therapy response in remission induction therapy of childhood ALL.

Pharmacogenomic and Pharmacotranscriptomic Profiling of Childhood Acute Lymphoblastic Leukemia: Paving the Way to Personalized Treatment.

Personalized medicine is focused on research disciplines which contribute to the individualization of therapy, like pharmacogenomics and pharmacotranscriptomics. Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. It is one of the pediatric malignancies with the highest cure rate, but still a lethal outcome due to therapy accounts for 1%⁻3% of deaths. Further improvement of treatment protocols is needed through the implementation of pharmacogenomics and pharmacotranscriptomics. Emerging high-throughput technologies, including microarrays and next-generation sequencing, have provided an enormous amount of molecular data with the potential to be implemented in childhood ALL treatment protocols. In the current review, we summarized the contribution of these novel technologies to the pharmacogenomics and pharmacotranscriptomics of childhood ALL. We have presented data on molecular markers responsible for the efficacy, side effects, and toxicity of the drugs commonly used for childhood ALL treatment, i.e., glucocorticoids, vincristine, asparaginase, anthracyclines, thiopurines, and methotrexate. Big data was generated using high-throughput technologies, but their implementation in clinical practice is poor. Research efforts should be focused on data analysis and designing prediction models using machine learning algorithms. Bioinformatics tools and the implementation of artificial i Lack of association of the CEP72 rs924607 TT genotype with intelligence are expected to open the door wide for personalized medicine in the clinical practice of childhood ALL.

Genetic predictors of celiac disease, lactose intolerance, and vitamin D function and presence of peptide morphins in urine of children with neurodevelopmental disorders.

Gastrointestinal disturbances, nutritional deficiencies, and food intolerances are frequently observed in children with neurodevelopmental disorders (NDD). To reveal possible association of celiac disease risk variants (HLA-DQ), lactose intolerance associated variant (LCT-13910C>T) as well as variant associated with vitamin D function (VDR FokI) with NDD, polymerase chain reaction-based methodology was used. Additionally, intestinal peptide permeability was estimated in NDD patients and healthy children by measuring the level of peptides in urine using high-performance liquid chromatography. Levels of opioid peptides, casomorphin 8, and gluten exorphin C were significantly elevated in urine samples of NDD patients (P = 0.004 and P = 0.005, respectively), but no association of genetic risk variants for celiac disease and lactose intolerance with NDD was found. Our results indicate that increased intestinal peptide permeability observed in analyzed NDD patients is not associated with genetic predictors of celiac disease or lactose intolerance. We have also found that FF genotype of VDR FokI and lower serum levels of vitamin D (25-OH) showed association with childhood autism (CHA), a subgroup of NDD. We hypothesize that vitamin D might be important for the development of CHA.

Pharmacogenomic markers of glucocorticoid response in the initial phase of remission induction therapy in childhood acute lymphoblastic leukemia.

Background Response to glucocorticoid (GC) monotherapy in the initial phase of remission induction treatment in childhood acute lymphoblastic leukemia (ALL) represents important biomarker of prognosis and outcome. We aimed to study variants in several pharmacogenes (NR3C1, GSTs and ABCB1) that could contribute to improvement of GC response through personalization of GC therapy. Methods Retrospective study enrolling 122 ALL patients was carried out to analyze variants of NR3C1 (rs33389, rs33388 and rs6198), GSTT1 (null genotype), GSTM1 (null genotype), GSTP1 (rs1695 and rs1138272) and ABCB1 (rs1128503, rs2032582 and rs1045642) genes using PCR-based methodology. The marker of GC response was blast count per microliter of peripheral blood on treatment day 8. We carried out analysis in which cut-off value for GC response was 1000 (according to Berlin-Frankfurt-Munster [BFM] protocol), as well as 100 or 0 blasts per microliter. Results Carriers of rare NR3C1 rs6198 GG genotype were more likely to have blast count over 1000, than the non-carriers (p = 0.030). NR3C1 CAA (rs33389-rs33388-rs6198) haplotype was associated with blast number below 1000 (p = 0.030). GSTP1 GC haplotype carriers were more likely to have blast number below 1000 (p = 0.036), below 100 (p = 0.028) and to be blast negative (p = 0.054), while GSTP1 GT haplotype and rs1138272 T allele carriers were more likely to be blasts positive (p = 0.034 and p = 0.024, respectively). ABCB1 CGT (rs1128503-rs2032582-rs1045642) haplotype carriers were more likely to be blast positive (p = 0.018). Conclusions Our results have shown that NR3C1 rs6198 variant and GSTP1 rs1695-rs1138272 haplotype are the most promising pharmacogenomic markers of GC response in ALL patients.

Importance of TLR9-IL23-IL17 axis in inflammatory bowel disease development: Gene expression profiling study.

BACKGROUND AND AIMS: Mucosal gene expression have not been fully enlightened in inflammatory bowel disease (IBD). Aim of this study was to define IL23A, IL17A, IL17F and TLR9 expression in different IBD phenotypes. METHODS: Evaluation of mRNA levels was performed in paired non-inflamed and inflamed mucosal biopsies of newly diagnosed 50 Crohn's disease (CD) and 54 ulcerative colitis (UC) patients by quantitative real-time PCR analysis. RESULTS: IL17A and IL17F expression levels were significantly increased in inflamed IBD mucosa. Inflamed CD ileal and UC mucosa showed increased IL23A, while only inflamed CD ileal samples showed increased TLR9 mRNA level. Correlation between analysed mRNAs levels and endoscopic and clinical disease activity were found in UC, but only with clinical activity in CD. CONCLUSION: Both CD and UC presented expression of Th17-associated genes. Nevertheless, expression profiles between different disease forms varies which should be taken into account for future research and therapeutics strategies.

Variants in TPMT, ITPA, ABCC4 and ABCB1 Genes As Predictors of 6-mercaptopurine Induced Toxicity in Children with Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia is the most common childhood malignancy. Optimal use of anti leukemic drugs has led to less toxicity and adverse reactions, and a higher survival rate. Thiopurine drugs, including 6-mercaptopurine, are mostly used as antileukemic medications in the maintenance phase of treatment for children with acute lymphoblastic leukemia. For those patients, TPMT genotype- tailored 6-mercaptopurine therapy is already implemented in the treatment protocols. We investigated the role of TPMT, ITPA, ABCC4 and ABCB1 genetic variants as predictors of outcome and 6-mercaptopurine induced toxicity during the maintenance phase of treatment in pediatric acute lymphoblastic leukemia. METHODS: Sixty-eight children with acute lymphoblastic leukemia were enrolled in this study. Patients have been treated according to ALL IC-BFM 2002 or ALL IC-BFM 2009 protocols. Toxicity and adverse events have been monitored via surrogate markers (off-therapy weeks, episodes of leu - ko penia and average 6-mercaptopurine dose) and a prob- abilistic model was employed to predict overall 6-mercaptopurine related toxicity. RESULTS: We confirmed that patients with acute lymphoblastic leukemia that carry inactive TPMT allele(s) require 6- mercaptopurine dose reduction. ITPA and ABCC4 genetic variants failed to show an association with 6-mercapto - purine induced toxicity during the maintenance phase. Carriers of ABCB1 variant allele experienced greater hepatotoxicity. The probabilistic model Neural net which considered all the analysed genetic variants was assessed to be the best prediction model. It was able to discriminate ALL patients with good and poor 6-mercaptopurin tolerance in 71% of cases (AUC=0.71). CONCLUSIONS: This study contributes to the design of a panel of pharmacogenetic markers for predicting thiopurineinduced toxicity in pediatric ALL.

Genetic and environmental factors significant for the presentation and development of inflammatory bowel disease.

OBJECTIVES: The aim of the study was to evaluate associations between inflammatory bowel disease (IBD) presentation and variants in NOD2, TLR4, TNF-α, IL-6, IL-1ß, and IL-RN genes in order to identify possible environmental factors that may affect IBD occurrence, investigate potential predictors for surgical treatment of IBD, and correlate the presence of granulomas in biopsy specimens with clinical characteristics of Crohn's disease (CD) patients. PATIENTS AND METHODS: We genotyped 167 IBD patients using PCR-based methodology and tested for disease genotype-phenotype associations. RESULTS: In CD patients ileal localization of disease was more frequent in NOD2 variant carriers. Ileal CD was associated with IL-6 GC+CC genotypes, identifying C allele as a possible marker of increased risk for ileal CD. In CD patients a positive family history for IBD was related to earlier onset of disease, higher risk for CD-related surgery, and appendectomy. CD patients who are TLR4 299Gly carriers are at higher risk for surgery at onset of the disease compared with TLR4 299Asp variant carriers. The presence of granuloma in biopsy specimens was more frequent in patients in whom a diagnosis of CD was made during emergency surgery. Multivariate analysis showed that CD carriers of the 299Gly allele had a 4.6-fold higher risk for emergency surgery before CD diagnosis is established compared with noncarriers, suggesting an aggressive disease course. Granuloma in endoscopic biopsies is detected 5.4-fold more frequently in patients treated surgically at the time of diagnosis. CONCLUSION: Genetic variants together with epidemiological and clinical data of IBD patients could potentially be used as predictors of the disease course.

New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

TPMT gene expression is increased during maintenance therapy in childhood acute lymphoblastic leukemia patients in a TPMT gene promoter variable number of tandem repeat-dependent manner.

AIMS: 6-mercaptopurine influences in vitro TPMT gene expression in a TPMT promoter variable number of tandem repeats (VNTR)-dependent manner. We studied TPMT expression following 6-mercaptopurine and methotrexate administration in childhood acute lymphoblastic leukemia (ALL) patients and the pharmacogenomic potential of the VNTR architecture. MATERIALS & METHODS: TPMT gene expression was determined in childhood ALL patients at diagnosis (n = 57) and during the maintenance therapy (n = 27). RESULTS: A threefold increase of TPMT gene expression was obtained during maintenance therapy, modulated by the architecture of the VNTR region. CONCLUSION: The TPMT promoter genetic variants need to be considered at the very beginning of the maintenance therapy for childhood ALL patients. The TPMT promoter VNTR region may serve as a pharmacogenomic biomarker when introducing thiopurine therapy.

Variations in inflammatory genes as molecular markers for prediction of inflammatory bowel disease occurrence.

OBJECTIVE: Research on inflammatory bowel disease (IBD) has highlighted genes involved in the regulation of inflammatory responses as contributors to disease pathogenesis. This study aimed to evaluate the associations between IBD and variations in NOD2, TLR4, TNF-α, IL-6, IL-1ß and IL-1RN genes, and to use the genetic data obtained in predictive modeling. METHODS: A total of 167 IBD patients and 101 healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Using the genotype data attained as the input to various classification algorithms, IBD prediction models were designed. The area under the receiver operating characteristic curve (AUROC) was used to measure their performance. RESULTS: Significant associations were found between Crohn's disease (CD) and minor NOD2 variants, as well as TLR4 299Gly, TNF-α G-308A, IL-6 G-174C and IL-1RN VNTR A2 variants, while ulcerative colitis (UC) was associated only with IL-1RN VNTR A2 variants. CD and UC showed highly significant difference in the allelic distribution of TNF-α G-308A, where the A allele was found to be related to CD, and the G allele to UC. A combined effect of patients' gender and TLR4 variants was observed among CD patients. When all analyzed genotype and gender data were used, prediction performance achieved a maximum AUROC of 0.690 for CD and 0.601 for UC dataset. CONCLUSION: Variations in the genes involved in immune regulation are genetic factors of importance in IBD susceptibility that could potentially be used as predictors of disease development.

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner.

AIM: TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. CONCLUSION: These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.