Genetic Variation In Genes Research Articles

Integrating rare genetic variants into pharmacogenetic drug response predictions

BackgroundVariability in genes implicated in drug pharmacokinetics or drug response can modulate treatment efficacy or predispose to adverse drug reactions. Besides common genetic polymorphisms, recent sequencing projects revealed a plethora of rare genetic variants in genes encoding proteins involved in drug metabolism, transport, and response.ResultsTo understand the global importance of rare pharmacogenetic gene variants, we mapped the variability in 208 pharmacogenes by analyzing exome sequencing data from 60,706 unrelated individuals and estimated the importance of rare and common genetic variants using a computational prediction framework optimized for pharmacogenetic assessments. Our analyses reveal that rare pharmacogenetic variants were strongly enriched in mutations predicted to cause functional alterations. For more than half of the pharmacogenes, rare variants account for the entire genetic variability. Each individual harbored on average a total of 40.6 putatively functional variants, rare variants accounting for 10.8% of these. Overall, the contribution of rare variants was found to be highly gene- and drug-specific. Using warfarin, simvastatin, voriconazole, olanzapine, and irinotecan as examples, we conclude that rare genetic variants likely account for a substantial part of the unexplained inter-individual differences in drug metabolism phenotypes.ConclusionsCombined, our data reveal high gene and drug specificity in the contributions of rare variants. We provide a proof-of-concept on how this information can be utilized to pinpoint genes for which sequencing-based genotyping can add important information to predict drug response, which provides useful information for the design of clinical trials in drug development and the personalization of pharmacological treatment.

Clinical Pharmacogenomics: Applications in Nephrology.

Pharmacogenomics is a tool for practitioners to provide precision pharmacotherapy using genomics. All providers are likely to encounter genomic data in practice with the expectation that they are able to successfully apply it to patient care. Pharmacogenomics tests for genetic variations in genes that are responsible for drug metabolism, transport, and targets of drug action. Variations can increase the risk for drug toxicity or poor efficacy. Pharmacogenomics can, therefore, be used to help select the best medication or aid in dosing. Nephrologists routinely treat cardiovascular disease and manage patients after kidney transplantation, two situations for which there are several high-evidence clinical recommendations for commonly used anticoagulants, antiplatelets, statins, and transplant medications. Successful use of pharmacogenomics in practice requires that providers are familiar with how to access and use pharmacogenomics resources. Similarly, clinical decision making related to whether to use existing data, whether to order testing, and if data should be used in practice is needed to deliver precision medicine. Pharmacogenomics is applicable to virtually every medical specialty, and nephrologists are well positioned to be implementation leaders.

Variation in coagulation and fibrinolysis genes evaluated for their contribution to cerebrovascular complications in adults with bacterial meningitis in the Netherlands

To study whether genetic variation in coagulation and fibrinolysis genes contributes to cerebrovascular complications in bacterial meningitis. We performed a nationwide prospective genetic association study in adult community-acquired bacterial meningitis patients. The exons and flanking regions of 16 candidate genes involved in coagulation and fibrinolysis pathways were sequenced. We analyzed whether genetic variation in these genes resulted in a higher risk of cerebrovascular complications, unfavorable outcome and differences in thrombocyte count on admission. From 2006 to 2011, a total of 1101 bacterial meningitis patients were identified of whom 622 supplied DNA for genotyping and passed genetic quality control steps. In 139 patients (22%) the episode of bacterial meningitis was complicated by cerebral infarction, and 188 (30%) had an unfavorable outcome. We identified the functional variant rs494860 in the protein Z (PROZ) gene as our strongest association with occurrence of cerebral infarction (odds ratio (OR) 0.49 (95% confidence interval 0.33-0.73), p = 5.2 × 10-4). After Bonferroni correction for multiple testing no genetic variant was significantly associated (p-value threshold 2.7 × 10-4). Our study suggests a functional genetic variation in the PROZ gene, rs494860, may be of importance in bacterial meningitis pathogenesis and cerebral infarction risk. Replication of this finding in other cohort studies populations is needed.

Patterns of geographic variation of thermal adapted candidate genes in Drosophila subobscura sex chromosome arrangements

BackgroundThe role of chromosomal arrangements in adaptation is supported by the repeatable clinal variation in inversion frequencies across continents in colonizing species such as Drosophila subobscura. However, there is a lack of knowledge on the genetic variation in genes within inversions, possibly targets of climatic selection, across a geographic latitudinal gradient. In the present study we analysed four candidate loci for thermal adaptation, located close to the breakpoints, in two chromosomal arrangements of the sex (A) chromosome of Drosophila subobscura with different thermal preferences. Individual chromosomes with A2 (the inverted arrangement considered warm adapted) or AST (the standard ancestral arrangement considered cold adapted) were sequenced across four European localities at varying latitudes, up to ~ 2500 Kms apart.ResultsImportantly, we found very low differentiation for each specific arrangement across populations as well as no clinal patterns of genomic variation. This suggests wide gene exchange along the cline. Differentiation between the sex chromosome arrangements was significant in the two more proximal regions relative to the AST orientation but not in the distal ones, independently of their location inside or outside the inversion. This can be possibly due to variation in the levels of gene flux and/or selection acting in these regions.ConclusionsGene flow appears to have homogenized the genetic content within-arrangement at a wide geographical scale, despite the expected diverse selective pressures in the specific natural environments of the different populations sampled. It is thus likely that the inversion frequency clines in this species are being maintained by local adaptation in face of gene flow. The differences between arrangements at non-coding regions might be associated with the previously observed differential gene expression in different thermal regimes. Higher resolution genomic scans for individual chromosomal arrangements performed over a large environmental gradient are needed to find the targets of selection and further elucidate the adaptive mechanisms maintaining chromosomal inversion polymorphisms.

Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome.

Silver-Russell syndrome (SRS) (mainly secondary to 11p15 molecular disruption) and Temple syndrome (TS) (secondary to 14q32.2 molecular disruption) are imprinting disorders with phenotypic (prenatal and postnatal growth retardation, early feeding difficulties) and molecular overlap. To describe the clinical overlap between SRS and TS and extensively study the molecular aspects of TS. We retrospectively collected data on 28 patients with disruption of the 14q32.2 imprinted region, identified in our center, and performed extensive molecular analysis. Seventeen (60.7%) patients showed loss of methylation of the MEG3/DLK1 intergenic differentially methylated region by epimutation. Eight (28.6%) patients had maternal uniparental disomy of chromosome 14 and three (10.7%) had a paternal deletion in 14q32.2. Most patients (72.7%) had a Netchine-Harbison SRS clinical scoring system ≥4/6, and consistent with a clinical diagnosis of SRS. The mean age at puberty onset was 7.2 years in girls and 9.6 years in boys; 37.5% had premature pubarche. The body mass index of all patients increased before pubarche and/or the onset of puberty. Multilocus analysis identified multiple methylation defects in 58.8% of patients. We identified four potentially damaging genetic variants in genes encoding proteins involved in the establishment or maintenance of DNA methylation. Most patients with 14q32.2 disruption fulfill the criteria for a clinical diagnosis of SRS. These clinical data suggest similar management of patients with TS and SRS, with special attention to their young age at the onset of puberty and early increase of body mass index.

Pharmacogenetics of Metabolic Genes of Anthracyclines in Acute Myeloid Leukemia.

Anthracyclines in combination with cytarabine have been the standard therapy for acute myeloid leukemia (AML) for decades with high efficacy. However, the majority of patients will show initial resistance or will relapse after initial complete remission. Genetic variability in genes involved in anthracyclines metabolic pathway could be one of the causes of the interindividual differences in clinical outcomes. A systematic review of published studies in AML cohorts was carried out in order to analyze the influence of polymorphisms in genes of anthracycline metabolism on efficacy and toxicity. Polymorphisms in the main enzymes of anthracyclines metabolism (CBR, AKR, NQO1, NOS3) have been related to lower enzymatic activity and higher cardiotoxicity. Moreover, variant alleles in the genes of carcinogens and chemotherapy neutralizing enzymes (GST, SULT, NADP(H) oxidase) have been associated with ROS generation and drug efficacy, influencing the survival rates and cardiac toxicities. In addition, genetic variability in the transporters of anthracyclines could affect the intake in cells, including influx (SLC28A3, SLC22A12, SLCO1B1) and efflux transporters (ABCB1, ABCC1, ABCC3, ABCG2). The knowledge of the role of pharmacogenetics in anthracyclines metabolism could explain the differences observed in their disposition in leukemic cells. These genetic variants are proposed biomarkers in clinical practice in order to individualize chemotherapy schemes, potentially increasing the effectiveness and reducing the toxicities.

Maternal polymorphisms in glutathione-related genes are associated with maternal mercury concentrations and early child neurodevelopment in a population with a fish-rich diet

IntroductionGlutathione (GSH) pathways play a key role the metabolism and elimination of the neurotoxicant methylmercury (MeHg). We hypothesized that maternal genetic variation linked to GSH pathways could influence MeHg concentrations in pregnant mothers and children and thereby also affect early life development. MethodsThe GCLM (rs41303970, C/T), GCLC (rs761142, T/G) and GSTP1 (rs1695, A/G) polymorphisms were genotyped in 1449 mothers in a prospective study of the Seychellois population with a diet rich in fish. Genotypes were analyzed in association with maternal hair and blood Hg, fetal blood Hg (cord blood Hg), as well as children's mental (MDI) and motor development (PDI; MDI and PDI assessed by Bayley Scales of Infant Development at 20 months). We also examined whether genotypes modified the association between Hg exposure and developmental outcomes. ResultsGCLC rs761142 TT homozygotes showed statistically higher mean maternal hair Hg (4.12 ppm) than G carriers (AG 3.73 and GG 3.52 ppm) (p = 0.037). For the combination of GCLC rs761142 and GCLM rs41303970, double homozygotes TT + CC showed higher hair Hg (4.40 ppm) than G + T carriers (3.44 ppm; p = 0.018). No associations were observed between GSTP1 rs1695 and maternal hair Hg or between any genotypes and maternal blood Hg or cord blood Hg. The maternal GSTP1 rs1695 rare allele (G) was associated with a lower MDI among children (β = −1.48, p = 0.048). We also observed some interactions: increasing Hg in maternal and cord blood was associated with lower PDI among GCLC rs761142 TT carriers; and increasing Hg in hair was associated with lower MDI among GSTP1 rs1695 GG carriers. ConclusionsMaternal genetic variation in genes involved in GSH synthesis is statistically associated with Hg concentrations in maternal hair, but not in maternal or fetal blood. We observed interactions that suggest maternal GSH genetics may modify associations between MeHg exposure and neurodevelopmental outcomes.

Genetic Variation in Genes Underlying Diverse Dementias May Explain a Small Proportion of Cases in the Alzheimer’s Disease Sequencing Project

Background/Aims: The Alzheimer’s Disease Sequencing Project (ADSP) aims to identify novel genes influencing Alzheimer’s disease (AD). Variants within genes known to cause dementias other than AD have previously been associated with AD risk. We describe evidence of co-segregation and associations between variants in dementia genes and clinically diagnosed AD within the ADSP. Methods: We summarize the properties of known pathogenic variants within dementia genes, describe the co-segregation of variants annotated as “pathogenic” in ClinVar and new candidates observed in ADSP families, and test for associations between rare variants in dementia genes in the ADSP case-control study. The participants were clinically evaluated for AD, and they represent European, Caribbean Hispanic, and isolate Dutch populations. Results/Conclusions: Pathogenic variants in dementia genes were predominantly rare and conserved coding changes. Pathogenic variants within ARSA, CSF1R, and GRN were observed, and candidate variants in GRN and CHMP2B were nominated in ADSP families. An independent case-control study provided evidence of an association between variants in TREM2, APOE, ARSA, CSF1R, PSEN1, and MAPT and risk of AD. Variants in genes which cause dementing disorders may influence the clinical diagnosis of AD in a small proportion of cases within the ADSP.

Genetic Variations in Genes of the Stress Response Pathway are Associated with Prolonged Abstinence from Heroin

This study assesses whether genetic variants in stress-related genes are associated with prolonged abstinence from heroin in subjects that are not in long-term methadone treatment. Frequencies of 117 polymorphisms in 30 genes were compared between subjects with history of heroin addiction, either without agonist treatment (n=129) or in methadone maintenance treatment (n=923). SNP rs1500 downstream of CRHBP and an interaction of SNPs rs10482672 (NR3C1) and rs4234955 (NPY1R/NPY5R) were significantly associated with prolonged abstinence without agonist treatment. This study suggests that variability in stress-related genes may contribute to the ability of certain subjects to remain in prolonged abstinence from heroin, possibly due to higher resilience to stress.

Interaction between polymorphisms in aspirin metabolic pathways, regular aspirin use and colorectal cancer risk: A case-control study in unselected white European populations.

Regular aspirin use is associated with reduced risk of colorectal cancer (CRC). Variation in aspirin’s chemoprevention efficacy has been attributed to the presence of single nucleotide polymorphisms (SNPs). We conducted a meta-analysis using two large population-based case-control datasets, the UK-Leeds Colorectal Cancer Study Group and the NIH-Colon Cancer Family Registry, having a combined total of 3325 cases and 2262 controls. The aim was to assess 42 candidate SNPs in 15 genes whose association with colorectal cancer risk was putatively modified by aspirin use, in the literature. Log odds ratios (ORs) and standard errors were estimated for each dataset separately using logistic regression adjusting for age, sex and study site, and dataset-specific results were combined using random effects meta-analysis. Meta-analysis showed association between SNPs rs6983267, rs11694911 and rs2302615 with CRC risk reduction (All P<0.05). Association for SNP rs6983267 in the CCAT2 gene only was noteworthy after multiple test correction (P = 0.001). Site-specific analysis showed association between SNPs rs1799853 and rs2302615 with reduced colon cancer risk only (P = 0.01 and P = 0.004, respectively), however neither reached significance threshold following multiple test correction. Meta-analysis of SNPs rs2070959 and rs1105879 in UGT1A6 gene showed interaction between aspirin use and CRC risk (Pinteraction = 0.01 and 0.02, respectively); stratification by aspirin use showed an association for decreased CRC risk for aspirin users having a wild-type genotype (rs2070959 OR = 0.77, 95% CI = 0.68–0.86; rs1105879 OR = 0.77 95% CI = 0.69–0.86) compared to variant allele cariers. The direction of the interaction however is in contrast to that published in studies on colorectal adenomas. Both SNPs showed potential site-specific interaction with aspirin use and colon cancer risk only (Pinteraction = 0.006 and 0.008, respectively), with the direction of association similar to that observed for CRC. Additionally, they showed interaction between any non-steroidal anti-inflammatory drugs (including aspirin) use and CRC risk (Pinteraction = 0.01 for both). All gene x environment (GxE) interactions however were not significant after multiple test correction. Candidate gene investigation indicated no evidence of GxE interaction between genetic variants in genes involved in aspirin pathways, regular aspirin use and colorectal cancer risk.

Redefining the Phenotype of Dental Caries

Dental caries is a multifactorial infectious disease and a major public health problem estimated to affect 60-90% of school children as well as a vast number of adults. The aim of this work was to define patterns of progression of the disease based on longitudinal data in contrast to using a cross-sectional assessment. dmft/DMFT scores were collected at ages 5, 12, 14, 16, 17, and 18 from 876 individuals. We tested our newly defined phenotypes for association with genetic variants in genes shown to be associated with caries. We generated genotyping data using Taqman chemistry in markers of genes involved in processes such as enamel formation and salivary contributions. Kallikrein 4 (KLK4) was found to show a significant association with the created phenotypes (p = 0.0008 in a recessive model for low caries experience in the primary dentition vs. high caries experience in the primary dentition, and p = 0.0004 in a recessive model for caries free primary dentition vs. high caries experience in the primary dentition).

Differential Association of Cx37 and Cx40 Genetic Variants in Atrial Fibrillation with and without Underlying Structural Heart Disease.

Atrial fibrillation (AF) appears in the presence or absence of structural heart disease. The majority of foci causing AF are located near the ostia of pulmonary veins (PVs), where cardiomyocytes and vascular smooth muscle cells interdigitate. Connexins (Cx) form gap junction channels and participate in action potential propagation. Genetic variants in genes encoding Cx40 and Cx37 affect their expression or function and may contribute to PV arrhythmogenicity. DNA was obtained from 196 patients with drug-resistant, symptomatic AF with and without structural heart disease, who were referred for percutaneous catheter ablation. Eighty-nine controls were matched for age, gender, hypertension, and BMI. Genotyping of the Cx40 −44G > A, Cx40 +71A > G, Cx40 −26A > G, and Cx37 1019C > T polymorphisms was performed. The promoter A Cx40 polymorphisms (−44G > A and +71A > G) showed no association with non-structural or structural AF. Distribution of the Cx40 promoter B polymorphism (−26A > G) was different in structural AF when compared to controls (p = 0.03). There was no significant difference with non-structural AF (p = 0.50). The distribution of the Cx37 1019C > T polymorphism was different in non-structural AF (p = 0.03) but not in structural AF (p = 0.08) when compared to controls. Our study describes for the first time an association of drug-resistant non-structural heart disease AF with the Cx37 1019C > T gene polymorphism. We also confirmed the association of the Cx40 − 26G > A polymorphism in patients with AF and structural disease.

Chapter 5 - Epigenetic mechanisms underlying nervous system diseases

Epigenetic mechanisms act as control systems for modulating genomic structure and activity in response to evolving profiles of cell-extrinsic, cell-cell, and cell-intrinsic signals. These dynamic processes are responsible for mediating cell- and tissue-specific gene expression and function and gene-gene and gene-environmental interactions. The major epigenetic mechanisms include DNA methylation and hydroxymethylation; histone protein posttranslational modifications, nucleosome remodeling/repositioning, and higher-order chromatin reorganization; noncoding RNA regulation; and RNA editing. These mechanisms are intimately involved in executing fundamental genomic programs, including gene transcription, posttranscriptional RNA processing and transport, translation, X-chromosome inactivation, genomic imprinting, retrotransposon regulation, DNA replication, and DNA repair and the maintenance of genomic stability. For the nervous system, epigenetics offers a novel and robust framework for explaining how brain development and aging occur, neural cellular diversity is generated, synaptic and neural network connectivity and plasticity are mediated, and complex cognitive and behavioral phenotypes are inherited transgenerationally. Epigenetic factors and processes are, not surprisingly, implicated in nervous system disease pathophysiology through several emerging paradigms - mutations and genetic variation in genes encoding epigenetic factors; impairments in epigenetic factor expression, localization, and function; epigenetic mechanisms modulating disease-associated factors and pathways; and the presence of deregulated epigenetic profiles in central and peripheral tissues.

The Pharmacogenetics of Immune-Modulating Therapy.

Immunosuppressive drugs are a prerequisite in organ transplantation to prevent rejection and are also widely used in inflammatory diseases such as inflammatory bowel disease (IBD) or also in some hematologic malignancies-depending on the mode of action. For thiopurine analogs the polymorphic thiopurine S-methyltransferase (TPMT) was early detected to be associated with thiopurine-induced leukopenia; recent studies identified also NUDT15 to be related to this severe side effect. For drugs like methotrexate and mycophenolate mofetil a number of ADME genes like UDP-glucuronosyltransferases (UGTs) and ABC efflux transporters were investigated, however, with partly contradicting results. For calcineurin inhibitors like cyclosporine and in particular tacrolimus however, cytochrome P450 3A4 and 3A5 variants were found to significantly affect the pharmacokinetics. Genetic variants in genes encoding relevant pharmacodynamic proteins, however, lacked compelling evidence to affect the clinical outcome. This chapter reviews the current evidence on the association of pharmacogenetic traits to dose finding and clinical outcome of small-molecule immunosuppressants. Moreover this chapter critically summarizes suitability to apply pharmacogenetics in clinical practice in order to optimize immunosuppressant therapy.

Data Mining: Applying the AD&FTD Mutation Database to Progranulin.

The online AD&FTD Mutation Database ( http://www.molgen.vib-ua.be/FTDmutations ) was conceived to meet the needs of a comprehensive knowledge base of genetic variations in genes associated with monogenic forms of Alzheimer's disease (AD) and frontotemporal dementia (FTD). Today, the AD&FTD Mutation Database provides curated, referenced information of 764 genetic variants in APP, PSEN1, and PSEN2 associated with AD and GRN, C9orf72, TBK1, MAPT, VCP, CHMP2B, TARDBP, and FUS associated with FTD and related diseases. In addition, the database stores demographic and clinicogenetic data of 1646 dementia families associated with these mutations. In FTD, the granulin (GRN) gene has the highest number of different mutations (79/231=34%) and the second highest number of associated FTD families after C9orf72. In addition to the detailed mutation and patient information, summary reports in tabular and graphical formats can be consulted. Further, all variants can be uploaded to the human genome browser for custom-designed analyses.

A genetic variant of NLRP1 gene is associated with asbestos body burden in patients with malignant pleural mesothelioma

ABSTRACTThe presence of asbestos bodies (ABs) in lung parenchyma is considered a histopathologic hallmark of past exposure to asbestos fibers, of which there was a population of longer fibers. The mechanisms underlying AB formation are complex, involving inflammatory responses and iron (Fe) metabolism. Thus, the responsiveness to AB formation is variable, with some individuals appearing to be poor AB formers. The aim of this study was to disclose the possible role of genetic variants of genes encoding inflammasome and iron metabolism proteins in the ability to form ABs in a population of 81 individuals from North East Italy, who died after having developed malignant pleural mesothelioma (MPM). This study included 86 genetic variants distributed in 10 genes involved in Fe metabolism and 7 genetic variants in two genes encoding for inflammasome molecules. Genotypes/haplotypes were compared according to the number of lung ABs. Data showed that the NLRP1 rs12150220 missense variant (H155L) was significantly correlated with numbers of ABs in MPM patients. Specifically, a low number of ABs was detected in individuals carrying the NLRP1 rs12150220 A/T genotype. Our findings suggest that the NLRP1 inflammasome might contribute in the development of lung ABs. It is postulated that the NLRP1 missense variant may be considered as one of the possible host genetic factors contributing to individual variability in coating efficiency, which needs to be taken when assessing occupational exposure to asbestos.

Surfactant proteins gene variants in premature newborn infants with severe respiratory distress syndrome

ObjectiveGenetic surfactant dysfunction causes respiratory failure in term and near-term newborn infants, but little is known of such condition in prematures. We evaluated genetic surfactant dysfunction in premature newborn infants with severe RDS.Patients and methodsA total of 68 preterm newborn infants with gestational age ≤32 weeks affected by unusually severe RDS were analysed for mutations in SFTPB, SFTPC and ABCA3. Therapies included oxygen supplementation, nasal CPAP, different modalities of ventilatory support, administration of exogenous surfactant, inhaled nitric oxide and steroids. Molecular analyses were performed on genomic DNA extracted from peripheral blood and Sanger sequencing of whole gene coding regions and intron junctions. In one case histology and electron microscopy on lung tissue was performed.ResultsHeterozygous previously described rare or novel variants in surfactant proteins genes ABCA3, SFTPB and SFTPC were identified in 24 newborn infants. In total, 11 infants died at age of 2 to 6 months. Ultrastructural analysis of lung tissue of one infant showed features suggesting ABCA3 dysfunction.DiscussionRare or novel genetic variants in genes encoding surfactant proteins were identified in a large proportion (35%) of premature newborn infants with particularly severe RDS. We speculate that interaction of developmental immaturity of surfactant production in association with abnormalities of surfactant metabolism of genetic origin may have a synergic worsening phenotypic effect.

Genetic profile of patients with early onset inflammatory bowel disease

Inflammatory Bowel disease (IBD) is a widespread pathological condition with clinical heterogeneity and with different levels of severity. Although IBD usually occurs in young adults, onset in childhood and infancy are described in patients within the 10th and second year of age. By genome-wide association studies and meta-analysis, several genetic loci have been identified associated with an increased risk of developing IBD in Western populations with variants that may alter the normal mucosal immunity in the gastrointestinal tract.The clinical complexity and the heterogeneity of the IBD phenotype probably reflect the presence of genetic heterogeneity where different genes or combinations of them may be involved, together with environmental factors. We hypothesized that patients with early onset IBD could have either more severe genetic variants in genes associated with IBD or multiple variants in different genes. Under the multifactorial diseases is crucial to consider the small contribution of a single variant in all not only to other small variations in the same gene but also in different genes belonging to the same pathway.We performed direct gene sequencing looking for 94 variations in NOD2, ATG16L1, IL23R, IL10R, IL10 and XIAP genes previously shown as correlated with IBD both in multifactorial and in Mendelian models. All variants identified are known in literature as being associated with IBD except for three variants in the genes NOD2, IL10 and IL10RB that even though present in online databases have never been involved in association studies on IBD patients. Moreover, we coupled genetic variants identification with an accurate “in silico” analysis to verify their predictive impact on the protein structure and function. The in-silico prediction of these variants results as benign therefore even if they exhibit a very low frequency in control population being benign, they cannot be considered pathogenic as monogenic disease but fall within the multifactorial range.The variants identified in our study partially reflect the association data described in the literature but there are no significant differences with the onset of disease (VEO vs EO-IBD).

A Protective Role of DNA Repair Genes Against Acute Graft Versus Host Disease in Children

Introduction:Acute Graft versus Host Disease (aGvHD) occurs in 20-50% of pediatric patients who undergo allogeneic stem cell transplantation. Despite advances in HLA-typing methods and post-transplant immune suppression, it remains a significant cause of mortality and morbidity. Conditioning regimens include alkylating agents that exert their effects through their ability to directly or indirectly damage DNA. Unfortunately normal cells are also damaged due to their rapid proliferation cycles, increasing the probability of treatment-related toxicities. The most vulnerable targets among these damaged tissues are the skin, the intestinal epithelium, and the liver: In this study, our aim was to find biomarkers for aGvHD by focussing on how inter-individual differences in DNA repair mechanisms due to genetic variants in genes encoding DNA repair proteins might affect toxicity.Methods:The study included 115 children that had undergone allo-HSCT at four different centers. All patients received a Busulfan(Bu)/Cyclophosphamide(Cy) conditioning regimen. Pharmacokinetic-guided dose adjustment was performed for Bu to obtain a concentration at the steady state (Css) between 600 and 900 ng/ml. Patients received 16 doses of Bu followed by, i.v. Cy (200mg/kg total dose, 80% of patients) or CyVP16 (120mg/kg total dose). Cyclosporine was given as GvHD prophylaxis, and Methotrexate (MTX) or steroids were added to bone marrow and cord blood transplantation, respectively. ATG was given to 75% of patients. HLA matching was as follows: MRD = 37%; MUD = 21%; MMRD = 4%; MMUD = 38%. Acute GvHD was diagnosed according to the 1994 consensus conference up to day 180 post HSCT. Peripheral blood was collected and the DNA was extracted prior to transplant. Fifty-one single nucleotide polymorphisms (SNPs) within seventeen DNA repair genes were chosen for investigation. Cumulative incidence analysis of aGvHD 2-4 was performed using Kaplan-Meier analysis and log-rank test. Multivariate Cox regression was performed to estimate the impact of genotype on clinical outcome in the presence of other covariates.Results:The most significant finding came from one SNP (rs10764881, G>A) located in the promoter of the MGMT gene. Patients with rs10764881 GG genotypes had a lower risk for aGvHD 2-4 incidence (Figure 1). This variant was not associated with any other treatment related toxicities nor relapses. Multivariate analysis including MGMT rs10764881 with known aGvHD risk covariates did not influence the model. In addition, from the expression analysis that we performed on 24 lymphoblastoid cell lines rs10764881 GG carriers showed 1.5 fold higher expression compared to AA or AG carriers (Figure 2). We were able to confirm experimentally with luciferase reporter constructs the impact of this variant on promoter function. Plasmids, which included the promoter sequence with rs10764881 demonstrated higher luciferase protein levels, compared to plasmids with a promoter sequence excluding rs10764881 (p= 0.01), suggesting the presence of an enhancement element close to the variant region (Figure 3). Electrophoretic mobility shift assays confirmed the presence of a Glucocorticoid Receptor Element (GRE) near to this variant. To understand whether the enhancing effects are related to corticosteroids, keratinocytes transfected with the gene reporter plasmids were stimulated with dexamethasone and luciferase expression was examined as previously. Exposing the cells with dexamethasone significantly increased protein expression of luciferase in both plasmids compared to their non-treated plasmid construct. The highest response was seen from the plasmid containing variant rs10764881 G (Figure 3). However, clinically we did not find clear differences between steroid and non-steroids recipients against rs10764881 with regards to aGvHD vulnerability (Figure 4).Conclusions:We hypothesize that the reason why variant rs10764881 GG might have a protective effect against aGvHD is due to its higher expression levels, resulting in more efficient DNA repair, in turn diminishing the immune response, reducing inflammation and hence causing less aGvHD. Thus aGvHD rs10764881 GG could potentially be a biomarker for aGvHD protection. The effects of steroids on MGMT expression needs to be addressed in future studies. [Display omitted] DisclosuresBader:Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding. Bittencourt:Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel Grant; Amgen Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy.

Role of Genetic Variations in Determining Treatment Outcome in Head and Neck Cancer

Worldwide, head and neck squamous cell carcinoma (HNSCC) is responsible for &gt;550,000 diagnoses and 380,000 deaths annually. It originates in the upper aerodigestive tract and has a multifactorial origin involving both genetic and lifestyle risk factors. The clinical management of HNSCC involves surgery, radiotherapy, and chemotherapy. Several studies point to the role of genetic variations in predicting drug efficacy and toxicity. Cancer pharmacogenomics has fast emerged as a new and promising field for the early identification of genetic markers that can predict drug response or toxicity, with the number of studies of genetic polymorphisms as prognostic factors of HNSCC treatment outcomes growing. The number of studies evaluating the association of candidate polymorphisms in drug-metabolising Phase I and II enzymes with treatment outcome far exceed the studies involving other candidate genes, such as those involved in drug metabolism, DNA repair, and cell cycle regulation. This review focusses on the relevance of genetic variations in genes, where the corresponding gene products play an important role in drug metabolism (TPMT, DPD), DNA repair (X-ray repair cross complementing 1), cell cycle (tumour protein P53), and carcinogenesis (matrix metalloproteinase 3 and 7), thereby contributing to the treatment outcome for HNSCC. This could greatly help clinicians in identifying genetic markers useful for the selection of optimal drugs, dose, and treatment duration on an individual basis, resulting in improved drug efficacy and decreased toxicity. However, further studies are needed in well characterised and larger HNSCC populations with proper validation of pharmacogenetic markers in experimental settings before application in clinical routine diagnostics.