Complete Linkage Disequilibrium Research Articles

Genome-Wide Association Study in Thai Tsunami Survivors Identified Risk Alleles for Posttraumatic Stress Disorder

Posttraumatic stress disorder (PTSD) is a psychiatric disorder found in individuals afflicted by a traumatic event. Multiple environmental and genetic factors can contribute to PTSD susceptibility. Since it is rare to find members of the same family afflicted by the same catastrophic event, it is not practical to determine PTSD susceptibility genes by a gene linkage analysis. A natural disaster, such as the 2004 Tsunami, provided us with a rare chance for a genetic analysis of PTSD. To identify SNPs associated with PTSD susceptibility, we conducted a genome-association study (GWAS) in Thai-Tsunami survivors. Initial phase of the study with 396 chronic PTSD patients and 457 controls, we identified top ninety SNPs (P -4), which were further assessed in the second phase with 395 chronic PTSD patients and 798 controls. Two SNPs (rs267950 and rs954406), were identified in the second phase, and subjected to fine mapping using a data set from both phases. SNP rs267943 showed the strongest association with PTSD susceptibility and was in complete linkage disequilibrium with SNP rs267950 with P = 6.15 × 10-8, OR = 1.46 and 95% CI = 1.19 - 1.79, reaching genome-wide significance. SNP rs267943 is located on chromosome 5 in the intron of the death-associated protein 1 (DAP1) gene and, when linked to a synthetic promoter, could regulate transcription. To our knowledge, this is the first GWAS for PTSD susceptibility in an Asian population which could provide an important insight into the genetic contribution of PTSD and may lead to new treatment strategies for PTSD.

Polymorphisms in NFKB1 and NFKBIA Genes Modulate the Risk of Developing Prostate Cancer among Han Chinese.

BackgroundNuclear factor kappa B (NF-κB) pathway proteins play an important role in modulating inflammation and other carcinogenic processes. Polymorphisms within NF-κB pathway genes may influence cancer risk. This study aimed to examine the association between NFKB19-4 ATTG ins→del, NFKBIA 3′ UTR A→G, -826CT and -881AG polymorphisms and prostate cancer risk among Chinese.Material/MethodsThe polymorphisms were genotyped via PCR-RFLP technique on 936 prostate cancer patients and 936 population-based healthy controls. Logistic regression model was used to measure the risk association present.ResultsWith the exception of NFKBIA 3′ UTR polymorphism, the heterozygous and mutant genotypes of the other polymorphisms were significantly associated with prostate cancer risk. For NFKB1 polymorphism, a decreased risk was observed, with adjusted OR: 0.69; 95% CI: 0.44, 0.98; P=0.01 (heterozygous) and adjusted OR: 0.60; 95% CI: 0.37, 0.91; P=0.02 (mutant). NFKBIA -826CT and -881AG polymorphisms were in complete linkage disequilibrium and shared the same risk association, with adjusted OR: 1.34; 95% CI: 1.09, 1.62; P=0.02 (heterozygous) and adjusted OR: 2.83; 95% CI: 1.79, 4.50; P=0.01 (mutants). Interestingly, the impact of the NFKB1 polymorphism was not present in nonsmokers and younger (<60 years) subjects (P<0.05).ConclusionsIn conclusion, polymorphisms in NFKB1 and NFKBIA genes may modulate the risk of developing prostate cancer among Chinese.

Polymorphisms of GLP-1 receptor gene and response to GLP-1 analogue in patients with poorly controlled type 2 diabetes.

Aim. The relationship between genetic polymorphisms of the glucagon-like peptide-1 (GLP-1) receptor (GLP1R) gene and unresponsiveness to GLP-1 analogue treatment in patients with poorly controlled type 2 diabetes mellitus (DM) is unclear. Methods. Thirty-six patients with poorly controlled type 2 DM were enrolled and they received six days of continuous subcutaneous insulin infusion for this study. After the normalization of blood glucose in the first 3 days, the patients then received a combination therapy with injections of the GLP-1 analogue, exenatide, for another 3 days. All 13 exons and intron-exon boundaries of the GLP1R gene were amplified to investigate the association. Results. The short tandem repeat at 8GA/7GA (rs5875654) had complete linkage disequilibrium (LD, with r 2 = 1) with single nucleotide polymorphism (SNP) rs761386. Quantitative trait loci analysis of GLP1R gene variation with clinical response of GLP1 analogue showed the missense rs3765467 and rs761386 significantly associated with changes in the standard deviation of plasma glucose (SDPGbaseline − SDPGtreatment with GLP-1 analogue) (P = 0.041 and 0.019, resp.). The reported P values became insignificant after multiple testing adjustments. Conclusion. The variable response to the GLP-1 analogue was not statistically correlated with polymorphisms of the GLP1R gene in patients with poorly controlled type 2 DM.

Polymorphisms of the bovine MC3R gene and their associations with body measurement traits and meat quality traits in Qinchuan cattle.

The melanocortin 3 receptor (MC3R) gene, which belongs to the rhodopsin-like family A of the G protein-coupled receptor family, plays a crucial role in feed efficiency and energy homeostasis. The aim of this study was to examine associations between bovine MC3R gene polymorphisms and body measurement traits (BMTs) and meat quality traits (MQTs). We identified three synonymous mutations (T429C, T537C, and T663C) in exon 1 of the MC3R gene in Chinese Qinchuan beef cattle (N = 271) by sequencing. D' and r(2) values revealed that these three SNPs were in strong linkage disequilibrium (LD) (r(2) > 0.33); the T429C and T537C SNPs were in complete LD (D' = 1 and r(2) = 1). Association analyses revealed that the SNPs were significantly associated with BMTs and MQTs in Qinchuan cattle. Individuals with the wild homozygotic genotypes g.TTTT and g.TT had significantly higher values of chest depth, heart girth, back fat thickness, intramuscular fat content, and loin muscle area than the mutant heterozygotic genotypes g.TCTC and g.TC. These results suggest that the MC3R gene affects MQTs in Qinchuan cattle, and that it may be a good candidate gene for marker-assisted selection.

Influence of MDM2 SNP309 and SNP285 status on the risk of cancer in the breast, prostate, lung and colon.

MDM2 is a key regulator of the p53 tumor suppressor protein and is overexpressed in many human cancers. Two single nucleotide polymorphisms (SNPs) located in the MDM2 intronic promoter (P2) have been found to exert biological function. The G-allele of SNP309T>G; rs2279744 increases MDM2 transcription and has been linked to increased cancer risk. In contrast, the less frequent SNP285G>C; rs117039649, which is in complete linkage disequilibrium with SNP309 (generating a SNP285C/309G variant haplotype), has been related to reduced MDM2 transcription and to reduced risk of breast, endometrial and ovarian cancer. In this large population-based case-control study, we genotyped SNP309 and SNP285 in 10,830 individuals, including cases with cancer of the breast (n=1,717), colon (n=1,532), lung (n=1,331) and prostate (n=2,501), as well as 3,749 non-cancer controls. We found a slightly reduced risk for lung cancer among individuals harboring the SNP309TG/GG genotypes compared to the SNP309TT genotype (OR= 0.86; CI = 0.67-0.98), but this association was restricted to women (OR = 0.77; CI = 0.63-0.95) and was not present among men (OR = 0.91; CI = 0.77-1.08). Consistent with previous findings, we found a reduced risk for breast cancer among individuals carrying the SNP285GC/309GG genotype versus the SNP285GG/309GG genotype (OR = 0.55; CI = 0.33-0.93). In conclusion, our data support the hypothesis that the effects of both SNP285 and SNP309 status are tissue dependent.

Cell-Free Hemoglobin, HMOX1 and APOL1 in Sickle Cell Nephropathy

We recently reported that hemoglobinuria is associated with chronic kidney disease (CKD) stage and progression in sickle cell disease (SCD) (Saraf et al. BJH 2014). To further investigate a potential role of cell-free hemoglobin in SCD nephropathy, we measured urinary concentrations of kidney injury molecule-1 (KIM-1), a biomarker of tubular injury, and nephrin, a marker of glomerular injury, in 32 University of Illinois at Chicago (UIC) patients. Urine KIM-1 concentration directly correlated with increasing urine cell-free hemoglobin concentration (P = 0.003) (Figure 1) but urine nephrin concentration did not. To determine biological responses of tubular cells to cell-free hemoglobin, we added lyophilized hemoglobin to cultured human kidney-2 (HK2) tubular cells. Supernatant KIM-1 concentrations increased progressively with increasing cell-free hemoglobin exposure (Figure 2) while total cell number and cell viability were stable. Using a fluorescein-labeled hemoglobin assay, we found that cell-free hemoglobin bound to HK2 cells in a competitive manner. To determine whether enzymes for metabolizing hemoglobin and protecting from reactive oxygen species are affected by exposure of HK2 cells to cell-free hemoglobin, we evaluated candidate gene expression using rt-PCR. The relative expression of heme oxygenase-1 (HMOX1) (Figure 3) progressively increased with increasing cell-free hemoglobin dose while expression of superoxide dismutase 1 and 2, catalase, glutathione reductase, and glutathione synthetase did not change.We then focused on variants of HMOX1 in a cohort of 247 UIC SCD patients. Genotyping was carried out using Affymetrix Axiom genome-wide Pan-African array in DNA isolated from peripheral blood mononuclear cells. Examination of 11 tag SNPs within +/-10 kb of HMOX1 identified a SNP in the promoter region of HMOX1 (rs743811, minor allele frequency 0.14) that had a significant association with CKD stage (β = -1.0, P = 0.00032) and non-significant associations of the same direction with end stage renal disease (ESRD) (β = -0.9, P = 0.2) and hemoglobinuria (β = -0.2, P = 0.6). Validation studies were conducted in 517 SCD patients from the Walk-Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk-PHaSST) cohort. Genotyping was performed using the Illumina Human 610-Quad SNP array and imputed using the HapMap II reference panels for HMOX1 tag SNPs. HMOX1 rs743811 had a significant associated with ESRD (β = -1.6, P = 0.014) and non-significant associations of a similar direction with CKD stage (β = -0.2, P = 0.3) and hemoglobinuria (β = -0.3, P = 0.3).Homozygosity or compound heterozygosity for the G1 and G2 variants of APOL1, encoding apolipoprotein 1, have been implicated in CKD in African Americans with and without SCD (Ashley-Koch et al. BJH 2011), and we therefore examined their association with hemoglobinuria-associated CKD in the UIC cohort. The S342G and I384M substitutions are in almost complete linkage disequilibrium and are termed G1; the deletion of two amino acids, N388 and Y389, is termed G2. The G2 variant was further imputed using reference panels of the 1000 Genomes Project. Homozygosity or compound heterozygosity of the G1/G2 variant was associated with hemoglobinuria (β = 2.0, P = 0.00018), CKD stage (β = 1.0, P = 0.022), and ESRD (β = 1.9, P = 0.036). For validation studies in Walk-PHaSST, the G1 and G2 variants of APOL1 were imputed using the 1000 Genomes Project. Homozygosity or compound heterozygosity of G1/G2 in the Walk-PHaSST cohort was associated with hemoglobinuria (β = 0.7, P = 0.021), but associations of a similar direction with CKD stage (β = 0.2, P = 0.6) and ESRD (β = 1.1, P = 0.3) were not statistically significant.Our findings are consistent with the possibility that cell-free hemoglobin contributes to sickle cell nephropathy through tubular injury. A SNP in the promoter region of HMOX1 is associated with CKD stage in the UIC cohort and ESRD in the Walk-PHaSST cohort, raising the possibility that altered HMOX1 expression can have a role in SCD-associated CKD. Our results also point to a novel association of the G1/G2 variants of APOL1 with cell-free hemoglobin-mediated CKD in SCD subjects. Future studies to explore the potential roles of HMOX1 and APOL1 in cell-free hemoglobin-associated sickle cell nephropathy are warranted. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Population Pharmacokinetics of Daunorubicin in AML: Influence on Clinical Outcome

Daunorubicin (Dnr) has been the choice of anthracycline in the induction chemotherapy of Acute Myeloid Leukemia (AML) along with Cytarabine for the past 4 decades. Drug resistance and relapse of the disease has been the major concern in the success of AML therapy. Several factors, including pharmacokinetics (PK) of Dnr could have impact on treatment outcome as pharmacokinetic effects might limit the amount of drug that reaches the tumor. In this study, we analyzed the factors influencing plasma PK of Daunorubicin and its major metabolite Daunorubicinol (DOL) in AML patients receiving induction chemotherapy and compared PK parameters with clinical outcome (relapse).Sixty four adult denovo AML patients were enrolled in the study after obtaining informed consent. Dnr was given as 1 hour bolus infusion at a dose of 60mg/m2/day for the first 3 days of induction chemotherapy. Peripheral blood were collected at 0, 15min, 1hr, 2hr, 4hr, 6hr and 24hr on the first day of induction chemotherapy. Plasma levels of Dnr and DOL were measured using high performance liquid chromatography coupled with fluorescence detector. Population PK was analyzed by nonlinear mixed effects modeling using Monolix version 4.3.2. Common single nucleotide polymorphisms in ABCB1, ABCG2, CBR1 and CBR3 genes were screened by PCR followed by either sequencing or restriction digestion. In vitro cytotoxicity of the primary AML cells to Dnr was assessed by MTT cell viability assay and IC50 was calculated using ADAPT5 software. The role of plasma Dnr PK on relapse of disease was estimated using nonparametric Mann-Whitney U test.Demographics of the patients enrolled in the study are listed in table 1. PK parameters of Dnr and DOL showed wide inter-individual variation (table2). Older patients had a trend towards lower Dnr clearance (CL) (p=0.09). Patients harboring FLT3-ITD mutations had higher CL than the patients without mutation though not reaching statistical significance. Patients with variants of SNPs rs20572 (exon3) and rs9024 (3’UTR) in CBR1 (which were in complete linkage disequilibrium) had a trend towards significantly higher Dnr AUC [median-0.2711; (0.04903-2.165) vs. median 0.2117; (0.0546-0.9036); p=0.06] than patients with wild-type.Among the 64 patients, complete remission (CR) was documented in 42 (65%) of which, 31% relapsed eventually [median time to relapse 328 (104-864) days]. AML patients who relapsed had significantly lower Dnr AUC [median 0.17 (0.049-2.165) vs. median 0.31 (0.1-1.74); p=0.009] and higher DOL AUC [median 8.67 (1.6-20.1) vs.4.99 (1.3-16.5); p=0.015].Also, relapsed patients had higher Dnr clearance (CL)[median -546.54 (41.5-1632)vs. 329.3 (42.9-944.5); p=0.011] and lower DOL CL[median-9.8 (4.9-56)vs. median- 19.7 (6.1-57); p=0.015]than patients who did not relapse. IC50 of Dnr was higher in patients who relapsed (median-0.7 (0.02-1.46) vs. median- 0.23 (0.007-1.11); p=0.056) than in patients who did not relapse.To the best of our knowledge, this is the first population PK report in AML which is designed to evaluate various factors which might influence Dnr and DOL PK in AML. Though the number is small, our results suggest that plasma PK of Dnr influences clinical outcome in AML. Lower systemic exposure of Dnr in relapsed patients suggests that amount of drug required to completely eradicate the leukemic cells is not achieved and escalating dose might be an option to decrease relapse rates. Cellular pharmacokinetics of Dnr in these patients is ongoing in the lab, which will be more informative and help in prognosticating and dose adjustment for better treatment outcome.This data provides a probable biological basis for the previously reported clinical benefit of decreased relapses following the increase of the dose of DNR in induction to 90mg/m2/day (Tallman et al. NEJM 2009).Wide inter-individual variation in Dnr PK also suggests that this increased dose may not be necessary for all patients. Further studies are required to confirm this finding in a larger cohort of patients and to work towards strategies for TDM of Dnr in AML [Display omitted] [Display omitted] DisclosuresSrivastava:Octapharma: Consultancy, Other.

Genetic Variation in NFATC2 Is Associated with a Higher Risk of Asparaginase Allergy

Asparaginase is an important drug for the treatment of acute lymphoblastic leukemia (ALL); however, hypersensitivity reactions to asparaginase can lead to suboptimal asparaginase exposure and to a higher risk of disease recurrence. Factors influencing the frequency of asparaginase hypersensitivity include asparaginase formulation, the schedule of asparaginase administration, and concurrent chemotherapy. In addition, using a candidate gene approach, our group previously identified a genetic association between HLA-DRB1*07:01 and asparaginase hypersensitivity in patients of European ancestry.[PMID 24970932] The objective of our current study was to use a genome-wide approach to identify additional genetic loci associated with hypersensitivity and to assess the relative importance of HLA-DRB1 as a genetic risk factor.Asparaginase hypersensitivity was assessed in children with ALL enrolled on SJCRH protocols Total XIIIA (n = 154), Total XV (n = 498) and Total XVI (n = 271) or Children’s Oncology Group (COG) protocols POG 9906 (n = 222) and AALL0232 (n = 2,163). Patients enrolled on protocols Total XIIIA, Total XV and POG 9906 received native E. coli asparaginase during treatment, whereas patients on Total XVI and AALL0232 received PEGylated E. coli asparaginase. Hypersensitivities were graded according to the NCI common toxicity criteria, and reactions of grade 2 and above were considered cases. Germline DNA was genotyped using the Affymetrix 500K, Affymetrix 6.0, or the Illumina Exome Beadchip array. Univariate analysis of risk factors associated with asparaginase hypersensitivity using the combined cohort of patients (n = 3,308) identified a significant positive association with treatment arm (P < 2.2x10-16; highest risk in patients enrolled on Total XV), native E coli asparaginase formulation (P < 2.2x10-16), age < 10 years (P = 3.3x10-4), male gender (P = 0.06), and racial ancestry (P = 2.1x10-4; highest risk among African ancestry). In multivariate analysis, treatment arm (P < 2.2x10-16), gender (P = 0.04), racial ancestry (P = 0.07), and ALL lineage (P = 0.003) remained significant risk factors.A multivariate logistic regression model adjusted for treatment arm, racial ancestry, gender, age, and ALL lineage was used to identify genomic variants associated with asparaginase hypersensitivity in the combined patient cohort. The minor allele of an intronic polymorphism in NFATC2 (rs6021191) was associated with hypersensitivity at the genome-wide significance threshold (P = 2.6x10-8, OR = 3.17). The minor allele frequency (MAF) for the NFATC2 high-risk variant was highest in non-European ancestries (MAFEuropean = 0.001, MAFAfrican = 0.142, MAFHispanic = 0.043, MAFAsian = 0.151, MAFOther = 0.034). Using RNA-seq data available from the ALL tumor samples of 65 SJCRH patients, we found that patients carrying the variant had higher expression of NFATC2 compared to non-carriers (P = 1.0x10-3). NFATC2 is known to play a critical role in inducing T-cell gene transcription during immune responses.The strongest association detected using SNPs from the Illumina Exome array was found in HLA-DRB1 (rs17885382, P = 4.0x10-6, OR = 1.65), and the SNP is in near complete linkage disequilibrium with the HLA-DRB1*07:01 allele, which we previously found associated with asparaginase hypersensitivity. A gene-level analysis was performed using SNP-set Kernel Association Test (SKAT), and the gene with the strongest association with hypersensitivity was PPT2, located in the HLA class III region on chromosome 6 (P = 3.70x10-6). To determine potential biological pathways differentially represented by the genes identified by our analysis, we used Ingenuity Pathway Analysis on the top 1% of genes associated with hypersensitivity as identified by our single SNP (68 genes) and gene level analysis (18 genes). We found an enrichment of genes (8 out of 21 genes, P = 2.6x10-5) within the top network involved in humoral immunity, suggesting that other genes regulating immune responses may also contribute to the risk of developing asparaginase hypersensitivity.In conclusion, the top inherited genetic loci (NFATC2, HLA-DRB1*0701, PPT2) associated with asparaginase hypersensitivity are directly involved in human immune response, and pathway analysis suggests that inherited variations in other genes involved in humoral immunity likely play a role in the development of asparaginase hypersensitivity. DisclosuresEvans:St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties. Relling:St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties.

Pharmacokinetics of Fludarabine in Patients with Aplastic Anemia Undergoing Hematopoietic Stem Cell Transplantation

▪Hematopoietic stem cell transplantation (HSCT) is the best treatment option for young patients with aplastic anemia. Conditioning with fludarabine (Flu) and cyclophosphamide (Cy) has been associated with improved long-term survival in patients undergoing HSCT (George et al, 2013). Graft versus host disease (GvHD), graft failure and infection associated mortality are still some of the major hurdles towards a successful outcome. Limited data available on the association between plasma Flu levels and HSCT outcome showed higher plasma Flu AUC levels to be a risk factor for non-relapse mortality. Fludarabine given intravenously as fludarabine mono-phosphate is readily converted to Flu by the enzyme ecto-5′-nucleotidase or NT5E and then is taken up into the cells by nucleoside transporters. NT5E mRNA expression and gene polymorphism in the lymphoblastoid cell lines have been shown associated with cytotoxicity to thiopurine drugs but its role on Flu pharmacokinetics (PK) is not known. We proposed to evaluate the population PK of Flu in patients with aplastic anemia undergoing HSCT and to test their influence on transplant outcome.Thirty patients diagnosed with aplastic anemia and consented to undergo HLA identical sibling HSCT using a Flu/Cy based conditioning regimen between January 2012 and February 2014 at the Department of Hematology, Christian Medical College, Vellore were included in the study (Table: Patient demographics). Pre-HSCT DNA was used to screen polymorphisms in the NT5E gene. Plasma was separated from the peripheral blood collected before (0hr) and 1,2,3,5,7 and 24 hrs after the infusion of fludarabine (30mg/m2/day x 6 days over 1 hr infusion) on days 1 and 4 and stored at -80oC until further analysis. Plasma fludarabine was analyzed using a LC-MS/MS based method and the concentration was expressed as ng/ml. Flu PK was estimated using a 2-compartment model with linear elimination. Inter-occasion variability (between dose 1, 4, 5, and 6 PK studies) on the volume (V) and elimination rate (k) was accounted for in the model. The covariates tested were: age, sex, body weight, BSA, disease type, and a polymorphism in NT5E gene. The population PK was analyzed using Monolix 4.3.2. The PK parameters AUC, CL, V and k were calculated for day 1 and day 4 of sampling. A limited sampling model (LSM) was developed with this data in order to reduce sample collection time points for future studies. The PK and PG parameters estimated are listed in Table. The influence of flu PK on clinical outcome parameters including overall survival, GvHD and rejection were estimated using Cox regression analysis.Flu PK showed wide inter-individual variation in patients with aplastic anemia receiving fludarabine based conditioning regimen (Table), which could be explained by the polymorphism in the gene converting the prodrug to active form of fludarabine. The NT5E 5’UTR variant rs2295890 was in complete linkage disequilibrium with 4 other SNPs namely: rs9450278, rs9450279, rs4599602, rs4458647 as seen both in patients and in normal healthy volunteers. Patients with variant genotype for rs2295890 showed significantly lower plasma Flu clearance compared to those with wild type genotype (p=0.0244) (Figure). Comparison of Flu PK parameters with previous studies is not possible due to heterogeneous population of patients, Flu dose and donor type included (Long Boyle et al, 2011; McCune et al, 2012). The LSM developed based on the current PK and using a D-optimality approach (via ADAPT 5) suggested a 4 time point sampling including 1 hr (end of infusion), 3, 7, and 24 hrs as a reasonable approach. This LSM will be validated by analyzing future samples.Of the 30 patients, 26 (86.6%) engrafted at a median of 14 days post BMT (range: 11-18) while 2 (6.6%) had primary graft failure and 2 died within the first 2 weeks of transplant. Grade 2-4 acute GVHD was seen in 6 (25%) with chronic GVHD in 28.5%. One patient had secondary graft failure. The Day 100 mortality was 26.6% and at present 19 (63.3%) are alive. None of the Flu PK parameters showed any significant association with engraftment, GVHD, rejection and mortality.This first study on Flu PK in a uniform cohort of patients with aplastic anemia undergoing HSCT shows that the PK of Flu is highly variable and there is genetic basis for this variation. The clinical significance of the variation in the Flu PK needs to be studied in a larger cohort of patients. [Display omitted] DisclosuresSrivastava:Octapharma: Consultancy, Other.

No Association Was Found Between MTHFR and MTHFD1 SNPs and Vitamin Levels in People with Increased and Normal Erythropoiesis, after Compulsory Flour Fortification with Folic Acid

Background The methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase (MTHFD1) enzymes play important roles in the metabolism of folate. MTHFR catalyses the reduction of 5,10-methylene-tetrahydrofolate (5,10-methylene-THF) to 5-methyl-THF and present a crucial role in the regulation of available folate to homocysteine remethylation, in a cobalamin (Cbl) dependent reaction. MTHFD1 has three distinct enzymatic activities in folate metabolism: the conversion of THF to 10-formyl-THF; the reversible conversion of 10-formyl-THF to 5,10-methenyl-THF; and the conversion of 5,10-methenyl-THF to 5,10-methylene-THF. The two enzymes were related with DNA synthesis and single nucleotides polymorphisms (SNPs) in their genes have been associated with lower folate levels. People who have increased cellular duplication could be affected by low folate levels. Hereditary spherocytosis (HS) patients had an increased erythropoiesis and need more amounts of acid folic. In Brazil, HS patients are usually treated with 5mg/day of folic acid, the only formulation available. The use of folic acid by β-thalassemia heterozygotes (β-TH) subjects is not common, and depends of the individual clinical feature. Furthermore, in Brazil, HS patients and β-TH people have been exposed to food fortification (wheat and corn flours) with 150 µg of FA since 2004. The influences of variants of MTHFR and MTHFD1 SNPs in people with increased erythropoiesis, especially in HS patients, are not known.Objective The aim of the study was to evaluate the interaction between MTHFR c. 677C>T and c. 1298A>C and MTHFD1 c. 1958G>A SNPs and the use or not of FA supplementation in HS patients. It was also our objective to compare the folate and Cbl levels according to genotypes for MTHFR and MTHFD1 SNPs in β-TH subjects and their controls.Methods Twenty-five patients with HS, 49 β-TH subjects and 98 healthy people exposed to mandatory fortification of wheat and corn flours with FA were included in this study. Serum folate and Cbl were determined by microbiologic assays. Genomic DNA was extracted from whole blood using commercial kit. The genotypes for MTHFR c. 1298A>C and MTHFD1 c.1958G>A SNPs were determined by Real Time PCR using TaqMan assays. PCR-RFLP was used for genotyping MTHFR c. 677C>T SNP. Haplotype frequencies (MTHFR c. 677C>T and c. 1298 A>G) and the standardized disequilibrium coefficient (D’) for pair-wise linkage disequilibrium were assessed by the Expectation-Maximization (EM) algorism using the Haploview Software. We considered linkage disequilibrium when D’ ≥0.50. The study of linkage disequilibrium was performed by calculating the coefficient D’ of Lewontin.Results The frequencies of genotypes for MTHFR (c. 677C>T and c. 1298A>C) and MTHFD1 c. 1958G>A SNPs were similar in HS, β-TH and control groups (p>0.05). When genotypes were grouped according to the presence of the mutated allele (CC vs. CT+TT for MTHFR c. 677C>T, AA vs. AC+CC for MTHFR c. 1298A>C and GG vs. GA+AA for MTHFD1 c. 1958G>A), no differences were also found (p>0.05). The MTHFR c.677C>T and c. 1298A>C SNPs are in complete linkage disequilibrium (D’=1.0). Regarding the correlation coefficient between the two loci (r2), the value found in this analysis was equal to 0.169 in HS versus control group and 0.205 in β-TH versus control group. The haplotype CA, which is formed by the ancestral allele of MTHFR c. 677C>T and c. 1298A>C SNPs respectively, was more frequent in controls (41.9%) than in the β-TH group (28.1%). However, this finding was not observed in HS group comared with control group. No association was found between genotypes for MTHFR c. 677C>T and c. 1298A>C and MTHFD1 c. 1958G>A and alterations in folate and Cbl levels in three groups. ANCOVA was used for evaluating the interaction between the use of FA supplementation and MTHFR c.677C>T SNP on folate levels in HS patients. Only FA supplementation was associated with increased serum folate, and no associations were observed with MTHFR SNP or interaction between both.Conclusion The SNPs were not associated with reduced serum folate levels in the three groups studied. This finding suggests that FA flour fortification in β-TH and controls, as well as, the supplementation plus fortification in HS group, could offset the effect of variants of SNPs, especially for MTHFR c.677C>T. DisclosuresNo relevant conflicts of interest to declare.

TSNARE1 polymorphisms are associated with schizophrenia susceptibility in Han Chinese.

t-SNARE domain containing 1 gene (TSNARE1) is located at human chromosome 8q24.3, and may play a crucial role in intracellular protein transport and synaptic transmission. Recently, a large-scale meta-analysis of genome-wide association study dataset identified that rs10098073 and rs4129585, two single nucleotide polymorphisms (SNPs) within TSNARE1, were closely associated with the risk of schizophrenia in Caucasians. However, this finding has not been validated in other populations or ethnic groups thus far. In the current study, we conducted a case-control study to confirm the association of these two SNPs with the schizophrenia risk in a Han Chinese population comprising 440 schizophrenia patients and 450 control subjects. According to the genotype data of Han Chinese from Beijing in 1,000 Genomes Project database, rs10098073 and rs4129585 were located in one haplotype block and were in almost complete linkage disequilibrium (D' = 1, r (2) ≥ 0.952). Therefore, only rs10098073 was selected for the subsequent analysis. We showed for the first time that the minor allele (A) of rs10098073 was associated with a reduced risk of schizophrenia (OR = 0.753; 95 % CI 0.613-0.924; P = 0.007). Furthermore, we found that the A allele of rs10098073 reduced the schizophrenia risk through a recessive manner (A/A vs. A/C + C/C, OR = 0.563; 95 % CI 0.357-0.89; P = 0.013, P Bonferroni corrected = 0.026) rather than a dominant manner (A/A + A/C vs. C/C, OR = 0.762; 95 % CI 0.586-0.992; P = 0.043, P Bonferroni corrected = 0.086). Taken together, these findings demonstrate a significant association between TSNARE1 polymorphisms and schizophrenia risk in a Han Chinese population, suggesting TSNARE1 may represent a susceptibility gene for this disease.

Two functional polymorphisms of ROCK2 enhance arterial stiffening through inhibiting its activity and expression

Derangement of Rho-associated kinases (ROCKs) has been related to coronary artery disease and stroke. ROCK2, rather than ROCK1, plays a predominant role in vascular contractility. The present study aims to test (1) the associations between ROCK2 single nucleotide polymorphisms (SNPs) and arterial stiffness, and (2) the molecular mechanism accounting for their effects. Stiffness parameters including beta (β), elasticity modulus (Ep) and pulse wave velocity (PWV) were obtained by carotid ultrasonography. Seven tagging SNPs of ROCK2 were initially genotyped in 856 subjects and significant SNPs were replicated in another group of 527 subjects. Two SNPs in complete linkage disequilibrium were found to be significantly associated with arterial stiffness. The major alleles of rs978906 (A allele) and rs9808232 (C allele) were associated with stiffer arteries. SNP rs978906 was predicted to influence microRNA(miR)-1183 binding to ROCK2, while rs9808232 causes amino acid substitution. To determine their functional impact, plasmid constructs carrying different alleles of the significant SNPs were created. Compared to rs978906G-allele constructs, cells transfected with rs978906A-allele constructs had higher baseline luciferase activities and were less responsive to miR-1183 changes. Oxidized-low density lipoprotein (Ox-LDL) suppressed miR-1183 levels and increased ROCK2 protein amounts. For rs9808232, cells transfected with C-allele constructs had significantly higher ROCK activities than those with A-allele constructs. Leukocyte ROCK activities were further measured in 52 healthy subjects. The average ROCK activity was highest in human subjects with CC genotype at rs9808232, followed by those with AC and lowest in AA. Taken together, the present study showed that two functional SNPs of ROCK2 increase susceptibility of arterial stiffness in the Chinese population. Non-synonymous SNP rs9808232 influences ROCK2 activity, while 3′ UTR SNP rs978906 affects the ROCK2 protein synthesis by interfering miR-1183 binding.

Genome-Wide Association Study for Certain Carcass Traits and Organ Weights in a Large White×Minzhu Intercross Porcine Population

Porcine carcass traits and organ weights have important economic roles in the swine industry. A total of 576 animals from a Large White×Minzhu intercross population were genotyped using the Illumina PorcineSNP60K Beadchip and were phenotyped for 10 traits, specifically, backfat thickness (6-7 libs), carcass length, carcass weight, foot weight, head weight, heart weight, leaf fat weight, liver weight, lung weight and slaughter body weight. The genome-wide association study (GWAS) was assessed by Genome Wide Rapid Association using the mixed model and regression-genomic control approach. A total of 31 single nucleotide polymorphisms (SNPs) (with the most significant SNP being MARC0033464, P value=6.80×10−13) were located in a 9.76-Mb (31.24-41.00 Mb) region on SSC7 and were found to be significantly associated with one or more carcass traits and organ weights. High percentage of phenotypic variance explanation was observed for each trait ranging from 31.21 to 67.42%. Linkage analysis revealed one haplotype block of 495 kb, in which the most significant SNP being MARC0033464 was contained, on SSC7 at complete linkage disequilibrium. Annotation of the pig reference genome suggested 6 genes (GRM4, HMGA1, NUDT3, RPS10, SPDEF and PACSIN1) in this candidate linkage disequilibrium (LD) interval. Functional analysis indicated that the HMGA1 gene presents the prime biological candidate for carcass traits and organ weights in pig, with potential application in breeding programs.

Novel single nucleotide polymorphism in CYP2C9 is associated with changes in warfarin clearance and CYP2C9 expression levels in African Americans

Warfarin is a widely used anticoagulant whose active S-enantiomer is primarily metabolized by the CYP2C9 enzyme. The CYP2C9*2 and CYP2C9*3 alleles are associated with lower warfarin dose requirement and decreased enzyme activity. In contrast, we previously identified a novel single-nucleotide polymorphism (SNP) (rs7089580A>T) in CYP2C9 that is associated with higher warfarin dose requirement in African Americans (AAs). In this study, we examine the effect of rs7089580 on warfarin pharmacokinetics and CYP2C9 expression in 63 AA patients and 32 AA liver tissues, respectively. We found oral clearance of S-warfarin to be higher among carriers of the minor rs7089580 allele (T) compared with wild-type homozygotes (3.73±1.46 vs 2.95±1.39mL/min; P=0.04). CYP2C9 messenger RNA expression in liver tissue was also higher among A/T and T/T genotypes compared with A/A (P<0.02). Our findings indicate that rs7089580 is associated with higher S-warfarin clearance and CYP2C9 expression and may help explain the higher dose requirement of warfarin in AAs. Furthermore, rs7089580 is in complete linkage disequilibrium with the promoter SNP rs12251841 in AAs, which may provide a biologically plausible explanation for the observed effect on CYP2C9 expression levels. Given the many clinically relevant substrates of CYP2C9, identifying polymorphisms that affect expression levels and metabolism across ethnicities is essential for individualization of doses with a narrow therapeutic index.

Association analyses of FGFR2 gene polymorphisms with femoral neck bone mineral density in Chinese Han population

Femoral neck (FN) bone mineral density (BMD) is the most important risk phenotype for osteoporosis and has been used as a reference standard for describing osteoporosis. Identification of genetic variations associated with FN BMD may provide potential targets for therapeutic studies. Given the important biological role of FGFR2 gene involved in bone, we tested the associations between FGFR2 polymorphisms and FN BMD in 1,300 Chinese Han subjects. Of the 28 total SNPs, 2 SNPs, namely rs11200014 and rs1078806, were significantly associated with FN BMD under dominant model (P = 0.0014 and 0.0012, respectively) after conservative Bonferroni correction. The two SNPs were in complete linkage disequilibrium. In addition, haplotype-based association tests identified two haplotypes significantly associated with FN BMD, including one haplotype in block 4 where the two SNPs located. However, different from previous studies in white older men, we did not detect any significant association in sex-stratified analyses. In summary, our findings suggest that the FGFR2 gene may play an important role in variation in FN BMD in Chinese Han population, independent of gender effects. Further studies performed in multiple and large samples are needed to elucidate the underlying molecular mechanism and pathophysiology of osteoporosis.

Genome-wide association study identified SNP on 15q24 associated with bladder cancer risk in Japanese population.

Through genome-wide association analysis and an independent replication study using a total of 1131 bladder cancer cases and 12 558 non-cancer controls of Japanese populations, we identified a susceptibility locus on chromosome 15q24. SNP rs11543198 was associated with bladder cancer risk with odds ratio (OR) of 1.41 and P-value of 4.03 × 10(-9). Subgroup analysis revealed rs11543198 to have a stronger effect in male smokers with OR of 1.66. SNP rs8041357, which is in complete linkage disequilibrium (r(2) = 1) with rs11543198, was also associated with bladder cancer risk in Europeans (P = 0.045 for an additive and P = 0.025 for a recessive model), despite much lower minor allele frequency in Europeans (3.7%) compared with the Japanese (22.2%). Imputational analysis in this region suggested CYP1A2, which metabolizes tobacco-derived carcinogen, as a causative candidate gene. We also confirmed the association of previously reported loci, namely SLC14A1, APOBEC3A, PSCA and MYC, with bladder cancer. Our finding implies the crucial roles of genetic variations on the chemically associated development of bladder cancer.

P.3.a.007 The interaction between NOS-I and NOS1AP in dendrite development: implications for schizophrenia

Using a pharmacogenetic approach in 185 schizophrenics who have been prospectively assessed for clozapine response, we have examined the hypothesis that polymorphisms in the 5-HT2A (HTR2A), and 5-HT2C (HTR2C) genes are involved in its variable response. A -1438 A→ G polymorphism in the putative promoter and a silent T→ C 102 substitution in HTR2A were in almost complete linkage disequilibrium, and neither was associated with response (T→ C 102 allele: χ2 = 0.02; 1 df, p = .90; genotype: χ2 = 0.02, 2 df, p = .99). A his452tyr HTR2A polymorphism was found to be associated with clozapine response (his452tyr allele: χ2 = 6.43, 1 df, p = .01 [p = .04, Bonferroni corrected]; genotype: χ2 = 6.54, 2 df, p = .04 [p = .16, Bonferroni corrected]). No HTR2A haplotype was associated with response. Interethnic differences were observed in the frequencies of the cys23ser HTR2C polymorphism. This polymorphism was not significantly associated with response in either of the ethnic groups (Caucasian and African American genotype: χ2 = 3.46, 2 df, p = .18; χ2 = .31, 2 df, p = .86, respectively). Although replication is required, the overall results suggest that the his452tyr HTR2A polymorphism may be involved in clozapine response.

Genome-wide association studies identify the loci for 5 exterior traits in a Large White × Minzhu pig population.

As one of the main breeding selection criteria, external appearance has special economic importance in the hog industry. In this study, an Illumina Porcine SNP60 BeadChip was used to conduct a genome-wide association study (GWAS) in 605 pigs of the F2 generation derived from a Large White × Minzhu intercross. Traits under study were abdominal circumference (AC), body height (BH), body length (BL), cannon bone circumference (CBC), chest depth (CD), chest width (CW), rump circumference (RC), rump width (RW), scapula width (SW), and waist width (WW). A total of 138 SNPs (the most significant being MARC0033464) on chromosome 7 were found to be associated with BH, BL, CBC, and RC (P-value = 4.15E-6). One SNP on chromosome 1 was found to be associated with CD at genome-wide significance levels. The percentage phenotypic variance of these significant SNPs ranged from 0.1–25.48%. Moreover, a conditional analysis revealed that the significant SNPs were derived from a single quantitative trait locus (QTL) and indicated additional chromosome-wide significant association for 25 SNPs on SSC4 (BL, CBC) and 9 SNPs on SSC7 (RC). Linkage analysis revealed two complete linkage disequilibrium haplotype blocks that contained seven and four SNPs, respectively. In block 1, the most significant SNP, MARC0033464, was present. Annotations from pig reference genome suggested six genes (GRM4, HMGA1, NUDT3, RPS10, SPDEF and PACSIN1) in block 1 (495 kb), and one gene (SCUBE3) in block 3 (124 kb). Functional analysis indicated that HMGA1 and SCUBE3 genes are the potential genes controlling BH, BL, and RC in pigs, with an application in breeding programs. We screened several candidate intervals and genes based on SNP location and gene function, and predicted their function using bioinformatics analyses.

SPP1 rs9138 variant contributes to the severity of radiological damage in anti-citrullinated protein autoantibody-negative rheumatoid arthritis

ObjectiveWe recently reported an association of the SPP1 rs9138 and rs11439060 functional variants with the risk of rheumatoid arthritis (RA), the association being greater in anti-citrullinated protein autoantibody (ACPA)-negative patients....

OP0193 The Rheumatoid Arthritis -Risk Locus CCR6 and Its Snp-Dependent Response to Estrogen: A Possible Genomic Link between Sex Hormones and the IL-17 Inflammatory Pathway

BackgroundThe CCR6-CCL20–mediated migration of Th17 cells to inflamed tissues may represent an important mechanism in the etiology of rheumatoid arthritis (RA). The CCR6 polymorphism rs3093023 is associated with RA disease...