TG Allele Research Articles

Influence of novel CYP2C-haplotype on proton pump inhibitor pharmacokinetics in children.

In this brief report, we provide an analysis of the influence of a novel CYP2C haplotype (CYP2C:TG) on proton pump inhibitor (PPI) pharmacokinetics (PK) in children. The CYP2C:TG haplotype has been proposed to be associated with increased CYP2C19 activity. We sought to determine if this CYP2C:TG haplotype resulted in similar alterations in metabolism for proton pump inhibitors, which are primarily metabolized by CYP2C19. In a cohort of 41 children aged 6-21 participating in a PPI pharmacokinetic study, effects of the CYP2C:TG allele were assessed by fitting two linear regression models for each of the six PK outcomes assessed, the second of which accounted for the presence of the CYP2C:TG allele. The difference in R2 values between the two models was computed to quantify the variability in the outcome that could be accounted for by the CYP2C:TG allele after adjustment for the CYP2C19 genotype. We found the CYP2C:TG haplotype to have no measurable additive impact on CYP2C19-mediated metabolism of PPIs invivo in older children and adolescents. The findings of this study do not support the clinical utility of routine testing for the CYP2C:TG haplotype to guide PPI dose adjustments in children.

CIRCULATING MICRORNA PROFILE IN OKINAWAN CENTENARIANS

Abstract Centenarians are a successful example of aging and extended longevity, exhibiting advanced regulation of biological mechanisms and homeostasis. Since human longevity is a complex field of study that navigates molecular and biological mechanisms that influence aging, we hypothesized that microRNAs, a class of small non-coding RNAs implicated in regulating gene expression at the post-transcriptional level, demonstrate altered expression profiles in circulation across young, middle-aged, and centenarian cohorts. We sequenced circulating microRNAs in Okinawan males and females < 40, 40-80, and >90 years of age accounting for FOXO3A genetic variations (TT and TG alleles) and validated the findings through RT-qPCR. We report 5 microRNAs exclusively upregulated in both male and female centenarians demonstrating predictive functional roles in TGF-β, FoxO, AMPK, Pi3K-Akt, and MAPK signaling pathways. Our findings suggest that these microRNAs upregulated in centenarians may provide novel insight into their enhanced longevity and warrant further exploration into their roles in human aging and longevity.

Impact of CYP2C:TG Haplotype on CYP2C19 Substrates Clearance In Vivo, Protein Content, and In Vitro Activity.

A novel haplotype composed of two non-coding variants, CYP2C18 NM_000772.3:c.*31T (rs2860840) and NM_000772.2:c.819+2182G (rs11188059), referred to as "CYP2C:TG," was recently associated with ultrarapid metabolism of various CYP2C19 substrates. As the underlying mechanism and clinical relevance of this effect remain uncertain, we analyzed existing in vivo and in vitro data to determine the magnitude of the CYP2C:TG haplotype effect. We assessed variability in pharmacokinetics of CYP2C19 substrates, including citalopram, sertraline, voriconazole, omeprazole, pantoprazole, and rabeprazole in 222 healthy volunteers receiving one of these six drugs. We also determined its impact on CYP2C8, CYP2C9, CYP2C18, and CYP2C19 protein abundance in 135 human liver tissue samples, and on CYP2C18/CYP2C19 activity in vitro using N-desmethyl atomoxetine formation. No effects were observed according to CYP2C:TG haplotype or to CYP2C19*1+TG alleles (i.e., CYP2C19 alleles containing the CYP2C:TG haplotype). In contrast, CYP2C19 intermediate (e.g., CYP2C19*1/*2) and poor metabolizers (e.g., CYP2C19*2/*2) showed significantly higher exposure in vivo, lower CYP2C19 protein abundance in human liver microsomes, and lower activity in vitro compared with normal, rapid (i.e., CYP2C19*1/*17), and ultrarapid metabolizers (i.e., CYP2C19*17/*17). Moreover, a tendency toward lower exposure was observed in ultrarapid metabolizers compared with rapid metabolizers and normal metabolizers. Furthermore, when the CYP2C19*17 allele was present, CYP2C18 protein abundance was increased suggesting that genetic variation in CYP2C19 may be relevant to the overall metabolism of certain drugs by regulating not only its expression levels, but also those of CYP2C18. Considering all available data, we conclude that there is insufficient evidence supporting clinical CYP2C:TG testing to inform drug therapy.

Impact of the novel CYP2C:TG haplotype and CYP2B6 variants on sertraline exposure in a large patient population.

Sertraline is a commonly used SSRI antidepressant drug, metabolized by CYP2C19 and CYP2B6, that exhibits a substantial interindividual variation in clinical response, of which only a part can be attributed to known genetic variants. In the current study we have examined the role of a newly discovered ultrarapid CYP2C:TG haplotype and CYP2B6 variants in order to identify the possible missing heritability for such variation in sertraline response in a large patient population (n = 840). Compared to the reference group (CYP2C19*1/*1, n = 160), sertraline exposure was increased by 128% in CYP2C19 PMs (n = 29, p < 0.001) but decreased by about 20% in CYP2C19 ultrarapid metabolizers (Ums) (homozygous carriers of CYP2C19*17 and/or CYP2C:TG haplotype) with the diplotypes CYP2C19*17/*17, CYP2C:TG/TG, or CYP2C19*17/CYP2C:TG (n = 135, p < 0.003, p = 0.022, p < 0.003, respectively). Interestingly, in patients carrying the increased function CYP2B6*4 allele, and also carrying the CYP2C19*17 and CYP2C:TG alleles (n = 10), sertraline exposure was 35.4% lower compared to the reference group, whereas in subjects being poor metabolizers (PM) in both the CYP2C19 and CYP2B6 gene, the sertraline concentrations were raised by 189%. In summary, the CYP2C19 variants including the CYP2C:TG haplotype had a significant impact on sertraline metabolism, as well as the CYP2B6*4, *6, and *9 alleles. Knowing the CYP2B6 and CYP2C19 genotype, including the CYP2C:TG haplotype status, can prospectively be useful to clinicians in making more appropriate sertraline dosing decisions

Evaluation of the frequency of ADIPOQ c.45 T>G and ADIPOQ c.276 G>T polymorphisms in adiponectin coding gene in girls with anorexia nervosa.

Anorexia nervosa (AN) is a serious chronic psychosomatic disorder, the essence of which are attempts by the sufferer to obtain a slim silhouette by deliberate weight loss (restrictive diet, strenuous physical exercise, provoking vomiting). The aetiology of this disorder is multifactorial. Genetic factors that influence the predisposition to AN have been sought. A broad meta-analysis points to a strong genetic correlation between AN and insulin resistance. Adiponectin (ADIPO) increases insulin sensitivity. In our pilot study we demonstrated that the TT genotype in locus ADIPOQ c.276 G>T of the ADIPO gene and a higher concentration of ADIPO in blood serum occurred significantly more frequently in 68 girls suffering from AN than in 38 healthy girls. The objective of this study was to evaluate the frequency of the occurrence of ADIPOQ c.45 T>G and ADIPOQ c.276 G>T in the ADIPO gene in a larger cohort of girls with AN and healthy girls, as well as an analysis of correlations between variants of the aforementioned polymorphisms and the levels of ADIPO in blood serum. The study covered 472 girls (age: 11-19 years): 308 with the restrictive form of AN (AN) and 164 healthy girls (C). The level of ADIPO in blood serum was determined by means of the ELISA method on a Bio-Vendor, LLC (Asheville, North Carolina, USA). The DNA isolation was carried out by means of Genomic Mini AX BLOOD (SPIN). The PCR reaction was carried out in a ThermoCycle T100 thermocycler. 80-150 ng of the studied DNA and relevant F and R starters were added to the reaction mixture. The reaction products were subjected to digestion by restriction enzymes and separated on agarose gels (RFLP). The distribution of genotypes in the polymorphic site ADP c.45 of the ADIPO gene and ADP c.276 was similar in both groups. In both groups the T allele was most frequent in locus ADIPOQ c.45 and the G allele in locus ADIPOQ c.276. In all the study subjects collectively (AN and C) a statistically significant negative correlation between the levels of ADIPO in blood serum on one hand and body weight (r = -0.46; p < 0.0001) and BMI (r = -0.67; p < 0.0001) on the other was demonstrated. Exclusively in the AN group a significant correlation between the level of ADIPO in blood and the distribution of TG, TT, and GG alleles in loci ADIPOQ c.45 and ADIPOQ c.276 was demonstrated (p = 0.0052 and p < 0.0001, respectively). The genotype in loci ADIPOQ c.45 and ADIPOQ c.276 of the ADIPO gene seems to have no effect on the predisposition to AN. Girls suffering from AN with the TT genotype in loci ADIPOQ c.45 and ADIPOQ c. 276 may demonstrate higher insulin sensitivity because they have significantly higher levels of ADIPO than girls suffering from AN with other genotypes. This may be suggestive of their better adaptation to the state of malnutrition, and it has a potential effect on treatment results.

Association of the Trough, Peak/Trough Ratio of Imatinib, Pyridine-N-Oxide Imatinib and ABCG2 SNPs 34 G>A and SLCO1B3 334 T>G With Imatinib Response in Egyptian Chronic Myeloid Leukemia Patients.

Imatinib mesylate (IM) is highly efficacious in the treatment of chronic myeloid leukemia (CML). Therapeutic drug monitoring and pharmacogenetic screening are affirmed for better management of IM therapy. The goal of this study was to gain a greater mechanistic understanding of the factors controlling variability in IM level and its relation to the response. One hundred and two patients with CML at chronic phase were recruited in this study. Blood samples were withdrawn at least 30 days after drug administration, and trough and peak concentrations of imatinib, N-des-methyl imatinib, and pyridine-N-oxide imatinib were determined by HPLC/MS/MS. Genetic polymorphism of the genes ABCG2 SNPs 34 G>A and 421C >A; ABCB1 SNPs 2677 G>A/T, 1236 C>T, 3435 C>T; SLCO1B3 SNPs 334 T>G and CYP3A5 were studied using PCR-RFLP technique. Our study presented significant higher trough IM (1,281 ± 578 ng/ml), lower Peak/Trough ratio, clearance (Cl), and elimination rate constant, ke, among patients who achieved favorable responses (N = 64) than those for patients who suffered unfavorable response (N = 37). The P/T ratio was the only significant independent factor affecting response, as the P/T ratio increased by one, the risk of unfavorable response increased by more than double as compared to favorable response with 95% CI (1.28–3.92, P = 00.005). Moreover, like the results of IM, the trough concentration of Pyridine-N-oxide imatinib was significantly higher (P = 0.01) and its P/T ratio was significantly lower (P = 0.008) in patients achieved favorable response than those without. The wild GG genotype of the ABCG2.34 G>A gene was associated with favorable response (P = 0.01), lower Cl, Ke and high plasma IM trough level than both (AA+GA) genotypes. ABCG2.421C >A (CC) genotype had a significantly higher plasma peak of IM, N-des-methyl imatinib and higher Css. The GG and TG alleles of the SLCO1B3.334 T>G gene were significantly correlated to favorable response, while the wild allele TT was linked to unfavorable response (P = 0.03). In conclusion, the trough and P/ T ratio for both IM and Pyridine-N-oxide imatinib, in addition to Polymorphism of ABCG2 SNPs 34 G>A and SLCO1B3.334 T>G gene, is a good predictor for response of IM in CML Egyptian patients.

Myc Regulates the Development and Anti-Tumor Immunity of Natural Killer Cells

Natural Killer (NK) cells play an essential role in the innate immunity. We recently reported that in aggressive NK cell leukemia, MYC is hyperactivated and responsible for the survival and proliferation of leukemic cells through the activation of JAK/STAT pathway (Huang et al., Cell Research 2017). However, the role of Myc in normal NK cell development or function has not been fully explored. Here we addressed the important question on whether Myc is required for NK cell development and its anti-tumor immunity.By crossing Mycf/f allele with Ncr1Cre allele, we generated a NK cell specific Myc conditional knock out (cKO) mouse model. Mycf/f mice were used as control. The percentages of NK1.1+DX5+ mature NK cells (mNK) decreased significantly in both the peripheral blood (PB) and spleen compared to the control mice. In addition, the percentages of terminally differentiated CD11b+CD27- cells within the mNK population decreased, while CD11b-CD27+ cells increased. The expression of terminal differentiation marker Klrg1 also significantly decreased. Further analyzing the NK cells in the bone marrow (BM) revealed the CD122+% and mNK% in the BM did not change. In contrast, the NK1.1-DX5-% in lin-CD122+ population (NK progenitors) increased by 1-fold. This suggested that the development of NK cells was impaired after Myc deletion.To investigate the effects of overexpressed Myc on normal NK cell development, we generated a MYC inducible mouse model (iMYC) by crossing Tg(tetO-MYC) allele with Ncr1Cre and Rosa26Loxp-Stop-Loxp-rtTA alleles. Mice without iMYC allele were used as controls. We transplanted the BM cells from the control or iMYC mice into lethally irradiated BoyJ recipients. We observed that mNK% in CD45.2+ cells increased significantly in liver and PB 3 weeks after the transplantation and dox induction. The ratio of CD27-:CD27+ cells in NK1.1+DX5+CD11b+ cells and Klrg1+mNK% also increased in all tissues analyzed (BM, liver, PB, spleen). In addition, Brdu incorporation rates were increased in spleen and BM NK cells from the iMYC mice. NK cells isolated from iMYC mice showed an enhanced proliferation ability in vitro by dox induction in a dose-dependent manner. These suggested MYC promoted the NK cell commitment, maturation and proliferation.To explore whether the NK cell cytotoxic functions were also affected by Myc, we measured the IFN-γ secretion and CD107a degranulation by PMA/ionomycin or YAC-1 stimulation. Both of the abilities were significantly impaired in Myc cKO mice. Furthermore, the direct lysis of YAC-1 cells by NK cells was also decreased. In contrast, the CD107a degranulation did not change in iMYC mice. The secretion of IFN-γ of splenic NK cells after PMA/Ionomycin stimulation was slightly decreased in iMYC mice, which may be due to the increased population of terminally differentiated NK cells with lower cytotoxicity. This suggested that the physiological level of Myc was important for the cytotoxic functions of NK cells.To investigate whether Myc deletion in NK cells affects the tumor surveillance, we used 2 syngeneic tumor models B16F10 (melanoma) and MC38 (colon adenocarcinoma). The lung metastatic dots were significantly increased in Myc cKO mice compared to control 2 weeks after the inoculation of B16F10. All of the mice in the Myc cKO group were dead on day 21 while the survival rates were 100% in the control group. The weights and volumes of tumors in mice subcutaneously inoculated with MC38 were also significantly higher in Myc cKO mice. This indicated a key role of Myc in NK cell mediated immunity.Finally, we performed transcriptome analysis of splenic NK cells sorted from Myc cKO mice and control mice. The top 2 enriched KEGG pathways in the down-regulated genes includes NK cell mediated cytotoxicity. Interestingly, the expression of Cish, which is an important NK cell anti-tumor immunity checkpoint, was significantly increased in the Myc cKO group, suggesting Myc may affect the NK cell functions through the downregulation of Cish.In summary, our data showed that Myc promoted NK cell development and proliferation. A physiological level of Myc was critical for NK cell cytotoxicity and anti-tumor immunity. Our data also indicated that targeting Myc could be a potential strategy in boosting NK cell-mediated immunotherapy or NK cell expansion ex vivo. Further studies are needed to uncover the underlying mechanism and explore the translational application. DisclosuresNo relevant conflicts of interest to declare.

Association of IL-27 rs153109 and rs17855750 Polymorphisms with Risk and Response to Therapy in Acute Lymphoblastic Leukemia.

Interleukin (IL)-27 is a cytokine with important anti-cancer activity. This study has evaluated the effects of IL-27 rs153109 and rs17855750 single nucleotide polymorphisms (SNPs) on risk of acute lymphoblastic leukemia (ALL) development, as well as their impact on prognosis and patient survival. A total of 200 patients and 210 healthy subjects were genotyped by polymerase chain reaction-restriction fragment length polymorphism. We observed a higher frequency of rs153109 AG and rs17855750 TG genotypes and allele G in patients compared to controls (p<0.001). Combined G variant genotypes (AG+GG and TG+GG) also conferred significantly greater risk of ALL. There was a significant correlation between the genotypes of both SNPs with event-free survival (EFS). Patients with GG genotypes of both SNPs and those of rs153109 AG and rs17855750 TG had a shorter EFS than patients with rs153109 AA and rs17855750 TT genotypes (p≤0.035). Combined G variant genotypes for both SNPs showed poorer response to therapy in all patients (p<0.027) as well as B-ALL (rs153109, p<0.001) and T-ALL (rs153109, p=0.048) patients. In multivariate analysis, rs153109 combined G variant genotype was associated with shorter EFS (relative risk=9.7, p=0.026). Among those who relapsed, 87.1% had the rs153109 AG genotype and 77.4% had the rs17855750 TG genotype (p<0.01). Patients had higher IL-27 serum levels compared to controls, but this did not differ between genotypes. In conclusion, the association of IL-27 rs153109 and rs17855750 polymorphisms with risk of ALL development and their impact on EFS suggested an important role for this cytokine in biology and response to ALL therapy.

Interferon λ3 gene (IL28B) is associated with spontaneous or treatment-induced viral clearance in hepatitisC virus-infected multitransfused patients with thalassemia.

Hepatitis C virus (HCV) is the major posttransfusion infection in multitransfused individuals in India with thalassemia major. To our knowledge, this study is the first conducted to correlate and comprehend the effects of the host interleukin (IL)28B gene polymorphism at loci rs12979860 and rs8099917 in spontaneous or interferon (IFN)-induced treatment response in the HCV-seroreactive individuals with thalassemia major. A total of 557 HCV-seroreactive individuals with thalassemia were processed for HCV viral genotyping and host IL28B single-nucleotide polymorphism analysis at loci rs12979860 and rs8099917. Of 557 individuals, 70.92% were found to be HCV RNA positive with Genotype 3 (95.18%) as predominant strain. A favorable CC allele at locus rs2979860 and TT allele at rs8099917 were 75.31 and 77.16%, respectively, which was strongly associated with spontaneous clearance of infection (p < 0.05). Of 85 IFN-treated cases, 56 achieved sustained virologic response (SVR) whereas 27 were relapsed cases. Among these patients who achieved SVR, a favorable CC/TT allele at rs12979860/rs8099917 was found to be predominant with 76.79 and 66.07%, respectively, whereas in the case of relapsed patients, unfavorable CT (55.56%) and TG (59.26%) alleles were found to be predominant. Additionally, low serum ferritin level was significantly associated with SVR. CC at rs12979860 and TT at rs8099917 was strongly associated with spontaneous clearance and SVR in the population with thalassemia. Low age group and low serum ferritin level are important cofactors. This allelic pattern will aid clinicians in making an informed decision about prognosis and therapeutic management.

Evaluation of the frequency of the IL-28 polymorphism (rs8099917) in patients with chronic hepatitis C using Zip nucleic acid probes, Kerman, Southeast of Iran.

Polymorphisms in the region of the interleukin IL-28 gene on chromosome 19 have been related with clearance of hepatitis C virus (HCV), a major human pathogen responsible for chronic hepatitis, cirrhosis and hepatocellular carcinoma. About 3% of the world's population is infected with HCV. The long-term response to therapy is influenced by many host and viral factors, and recent evidence has indicated that some host genetic polymorphisms related to IL-28 are the most powerful predictors of virological response in patients with HCV. This study assessed frequency of the IL-28 polymorphism (rs8099917) in 50 patients (39 men and 11 women ) with chronic hepatitis C using ZNA probe real time PCR new method . All patients were tested for genotype of HCV and the HCV viral load. In parallel, the levels of SGOT, SGPT and ALK enzymes were assessed. Treatment using Peg-interferon alpha with ribavirin was conducted for patients and subsequently samples were collected to detect any change in viral load or liver enzyme rates. The overall frequency of the TT allele is 74%, TG allele 20% and GG allele 6% and the percent of patients who had T allele was 84%. Clear reduction in viral load and liver enzymes was reported in patients with the T allele. Especially for genotype 1 which is relatively resistant to treatment, these alleles may have a role in this decline. In conclusion, we showed that IL-28 polymorphism rs8099917 strongly predicts virological response in HCV infection and that real-time PCR with Zip nucleic acid probes is a sensitive, specific and rapid detection method for detection of SNPs which will be essential for monitoring patients undergoing antiviral therapy.

Association of interleukin 28B polymorphism and mutations in the NS5A region of hepatitis C virus genotype 2 with interferon responsiveness.

The single nucleotide polymorphism (SNP) of interleukin 28B (IL28B) and the mutations in the NS5A region of hepatitis C virus (HCV) genotype 1 have been associated with response to interferon (IFN) therapy. However, these relationships in patients with HCV genotype 2 are not well understood. The aim of this study was to investigate whether the SNP of IL28B (rs8099917) and amino acid substitutions in the NS5A region in patients with HCV genotype 2 affect the response to IFN and ribavirin combination therapy. The study enrolled 286 patients with chronic hepatitis C genotype 2. Patients received pegylated-IFN-alpha 2b once each week plus oral ribavirin daily for 24 weeks. Of the 286 patients, 215 (75.2%) achieved sustained virologic response (SVR). Rate of SVR was similar in patients with IL28B TT allele (76%) and those with TG or GG alleles (72%). Patients with SVR were younger than those without SVR (P < 0.001). SVR was achieved in 65.9% of patients with wild-type IFN sensitivity-determining region (ISDR) and 83.5% of patients with mutant type (P < 0.001). There were no significant differences in other factors, including sex, alanine aminotransferase, platelet count, HCV viral load, HCV genotype, and IL28B genotype. The factors related to SVR on multivariate analysis were age (P = 0.019) and ISDR (P = 0.003). ISDR sequence variations are significantly associated with IFN responsiveness in patients with HCV genotype 2. The SNP of IL28B was not associated with SVR in patients with HCV genotype 2.

Impact of host IL28B rs12979860, rs8099917 in interferon responsiveness and advanced liver disease in chronic genotype 3 hepatitis C patients.

Background and AimsGenetic polymorphisms near interleukin 28B gene are associated with spontaneous and treatment induced clearance of hepatitis C virus (HCV). Our objective was to evaluate the impact of interleukin 28B single nucleotide polymorphism (rs12979860, rs8099917) variability in HCV genotype 3 infected populations.Methods400 hepatitis C seroreactive patients from different population groups in Eastern and North Eastern part of India were assessed for host and viral genotypic analysis. 83 HCV genotype 3 infected patients were administered pegylated interferon- ribavirin therapy. Viral genotyping was performed using nested reverse transcriptase-PCR followed by direct sequencing methods. Host interleukin 28B genotyping was performed using real-time PCR based single nucleotide polymorphism analysis.ResultsOut of 400 hepatitis C seroreactive individuals, 73.25% were found to be RNA positive. HCV genotype 3 (65.87%) was found to be the major circulating strain in this region followed by genotype 1 (32.08%). rs12979860 CC genotype was significantly associated with sustained virological response in HCV genotype 3 infected population. In patients achieving rapid virological response, favourable CC/TT allele at rs12979860, rs8099917 was found to be predominant at both the alleles at 77%, 73.2% respectively; whereas in case of patients with relapsed HCV infection CT, TG alleles were found to be predominant. Additionally, CC genotypes at rs12979860 were found to be associated with sustained virological response in patients with high viral load (OR = 6.75, 0.05<p). HCV unfavourable rs12979860 TT, rs8099917 GG alleles were present in 34%, 27.6% patients with relapsed HCV infection respectively. Also unfavourable CT, TG genotypes were found to be predominant in patients with advanced stages of liver disease.ConclusionCC, TT the two favourable markers at SNPs rs12979860 and rs8099917 are strongly associated with sustained virological response in genotype 3 infected populations. This information will aid clinicians to effectively design response based treatment regimen.

CD24 single nucleotide polymorphisms and cancer risk.

OR = 0.70; 95 % CI, 0.44-1.12; P OR = 0.135; del/TG vs. OR = 0.91; 95 % CI, 0.80-1.04, P OR = 0.180; del/del + del/TG vs. OR = 0.90; 95 % CI, 0.79-1.03; P OR = 0.123; del/del vs. TG/TG + del/TG: OR = 0.69; 95 % CI, 0.44-1.08, P OR = 0.105). Our study firstly provides the evidence that SNPs (rs52812045 and rs3838646) of CD24 may not modify the risk of cancer. Nonetheless, more individual studies with high quality are needed for further elucidation.

Abstract C31: Molecular profiling in patients with radioactive iodine-refractory thyroid cancers using targeted mutation analysis.

Abstract Background: Thyroid cancer is the most common neoplasia of the endocrine system, accounting for 1%-3% of all cancers. Differentiated thyroid cancer (DTC) accounts for ∼93% of all thyroid cancers. While most cases of DTC are treated with surgery followed by radioactive iodine (RAI) ablation, 5-15% of DTC become RAI-refractory (RAI-R) resulting in a poorer prognosis and limited treatment options. In an attempt to use genetic information to predict prognosis in patients RAI-R DTC, we designed and implemented a mass spectrometry based genotyping assay panel encompassing the most significant oncogenes in thyroid cancer. Germline mutations associated with thyroid hormone dysgenesis were also included in the panel to examine their potential role in thyroid cancer prognosis. Methods: Somatic and germline mutations were evaluated in tumor DNA using targeted mutation analysis by Sequenom MassARRAY iPLEX platform. A total of 321 assays interrogating 212 somatic mutations and 113 germline mutations in 44 genes were included in a thyroid-cancer mutational panel. Genotype-calling was manually done by the Sequenom Typer 4.0 software. Statistical significance in categorical data was determined by a χ2 test using SPSS 11.5. Results: Of 80 patients with RAI-R DTC, 37 were male and 43 were female. Median age at diagnosis was 59 years (range 27-86). Somatic mutations were identified in 38 (47.5%) patients; mutated genes included BRAF, RAS, TP53, MET, PIK3CA, GNAS and TPO. The BRAF V600E mutation was the most prevalent somatic mutation (28.8%) and was significantly associated with regional lymph nodes metastasis (P=0.02). RAS mutations were found in 9.9% and TP53 in 6.2% of patients, while somatic mutations in other genes were rare (&amp;lt;2.5%). Germline mutations in 5 genes, including SLC5A5, SLC26A4, IGF1R, RAP1GAP and TG, were identified in tumor DNA. Only the TG polymorphism M1028V was detected at very high allele frequency of 61.9% while mutations in other 4 genes were detected at low allele frequencies (&amp;lt;3%). As expected, homozygous for the TG mutation produced one and heterozygous produced two MassARRAY peaks. Notably the detected allele frequency for TG M1028V in the RAI-R DTC group analyzed is significantly higher (61.9% vs 50.9%, P=0.017) compared to previously published population allele frequency (Collins et al, 2004). Within the group analyzed, 42.5% of patients were homozygous for TG M1028V accounting for its high allele frequency. The TG M1028V was associated significantly with such clinicopathologic parameters, as regional lymph nodes metastasis (P&amp;lt;0.01) and lymphatic invasion in primary tumor (P=0.018). Conclusion: These data suggest that in addition to well characterized activating BRAF V600E mutation, the germline TG allele, M1028V, may have clinical significance in thyroid cancer. This is first time report that a specific TG allele may be prognostic for patients with RAI-R DTC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C31. Citation Format: Tatyana Appelbaum, Waixing Tang, Stephen Stopenski, Marcia Brose. Molecular profiling in patients with radioactive iodine-refractory thyroid cancers using targeted mutation analysis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C31.

The Origin of Spelt and Free-Threshing Hexaploid Wheat

It is widely believed that hexaploid wheat originated via hybridization of hulled tetraploid emmer with Aegilops tauschii (genomes DD) and that the nascent hexaploid was spelt, from which free-threshing wheat evolved by mutations. To reassess the role of spelt in the evolution of Triticum aestivum, 4 disomic substitution lines of Ae. tauschii chromosome 2D in Chinese Spring wheat were developed and one of them was used to map the Tg locus, which controls glume tenacity in Ae. tauschii, relative to simple sequence repeat (SSR) and expressed sequence tag loci on wheat chromosome 2D. The segregation of SSR markers was used to assess the presence of Tg alleles in 11 accessions of spelt, both from Europe and from Asia. Ten of them had an inactive tg allele in the D genome and most had an active Tg allele in the B genome. This is consistent with spelt being derived from free-threshing hexaploid wheat by hybridization of free-threshing wheat with hulled emmer. It is proposed that the tetraploid parent of hexaploid wheat was not hulled emmer but a free-threshing form of tetraploid wheat.

Lack of association of Klotho gene variants with valvular and vascular calcification in Caucasians: a candidate gene study of the Framingham Offspring Cohort

Valvular and vascular calcification are important early aging phenotypes and represent risk factors for cardiovascular morbidity and mortality. Klotho is a gene primarily expressed in the kidney that has an important role in calcium-phosphate homeostasis. The functional KL-VS variant of Klotho has been associated with aging and cardiovascular disease in human studies, but its role in valvular and vascular calcification remains unknown. We performed a candidate gene study in the Framingham Offspring Cohort to evaluate the effect of KL-VS variant of the Klotho gene on valvular calcification. We analyzed the Klotho KL-VS genotype (rs9536314) from the Affymetrix 550K genome-wide dataset, distributed by dbGAP, on 1389 cases and 2139 controls from the Framingham Heart Study Offspring Cohort. Allele and genotype frequencies were compared between cases and controls. Valvular calcification was defined as presence of calcification on the mitral annulus or the aortic valve as determined by echocardiography. A sensitivity analysis of coronary artery calcification by electron beam computed tomography was performed on 1363 patients. The frequency of the TT versus the TG allele was not different between the cases and the controls (39 versus 41%). The KL-VS variant of Klotho was not associated with valvular or vascular calcification, despite adequate power to detect association (86% for odds ratios ≥1.2). In sensitivity analyses, no association (P > 0.001) between other common variants of Klotho, β-Klotho or fibroblast growth factor-23 and the end points of valvular or vascular calcification was observed. In our adequately powered candidate gene study, we did not observe an association with the functional KL-VS variant of Klotho and presence of valvular or vascular calcification. Future studies aimed at combining cohorts with echocardiographic phenotypes need to be conducted to identify genetic variants associated with valvular calcification.

Two Distinct Populations of H Chain-Edited B Cells Show Differential Surrogate L Chain Dependence

Developing autoreactive B cells may edit (change) their specificity by secondary H or L chain gene rearrangement. Recently, using mice hemizygous for a site-directed VDJH and VJkappa transgene (tg) encoding an autoreactive Ab, we reported ongoing L chain editing not only in bone marrow cells with a pre-B/immature B cell phenotype but also in immature/transitional splenic B cells. Using the same transgenic model, we report here that editing at the H chain locus appears to occur exclusively in bone marrow cells with a pro-B phenotype. H chain editing is shown to involve VH replacement at the tg allele or VH rearrangement at the wild-type (wt) allele when the tg is inactivated by nonproductive VH replacement. VH replacement/rearrangement at the tg/wt alleles was found to entail diverse usage of VH genes. Whereas the development of edited B cells expressing the wt allele was dependent on the lambda5 component of the surrogate L chain, the development of B cells expressing the tg allele, including those with VH replacement, appeared to be lambda5 independent. We suggest that the unique CDR3 region of the tg-encoded muH chain is responsible for the lambda5 independence of tg-expressing B cells.

Tolerance to DNA in (NZB x NZW)F1 mice that inherit an anti-DNA V(H) as a conventional micro H chain transgene but not as a V(H) knock-in transgene.

Lupus-prone (NZB x NZW)F(1) (BWF(1)) mice were made transgenic (Tg) for an anti-DNA Ab inherited either as a conventional V(H)3H9- micro H chain Tg (3H9- micro ) with or without a conventional V(kappa)8-kappa Tg, or a V(H)3H9 V(H) knock-in Tg allele (3H9R) with or without a V(kappa)4 V(kappa) knock-in Tg allele (V(kappa)4R). V(H)3H9 yields an anti-DNA Ab with most L chains including an anti-ssDNA with the V(kappa)8 Tg and an anti-dsDNA with the V(kappa)4 Tg. BWF(1) mice that inherited the conventional 3H9- micro had normal serum IgM, little to none of which was encoded by 3H9- micro, and only a small percentage of those mice had serum anti-DNA, none of which was transgene encoded. B cells expressing the conventional 3H9- micro Tg were anergic. BWF(1) mice that inherited the knock-in 3H9R Tg allele also had normal serum IgM, one-half of which was encoded by 3H9R, and produced anti-DNA encoded by the Tg allele. Most B cells expressing the knock-in 3H9R Tg also had an anergic phenotype. The results indicate that autoimmune-prone BWF(1) mice initially develop effective B cell tolerance to DNA through anergy, and anergy was sustained in 3H9- micro Tg peripheral B cells but not in 3H9R Tg B cells. B cells expressing the 3H9R knock-in Tg allele were able to achieve an activation threshold that B cells expressing the 3H9- micro conventional Tg could not. The maintenance of B cell tolerance to DNA in autoimmune-prone BWF(1) mice appears to differ from both normal mice and autoimmune-prone MRL(lpr/lpr) mice.

PCR Method for Genotyping and Zygosity-Testing of RasH2 Transgenic Mice

In short-term carcinogenicity testing using CB6F1-TgrasH2 mice, sibling nonTgrasH2 mice are used as a negative control. However, selection of TgrasH2 and nonTgrasH2 mice has been performed by PCR with only transgene specific primers by the conventional method. Therefore, the conventional method involves the risk of false negative results due to reaction failure, and contamination with TgrasH2 mice in the control mice group. Based on the nucleotide sequence information around the pre-integration site, we developed a genotyping method for distinguishing not only TgrasH2 mice (hemizygous for the Tg allele) but also nonTgrasH2 (homozygous for the nonTg allele) in a positive manner.

Unusual scarcity of restriction site polymorphism in the human thyroglobulin gene. A linkage study suggesting autosomal dominance of a defective thyroglobulin allele.

Chromosomal DNA prepared from 90 unrelated individuals, mainly of Caucasian origin, was screened for restriction fragment length polymorphisms in the 3' 220 kilobase pairs (kb) of the human thyroglobulin (Tg) gene. The probes used were Tg cDNA fragments and subcloned single-copy genomic segments, isolated from a human cosmid library. All in all, 1164 nucleotides were screened using 15 different restriction enzymes. The average number of nucleotides screened was 354 per individual. Only one polymorphism was found in these 1164 nucleotides, with a minor allele frequency of 2.2%. This polymorphism, which is located in an intervening sequence, was found in healthy individuals and in a family with hereditary congenital hypothyroidism due to a defect in the synthesis and structure of thyroglobulin. The Mendelian segregation of polymorphism and goiter in ten family members suggests that the rare variant is linked to a normal Tg allele and provides strong evidence for autosomal dominant inheritance of this Tg synthesis defect.