Silent Single Nucleotide Polymorphism Research Articles

A Novel Missense Single Nucleotide Polymorphism in the GREM1 Gene is Highly Associated with Higher Reproductive Traits in Awassi Sheep.

GREM1 (gremlin1) is a known inhibitor for BMP15 (bone morphogenetic protein 15) family, but its genetic diversity in sheep is unknown. The present study was conducted to analyze the polymorphism of GREM1 gene using PCR- single-strand conformation polymorphism (SSCP) and DNA sequencing methods and to assess the possible association of GREM1 gene polymorphism with reproductive traits in Awassi ewes. A total of 224 ewes, 124 producing singles and 100 producing twins, were included in the study. Two SSCP patterns were detected in two amplified loci within the exon 2. Two exonic novel single nucleotide polymorphism (SNP)s were identified, c.74T > G (the silent SNP p.Met123 =) and c.30T > A with (the missense SNP p.Ile237Phe). Statistical analyses indicated a non-significant (P > 0.05) association of p.Met123 = with the analyzed reproductive traits of fecundity, prolificacy, litter size, and twinning rate. Meanwhile, p.Ile237Phe SNP exhibited a highly significant (P < 0.01) association with the measured reproductive traits, in which ewes with TA genotype (with p.Ile237Phe SNP) exhibited higher litter size, twinning ratio, fecundity, and prolificacy than those with TT genotype (without p.Ile237Phe SNP). The deleterious impact of p.Ile237Phe SNP was observed by the means of ten different state-of-the-art in silico tools that predicted a highly damaging effect of p.Ile237Phe SNP on the structure, function, and stability of gremlin1. In conclusion, the results of our study suggest that p.Ile237Phe SNP has a remarkable negative impact on the gremlin1 structure, function, and stability. Since gremlin1 is a known inhibitor of reproductive performance, a consequent higher reproductive performance was observed in ewes with damaged gremlin1 (with p.Ile237Phe SNP) than those with non-damaged gremlin1 (without p.Ile237Phe SNP). Therefore, it can be stated that the implementation of the novel p.Ile237Phe SNP in the GREM1 gene could be a useful marker in marker-assisted selection. This manuscript is the first one to describe GREM1 gene variations in sheep.

The association of codon 392 polymorphism in ESR2 gene with breast cancer in Iran

Epidemiological studies revealed that the pattern of age onset of breast cancer in the Middle East region differ from those in Caucasians. Therefore, genomic data for ESR2 (ER-?) is of the value in the clinical setting for that ethnic group and the aim of the current investigation is investigated whether polymorphisms in the ER-? gene are associated with breast cancer risk among Iranian women. The coding sequence in Exon 7 at ESR2 gene was looking for any variation among the Iranian breast cancer women by SSCP-PCR method. In codon 392 of exon 7, showed a silent SNP (silent single nucleotide polymorphism). The frequency of allele G in codon 392 (CTC ? CTG was found only in cases (5.7%). We also found that allele G in codon 392 (C1176G) had direct association with development of lymph node metastasis in breast cancer. The current results suggest that ESR2 SNP in exon 7, codon 392 is associated with various aspects of breast cancer in Iran. ESR2 gene structure determination, in presurgical evaluation, might be a useful marker in predicting familial breast cancer and metastasis in LN.

BIM deletion polymorphism accounts for lack of favorable outcome in Japanese females with follicular lymphoma

Deletion polymorphism of BCL-2-like protein 11 (BIM) is specifically found in East Asia. To explain some epidemiological discrepancies between Asian and Western countries, we analyzed a silent single nucleotide polymorphism (SNP) in exon 5 (c465C > T) and a deletion site (2903 bp) in intron 2 in 77 patients with follicular lymphoma by the Q-invader method using PCR. In females, 5-year progression-free survivals (PFS) were 20.0% in the BIM deletion group, 66.7% in the SNP group and 81.5% in the wild-type (WT) group (p = .0012). In the WT group, 5-year PFS was 40.4% in males (p = .0448 vs. female PFS). This tendency was strengthened in patients receiving rituximab (26.9% vs. 84.2%, p = .006). Superior PFS in the WT females in Japan was comparable with the results of cohort studies in the United States and Sweden. Favorable prognosis in Japanese females may be masked by the BIM deletion polymorphism.

Tenascin-XB (TNXB) amino acid substitution E2004G is associated with mature weight and milk score in American Rambouillet, Targhee, Polypay, and Suffolk sheep

Sheep are economically important worldwide, and growth-associated genetic variants, such as a TNXB charged amino acid substitution E2004G and a silent DGAT1 single nucleotide polymorphism (rs409119650), could improve sheep profitability. However, both were identified in single reports using small groups of sheep from outside the U.S. We evaluated 896 U.S. sheep from one location to investigate an association of TNXB E2004G and DGAT1 rs409119650 with growth and lifetime production. For TNXB E2004G, glutamic acid homozygotes had greater body weights in spring and fall at ages 3 and 4 (all P ≤ 0.05) and greater milk scores at ages 3 and 4 (P < 0.05). These data provide the first report of an association with milk score and confirm a prior report showing association of the ancestral glutamic acid allele with increased growth. While DGAT1 rs409119650 was associated with increased 4-year-old body weight and lifetime greasy fleece weight (P < 0.05), these results should be interpreted with caution given a low minor allele frequency (4.9%). Overall, confirmation that TNXB E2004G is associated with increased mature weight in divergent breeds and multiple countries may suggest a useful role in selective breeding programs.

Association of MSTN gene polymorphism (C354T) with performance traits in raccoon dogs

The aim of the study was to identify polymorphisms within nuclear DNA genes and determine their association with the performance traits of farmed raccoon dogs. The study involved 354 animals and their breeding documentation data. Myostatin (MSTN), insulin-like growth factor 1 (IGF1), and growth hormone (GHR) genes were amplified, sequenced, and subjected to bioinformatics and statistical analysis. Estimation of variance components was performed with the residual maximum likelihood (REML) method, and best linear unbiased prediction (BLUP) of the breeding values was carried out. The predictors of the traits of direct additive, maternal additive, and random-specific maternal environmental effects were analysed. Within the nucleotide sequences of the analysed genes, one silent single nucleotide polymorphism (SNP) was identified in exon 1 of the MSTN gene. The analysis of the variance of the fixed-specific maternal environmental effect revealed statistically significant differences in the body weight among raccoon dogs with alternative polymorphisms. The mean effects of the estimators of polymorphism addition and dominance measured by MSTN gene regression had mostly a significant impact on the level of the traits estimated in animals. There was statistically significant association of the polymorphism in the MSTN gene with the body weight in raccoon dogs, which validates selection thereof as a candidate gene for this economically most important performance trait of raccoon dogs.

A silent mutation in human alpha-A crystallin gene in patients with age-related nuclear or cortical cataract.

A cataract is a complex multifactorial disease that results from alterations in the cellular architecture, i.e. lens proteins. Genes associated with the development of lens include crystallin genes. Although crystallins are highly conserved proteins among vertebrates, a significant number of polymorphisms exist in human population. In this study, we screened for polymorphisms in crystallin alpha A (CRYAA) and alpha B (CRYAB) genes in 200 patients over 40 years of age, diagnosed with age-related cataract (ARC; nuclear and cortical cataracts). Genomic DNA was extracted from the peripheral blood. The coding regions of the CRYAA and CRYAB gene were amplified using polymerase chain reaction and subjected to restriction digestion. Restriction fragment length polymorphism (RFLP) was performed using known restriction enzymes for CRYAA and CRYAB genes. Denaturing high performance liquid chromatography and direct sequencing were performed to detect sequence variation in CRYAA gene. In silico analysis of secondary CRYAA mRNA structure was performed using CLC RNA Workbench. RFLP analysis did not show any changes in the restriction sites of CRYAA and CRYAB genes. In 6 patients (4 patients with nuclear cataract and 2 with cortical cataract), sequence analysis of the exon 1 in the CRYAA gene showed a silent single nucleotide polymorphism [D2D] (CRYAA: C to T transition). One of the patients with nuclear cataract was homozygous for this allele. The in silico analysis revealed that D2D mutation results in a compact CRYAA mRNA secondary structure, while the wild type CRYAA mRNA has a weak or loose secondary structure. D2D mutation in the CRYAA gene may be an additional risk factor for progression of ARC.

Abstract 5464: Host variation in OATP1B1 is associated with treatment outcome in pediatric AML

Abstract Improvements in survival have been achieved for children and adolescents with acute myeloid leukemia (AML), with the 5-year survival rate increasing between 1975 and 2010 from &amp;lt;20% to 70%. However in the past decade, outcome has not improved. Therapeutic approach in AML consists of intensive combination chemotherapy and hematopoietic stem cell transplantation. Although several prognostic factors have been identified, current predictive analysis remains inadequate. We performed a pharmacogenetic-association study on leukemic and/or germline DNA of 164 pediatric patients with AML receiving cytarabine (Ara-C), daunorubicin (Dauno), etoposide (VP16) and mitoxantrone (MTZ) using the DMET platform involving 1,936 variants in 225 genes related to drug metabolism and transport. After adjusting for age, risk group and treatment arm, among the top-ranking variants associated with event-free survival (P = 0.0060) and overall survival (P = 0.012) was a silent single-nucleotide polymorphism (SNP) in SLCO1B1 (c.597C&amp;gt;T), a gene encoding the hepatocellular uptake transporter OATP1B1. This SNP was not correlated with gene expression in leukemic blasts (P = 0.58), suggesting the association might be due to a host rather than a leukemia effect. Since this SNP is in strong linkage disequilibrium (P&amp;lt;0.0001) with two non-synonymous coding variants in SLCO1B1 that encode known functional variants OATP1B1*1B (Asn130Asp) and OATP1B1*5 (Val174Ala), we hypothesized that one or more of the AML drugs may be a substrate of OATP1B1. In vitro transport studies using mammalian cells stably transfected with OATP1B1*1A (wild-type) revealed that the intracellular uptake of Ara-C, Dauno, VP16 and MTZ was enhanced compared with control cells in a time- and concentration-dependent manner. Moreover, the efficiency of drug transport was impaired in cells expressing either OATP1B1*1B or OATP1B1*5, or both (OATP1B1*15) compared with OATP1B1*1A. To test whether Ara-C, Dauno, VP16 and/or MTZ are transported by OATP1B-type carriers in vivo, we determined the pharmacokinetic profile of these agents (1-100 mg/kg; i.v. or i.p.) in mice deficient in the ortholog transporter Oatp1b2 [Oatp1b2(-/-) mice]. We found the concentrations of Dauno, VP16 and MTZ in livers of Oatp1b2(-/-) mice were reduced (1.4-2.8 fold) compared to wild-type mice, and this phenomenon was accompanied by significant increases in plasma area under the curve. Despite being a transported substrate of OATP1B1*1A and Oatp1b2 in vitro (4.7-fold vs control), plasma concentrations of Ara-C were not affected by Oatp1b2 deficiency, which may reflect a differential contribution of the liver in drug elimination between different species. Collectively, these findings indicate an important role for OATP1B1 in the systemic pharmacokinetics of multiple drugs used in the treatment of pediatric AML and suggest that inherited variability in host transporter function may indirectly regulate the effectiveness of therapy. Citation Format: Christina D. Drenberg, Stanley Pounds, Lei Shi, Shelley Orwick, Lie Li, Shuiying Hu, Alice Gibson, Raul Ribeiro, Jeffrey Rubnitz, Alex Sparreboom, Sharyn D. Baker. Host variation in OATP1B1 is associated with treatment outcome in pediatric AML. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5464. doi:10.1158/1538-7445.AM2015-5464

Genomic and single nucleotide polymorphism analysis of infectious bronchitis coronavirus

Infectious bronchitis virus (IBV) is a Gammacoronavirus that causes a highly contagious respiratory disease in chickens. A QX-like strain was analysed by high-throughput Illumina sequencing and genetic variation across the entire viral genome was explored at the sub-consensus level by single nucleotide polymorphism (SNP) analysis. Thirteen open reading frames (ORFs) in the order 5′-UTR-1a-1ab-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′UTR were predicted. The relative frequencies of missense: silent SNPs were calculated to obtain a comparative measure of variability in specific genes. The most variable ORFs in descending order were E, 3b, 5′UTR, N, 1a, S, 1ab, M, 4c, 5a, 6b. The E and 3b protein products play key roles in coronavirus virulence, and RNA folding demonstrated that the mutations in the 5′UTR did not alter the predicted secondary structure. The frequency of SNPs in the Spike (S) protein ORF of 0.67% was below the genomic average of 0.76%. Only three SNPS were identified in the S1 subunit, none of which were located in hypervariable region (HVR) 1 or HVR2. The S2 subunit was considerably more variable containing 87% of the polymorphisms detected across the entire S protein. The S2 subunit also contained a previously unreported multi-A insertion site and a stretch of four consecutive mutated amino acids, which mapped to the stalk region of the spike protein. Template-based protein structure modelling produced the first theoretical model of the IBV spike monomer. Given the lack of diversity observed at the sub-consensus level, the tenet that the HVRs in the S1 subunit are very tolerant of amino acid changes produced by genetic drift is questioned.

A Single Nucleotide Polymorphism in cBIM Is Associated with a Slower Achievement of Major Molecular Response in Chronic Myeloid Leukaemia Treated with Imatinib

PurposeBIM is essential for the response to tyrosine-kinase inhibitors (TKI) in chronic myeloid leukaemia (CML) patients. Recently, a deletion polymorphism in intron 2 of the BIM gene was demonstrated to confer an intrinsic TKI resistance in Asian patients. The present study aimed at identifying mutations in the BIM sequence that could lead to imatinib resistance independently of BCR-ABL mutations.Experimental DesignBIM coding sequence analysis was performed in 72 imatinib-treated CML patients from a French population of our centre and in 29 healthy controls (reference population) as a case-control study. Real-time quantitative PCR (RT qPCR) was performed to assess Bim expression in our reference population.ResultsNo mutation with amino-acid change was found in the BIM coding sequence. However, we observed a silent single nucleotide polymorphism (SNP) c465C>T (rs724710). A strong statistical link was found between the presence of the T allele and the high Sokal risk group (p = 0.0065). T allele frequency was higher in non responsive patients than in the reference population (p = 0.0049). Similarly, this T allele was associated with the mutation frequency on the tyrosine kinase domain of BCR-ABL (p<0.001) and the presence of the T allele significantly lengthened the time to achieve a major molecular response (MMR). Finally, the presence of the T allele was related to a decreased basal expression of the Bim mRNA in the circulating mononuclear cells of healthy controls.ConclusionThese results suggest that the analysis of the c465C>T SNP of BIM could be useful for predicting the outcome of imatinib-treated CML patients.

A Single Nucleotide Polymorphism in the Coding Sequence of BIM Is Associated with Poor Prognostic in Chronic Myeloid Leukaemia Treated by Imatinib

AbstractAbstract 1683BIM is a pro-apoptotic protein essential to tyrosine kinase inhibitors (TKI) response in chronic myeloid leukaemia (CML) patients. Thus, mutations in BIM sequence could lead to imatinib resistance independently of BCR-ABL mutations. Recently, a deletion polymorphism in intron 2 of BIM was demonstrated to confer intrinsic TKI resistance in asiatic patients (Ng et al, nature medicine, 2012). However the Caucasian population is not concerned by this intronic deletion polymorphism. So, in the current study we performed BIM coding sequence analysis in 72 CML imatinib-treated patients and among them 40 were resistant to imatinib in our center from French population of patients. For twenty patients, the disease evolved in either acceleration or blastic phases at least once during the study. We did not find any mutation with amino-acid change in the BIM coding sequence. However, we observed a silent single nucleotide polymorphism (SNP) c465C>T (rs724710) in the region coding for the active BH3 domain. A strong statistic link was found between the presence of the T allele of this polymorphism and the Sokal high risk group (p=0.0065). The frequency of the T allele was found higher in non responsive patients than in a local healthy donor reference population (p=0.0049). Similarly, the analysis showed an association of this T allele with the frequency of mutations on the tyrosine kinase domain of BCR-ABL (p<0.001) but not with additional chromosomal alterations. Moreover, the presence of the allele T significantly lengthened the time of achievement of major molecular remission. Finally, the presence of the T allele was found related to a decreased basal expression of the Bim mRNA in the circulating mononuclear cells of healthy donors. These results suggest that analysis of the c465C>T SNP of BIM could be useful for predicting the outcome of CML patients under imatinib treatment. Disclosures:Mahon:Novartis Pharma: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy.

Progesterone receptor variants associated with the PROGINS haplotype exhibit functional properties similar to those of wild-type progesterone receptor

The progesterone receptor (PR) is a ligand-activated transcription factor existing in two isoforms, A (PRA) and B (PRB), resulting from alternative promoter usage. It has long been speculated that genetic variants of PR are associated with the risk for various benign and malignant diseases, but data from clinical trials and in-vitro studies remain contradictory. The most extensively studied variant is termed PROGINS and consists of an intronic 320-bp Alu insertion and two coding (Ser344Thr, Val660Leu) and one silent single nucleotide polymorphism in complete linkage disequilibrium (allele frequency in Caucasians 9-19%). Our study aimed at elucidating the functional consequences of the PROGINS-associated single nucleotide polymorphisms of PRA and PRB (i.e. Thr344 and Leu660) as compared with wild-type PR (Ser344, Val660). The two PRA and two PRB full-length receptor variants were expressed by adenovirus in the PR-negative human breast cancer cell line T47D-Y and assayed with respect to transactivational properties, c-src activation, combined net mRNA and protein stability and hormone-binding characteristics. In all experiments the wild-type PR and the PROGINS variant were undistinguishable. Though there still might be tissue specific effects of the variants, our data indicate that these common PR variants do not functionally differ, which may provide a basis to explain the heterogeneous outcome of association studies.

Genetic basis of rifampicin resistance in methicillin-resistant Staphylococcus aureus suggests clonal expansion in hospitals in Cape Town, South Africa

BackgroundSince 2001, several studies have reported high rifampicin resistance rates (45 - 100%) among methicillin-resistant Staphylococcus aureus (MRSA) isolates from South Africa. The authors previously characterised 100 MRSA isolates from hospitals in Cape Town, South Africa; forty-five percent of these isolates were rifampicin-resistant. The majority (44/45) corresponded to ST612-MRSA-IV, which is prevalent in South Africa, but has not been reported frequently elsewhere. The remaining rifampicin-resistant isolate corresponded to ST5-MRSA-I. The aim of this study was to investigate further the prevalence and genetic basis of rifampicin-resistance in MRSA isolates from hospitals in Cape Town.ResultsBetween July 2007 and June 2011, the prevalence of rifampicin-resistant MRSA in hospitals in Cape Town ranged from 39.7% to 46.4%. Based on the results of the aforementioned study, nine ST612-MRSA-IV isolates, the rifampicin-resistant ST5-MRSA-I isolate, and two rifampicin-susceptible MRSA isolates were investigated. Four previously described ST612-MRSA-IV isolates, including two each from South Africa and Australia, were also included.The ST5-MRSA-I isolate carried a single mutational change, H481Y, commonly associated with high-level rifampicin resistance. All ST612-MRSA-IV isolates carried an uncommon double amino acid substitution in RpoB, H481N, I527M, whilst one of the Australian ST612-MRSA-IV isolates carried an additional mutation within rpoB, representing a novel rpoB genotype: H481N, I527M, K579R. All ST612-MRSA-IV isolates also shared a unique silent single nucleotide polymorphism (SNP) within rpoB.ConclusionsThat local ST612-MRSA-IV isolates described here share an uncommon rpoB genotype and a unique silent SNP suggests this clone may have undergone clonal expansion in hospitals in Cape Town. Further, the data suggest that these isolates may be related to rifampicin-resistant ST612-MRSA-IV previously described in South Africa and Australia.

Prognostic Impact of Wilms Tumor 1 Single Nucleotide Polymorphism rs16754 In Older Patients with Acute Myeloid Leukemia

AbstractAbstract 2701The Wilms tumor 1 (WT1) gene, located at chromosome band 11p13, encodes a transcriptional regulator involved in normal hematopoietic development. The role of WT1 in acute myeloid leukemia (AML) has been underlined by the finding of WT1 overexpression in most AML cases and WT1 gene mutations in approximately 10% of AML patients. Recently, the minor allele of the silent Arg301 single nucleotide polymorphism (SNP) (rs16754), which is a 903A>G substitution in WT1 exon 7, was suggested to predict a favorable prognosis in adult patients with cytogenetically normal (CN) acute myeloid leukemia (AML). In contrast, no prognostic impact of this SNP was found in another study performed in pediatric AML. Our aim was to evaluate the frequency, the main associated features and the prognostic significance of WT1 SNP rs16754 in elderly patients with AML. Diagnostic bone marrow or peripheral blood samples were analyzed from 266 patients (age 50 to 70 years) with previously untreated de novo AML included in the French ALFA-9801 trial. WT1 SNP rs16754 and gene mutations of NPM1, CEBPA, FLT3 (internal tandem duplication, ITD), WT1 (exon 7 and 9), IDH1 and IDH2 (exon 4) were screened on genomic DNA by direct sequencing and fragment-length analysis for the detection of small insertion/deletion. The minor allele of WT1 SNP rs16754 was found in 85 (32%) out of 266 cases. Patients heterozygous or homozygous for minor allele (WT1AG or WT1GG) and patients homozygous for the major allele (WT1AA) did not significantly differ in terms of age, gender, white blood cell (WBC) count, FAB subtypes, cytogenetic categories, and gene mutations when those mutations are considered separately. However, among CN-AML, the frequency of the favorable genotype defined by the presence of NPM1 mutation or CEBPA mutation without neither FLT3-ITD nor IDH1 mutation was significantly lower in patients with at least one minor allele than in patients with two major alleles (5% vs 23% of CN-AML, p=0.02). In univariate analysis, complete remission rate was found similar between patients with at least one minor allele and patients with two major alleles (76% vs 75%, p=0.88). Patients with at least one minor allele tended to have a shorter median delay to relapse (7 vs 12.2 months, p=0.06) and had a significantly shorter overall survival compared to patients with the two major alleles (5-year OS, 14% vs 26%, p=0.02). In the subset of CN-AML (n=113), the presence of at least one minor allele was also associated with a shorter median delay to relapse (6.9 vs 12.5 months, p=0.02) but only a trend regarding overall survival was observed (5-year OS, 17% vs 30%, p=0.1). In multivariate analysis considering age, WBC count, cytogenetics (favorable, intermediate and unfavorable categories) as covariates, WT1 SNP rs16754 status was found to be an independent prognostic factor for relapse risk (HR 1.56, 95% CI 1.06 to 2.30, p=0.03) and overall survival (HR 1.54, 95% CI 1.08 to 2.20, p=0.02). Thus, in our cohort of older AML patients, the minor allele of WT1 SNP rs16754 appears to confer a relatively poor prognosis, which is in contradiction to what has been reported so far. Overall, our data suggest that WT1 SNP rs16754 is an interesting marker that may contribute to refine prognosis in AML, at least in this age group of patients. Further investigations are needed to clarify the relationship between WT1 SNP rs16754 and treatment outcome in AML and elucidate the biological effects of this SNP. Disclosures:No relevant conflicts of interest to declare.

Estrogen Receptor-Beta Gene Polymorphism in women with Breast Cancer at the Imam Khomeini Hospital Complex, Iran

ER-alpha and ER-beta genes have been proven to play a significant role in breast cancer. Epidemiologic studies have revealed that age-incidence patterns of breast cancer in Middle East differ from those in the Western countries. Two selected coding regions in the ER-β gene (exons 3 and 7) were scanned in Iranian women with breast cancer (150) and in healthy individuals (147). PCR single-strand conformation polymorphism was performed. A site of silent single nucleotide polymorphism was found only on exon 7. The SNP was found only in breast cancer patients (5.7%) (χ2 = 17.122, P = 0.01). Codon 392 (C1176G) of allele 1 was found to have direct association with the occurrence of lymph node metastasis. Our data suggest that ER-β polymorphism in exon 7 codon 392 (C1176G) is correlated with various aspects of breast cancer and lymph node metastasis in our group of patients.

A novel single-nucleotide polymorphism of thevisfatin gene and its associations with performance traits in the chicken

Visfatin is a peptide that is predominantly expressed in visceral adipose tissue and is hypothesized to be related to obesity and insulin resistance. In this study, a novel silent single-nucleotide polymorphism (SNP) was found in exon 7 of the chicken visfatin gene (also known as PBEF1) by single-stranded conformation polymorphism (SSCP) and DNA sequencing. In total, 836 chickens forming an F2 resource population of Gushi chicken crossed with Anka broiler were genotyped by XbaI forced RFLP, and the associations of this polymorphism with chicken growth, carcass characteristics, and meat quality were analyzed. Significant associations were found between the polymorphism and 4-week body weight (BW4), 6-week body weight (BW6), 4-week body slanting length (BSL4), fat bandwidth (FBW), breast muscle water loss rate (BWLR) and breast muscle fiber density (BFD) (P < 0.05), as well as 4-week breastbone length (BBL4) (P < 0.01). These observations suggested that the polymorphism in exon7 of the visfatin gene had significant effects on the early growth traits of chicken.

Single Nucleotide Polymorphism Analysis of the Major Tripartite Multidrug Efflux Pump of Escherichia coli : Functional Conservation in Disparate Animal Reservoirs despite Exposure to Antimicrobial Chemotherapy

AcrAB-TolC imparts a strong intrinsic resistance phenotype to many clinically significant molecules in Escherichia coli. This complex is composed of a pump, AcrB, and a periplasmic protein, AcrA, that exports substrates through a common outer membrane porin, TolC. A sequence survey of the pump-specific components, acrA and acrB, was conducted on three discrete animal reservoirs: rodents, bovines, and catfish. Although two of the reservoirs (bovine and catfish) were agrarian, and antibiotic use (ceftiofur and oxytetracycline/Romet 30, respectively) was reported for them, the vast majority of structural polymorphisms were silent except for T104A (AcrA) and Q733R (AcrB), found in certain bovine-derived strains. Overall, the genes were well conserved, with high ratios of synonymous to nonsynonymous substitutions (d(S)/d(N) ratios), consistent with or, in the case of acrB, better than those of standard multilocus sequence typing (MLST) loci. Furthermore, predicted recombination points from single nucleotide polymorphism (SNP) patterns in acrB support a modular evolution of transporter proteins, consistent with an ancient origin. However, functional studies with clones representing the major silent SNPs and the nonsilent mutation in acrB failed to generate significant differences in resistance to a range of common efflux pump substrates. Interestingly, a comparison between log-phase acrA and acrB expression profiles yielded inconsistent trends, with acrB expression increasing modestly (<1.8-fold) in many strains from the antibiotic-enriched pools. Our results suggest that structural polymorphisms in this major efflux pump system may not contribute significantly to adaptive resistance by altering function or substrate specificity but may have a potential use in improving phylogenetic relationships and/or source tracking.

Expression of candidate tumor suppressor gene ING2 is lost in non-small cell lung carcinoma

ING2 is a candidate tumor suppressor gene involved in cell cycle control, apoptosis and senescence. Furthermore, we have recently shown that loss of ING2 expression is associated with increased genome instability. We investigated its status in a series of 120 non-small cell lung cancer (NSCLC) by using immunohistochemistry (IHC). The results showed that ING2 protein expression is downregulated in more than 50% of NSCLC, with a higher frequency in adenocarcinoma (ADK) as compared to squamous cell carcinoma (SCC) (68% versus 45%, P = 0.021). Loss of ING2 expression occurs in a high proportion of tumors from stage I and was not associated with patient's gender, age and 5-year survival. When investigating the possible mechanisms responsible for the decrease of ING2 expression, we did not observe any loss of heterozygosity or mutation in the ING2 gene. However, in 95% of the cases examined, we identified a silent single nucleotide polymorphism (SNP). By using quantitative RT-PCR, we found that ING2 loss of expression may be due to the decrease of its mRNA level. Analysis of CpG islands present in the promoter region of the ING2 gene did not allow for the detection of methylation. Mechanistically, although p53 can regulate ING2 transcription and ING2 enhances p53 activity, no correlation between ING2 and p53 IHC status was observed. Overall, these results indicate that loss of ING2 expression could contribute to lung tumorigenesis independently of p53.

A Comparison of Coding Sequence and Cytogenetic Localization of the Myostatin Gene in the Dog, Red Fox, Arctic Fox and Chinese Raccoon Dog

The gene encoding myostatin (MSTN), due to its crucial function for growth of skeletal muscle mass, is an important candidate for muscularity. In this study we analyzed the nucleotide sequence and FISH localization of this gene in 4 canids, including 3 farm species. The nucleotide sequence of the MSTN coding fragment turned out to be highly conserved, since its identity among the studied species was very high and varied between 99.4 and 99.7%. Only 1, widely spread, silent single nucleotide polymorphism (SNP) was found in exon 1 of the Chinese raccoon dog. The MSTN gene was localized close to the centromere in one-armed chromosomes of the dog (37q11) and bi-armed chromosomes of the red fox (16p11) and arctic fox (10q11), with an exception of the Chinese raccoon dog chromosome (2q14–q21). This chromosome is orthologous to 3 canine chromosomes and thus the MSTN was found more interstitially. Our results are in agreement with the hypothesis that karyotypes of the canids evolved mainly through centric fusion/fission events, while tandem fusions occurred rarely.

Mutation and Single-Nucleotide Polymorphism (rs16754) in Wilms Tumor-1 Gene Are Independent Prognostic Factors in Acute Myeloid Leukemia.

Abstract 995Poster Board I-17Several reports have suggested that mutations in the Wilms tumor 1 gene (WT1) represent an adverse prognostic factor in acute myeloid leukemia (AML). Here we examined the associations of WT1 mutations in exons 7 and 9 and the silent R301 single-nucleotide polymorphism (SNP) in exon 7 (A903G; NCBI dbSNP reference ID: rs16754) with outcome in AML patients treated at a single institution. Peripheral blood plasma and bone marrow samples from 174 newly diagnosed AML patients were tested for WT1 mutations in exons 7 and 9 by sequencing and fragment-length analysis for the detection of small deletions/insertions. Sequencing provided information on the specific genotype of the rs16754 SNP. The findings were correlated with outcome and other laboratory findings. WT1 mutation was detected in 7 of 50 (14%) AML patients <50 years of age and in 5 of 124 (4%) patients >50. The silent R301 SNP was detected at frequencies of 4% for GG, 26% for GA, and 70% for AA genotypes, but there was no difference with age. A similar SNP genotype distribution was detected in normal control subjects. WT1 mutations were associated with higher white cell count (P=0.01) and higher percentage of blasts in bone marrow (P=0.03) and peripheral blood (P=0.009). In addition, WT1 mutation was significantly associated with FLT3 mutation (P=0.002) but not NPM1 mutation (P=0.8). WT1 mutation was also significantly associated with shorter survival (P=0.025), event-free survival (P=0.002), and complete remission duration (P=0.002) in patients <50 years of age, but not in older patients. The association with shorter survival persisted when only patients with intermediate cytogenetics were considered (P=0.03). There was no correlation between WT1 mutation and response to therapy. The most striking correlation was between the presence of the GG genotype at R301 and longer survival, irrespective of age (see survival chart below). There was no difference in survival between the AA and GA groups. To the best of our knowledge, this is the first report describing correlation between survival duration and an SNP in the WT1 gene. While we confirm that the presence of WT1 mutation is associated with poor outcome in young AML patients, our data suggest that there is no clinical value in testing patients older than 50 for WT1 mutation, but that there may be value in testing adult patients of all age groups for the SNP polymorphism at R301 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The GNAS 393 T &gt; C Polymorphism and the Blood Pressure Response Immediately Following Aerobic Exercise Among Men with Elevated Blood Pressure

A common silent single nucleotide polymorphism (393 T>C) in exon 5 of the guanine nucleotide-binding pro- tein systemsubunit (GNAS) gene associates with hypertension, and altered autonomic nervous system function and re- sponse to � -blockade. We examined the effects of GNAS 393 T>C on the acute post-exercise BP response among 48 Caucasian men (mean ± SEM, 43.7±1.4 yr) with hypertension (145.1±1.5/85.5±1.1 mmHg). Subjects self disclosed a fam- ily history of hypertension. Experiments were non-exercise control and 2 exercise bouts at 40% (LIGHT) and 60% (MODERATE) of peak oxygen uptake. Subjects left the laboratory with an ambulatory BP monitor. Genotypes were de- tected using polymerase chain reaction and restriction enzyme digestion. Repeated measure ANCOVA tested if BP dif- fered over time among experiments and GNAS genotypes (n=37, TT/TC; n=11, CC). Systolic BP increased 8.0+3.6 mmHg less (p 0.05). Most men with the GNAS T 393 allele and a family history of hyperten- sion had lower BP after LIGHT (18/20) vs. non-exercise control; whereas 64% of men with the GNAS CC genotype did not have lower BP after LIGHT (7/11), independent of family history of hypertension (p<0.01). Men with the GNAS T 393 allele, a family history of hypertension, and high BP appear to experience the antihypertensive effects of lower intensity, aerobic exercise more so than men with the GNAS CC genotype.