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author:("Liu, jinhua")
1.  Imputation of the Rare HOXB13 G84E Mutation and Cancer Risk in a Large Population-Based Cohort 
PLoS Genetics  2015;11(1):e1004930.
An efficient approach to characterizing the disease burden of rare genetic variants is to impute them into large well-phenotyped cohorts with existing genome-wide genotype data using large sequenced referenced panels. The success of this approach hinges on the accuracy of rare variant imputation, which remains controversial. For example, a recent study suggested that one cannot adequately impute the HOXB13 G84E mutation associated with prostate cancer risk (carrier frequency of 0.0034 in European ancestry participants in the 1000 Genomes Project). We show here that—by utilizing the 1000 Genomes Project data plus an enriched reference panel of mutation carriers—we were able to accurately impute the G84E mutation into a large cohort of 83,285 non-Hispanic White participants from the Kaiser Permanente Research Program on Genes, Environment and Health Genetic Epidemiology Research on Adult Health and Aging cohort. Imputation authenticity was confirmed via a novel classification and regression tree method, and then empirically validated analyzing a subset of these subjects plus an additional 1,789 men from the California Men’s Health Study specifically genotyped for the G84E mutation (r2 = 0.57, 95% CI = 0.37–0.77). We then show the value of this approach by using the imputed data to investigate the impact of the G84E mutation on age-specific prostate cancer risk and on risk of fourteen other cancers in the cohort. The age-specific risk of prostate cancer among G84E mutation carriers was higher than among non-carriers, and this difference increased with age. Risk estimates from Kaplan-Meier curves were 36.7% versus 13.6% by age 72, and 64.2% versus 24.2% by age 80, for G84E mutation carriers and non-carriers, respectively (p = 3.4×10−12). The G84E mutation was also suggestively associated with an increase in risk for the following cancer sites by approximately 50% in a pleiotropic manner: breast, non-Hodgkin’s lymphoma, kidney, bladder, melanoma, endometrium, and pancreas (p = 0.042).
Author Summary
An efficient approach to characterizing the disease burden of rare genetic variants is to impute them into existing well-phenotyped cohorts with genome-wide data by using large sequenced reference panels; however, the efficacy of this approach remains controversial. A recent study suggested that it is not possible to impute the rare HOXB13 G84E variant using neighboring SNP markers. We show that by using an enriched reference sequenced sample of 22 mutation carriers, we were able to impute this mutation into a large cohort of 83,285 non-Hispanic White individuals from the Kaiser Permanente Research Program on Genes, Environment, and Health Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort. The imputation was confirmed via a novel classification and regression tree method, and then empirically validated by direct mutation genotyping of a subset of 1,673 of these individuals in addition to 1,789 other men from Kaiser. Using the same GERA cohort, we then confirmed that the G84E mutation is associated with increased risk of prostate cancer, and estimated the age-specific risk for carriers of the mutation. Finally, we obtained evidence that the mutation is associated with additional types of cancer in the GERA cohort.
PMCID: PMC4309593  PMID: 25629170
2.  Cytoplasmic and/or Nuclear Expression of β-Catenin Correlate with Poor Prognosis and Unfavorable Clinicopathological Factors in Hepatocellular Carcinoma: A Meta-Analysis 
PLoS ONE  2014;9(11):e111885.
The β-catenin is an important effector in WNT/β-catenin signaling pathway, which exerts a crucial role in the development and progression of hepatocellular carcinoma (HCC). Some researchers have suggested that the overexpression of β-catenin in cytoplasm and/or nucleus was closely correlated to metastasis, poor differentiation and malignant phenotype of HCC while some other researchers hold opposite point. So far, no consensus was obtained on the prognostic and clinicopathological significance of cytoplasmic/nuclear β-catenin overexpression for HCCs.
Systematic strategies were applied to search eligible studies in all available databases. Subgroup analyses, sensitivity analyses and multivariate analysis were performed. In this meta-analysis, we utilized either fixed- or random-effects model to calculate the pooled odds ratios (OR) and its 95% confidence intervals (CI).
A total of 22 studies containing 2334 cases were enrolled in this meta-analysis. Pooled data suggested that accumulation of β-catenin in cytoplasm and/or nucleus significantly correlated with poor 1-, 3- and 5-year OS and RFS. Moreover, nuclear accumulation combined with cytoplasmic accumulation of β-catenin tended to be associated with dismal metastasis and vascular invasion while cytoplasmic or nuclear expression alone showed no significant effect. Besides, no significant association was observed between cytoplasmic and/or nuclear β-catenin expression and poor differentiation grade, advanced TNM stage, liver cirrhosis, tumor size, tumor encapsulation, AFP and etiologies. Additional subgroup analysis by origin suggested that the prognostic value and clinicopathological significance of cytoplasmic and/or nuclear β-catenin expression was more validated in Asian population. Multivariate analyses of factors showed that cytoplasmic and/or nuclear β-catenin expression, as well as TNM stage, metastasis and tumor size, was an independent risk factors for OS and RFS.
Cytoplasmic and/or nuclear accumulation of β-catenin, as an independent prognostic factor, significantly associated with poor prognosis and deeper invasion of HCC, and could serve as a valuable prognostic predictor for HCC.
PMCID: PMC4234306  PMID: 25401330
3.  PRAK Interacts with DJ-1 and Prevents Oxidative Stress-Induced Cell Death 
As a core member of p38 MAPK signal transduction pathway, p38 regulated/activated kinase (PRAK) is activated by cellular stresses. However, the function of PRAK and its downstream interacting partner remain undefined. Using a yeast two-hybrid system, we identified DJ-1 as a potential PRAK interacting protein. We further verified that DJ-1 bound to PRAK in vitro and in vivo and colocalized with PRAK in the nuclei of NIH3T3 cells. Furthermore, following H2O2 stimulation the majority of endogenous DJ-1 in PRAK+/+ cells still remained in the nucleus, whereas most DJ-1 in PRAK−/− cells translocated from the nucleus into the cytoplasm, indicating that PRAK is essential for DJ-1 to localize in the nucleus. In addition, PRAK-associated phosphorylation of DJ-1 was observed in vitro and in vivo of H2O2-challenged PRAK+/+ cells. Cytoplasmic translocation of DJ-1 in H2O2-treated PRAK−/− cells lost its ability to sequester Daxx, a death protein, in the nucleus, and as a result, Daxx gained access to the cytoplasm and triggered cell death. These data highlight that DJ-1 is the downstream interacting target for PRAK, and in response to oxidative stress PRAK may exert a cytoprotective effect by facilitating DJ-1 to sequester Daxx in the nucleus, thus preventing cell death.
PMCID: PMC4212658  PMID: 25383140
4.  Components of the Cultivated Red Seaweed Chondrus crispus Enhance the Immune Response of Caenorhabditis elegans to Pseudomonas aeruginosa through the pmk-1, daf-2/daf-16, and skn-1 Pathways 
Applied and Environmental Microbiology  2013;79(23):7343-7350.
Marine macroalgae are rich in bioactive compounds that can, when consumed, impart beneficial effects on animal and human health. The red seaweed Chondrus crispus has been reported to have a wide range of health-promoting activities, such as antitumor and antiviral activities. Using a Caenorhabditis elegans infection model, we show that C. crispus water extract (CCWE) enhances host immunity and suppresses the expression of quorum sensing (QS) and the virulence factors of Pseudomonas aeruginosa (strain PA14). Supplementation of nematode growth medium with CCWE induced the expression of C. elegans innate immune genes, such as irg-1, irg-2, F49F1.6, hsf-1, K05D8.5, F56D6.2, C29F3.7, F28D1.3, F38A1.5 ZK6.7, lys-1, spp-1, and abf-1, by more than 2-fold, while T20G5.7 was not affected. Additionally, CCWE suppressed the expression of PA14 QS genes and virulence factors, although it did not affect the growth of the bacteria. These effects correlated with a 28% reduction in the PA14-inflicted killing of C. elegans. Kappa-carrageenan (K-CGN), a major component of CCWE, was shown to play an important role in the enhancement of host immunity. Using C. elegans mutants, we identified that pmk-1, daf-2/daf-16, and skn-1 are essential in the K-CGN-induced host immune response. In view of the conservation of innate immune pathways between C. elegans and humans, the results of this study suggest that water-soluble components of C. crispus may also play a health-promoting role in higher animals and humans.
PMCID: PMC3837755  PMID: 24056462
5.  HOXB13 Mutation and Prostate Cancer: Studies of Siblings and Aggressive Disease 
Recent work detected for the first time a high-risk prostate cancer mutation, in homeobox B13 (HOXB13) among European-Americans.
We further evaluated this G84E missense mutation (rs138213197) in two genetic association studies of prostate cancer: a family-based study of brothers and a case-control study of more aggressive disease (N=2,665 total). We then calculated overall impact of this mutation by pooling all published studies of European-Americans.
In our studies the mutation was found exclusively among men with prostate cancer (carrier frequency=1.48%) or unaffected brothers of cases carrying the mutation (frequency=0.34%), and carrying the mutation gave an odds ratio for disease=4.79 (P=0.01). The G84E mutation was more common among men with an earlier age of onset (≤55 years) or a family history of prostate cancer. We also observed for the first time an African-American case carrying the G84E mutation, although at HOXB13 both of his chromosomes were of European-American ancestry. The pooled analysis also indicated that carrying the G84E mutation results in an almost five-fold increase in risk of prostate cancer (P=3.5×10−17), and this risk is even higher among cases with an early age of prostate cancer onset (≤55 years) or a family history of disease: a test of heterogeneity across these strata gives P<1×10−5.
The HOXB13 mutation substantially increases risk of early onset, familial prostate cancer in European-American men. Impact: Testing for the G84E mutation in men with a positive family history may help distinguish those who merit more regular screening for prostate cancer.
PMCID: PMC3617049  PMID: 23396964
Familial Cancer; Genetic Association; Homeobox; Functional Mutation; Prostate Cancer
6.  Confounding and Heterogeneity in Genetic Association Studies with Admixed Populations 
American Journal of Epidemiology  2013;177(4):351-360.
Association studies among admixed populations pose many challenges including confounding of genetic effects due to population substructure and heterogeneity due to different patterns of linkage disequilibrium (LD). We use simulations to investigate controlling for confounding by indicators of global ancestry and the impact of including a covariate for local ancestry. In addition, we investigate the use of an interaction term between a single-nucleotide polymorphism (SNP) and local ancestry to capture heterogeneity in SNP effects. Although adjustment for global ancestry can control for confounding, additional adjustment for local ancestry may increase power when the induced admixture LD is in the opposite direction as the LD in the ancestral population. However, if the induced LD is in the same direction, there is the potential for reduced power because of overadjustment. Furthermore, the inclusion of a SNP by local ancestry interaction term can increase power when there is substantial differential LD between ancestry populations. We examine these approaches in genome-wide data using the University of Southern California's Children's Health Study investigating asthma risk. The analysis highlights rs10519951 (P = 8.5 × 10−7), a SNP lacking any evidence of association from a conventional analysis (P = 0.5).
PMCID: PMC3626055  PMID: 23334005
confounding; genetic association studies; genome-wide association studies; heterogeneity; linkage disequilibrium; population stratification
7.  Nicotinic Acetylcholine Receptor Variation and Response to Smoking Cessation Therapies 
Pharmacogenetics and genomics  2013;23(2):94-103.
Evaluate nicotinic acetycholine receptor (nAChR) single nucleotide polymorphism (SNP) association with seven day point prevalence abstinence (abstinence) in randomized clinical trials of smoking cessation therapies (RCTs) in individuals grouped by pharmacotherapy randomization to inform the development of personalized smoking cessation therapy.
We quantified association of four SNPs at three nAChRs with abstinence in eight RCTs. Participants were 2,633 outpatient treatment-seeking, self-identified European ancestry individuals smoking ≥10 cigarettes per day, recruited via advertisement, prescribed pharmacotherapy, and provided with behavioral therapy. Interventions included nicotine replacement therapy (NRT), bupropion, varenicline, placebo or combined NRT and bupropion, and five modes of group and individual behavioral therapy. Outcome measures tested in multivariate logistic regression were end of treatment (EOT) and six month (6MO) abstinence, with demographic, behavioral and genetic covariates.
“Risk” alleles previously associated with smoking heaviness were significantly (P<0.05) associated with reduced abstinence in the placebo pharmacotherapy group (PG) at 6MO [for rs588765 OR (95%CI) 0.41 (0.17–0.99)], and at EOT and at 6MO [for rs1051730, 0.42 (0.19–0.93) and 0.31 (0.12–0.80)], and with increased abstinence in the NRT PG at 6MO [for rs588765 2.07 (1.11–3.87) and for rs1051730 2.54 (1.29–4.99)]. We observed significant heterogeneity in rs1051730 effects (F=2.48, P=0.021) between PGs.
chr15q25.1 nAChR SNP risk alleles for smoking heaviness significantly increase relapse with placebo treatment and significantly increase abstinence with NRT. These SNP-PG associations require replication in independent samples for validation, and testing in larger sample sizes to evaluate whether similar effects occur in other PGs.
PMCID: PMC3563676  PMID: 23249876
logistic regression; mediation analysis; nAChR variation; nicotine dependence; pharmacotherapy; randomized clinical trials
8.  Development of a Vero cell DNA reference standard for residual DNA measurement in China 
This collaborative study developed a Vero cell DNA reference for standardizing dot blot hybridization, an assay widely employed to measure residual DNA contents of viral vaccines prepared with Vero cells. High purity of Vero cell DNA was extracted and characterized by Hind III enzyme digestion and DNA sequencing. Then, with a cooperative calibration, the concentration of Vero cell DNA reference bulk solution was determined (64.0 ± 1.9 μg/mL, OD260/OD280 = 1.87) and diluted (40 ng/mL) with Tris-EDTA buffer containing bovine serum albumin as freeze-dried excipients. With industrial filling apparatus, the diluted bulk was loaded into ampoules (0.5 mL each) which were heat sealed after nitrogen filling. Finally, a collaborative study showed that the Vero cell DNA reference could reach a sensitivity of 1 to 5 pg/dot and maintained good stability after accelerated destruction test. The successful establishment of the Vero cell DNA quantitative reference will facilitate the standardization of dot blot hybridization for testing residual host cell DNA.
PMCID: PMC3859766  PMID: 23291952
Vero cell genomic DNA; quantitative reference standard; dot blot; standardization
9.  Further Replication Studies of the EVE Consortium Meta-Analysis Identifies Two Asthma Risk Loci in European Americans 
Genome-wide association studies of asthma have implicated many genetic risk factors, with well-replicated associations at approximately 10 loci that account for only a small proportion of the genetic risk.
We aimed to identify additional asthma risk loci by performing an extensive replication study of the results from the EVE Consortium meta-analysis.
We selected 3186 SNPs for replication based on the p-values from the EVE Consortium meta-analysis. These SNPs were genotyped in ethnically diverse replication samples from nine different studies, totaling to 7202 cases, 6426 controls, and 507 case-parent trios. Association analyses were conducted within each participating study and the resulting test statistics were combined in a meta-analysis.
Two novel associations were replicated in European Americans: rs1061477 in the KLK3 gene on chromosome 19 (combined OR = 1.18; 95% CI 1.10 – 1.25) and rs9570077 (combined OR =1.20 95% CI 1.12–1.29) on chromosome 13q21. We could not replicate any additional associations in the African American or Latino individuals.
This extended replication study identified two additional asthma risk loci in populations of European descent. The absence of additional loci for African Americans and Latino individuals highlights the difficulty in replicating associations in admixed populations.
PMCID: PMC3666859  PMID: 23040885
Asthma; genetic risk factors; meta-analysis; KLK3
10.  Gender Stratified Gene and Gene–Treatment Interactions in Smoking Cessation 
The pharmacogenomics journal  2011;12(6):521-532.
We conducted gender-stratified analyses on a systems-based candidate gene study of 53 regions involved in nicotinic response and the brain-reward pathway in two randomized clinical trials of smoking cessation treatments (placebo, bupropion, transdermal and nasal spray nicotine replacement therapy). We adjusted P-values for multiple correlated tests, and used a Bonferroni corrected α-level of 5 × 10−4 to determine system-wide significance. Four SNPs (rs12021667, rs12027267, rs6702335, rs12039988; r2>0.98) in erythrocyte membrane protein band 4.1 (EPB41) had a significant male-specific marginal association with smoking abstinence (OR=0.5; 95% CI 0.3–0.6) at end of treatment (adjusted P<6 × 10−5). rs806365 in cannabinoid receptor 1 (CNR1) had a significant male-specific gene-treatment interaction at 6-month follow-up (adjusted P=3.9 × 10−5); within males using nasal spray, rs806365 was associated with a decrease in odds of abstinence (OR=0.04; 95% CI 0.01–0.2). While the role of CNR1 in substance abuse has been well studied, we report EPB41 for the first time in the nicotine literature.
PMCID: PMC3208134  PMID: 21808284
Genetic association studies; heterogeneity; smoking cessation
11.  Dopamine genes and nicotine dependence in treatment seeking and community smokers 
We utilized a cohort of 828 treatment seeking self-identified white cigarette smokers (50% female) to rank candidate gene single nucleotide polymorphisms (SNPs) associated with the Fagerström Test for Nicotine Dependence (FTND), a measure of nicotine dependence which assesses quantity of cigarettes smoked and time- and place-dependent characteristics of the respondent’s smoking behavior. 1123 SNPs at 55 autosomal candidate genes, nicotinic acetylcholine receptors and genes involved in dopaminergic function, were tested for association to baseline FTND scores adjusted for age, depression, education, sex and study site. SNP P values were adjusted for the number of transmission models, the number of SNPs tested per candidate gene, and their intragenic correlation. DRD2, SLC6A3 and NR4A2 SNPs with adjusted P values < 0.10 were considered sufficiently noteworthy to justify further genetic, bioinformatic and literature analyses. Each independent signal among the top-ranked SNPs accounted for ~1% of the FTND variance in this sample. The DRD2 SNP appears to represent a novel association with nicotine dependence. The SLC6A3 SNPs have previously been shown to be associated with SLC6A3 transcription or dopamine transporter density in vitro, in vivo and ex vivo. Analysis of SLC6A3 and NR4A2 SNPs identified a statistically significant gene-gene interaction (P=0.001), consistent with in vitro evidence that the NR4A2 protein product (NURR1) regulates SLC6A3 transcription. A community cohort of N=175 multiplex ever smoking pedigrees (N=423 ever smokers) provided nominal evidence for association with the FTND at these top ranked SNPs, uncorrected for multiple comparisons.
PMCID: PMC3558036  PMID: 19494806
dopamine transporter; Fagerström Test for Nicotine Dependence; single nucleotide polymorphism; candidate gene association scan; gene-gene interaction
12.  Association of the Calcyon Neuron-Specific Vesicular Protein Gene (CALY) With Adolescent Smoking Initiation in China and California 
American Journal of Epidemiology  2011;173(9):1039-1048.
Although previous investigations have indicated a role for genetic factors in smoking initiation, the underlying genetic mechanisms are still unknown. In 2,339 adolescents from a Chinese Han population in the Wuhan Smoking Prevention Trial (Wuhan, China, 1998–1999), the authors explored the association of 57 genes in the dopamine pathway with smoking initiation. Using a conservative approach for declaring significance, positive findings were further examined in an independent sample of 603 Caucasian adolescents followed for up to 10 years as part of the Children's Health Study (Southern California, 1993–2009). The authors identified 1 single nucleotide polymorphism (rs2298122) in the calcyon neuron-specific vesicular protein gene (CALY) that was positively associated with smoking initiation in females (odds ratio = 2.21, 95% confidence interval: 1.49, 3.27; P = 8.4 × 10−5) in the Wuhan Smoking Prevention Trial cohort, and they replicated the association in females from the Children's Health Study cohort (hazard rate ratio = 2.05, 95% confidence interval: 1.27, 3.31; P = 0.003). These results suggest that the CALY gene may influence smoking initiation in adolescents, although the potential roles of underlying psychological characteristics that may be components of the smoking-initiation phenotype, such as impulsivity or novelty-seeking, remain to be explored.
PMCID: PMC3121219  PMID: 21415033
adolescent; dopamine; genetic association studies; smoking
13.  Investigation on Mycobacterium tuberculosis Diversity in China and the Origin of the Beijing Clade 
PLoS ONE  2011;6(12):e29190.
Investigation of the genetic diversity of Mycobacterium tuberculosis in China has shown that Beijing genotype strains play a dominant role in the tuberculosis (TB) epidemic. In order to examine the strain diversity in the whole country, and to study the evolutionary development of Beijing strains, we sought to genotype a large collection of isolates using different methods.
Methodology/Principal Findings
We applied a 15-loci VNTR typing analysis on 1,586 isolates from the Beijing municipality and 12 Chinese provinces or autonomous regions. The data was compared to that of 900 isolates from various other worldwide geographic regions outside of China. A total of 1,162/1,586 (73.2%) of the isolates, distributed into 472 VNTR types, were found to belong to the Beijing genotype family and this represented 56 to 94% of the isolates in each of the localizations. VNTR typing revealed that the majority of the non-Beijing isolates fall into two genotype families, which represented 17% of the total number of isolates, and seem largely restricted to China. A small number of East African Indian genotype strains was also observed in this collection. Ancient Beijing strains with an intact region of difference (RD) 181, as well as strains presumably resembling ancestors of the whole Beijing genotype family, were mainly found in the Guangxi autonomous region.
This is the largest M. tuberculosis VNTR-based genotyping study performed in China to date. The high percentage of Beijing isolates in the whole country and the presence in the South of strains representing early branching points may be an indication that the Beijing lineage originated from China, probably in the Guangxi region. Two modern lineages are shown here to represent the majority of non-Beijing Chinese isolates. The observed geographic distribution of the different lineages within China suggests that natural frontiers are major factors in their diffusion.
PMCID: PMC3248407  PMID: 22220207
14.  Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China▿ †  
Journal of Clinical Microbiology  2010;48(11):4102-4106.
A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study.
PMCID: PMC3020837  PMID: 20739484
15.  Integrative Identification of Arabidopsis Mitochondrial Proteome and Its Function Exploitation through Protein Interaction Network 
PLoS ONE  2011;6(1):e16022.
Mitochondria are major players on the production of energy, and host several key reactions involved in basic metabolism and biosynthesis of essential molecules. Currently, the majority of nucleus-encoded mitochondrial proteins are unknown even for model plant Arabidopsis. We reported a computational framework for predicting Arabidopsis mitochondrial proteins based on a probabilistic model, called Naive Bayesian Network, which integrates disparate genomic data generated from eight bioinformatics tools, multiple orthologous mappings, protein domain properties and co-expression patterns using 1,027 microarray profiles. Through this approach, we predicted 2,311 candidate mitochondrial proteins with 84.67% accuracy and 2.53% FPR performances. Together with those experimental confirmed proteins, 2,585 mitochondria proteins (named CoreMitoP) were identified, we explored those proteins with unknown functions based on protein-protein interaction network (PIN) and annotated novel functions for 26.65% CoreMitoP proteins. Moreover, we found newly predicted mitochondrial proteins embedded in particular subnetworks of the PIN, mainly functioning in response to diverse environmental stresses, like salt, draught, cold, and wound etc. Candidate mitochondrial proteins involved in those physiological acitivites provide useful targets for further investigation. Assigned functions also provide comprehensive information for Arabidopsis mitochondrial proteome.
PMCID: PMC3031521  PMID: 21297957
16.  Antisense Transcription in Gammaretroviruses as a Mechanism of Insertional Activation of Host Genes▿  
Journal of Virology  2010;84(8):3780-3788.
Transcription of retroviruses is initiated at the U3-R region boundary in the integrated provirus and continues unidirectionally to produce genomic and mRNA products of positive polarity. Several studies have recently demonstrated the existence of naturally occurring protein-encoding transcripts of negative polarity in complex retroviruses. We report here on the identification of transcripts of negative polarity in simple murine leukemia virus (MLV). In T-cell and B-cell lymphomas induced by SL3-3 and Akv MLV, antisense transcripts initiated in the U3 region of the proviral 5′ long terminal repeat (LTR) and continued into the cellular proto-oncogenes Jdp2 and Bach2 to create chimeric transcripts consisting of viral and host sequence. The phenomenon was validated in vivo using a knock-in mouse model homozygous for a single LTR at a position known to activate Nras in B-cell lymphomas. A 5′ rapid amplification of cDNA ends (RACE) analysis indicated a broad spectrum of initiation sites within the U3 region of the 5′ LTR. Our data show for the first time transcriptional activity of negative polarity initiating in the U3 region of simple retroviruses and suggest a novel mechanism of insertional activation of host genes. Elucidation of the nature and potential regulatory role of 5′ LTR antisense transcription will be relevant to the design of therapeutic vectors and may contribute to the increasing recognition of pervasive eukaryotic transcription.
PMCID: PMC2849499  PMID: 20130045
17.  Identification of novel Bach2 transcripts and protein isoforms through tagging analysis of retroviral integrations in B-cell lymphomas 
The Bach2 gene functions as a transcriptional repressor in B-cells, showing high expression level only before the plasma cell stage. Several lines of evidence indicate that Bach2 is a B-cell specific tumor suppressor. We here address patterns of insertional mutagenesis and expression of Bach2 is a murine retroviral model of B-cell lymphoma induction.
We report that the Bach2 gene is a target of proviral integrations in B-cell lymphomas induced by murine leukemia virus. An alternative Bach2 promoter was identified within intron 2 and this promoter was activated in one of the tumors harboring proviral integration. The alternative promoter was active in both normal and tumor tissue and the tissue specificity of the two Bach2 promoters was similar. Three different alternatively used Bach2 terminal exons were identified to be located in intron 4. The inclusion of these exons resulted in the generation of Bach2 mRNA with open reading frames lacking the bZIP DNA binding domain present in the normal Bach2 protein, but retaining a partial BTB protein dimerization domain. Such Bach2 protein was excluded from the cell nucleus.
We have identified an alternative promoter and new protein isoforms of Bach2. Our data imply that activation of an alternative promoter by proviral integration serves as a possible mechanism of up-regulation of the Bach2 gene with a potential role in B-cell lymphomagenesis. The finding of novel Bach2 transcripts and protein isoforms will facilitate a better insight into the normal and pathophysiological regulation of the Bach2 gene.
PMCID: PMC2635362  PMID: 19159451
18.  Nicotinic acetylcholine receptor β2 subunit gene implicated in a systems-based candidate gene study of smoking cessation 
Human Molecular Genetics  2008;17(18):2834-2848.
Although the efficacy of pharmacotherapy for tobacco dependence has been previously demonstrated, there is substantial variability among individuals in treatment response. We performed a systems-based candidate gene study of 1295 single nucleotide polymorphisms (SNPs) in 58 genes within the neuronal nicotinic receptor and dopamine systems to investigate their role in smoking cessation in a bupropion placebo-controlled randomized clinical trial. Putative functional variants were supplemented with tagSNPs within each gene. We used global tests of main effects and treatment interactions, adjusting the P-values for multiple correlated tests. An SNP (rs2072661) in the 3′ UTR region of the β2 nicotinic acetylcholine receptor subunit (CHRNB2) has an impact on abstinence rates at the end of treatment (adjusted P = 0.01) and after a 6-month follow-up period (adjusted P = 0.0002). This latter P-value is also significant with adjustment for the number of genes tested. Independent of treatment at 6-month follow-up, individuals carrying the minor allele have substantially decreased the odds of quitting (OR = 0.31; 95% CI 0.18–0.55). Effect of estimates indicate that the treatment is more effective for individuals with the wild-type (OR = 2.14, 95% CI 1.20–3.81) compared with individuals carrying the minor allele (OR = 0.83, 95% CI 0.32–2.19), although this difference is only suggestive (P = 0.10). Furthermore, this SNP demonstrated a role in the time to relapse (P = 0.0002) and an impact on withdrawal symptoms at target quit date (TQD) (P = 0.0009). Overall, while our results indicate strong evidence for CHRNB2 in ability to quit smoking, these results require replication in an independent sample.
PMCID: PMC2525499  PMID: 18593715

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