AG Variant Research Articles

Short communication: Molecular epidemiology, phylogeny, and phylodynamics of CRF63_02A1, a recently originated HIV-1 circulating recombinant form spreading in Siberia.

The HIV-1 epidemic in Russia is dominated by the former Soviet Union subtype A (A(FSU)) variant, but other genetic forms are circulating in the country. One is the recently described CRF63_02A1, derived from recombination between a CRF02_AG variant circulating in Central Asia and A(FSU), which has spread in the Novosibirsk region, Siberia. Here we phylogenetically analyze pol and env segments from 24 HIV-1 samples from the Novosibirsk region collected in 2013, with characterization of three new near full-length genome CRF63_02A1 sequences, and estimate the time of the most recent common ancestor (tMRCA) and the demographic growth of CRF63_02A1 using a Bayesian method. The analyses revealed that CRF63_02A1 is highly predominant in the Novosibirsk region (81.2% in pol sequences) and is transmitted both among injecting drug users and by heterosexual contact. Similarity searches with database sequences combined with phylogenetic analyses show that CRF63_02A1 is circulating in East Kazakhstan and the Eastern area of Russia bordering China. The analyses of near full-length genome sequences show that its mosaic structure is more complex than reported, with 18 breakpoints. The tMRCA of CRF63_02A1 was estimated around 2006, with exponential growth in 2008-2009 and subsequent stabilization. These results provide new insights into the molecular epidemiology, phylogeny, and phylodynamics of CRF63_02A1.

Chromosome 9p21 rs10757278 polymorphism is associated with the risk of metabolic syndrome

Metabolic syndrome (MetS) is a common multifactorial disorder that involves abdominal obesity, dyslipidemia, hypertension, and hyperglycemia. Genome-wide association studies have identified a major risk locus for coronary artery disease and myocardial infarction on chromosome 9p21. Here, we examined the frequency of single nucleotide polymorphisms (SNPs) on chromosome 9p21 in a sample of Turkish patients with MetS and further investigated the correlation between regional SNPs, haplotypes, and MetS. The real-time polymerase chain reaction (RT-PCR) was used to analyze 4 SNPs (rs10757274 A/G, rs2383207 A/G, rs10757278 A/G, rs1333049 C/G) in 291 MetS patients and 247 controls. Analysis of 4 SNPs revealed a significant difference in the genotype distribution for rs2383207, rs10757278, and rs1333049 between MetS patients and controls (p = 0.041, p = 0.005, p = 0.023, respectively) but not for rs10757274 (p = 0.211). MetS and control allelic frequencies for rs2383207, rs10757278, and rs1333049 were statistically different (p < 0.05). The rs2383207 AG variant, was identified as a MetS risk factor (p = 0.012, OR = 33.271; 95 % CI: 2.193-504.805) and the AA haplotype in block 1 and the GC, AG haplotypes in block 2 were associated with MetS (χ(2) = 3.875, p = 0.049; χ(2) = 9.334, p = 0.0022; χ (2) = 9.134, p = 0.0025, respectively). In this study, we found that chromosome 9p21 SNP rs10757278 and related haplotypes correlate with MetS risk. This is the first report showing an association between a 9p21 variant and MetS and suggests that rs10757278 polymorphism may confer increased risk for disease.

Analysis of HIV‐1 primary drug resistance in Kazakhstan

Purpose of the studyMonitoring of primary resistance in HIV‐1 variants circulating in Russia and FSU countries is the important task of HIV infection molecular epidemiology. The data of HIV molecular epidemiology are absent or limited in many FSU countries. IDU‐A variant of HIV‐1 subtype A has been dominating in Russia (&gt;90%) and other FSU countries since 1996. Additionally, the Central Asian region (e.g. Kazakhstan and Uzbekistan) is characterized by relatively wide spread of CRF02_AG recombinant. The aim of our study was the analysis of HIV primary drug resistance in HIV‐1 variants from Kazakhstan.MethodsBlood collection from the HIV‐infected naïve patients was carried out by local specialists. The study was performed with the informed consent of patients. All sequences of pol gene were analyzed by COMETv0.1 and HIVdb on‐line programs. The phylogenetic analysis and tropism testing were carried out by MEGA4.0 and geno2pheno.Summary of results51 PBMC samples were analyzed. According to pol gene phylogenetic analysis and genotyping 24 (47.0%) samples belonged to recombinant CRF02_AG; 25 (49.0%)‐to subtype A1; 2 (3.9%)‐to CRF03_AB circulating in Russia and some FSU countries. Only one subtype A1 sample had D30N mutation in Pro‐region that cause high‐level resistance to NFV. All AG‐samples had K20I substitution which is characteristic for HIV‐1 subtype A and G and is believed to be associated with resistance to LPV and NFV. In addition, we found that 2 AG‐samples had L10V and L76I substitutions, accordingly. Two A1‐subtype samples studied had L10I and K43T, accordingly. A62V characteristic mutation in RT‐region was found in 12 (48%) subtype A1‐samples. One of them had M184I mutation as well. Besides, we found 3 A1‐samples harboring NNRTIs resistance mutations‐K103N, G190S, K238N, accordingly. Further we analyzed IN region of pol‐gene in 16 and env‐gene in 23 samples studied. We found only one E157Q mutation (in CRF03_AB sample) in IN region. As to tropism, only two subtype A1 and 1 CRF03_AB samples belonged to X4/X4R5 variant, all these A1‐subtype samples harbored A62V and one of them‐G190S in RT region of pol‐gene; the other samples were treated as R5 variants.ConclusionsOur data demonstrated the low level of HIV primary drug resistance in Kazakhstan. HIV‐1 subtype A variant dominates on this territory, but CRF02_AG recombinant is rather widespread as well. CRF02_AG variant studied has characteristic features in Pro region compared with IDU‐A.

The insulin-like growth factor-1 gene is associated with cerebral infarction in Japanese subjects

Atherosclerosis leads to cerebral infarction (CI) and the insulin/insulin-like growth factor-1 (IGF1) signaling pathway plays an important role in this process during adult life. The purpose of this study was to investigate the relationship between the human IGF1 gene and CI in the Japanese population via a case-control study that also included a separate analysis of the two gender groups. A total of 155 CI patients and 316 controls were genotyped for six single nucleotide polymorphisms (SNPs) of the human IGF1 gene (rs2162679, rs7956547, rs2288378, rs2072592, rs978458 and rs6218). All data were analyzed for three separate groups: the total subjects, men and women. The logistic regression analysis revealed that the GG + AG variant of rs2162679 (P = 0.047), the AA + GA variant of rs2072592 (P = 0.005) and the CC + TC variant of rs6218 (P = 0.015) exhibited a protective effect for CI in the total subject group. For the women and the total subjects groups, the overall distribution of the haplotype established by rs7956547-rs978458 was significantly different between the CI patients and the non-CI subjects. For the total subjects, the frequency of the T-G haplotype (rs7956547-rs978458) was also significantly higher (P = 0.034), whereas the frequency of the T-A haplotype (rs7956547-rs978458) was significantly lower (P = 0.008) in the CI patients versus the non-CI subjects. For women, the frequency of the T-A haplotype (rs7956547-rs978458) was significantly lower (P = 0.021) in the CI patients as compared with the non-CI subjects. The specific SNPs and haplotypes can be utilized as genetic markers for CI resistance or CI risk.

Non-synonymous polymorphism (Gln261Arg) of 12-lipoxygenase in colorectal and thyroid cancers

12-lipoxygenase (12-LOX) pathway which produces 12-HETE and hepoxiline HXA3 and HXB3, and induces production of reactive oxygen species and inflammation is increasingly being implicated in variety of cancers, including those of colorectal and thyroid cancers. Hence, we examined whether the functional polymorphism of 12-LOX (mRNA A835G; Protein Gln261Arg) has any association with human colorectal and thyroid cancers. In this communication, we report that the mutation is linked to colorectal cancer and thyroid cancers. Further, we also observed that the heterozygous mutant (AG) is more prevalent in females than in males. Frequencies of AA, AG and GG, respectively were 62.5, 36.2 and 1.3% in controls, 36.5, 61.5 and 2.0% in colorectal cancer cases and 35.6, 62.4 and 2.0% in thyroid cancer cases. The results obtained suggested a significant association of the heterogenous variant (AG) with the cancers. Relative risk of the cancers with the presence of the AG variant was found to be 2.9 and 4.0 for colorectal and thyroid cancers, respectively. However, the association of the variant (AG) was significant only in male colorectal cancer patients but not in female patients. On the other hand, prevalence of the AG variant is significantly higher in control females than in male control subjects. To the best of our knowledge, this is the first study that links the 12-LOX gene polymorphism with thyroid cancer and reveals a gender bias in the prevalence of the polymorphic variants in controls and colorectal cancer patients.

Genetic polymorphisms and obesity influence estradiol decline during the menopause

Obesity and genetic variation in aromatase and type 1 17-β hydroxysteroid dehydrogenase (HSD) could influence the E2 trajectory of decline during the menopause transition. E2 trajectories during the menopause transition (phenotype) were identified using 5934 data points acquired annually from 681 women in Study of Women's Health across the Nation (SWAN), a multiethnic study of the mid-life. E2 trajectories were related to CYP19 and type I 17-βHSD single-nucleotide polymorphisms (SNPs) and obesity. (log) E2 trajectories began to decline precipitously 2 years before the final menstrual period (FMP). The trajectory of the (log) E2 decline varied with genotypes and obesity. (log) E2 rates of decline were greater in nonobese women than in obese women, P < 0·05. Women with the CYP19rs936306 CT variant had (log) E2 rate of decline that was 54% as rapid as the rate of decline of women with the TT variant, P < 0·05. (log) E2 rate of decline in women with the CYP19rs749292 GG variant was two-thirds the rate of (log) E2 decline in women with the AG variant, P < 0·05. (log) Rates of E2 decline with 17-βHSD SNPs (rs2830, rs592389, and rs615942) varied according to genotype within obesity groups. Within each obesity group, (log) E2 rate of decline was greater in heterozygous variants and much less in homozygotes (P < 0·05). Obese women with selected CYP19 and 17-β HSD gene variants had remarkably different E2 trajectories around the FMP, resulting in different postmenopausal E2 levels. The rate of the E2 decline and the subsequent postmenopausal E2 levels may be relevant to oestrogen-sensitive chronic diseases including cancers.

M9.2 Dissecting the allelic architecture of ERAP2: a novel preeclampsia susceptibility gene

To study single nucleotide polymorphisms (SNPs) involved in angiogenesis (VEGF, PLGF, VEGFR1, VEGFR2, HIF-1α) and plasma levels of the corresponding proteins (VEGF, PLGF, sVEGFR1, sVEGFR2) in women with and without endometriosis.Allele frequencies of vascular endothelial growth factor (VEGF) pathway SNPs and plasma levels of the corresponding proteins were investigated in patients with endometriosis and in controls.University hospital.Samples of DNA from 1,931 Caucasian patients were included (1,109 patients with endometriosis and 822 controls). An additional study group included 973 DNA samples from volunteers, self-reported to be healthy without laparoscopic evaluation.Women who underwent a laparoscopy for subfertility and/or pain and healthy volunteers without laparoscopic evaluation.Functional SNPs of the VEGF, VEGFR1, VEGFR2, HIF-1α genes and Hap Map tagging SNPs of the PLGF gene were genotyped by using iPLEX technology on a Sequenom MassArray and TaqMan SNP Genotyping Assay. The VEGF levels were determined in ethylenediaminetetraacetic acid plasma samples by using Bio-Plex Protein Array System. PLGF, sVEGFR1, and sVEGFR2 levels were measured in ethylenediaminetetraacetic acid plasma samples by using ELISA Quantikine kits.A significant association was found between the rs2268613 polymorphism in the PLGF gene and PLGF plasma levels. In all study subjects, women with the AA variant of the rs2268613 PLGF gene had significantly lower PLGF plasma levels (median [interquartile range] 9.36 [8.19–10.43] pg/mL) than those with the AG variant (12.1 [11.81–20.84] pg/mL; Pa=.0085, Pb=.04), both before and after multiple testing. Plasma levels of VEGF were elevated in endometriosis patients (especially in minimal–mild endometriosis during the menstrual cycle phase) compared with laparoscopic controls but had a moderate diagnostic performance (area under the curve, 0.73) in this discovery dataset. At a cut-off plasma level of VEGF >3.88 pg/mL, minimal–mild stages of endometriosis were diagnosed with a sensitivity of 74% and a specificity of 80% during the menstrual phase of cycle. The associations between the presence of endometriosis and SNPs in PLGF (rs2268614), HIF-1α (rs11549465), and VEGFR1 (rs9582036) genes lost statistical significance after multiple testing.Genetic variants in the PLGF rs2268613 gene may influence plasma levels of the corresponding protein. Plasma levels of VEGF were elevated in endometriosis patients compared with controls. The associations between the presence of endometriosis and SNPs in PLGF (rs2268614), HIF-1α (rs11549465), and VEGFR1 (rs9582036) genes lost statistical significance after multiple testing.

Abstract 2839: Effects of CDKN2A genetic polymorphisms on DNA methylation in head and neck cancer

Abstract Gene polymorphism has been found to be associated with the susceptibility to cancer or its severity as exemplified in the CDKN2A gene. CDKN2A regulates G1 arrest and plays an important role in head and neck cancer (HNSCC) carcinogenesis. Deregulated tumor expression of CDKN2A has previously been described in association with HNSCC clinical progression. Aberrant methylation of CDKN2A is common gene silencing mechanism in HNSCC. CDKN2A is polymorphic, with variant alleles G/A442, C/T 540, and C/G 500 associated with cancer. Previously we have shown that in the Puerto Rican population the G/A 442, GG variant is associated with an increased head and neck cancer risk (OR, 4.09). In the present study, we examined the CDKN2A G/A442, C/T 540 and C/G 500 polymorphisms using PCR-RFLP and the pattern of methylation of the CDKN2A gene by methylation-specific PCR or Methylight assay in 152 HNSCC. The allelic frequencies were compared to a control group of 69 age - matched healthy blood donors. CpG island methylation for CDKN2A was found in 45% HNSCCs. The G/A 442, GG variant was more common in tumors of the larynx (63%). A significant correlation between CDKN2A G/A442 and head and neck cancer was indicated (r = 0.24, P = 0.02). We found no association between CDKN2A gene hypermethylation and CDKN2A C/T 540 and C/G 500 polymorphisms, However, we confirmed that individuals carrying the AG variant in G/A 442 gene had a higher frequency of hypermethylation in HNSCCs. Our results suggest that G/A 442 polymorphisms of CDKN2A gene may affect the methylation status. Functional and bioinformatic studies to clarify these results are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2839.

CYP1A1, smoking and venous thromboembolism

Since CYP1A1 enzyme is involved in metabolism of tobacco carcinogens, the CYP1A1 gene may be of relevance to smoking-induced differences in the risks of venous thromboembolism (VTE). We conducted a case-control study to investigate genetic polymorphisms in CYP1A1 that might modify the risk of developing VTE. A total of 425 Chinese patients with VTE and 527 VTE-free control individuals, matched by age and gender, were included in this analysis. The MspI and Ile462Val polymorphisms in CYP1A1 gene were analysed using the Amplification Refractory Mutation System (ARMS) method. The Ile462Val AG variant and combined AG and GG variant was significantly associated with VTE, adjusted for age, gender, weight and contraceptives (OR=1.362, 95%CI 1.026, 1.809, p=0.033; OR=1.420, 95% CI 1.081, 1.865, p=0.012, respectively); The AG and GG combined variant was still significantly associated with VTE when adjusting further for smoking (OR=1.344, 95%CI 1.019,1.772, p=0.036) A more than two-fold increase for VTE was associated with the Ile462Val combined variant of AG+GG (OR of 2.805, 95% CI 1.250, 6.293, p=0.012) in the smokers. Genetic variations of CYP1A1 Ile462Val contribute to susceptibility to smoking-induced VTE in the Chinese populations. A two-fold increase in the risk in the smokers in the patients who carry CYP1A1 Ile462Val variant alleles has demonstrated the importance of gene-environment interactions in the development of this disease.

Genetic polymorphisms in double-strand break DNA repair genes associated with risk of oral premalignant lesions

Oral premalignant lesions (OPLs) are early genetic events en route to oral cancer. To identify individuals susceptible to OPL is critical to the prevention of oral cancer. In a case–control study consisting of 147 patients with histologically confirmed OPL and 147 matched controls, we evaluated the associations of 10 genetic variants in nine genes of the double-strand break (DSB) DNA repair pathway with OPL risk. The most notable finding was an intronic polymorphism (A17893G) of the XRCC3 gene. Compared with the homozygous wild-type AA genotype, the odds ratio (OR) (95% confidence interval [CI]) for the heterozygous AG and homozygous variant GG genotype was 0.85 (0.49–1.48) and 0.18 (0.07–0.47), respectively ( P for trend = 0.002). In addition, compared with the most common A–C haplotypes of XRCC3 (in the order of A17893G-T241M), the G–C haplotypes were associated with a significantly decreased risk of OPL (OR = 0.40, 95% CI 0.23–0.68). Moreover, compared with individuals without the G–C haplotype, the OR was 1.04 (0.56–1.95) and 0.20 (0.08–0.51) for subjects with one copy and two copies of the G–C haplotypes, respectively ( P for trend = 0.005). Classification and regression tree (CART) analysis further revealed potential high-order gene–gene and gene–environmental interactions and categorised subjects into different risk groups according to their specific polymorphic signatures. Overall, our study provides the first epidemiological evidence supporting a connection between DSB gene variants and OPL development. Our data also suggest that the effects of high-order interactions should be taken into consideration when evaluating OPL predisposition.

MMP‐9 haplotypes and carotid artery atherosclerosis: An association study introducing a novel multicolour multiplex RealTime PCR protocol

Among other matrix metalloproteinases (MMPs), gelatinase B (MMP-9) is discussed to be associated with the pathogenesis of vascular diseases. Two single nucleotide polymorphisms (SNPs) of the MMP-9 gene, C-1562T in the promoter region and a G/A transition in exon 6 (R + 279Q), have been addressed in previous association studies which, however, produced conflicting results. A novel multiplex RealTime PCR protocol for the fast and simultaneous detection of both polymorphisms is presented, which was used for genotyping 1737 participants of a prospective study investigating genetic factors influencing the progression of atherosclerosis. Haplotype analysis revealed -1562C/+279Q as the major haplotype in this population. Allelic distribution of the C-1562T polymorphism was consistent with data published for similar cohorts; however, we found that R + 279Q allelic distribution appears to vary significantly among Caucasian populations. Considering clinical data available from 1487 participants, we found significant associations between the presence of atherosclerotic plaque and the CA-haplotype in men (P = 0.028, phi = 0.08), and between the AG variant of exon 6 and common carotid artery intima-media thickness (CIMT) in women (P = 0.004, Eta(2) = 0.019). In summary, our results demonstrate associations of MMP-9 genotypes with different stages of carotid atherosclerosis.

Bactericidal permeability increasing protein gene variants in children with sepsis

To evaluate the role of genetic polymorphisms of the bactericidal permeability increasing protein (BPI) in pediatric patients with sepsis. Prospective, single-center, case-control study at the pediatric intensive care unit (PICU) of a university hospital. 345 consecutive pediatric patients admitted to the PICU with fever, systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, or multiple organ distress syndrome (MODS). DNA was isolated and two BPI gene polymorphisms BPI (G545 > C) Taq and BPI (A645[ > G) 216 were studied in patients and compared with healthy controls. Genetic analysis of the BPI Taq gene revealed significant differences between healthy controls and the subgroup of febrile patients (p = 0.0243), the subgroup of SIRS and sepsis (p = 0.0101), and the subgroup of severe sepsis, septic shock, and MODS (p = 0.0027), respectively. No statistically significant differences for the BPI 216 gene polymorphism were found between patient and healthy control groups. A statistically significant predisposition to Gram-negative sepsis in patients carrying the BPI Taq GG variant together with the BPI 216 AG or GG variant was revealed (p = 0.0081), and these haplotypes were also associated with death due to sepsis-related complications. BPI Taq gene polymorphism is the accurate predictor of the severity of sepsis in children admitted to the PICU.

No association of the A260G and A386G DAZL single nucleotide polymorphisms with male infertility in a Caucasian population

The human DAZ gene family includes two autosomal genes, BOULE and DAZL, and a Y-chromosomal DAZ gene cluster. All are RNA-binding proteins and assumed to be master regulators of germline gene expression. We have investigated the impact of two DAZL polymorphisms, located at nucleotide positions 260 (SNP 260) and 386 (SNP 386), on the fertility of Caucasian men. These single nucleotide polymorphisms (SNPs) have been described previously to be associated with spermatogenic failure. Blood samples were collected and genomic DNA was extracted from 165 normozoospermic men and 202 oligo- or azoospermic patients, of whom 28 displayed an AZFc deletion. The frequencies of A or G allelic variants in SNP 260 and 386 were analysed via TaqMan allelic discrimination assays. In both cases, the A to G transition leads to a threonine to alanine change. A total of 24.2% of the controls showed a heterozygous nucleotide variant (AG) for the SNP 260 and the remaining 75.8% were homozygous for A. In the AZFc-deleted group, this distribution was significantly different, with 39.3% for AG, 57.1% for AA and 3.6% for GG. However, the increased heterozygosity was not correlated with sperm counts and morphology. The patients without deletions displayed a similar allelic pattern to the controls (24.1% AG/75.9% AA). For SNP 386, only the AA nucleotide variant was found in all subjects studied and in no case was the previously described heterozygous AG variant found. In a selected Caucasian population, the DAZL SNP 386 is completely absent and SNP 260 is not associated with spermatogenic failure and therefore does not represent a molecular marker for genetic diagnosis of male infertility.

Heterozygote AG variant of −596 A/G IL-6 gene polymorphism is a marker for cutaneous T-cell lymphoma (CTCL)

The aim of the study was to investigate possible associations of IL-6 gene polymorphisms (−596 A/G and −174 C/G) with cutaneous T-cell lymphoma (CTCL). In the case-control study, genotype distributions and allelic frequencies in two promoter IL-6 gene polymorphisms in the group of 63 Czech patients with CTCL were compared to those of 105 control non-CTCL subjects matched for age and sex. The IL-6 gene polymorphisms were determined by PCR with following restriction analysis. A significant difference of −596 A/G IL-6 genotype distribution was found between the CTCL patients and the controls ( P = 0.002) with almost threefold odds ratio for the heterozygote (AG) genotype in CTCL patients (OR = 2.64, P = 0.002). No significant differences in genotype distribution and/or allelic frequency of functional −174 C/G IL-6 gene polymorphism were observed. The double heterozygote AGCG of both IL-6 promoter polymorphisms was associated with CTCL (OR = 2.24, 95% confidence interval 1.17–4.28, P = 0.01). Thus, the heterozygote variant of −596 A/G promoter IL-6 polymorphism could be considered as a genotype marker for CTCL.

Biological and genetic characteristics of HIV infections in Cameroon reveals dual group M and O infections and a correlation between SI-inducing phenotype of the predominant CRF02_AG variant and disease stage

In Yaounde, Cameroon, HIV-1 group-specific V3 serology on 1469 HIV-positive samples collected between 1996 and 2001 revealed that group O infections remained constant around 1% for 6 years. Only one group N sample was identified and 4.3% reacted with group M and O peptides. Although the sensitivity of the group-specific polymerase chain reaction (PCR) in two genomic regions was not optimal, we confirmed, in at least 6 of 49 (12.2%) dual O/M seropositive samples and in 1 of 9 group O samples, dual infection with group O and M viruses ( n = 4) or with group O or M virus and an intergroup recombinant virus ( n = 3). Partial env (V3–V5) sequences on a subset of 295 samples showed that at least eight subtypes and five circulating recombinant forms (CRFs) of HIV-1 group M co-circulate; more than 60% were CRF02_AG and 11% had discordant subtype/CRF designations between env and gag. Similarly as for subtype B, the proportion of syncytium-inducing strains increased when CD4 counts were low in CRF02_AG-infected patients. The V3-loop charge was significantly lower for non-syncytium-inducing strains than for syncytium-inducing strains but cannot be used as an individual marker to predict phenotype. The two predominant HIV-1 variants in Africa, CRF02_AG and subtype C, thus have different biological characteristics.

Suppression of insulin-like growth factor type I receptor by a triple-helix strategy inhibits IGF-I transcription and tumorigenic potential of rat C6 glioblastoma cells

Homopurine (AG) and homopyrimidine (CT) oligodeoxyribonucleotides predicted to form triple-helical (triplex) structures have been shown to specifically suppress gene expression when supplied to cultured cells. Here we present evidence that homopurine RNA (effector) sequences designed to form a triplex with a homopurine. homopyrimidine sequence 3' to the termination codon of the insulin-like growth factor type I receptor (IGF-IR) structural gene can efficiently suppress IGF-IR gene transcription. Transfection vectors were constructed to drive transcription of either AG or CT variant triplex-forming strands. To increase the probability of obtaining stable transfectants with adequate expression of effector sequences, these were designed to be transcribed together with cDNA sequences conferring neomycin resistance as a fusion transcript. Rat C6 glioblastoma cells transfected with the AG variant showed dramatic reduction of IGF-IR transcripts compared with untransfected cells. The AG transfectants also exhibited marked down-regulation of the IGF-I, and an enhanced accumulation of serine protease inhibitor nexin-I mRNA. Similar changes in gene expression were observed following transfection of C6 cells with constructs transcribing antisense RNA to IGF-IR transcripts, but were not observed in C6 cells transfected with either the CT triplex variant or with vector lacking triplex-forming sequences. Moreover, C6 cells transfected with AG triplex variant displayed a dramatic inhibition of tumor growth when injected into nude mice. The results suggest that a triple-helix strategy can be used to inhibit transcription elongation of the IGF-IR gene, and emphasize the efficacy of triplex-mediated gene inhibition in an animal model.