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Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes.

ABSTRACT: AIM:To evaluate a novel biosensor-based microarray (BBM) assay for detecting rs12979860 and rs8099917 genotypes. METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed, constructed and arrayed on an optical biosensor to develop a microarray assay. Two sets of primers were used in a one tube polymerase chain reaction (PCR) system to amplify two target fragments simultaneously. The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards. In addition to rehybridization of four probes of known sequence, a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested. The target fragments of all 40 samples were amplified in a 50 ?L PCR system. Ten ?L of each amplicon was tested by BBM assay, and another 40 ?L was used for sequencing. The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient. The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10(3)-10(4) white cells/?L. RESULTS:As shown by polyacrylamide gel electrophoresis, two target segments of the interleukin 28B-associated polymorphisms (SNPs) were successfully amplified in the one-tube PCR system. The lengths of the two amplified fragments were consistent with the known length of the target sequences, 137 and 159 bps. After hybridization of the PCR amplicons with the probes located on the BBM array, the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye. The signals were distinct from each other, could be interpreted visually, and accurately recorded using an ordinary digital camera. To evaluate the specificity of the assay, both the plasmids and clinical samples were applied to the microarray. First, 30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray. Full agreement between plasmids and the BBM assay was observed, with 30/30 correct matches (100%). The kappa value for the BBM assay with plasmids was 1.00 (P < 0.05). For the 40 clinical blood samples, the BBM assay hybridization and direct sequencing results were compared for each amplicon. For patient blood samples, agreement was 28/28 for rs8099917T/T, 9/11 for rs8099917T/G, 1/1 for rs8099917G/G, 24/24 for rs12979860C/C, 11/14 for rs12979860C/T, and 2/2 for rs12979860T/T. Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes: 2/11 rs8099917T/G and 3/14 rs12979860C/T. The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%, respectively; and the corresponding kappa values were 0.88 and 0.85 (A kappa value > 0.75 was defined as substantial agreement). The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles. The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10(2) white blood cells/?L. CONCLUSION:This biosensor microarray assay was highly specific, sensitive, rapid and easy to perform. It is compatible with clinical practice for detection of rs8099917 and rs12979860.

SUBMITTER: Li PY

PROVIDER: S-EPMC3508644 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes.

Li Pei-Yuan PY Zhou Xiao-Jun XJ Yao Lan L Fang Xin-Hua XH Ren Jiang-Nan JN Song Jia-Wu JW

World journal of gastroenterology 20121101 44

<h4>Aim</h4>To evaluate a novel biosensor-based microarray (BBM) assay for detecting rs12979860 and rs8099917 genotypes.<h4>Methods</h4>Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed, constructed and arrayed on an optical biosensor to develop a microarray assay. Two sets of primers were used in a one tube polymerase chain reaction (PCR) system to amplify two target fragments simultaneously. The biosensor microarray containe ...[more]

PMID: 23197895

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Project description:BackgroundHost single-nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) locus are associated with sustained virological response to antiviral therapy and with spontaneous Hepatitis C Virus (HCV) clearance. Prevalence of these SNPs varies depending on ethnicity. The impact of IL28B SNPs in HCV-infected patients is currently unknown in Uruguay. Therefore, the aim of this study was to evaluate and compare the distribution of polymorphisms in the IL28B gene (rs12979860 and rs8099917) among HCV-infected patients and healthy individuals in Uruguay and thus assess their possible association with the establishment of HCV infection.MethodsDNA was recovered from 92 non-infected individuals and 78 HCV-infected patients and SNPs were determined by RFLP and allelic discrimination by real-time PCR.ResultsThe distribution of rs12979860 genotypes for the infected population was 29.5%-CC, 47.4%-CT and 23.1%-TT and for the control group 45.7%, 42.4% and 11.9%, respectively. Prevalence in both infected and uninfected individuals is similar to that reported in other countries with admixed populations. The distribution of rs8099917 genotypes for the infected population was 57.7%-TT, 27.2%-TG and 14.1%-GG and for the control group 60.9%, 33.7% and 5.4%, respectively. The comparison of rs12979860 genotype distribution between the two populations evidenced a higher prevalence of the favourable genotype (CC) in the uninfected control group (p < 0.05). Additionally, results generated using logistic regression analysis show that individuals carrying rs12979860-TT or CT genotypes have a higher likelihood of developing chronic hepatitis upon infection with HCV, when compared to CC carriers, considering rs8099917 genotype as constant.ConclusionPatients with HCV infection have a statistically significant lower prevalence of the favourable rs12979860 genotype when compared to uninfected individuals; therefore we can establish that only IL28B rs12979860-CT and TT genotypes seem to contribute to the occurrence of chronic HCV infection in the cohort of Uruguayan population studied. Considering that a trend towards a higher frequency of "good" response genotypes was observed in responder patients, we believe that IL28B rs12979860 genotyping could be a useful tool for predicting different therapies outcome, including in the DAA era.

Project description:BackgroundThis meta-analysis aimed to assess available evidence on possible associations of interleukin-28B polymorphisms rs8099917 and rs12979860 with development of hepatitis virus-related hepatocellular carcinoma (HCC).MethodsPubMed, EMBASE, Google Scholar, and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. Meta-analyses were performed to examine the association of interleukin-28B rs8099917 G/T and rs12979860 T/C polymorphisms with development of hepatitis virus-related HCC. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated.ResultsA total of ten studies involving 2,529 cases and 2,412 controls were included. The G-allele and GT genotype of rs8099917 were significantly associated with increased risk of hepatitis B virus (HBV)-related HCC (allelic model, OR 1.49, 95% CI 1.13-1.96, P=0.005; heterozygous model, OR 1.39, 95% CI 1.04-1.88, P=0.03). Conversely, the TT genotype was found to be significantly associated with lower risk of HBV-related HCC (dominant model, OR 0.68, 95% CI 0.51-0.91, P=0.01). Similar results were observed in the subgroup of Chinese patients and controls. In the pooled data set, the T-allele and TT genotype of rs12979860 showed a significant association with increased HCC risk (allelic model, OR 1.36, 95% CI 1.05-1.78, P=0.02; recessive model, OR 1.75, 95% CI 1.28-2.39, P=0.005; homozygous model, OR 1.99, 95% CI 1.41-2.80, P<0.001). Subgroup analysis based on ethnicity and etiology showed rs12979860 polymorphism to be significantly associated with HCC risk in Caucasians, especially hepatitis C virus-related HCC, according to all five genetic models. In contrast, only the TT genotype of rs12979860 was found to be significantly associated with increased risk of HBV-related HCC, especially in Asians.ConclusionThe G-allele of rs8099917 may confer elevated risk of HBV-related HCC, while the wild-type TT genotype may protect against the disease. The T-allele of rs12979860 may increase the risk of HCC, in Caucasians, especially hepatitis C virus-related HCC. The TT genotype of rs12979860 may confer increased risk of HBV-related HCC, especially in Asians. These conclusions should be verified in large, well-designed studies.

Project description:IntroductionRecent studies have demonstrated the role of the interleukin 28B (IL28B) polymorphisms in predicting treatment induced and spontaneous clearance from Hepatitis C virus (HCV) infection, suggesting the possibility of tailored therapy in HCV infected patients. Genome-wide association studies have shown that single nucleotide polymorphisms (SNPs) near IL 28B gene on chromosome 19 are strong predictors of sustained virologic response (SVR) to pegylated interferon and ribavirin. This study was aimed at analyzing the co-prevalence of two common and clinically significant SNPs in a cohort of Ligurian patients.MethodsTwo SNPs (rs12979860, rs8099917) were genotyped in the IL28B locus from 175 DNA samples collected from HCV-infected consecutive patients in a Laboratory of Liguria Region, northern Italy. A real-time polymerase chain reaction in a Corbett Research Termocycler (Rotor Gene 3000A) by fluorescent probes (Fast Set IL 28B, Arrow Diagnostics) was used for the detection, according to the manufacturer's instructions.ResultsCarriers of rs12979860CT genotype predominated (87/175, 50%), homozygotes for allele C were 68/175 (39%) and the remaining were homozygotes for IFN-resistant allele T (11%). As for the rs8099917 SNP, genotypes were thus distributed: 96/175 (55%) carried the rs8099917 TT genotype, whereas 70/175 (40%) and 9/175 (5%), were heterozygotes or homozygotes for the G allele. The variants rs12979860CC and rs8099917TT were found in 39% and 54% of overall patients with HCV genotype 1, respectively. The combined assessment of examined SNPs resulted in three most prevalent genotypes (rs12979860CC/rs8099917TT, rs12979860CT/rs8099917TG and rs12979860CT/rs8099917TT) with a frequency of 35%, 31% and 18%, respectively.DiscussionRecent findings demonstrated that in carriers of rs12979860CT the determination of additional genotype of rs8099917 SNP could significantly improve the prediction of SVR. In our study cohort carriers of rs12979860CT represented 50% of all patients, who could take advantage with respect to SVR prediction by further determination of the rs8099917 SNP. The simultaneous genotyping of two IL28B SNPs should thus be recommended in HCV infected patients prior to treatment initiation.

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

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Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes.

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Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes.

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