ITPA gene polymorphisms significantly affect hemoglobin decline and treatment outcomes in patients coinfected with HIV and HCV

Published studies have described a strong association with a single‐nucleotide polymorphism (SNP) in the inosine triphosphate pyrophosphatase (ITPA) gene and ribavirin (RBV)‐induced hemolytic anemia in HCV‐infected patients receiving pegylated interferon (pegIFN) and RBV. This study sought to evaluate the effect of these polymorphisms on anemia, hemoglobin reduction, HCV kinetics, and treatment outcomes. Sixty‐three patients coinfected with HIV and HCV and 58 patients infected with HCV only were treated with pegIFN/RBV were genotyped using the ABI TaqMan allelic discrimination kit for the 2 ITPA SNP variants rs1127354 and rs7270101. A composite variable of ITPA deficiency using both SNPs was created as previously reported. Statistical analysis was performed using Mann‐Whitney test or Chi square/Fishers exact test for categorical data and mixed model analysis for multiple variables. Thirty‐five patients (30%) were predicted to have reduced ITPA activity. ITPA deficiency was found to be protective against the development of hemoglobin reduction >3 g/dl over the course of treatment. The rates of hemoglobin reduction >3 g/dl decreased in correlation with the severity of ITPA deficiency. ITPA deficiency was associated with slower hemoglobin decline early in treatment (week 4, P = 0.020) and rapid virologic response (RVR) at week 4 (P = 0.017) in patients coinfected with HIV and HCV. ITPA polymorphisms are associated with hemoglobin decline and in patients coinfected with HIV and HCV it is also associated with early virologic outcomes. Determination of ITPA polymorphisms may allow prediction of RBV‐induced anemia and earlier initiation of supportive care to ensure optimal therapeutic outcomes. J. Med. Virol. 84: 1106–1114, 2012. Published 2012. This is a US Government work and as such is in the public domain in the United States of America.     

The rs8099917 polymorphism, when determined by a suitable genotyping method, is a better predictor for response to pegylated alpha interferon/ribavirin therapy in Japanese patients than other single nucleotide polymorphisms associated with interleukin-28B

We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.     

Association of IL28B gene polymorphism with development of hepatocellular carcinoma in Japanese patients with chronic hepatitis C virus infection

IL28B single nucleotide polymorphisms (SNPs) are associated with spontaneous and treatment-induced elimination of hepatitis C virus (HCV). To assess whether the IL28B rs8099917 SNP also affects the progression of chronic HCV infection, we genotyped 511 Japanese HCV patients, including 69 with hepatocellular carcinoma (HCC). The T/T genotype of rs8099917 was not associated with the development of HCC (p = 0.623), although stepwise logistic regression analysis showed that liver cirrhosis, age greater than 68 years, and serum albumin <4.2 mg/dl were associated with HCC onset. It appears that the IL28B SNP does not directly influence hepatocarcinogenesis in chronic HCV infection.     

广东地区慢性丙型肝炎患者IL28B基因多态性与抗病毒疗效的关系

目的 研究广东地区慢性丙型肝炎患者（CHC）白细胞介素28B（IL28B）基因单核苷酸多态性（single nucleotide polymorphism,SNP）的分布特点及其与抗病毒疗效之间的关系,为合理选择经济有效的个体化抗病毒治疗方案提供依据,提高CHC的治疗应答率.方法 对74例CHC患者给予聚乙二醇干扰素（PEG-IFN）联合利巴韦林（RBV）抗病毒治疗,疗程48周或72周,停药后随访24周.通过PCR测序,检测所有患者的IL28B（rs8099917、rs12979860、rs12980275）位点SNP,以快速病毒学应答率（RVR）及持续病毒学应答率（SVR）作为疗效的主要评价指标,比较IL28B不同基因型与抗病毒疗效的关系,评估IL28 BSNP在CHC患者治疗应答中的作用.结果 74例患者中,rs8099917位点基因型为TT、TG、GG各63（85.1％）、11（14.9％）、0（0％）例,rs12979860位点基因型为CC、CT、TT各60（81.1％）、14（18.9％）、0（0％）例,rs12980275位点基因型为AA、AG、GG各57 （77.0％）、17（23.0％）、0（0％）例.对HCV 1型患者,上述三个位点中仅rs12979860 CC型与SVR有关,结果有统计学差异（SVR组vs NonSVR组,88.4％ vs 58.3％,P＜0.05）;对非HCV1型患者,rs8099917、rs12979860、rs12980275三个位点与RVR和SVR无关,结果无统计学差异（P＞0.05）.结论 广东地区CHC患者IL28B基因rs8099917、rs12979860、rs12980275位点分别以TT、CC、AA为主;对于HCV 1型CHC患者,rs12979860位点的基因型可以作为治疗前SVR的一个重要预测因子.     

No association between the IL28B SNP and response to peginterferon plus ribavirin combination treatment in Korean chronic hepatitis C patients

Background/Aims

There are few available data regarding the association between the single nucleotide polymorphisms (SNPs) of the gene encoding interleukin 28B (IL28B) and a sustained virologic response (SVR) to peginterferon (PEG-IFN) plus ribavirin (RBV) therapy in Korean chronic hepatitis C patients.

Methods

This was a retrospective cohort study of 156 patients with chronic hepatitis C virus (HCV) infection who received combination treatment of PEG-IFN plus RBV. Blood samples from these patients were analyzed to identify the IL28B SNPs at rs12979860, rs12980275, rs8099917, and rs8103142. Association analyses were performed to evaluate the relationships between each IL28B SNP and SVRs.

Results

Seventy six patients with HCV genotype 1 and 80 with genotype non-1 were enrolled. The frequencies of rs12979860 CC and CT genotypes were 90.4% and 9.6%, respectively; those of rs12980275 AA and AG genotypes were 87.2% and 12.8%, respectively; those of rs8099917 TT and TG genotypes were 92.3% and 7.7%, respectively; and those of rs8103142 TT and CT genotypes were 90.4% and 9.6%, respectively. Among the patients with HCV genotype 1, the SVR rates were 69.7% and 80.0% for rs12979860 CC and CT, respectively (P=0.71). Among the HCV genotype non-1 patients, SVR rates were 88.0% and 100% for rs12979860 CC and CT (P=1.00), respectively.

Conclusions

Genotypes of the IL28B SNP that are known to be favorable were present in most of the Korean patients with chronic hepatitis C in this study. Moreover, the IL28B SNP did not influence the SVR rate in either the HCV genotype 1 or non-1 patients. Therefore, IL28B SNP analysis might be not useful for the initial assessment, prediction of treatment outcomes, or treatment decision-making of Korean chronic hepatitis C patients.     

基于扩增阻碍突变系统实时荧光PCR的IL28B基因SNP位点联合检测方法的建立

目的 建立基于扩增阻碍突变系统和实时荧光定量PCR相结合的IL28B基因多SNP位点联合检测方法,为进一步研究丙型肝炎患者抗病毒治疗效果提供技术平台.方法 分别构建包含IL28B基因的rs12979860和rs8099917两个SNP位点的野生型和突变型质粒标准品;采用ARMS技术,结合Taqman探针技术,建立能够同时检测上述两个SNP位点的实时荧光ARMS-PCR方法.然后采用本方法检测60例丙型肝炎住院患者血清标本并以基因测序结果为金标准进行方法学评价.结果 实时荧光ARMS-PCR能够有效的鉴别rs 12979860位点的CC、CT、TT基因型和rs8099917位点的TT、TG和GG基因型,通过和测序结果进行比对,二者符合率为100％ （P ＜0.05）.结论 本研究建立的双重实时荧光ARMS-PCR能够快速可靠的对IL28B的rs12979860和rs8099917位点进行基因型多态性的联合检测,为慢性丙型肝炎患者实现个体化治疗奠定基础.     

Common genetic polymorphism of ITPA gene affects ribavirin-induced anemia and effect of peg-interferon plus ribavirin therapy

An association between a single nucleotide polymorphism (SNP) in the inosine triphosphate pyrophosphatase (ITPA) gene and reduction of hemoglobin during peg‐interferon plus ribavirin combination therapy for patients with chronic hepatitis C virus (HCV) infection has been reported. However, the effect of the SNP on outcome of therapy has not been fully elucidated. Factors associated with anemia during combination therapy, including rs1127354 genotype, were analyzed in 1,002 treated patients. The effect of the SNP on outcome of therapy was analyzed in a subset of 830 patients with genotype 1. A rapid initial decrease in hemoglobin levels was observed in patients with rs1127354 genotype CC compared with a slow decrease in non‐CC patients. Cumulative reduction of ribavirin was significantly more frequent in genotype CC patients than non‐CC patients (odds ratio 1.928, P = 8.6 × 10^?8). The frequency of patients who received at least the recommended 80% of scheduled ribavirin was significantly lower among genotype CC patients, especially among those who had pretreatment hemoglobin levels between 13.5 and 15 g/dl (P < 0.03), and the sustained viral response rate was significantly lower in this group of patients. Independent predictive factors for sustained virological response included a SNP in the IL28B locus (rs809991), age, fibrosis, ITPA SNP rs1127354 as well as pretreatment hemoglobin levels. Our data suggests that measures to prevent anemia should be considered for patients who have pretreatment hemoglobin levels less than 13.5 g/dl or who have rs1127354 genotype CC and pretreatment hemoglobin levels between 13.5 and 15 g/dl. J. Med. Virol. 83:1048–1057, 2011. © 2011 Wiley‐Liss, Inc.     

Genetic variant of IL28B rs12979860, as predictive marker of interferon‐based therapy in Pakistani population

Hepatitis C virus (HCV) genotypes and genetic variants of interleukin 28B (IL28B) are significantly associated with interferon plus ribavirin treatment of HCV infection. We investigated the distribution of HCV genotypes and single‐nucleotide polymorphisms (SNPs) of IL28B (rs12979860 and rs8099917) in Pakistani population. IL28B genotyping was performed by allele‐specific PCR and restriction fragment length polymorphism PCR in 140 chronic hepatitis C patients (CHC) and 120 healthy controls. HCV genotype 3 (HCVG3) was the most prevalent genotype, 71.4% (n = 100/140) and with the highest treatment response of 90% (n = 90/100). The overall treatment response of all the HCV genotypes was 82% (n = 115/140). The distribution of IL28B rs12979860CC genotype in treatment responder and non‐responder groups was 40.8% (n = 47/115) and 16% (n = 4/25) respectively. IL28B rs12979860CC genotype demonstrated a significant correlation (p = 0.019) with interferon‐based therapy of HCV infection. However, there was no observed association of IL28B rs8099917 polymorphism with treatment response in CHC patients (p = 0.264). In conclusion, HCV genotypes and IL28B rs12979860 are predictive markers for the efficiency of interferon plus ribavirin combinational therapy of HCV infection. We recommend the inclusion of testing for these markers in the clinical criteria for decision making for HCV therapy in Pakistani population.     

Association of interleukin-28B rs12979860 and rs8099917 polymorphisms with sustained viral response in hepatitis C virus genotype 1 and 3 infected patients from the Indian subcontinent

Background: Polymorphisms of the IL28B gene (rs12979860 and rs8099917) have been shown to impact treatment responses in hepatitis C virus (HCV) infected patients. The association of these polymorphisms with sustained viral response (SVR) has been studied in HCV genotype 3 infected patients in India, but not in genotype 1. Objectives: This study aimed to determine the association of IL28B gene polymorphisms and other host and viral factors with treatment response in patients with HCV genotype 1 and 3 infection. Materials and Methods: DNA from 42 HCV-infected patients on antiviral therapy was analysed for the IL28B polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Bidirectional sequencing was performed on a subset of samples for verification of PCR-RFLP results. Information on age, weight, height, diabetic status, pre-treatment viral load and alanine aminotransferase (ALT) levels was obtained from clinical records. The IL28B genotypes and the other factors were analysed for their association with SVR. Results: The frequency distribution of rs12979860 CC/CT/TT genotypes was found to be 66.7%, 26.2% and 7.1%, respectively. For rs8099917 genotype, the TT/GT/GG distribution was 73.8%, 21.4% and 4.8%, respectively. SVR was seen in 61.9% of cases (55.6% in genotype 1 and 62.5% in genotype 3). CC genotype at rs12979860 and TT genotype at rs8099917 were significantly higher in responders (P = 0.013 and 0.042, respectively). Lower baseline ALT and rapid viral response were also found to be associated with SVR. On logistic regression analysis, CC genotype at rs12979860 emerged as the most powerful predictor of treatment response. Conclusion: IL28B polymorphisms are strong predictors of SVR in patients from the Indian subcontinent infected with HCV genotype 3 and genotype 1.     

变性高效液相色谱与直接测序法在单核苷酸多态性检测中的应用比较

目的 了解变性高效液相色谱（denaturing high performance liquid chromatography,DHPLC)方法,在人类基因组单核苷酸多态性（single nucleotide polymorphism,SNP)检测中的灵敏度和精确度。方法 应用DHPLC方法检测了41份样本,并与直接测序法相比较。结果 在41个样本的检测中,DHPLC的检出率达到了100%,在大规模样本的杂合子筛检中,检测的错误率几乎为0。结论 DHPLC技术是一项可用于未知SNP的检测,尤其是在人群中大规模筛查已知SNP的有效手段。     

Role of interleukin 28-B in the spontaneous and treatment-related clearance of HCV infection in patients with chronic HBV/HCV dual infection

The purpose of this investigation was to evaluate the role of IL28-B polymorphism in the clearance of hepatitis C virus (HCV) in chronic hepatitis B virus (HBV)/HCV coinfection during a long-term follow-up. Thirty-four consecutive patients with HBV surface antigen (HBsAg)-positive/anti-HCV-positive chronic hepatitis were retrospectively enrolled at their first liver biopsy (LB). For all patients, a documented clinical, serological and virological follow-up of at least 3 years (range 3–16 years) after LB and a sample of whole blood for genetic evaluation were available. Of the 24 patients with detectable serum HBV-DNA and HCV-RNA at their first observation, three cleared both HBV-DNA and HCV-RNA, 12 HCV-RNA and five HBV-DNA. Of the seven HBV DNA-positive/HCV RNA-negative patients at enrolment, three cleared HBV-DNA and one remained HBV DNA-positive and became HCV RNA-positive. All three HBV DNA-negative/HCV RNA-positive patients remained unchanged. Compared with the 12 patients with HCV persistence, the 15 patients who cleared HCV were younger, had lower serum alanine aminotransferase (ALT), HCV load, and histological activity index (HAI) and fibrosis score, more frequently had IL28-B CC variant, had been receiving an interferon-based treatment and less frequently cleared serum HBV-DNA. To investigate the relationship between the IL28-B variants and clearance of HCV, excluding the confounding effect of interferon-based treatment, the Mantel–Haenszel test was used, which indicated an association between HCV clearance and IL28-B variants (p?=?0.009). In chronic HBV/HCV coinfection, a long-term follow-up showed a frequent spontaneous or treatment-related clearance of active replication of one or both viruses and identified the IL28-B CC genotype as an independent predictor of HCV clearance.     

Polymorphism in interferon λ3/interleukin-28B gene and risk to noncirrhotic chronic hepatitis C genotype 3 virus infection and its effect on the response to combined daclatasvir and sofosbuvir therapy

Hepatitis C virus (HCV) infection is a considerable public-health problem and an important cause of liver disease with about 71 million people infected worldwide and more than 399 000 people die every year from hepatitis C-related liver diseases. The present study was, therefore, initiated to investigate the association of polymorphism in interferon λ3 (IFNL3) also known as interleukin-28B (IL-28B) gene with chronic HCV infection and association of these polymorphic variants with the combination daclatasvir and sofosbuvir HCV therapy response. Genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in a total of 250 chronic HCV genotype three patients and 500 number of healthy controls. Our data revealed that the TT (minor) genotype of IFNL3 (rs12979860) and GG (minor) genotype of IFNL3 (rs8099917) exhibited a significant association with chronic HCV genotype 3 infection when compared with controls. The results of treatment response showed that CC (major) genotype of IFNL3 (rs12979860) and TT (major) genotype of IFNL3 (rs8099917) are associated with the likelihood of achieving a higher sustained virological response (SVR), to combined daclatasvir and sofosbuvir therapy, in genotype 3-infected HCV patients, whereas the individuals with TT (minor) genotype of IFNL3 (rs12979860) and GG (minor) genotype of IFNL3 (rs8099917) are more susceptible to chronic HCV infection and treatment relapse, suggesting a role of IFNL3 (rs12979860) and (rs8099917) in the treatment outcome of combined daclatasvir and sofosbuvir therapy in chronic HCV genotype 3 infection.     

Superiority of Denaturing High Performance Liquid Chromatography over single-stranded conformation and conformation-sensitive gel electrophoresis for mutation detection in TSC2

We evaluated denaturing high pressure liquid chromatography (DHPLC) as a scanning method for mutation detection in TSC2 , and compared it to conformation-sensitive gel electrophoresis (CSGE) and single-stranded conformation polymorphism analysis (SSCP). The first 20 exons of TSC2 were amplified from 84 TSC patients and screened initially by CSGE and then by DHPLC. Optimization of DHPLC analysis of each exon was carried out by design of primers with minimum variation in the melting temperature of the amplicon, and titration of both elution gradient and temperature. CSGE analysis identified 40 shifts (21 unique) in the 84 patients and 20 exons. All of these variants were detected by DHPLC, and an additional 27 changes (14 unique) were identified. Overall 15 of 28 (54%) unique single base substitutions were detected by CSGE; all were detected by DHPLC. 25 definite or probable mutations were found in these 84 patients (30%) in exons 1–20 of TSC2 . In a subsequent blinded analysis of 15 samples with 18 distinct TSC2 sequence variants originally detected by SSCP in another centre, all variants were detected by DHPLC except one where the variation occurred within the primer. Ten other (7 unique) sequence variants were detected in these samples which had not been detected by SSCP. Overall, 11 of 16 (69%) unique single base substitutions were detected by SSCP; all were detected by DHPLC. We conclude that DHPLC is superior to both CSGE and SSCP for detection of DNA sequence variation in TSC2 , particularly for single base substitution mutations.     

A single nucleotide polymorphism in IL28B affects viral evolution of hepatitis C quasispecies after pegylated interferon and ribavirin therapy

Interleukin‐28B (IL28B) polymorphisms are associated with viral response to peginterferon and ribavirin (RBV) in chronic hepatitis C (HCV). Their recognition represents a breakthrough in the understanding of the role of the host in viral eradication. How these polymorphisms determine viral eradication is unknown. The IL‐28B variants are hypothesized to have a differential impact on HCV quasispecies evolution during treatment with pegylated interferon (PEG‐IFN) and RBV. In this study, HCV RNA levels were measured at early time points in 33 naïve genotype 1 hepatitis C patients and clonal analysis of the entire NS5A region was performed on sera from baseline and Day 7. Site rs12979860 polymorphisms were determined by direct sequencing of PCR products and classified into CC, CT, and TT and were identified in 13, 11, and 9 patients, respectively. The CC polymorphism more commonly was seen in Whites versus Blacks [12/21 (57%) vs. 1/12 (8%), P = 0.009] and HIV‐infected versus mono‐infected [13/25 (52%) vs. 0/8 (0%), P = 0.009]. Patients with CC and non‐CC had similar baseline viral loads. More patients with the CC polymorphism had amino acid substitutions in NS5A compared to non‐CC patients. Despite similar baseline viral diversity, by Day 7, significantly more patients with CC had higher non‐synonymous substitution values compared to non‐CC (P = 0.02). Chronic hepatitis C patients with the CC IL28B polymorphism have a higher number of amino acid substitutions in the NS5A region and early viral evolution due to greater interferon induced selective pressure during this critical period of treatment. J. Med. Virol. 84:1913–1919, 2012. © 2012 Wiley Periodicals, Inc.     

Interleukin 28A.rs12980602 and interleukin 28B.rs8103142 genotypes could be protective against HCV infection among Egyptians

Previous studies showed that interleukin (IL)-28B gene polymorphisms were associated with hepatitis C Virus (HCV) infection and treatment outcomes. We tested whether single-nucleotide polymorphisms (SNPs) in IL-28A and IL-28B are associated with HCV infection among Egyptians with HCV genotype 4 infections. We enrolled 144 chronic HCV patients, 72 spontaneously resolved HCV subjects, and 69 healthy controls. Four SNPs in IL-28A and IL-28B genes (IL-28A.rs12980602, IL-28B.rs12979860, IL-28B.rs8099917, and IL-28B.rs8103142) were genotyped. The most frequent IL-28B haplotype “TCT” was significantly more frequent in HCV-infected subjects than in HCV negative subjects (62.2% vs. 48.6%, respectively; p = 0.005). The frequency of IL-28A.rs12980602 “T” allele was significantly higher than the “C” allele in healthy controls compared to HCV-infected subjects (p < 0.001) with the “TT” genotype significantly higher in healthy controls compared to HCV-infected subjects (p < 0.001) with no association with viral load (p = 0.11) among chronically infected subjects. The results, also, confirmed the previous role of IL-28B SNPs in predicting HCV infection outcome. Importantly, IL-28B.rs8099917 “TT” genotype was significantly associated with low viral load in HCV-infected subjects, while the remaining three SNPs did not. The three IL-28B SNPs were in linkage disequilibrium (D′ > 0.68; r² > 0.43) for all comparisons in HCV patients, while there was no linkage disequilibrium of IL-28A polymorphisms and the three IL-28B SNPs. In conclusion, IL-28A.rs12980602 and IL-28B.rs8103142 TT genotype could be protective against HCV infection. Also, IL-28B.rs12979860, IL-28B.rs8099917, and IL-28B.rs8103142 SNPs predicted the outcome of HCV infection among genotype-4-infected Egyptians. Moreover, IL-28B.rs8099917 SNP affected the viral load in chronic HCV patients.

     

Real-time polymerase chain reaction assay based on high-resolution melting analysis for the determination of the rs12979860 polymorphism involved in hepatitis C treatment response

Treatment of chronic hepatitis C is associated with varying success rates, substantial medical costs and serious side effects. Several host polymorphisms have been identified near the IL28B gene, of which the homozygous rs12979860 CC was found to be associated with significantly favourable treatment outcome. To determine accurately the presence of this variant, a real-time PCR assay with detection based on post-PCR high-resolution melting analysis (HRM) was developed. The assay, performed on a Roche LightCycler 480, was able to differentiate among all three rs12979860 variants (CC, TT, CT) across a dynamic range of four orders of magnitude (10³-10⁷). The sensitivity of the assay was determined at a 95% detection level to be 44.6 copies/reaction for the CC variant, 57.1 copies/reaction for the TT variant and 47.4 copies/reaction for the CT variant. Input DNA amount above 10³ copies/reaction is recommended for clinical samples to ensure optimal performance. Clinical performance was assessed on 60 clinical samples by comparative testing using the assay and Sanger sequencing. Concordant results were obtained for all 60 samples, showing high specificity of the assay. The novel assay can be easily added to the testing repertoire of virological laboratories, providing additional information for physicians treating chronic hepatitis C patients.     

Prediction of response to peginterferon-alfa-2b plus ribavirin therapy in Japanese patients infected with hepatitis C virus genotype 1b

Variation at the IL‐28B locus was recently reported to be a significant predictive factor of viral response to pegylated‐interferon plus ribavirin combination therapy against chronic hepatitis C. Predictive factors for the effect of therapy, including IL‐28B polymorphism rs8099917 and viral and clinical factors were investigated. A total of 288 patients were enrolled who were chronically infected with hepatitis C virus (HCV) genotype 1b and treated with combination therapy. Among them, 87 patients completed 48 weeks of therapy without dose reduction or discontinuation. In multivariate regression analysis, the rs8099917 TT genotype was the only independent factor significantly associated with sustained viral response (P = 0.016, OR 61.5), whereas substitutions at amino acid 70 (aa 70) of the HCV core protein (P = 0.038, OR 5.9) and non‐TT genotypes (P = 0.002, OR 17.2) were associated with nonvirological response. Both factors were also associated with viral dynamics during the initial stage of the therapy. Correlation analysis revealed that rs8099917 genotype was correlated with γ‐glutamyl transpeptidase, hyaluronic acid, and HCV core aa 70. In conclusion, host (IL‐28B polymorphism) and viral (aa 70) factors independently affect response to combination therapy. J. Med. Virol. 83:981–988, 2011. © 2011 Wiley‐Liss, Inc.     

Impact of IL28B gene polymorphisms rs8099917 and rs12980275 on response to pegylated interferon-α/ribavirin therapy in chronic hepatitis C genotype 4 patients

Host genetic factors may predict the outcome and treatment response in hepatitis C virus (HCV) infection. One of these factors is the single nucleotide polymorphisms of the interleukin 28B (IL28B) gene. We sought to evaluate the outcome of pegylated interferon and ribavirin therapy in association with IL-28B rs8099917 and rs12980275 in patients infected with HCV genotype 4. A total of 180 patients with chronic hepatitis C were selected from Egyptians who have received combined therapy with pegylated interferon and ribavirin for 6 months and their response was evaluated after follow-up at 0, 6, 12, 24 and 48 weeks from the beginning of the therapy. Blood samples were collected from responders and non-responders. Genomic DNA was extracted from whole blood and genotyping was carried out by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Our results showed that TT genotype of rs8099917 was associated with higher sustained viral response (SVR) rates and G allele represented a risk factor for failure of response (OR = 3.7, CI = 1.8:7.64) while rs12980275 was not significantly associated with SVR in genotype 4 Egyptian patients. The determination of IL-28B SNPs may be useful in enhancing correct prediction of SVR achievement in treating this group of genotype 4 patients.     

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease

AIMS: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS: The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.     

Random mutagenesis-PCR to introduce alterations into defined DNA sequences for validation of SNP and mutation detection methods

Sensitive and high throughput techniques are required for the detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs) and mutations. One problem, common to all methods of SNP and mutation detection, is that experimental conditions required for detection of DNA sequence variants depend on the specific DNA sequence to be analyzed. Although algorithms and other calculations have been developed to predict the experimental conditions required to detect DNA sequence variation in a specific DNA sequence, these algorithms do not always provide reliable information and experimental conditions for SNP and mutation detection must be devised empirically. Determination of experimental conditions for detection of DNA sequence variation is difficult when samples containing only wild type sequence are available. When patient derived positive controls are used, increasingly there are valid concerns about commercial ownership and patient privacy. This report presents a rapid and efficient method, employing random mutagenesis-PCR (RM-PCR) using low fidelity DNA polymerase, to randomly introduce single and multiple base substitutions and deletions into DNA sequences of interest. Clones with sequence changes were used to validate denaturing HPLC (DHPLC) algorithm predictions, optimize conditions for mutation detection in exon 15 of the tyrosine kinase domain of the MET proto-oncogene, and to confirm the association between specific DNA sequence changes and unique DHPLC chromatographic profiles (signatures). Finally, DNA from 33 papillary renal carcinoma (PRC) patients was screened for mutations in exon 15 of MET using "validated" DHPLC conditions as a proof of principle application of RM-PCR. Use of RM-PCR for DHPLC and other SNP/mutation detection methods is discussed along with challenges associated with detecting sequence alterations in mixed tumor/normal tissue, pooled samples, and from regions of the genome that have been amplified during tumorigenesis or duplicated during evolution. Hum Mutat 17:210-219, 2001. Published 2001 Wiley-Liss, Inc.