Michie Hashimoto

Genetic Polymorphism of the MxA Gene Promoter and Interferon Responsiveness of Hepatitis C Patients: Revisited by Analyzing Two SNP Sites (–123 and –88) in vivo and in vitro

We have previously reported a single nucleotide polymorphism (SNP) at nucleotide (nt) position –88 (G or T) within an interferon-stimulated response element-like sequence in the promoter region of the MxA gene, which correlated with responsiveness of hepatitis C patients to interferon. Upstream of it, we then identified another SNP (C or A at nt –123) and investigated whether this SNP also correlates with interferon responsiveness. The two SNPs showed a high linkage to each other: all the individuals having G at –88 had C at –123, and 73% of those having T at –88 had A at –123. As was expected from this observation, the SNP at –123 also exhibited a correlation with interferon responsiveness (C/C homozygotes were more frequent among nonresponders than among responders: 65% of 107 vs. 40% of 52, p = 0.0028). These in vivo data from patients were further supported by results from in vitro experiments. The MxA promoter sequence with A at –123 and T at –88 showed about 4-fold higher activity of upregulating the downstream reporter gene than that with C at –123 and G at –88, in a luciferase reporter assay.

SNPs in the promoter region of the osteopontin gene as a marker predicting the efficacy of interferon-based therapies in patients with chronic hepatitis C.

BackgroundThe T-helper (Th)1 immune reaction is essential for the eradication of hepatitis C virus (HCV) during interferon (IFN) therapy in patients with chronic hepatitis C. Osteopontin is a cytokine crucial for the initiation of the Th1 response. Recently, we identified four single-nucleotide polymorphisms (SNPs) in the promoter region of the osteopontin gene (OPN), at nucleotide (nt) -155, -443, -616, and -1748, and suggested that the SNP at nt -443 was a marker reflecting hepatitis activity in patients with HCV. Therefore, we examined the possibility that SNPs in OPN were also markers predicting the therapeutic efficacy of IFN in patients with chronic hepatitis C.MethodsBlood was collected from 77 patients with chronic hepatitis C who had received either IFN monotherapy or IFN-ribavirin combination therapy (IFN-based therapies). SNPs in OPN, MxA, MBL, and LMP7 were analyzed by Invader assay.ResultsPromoter SNPs of OPN at nt -155, -616, and -1748 showed linkage disequilibrium at 100% to each other. Sustained virological response (SVR) was observed in 58% of all patients. The SVR rate was higher in patients with the G/G or G/A alleles in the OPN promoter SNP at nt -1748 than in those with A/A (85% vs 45%; P < 0.05). The SVR rate was also higher in patients with T/T at nt -443 than in those with C/C or C/T (86% vs 47%; P < 0.05). Such differences were particularly evident in patients with HCV genotype 1b who had a pretreatment viral load greater than 100 KIU/ml. All the patients who had G/G or G/A at nt -1748 and T/T at nt -443 obtained an SVR. On the other hand, there was no relationship between the efficacy of IFN-based therapies and SNPs in MxA, MBL, and LMP7, which had been shown to have association with the response to IFN monotherapies.ConclusionsSNPs in the promoter region of OPN may be useful as a marker to predict the efficacy of IFN-based therapies in patients with chronic hepatitis C, and further investigation regarding their real significance is warranted in a large series of patients.

The dinucleotide microsatellite polymorphism of the IFNAR1 gene promoter correlates with responsiveness of hepatitis C patients to interferon

We studied a possible correlation between the dinucleotide microsatellite polymorphism of the interferon alpha/beta receptor-1 gene (IFNAR1) and responsiveness of hepatitis C patients to interferon therapy. The length of GT-repeat (n of (GT)(n)) in the IFNAR1 promoter was determined in 157 patients, of whom 50 were responders and 107 were nonresponders to interferon. The genotypes 5/5 (i.e. homozygotes of (GT)(5)) and 5/14 (i.e. heterozygotes of (GT)(5) and (GT)(14)) were more frequently found in responders than in nonresponders: 80 versus 58%, P=0.008. Thus, determining (GT)(n) of the IFNAR1 promoter may help predict treatment outcome in patients treated with interferon alpha and/or beta. In addition, we sought for genetic polymorphism in the promoter region of the interferon alpha/beta receptor-2 gene (IFNAR2), and found single nucleotide polymorphisms (SNPs) at -134 and -75. But these SNPs did not show a clinical significance, as compared with the GT-repeat in IFNAR1, in the context of interferon responsiveness.

Construction of an electrochemical DNA chip for simultaneous genotyping of single nucleotide polymorphisms.

An electrochemical DNA chip was constructed for simultaneous genotyping of single nucleotide polymorphisms (SNPs) using genomic DNA extracted from blood samples. This chip consisted of electrodes located on a single piece of substrate and allele-specific oligonucleotide probes on the electrodes. As a first application, the 4 SNPs (MxA[-88], MxA[-123], MBL[X/Y], and MBL[A/B]), which have association with the efficacy of interferon therapy for HCV patient, were genotyped on the new DNA chip. Following hybridization of PCR products containing the 4 types of fragments, washing, bisbenzimide H33258 (Hoechst 33258) reaction and electrochemical analyses, 59 blood samples were genotyped by the chip method simultaneously. All procedures were completed within 2 h and the results were 100% concordant with those by the direct sequence method. The electrochemical DNA chip is expected to be a practical tool for SNPs genotyping.

Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip.

Abstract We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.

Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.