Fluorescent Single-strand Conformation Polymorphism Research Articles

Новые мутации гена рецептора липопротеинов низкой плотности у пациентов с семейной гиперхолестеринемией из Петрозаводска

Using an automated fluorescent single-strand conformation polymorphism (SSCP) analysis of the entire coding region, promoter zone, and exon-intron junctions of the low-density lipoprotein (LDL) receptor gene, we examined 80 DNA samples of patients with familial hypercholesterolemia (FH) from Petrozavodsk. We revealed mutations that might cause FH in five probands, including FH-North Karelia (c.925-931del7) mutation and four previously unknown mutations. These novel mutations included a transversion (c.618T>G (p.S206R), one nucleotide insertion c.195_196insT (p.FsV66:D129X), a complex gene rearrangement c.192del10/ins8 (p.FsS65:D129X), and a single nucleotide deletion c.2191delG (p.FsV731:V736X). Three out of four novel mutations produce an open reading frame shift and the premature termination of translation. An analysis of the cDNA sequence of the LDL receptor showed that this might result in the formation of a transmembrane-domain-deficient receptor that is unable to bind and internalize the ligand. Our results suggest the absence of a strong founder effect associated with FH in the Petrozavodsk population.

Identification of KCNJ15 as a Susceptibility Gene in Asian Patients with Type 2 Diabetes Mellitus

Recent advances in genome research have enabled the identification of new genomic variations that are associated with type 2 diabetes mellitus (T2DM). Via fine mapping of SNPs in a candidate region of chromosome 21q, the current study identifies potassium inwardly-rectifying channel, subfamily J, member 15 (KCNJ15) as a new T2DM susceptibility gene. KCNJ15 is expressed in the beta cell of the pancreas, and a synonymous SNP, rs3746876, in exon 4 (C566T) of this gene, with T allele frequency among control subjects of 3.1%, showed a significant association with T2DM affecting lean individuals in three independent Japanese sample sets (p = 2.5 x 10(-7), odds ratio [OR] = 2.54, 95% confidence interval [CI] = 1.76-3.67) and with unstratified T2DM (p = 6.7 x 10(-6), OR = 1.76, 95% CI = 1.37-2.25). The diabetes risk allele frequency was, however, very low among Europeans in whom no association between this variant and T2DM could be shown. Functional analysis in human embryonic kidney 293 cells demonstrated that the risk allele of the synonymous SNP in exon 4 increased KCNJ15 expression via increased mRNA stability, which resulted in the higher expression of protein as compared to that of the nonrisk allele. We also showed that KCNJ15 is expressed in human pancreatic beta cells. In conclusion, we demonstrated a significant association between a synonymous variant in KCNJ15 and T2DM in lean Japanese patients with T2DM, suggesting that KCNJ15 is a previously unreported susceptibility gene for T2DM among Asians.

Glacial refugia and recolonization pathways in the brown seaweed Fucus serratus.

The last glacial maximum (20,000-18,000 years ago) dramatically affected extant distributions of virtually all northern European biota. Locations of refugia and postglacial recolonization pathways were examined in Fucus serratus (Heterokontophyta; Fucaceae) using a highly variable intergenic spacer developed from the complete mitochondrial genome of Fucus vesiculosus. Over 1,500 samples from the entire range of F. serratus were analysed using fluorescent single strand conformation polymorphism. A total of 28 mtDNA haplotypes was identified and sequenced. Three refugia were recognized based on high haplotype diversities and the presence of endemic haplotypes: southwest Ireland, the northern Brittany-Hurd Deep area of the English Channel, and the northwest Iberian Peninsula. The Irish refugium was the source for a recolonization sweep involving a single haplotype via northern Scotland and throughout Scandinavia, whereas recolonization from the Brittany-Hurd Deep refugium was more limited, probably because of unsuitable soft-bottom habitat in the Bay of Biscay and along the Belgian and Dutch coasts. The Iberian populations reflect a remnant refugium at the present-day southern boundary of the species range. A generalized skyline plot suggested exponential population expansion beginning in the mid-Pleistocene with maximal growth during the Eems interglacial 128,000-67,000 years ago, implying that the last glacial maximum mainly shaped population distributions rather than demography.

Complexity of the HVR‐1 quasispecies and disease activity in patients with hepatitis C

Hepatitis C virus (HCV) easily undergoes genomic changes, especially in the hypervariable region (HVR) in the N-terminus of the E2/NS1 region. The quasispecies nature of HCV may have important biological implications in relation to viral persistence; however, the relationship between disease activity of chronic HCV infection and development of the genomic complexity have yielded conflicting results. We explored the changes in the complexity of the HVR-1 in the natural course of chronic HCV infection with and without elevation of serum alanine transaminase (ALT) levels. Ten patients with chronic hepatitis C proven by liver biopsy, who showed persistent elevation of the serum ALT levels, and 15 patients with chronic HCV infection and persistently normal serum ALT levels (PNAL) were enrolled in this study. The number of the HCV quasispecies was determined twice for each patient at an interval of mean 2.5 years by fluorescence single-strand conformation polymorphism and sequence analysis. There was no significant difference in the changes in the number of quasispecies during the follow-up period between chronic hepatitis C and PNAL. There was also no significant difference in the change in the number of variable nucleotides sites between the two groups. In these patients, the number of quasispecies and the diversity of HVR-1 were correlated with platelet counts and serum hyaluronic acid levels previously shown to be associated with disease progression. Our results suggested that the disease activity is not always related to the generation of the HVR-1 quasispecies complexity.

Toward a fluorescent single-strand conformation polymorphism technique that detects all mutations: F-DOVAM-S

Although DOVAM-S (detection of virtually all mutations–SSCP) in effect detects all mutations and is less costly than direct sequencing, the technique currently requires the use of radioactivity. F-DOVAM-S (fluorescent DOVAM-S) was developed to replace the isotopic label with fluorescence and to increase throughput via dye color multiplexing. As proof of principle, two multitemperature slab gel electrophoresis conditions were evaluated through the blinded analysis of mutations in the factor IX (FIX) genes of 88 hemophilia B (HB) patients and 7 wild-type controls. Using only two conditions, it was determined that F-DOVAM-S had a detection sensitivity of 97%. It is anticipated that when three or four optimized conditions are employed, F-DOVAM-S will detect all mutations. Three patient samples were multiplexed per well using three different fluorescent dyes (6FAM, VIC, and NED), demonstrating that it is possible to analyze up to 44 kb of diploid, color-coded amplification product per gel lane. This value corresponds to a throughput of approximately 4 Mb of DNA analyzed per 96-well gel, which is approximately triple that of conventional radiolabeled DOVAM-S. Throughput is further enhanced by the rapidity at which the fluorescent signal can be captured and the resultant multicolor chromatograms analyzed. Given these data, F-DOVAM-S has the potential to be a particularly powerful technology for clinical diagnosis because it allows the mutation analysis of multiple patients to be performed within 24 h.

Allelic Losses of Chromosome 10 in Glioma Tissues Detected by Quantitative Single-Strand Conformation Polymorphism Analysis

Detection of loss of heterozygosity (LOH) in clinical tissue samples is frequently difficult because samples are usually contaminated with noncancerous cells or because tumor cells in single tissues have genetic heterogeneity, and the precision of available techniques is insufficient for reliable analysis in such materials. We hypothesized that single-strand conformation polymorphism (SSCP) analysis can precisely quantify the gene dosage in mixed samples and is suitable for detection of LOH in clinical tissue samples. We assessed the accuracy of a fluorescent SSCP method for the quantification of single-nucleotide polymorphism (SNP) alleles, using DNAs that were composed of cancerous DNA mixed with noncancerous DNA at various ratios. We applied this method to precisely characterize LOH in glioma tissue samples, using 96 SNPs that were evenly distributed throughout chromosome 10. LOH could be detected even in the cancerous DNA heavily contaminated (up to 80%) with noncancerous DNA. Using this method, we obtained LOH profiles of 56 gliomas with resolution at the SNP level (i.e., 1.5-Mbp interval). Anaplastic astrocytomas exhibited both 10p and 10q LOH, whereas diffuse astrocytomas frequently (63% of the cases) exhibited loss of 10p alone. We also found a possible new LOH region (around 10p13) in gliomas. The present method is effective for precise mapping of LOH region in surgically obtained tumor tissues and could be applicable to the genetic diagnosis of cancers other than gliomas.

Comparison of Two Nonradioactive, Single-Strand Conformation Polymorphism Electrophoretic Methods for Identification of rpoB Mutations in Rifampin- Resistant Isolates of Mycobacterium tuberculosis.

Background: Diverse mutations in an 81 bp region of the rpoB gene are found in approximately 95% of rifampin-resistant (RIFr) Mycobacterium tuberculosis isolates. Various methods to detect these mutations have been evaluated for their usefulness as rapid screens for rifampin resistance. Methods and Results: Two nonradioactive variations of single-strand conformation polymorphism (SSCP) electrophoresis were optimized and evaluated for their ability to distinguish nine rpoB mutations present in a collection of 51 RIFr M. tuberculosis isolates. One of the methods used polymerase chain reaction products (128 bp) encompassing the 81 bp region of the rpoB gene, which were denatured in the presence of methyl mercury hydroxide, subjected to polyacrylamide gel electrophoresis (PAGE) and detected by staining with ethidium bromide. For the second method, fluorogenically labeled primers were used to generate products that were electrophoresed in an ABI Model 310 Genetic Analyzer equipped with a 3% GeneScan Polymer Column (Applied Biosystems Inc; Foster City, CA). Mobility shifts for all nine mutations were clearly discernible from the wild-type pattern by methods when tested in blind analyses. When an additional 30 isolates were tested by both SSCP methods in a blinded fashion, correlations with RIF susceptibility testing were complete for susceptible and homogeneously resistant isolates. Among three isolates with heterogeneously resistant populations, however, two were correctly identified by fluorescent SSCP compared with one by the PAGE SSCP method. Subpopulations of the His 526-->Tyr rpoB mutant, which is frequently encountered among RIFr strains, could be detected using templates prepared from mixtures of broth cultures with a susceptible strain. Conclusions: SSCP electrophoresis is useful for rapid screening for RIF resistance in susceptible and fully resistant isolates of M. tuberculosis. However, conventional susceptibility testing is still necessary for two reasons: (1) <100% of RIF strains have mutations in the 81 bp hotspot rpoB genomic region, and (2) SSCP may not offer sufficient sensitivity to detect clinically important emergent mutant subpopulations, especially those present as <10% of the total population in a sample. Whereas PAGE SSCP is less costly than fluorescent SSCP, the latter method is somewhat easier to perform and generates quantitative data.

Mode of HCV infection examined by polymorphism of hypervariable region‐1 in cases of acute hepatitis C after accidental exposure to blood‐borne pathogens

Acute hepatitis C is known to respond better to interferon therapy than chronic hepatitis C. The reason for this difference remains unclear. The present study was undertaken to examine HCV quasispecies in blood from patients with acute hepatitis C caused by accidental exposure to blood-borne pathogens and in blood from the source patients. Three patients who developed hepatitis C (recipient patients; R-Pt.) and two patients who served as a source of HCV infection (source patients; S-Pt.) were the subjects of this study. The number of quasispecies and the genetic diversity in hypervariable region-1 (HVR-1) were examined on the basis of fluorescence single-strand conformation polymorphism and sequence analysis (FSSA). On the day of the accident, the number of quasispecies and genetic diversity were 13 and 36 in S-Pt.1 and 6 and 20 in S-Pt.3, respectively. At the time of diagnosis of acute hepatitis, the number of quasispecies and nucleotide diversity were 2 and 2 in R-Pt.1, 2 and 0 in R-Pt.2, and 4 and 0 in R-Pt.3, respectively. Immediately before the start of treatment, the number of quasispecies and genetic diversity were 4 and 4 in R-Pt.1, 2 and 0 in R-Pt.2, and 3 and 0 in R-Pt.3., respectively. In three R-Pts, interferon therapy resulted in eradication of HCV. These findings indicate that in the early stage of HCV infection, only a portion of HCV transmitted from S-Pts to R-Pts can proliferate. The low number of quasispecies of HCV appears to be one of the reasons why acute hepatitis responds well to interferon therapy.

Genetic variants of insulin receptor substrate-1 (IRS-1) in syndromes of severe insulin resistance. Functional analysis of Ala513Pro and Gly1158Glu IRS-1.

To define further the role of IRS-1 mutations in human syndromes of severe insulin resistance. The IRS-1 gene was scanned for mutations in 83 unrelated affected subjects and 47 unaffected individuals using fluorescent single-strand conformation polymorphism (fSSCP) analysis. A novel heterozygous mutation, Gly1158Glu, was found in one affected subject. Four and two subjects were heterozygous for the previously reported variants Gly972Arg and Ala513Pro, respectively. The previously identified variant Gly819Arg was found in one affected and one unaffected subject. While Gly972Arg has been described to alter the signalling properties of IRS-1, no functional studies of Ala513Pro or Gly1158Glu have been reported. Chinese hamster ovary (CHO) cells stably over-expressing the insulin receptor were transiently transfected with vectors expressing either wild-type, Glu1158 or Pro513 IRS-1. A modest increase in insulin-stimulated tyrosine phosphorylation of Glu1158 IRS-1 was observed. However, this did not result in any significant change in the association of Grb2 or the p85 alpha subunit of PI3-kinase or of PI3-kinase activity. In parallel studies, the Pro513 IRS-1 variant was indistinguishable from wild-type IRS-1. While subtle effects of these variants cannot be excluded in this system, it is unlikely that these variants are responsible for the extreme insulin resistance seen in the subjects harbouring them. Although IRS proteins play a central role in insulin signalling, functionally significant mutations in the IRS-1 gene are a rare cause of human syndromes of severe insulin resistance.

Fluorescent SSCP of overlapping fragments (FSSCP-OF): a highly sensitive method for the screening of mitochondrial DNA variation

The mtDNA analysis (mtDNA) is increasingly being demanded for forensic purposes due to the fact that many times the use of standard nuclear marker fails to analyze degraded samples (such as bones) and specially for the analysis of hair shafts (a common sample in the crime scene). However, analysis of mtDNA sequencing implies a great lab effort when a high number of samples must be analyzed. The present work introduces a novel and reliable method for the screening of mtDNA variation in the first and second hypervariables (HV1 and HV2) regions which we have denominated fluorescent single strand conformation polymorphism (SSCP) of overlapping fragments (FSSCP-OF). FSSCP-OF is based on the basic theory of SSCP analysis and combines two complementary strategies: the use of PCR amplified overlapping fragments and fluorescent detection technology. The overlap region contains a high percentage (50%) of the d-loop mtDNA variation and for this reason, the probability to detect a polymorphic position by SSCP analysis is clearly increased in comparison to conventional SSCP methods due to the fact that the same polymorphic position is usually placed in a different “relative” position in the two overlapped fragments. The use of multicolor fluorescent technology allows also the multiplex amplification of overlapping fragment and its subsequent analysis in an automatic sequencer. We have analyzed 50 samples of unrelated individuals through the FSSCP-OF technique and we have found that using this methodology the probability to distinguish two samples with different sequences is close to 100%. FSSCP-OF has other important advantages with respect to previous screening methods, such as the automation and standardization of the protocols, which is of special interest for the forensic routine.

Detection of avocado sunblotch viroid variants using fluorescent single-strand conformation polymorphism analysis.

A specific reverse transcription-polymerase chain reaction (RT-PCR) protocol has been developed for routine detection of avocado sunblotch viroid (ASBVd). Modifications in this diagnostic technique were made to enable fluorescent detection and variant identification using automated capillary electrophoresis (CE) and fluorescent single-strand conformation polymorphism (SSCP) analysis. Sixteen sequence variants characterized in a previous study were analyzed using CE-SSCP on two ABI 310 Genetic Analyzers. Significant differences were detected between data obtained from the two ABI 310 Genetic Analyzers indicating that an internal control must be run concurrently with the samples. The 16 variants could be classified into 11 groups based on the SSCP patterns. The statistical analysis of the migration rate data provided support for the visual differences in SSCP patterns. The use of SSCP in the ASBVd assay is easily accomplished and gives an estimate of the number of variants in crude samples extracted from infected avocado plants.

Direct comparison of detection systems used for the development of single-cell genetic tests in preimplantation genetic diagnosis.

Single-cell polymerase chain reaction (PCR) requires efficient amplification and accurate detection. We compare the accuracy of heteroduplex, fluorescent-fragment, and fluorescent single-strand conformation polymorphism (F-SSCP) analysis as detection systems for analysis of a PCR assay developed for preimplantation genetic diagnosis. A single-cell, fluorescent multiplex PCR assay was developed for the cystic fibrosis delta F508 mutation and the short tandem repeat, D21S11. Detection systems were compared by analyzing blinded PCR products. Amplification rates for cystic fibrosis were 89% by heteroduplex and 91% by fragment analysis, while it was 72% for D21S11 by fragment analysis. No difference in allele dropout was detected for cystic fibrosis by any method (2%). Overall accuracy was high, > 97%, although SSCP was the least accurate. Heteroduplex and fragment analysis proved equal in the diagnosis of a single amplified locus. We determined that fragment analysis allows maximal accuracy of detection and permits analysis of a second loci, controlling for DNA contamination and allelic dropout.

A rapid fluorescence based multiplex polymerase chain reaction--single-strand conformation polymorphism method for p53 mutation detection.

We have developed a fast single-strand conformation polymorphism (SSCP) technique to screen for mutations and polymorphisms in exons 5-8 of the human tumor suppressor gene p53. We use multiplex polymerase chain reaction (PCR) to amplify the four exons in one single PCR reaction and then fluorescent SSCP for screening. p53 fragments are labeled with three different colors and a fourth color is used for an internal size marker calibrating the gel. The method was evaluated in two ways: (i) 16 different cell lines with known mutations were tested blindly for band-shifts with SSCP, and (ii) 32 human urinary bladder cancer samples were screened for mutations using the present technique. After screening for mutations all exons from all samples were sequenced, both sense as well as antisense strands. Evaluating the method with four different gels shows that 21/23 mutations and polymorphism were detected in the cell lines and that 10/10 mutations and polymorphisms were detected in the patient samples. Sensitivity, specificity, positive and negative predictive values were 91/100%, 88/ 97%. 78/77% and 96/100% for cell lines / patient samples, respectively. Sensitivity, using one SSCP gel only, was 87% (20/23) for cell lines and 90% (9/10) for patient samples. We conclude that our modified SSCP technique is efficient and has a sensitivity close to 100% in detecting mutations.

A high-density linkage map of the RN region in pigs

The porcine RN locus affects muscle glycogen content and meat quality. We previously mapped the RN locus to chromosome 15. This study describes the identification of polymorphisms for four class I and four class II markers located in the RN region. Resource families were genotyped with F-SSCP markers (fluorescent single strand conformation polymorphism) and microsatellite markers. Subsequent multipoint linkage analysis revealed the order FN1-IGFBP5-S1000-S1001-IL8RB-VIL1-RN-Sw936-Sw906. The gene order is identical to the previously reported porcine RH map of the same region. The described map will facilitate positional cloning of the RN gene.

Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations.

We have developed a fluorescence-based single strand conformation polymorphism (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer for unique color detection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell lines with reported mutations in p53 exons 5-8 were analyzed by SSCP using various gel conditions. The sensitivity for mutation detection was 95% for all cell lines studied, and no false positives were seen in 10 normal DNA samples for all four exons. Experiments mixing known amounts of tumor and normal DNA showed that mutations were detected even when tumor DNA was mixed with 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a useful tool in cancer research, where screening large numbers of samples for p53 mutations is desired.

Mapping of 16 ESTs expressed in the bovine mammary gland during lactation.

A bovine mammary gland cDNA-library was used to characterize and map genes expressed during lactation. Fifty cDNA clones selected by differential hybridization were sequenced from both ends, and sequences were examined for similarities with database sequences. For 34 of the transcripts, the sequences showed more than 80% similarity to previously characterized genes or expressed sequence tags (ESTs). Twenty cDNAs that could be of interest as candidate genes for milk production are selected for genetic or chromosomal mapping. Twenty-three out of the 39 designed primer pairs representing 16 cDNA clones amplified the expected fragments and were used for subsequent fluorescent single-strand conformation polymorphism analysis (F-SSCP) in the International Bovine Reference Panel families (IBRP). Ten polymorphic loci could be identified and used to genotype the IBRP animals, and nine of them were subsequently genetically mapped on nine chromosomes. In addition, eight loci from the 16 cDNA clones could be mapped by somatic cell hybrids, bringing the total number of mapped genes to 16, one of which was mapped genetically as well as physically. The mapped mammary gland ESTs are potentially useful for cloning economic trait loci by a positional candidate gene cloning approach.

Routine analysis of p53 mutation in clinical breast tumor specimens using fluorescence-based polymerase chain reaction and single strand conformation polymorphism.

Improved prognostic and predictive markers in breast cancer management would help considerably in therapeutic decision making, particularly in patients with early-stage breast cancer. Tumor factors currently used for prognostication and management decisions are tumor size, histologic type and grade, axillary lymph node status, and estrogen receptor content. The discovery of various somatic genetic alterations in breast cancer has raised the possibility that these may provide additional and independent prognostic and predictive information. Alterations of the p53 tumor suppressor gene in particular have received the most attention as potential prognostic and predictive factors. In multivariate analysis, p53 gene mutation is consistently associated with a two- to threefold increased risk of relapse and death from breast cancer. One of the major reasons preventing the introduction of p53 mutation as a routine marker to assist in therapeutic decision making is the lack of a simple, reproducible, and inexpensive assay. In the present study the authors optimized a polymerase chain reaction-based mutation screening method, fluorescence-single strand conformation polymorphism (F-SSCP), that allows p53 status to be assessed accurately and reproducibly in routinely handled, formalin-fixed and paraffin-embedded tumor specimens. The frequency of p53 mutation observed using F-SSCP in a consecutive series of invasive ductal breast carcinomas was 17% (28/164). The authors propose that the prognostic and predictive values of p53 mutation in breast cancer should be further evaluated in prospective, randomized studies using this standardized technique.

A high-density linkage map of the RN region in pigs.

The porcine RN locus affects muscle glycogen content and meat quality. We previously mapped the RN locus to chromosome 15. This study describes the identification of polymorphisms for four class I and four class II markers located in the RN region. Resource families were genotyped with F-SSCP markers (fluorescent single strand conformation polymorphism) and microsatellite markers. Subsequent multipoint linkage analysis revealed the order FN1-IGFBP5-S1000-S1001-IL8RB-VIL1-RN-Sw936-Sw906. The gene order is identical to the previously reported porcine RH map of the same region. The described map will facilitate positional cloning of the RN gene.

Apical hypertrophic cardiomyopathy and hepatitis C virus infection.

The familial form of hypertrophic cardiomyopathy (HCM) is attributed to mutations in the genes for contractile proteins, but the etiology of non-familial form remains unknown. This study was designed to examine the clinical features, histopathologic changes, and hepatitis C virus (HCV) genomes in patients with HCM associated with HCV infection. Anti-HCV antibody was present in the sera of 9 of 65 patients (13.8%) with HCM versus 2.41% in a control population of voluntary blood donors in Japan, a statistically significant difference (p<0.0001). Among these 9 patients, 6 had ace-of-spades-shaped deformities of the left ventricle with apical hypertrophy. Myocardial fibrosis was found in all patients, and mild cellular infiltration was observed in 5 patients. Type 1b HCV RNA was present in the sera of 5 of the 9 patients. The copy number of HCV was 5.5x10(3)-8.6x10(5) genomes/ml serum, and multiple clones of HCV were detected in the sera of each patient by an analysis of the hypervariable regions using fluorescent single-strand conformation polymorphism. Positive strands of HCV were found in the hearts of 5 patients, and negative strands in the hearts of 2 patients. A high prevalence of HCV infection was found in patients with HCM, particularly of the apical variety, suggesting that HCV is an important causal agent in the pathogenesis of the disease.

Quasispecies nature of hepatitis C virus (HCV) in patients with chronic hepatitis C with mixed HCV subtypes

The quasispecies nature of hepatitis C virus (HCV) in patients with mixed HCV subtype infection was compared with that in patients with single HCV subtype infection. The number of HCV quasispecies was compared between 35 patients with mixed HCV subtype infection and 83 patients with single subtype infection. Subtype was determined by primers deduced from the core region and by line probe assay respectively. The number of quasispecies was evaluated by polymerase chain reaction amplification of hypervariable region 1 and by fluorescence single-strand conformation polymorphism analysis. There was no difference in clinical background between patients with mixed subtype infection and patients with single subtype infection. The number of quasispecies in patients with multiple subtype HCV infection was larger than in patients with single subtype HCV infection. The immunologic environments which allow the coexistence of more HCV quasispecies in patients with multiple HCV subtype infection differs from that in patients with single HCV subtype infection.