Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Genotypes”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
PubMed1994

Sequence analysis of the core gene of 14 hepatitis C virus genotypes.

We previously sequenced the 5' noncoding region of 44 isolates of hepatitis C virus (HCV), as well as the envelope 1 (E1) gene of 51 HCV isolates, and provided evidence for the existence of at least 6 major genetic groups consisting of at least 12 minor genotypes of HCV (i.e., genotypes I/1a, II/1b, III/2a, IV/2b, 2c, V/3a, 4a-4d, 5a, and 6a). We now report the complete nucleotide sequence of the putative core (C) gene of 52 HCV isolates that represent all of these 12 genotypes as well as two additional genotypes provisionally designated 4e and 4f that we identified in this study. The phylogenetic analysis of the C gene sequences was in agreement with that of the E1 gene sequences. A major division in the genetic distance was observed between HCV isolates of genotype 2 and those of the other genotypes in analysis of both the E1 and C genes. The C gene sequences of 9 genotypes have not been reported previously (i.e., genotypes 2c, 4a-4f, 5a, and 6a). Our analysis indicates that the C gene-based methods currently used to determine the HCV genotype, such as PCR with genotype-specific primers, should be revised in light of these data. We found that the predicted C gene was exactly 573 nt long in all 52 HCV isolates, with an N-terminal start codon and no in-frame stop codons. The nucleotide and predicted amino acid identities of the C gene sequences were in the range of 79.4-99.0% and 85.3-100%, respectively. Furthermore, we mapped universally conserved, as well as genotype-specific, nucleotide and deduced amino acid sequences of the C gene. The predicted C proteins of the different HCV genotypes shared the following features: (i) high content of proline residues, (ii) high content of arginine and lysine residues located primarily in three domains with 10 such residues invariant at positions 39-62, (iii) a cluster of 5 conserved tryptophan residues, (iv) two nuclear localization signals and a DNA-binding motif, (v) a potential phosphorylation site with a serine-proline motif, and (vi) three conserved hydrophilic domains that have been shown by others to contain immunogenic epitopes. Thus, we have extended analysis of the predicted C protein of HCV to all of the recognized genotypes, confirmed the existence of highly conserved regions of this important structural protein, and demonstrated that the genetic relatedness of HCV isolates is equivalent when analyzing the most conserved (i.e., C) and the most variable (i.e., E1) genes of the HCV genome.

Amino Acid Sequence↗
PubMed2005

Protection against chronic hepatitis C virus infection after rechallenge with homologous, but not heterologous, genotypes in a chimpanzee model.

An open question for hepatitis C virus (HCV) vaccine development is whether the various genotypes of this virus protect against the development of chronic infection after heterologous infection with different genotypes. We approached this question by challenging chimpanzees that had recovered from HCV genotype 1a or 1b infection with 6 heterologous genotypes as well as with a homologous genotype (for chimpanzees originally infected with genotype 1a). All 9 chimpanzees rechallenged with a homologous genotype developed self-limited infections. Of 11 chimpanzees challenged with 100 chimpanzee infectious doses of heterologous genotypes, 6 developed self-limited infections, with peak viral loads in acute-phase serum that were ~5-fold lower than those seen during primary infections. One chimpanzee (which had recovered from genotype 1b infection and was rechallenged with genotype 6a) did not develop viremia but did show an anamnestic cell-mediated immune response after rechallenge. Four of the 11 chimpanzees rechallenged with heterologous genotypes developed chronic infections with the genotypes used for rechallenge. These findings suggest that a universally protective HCV vaccine may need to incorporate epitopes from multiple genotypes.

Animals↗
PubMed2006

Predictors of liver fibrosis in HIV-infected patients with chronic hepatitis C virus (HCV) infection: assessment using transient elastometry and the role of HCV genotype 3.

BACKGROUND: Liver fibrosis is accelerated in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The reasons for this faster liver disease progression are unclear, although higher plasma HCV RNA levels and distinct HCV genotype distribution in this population, compared with in HCV-monoinfected subjects, could play a role. METHODS: Liver fibrosis was assessed using elastometry in all consecutive HIV-infected patients with chronic hepatitis C who attended our institution (Hospital Carlos III, Madrid) during the past 12 months. Hepatic stiffness was measured in kiloPascal units (kPa) and was interpreted on the basis of Metavir score: no or mild fibrosis (score, F0-F1) when liver stiffness is < or =7.1 kPa, and fibrosis with septa or cirrhosis (F2-F4) when >7.1 kPa. RESULTS: A total of 283 patients (71% were male; mean age, 42 years; 94% were injection drug users and 94% were receiving antiretrovirals; mean CD4 cell count, 554 cells/microL; 72% with plasma HIV RNA level of <50 copies/mL) were analyzed. The mean alanine aminotransferase level was 68 IU/L, and the mean plasma HCV RNA level was 5.9 log IU/mL. HCV genotype distribution was as follows: genotype 1, 60% of patients; genotype 2, 2%; genotype 3, 26%; and genotype 4, 12%. Overall, 164 (58%) of the patients had scores indicating advanced liver fibrosis (F2-F4), as determined using elastometry. In the univariate and multivariate analyses, respectively, a significant odds ratio (OR) for score F2-F4 was found for HCV genotype 3, compared with the other genotypes (OR, 1.9 [95% confidence interval {CI}, 1.1-3.4] vs. 4.3 [95% CI, 1.4-13.3]); for older age (OR, 1.1 [95% CI, 1.03-1.17] vs. 1.1 [95% CI, 1.01-1.25]); and for elevated alanine aminotransferase levels (OR, 1.02 [95% CI, 1.01-1.03] vs. 1.03 [95% CI, 1.01-1.04]). Although patients with HCV genotype 1 had higher mean serum HCV RNA levels than did those with HCV genotype 3 (6.1 log IU/mL vs. 5.7 log IU/mL; P=.01), patients with HCV genotype 3 tended to have F2-F4 scores more frequently than did those with HCV genotype 1 (69% vs. 58%; P = not significant). CONCLUSIONS: HCV genotype 3, older age, and elevated alanine aminotransferase levels are independent predictors of advanced liver fibrosis in HCV-HIV-coinfected patients.

Adult↗
PubMed2006

Effect of MTHFR C677T genotype on survival in type 2 diabetes patients with end-stage diabetic nephropathy.

BACKGROUND: The MTHFR C677T single nucleotide polymorphism TT genotype is associated with increased levels of plasma homocysteine and possibly an effect on cardiovascular mortality. We evaluated the effect of C677T genotype on mortality in a large end-stage renal disease (ESRD) cohort. METHODS: C677T genotype was determined in 439 Caucasians with end-stage diabetic nephropathy (DNP) (cases) recruited from 30 dialysis centres in Southern Germany. A total of 482 type 2 diabetes patients without DNP (no microalbuminuria) at inclusion served as a genotype control collective. Patients were prospectively followed for 4 years. Primary endpoint was all-cause mortality. RESULTS: In contrast to controls, the genotype distribution in cases was not in Hardy-Weinberg equilibrium (HWE, P = 0.003), due to a less than expected number of patients with the TT genotype. The requirements of HWE were met in cases with < 2 years dialysis therapy prior to study inclusion (n = 219). TT genotype was associated with a decreased body mass index (P = 0.002) and long diabetes duration in dialysis patients (P = 0.03). However, TT genotype was not associated with an increased risk of all-cause or cardiac mortality in the total dialysis collective or the subgroup. Also, we observed no association of MTHFR genotype with cardiovascular morbidity in cases or controls (P > 0.05), or with an increased rate of progression to novel microalbuminuria. CONCLUSION: MTHFR 677TT genotype was significantly underrepresented in patients with ESRD in our study, but was not associated with premature mortality in these patients. We found no evidence for survival bias due to C677T genotype in the ESRD cohort, or bias due to genetically determined accelerated progression to novel microalbuminuria in the controls. However, we cannot exclude that the TT genotype protects from progression from microalbuminuria to more advanced stages of DNP, or that TT genotype is associated with premature mortality before a patient progresses to ESRD.

Cohort Studies↗
PubMed2005

Evaluation of slower-growing broiler genotypes grown with and without outdoor access: meat quality.

Consumer interest in organic and natural poultry products raised with outdoor access is growing. An experiment was conducted to assess the effects of outdoor access and genotype on meat quality. One slow-growing genotype (S), 2 medium-growing genotypes (M1 and M2), and a commercial fast-growing genotype (F) were raised (straight-run) for 81, 67, or 53 d, respectively. The placement date was staggered in order to achieve a similar final body weight. Each genotype was assigned to 3 pens of 24 birds each and raised in indoor floor pens in a naturally ventilated facility; the S and F genotypes were also assigned to 2 floor pens with outdoor access containing 36 birds each. All birds were provided with the same starter, grower, and finisher feeds, and birds were commercially processed. Pectoralis samples were collected at 6 h postmortem for proximate analysis and evaluation of meat quality. The principal effect of outdoor access was to make the meat more yellow in the case of the S genotype (P < 0.05) although not the F genotype (P > 0.05). Drip loss and cook loss (%) were affected (P < 0.05) by genotype, with the highest losses occurring with the S genotype and the lowest losses occurring with the F and M genotypes. Tenderness was affected (P < 0.05) by gender as well as production system but only in the F birds. Pectoralis dry matter (%), fat (%), and ash (%) were largely unaffected (P > 0.05) by genotype or outdoor access. These data indicate that meat quality differences exist among genotypes with very different growth rates and reared with or without outdoor access.

Animals↗
PubMed2005

Evaluation of slower-growing broiler genotypes grown with and without outdoor access: growth performance and carcass yield.

Consumer interest in organic and free-range poultry production is growing. An experiment was conducted to assess the impact of genotype and outdoor access on growth rate and carcass yield. One slow-growing genotype (S), 2 medium-growing genotypes (M1 and M2), and a commercial fast-growing genotype (F) were raised (straight-run) for 81, 67, and 53 d, respectively. The placement date was staggered in order to achieve a similar final body weight and each genotype was processed on the same day. Each genotype was assigned to 3 pens of 24 birds each and raised in indoor floor pens in a curtain-sided house with ventilation fans; the S and F genotypes were also assigned to 2 floor pens with outdoor access (during daylight hours) containing 36 birds each. All birds were provided with the same starter, grower, and finisher feeds, and birds were commercially processed. Weight gain was similar (P > 0.05) among genotypes, but males gained more weight (P < 0.05) than females. The S and F genotypes had the highest and lowest (P < 0.05) feed intakes and, consequently, the lowest and highest (P < 0.05) feed efficiencies, respectively. The F genotype had the greatest (P < 0.05) breast yield (%) and the lowest (P < 0.05) wing yield (%). The S genotype exhibited the lowest (P < 0.05) breast yield (%) and the greatest leg quarter yield (%). Birds given outdoor access had greater (P < 0.05) bone strength in the tibia, and the F genotype had highest (P < 0.05) bone strength. These data indicate that substantial growth performance and yield differences exist among genotypes in alternative poultry systems.

Animals↗
PubMed2004

Infection with concurrent multiple hepatitis C virus genotypes is associated with faster HIV disease progression.

OBJECTIVE: To elucidate the importance of hepatitis C Virus (HCV) genotype in HIV disease progression. DESIGN: This study was conducted among 126 HIV/HCV co-infected drug users with a known interval of HIV seroconversion whose HCV genotype was known early in HIV infection. Both clinical progression (to AIDS) and immunological progression (to a CD4+ T-cell count of 200 x 10(6) cells/l) by HCV genotype were studied using Cox proportional hazards analysis. RESULTS: The median duration of follow-up was 7.3 years [interquartile range (IQR), 4.6-10.1 years]. The majority of the HCV infections concerned genotype 1 and genotype 3; The distribution was: HCV type 1: 48%, HCV type 3: 34%, HCV type 4: 13%, multiple HCV types: 5%. Concurrent multiple infections consisted of HCV genotypes 1b+3a, 1b+4 and 3a+4. HCV genotype 1 and multiple HCV genotype infections were associated with faster immunological progression [hazard ratio (HR), 2.02; 95% confidence interval (CI), 1.04-3.92 and HR, 2.74; 95% CI, 0.95-7.90, respectively]. Multiple HCV genotype infection was also associated with faster clinical progression (HR, 3.36; 95% CI, 0.82-13.79). These hazard ratios increased further and were all significant when analyses were limited to data in the pre-HAART era (HR, 3.92; 95% CI, 1.51-10.20; HR, 4.38; 95% CI, 1.04-18.40 and HR, 6.54; 95% CI, 1.39-30.76, respectively). CONCLUSION: HIV disease progression differs by HCV genotype and is especially faster in individuals whose HCV infection involves more than one HCV genotype. The effect of HCV genotype on HIV progression was greater in the pre-highly active antiretroviral therapy (HAART) era, suggesting that the effectiveness of HAART may diminish the effect of HCV genotype on HIV disease progression.

AIDS-Related Opportunistic Infections↗
PubMed2007

Clinicopathological differences between hepatitis B viral genotype B- and C-related resectable hepatocellular carcinoma.

Clinical and pathogenic differences exist between hepatitis B viral (HBV) genotypes B and C, and genotype C has a higher risk of hepatocellular carcinoma (HCC) development than genotype B. The aim of this study was to investigate whether HBV genotypes B and C influence the clinicopathological features of patients with resectable HCC. Stored serum samples from 193 patients with resectable HBV-related HCC were tested for HBV genotypes by a molecular method. Of 193 patients undergoing resection of HCC, 107 (55%) and 86 (45%) were infected with genotypes B and C, respectively. Compared with genotype C patients, genotype B patients were less likely to be associated with liver cirrhosis (33%vs 51%, P = 0.01). Pathologically, genotype B patients had a higher rate of solitary tumour (94%vs 86%, P = 0.048) and more satellite nodules (22%vs 12%, P = 0.05) than genotype C patients. Our results indicate that genotype B-related HCC is less associated with liver cirrhosis and has a higher frequency of solitary tumour as well as more satellite nodules than genotype C-related HCC. These characteristics may contribute to the recurrence patterns and prognosis of HBV-related HCC in patients with genotype B or C infection.

Alanine Transaminase↗
PubMed2004

Genotype distribution of Candida albicans isolates by 25S intron analysis with regard to invasiveness.

The aim of this study was to genotype Candida albicans strains isolated from patients with invasive and non-invasive deep-seated infections. For this purpose, 301 C. albicans isolates (81 invasive and 220 non-invasive) were genotyped by using specific PCR primers designed to span the transposable group I intron of the 25S rDNA gene. Fifty-three of the 81 invasive isolates were genotype A (65.4%), eight were genotype B (9.9%) and 20 were genotype C (24.7%), while 98 of the 220 non-invasive isolates were genotype A (44.6%), 46 were genotype B (20.9%) and 76 were genotype C (34.5%). Genotype A was more prevalent among invasive isolates and genotypes B and C were more prevalent among non-invasive isolates (P = 0.0046). Genotypes D and E which represent C. dubliniensis were not found. These results indicate that there may be a relationship between C. albicans genotypes and invasiveness; genotype A being more invasive than others. The presence or absence of the transposable group I intron in the 25S rDNA gene may be important in determining the invasiveness of C. albicans.

Adolescent↗
PubMed1999

Hepatitis C virus genotypes and viremia and hepatocellular carcinoma in the United States.

OBJECTIVE: Hepatitis C virus (HCV) is a well recognized cause of hepatocellular carcinoma (HCC). The pathogenic significance of HCV genotypes in hepatocarcinogenesis is undefined. The aim of this study was to investigate the genotypic distribution and viremic level of HCV in patients with HCV-associated cirrhosis with or without HCC. METHODS: A total of 28 HCV-infected patients with HCC (HCC+) and 38 patients with HCV-associated cirrhosis without HCC (HCC-) were studied. HCV genotype was assessed by the genotype-specific polymerase chain reaction (PCR) method of Okamoto and restriction fragment length polymorphism (RFLP) of the 5' untranslated region (5' UTR). Hepatitis C viremia was quantitated with the branched-chain DNA (bDNA) assay. RESULTS: Using the Okamoto method, we found genotype 1b in 64% of the HCC+ group and 74% of the HCC- group, 36% of the HCC+ group and 16% of the HCC- group were coinfected with a combination of genotype 1b and another genotype. Using the RFLP method, we found genotype 1b in 41% of the HCC+ group and in 24% of the HCC- group. Other genotypes accounted for 18% of the HCC+ group and 55% of the HCC- group; no combination genotypes were identified. Poor concordance occurred between the two genotyping methods. Mean bDNA levels were not significantly different between the two groups. CONCLUSIONS: Our study demonstrates that no particular HCV genotypes were associated with HCC and genotype did not appear to influence the development of HCV-associated HCC.

Carcinoma, Hepatocellular↗
PubMed2002

Genotyping of Helicobacter pylori in paraffin-embedded gastric biopsy specimens: relation to histological parameters and effects on therapy.

OBJECTIVES: Colonization with Helicobacterpylori can lead to GI disease. Bacterial genotypes and host factors, such as acid production, can influence the progress of disease. We investigated H. pylori genotypes and histological parameters in the same paraffin-embedded gastric biopsy specimens. METHODS: Paraffin-embedded antrum and corpus biopsy samples from 75 gastroesophageal reflux disease patients were histologically examined and tested for H. pylori vacA (s and m regions), cagA, and iceA genotypes. Patients were investigated at baseline (58 H. pylori positive and 17 H. pylori negative) and after treatment with omeprazole with or without additional antibiotic therapy. RESULTS: Genotyping at baseline was complete in 52 (90%) of the 58 H. pylori positive patients. Multiple genotypes were detected in eight (14%) of these. Genotypes were highly consistent between antrum and corpus biopsy specimens at baseline and in follow-up samples. Genotypes from paraffin sections matched those from corresponding cultured strains in 10 selected cases. In the antrum, the degree of inflammation was associated with vacA s1 and cagA+ genotypes, and the degree of neutrophil activity was associated with the cagA+ genotype. In the corpus, the degree of inflammation was significantly associated with vacA s1, cagA+, and iceA1 genotypes and the degree of atrophy was associated with vacA s1, m1, and cagA+ genotypes, whereas the degree of neutrophil activity was associated with vacA s1 and cagA+ genotypes. vacA s2 and cagA-strains appeared more resistant to antibiotic therapy, irrespective of resistance to clarithromycin. CONCLUSIONS: Our findings confirm the relevance of the H. pylori genotypes for the severity of gastric disease and the efficacy of antibiotic therapy.

Amoxicillin↗
PubMed1997

Genotypic distribution of hepatitis C virus in different regions of Thailand.

The genotypic distribution of hepatitis C virus (HCV) isolated from blood donors from four major regions of Thailand was studied by reverse hybridization assays. PCR-amplified products from the 5' noncoding and core regions of the viral genome were hybridized to genotype- and subtype-specific probes which were immobilized on the nitrocellulose membrane. Of 332 anti-HCV-positive plasma samples studied, 71% contained HCV RNA. HCV genotype 3a was the most prevalent genotype (39%), followed by genotype 1b (20%) and genotype 6 group variants (18%). HCV genotype 1a was identified among 9% of all isolates. Other genotypes (genotype 1 which was neither 1a nor 1b, genotype 3b, and an unclassified genotype) were uncommon. There was no difference in the mean age of the donors infected with different HCV genotypes. The genotypic distribution pattern of HCV was similar among HCV isolates from different regions of Thailand.

Genes, Viral↗
PubMed2003

Molecular phylogeny of the psittacid herpesviruses causing Pacheco's disease: correlation of genotype with phenotypic expression.

Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species.

Alphaherpesvirinae↗
PubMed2005

Hepatitis C virus (HCV) genotypes in 373 Italian children with HCV infection: changing distribution and correlation with clinical features and outcome.

BACKGROUND AND AIMS: Little is known of hepatitis C virus (HCV) genotypes in HCV infected children. This retrospective, multicentre study investigated genotype distribution and correlation with clinical features and outcome in a large series of Italian children. METHODS: Between 1990 and 2002, 373 HCV RNA positive children, consecutively recruited in 15 centres, were assayed for genotypes by a commercial line probe assay. RESULTS: The following genotype distribution pattern was recorded: genotype 1b = 41%; 1a = 20%; 2 = 17%; 3 = 14.5%; 4 = 5%; other = 2.5%. The prevalence of genotypes 1b and 2 decreased significantly (p<0.001) among children born from 1990 onwards compared with older children (46% v 70%) while the rate of genotypes 3 and 4 increased significantly (from 8% to 30%). Children infected with genotype 3 had the highest alanine aminotransferase levels and the highest rate of spontaneous viraemia clearance within the first three years of life (32% v 3% in children with genotype 1; p<0.001). Of 96 children enrolled in interferon trials during the survey, 22% definitely lost HCV RNA, including 57% of those with genotypes 2 and 3. CONCLUSION: HCV genotypes 1 and 2 are still prevalent among infected adolescents and young adults in Italy but rates of infection with genotypes 3 and 4 are rapidly increasing among children. These changes could modify the clinical pattern of hepatitis C in forthcoming years as children infected with genotype 3 have the best chance of spontaneous viraemia clearance early in life, and respond to interferon in a high proportion of cases.

Adolescent↗
PubMed2004

Tradeoff between no-call reduction in genotyping error rate and loss of sample size for genetic case/control association studies.

Single nucleotide polymorphisms (SNP) may be genotyped for use in case-control designs to test for association between a SNP marker and a disease using a 2 x 3 chi-squared test of independence. Genotyping is often based on underlying continuous measurements, which are classified into genotypes. A "no-call" procedure is sometimes used in which borderline observations are not classified. This procedure has the simultaneous effect of reducing the genotype error rate and the expected number of genotypes observed. Both quantities affect the power of the statistic. We develop methods for calculating the genotype error rate, the expected number of genotypes observed, and the expected power of the resulting test as a function of the no-call procedure. We examine the statistical properties of the chi-squared test using a no-call procedure when the underlying continuous measure of genotype classification is a three-component mixture of univariate normal distributions under a range of parameter specifications. The genotype error rate decreases as the no-call region is increased. The expected number of observations genotyped also decreases. Our key finding is that the expected power of the chi-squared test is not sensitive to the no-call procedure. That is, the benefits of reduced genotype error rate are almost exactly balanced by the losses due to reduced genotype observations. For an underlying univariate normal mixture of genotype classification to be analyzed with a 2 x 3 chi-squared test, there is little, if any, increase in power using a no-call procedure.

Case-Control Studies↗
PubMed2005

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance.

BACKGROUND: Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1-200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR Identifiler panel) and N = 49 SNP (TaqMan) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. RESULTS: The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. CONCLUSION: The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR Identifiler panel from wgaDNA equivalent to that of gDNA.

Biotechnology↗
PubMed2005

Geographic distribution of hepatitis C virus genotypes in Brazil.

Brazil is a country of continental dimension with a population of different ethnic backgrounds. Thus, a wide variation in the frequencies of hepatitis C virus (HCV) genotypes is expected to occur. To address this point, 1,688 sequential samples from chronic HCV patients were analyzed. HCV-RNA was amplified by the RT-PCR from blood samples collected from 1995 to 2000 at different laboratories located in different cities from all Brazilian States. Samples were collected in tubes containing a gel separator, centrifuged in the site of collection and sent by express mail in a refrigerated container to Laboratório Bioquímico Jardim Paulista, São Paulo, SP, Brazil. HCV-RNA was extracted from serum and submitted to RT and nested PCR using standard procedures. Nested PCR products were submitted to cycle sequencing reactions without prior purification. Sequences were analyzed for genotype determination and the following frequencies were found: 64.9% (1,095) for genotype 1, 4.6% (78) for genotype 2, 30.2% (510) for genotype 3, 0.2% (3) for genotype 4, and 0.1% (2) for genotype 5. The frequencies of HCV genotypes were statistically different among Brazilian regions (P = 0.00017). In all regions, genotype 1 was the most frequent (51.7 to 74.1%), reaching the highest value in the North; genotype 2 was more prevalent in the Center-West region (11.4%), especially in Mato Grosso State (25.8%), while genotype 3 was more common in the South (43.2%). Genotypes 4 and 5 were rarely found and only in the Southeast, in São Paulo State. The present data indicate the need for careful epidemiological surveys throughout Brazil since knowing the frequency and distribution of the genotypes would provide key information for understanding the spread of HCV.

5' Untranslated Regions↗
PubMedDate not supplied

[Role of various virus genotypes in progression of chronic hepatitis C].

The relation between HCV genotypes and the progression of chronic hepatitis is still unknown. Some studies implied more pathogenic effect of genotype 1b for the severity of liver inflammation. However, other studies did not show the association between HCV genotype 1b and the severe outcome of HCV infection. The aim of this study was to determine the most frequent genotypes in this environment and their influence on hepatitis C severity. The investigation included 34 patients with histologically confirmed chronic hepatitis C, aged 20-65 (mean 35.0 years). On the basis of pathohistological findings, applying the modern classification, the disease activity was graded as: minimal (A1) moderate (A2) and severe (A3). The extent of fibrosis was marked as: absent (F0), mild (F1), moderate (F2) and severe (F3). Genotyping was performed by nested PCR with type-specific primers and LIPA test and verified by sequencing. The most prevailing genotype in our group of patients was 1b (44.1%), followed by genotype 3a (26.4%), genotype 1a (11.7%) and 2a (2.8%). Five patients had mixed genotypes (four 3a/1b, and one 1a/1b). The severity of liver cell necrosis, measured by alanintransferase (ALT) levels in serum was not related to any of HCV genotypes. There was no statistically significant difference between histological disease activity in relation to HCV genotypes. Stage of the disease was not significantly related to the HCV genotypes. There was a strong association between the degree of fibrosis and the age of patients (p < 0.01). These results could indicate that the determination of HCV genotypes was not useful in the estimation of disease severity and that liver biopsy was the most important for the prognosis of the disease.

Adult↗