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Comparison of capillary electrophoresis sequencing with the new CEQ 2000 DNA Analysis System to conventional gel based systems for HIV drug resistance analysis.

To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an 'in house' protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel plate-based systems. Plasma from 20 HAART-treated patients with virological failure were analyzed for protease (PR) and reverse transcriptase (RT) genes. A total of 520 RT codons (26/patient) and 360 PR codons (18/patient) related to antiretroviral drug resistance were evaluated. The overall agreement between CEQ 2000 and Prism-377 results was 100% for the RT and PR primary and secondary mutations. The overall agreement between CEQ 2000 and TruGene was 100% for primary and > or =97% for secondary mutations. Discrepant results would have never led to errors in genotype interpretation. Performances for a 24 patients/week/one technician genotyping throughput were analyzed. For Prism-377, TruGene and CEQ 2000, manual processing required 5, 4 and 2,5 days, sequence data analysis needed additional 3, 1 and 2 days and cost/patient was approximately 49, 214 and 39 $, respectively. The CEQ 2000 sequencer offers a reliable alternative for fast and cost effective HIV drug resistance analysis.

Anti-HIV Agents↗

Comparison of nevirapine (NVP) resistance in Ugandan women 7 days vs. 6-8 weeks after single-dose nvp prophylaxis: HIVNET 012.

We compared nevirapine (NVP) resistance (NVPR) mutations in maternal plasma 7 days vs. 6-8 weeks after single-dose NVP prophylaxis. In the HIVNET 012 trial, Ugandan women received a single dose of NVP in labor for prevention of HIV-1 mother-to-child transmission. NVPR mutations were detected in 70 (25%) of 279 women 6-8 weeks after NVP. Samples collected 7 days after NVP were analyzed from a subset of those 279 women. Genotyping was performed with the ViroSeq HIV-1 Genotyping System. NVPR was analyzed using paired samples from 7 days and 6-8 weeks after NVP. Sixty-five women had genotyping results obtained for samples collected at both 7 days and 6-8 weeks post-NVP. Twenty-one (32%) of those women had NVPR mutations detected in one or both samples. This included three women with NVPR at 7 days only, seven with NVPR at 6-8 weeks only, and 11 with NVPR at both time points. Eight women had >1 NVPR mutation detected 7 days after NVP. Y181C was the most common NVPR mutation detected at 7 days, whereas K103N was the most common NVPR mutation detected at 6-8 weeks. We conclude that NVPR may be detected in women as early as 7 days after single-dose NVP. Complex patterns of NVPR are detected in some women. The Y181C NVPR mutation often fades from detection by 6-8 weeks. In contrast, the K103N mutation emerges more slowly, but often remains detectable 6-8 weeks after NVP.

Amino Acid Substitution↗

Limited evolution in the HIV type 1 pol region among acute seroconverters in Pune, India.

India has the second largest burden of HIV-1-infected persons worldwide. Access to antiretroviral drugs in India is increasing. We analyzed HIV-1 protease and reverse transcriptase sequences in 12 acute seroconverters from Pune, India, and evaluated HIV-1 evolution in these individuals over time. HIV-1 genotyping was performed with the ViroSeq HIV-1 Genotyping System. Baseline samples, collected between 1999 and 2001, had viral loads from 3,523 to 8,556,280 copies/ml. All subjects had subtype C HIV-1. None of the samples had primary drug resistance mutations. The sequence identity between baseline and 1-year samples ranged from 99.7% to 99.9%, and between baseline and 2-year samples ranged from 99.4% to 100%. Most of the nucleotide changes were silent (synonymous). Amino acid substitutions were rare, and varied from subject to subject. In this cohort, drug resistance was not observed and evolution in the pol region was very limited during the first 2 years of infection.

Acute Disease↗

Arrayed primer extension: a robust and reliable genotyping platform for the diagnosis of single gene disorders: beta-thalassemia and thiopurine methyltransferase deficiency.

Mutation screenings, which were conventionally carried out individually because of different assay conditions, are usually time consuming and not cost effective. Using microarray technology, simultaneous molecular diagnosis of multiple mutations on a single platform is possible. To evaluate this idea, we developed a DNA chip platform to simultaneously detect 23 mutations of the beta-globin gene and 9 mutations of thiopurine methyltransferase (TPMT) gene based on the principle of arrayed primer extension (APEX). A blinded test consisting of 200 DNA samples with known genotypes was performed to validate this strategy. High genotyping accuracy of 97.3% and 100% for beta-globin and TPMT genes, respectively, were achieved. Further analysis on the fluorescent intensity demonstrated clear separation between the real signal and the background noise, which enabled us to set two cutoff values (V(lower) = 4.0 and V(upper) = 12.0) to determine the genotype quantitatively. Our results showed that APEX is a highly reliable genotyping strategy to detect mutations that cause beta-thalassemia or TPMT enzyme deficiency.

Base Sequence↗

Genetic polymorphism in the phenobarbital-responsive enhancer module of the UDP-glucuronosyltransferase 1A1 gene and irinotecan toxicity.

Genetic polymorphism of the UDP-glucuronosyltransferase (UGT) 1A1 gene is associated with the decreased glucuronidation activity of an active metabolite of irinotecan, SN-38, and UGT1A1*28 has been shown as a predictive factor for irinotecan toxicity. The phenobarbital-responsive enhancer module (PBREM) of the UGT1A1 promoter region has been reportedly associated with the transcriptional activity of the gene. We investigated whether the polymorphism of PBREM (T-3279G) would affect inter-patient variations in sensitivity to irinotecan toxicity. The study population comprised 119 cancer patients who had received irinotecan. We reviewed their clinical records, including patient characteristics, and observed their toxicity levels following irinotecan infusion. Genotyping was performed by sequencing analyses. Logistic regression analyses were performed to assess the relationship between genotypes and irinotecan toxicity. We identified the homozygotes of the reference allele for T-3279G in 68 patients, the heterozygotes in 37, and the homozygotes for the variant in 14. Logistic regression analysis indicated a significant association between the homozygotes for T-3279G and the severe toxicity (odds ratio 5.80; 95% confidence interval 1.67-20.1). However, multivariate analysis, including the data of UGT1A1*28 polymorphism, revealed a diminution of the association due to a highly significant linkage disequilibrium between these polymorphisms. Our results suggest that a highly significant linkage disequilibrium exists between T-3279G and UGT1A1*28 polymorphisms, and that the variants of T-3279G and UGT1A1*28 cooperatively decrease transcriptional activity of the UGT1A1 promoter. The determination of T-3279G and UGT1A1*28 genotypes might be clinically useful in predicting severe irinotecan toxicity in cancer patients.

Adult↗

The 825C/T polymorphism of the G-protein subunit beta3 is not related to hypertension.

A polymorphism at position 825 (C-->T) of the cDNA that encodes the beta3 subunit (GNB3) of the pertussis toxin-sensitive G protein was recently shown to be associated with human hypertension. To verify this finding and to investigate whether this polymorphism could also be associated with coronary heart disease, we analyzed the GNB3 variant in subjects from 2 previously described studies: Projet d'Etude des Gènes de l'hypertension Artérielle Sévère à modérée Essentielle (PEGASE), a case-control study of moderate to severe hypertension (681 cases and 308 controls), and Etude Cas-Témoins de l'Infarctus du Myocarde (ECTIM), a case-control study of myocardial infarction (MI) (564 cases and 633 controls). Genotyping was performed with allele-specific oligonucleotides. Genotype and allele frequencies were in Hardy-Weinberg equilibrium in all groups. Allele and genotype frequencies did not differ significantly between case patients with essential hypertension or MI and control subjects. In the ECTIM study, the 825T allele frequencies in cases and controls from Belfast, Northern Ireland, were 0.31 and 0.30 (P=0.79), respectively; the corresponding frequencies in cases and controls from France were 0.33 and 0.31 (P=0.30), respectively. In the PEGASE study, the 825T allele frequency was 0.35 in female and male cases and 0.31 in male normotensive controls (P=0.12). The odds ratios for hypertension (PEGASE) and MI (ECTIM) associated with T-allele carrying were 1.23 (95% confidence interval, 0.94 to 1.62; P=0.13) and 1.11 (95% confidence interval, 0.88 to 1.39; P=0.37), respectively. There was no association of the GNB3 polymorphism with early onset of hypertension, familial history of hypertension, or blood pressure level. We conclude that the 825C/T polymorphism of the GNB3 gene did not contribute in any important way to the risk of essential hypertension or MI in these studies.

Adult↗

Polymorphisms and the pocketbook: the cost-effectiveness of cytochrome P450 2C19 genotyping in the eradication of Helicobacter pylori infection associated with duodenal ulcer.

The clinical outcome of duodenal ulcer treated with proton pump inhibitor (PPI)-based, anti-Helicobacter pylori (H.p.) regimens varies according to cytochrome P450 2C19 (CYP2C19) genotype. CYP2C19 genotypes differ markedly in peoples of Pacific Rim descent compared with another ethnicity. The authors sought to determine the specific impact that these factors have on the cost-effectiveness of duodenal ulcer management. Their model consisted of two patient cohorts with Helicobacter pylori and duodenal ulcer, trichotomized into CYP2C19 homozygous extensive metabolizers (EMs), heterozygous EMs, and poor metabolizers (PMs), altering the anti-H.p. regimen in the genotyped cohort only. The authors took the perspective of a third-party payer, and the denominator was ulcer episode prevented. In the reference case, the use of CYP2C19 genotyping prior to initiating anti-H.p. therapy was dominant (costs were saved with each ulcer episode prevented) in all geographic regions of the United States. The subsequent break-even analysis showed a range of 89.20 dollars to 118.96 dollars--from Hawaii to the Midwest, respectively--required to eliminate the cost-savings from each genotype test performed. Using probabilities most unfavorable to genotyping, the variation of peoples with Pacific Rim origins from 0% to 100% altered the cost-effectiveness from 495 dollars to 2125 dollars per ulcer event prevented, respectively. The results suggest that treatment decisions for H.p. infection that are based on a patient's CYP2C19 genotype decreases expenses for health plans implementing testing. This analysis provides an economic basis to support recent calls to expand this technology into routine clinical care to prevent toxicity of narrow therapeutic index drugs.

Aryl Hydrocarbon Hydroxylases↗

Influence of IFNG rs2069709 on the severity of varicella in children.

OBJECTIVE: Aim: To assess the association between IFNG rs2069709 genotypes (CC, AC), as well as demographic factors such as sex and age, with the risk of complicated varicella in children. PATIENTS AND METHODS: Materials and Methods: The study included 132 children with confirmed varicella. Genotyping was performed using PCR with the TaqMan Genotyping Assay. Statistical analysis comprised the χ2 test, allele frequency evaluation, and logistic regression to determine the effects of genotype, sex, and age on disease severity. RESULTS: Results: The AC genotype was detected in 67.2% of children, while the CC genotype accounted for 20.3%; samples lacking genotype data (12.5%) were excluded from analysis. The C allele predominated in the uncomplicated group (64.2%), whereas the A allele was more frequent among children with complicated varicella (41.3%). Logistic regression demonstrated a tendency toward increased risk of complicated disease in AC carriers (OR=2.05; p=0.10). Sex and age showed no significant association with disease severity (p>0.05). Although statistical significance was not reached, the findings indicate that variability in the IFNG locus may influence individual immune responses to varicella-zoster virus. CONCLUSION: Conclusions: The IFNG rs2069709 C allele may act as a protective factor against severe varicella, while the A allele may increase susceptibility to complications. The observed trend for the AC genotype highlights the potential role of genetic markers in predicting disease course. Larger studies are required to confirm these associations and clarify their underlying mechanisms.

Humans↗

Water relations and leaf growth rate of three Agropyron genotypes under water stress.

The effects of water stress on leaf water relations and growth are reported for three perennial tussock grass genotypes under glasshouse conditions. Studies were performed in genotypes El Palmar INTA and Selección Anguil of Agropyron scabrifolium (Döell) Parodi, and El Vizcachero of A. elongatum (Host) Beauv. Agropyron scabrifolium El Palmar INTA is native to a region with warm-temperate and humid climate without a dry season, and an average annual precipitation of 900 mm. Agropyron scabrifolium Selección Anguil comes from a region with a sub-humid, dry to semiarid climate and a mean annual precipitation of 600 mm. Agropyron elongatum is a widespread forage in semiarid Argentina with well-known water stress resistance. A mild water stress treatment was imposed slowly; plants reached a minimum pre-dawn leaf water potential of about -1.83 MPa by day 21 after watering was withheld. In all genotypes, water stress led to a reduction of leaf growth. There was a tendency for a greater epicuticular wax accumulation on water-stressed plants of A. scabrifolium Selección Anguil and A. elongatum than on those of A. scabrifolium El Palmar INTA. This may have contributed to obtain greater turgor pressures and relative water contents in the first two than in the later genotype. In turn, this may have contributed to determine smaller leaf growth rate reductions in A. scabrifolium Selección Anguil and A. elongatum than in A. scabrifolium El Palmar INTA under water stress. This study demonstrated variation in water stress resistance between genotypes in A. scabrifolium, and between A. scabrifolium Selección Anguil and A. elongatum versus A. scabrifolium El Palmar INTA, which was related to their differential responses in water relations.

Agriculture↗

A case-control study in IL6 and TGFB1 gene polymorphisms and recurrent spontaneous abortion in southern Brazilian patients.

PROBLEM: A high proportion of recurrent spontaneous abortions (RSA) remains unexplained. Cytokine genotyping has been investigated. We studied the relationship between unexplained RSA, IL6 (-174 G-->C) and TGFB1 (+869, T-->C; +915, G-->C) gene polymorphisms. METHOD OF STUDY: The case-control study composed of 57 south Brazilian women, with unexplained RSA and 74 controls carefully matched was performed. Cytokine genotyping was performed by the polymerase chain reaction-sequence specific primer method, using the 'Cytokine Genotyping Tray'. RESULTS: The results showed that the genotypic frequencies did not differ from samples for TGFB1 gene. In relation to IL6 gene polymorphism there was a statistical difference in genotypic distribution between samples (P < or = 0.025). The frequency of the C/C genotype was increased in women with RSA in comparison with the frequency observed in controls: 18% versus 4% (P = 0.01). CONCLUSIONS: This result strengthened the importance of IL6 genotypes in the pathogenesis of RSA of unknown cause in the south Brazilian population.

Abortion, Habitual↗

Genetic markers may predict disease behavior in patients with ulcerative colitis.

BACKGROUND & AIMS: Recent studies have suggested that HLA DRB1*0103 and allele 2 of the interleukin 1 receptor antagonist (IL-1RA) gene predict severe and extensive ulcerative colitis, respectively. The aim of this study was to test these hypotheses in patients undergoing surgery for their colitis. METHODS: HLA DRB1 and DQB1 genotyping was performed in 99 patients and 472 controls. Genotyping for polymorphisms of genes encoding tumor necrosis factor alpha and IL-1RA was performed in 107 patients and 89 controls. Measurement of antineutrophil cytoplasmic antibody (ANCA) was performed in 72 patients and 58 healthy subjects by fixed neutrophil enzyme-linked immunosorbent assay and indirect immunofluorescence. RESULTS: The DRB1*0103 allele was increased in patients (14.1% vs. 3.2% in controls; P < 1 x 10[-5]). This association was greatest in patients with extensive disease (15.8%; P < 0.0001) or extraintestinal manifestations (22.8%; P < 0.0001): mouth ulcers (25.8%; P < 0.0001), arthritis (27.2%; P < 0.0001), and uveitis (35.7%; P < 0.0001). The DRB1*04 alleles were reduced in patients (P = 0.005). Differences were noted between extensive and distal disease in the frequency of allele 2 of IL-1RA (10.9% in distal vs. 28.6% in extensive; P = 0.01) and allele 2 homozygosity. ANCA was detected in 76.4% of patients. Carriage of IL-1RA allele 2 and tumor necrosis factor 2 allele was increased in ANCA-positive patients. CONCLUSIONS: Genetic markers may predict disease behavior in ulcerative colitis.

Adolescent↗

The search for genotypes that underlie human performance phenotypes.

For a species spread throughout the world, humans are remarkably invariant; yet there has always been more interest in the slight differences between individuals than in the great commonality. This is especially true in athletic endeavours, where nearly immeasurable differences in performance can separate the winner from the rest of the competitors. There is little doubt that performance is influenced by environment, as the effects of diet and training on athletic ability have long been known, if not completely understood; however, the contribution of an individual's genetic make-up is less clear. The dominance of particular nationalities, ethnic groups, or families in various sporting events is often perceived as evidence that heritage (biological or cultural), plays a role in the development of athletic skills. Further complicating the issue are the interactions between genetic background and environment, as both of these fundamental arbiters of development rarely act independently. Despite the complexity of the problem, numerous researchers have attempted to elucidate the effects of genetic background on physical performance and, more recently, to identify the specific genetic variants that contribute to performance. This article reviews some of these studies with a focus on the methodologies employed.

Adaptation, Physiological↗

The incidence of genotypes at codon 171 of the prion protein gene (PRNP) in five breeds of sheep and production traits of ewes associated with those genotypes.

Scrapie is one of several transmissible spongiform encephalopathies of livestock. Disease susceptibility is linked to polymorphisms in the normal prion protein gene that encodes the mammalian prion precursor. Codon 171 of this gene is a major determinant of scrapie susceptibility. Selection for arginine (R) at codon 171 is encouraged by the USDA to decrease the incidence of scrapie. Objectives of this study were to determine the frequency of R allele variants at codon 171 in a sample of sheep from five breeds (Columbia, Hampshire, Rambouillet, Suffolk, and Targhee) and western white-faced commercial ewes and to determine whether the R allele is associated with ewe and lamb production traits. Genotyping was performed on 532 ewes and 901 lambs from the University of Wyoming flock, in addition to 820 rams from 52 sheep producers from Wyoming and surrounding areas, using a DNA mismatch assay that discriminated the R allele from others at codon 171. Genotyping was performed by DNA sequencing on 127 rams representing all breeds, except Hampshire from the USDA Sheep Experiment Station at Dubois, ID. The 171R allele was found in all five breeds and in the commercial western white-faced ewes. Genotype frequencies varied (P < 0.001) by breed in ewe and ram populations. Influence of R-allele frequency on ewe lambing records and individual lamb records was analyzed for Columbia (62, 161, 121), Hampshire (89, 193, 162), Rambouillet (87, 179, 133), Suffolk (67, 178, 161), and commercial sheep (227, 463, 324) for numbers of ewes, total number of ewe production records, and individual lamb records, respectively. Suffolk ewes without the R allele (non-R/non-R) gave birth to more (P or= 0.08) by ewe genotype. Lamb birth and weaning weights were not influenced (P >or= 0.12) by lamb genotype in any of the breeds or in the commercial flock. In this population, ultimate lamb production was only influenced by genotype at codon 171 in the Suffolk flock.

Animals↗

PopGLen-a Snakemake pipeline for performing population genomic analyses using genotype likelihood-based methods.

SUMMARY: PopGLen is a Snakemake workflow for performing population genomic analyses within a genotype-likelihood framework, integrating steps for raw sequence processing of both historical and modern DNA, quality control, multiple filtering schemes, and population genomic analysis. Currently, the population genomic analyses included allow for estimating linkage disequilibrium, kinship, genetic diversity, genetic differentiation, population structure, inbreeding, and allele frequencies. Through Snakemake, it is highly scalable, and all steps of the workflow are automated, with results compiled into an HTML report. PopGLen provides an efficient, customizable, and reproducible option for analyzing population genomic datasets across a wide variety of organisms. AVAILABILITY AND IMPLEMENTATION: PopGLen is available under GPLv3 with code, documentation, and a tutorial at https://github.com/zjnolen/PopGLen. An example HTML report using the tutorial dataset is included in the Supplementary Material.

Software↗

Performance of phenotypic and genotypic methods to determine the clinical relevance of serial blood isolates of staphylococcus epidermidis in patients with septicemia.

Five typing methods, including biotyping (API ID32; BioMérieux, Marcy l'Etoile, France), quantitative antibiogram typing based on actual zone sizes, plasmid typing, randomly amplified polymorphic DNA (RAPD) analysis (with primer M13 and primer set ERIC-2-1026), and pulsed-field gel electrophoresis (PFGE), were compared with a previously performed method of DNA fingerprinting by AFLP (amplified fragment length polymorphism analysis) for their performance in the typing of blood isolates of Staphylococcus epidermidis. Sixteen epidemiologically unrelated strains and 11 sets of four blood culture isolates from 11 patients with septicemia were used. The stabilities and reproducibilities of the patterns, the discriminatory capacities of the methods, and the ability to apply the methods to blood culture isolates were used as performance criteria. All strains tested were typeable by each method, and the patterns were stable and reproducible. The numbers of different types within the collection of 16 epidemiologically different isolates were 5 by biotyping, 14 by antibiogram typing, 4 by plasmid typing, 9 by the RAPD assay (combination of results with primer M13 and primer set ERIC-2-1026), and 16 by PFGE. Within the 11 sets of four blood culture isolates the types found by quantitative antibiogram typing, plasmid typing, and PFGE were unique for each set, whereas by biotyping and RAPD analysis some types were observed in more than one set. The results of biotyping did not correspond with the results of the other methods or the results of AFLP. For 6 of the 11 sets, the results of all methods except those of biotyping corresponded completely. Quantitative antibiogram typing, PFGE, and AFLP proved to be the most accurate of the six typing methods tested.

Bacteremia↗

Molecular epidemiology of multidrug-resistant tuberculosis, New York City, 1995-1997.

From January 1, 1995, to December 31, 1997, we reviewed records of all New York City patients who had multidrug-resistant tuberculosis (MDRTB); we performed insertion sequence (IS) 6110-based DNA genotyping on the isolates. Secondary genotyping was performed for low IS6110 copy band strains. Patients with identical DNA pattern strains were considered clustered. From 1995 through 1997, MDRTB was diagnosed in 241 patients; 217 (90%) had no prior treatment history, and 166 (68.9%) were born in the United States or Puerto Rico. Compared with non-MDRTB patients, MDRTB patients were more likely to be born in the United States, have HIV infection, and work in health care. Genotyping results were available for 234 patients; 153 (65.4%) were clustered, 126 (82.3%) of them in eight clusters of >or=4 patients. Epidemiologic links were identified for 30 (12.8%) patients; most had been exposed to patients diagnosed before the study period. These strains were likely transmitted in the early 1990 s when MDRTB outbreaks and tuberculosis transmission were widespread in New York.

Adolescent↗

Persistence of multidrug-resistant HIV-1 without antiretroviral treatment 2 years after sexual transmission.

OBJECTIVES: To understand the virological mechanisms of 2-year persistence of multidrug-resistant virus without selective antiretroviral pressure in HIV-1-infected patients. PATIENTS AND METHODS: Two patients were contaminated recently by their HIV-1-infected partners, who had received, before the transmission, all available antiretroviral drugs and who exhibited a severe therapeutic failure. The resistance mutations analysis was performed by clonal sequencing of 1.2 kb of pol gene in plasma of index and sources patients. Sequencing of HIV-1 DNA was performed in PBMCs of index patients. RESULTS: Genotypic testing performed in index patients at time of seroconversion showed resistance mutations to three classes of drugs. All mutations were linked on the same viral genome and all quasispecies carried all mutations. No wild-type virus was detected. The same results were found in source patients and showed that all mutations were transmitted. In the index patients, all mutations persisted over 2 years without antiretroviral treatment. Moreover, the resistance mutations were all archived in the cellular reservoir. Viral load and CD4 count of index patients remained unchanged during 2 years of follow-up. DISCUSSION: Only multidrug-resistant viruses were detected in the source patients and could be transmitted in index patients. In the latter, an expansion of predominant multidrug-resistant quasispecies and the 'archival' of all mutations were observed. These results explain the persistence of mutations and suggest that it is highly difficult to return to a wild-type viral population, sensitive to an antiretroviral treatment. The treatment of index patients is limited and the major risk is the transmission of these multidrug-resistant viruses. This work was presented in part in the XII International HIV Drug Resistance Workshop, Los Cabos, Mexico, June 2003; and in the 2nd IAS Conference on HIV Pathogenesis & Treatment, Paris, France, July 2003.

Adult↗