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1.  Analysis of BRCA1 Variants in Double-Strand Break Repair by Homologous Recombination and Single-Strand Annealing 
Human mutation  2012;34(3):439-445.
Missense substitutions of uncertain clinical significance in the BRCA1 gene are a vexing problem in genetic counseling for women who have a family history of breast cancer. In this study, we evaluated the functions of 29 missense substitutions of BRCA1 in two DNA repair pathways. Repair of double-strand breaks by homology-directed recombination (HDR) had been previously analyzed for 16 of these BRCA1 variants, and 13 more variants were analyzed in this study. All 29 variants were also analyzed for function in double-strand break repair by the single-strand annealing (SSA) pathway. We found that among the pathogenic mutations in BRCA1, all were defective for DNA repair by either pathway. The HDR assay was accurate because all pathogenic mutants were defective for HDR, and all nonpathogenic variants were fully functional for HDR. Repair by SSA accurately identified pathogenic mutants, but several nonpathogenic variants were scored as defective or partially defective. These results indicated that specific amino acid residues of the BRCA1 protein have different effects in the two related DNA repair pathways, and these results validate the HDR assay as highly correlative with BRCA1-associated breast cancer.
PMCID: PMC3906639  PMID: 23161852
BRCA1; homologous recombination; single-strand annealing; centrosome; VUS
2.  Use of a High Resolution Melting Assay to Analyze HIV Diversity in HIV-infected Ugandan Children 
We used a novel high resolution melting (HRM) diversity assay to analyze HIV diversity in Ugandan children (ages 0.6 to 12.4 years) who were enrolled in an observational study of antiretroviral treatment (ART). Children were maintained on ART if they were clinically and immunologically stable.
HIV diversity was measured prior to ART (baseline) in 76 children and after 48 or 96 weeks of ART in 14 children who were not virally suppressed. HIV diversity (expressed as HRM scores) was measured in six regions of the HIV genome (two in gag, one in pol, three in env).
Higher baseline HRM scores were significantly associated with older age (≥ 2 years, P ≤ 0.001 for all six regions). HRM scores from different regions were weakly correlated. Higher baseline HRM scores in three regions (one in gag, two in env) were associated with ART failure. HIV diversity was lower in four regions (two in gag, one in pol, one in env) after 48 to 96 weeks of non-suppressive ART compared to baseline.
Higher levels of HIV diversity were observed in older children prior to ART and higher levels of diversity in some regions of the HIV genome were associated with ART failure. Prolonged exposure to non-suppressive ART was associated with a significant decrease in viral diversity in selected regions of the HIV genome.
PMCID: PMC3473149  PMID: 22785048
HIV; diversity; children; antiretroviral therapy
3.  Use of a High Resolution Melting (HRM) Assay to Compare Gag, Pol, and Env Diversity in Adults with Different Stages of HIV Infection 
PLoS ONE  2011;6(11):e27211.
Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual.
HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14–540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env.
Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection.
The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined.
PMCID: PMC3206918  PMID: 22073290
4.  Analysis of HIV Diversity Using a High-Resolution Melting Assay 
HIV viruses are usually genetically homogeneous shortly after infection, and become more heterogeneous over time. We developed a high-resolution melting (HRM) assay to analyze HIV diversity without sequencing. Plasma samples from the HIVNET 012 trial were obtained from nine Ugandan mother–infant pairs. DNA amplified from the HIV gag region was analyzed to determine the number of degrees over which the DNA melted (HRM score). HRM gag DNA was also cloned and sequenced (50 clones/mother; 20 clones/infant). The median HRM score for infants (4.3, range 4.2–5.3) was higher than that for control plasmids (3.4, range 3.2–3.8, p < 0.001) and lower than that for mothers (5.7, range 4.4–7.7, p = 0.005, exact Wilcoxon rank sum test). The intraclass correlation coefficient reflecting assay reproducibility was 94% (95% CI: 89–98%). HRM scores were also compared to sequenced-based measures of HIV diversity; higher HRM scores were associated with higher genetic diversity (p < 0.001), complexity (p = 0.009), and Shannon entropy (p = 0.022), but not with length variation (p = 0.111). The HRM assay provides a novel, rapid method for assessing HIV diversity without sequencing. This assay could be applied to any region of the HIV genome or to other genetic systems that exhibit DNA diversity.
PMCID: PMC2920076  PMID: 20666583
5.  Analysis of Drug Resistance in Children Receiving Antiretroviral Therapy for Treatment of HIV-1 Infection in Uganda 
We analyzed drug resistance in HIV-infected Ugandan children who received antiretroviral therapy in a prospective, observational study (2004–2006); some children had prior single-dose nevirapine (sdNVP) exposure. Children received stavudine (d4T), lamivudine (3TC), and nevirapine (NVP); treatment was continued if they were clinically and immunologically stable. Samples with >1,000 copies/ml HIV RNA were analyzed by using the ViroSeq HIV Genotyping System (ViroSeq). Subtype A and D pretreatment samples also were analyzed with the LigAmp assay (for K103N, Y181C, and G190A). ViroSeq results were obtained for 74 pretreatment samples (35 from sdNVP-exposed children (median age, 19 months) and 39 from sdNVP-unexposed children (median age, 84 months). This included 39 subtype A, 22 subtype D, 1 subtype C, and 12 inter-subtype recombinant samples. One sample had nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance, one had nucleoside reverse transcriptase inhibitor (NRTI) resistance, and three had protease inhibitor (PI) resistance. Y181C was detected by using LigAmp in five pretreatment samples [four (14.8%) of 37 samples from sdNVP-exposed children, one (4.2%) of 24 samples from children without prior sdNVP exposure; p = 0.35]. Among children who were not virally suppressed at 48 weeks of treatment, all 12 tested had NNRTI resistance, as well as resistance to 3TC and emtricitibine (FTC); three had resistance to other NRTIs. Seven of those children had a ViroSeq result at 96 weeks of treatment; four of the seven acquired resistance to additional NRTIs by 96 weeks. In Uganda, clinically and immunologically stable children receiving nonsuppressive antiretroviral treatment regimens are at risk for development of drug resistance.
PMCID: PMC2875950  PMID: 20455758
6.  Association of HIV Diversity and Survival in HIV-Infected Ugandan Infants 
PLoS ONE  2011;6(4):e18642.
The level of viral diversity in an HIV-infected individual can change during the course of HIV infection, reflecting mutagenesis during viral replication and selection of viral variants by immune and other selective pressures. Differences in the level of viral diversity in HIV-infected infants may reflect differences in viral dynamics, immune responses, or other factors that may also influence HIV disease progression. We used a novel high resolution melting (HRM) assay to measure HIV diversity in Ugandan infants and examined the relationship between diversity and survival through 5 years of age.
Plasma samples were obtained from 31 HIV-infected infants (HIVNET 012 trial). The HRM assay was used to measure diversity in two regions in the gag gene (Gag1 and Gag2) and one region in the pol gene (Pol).
HRM scores in all three regions increased with age from 6–8 weeks to 12–18 months (for Gag1: P = 0.005; for Gag2: P = 0.006; for Pol: P = 0.016). Higher HRM scores at 6–8 weeks of age (scores above the 75th percentile) were associated with an increased risk of death by 5 years of age (for Pol: P = 0.005; for Gag1/Gag2 (mean of two scores): P = 0.003; for Gag1/Gag2/Pol (mean of three scores): P = 0.002). We did not find an association between HRM scores and other clinical and laboratory variables.
Genetic diversity in HIV gag and pol measured using the HRM assay was typically low near birth and increased over time. Higher HIV diversity in these regions at 6–8 weeks of age was associated with a significantly increased risk of death by 5 years of age.
PMCID: PMC3077388  PMID: 21533179

Results 1-6 (6)