Analysis of human immunodeficiency virus type 1 drug resistance in children receiving nucleoside analogue reverse-transcriptase inhibitors plus nevirapine, nelfinavir, or ritonavir (Pediatric AIDS Clinical Trials Group 377).

In Pediatric AIDS Clinical Trials Group 377, antiretroviral therapy-experienced children were randomized to 4 treatment arms that included different combinations of stavudine, lamivudine (3TC), nevirapine (Nvp), nelfinavir (Nfv), and ritonavir (Rtv). Previous treatment with zidovudine (Zdv), didanosine (ddI), or zalcitabine (ddC) was acceptable. Drug resistance mutations were assessed before study treatment (baseline) and at virologic failure. Zdv, ddI, and ddC mutations were detected frequently at baseline but were not associated with virologic failure. Children with drug resistance mutations at baseline had greater reductions in virus load over time than did children who did not. Nvp and 3TC mutations were detected frequently at virologic failure, and Nvp mutations were more common among children receiving 3-drug versus 4-drug Nvp-containing regimens. Children who were maintained on their study regimen after virologic failure accumulated additional Nvp and 3TC mutations plus Rtv and Nfv mutations. However, Rtv and Nfv mutations were detected at unexpectedly low rates.

Performance of the applied biosystems ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for sequence-based analysis of HIV-1 in pediatric plasma samples.

The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.

Segregation analysis of urinary albumin excretion in families with type 2 diabetes.

Elevated urinary albumin excretion (UAE) is a predictor of the development of nephropathy and cardiovascular mortality. To study whether genetic factors may determine UAE, we examined familial aggregation of UAE in 96 large multigenerational pedigrees ascertained for type 2 diabetes. A total of 1,269 subjects had UAE measured as the urinary albumin-to-creatinine ratio (ACR). This included 630 subjects with type 2 diabetes and 639 subjects without diabetes. A significant correlation (Spearman's correlation 0.34, P < 0.001) was found between the median ACR values determined separately in nondiabetic and diabetic members of the same family. To determine whether this familial aggregation of ACR could be explained by the transmission of 1 or more major genes and thus be suitable for gene mapping studies, segregation analyses were performed. In these analyses, ACR was modeled as a continuous trait with the inclusion of age, sex, and duration of diabetes as covariates. Likelihood ratio tests were performed to test competing hypotheses, and Akaike's information criterion was used to determine the most parsimonious models. The Mendelian model with multifactorial inheritance was supported more strongly than Mendelian inheritance alone. These analyses suggested that the best model for ACR levels was multifactorial with evidence for a common major gene. When the analyses were repeated for diabetic subjects only, the evidence for Mendelian inheritance was improved, although a single major locus with additional multifactorial effects was more strongly supported. The results from the current study suggest that levels of UAE are determined by a mixture of genes with large and small effects as well as other measured covariates, such as diabetes.

Progression of microalbuminuria to proteinuria in type 1 diabetes: nonlinear relationship with hyperglycemia.

While small clinical trials have shown that improved glycemic control reduces the risk of progression of microalbuminuria to proteinuria, two recent clinical trials did not confirm this finding. We sought to reconcile the contradictory evidence by examining the dose-response relationship between hyperglycemia and progression of microalbuminuria to proteinuria in individuals with type 1 diabetes and microalbuminuria (n = 312) who were followed for 4 years with repeated assessments of urinary albumin excretion. Since 33 patients did not participate in follow-up (10.6%), data for 279 patients were analyzed. Urinary albumin excretion level worsened to proteinuria in 40 (4.1 per 100 person-years). To examine the dose-response relationship, baseline HbA1c was divided into four roughly equal groups using the cut points 8, 9, and 10%. The incidence rate varied significantly among the four groups (P = 0.008). Among those with HbA1c <8.0%, the incidence rate of progression was only 1.3 per 100 person-years, while it was 5.1, 4.2, and 6.7 per 100 person-years in the three other groups. We used generalized additive models to examine the dose-response curve using HbA1c as a continuous variable and found that the risk of progression rises steeply between an HbA1c of 7.5-8.5% and then remains approximately constant across higher levels. In conclusion, the results of this study suggest that, in patients with microalbuminuria, the risk of progression to overt proteinuria can be reduced by improved glycemic control only if the HbA1c is maintained below 8.5%. Moreover, below that value, the risk declines as the level of HbA1c decreases.

Risk of advanced diabetic nephropathy in type 1 diabetes is associated with endothelial nitric oxide synthase gene polymorphism.

UNLABELLED: Risk of advanced diabetic nephropathy in type 1 diabetes is associated with endothelial nitric oxide synthase gene polymorphism. BACKGROUND: Polymorphisms in the endothelial nitric oxide synthase gene (eNOS) may be implicated in the development of nephropathy in patients with type 1 or insulin-dependent diabetes mellitus (IDDM). METHODS: Three groups of IDDM patients were selected to study this hypothesis: cases with advanced diabetic nephropathy (N = 78), cases with overt proteinuria but normal serum creatinine (N = 74), and controls with normoalbuminuria despite 15 years of diabetes (N = 195). Parents of 132 cases and 53 controls were also examined and were used for the transmission disequilibrium test, a family-based study design to test association. RESULTS: We examined four eNOS polymorphisms, and two were associated with diabetic nephropathy in the case-control comparisons: a T to C substitution in the promoter at position -786 and the a-deletion/b-insertion in intron 4. For the former, the risk of developing advanced nephropathy was higher for C allele homozygotes than for the other two genotypes (odds ratio 2.8, 95% CI, 1.4 to 5.6). For the latter polymorphism, it was the a-deletion carriers that had the higher risk (odds ratio 2.3, 95% CI, 1.3 to 4.0) in comparison with noncarriers. Both polymorphisms were analyzed together as haplotypes in a family-based study using the transmission disequilibrium test. The C/a-deletion haplotype was transmitted from heterozygous parents to cases with advanced diabetic nephropathy with a significantly higher frequency than expected (P = 0.004). CONCLUSION: The findings of the case-control and family-based studies demonstrate clearly that DNA sequence differences in eNOS influence the risk of advanced nephropathy in type 1 diabetes.

Novel mutations and a mutational hotspot in the MODY3 gene.

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha) located on chromosome 12q. We have identified four novel HNF-1alpha missense mutations in MODY3 families. In four additional and unrelated families, we observed an identical insertion mutation that had occurred in a polycytidine tract in exon 4. Among those families, one exhibited a de novo mutation at this location. We propose that instability of this sequence represents a general mutational mechanism in MODY3. We observed no HNF-1alpha mutations among 86 unrelated late-onset diabetic patients with relative insulin deficiency. Hence mutations in this gene appear to be most strongly associated with early-onset diabetes.