Extended spectrum of HIV-1 reverse transcriptase mutations in patients receiving multiple nucleoside analog inhibitors

Abstract

Objective: To characterize reverse transcriptase (RT) mutations by their association with extent of nucleoside RT inhibitor (NRTI) therapy. To identify mutational clusters in RT sequences from persons receiving multiple NRTI.

Design: A total of 1210 RT sequences from persons with known antiretroviral therapy were analyzed: 641 new sequences were performed at Stanford University Hospital; 569 were previously published.

Methods: Chi-square tests and logistic regression were done to identify associations between mutations and NRTI therapy. Correlation studies were done to identify mutational clusters. The Benjamini-Hochberg procedure was used to correct for multiple comparisons.

Results: Mutations at 26 positions were significantly associated with NRTI including 17 known resistance mutations (positions 41, 44, 62, 65, 67, 69, 70, 74, 75, 77, 116, 118, 151, 184, 210, 215, 219) and nine previously unreported mutations (positions 20, 39, 43, 203, 208, 218, 221, 223, 228). The nine new mutations correlated linearly with number of NRTI; 777 out of 817 (95%) instances occurred with known drug resistance mutations. Positions 203, 208, 218, 221, 223, and 228 were conserved in untreated persons; positions 20, 39, and 43 were polymorphic. Most NRTI-associated mutations clustered into three groups: (i) 62, 65, 75, 77, 115, 116, 151; (ii) 41, 43, 44, 118, 208, 210, 215, 223; (iii) 67, 69, 70, 218, 219, 228.

Conclusions: Mutations at nine previously unreported positions are associated with NRTI therapy. These mutations are probably accessory because they occur almost exclusively with known drug resistance mutations. Most NRTI mutations group into one of three clusters, although several (e.g., M184V) occur in multiple mutational contexts.

Fig. 1. Median number of differences from consensus B (mutations, grey bars) and known drug resistance mutations (black bars) according to the number of NRTI received

The patients receiving three to four and five or more NRTI were more likely to have also received NNRTI. The median number of NRTI mutations for these two groups was four and six, respectively. The median number of NNRTI mutations for both groups was one.

Fig. 2. Mutation frequency at RT positions 1-240 according to the number of NRTI received in NNRTI-naive patients (with the exception of positions 65, 221, and 223, which include data from all patients)

Each bar represents one of the subgroups as defined in Fig. 1, namely 0, 1-2, 3-4, and 5+ NRTI. At each position, a chi-square test of independence was performed to determine if there was an association between NRTI treatment and a mutation at the given position. Positions that were significantly associated with therapy using an FDR of 0.01 (chi-square analysis) are boxed with a solid line; those that were significant using an FDR of 0.05 are boxed with a dotted line. Positions that were significantly associated with increasing drug therapy (logistic regression) using an FDR of 0.01 are marked with two arrows; those that were significant using an FDR of 0.05 are marked with one arrow.

Fig. 3. Correlations among the 18 known NRTI resistance mutations as well as the nine newly identified mutations

Blue lines indicate positions with positive correlation coefficients > 0.27. Red lines indicate positions with negative correlation coefficient. The width of each line is linearly proportional to the value of the correlation coefficient. All positive correlations were statistically significant at P = 0.05 using a Bonferroni correction for 351 pairwise comparisons.

Fig. 4. Principal component analysis

This figure shows the 27 positions that have been either previously reported as associated with NRTI therapy or found to be associated in this study. The graph is a two-dimensional projection of the distances among the 27 positions, where the similarity between any two positions is measured by their binary (phi) correlation coefficient among patients who have received three or more NRTI. Positions that are close together on the graph are those with a high degree of comutation in patients, whereas positions that are far apart are those with a low or negative degree of co-mutation.