The results presented here indicate that codon 69 mutations have a larger role in NRTI resistance than currently thought. The T69D mutation and its association with ddC resistance were originally presented by Fitzgibbon et al. (9
). While the existence of the T69N mutation in NRTI-treated patients has been mentioned previously (18
), current literature and genotype interpretation guidelines associate codon 69 only with ddC resistance. While our database analysis indicates that codon 69 changes are associated with ddC treatment, a substantial proportion of patients develops codon 69 changes without ddC experience. Also, codon 69 changes appeared at only a slightly lower frequency than those of several other NRTI-associated mutations, even though ddC was used by fewer patients than those using other NRTI. These data suggest that codon 69 changes may be selected by or maintained during treatment histories that do not include ddC.
The database analysis showed some significant associations of codon 69 variants with other RT gene mutations. The T69N mutation was found in the database analysis to be less likely to be associated with M41L, L210W, and T215Y. These data suggest that this mutation may be sufficiently contributing to AZT resistance with D67N, K70R, and T215F without requiring the mutations generally required to generate high-level AZT resistance (i.e., M41L, L210W, and T215Y) (12
). While the changes in drug susceptibility in this study were small, recent studies have shown that even relatively modest susceptibility changes can correlate with clinical outcome (6
Two other significant mutational associations were seen with codon 69 mutations. The T69I mutation was always associated with the K65R mutation and almost always found with the Q151M mutation. However, not all patients with Q151M have the T69I mutation. The T69I mutation may be a requirement for these specific HIV strains to overcome spatial constraints in the RT enzyme carrying other mutations and polymorphisms. Alternatively, the T69I mutation may be involved in modulating susceptibility to one or more NRTI. Over half of the T69S mutations were primarily found in insert- or deletion-containing strains. Patients with T69S but without an insertion or deletion had mutation frequencies similar to those of NRTI-treated patients with wild-type codon 69 (data not shown). Again, this codon 69 variant may have an impact on enzyme fitness in the presence or absence of inserts or deletions and/or contribute to reduced susceptibility to NRTI alone or in association with other mutations.
In vitro susceptibility data showed that codon 69 variants had reduced susceptibility to NRTI. No other studies have directly studied point mutations at codon 69 other than T69D (9
). These results indicate that codon 69 changes can impact drug susceptibility beyond ddC in an otherwise wild-type setting. Some mutations have been shown to modulate susceptibility conferred by other mutations (15
). Most occurrences of codon 69 mutations were with other NRTI mutations. Our laboratory work did not examine the interaction of other mutations with the codon 69 changes in susceptibility assays, for example, the impact of codon 69 variants on AZT susceptibility in the presence of AZT resistance mutations and M184V/I. The large number of possible mutation combinations and associated polymorphisms makes this analysis best suited for large phenotypic assay databases containing results from clinical isolates. These databases would be able to genotypically match large numbers of isolates except for the codon 69 mutations and statistically analyze the difference between drug susceptibility in the presence or absence of codon 69 changes. This “virtual phenotype” analysis is currently being applied in research and clinical settings (13
The treatment regimens that select for the different codon 69 mutations were examined and reported (Table ). AZT monotherapy was a relatively common regimen selecting for codon 69 changes, as was AZT-3TC combination therapy. These observations could not be statistically validated, as a majority of patients had extensive and complicated treatment histories of varied duration that prevent treatment groups of reasonable numbers from being assembled. While the total amount of data is quite large, some treatment regimens are underrepresented. These factors limit the ability of database information to significantly address certain questions; however examination of such data can be useful to identify hypotheses that can be evaluated in well-controlled trials or data sets.
The results presented here suggest that codon 69 changes have a wider impact on NRTI resistance than currently thought. In clinical isolates, codon 69 changes develop and persist across a wide range of treatment regimens. To date, codon 69 mutations have not been reported to directly affect clinical outcome. Since the Stanford database does not collect viral load or CD4 measurements on patients, such analysis could not be performed in this report. However, the results presented here suggest that genotypic analysis of clinical outcome in randomized trials should include all variants of codon 69 and not restricted to T69D. While current understanding of the relationships between genotype, resistance, and clinical outcome has been shown to be beneficial for patient management (2
), further enhancement of this knowledge will undoubtedly increase the long-term efficacy of antiretroviral treatment regimens.