(a) COX histochemistry from a representative healthy control subject (HIV−) showing normal COX activity, whereas a nucleoside analog treated HIV-infected patient (HIV+/NRTI+) shows multiple COX-deficient fibers (counterstained blue by residual SDH (succinate dehydrogenase) activity). (b) COX defects observed in each subject group (HIV+/NRTI−, HIV-infected treatment-naïve subjects; each dot represents an individual patient biopsy; ≥500 fibers sampled per biopsy).

(a) Mitochondrial DNA (mtDNA) content in individual COX (cytochrome c oxidase) deficient muscle fibers from nucleoside analogue treated HIV-infected (HIV+/NRTI+) subjects, expressed relative to mtDNA content in adjacent fibers of normal COX activity from the same subject. A few fibers show reduced mtDNA content, whereas the majority show increased content (geometric mean, 2.1 fold proliferation; maximum 21.3 fold; p <0.001 for difference in mean mtDNA content between COX deficient and normal fibers). (b) The majority of COX deficient fibers (COX−) contained high percentage levels of mtDNA containing a large-scale deletion of the major arc, causing the COX defect; whereas no deleted mtDNA was detected in adjacent COX positive fibers (COX+) (P <0.001). (c) Schematic representation of mtDNA large-scale deletion break-points in COX deficient fibers from HIV+/NRTI+ patients relative to the mtDNA gene positions(tRNA and rRNA not shown). Each line represents an individual deleted region. O_L, origin of light chain replication; O_H, origin of heavy chain replication. (n=15 fibers from 4 patients).

HIV+/NRTI+, HIV-infected, nucleoside analogue exposed; HIV+/NRTI−, HIV-infected, treatment-naïve. Dotted line represents lower threshold of assay. NRTI treated subjects showed significantly higher mean levels of CD than untreated subjects (HIV+/NRTI+ (mean ±SEM), −3.45 ±0.25 log₁₀(/mtDNA); HIV+/NRTI−, −4.56 ±0.31 log₁₀(/mtDNA); p=0.012). Box and whisker plot.

UDS (Roche 454 FLX GS) shows no difference in burden of low-level mtDNA point variants (exceeding 0.2% frequency) between HIV-infected nucleoside analog treated (HIV+/NRTI+, n=8), HIV-infected treatment-naïve (HIV+/NRTI−, n=4), and control (HIV−, n=4) subjects in two amplicons located in mtDNA hypervariable segment 2 (MT-HV2) and mtDNA COX subunit 3 (MT-CO3). In contrast, positive control subjects with inherited POLG defects (POLG, n=4) show increased burden of low-level mutations compared with healthy controls in MT-HV2 (OR 2.33, p =0.002).

Using a validated computer model of mtDNA replication based solely on experimentally derived parameters ²², we incorporated a finite period of partial replication failure due to the mtDNA chain-terminating effects of NRTI exposure ⁹, assigning a probability of failure per mtDNA replication event. All other parameters remained constant, including the de novo mutation rate ²². 2000 cells were simulated for 80 years. (a) The amount of mtDNA depletion during the NRTI exposure period caused by 25% and 45% probability of replication failure between 20 and 30 years-of-age. (>50% failure led to the complete loss of mtDNA). The range of mtDNA depletion predicted is in keeping with published in vivo data^12,23. (b) This led to a persistent increase in the frequency of COX (cytochrome c oxidase) deficient cells through the accelerated clonal expansion of pre-existing somatic mtDNA mutations. (c) Direct simulation of the effects of NRTI exposure within our study population (two different periods, 10 and 3 years, starting at age 20, of replication failure with 45% probability). The range of COX defects predicted closely fits our empiric data. (d) Late exposure (40 – 50 years) had a more pronounced effect than early exposure (20 – 30 years) (with 45% probability of replication failure) due to the higher number of pre-existing (age-related) somatic mtDNA mutations at the time of exposure.