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Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations
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Download the PDF here
Download the PDF here
Download the PDF here
Nature Genetics June 26 2011
The rapid clonal expansion of somatic mtDNA mutations we observed in NRTI-treated HIV-infected patients provides a plausible mechanism for accelerated aging in treated HIV infection. This is potentially of great importance for the millions of HIV-infected patients in the developing world where these drugs remain the mainstay of therapy and adds weight to a causal role for somatic mtDNA.....we explored the effect of timing of NRTI exposure and showed that later periods of therapy predicted a higher frequency of COX deficiency (Fig. 5d). This is because of older subjects harboring a greater number of age-related somatic mtDNA mutations than younger subjects, which rapidly clonally segregate during NRTI therapy.. This is in keeping with the observation that mitochondrially mediated clinical complications of NRTI therapy appear to be more common in older individuals24. Finally we mod- eled the longer-term effects of treatment. Using this approach, an HIV-infected individual treated with NRTIs during their third decade is predicted to develop ~5% COX-deficient cells by age 60 (Fig. 5b-d). This is similar to or exceeds that seen in the healthy very old4......Given that NRTI-treated subjects showed high-level COX defects (up to 10% of fibers) which contained clonal mutated mtDNA spe- cies, one explanation for our findings is accelerated segregation of preexisting (age-associated) mtDNA mutations caused by NRTI treat- ment rather than de novo somatic mutation.
Brendan A I Payne1,2, Ian J Wilson1, Charlotte A Hateley1, Rita Horvath1, Mauro Santibanez-Koref1, David C Samuels3, D Ashley Price2 & Patrick F Chinnery1
1Mitochondrial Research Group, Institute of Genetic Medicine, Newcastle University, Newcastle-upon-Tyne, UK. 2Department of Infection and Tropical Medicine, Royal Victoria Infirmary, Newcastle-upon-Tyne, UK. 3Centre for Human Genetics Research, Vanderbilt University, Nashville, Tennessee, USA. Correspondence should be addressed to P.F.C. (p.f.chinnery@ncl.ac.uk).
There is emerging evidence that people with successfully treated HIV infection age prematurely, leading to progressive multi-organ disease1, but the reasons for this are not known. Here we show that patients treated with commonly used nucleoside analog anti-retroviral drugs progressively accumulate somatic mitochondrial DNA (mtDNA) mutations, mirroring those seen much later in life caused by normal aging2, 3. Ultra-deep re-sequencing by synthesis, combined with single-cell analyses, suggests that the increase in somatic mutation is not caused by increased mutagenesis but might instead be caused by accelerated mtDNA turnover. This leads to the clonal expansion of preexisting age-related somatic mtDNA mutations and a biochemical defect that can affect up to 10% of cells. These observations add weight to the role of somatic mtDNA mutations in the aging process and raise the specter of progressive iatrogenic mitochondrial genetic disease emerging over the next decade.
Somatic mtDNA mutations accumulate in individual cells during normal human aging, leading to cellular bio-energetic defects of oxidative phos- phorylation2,4. Transgenic mice with a defective mtDNA polymerase (pol γ) accumulate secondary mtDNA mutations and a prematurely aged phenotype3, but it is still not clear whether the mtDNA mutations are a cause or a consequence of aging in humans. Accelerated senescence has recently been described in humans with successfully treated HIV infection1. These patients become frail at an early age, decline physi- ologically5,6 and acquire age-associated degenerative disorders affecting the cardiovascular system and the brain leading to dementia7,8. Several nucleoside analog reverse transcriptase inhibitor anti-retroviral drugs (NRTIs) used in the treatment of HIV inhibit the function of pol γ 9, rais- ing the possibility that drug treatment contributes to the accelerated aging phenotype through mtDNA damage. NRTIs are well known to cause an acute, temporary and reversible reduction in the amount of mtDNA (mtDNA depletion), and one previous study detected mtDNA deletions in patients being actively treated with NRTIs10-12. However, no previous studies have looked at the possibility of irreversible long-term effects of the drugs on mtDNA m-utations after NRTI treatment has ceased.
We studied skeletal muscle from 33 HIV-infected adults, all aged 50 years or under, stratified by lifetime exposure to NRTIs previously shown to affect pol γ in vitro9 (Online Methods and Supplementary Table 1), and 10 HIV-uninfected healthy controls (HIV-) of comparable age. We initially looked for a defect of mitochondrial oxi- dative phosphorylation within individual cells using cytochrome c oxidase-succinate dehydrogenase (COX-SDH) histochemistry. Cellular COX defects would not be expected in this younger subject group (<0.5%)4.The frequency of COX-deficient muscle fibers in HIV-infected non-treated (treatment-naïve, HIV+/NRTI-) subjects (n = 12) was indistinguishable from that observed in HIV- controls, with the majority having no COX-deficient fibers. By contrast, NRTI- exposed (HIV+/NRTI+) subjects (n = 21) had an increased frequency of COX-deficient muscle fibers (maximum 9.8%, P = 0.047), reaching or exceeding levels expected in healthy elderly individuals4 (Fig. 1). The severity of the COX defect was strongly predicted by cumula- tive lifetime NRTI exposure, rather than therapy at the time of study, implicating a persistent and cumulative mitochondrial defect (r2 = 87%, P < 0.001; Supplementary Fig. 1).
We then defined the molecular basis for the COX deficiency observed in NRTI-exposed subjects. We first excluded persistent mtDNA depletion. The mtDNA content in homogenized skeletal muscle did not differ between HIV+/NRTI+ and HIV+/NRTI- patients (Supplementary Fig. 2). In keeping with this, the analysis of individual laser-captured single muscle fibers (n = 128) showed that only a small minority of COX-deficient fibers (6 out of 70, or 9%) from NRTI-treated patients had mtDNA depletion compared to adjacent fibers with normal COX activity. By contrast, the vast major- ity of the isolated COX-deficient fibers contained markedly increased amounts of mtDNA (geometric mean of 2.1-fold proliferation, maxi- mum 21.3-fold; P < 0.001 for difference in mean mtDNA content between COX-deficient and normal fibers) (Fig. 2a). Focal mtDNA proliferation is often seen in association with pathogenic mtDNA mutations. In keeping with this, the majority of the COX-deficient fibers analyzed (40 out of 70 fibers from 12 HIV+/NRTI+ patients) showed high percentage levels of mtDNA molecules containing large- scale deletion mutations, exceeding the percentage level of mutation required to cause a COX defect (~60%13). We detected no deletion mutations in adjacent skeletal muscle fibers (n = 58) with normal COX activity. Analysis of the mtDNA deletion break points (n = 15 fibers from four HIV+/NRTI+ patients) revealed different deletions in different fibers, all of which were clonal within individual fibers. Most of the clonally expanded deletions were unique; the only deletion observed more than once was the mt.σ4977 'common deletion', the commonest age-associated somatic mtDNA mutation14,15 (Fig. 2b,c and Supplementary Table 2).
Although less common than large-scale deletion mutations, mtDNA point mutations are also found in COX-deficient fibers from healthy aged subjects16. In keeping with this, in the NRTI-treated patients, we found COX-deficient fibers not containing a deletion to harbor non- synonymous somatic mtDNA point mutations (5 out of 29 fibers). These mutations are predicted to alter a highly conserved amino acid and have not previously been described as inherited polymorphic variants in 5,140 humans (Table 1) (one variant, 12797T>C, had been observed as a somatic variant in a single human sequence)17 and thus provide an explanation for the associated cellular COX defect. Other fibers con- tained high levels of noncoding control-region (nt 16,024 to nt 576) variants, which were previously described in healthy aged humans.
We then estimated the total burden of mtDNA deletion muta- tions at the whole-tissue level. The proportion of mtDNA molecules containing the mt.σ4977 'common deletion' was significantly higher in NRTI-treated patients compared with untreated patients (HIV+/ NRTI+ (mean ± s.e.m.), -3.45 ± 0.25 log10(/mtDNA); HIV+/NRTI-, -4.56 ± 0.31 log10(/mtDNA); P = 0.012) (Fig. 3) and were comparable with those previously reported in very elderly healthy subjects18. Furthermore, the proportion of COX-deficient muscle fibers from NRTI-treated subjects which contained mt.σ4977 was very similar to that reported in healthy aged individuals15. Pathogenic muta- tions within single fibers (of which the majority were deletions) were accompanied by proliferation of mtDNA, which occurs in an attempt to maintain adequate levels of wild-type mtDNA, as shown previously19. As a result, mutated mtDNA also proliferates within the fiber. Over time, this will lead to a detectable increase in the level of deletions at the whole-tissue level.
To estimate the relative burden of mtDNA point mutations between treatment groups in homogenized skeletal muscle, we designed an ultra-deep re-sequencing by synthesis (UDS) assay using FLX GS technology (Roche 454). First, we carried out a series of control exper- iments to show the sensitivity of UDS to detect mtDNA point variants. We initially established that UDS of an mtDNA template did not generate an intrinsically different signal when compared to a nuclear DNA template by sequencing amplicons of cloned autosomal and mitochondrial DNA fragments as well as an autosomal DNA ampli- con from genomic DNA (Supplementary Table 3). By this approach, we confirmed a very low background noise level for the UDS assay (Online Methods and Supplementary Fig. 3). As a positive control, we then compared two mtDNA amplicons from skeletal muscle DNA of POLG patients (n = 4), individuals known to harbor high levels of somatic mtDNA point mutations20,21. One mtDNA amplicon was in the hypervariable noncoding control region (MT-HV2) predicted from 5,140 population-level sequences17 to have a high mutation rate, and one was in a highly conserved mtDNA coding region (MT-CO3). Mean coverage was 5,892 sequence reads per amplicon in each direc- tion. Consistent with an error-prone pol γ, these subjects showed an increase in mtDNA point variants detectable at >0.2% frequency in the MT-HV2 amplicon (OR = 2.33, P = 0.002) (Fig. 4) when com- pared to healthy controls (n = 4). We detected no increase in vari- ants in the MT-CO3 amplicon. These findings were confirmed on replicate samples (Supplementary Fig. 4). When we studied skeletal muscle mtDNA from the HIV+/NRTI+ subjects (n = 8), the overall burden of point variants within each amplicon was indistinguish- able from HIV+/NRTI- subjects (n = 4) and healthy HIV- controls (n = 4), all of comparable age (OR = 1.08, P = 0.79 for comparison of HIV+/NRTI+ and HIV- for MT-HV2). Furthermore, there was no correlation between COX defect in HIV+/NRTI+ subjects (range up to 10%) and mutation burden on the UDS assay.
Given that NRTI-treated subjects showed high-level COX defects (up to 10% of fibers) which contained clonal mutated mtDNA spe- cies, one explanation for our findings is accelerated segregation of preexisting (age-associated) mtDNA mutations caused by NRTI treat- ment rather than de novo somatic mutation. In contrast, the POLG subjects showed a significant increase in point mutation burden in the UDS assay (although only in MT-HV2, P = 0.002) but a low propor- tion of COX-deficient fibers. Although the UDS data does not exclude the possibility of a slight increase in mutagenesis in NRTI-exposed subjects, it would not be of the level predicted to be required (>100- fold increase22) to cause the observed COX defects.
To determine whether accelerated clonal expansion was a plausi- ble explanation for our findings, we used an established computa- tional model based solely on experimentally derived parameters22 and simulated the effects of NRTI-induced chain-termination during mtDNA replication9. The de novo mutation rate was not altered from the original model of aging muscle. A finite NRTI exposure predicted a period of temporary mtDNA depletion which was concordant with reported mtDNA levels12,23 and the COX defects observed12 in acutely treated HIV patients. This resulted in accelerated clonal expansion of preexisting mtDNA mutations and led to an irreversible increase in the frequency of COX-deficient muscle fibers (Fig. 5a,b). The severity of predicted COX defect was dependent on the degree of replication failure and the duration of exposure (Fig. 5b,c), which is in keeping with our observations in patient muscle that had suggested a strong dependence on these factors (Supplementary Fig. 1). In silico mod- eling is thus consistent with the hypothesis that accelerated clonal expansion of preexisting (age-associated) mtDNA somatic mutations is sufficient to explain our observations in NRTI-treated subjects. Having established the model, we explored the effect of timing of NRTI exposure and showed that later periods of therapy predicted a higher frequency of COX deficiency (Fig. 5d). This is because of older subjects harboring a greater number of age-related somatic mtDNA mutations than younger subjects, which rapidly clonally segregate during NRTI therapy. This is in keeping with the observation that mitochondrially mediated clinical complications of NRTI therapy appear to be more common in older individuals24. Finally we mod- eled the longer-term effects of treatment. Using this approach, an HIV-infected individual treated with NRTIs during their third decade is predicted to develop ~5% COX-deficient cells by age 60 (Fig. 5b-d). This is similar to or exceeds that seen in the healthy very old4.
Although the UDS data for mtDNA point mutations support the hypothesis of accelerated clonal expansion of preexisting age- related mutations rather than increased mutagenesis, it is possible that additional mechanisms may be involved for mtDNA large-scale deletions, including a replicative advantage favoring deleted molecules25. Furthermore, although UDS provides great depth of mutational analysis, it is analogous to the PCR-cloning method of mutation rate determination and as such will tend to exaggerate an estimate of the mutation rate26,27.
The rapid clonal expansion of somatic mtDNA mutations we observed in NRTI-treated HIV-infected patients provides a plausible mechanism for accelerated aging in treated HIV infection. This is potentially of great importance for the millions of HIV-infected patients in the developing world where these drugs remain the mainstay of therapy and adds weight to a causal role for somatic mtDNA
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