Abstract Introduction: Acquired mitochondrial DNA mutations (mtDNA heteroplasmy) and changes in mtDNA copy number (mtDNA-CN) are indicators of aging and inflammation ('inflammaging') in various diseases. In sickle cell disease (SCD), systemic inflammation is a key factor driving the disease progression, and patients with the more severe genotypes (HbSS and HbSb0Thal) display higher levels of inflammation. Our previous research demonstrated that patients with HbSS and HbSβ0Thal exhibit a greater burden of mtDNA heteroplasmy and higher mtDNA-CN levels. We also identified specific 'hotspot' regions in the mitochondrial genome, which are more prone to acquiring mutations. In this longitudinal study, we used the humanized Townes SCD mouse model to explore how dysfunctional mtDNA accumulates over time and if the rate of accumulation is influenced by severity of the SCD genotype. Methods: The study included 60 Townes SCD mice: 20 HbAA (controls), 20 HbAS (sickle cell trait), and 20 HbSS (sickle mice). All groups included an equal number of littermate male and female mice. We collected 100ul- 200ul of blood from anesthetized mice at 8 time points: weeks 16 (T1), 27 (T2), 39 (T3), 58 (T4), 65 (T5), 69 (T6), 71 (T7) and 73 (T8). Genomic DNA extracted from the whole blood, was used to enrich mtDNA and then subjected to deep sequencing. The analysis of mtDNA heteroplasmy utilized LoFreq variant caller (allele frequency filter 2%). SIFT prediction was utilized to identify deleterious mtDNA heteroplasmies. Additionally, the remaining genomic DNA was whole genome sequenced (WGS) to estimate mtDNA-CN. For mtDNA heteroplasmy comparison, changes from the baseline (T1) were derived for each mouse, and the mean percentage change was determined for each genotype at each timepoint. Analysis of deleterious mtDNA mutations and mtDNA-CN were conducted using aggregated group means at each time point, due to the lack of consistently paired samples across the time points. Statistical analyses were conducted with GraphPad Prism. Results: The study is ongoing.At week 73 (T8), 22 mice (11 HbAA, 8 HbAS, 3 HbSS) were alive. HbSS mice had a higher mortality rate compared to HbAA and HbAS mice (p=0.0177). mtDNA mutational burden analysis for the initial 3 time points (T1, T2, T3) containing a total of 176 samples has been completed to date. Over time, the mtDNA heteroplasmy burden increased remarkably, rising by 6.38% at T2 (p=n.s) and 18.38% at T3 (p=0.0368) compared to baseline (T1). HbSS mice displayed an accelerated increase in mtDNA heteroplasmy burden, showing a 42.47% rise at T3 compared to 12.5% at T2 and T1 (p=0.0062 and p=0.0006, respectively) while HbAA and HbAS mice showed minimal changes (p=ns). Deleterious mtDNA heteroplasmy also expanded over time, with increases of 20.83% at T2 and 31.70% at T3 compared to T1, with a dramatic escalation in HbSS mice, 71.43% increase at T2, and 200% at T3 compared to T1. In HbSS mice, the burden of deleterious heteroplasmies at T3 was significantly higher than at baseline (p=0.0015), while that in HbAS and HbAA were relatively stable over time. All identified deleterious heteroplasmies (n=165) were found in mitochondrial genes, ATP8, ATP6, and ND2. This study also observed a decrease in mtDNA-CN at T3 compared to T2 and T1. Of the 176 samples collected, 130 were WGS, 46 were excluded due to insufficient DNA. Analysis of 45 paired samples with two time points showed a significant reduction from T-Young (average of T1 and T2; mean mtDNA-CN=1580) to T-Old (T3; mean mtDNA-CN=1438) (p=0.026). There was an overall declining trend in mtDNA-CN with age, with HbAA mice showing significantly greater reduction compared to HbAS and HbSS mice (p 0.05). Conclusions: The study reveals that in Townes mice, the mtDNA heteroplasmy burden increases with aging, with HbSS mice showing a faster accumulation of mtDNA mutations and a pronounced rise in deleterious mtDNA mutations, primarily found in genes related to oxidative phosphorylation. Our findings also suggest a potential threshold effect, when the mutational burden escalates dramatically. Completion of the longitudinal mtDNA analysis until death of all HbSS mice in this cohort will provide insights on how mtDNA mutations accumulate and their correlation with sickle cell pathology.
Wang et al. (Mon,) studied this question.