Neutral mitochondrial heteroplasmy and the influence of aging

Abstract

The development and maintenance of mitochondrial heteroplasmy has important consequences for both health and heredity. Previous studies using pathogenic mutations have shown considerable variability between maternally related individuals and studies of several D-loop polymorphisms have suggested a relationship between heteroplasmy and somatic aging. To broadly explore the variation of human heteroplasmy and to clarify the dynamics of somatic heteroplasmy over the course of lifespan, we analyzed mitochondrial sequence variation across a range of ages. We utilized array-generated single-nucleotide polymorphism data that were well correlated with independent measures of heteroplasmy. Significant levels of heteroplasmy were identified at 0.24% of sites evaluated. By examining mother-child pairs, we found that heteroplasmy was inherited (30%) but could occur de novo in offspring or, conversely, be present in mothers but eliminated in their children (70%). Cumulatively, mitochondrial heteroplasmy across the genome increased significantly with advanced age (r = 0.224, P =8 × 10(-30)). Surprisingly, changes in heteroplasmy were not uniform with some sites demonstrating a loss of variation (increased homoplasmy) with aging. These data suggest that both mutation and selective pressure affect blood mitochondrial DNA sequence over the course of the human lifespan and reveal the unexpectedly dynamic nature of human heteroplasmy.

Figure 1.

Validation of array-based genotyping using RFLP analysis or quantitative real-time PCR. (A) DNA from individuals with varying G16129A heteroplasmy detected by the SNP array was amplified and digested with KpnI, which cuts the G16129 allele (upper panel). The array-determined fraction of G16129 heteroplasmy is displayed above and the calculated last-hot-start heteroplasmy value is displayed below. Mixtures of plasmid DNA with the indicated fraction of G16129 were analyzed to create a standard curve shown below. Ladder (L) and no template (NT) lanes are also provided. (B) qPCR for heteroplasmy at 1440 was performed on previously array-analyzed samples and the results were correlated (R²= 0.99). The inset shows a magnification of the values close to 1.0.

Figure 2.

Higher levels of maternal heteroplasmy are more frequently inherited. Maternal and child genotype values are plotted for any SNP pair in which either mother or child exceeds the heteroplasmy threshold (n = 194). Mothers who transmitted their heteroplasmy or had offspring with homoplasmy for the other allele (black squares) were significantly more heteroplasmic than mothers who did not transmit heteroplasmy (grey squares) (17.9 versus 11.1%, P< 1× 10⁻⁴ by t-test).

Figure 3.

Changes in heteroplasmy across the mitochondrial genome with age. (A) The aggregate heteroplasmy at all 138 SNPs was calculated and plotted against decadal age using a box plot. Error bars indicate one standard deviation from the mean. There are increased levels of mutation with increasing age (r = 0.224, P =8 ×10⁻³⁰). (B) Heteroplasmy at 13650 in individuals with the major C-allele (n = 2458) was plotted against age in decades (r = 0.27, P< 0.0002). (C) Heteroplasmy at 10915 in individuals with the major T-allele (n = 2459) was plotted against age in decades (r = −0.38, P <0.0002).

Figure 4.

Evidence for selective pressure on polymorphisms during lifespan. Heteroplasmy is plotted for individuals who predominantly have the major G-allele (A; n = 2428) or the minor A-allele (B; n = 58) at 1440. The two populations show a convergence towards G1440. A similar convergence towards the G-allele is seen for the major (C) and minor (D) populations of 11 467.