The mitochondrial DNA control region shows genetically correlated levels of heteroplasmy in leukocytes of centenarians and their offspring

Abstract

Background: Studies on heteroplasmy occurring in the mitochondrial DNA (mtDNA) control region (CR) in leukocytes of centenarians and younger subjects have shown that the C150T somatic transition is over-represented in centenarians. However, whether the occurrence/accumulation of heteroplasmy is a phenotypic consequence of extreme ageing or a genetically controlled event that may favor longevity is a question that deserves further attention. To clarify this point, we set up a Denaturing High Performance Liquid Chromatography (DHPLC) protocol to quantify mtDNA CR heteroplasmy. We then analyzed heteroplasmy in leukocytes of centenarians (100 subjects), their offspring and nieces/nephews (200 subjects, age-range 65-80 years, median age 70 years), and in leukocytes of 114 control subjects sex- and age-matched with the relatives of centenarians.

Results: The centenarians and their descendants, despite the different ages, showed similar levels of heteroplasmy which were significantly higher than levels in controls. In addition we found that heteroplasmy levels were significantly correlated in parent-offspring pairs (r = 0.263; p = 0.009), but were independent of mtDNA inherited variability (haplogroup and sequence analyses).

Conclusion: Our findings suggest that the high degree of heteroplasmy observed in centenarians is genetically controlled, and that such genetic control is independent of mtDNA variability and likely due to the nuclear genome.

Figure 1

DHPLC reference curve assembled from clones of mtDNA CR (16531 nt -261 nt) including either C150 or T150. The two clones were combined to generate samples having heteroplasmy levels of 0%, 5%, 10%, 20%, 30%, 40%, 50 %. Het/Tot is the ratio between the height of the Heteroplasmic peak and the Total height of homoplasmic plus heteroplasmic peaks. Bars denote the standard deviation in triplicate experiments. The observed values were used to fit a 2^nd degree polynomial function y = β₁x + β₂x².

Figure 2

Histograms showing mtDNA CR heteroplasmy in the four sample groups. Heteroplasmy is estimated from the DHPLC reference curve reported in Fig.1.

Figure 3

Linear regression between heteroplasmy levels in centenarians (x axis) and their offspring (y axis). Log transformed values were used in order normalise the distribution. The regression line showed r = 0.263 (p = 0.009).

Figure 4

Linear regression of heteroplasmy levels in parent-offspring pairs according to the sex of the parent: (a) male centenarian parents; (b) female centenarian parents. The regression lines showed r = -0.053 (p = 0.704) and r = 0.456 (p = 0.001) in (a) and (b), respectively.

Figure 5

DHPLC profiles observed in the samples having levels of heteroplasmy equal to or higher than 25%. The sequence which characterizes each profile is shown on the right.