Non-random mtDNA segregation patterns indicate a metastable heteroplasmic segregation unit in m.3243A>G cybrid cells

Abstract

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.

Figure 1. The metastable-segregation-unit working model.

(A) Schematic representation of the mitotic consequences of a stable heteroplasmic mtDNA segregation unit. The hypothetical founding cell is 50% heteroplasmic with 72 mtDNA molecules arranged in 9 segregation units each with 8 mtDNA’s, 4 of which are variant. Upon their faithful replication and partitioning to daughter cells for N mitosis, all 2^N descendant cells acquire the same 50% heteroplasmy level. (B) Schematic representation of the mitotic consequences after a metastable event in a cell from (A) such that one of its units has 6 instead of 4 variant mtDNA molecules. Upon random mitotic segregation of the units, 1/9^th of the cells will become fixed at 75% heteroplasmy and 8/9^th at 50%. Other scenarios can be envisaged. For example, a random redistribution of all mtDNAs in a cell from (A) will result in units with 0/8^th, 1/8^th ….7/8^th, 8/8^th mutation loads with binomially distributed frequencies. For simplicity, a uniform heteroplasmic segregation unit with identical number of copies of mtDNA is depicted. Slight variation in mtDNA copy number per unit in a cell will rapidly evolve through random mitotic segregation of these units into multiple cell types with each another uniform heteroplasmic segregation unit (like in B).

Figure 2. Heteroplasmy evolution of cybrid clone V_50.

(A) Average cellular mutation load decreases with increasing passage number of clone V_50. Conventional gel-based PCR-RFLP was used for quantitation of the m.3243A>G heteroplasmy. (B) Relative mutation load PCR-RFMT histograms of flow sorted single cells of selected passages of clone V_50. n refers to the number of single cells evaluated in the histogram. (C) Frequency histograms of m.3243A>G Padlock/RCA mutation loads of cells in V_50 P52 at increasing stringency of the dots/cell number criterion, cells with >0 (all), ≥20 and ≥40 dots/cell. See Figure S1 for choice of stringency and bin size. (D) Microscopic image of V_50 P52 after m.3243A>G Padlock/RCA in situ genotyping. Note in this microscopic field the presence of homoplasmic wild type cells (arrow heads) amidst the heteroplasmic cells. See Figure S2 for discussion on the number and nature of the signals.

Figure 3. Evolution of bulk mtDNA mutation load during long term continuous culture of 11 cybrid clones.

Average m.3243A>G mutation loads were determined with PCR/RFMT on bulk DNA in triplicate. The size of the data marker masks error bars of most data points.

Figure 4. Cellular heteroplasmy evolution of clone G_4.21 by Padlock/RCA analysis.

The m.3243A>G mutation load of single cells was determined by Padlock/RCA in passages 15 (A), 42 (B) and 71 (C). Dots/cell number may reflect detection efficiency, but also the actual segregation unit number. Since detection efficiency is arguable low (Supplemental Figure S2), in the order ∼5%, cells with a wide range of dots/cell number will be observed, independent of true variations in segregation unit number. The frequency histograms of the three passages are shown separately at increasing stringency of the dots/cell criterion (>0 (all) up to ≥80 dots/cell) to emphasize that the peaks and shoulders are genuine. Thus, the outcome of the experiment does not change substantially with the smaller amount of cells with larger dots/cell (≥60 or ≥80), which makes calculation of mutation load more solid (See also Figure S1 for choice of stringency and bin size). Due to the choice for a 5% bin, it is not apparent that the cells in the 95–100% bin of P15 are for the very great majority heteroplasmic: in that bin, 94% of the cells with ≥40 dots/cell and 92% of the cells ≥80 dots/cell bin had in fact one or more green (wild type mtDNA) present.

Figure 5. Cellular heteroplasmy evolution of V_50 P48 sub-clone V_3.18 by Padlock/RCA analysis.

m.3243A>G mutation load of single cells of the sub-clone V_3.18 derived from V_50 P48 was assessed with Padlock/RCA. Relative frequency histograms of cells in 7 passages are shown. Since 3 of the 7 passages did not meet the criterion of >30% of the cells having ≥40 dots/cell (see Figure S1), the relative frequency histograms are therefore shown at ≥20 dots/cell stringency and with 10% bin. n refers to the number of single cells evaluated in the histogram whereas numbers in parentheses represent the total number of cells analyzed.

Figure 6. Genomic instability of the 143B host nucleus as revealed by array-CGH.

Array-CGH was at 1 Mb resolution with a BAC/PAC array using genomic DNAs from V_50 P62 and V_50 P6 as test DNA and reference DNA respectively. Red, blue and green dots represent, respectively, copy number change, no copy number change and natural copy number variations. Numbers on the X-axis represent human chromosome numbers.

Figure 7. Cellular heteroplasmy evolution of V_50 P48 sub-clone V_3.2.

(A) Relative m.3243A>G Padlock/RCA mutation load frequency histograms of cells in passages 1, 12 and 81 of sub-clone V_3.2. Histograms from cells with ≥60 dots/cell had similar shapes. Numbers in parentheses represent the total number of cells analyzed. (B) Relative mutation load frequency histograms of cells in passages 1, 12 and 81 of V_50 P42 sub-clone V_3.2 generated by computer simulation of random segregation (mtDNA copy number input 1,800/cell). Additional computer simulations showed that an mtDNA copy number input of 12,000 is required to explain the experimental distribution at P81 by random mtDNA segregation.

Figure 8. Cellular heteroplasmy evolution of clone G_55.2.

(A) Relative m.3243A>G mutation load frequency histograms of cells in multiple passages of G_55.2. With exception of P32, histograms were produced by PCR/RFMT of flow-sorted single cells. Mutation loads of cells in P32 were measured by Padlock/RCA using cells ≥40 dots/cell, which represented 80% of the analyzed cells. SDs for the single cell PCR/RFMT mutation load determinations of P1–P25 ranged from 4 to 8%. (B) Relative mutation load frequency histograms of cells in passages P1–P32 of clone G_55.2 generated by computer simulation of random segregation.