Positive germline selection of mtDNA mutations: evidence from the oocyte.

Purifying selection of mtDNA mutations is a vital process that cleanses the mitochondrial genome of detrimental variants that may endanger individuals and populations. A common measure of purifying selection is the increase of average synonymity by reduction the proportion of mostly detrimental non-synonymous mutations. The mechanisms underlying purifying selection are still debated. The Makova group has recently published high-fidelity analysis of mtDNA mutations in individual human oocytes (Arbeithuber et al., 2025). The authors observed a decrease in the proportion of potentially detrimental coding and conservative mutations at higher mutant fractions (MFs) and interpreted this as purifying selection removing detrimental mutations at higher MFs. We noted, however, that, in contrast to what would be expected under purifying selection, the synonymity of oocyte mutations was very low and decreased, rather than increased, at higher MFs. We hypothesized that this inconsistency resulted from non-synonymous mutations being prone to strong positive selection which erroneously made coding mutations appear negatively selected in comparison. In support of our hypothesis, we show that non-coding oocytes mutations indeed are under strong positive selection. To alleviate this setback, we reanalyzed the data using a new metric of intracellular clonal selection and neutral synonymous mutations as the reference. We demonstrated that coding mutations are in fact under prevailing positive selection. This is in line with previous estimates of positive selection in primordial germ cells (PGCs) and in mother-child pairs. Importantly, "prevailing positive selection" does not imply the absence of negative selection. We show that specific types of mutations may be under prevailing purifying selection (e.g., the Co1 gene). Of note, this prevailing positive selection pertains only to the most recent, germline mtDNA mutations which have not been yet inherited into the next generation. Purifying selection steps in as germline mutations proceed to subsequent generations. The implications of these findings and the potential benefits of positive selection of detrimental mtDNA mutations are discussed. Graphical summaryGermline mtDNA mutations fuel evolution, shape population genomics, and cause mitochondrial disease. Yet it remains unresolved whether mtDNA selection in the germline is predominantly purifying or positive. A recent high-fidelity single-oocyte study (Arbeithuber et al., 2025) reported purifying(negative) selection on germline mutations at elevated mutant fractions (MFs). However, the low synonymity of oocyte mutations and concerns about using non-coding mutations as a reference for estimating selection prompted us to reanalyze the data. In single cells, mtDNA mutations are subject to genetic drift which randomly expands mtDNA clones. In this context, selection means that some mutant clones expand systematically faster (positive selection), or slower/get lost (purifying selection) than expected by random drift. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/697248v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@96fc4eorg.highwire.dtl.DTLVardef@8e717eorg.highwire.dtl.DTLVardef@1bd38b8org.highwire.dtl.DTLVardef@1d6cc69_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure A.C_FLOATNO Cumulative proportion curves of relevant types of mutations (color coded). The corresponding selection metrics,[S] 0.01, and Monte-Carlo p-values are shown, analyses for other thresholds: tables 2 and S1 C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@1063ef6org.highwire.dtl.DTLVardef@10fa4b2org.highwire.dtl.DTLVardef@5e13corg.highwire.dtl.DTLVardef@64afaorg.highwire.dtl.DTLVardef@12484f3_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable S1.C_FLOATNO C_TBL In Figure 1, datapoints represent clones of mutations ranked by their size and plotted vs. their cumulative contribution to the mutational pool (in reverse order). The resulting cumulative curves represent the collective expansion of clones of mutations of each type. As previously shown by direct simulations (Franco et al., 2025), the slope of the curve qualitatively depicts the relative intensity of clonal expansion. The green curve consists of synonymous (neutral) mutations and thus defines the trajectory of expansion driven by neutral genetic drift. Curves that diverge upward from the neutral green curve imply faster-than-neutral expansion, i.e., positive selection in corresponding mutation types, and those that diverge downward (grey Co1 curve) imply negative selection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/697248v1_fig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@9483a5org.highwire.dtl.DTLVardef@4efc4dorg.highwire.dtl.DTLVardef@1964229org.highwire.dtl.DTLVardef@1d1c5d9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Cumulative proportion curves of relevant types of mutations (color coded). C_FIG To estimate selection, we first defined extent of clonal expansion,[E] t (mutant class), a measure of overall clonal expansion, i.e., the proportion of aggregate fraction of mutants of a particular class in clones that exceeded a specific size (i.e., MF). For example,[E] 0.01(coding) is the ratio of aggregate mutant fraction of all large clones (MF>0.01) of coding mutations, divided by the aggregate mutant fraction of all coding mutations.[E] t changes with t, but at each t, it permits us to compare the extent of expansion between different classes of mutations (e.g., coding vs. non-coding). Selection at the intracellular level manifests as an acceleration or deceleration of the expansion (or loss) of mutant clones relative to the expansion expected under random drift, as represented by synonymous mutations. Accordingly, \a measure of clonal selection for a tested mutation class, denoted[S] t(tested), is defined as the excess clonal expansion in the tested mutations over expansion of synonymous mutations, normalized by the expansion of synonymous mutations: O_FD O_INLINEFIG[Formula 1]C_INLINEFIGM_FD(1)C_FD In Fig.1, [S]0.01 and p-values demonstrate: O_LIStrong positive selection of noncoding mutations (blue). C_LIO_LIPositive selection of coding mutations (orange). C_LIO_LIA higher positive selection in conservative (i.e., more detrimental) coding mutations (red). So, selection may be driven by the detrimental effects of mtDNA mutations. C_LIO_LINegative selection in the Co1 gene (grey). Thus, negative/purifying selection does exist, but dominates only in specific small regions (like Co1). C_LIO_LISelection in non-coding mutations starts at low mutant fractions, coding at higher mutant fractions. C_LI This confirms the prevalence of positive selection and clarifies why Arbeithuber et al. perceived selection as purifying. The authors compared coding to non-coding mutations. The latter are under stronger positive selection than coding mutations. Thus, coding mutations appear to be under relative negative selection, but only in comparison to non-synonymous mutations, not in absolute, real terms. Note that positive selection does not necessarily proceed in oocytes. Some of the mutations present in oocytes originate in primordial germ cells (PGCs), where they may also have been under selection before being passed on to oocytes. Indeed, positive selection in PGCs has been demonstrated previously (Fleischmann et al., 2024). Finally, positive selection of detrimental mutations in oocytes may seem surprising from an evolutionary perspective. A possible explanation is that the detrimental effects of mtDNA mutations usually do not show up till MF surpasses a physiological threshold. Thus, positive expansion may expose the detrimental phenotype of mutations and assist in removing carrier cells, embryos, or individuals, thus reducing burden on the mother. In line with this, purifying selection become prevalent among inherited mtDNA mutations.