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. ### Competing Interest Statement The authors have declared no competing interest.

The resilience of the mitochondrial genome (mtDNA) to a high mutational pressure depends, in part, on negative purifying selection in the germline. A paradigm in the field has been that such selection, at least in part, takes place in primordial germ cells (PGCs). Specifically, Floros et al. (Nature Cell Biology 20: 144-51) reported an increase in the synonymity of mtDNA mutations (a sign of purifying selection) between early-stage and late-stage PGCs. We re-analyzed Floros' et al. data and determined that their mutational dataset was significantly contaminated with single nucleotide variants (SNVs) derived from a nuclear sequence of mtDNA origin (NUMT) located on chromosome 5. Contamination was caused by co-amplification of the NUMT sequence by crossspecific PCR primers. Importantly, when we removed NUMT-derived SNVs, the evidence of purifying selection was abolished. In addition to bulk PGCs, Floros et al. reported the analysis of single-cell late-stage PGCs, which were amplified with different sets of PCR primers that cannot amplify the NUMT sequence. Accordingly, there were no NUMT-derived SNVs among single PGC mutations. Interestingly, single PGC mutations show a decrease of synonymity with increased intracellular mutant fraction. More specifically, nonsynonymous mutations show faster intracellular genetic drift towards higher mutant fraction than synonymous ones. This pattern is incompatible with predominantly negative selection. This suggests that germline selection of mtDNA mutations is a complex phenomenon and that the part of this process that takes place in PGCs may be predominantly positive. However counterintuitive, positive germline selection of detrimental mtDNA mutations has been reported previously and potentially may be evolutionarily advantageous.

The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different organisms is still incomprehensible. Since mitochondria are responsible for aerobic respiration, it is expected that mtDNA mutational spectrum is affected by oxidative damage. Assuming that oxidative damage increases with age, we analyse mtDNA mutagenesis of different species in regards to their generation length. Analysing, (i) dozens of thousands of somatic mtDNA mutations in samples of different ages (ii) 70053 polymorphic synonymous mtDNA substitutions reconstructed in 424 mammalian species with different generation lengths and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of A(H) > G(H) substitutions (H: heavy strand notation) is twice bigger in species with high versus low generation length making their mtDNA more A(H) poor and G(H) rich. Considering that A(H) > G(H) substitutions are also sensitive to the time spent single-stranded (TSSS) during asynchronous mtDNA replication we demonstrated that A(H) > G(H) substitution rate is a function of both species-specific generation length and position-specific TSSS. We propose that A(H) > G(H) is a mitochondria-specific signature of oxidative damage associated with both aging and TSSS.

Background Third-generation sequencing offers some advantages over next-generation sequencing predecessors, but with the caveat of harboring a much higher error rate. Clustering-related sequences is an essential task in modern biology. To accurately cluster sequences rich in errors, error type and frequency need to be accounted for. Levenshtein distance is a well-established mathematical algorithm for measuring the edit distance between words and can specifically weight insertions, deletions and substitutions. However, there are drawbacks to using Levenshtein distance in a biological context and hence has rarely been used for this purpose. We present novel modifications to the Levenshtein distance algorithm to optimize it for clustering error-rich biological sequencing data. Results We successfully introduced a bidirectional frameshift allowance with end-user determined accommodation caps combined with weighted error discrimination. Furthermore, our modifications dramatically improved the computational speed of Levenstein distance. For simulated ONT MinION and PacBio Sequel datasets, the average clustering sensitivity for 3GOLD was 41.45% (S.D. 10.39) higher than Sequence-Levenstein distance, 52.14% (S.D. 9.43) higher than Levenshtein distance, 55.93% (S.D. 8.67) higher than Starcode, 42.68% (S.D. 8.09) higher than CD-HIT-EST and 61.49% (S.D. 7.81) higher than DNACLUST. For biological ONT MinION data, 3GOLD clustering sensitivity was 27.99% higher than Sequence-Levenstein distance, 52.76% higher than Levenshtein distance, 56.39% higher than Starcode, 48% higher than CD-HIT-EST and 70.4% higher than DNACLUST. Conclusion Our modifications to Levenshtein distance have improved its speed and accuracy compared to the classic Levenshtein distance, Sequence-Levenshtein distance and other commonly used clustering approaches on simulated and biological third-generation sequenced datasets. Our clustering approach is appropriate for datasets of unknown cluster centroids, such as those generated with unique molecular identifiers as well as known centroids such as barcoded datasets. A strength of our approach is high accuracy in resolving small clusters and mitigating the number of singletons.

The hypothesis that the evolution of humans involves hybridization between diverged species has been actively debated in recent years. We present the following novel evidence in support of this hypothesis: the analysis of nuclear pseudogenes of mtDNA (“NUMTs”). NUMTs are considered “mtDNA fossils” as they preserve sequences of ancient mtDNA and thus carry unique information about ancestral populations. Our comparison of a NUMT sequence shared by humans, chimpanzees, and gorillas with their mtDNAs implies that, around the time of divergence between humans and chimpanzees, our evolutionary history involved the interbreeding of individuals whose mtDNA had diverged as much as ~4.5 Myr prior. This large divergence suggests a distant interspecies hybridization. Additionally, analysis of two other NUMTs suggests that such events occur repeatedly. Our findings suggest a complex pattern of speciation in primate/human ancestors and provide one potential explanation for the mosaic nature of fossil morphology found at the emergence of the hominin lineage. A preliminary version of this manuscript was uploaded to the preprint server BioRxiv in 2017 (10.1101/134502).

The resilience of the mitochondrial genome to a high mutational pressure depends, in part, on purifying selection against detrimental mutations in the germline. It is crucial to understand the mechanisms of this process. Recently, Floros et al. concluded that much of the purifying selection takes place during the proliferation of primordial germ cells (PGCs) because, according to their analysis, the synonymity of mutations in late PGCs was seemingly increased compared to those in early PGCs. We re-analyzed the Floros et al. mutational data and discovered a high proportion of sequence variants that are not true mutations, but originate from NUMTs, the latter of which are segments of mitochondrial DNA (mtDNA) inserted into nuclear DNA, up to millions of years ago. This is a well-known artifact in mtDNA mutational analysis. Removal of these artifacts from the Floros et al. dataset abolishes the reported effect of purifying selection in PGCs. We therefore conclude that the mechanism of germline selection of mtDNA mutations remains open for debate, and more research is needed to fully elucidate the timing and nature of this process.

The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different tissues and organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra may be affected by the oxidative damage which is increasing with organismal aging. However, the well-documented mutational signature of the oxidative damage, G>T substitutions, is typical only for the nuclear genome while it is extremely rare in mtDNA. Thus it is still unclear if there is a mitochondria-specific mutational signature of the oxidative damage. Here, reconstructing mtDNA mutational spectra for 424 mammalian species with variable generation length which is a proxy for oocyte age, we observed that the frequency of AH>GH substitutions (H - heavy chain notation) is positively correlated with organismal longevity. This mutational bias from AH to GH significantly affected the nucleotide content of analyzed 650 complete mammalian mitochondrial genomes, where fourfold degenerative synonymous positions of long-lived species become more AH poor and GH rich. Because (i) A>G is a substitution, typical for mtDNA; (ii) it is characterized by very strong asymmetry: A>G is several-fold more frequent on a heavy chain as compared to the light one; (iii) it is sensitive to the time being single-stranded during mtDNA asynchronous replication; (iv) it is associated with oxidative damage of single-stranded DNA in recent experimental studies we propose that A>G is a novel mutational signature of age-associated oxidative damage of single-stranded mtDNA. The described association of the mtDNA mutational spectra with a species-specific life-history trait can significantly affect general patterns of molecular evolution of mtDNA.

It is conventionally assumed that detrimental mitochondrial DNA (mtDNA) mutations generally are under negative purifying selection in the germline, i.e., that children on average carry a lower burden than mothers. We asked whether, at least for some detrimental mutations, this may not be the case. We chose the infamous 3243G>A mutation, which disrupts mitochondrial function and causes a variety of serious neuromuscular disorders including a severe mitochondrial disease, MELAS. This mutation occurs frequently (detectable in as many as 1 in 500 individuals), and is well studied. To determine whether this mutation is under selection, the mutation level in kids is compared to that in mothers. However, it has been shown that the level of the 3243G>A mutation appears to exponentially decrease with age in blood, where it is commonly measured (~2% per year). This estimation is based on a biologically sensible numerical model that simulates random drift in blood stem cells and removes cells that reach a lethal threshold (Rajasimha, 2008). This model asymptotically converges to an exponential function that describes a decline of 2% heteroplasmy per year. Thus, for fair comparison, kid/mom heteroplasmy ratios need to be corrected for the age difference. With the conventional 2% per year correction, no significant difference between moms and kids could be detected.There are indications, however, that the change of the 3243G>A level with age may be more complex than simple exponent. It may depend on initial heteroplasmy levels and the age. Indeed, we demonstrate that the 2% model predictions are systematically under‐estimates, and that several predictive models that include heteroplasmy and age as variables ensure better fit of the data and decrease this negative bias. One such model is an adaptation of the original numerical model (Rajasimha, 2008). If its asymptotic behavior is not unconditionally assumed, this model explicitly includes heteroplasmy and age as variables. This non‐asymptotic model produces a better fit and corrects the negative bias.We reasoned that a negative bias in prediction might have obscured positive selection on 3243G>A in the germline if it existed. To clarify the issue, we used the non‐asymptotic numerical model to predict the children’s heteroplasmy levels at their mothers’ age. Indeed, in preliminary simulations with this improved correction, a statistically significant positive germline selection of 3243G>A was demonstrated. More analysis is needed to confirm this initial result.We note that, interestingly, if such positive selection of pathogenic mtDNA mutations existed, it might have had an adaptive purpose. Our simulations demonstrate that under realistic conditions, positive selection in the germline may promote removal of detrimental mtDNA mutations at the population level and thus help to evade the dangers of the infamous Muller’s ratchet.

Mutational spectrum of the mitochondrial genome (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different tissues and organisms is still incomprehensible. Since mitochondria is tightly involved in energy production, we expect that mtDNA mutational spectra can reflect the level of cellular aerobic metabolism, which varies in different tissues. Analyzing a collection of somatic mtDNA mutations from human cancers, de novo mtDNA germline mutations from the human mother-offspring pairs, as well as mtDNA substitutions in hundreds of mammalian species, we observed that the frequency of AH>GH (heavy strand notation) substitutions is positively correlated with cellular and organismal longevity. For example, epithelium, oocytes of young mothers and mice have decreased AH>GH frequencies. We propose that AH>GH is a marker of cellular and organismal age, which is driven by oxidative damage of the single-stranded mtDNA during replication. Graphical abstract

Nucleic acid sequence analyses are fundamental to all aspects of biological research, spanning aging, mitochondrial DNA (mtDNA) and cancer, as well as microbial and viral evolution. Over the past several years, significant improvements in DNA sequencing, including consensus sequence analysis, have proven invaluable for high-throughput studies. However, all current DNA sequencing platforms have limited utility for studies of complex mixtures or of individual long molecules, the latter of which is crucial to understanding evolution and consequences of single nucleotide variants and their combinations. Here we report a new technology termed LUCS (Long-molecule UMI-driven Consensus Sequencing), in which reads from third-generation sequencing are aggregated by unique molecular identifiers (UMIs) specific for each individual DNA molecule. This enables in-silico reconstruction of highly accurate consensus reads of each DNA molecule independent of other molecules in the sample. Additionally, use of two UMIs enables detection of artificial recombinants (chimeras). As proof of concept, we show that application of LUCS to assessment of mitochondrial genomes in complex mixtures from single cells was associated with an error rate of 1X10-4 errors/nucleotide. Thus, LUCS represents a major step forward in DNA sequencing that offers high-throughput capacity and high-accuracy reads in studies of long DNA templates and nucleotide variants in heterogenous samples.

Whether mtDNA replication occurs via strand‐symmetric or strand‐asymmetric mechanism has been a matter of controversy. The asymmetric model predicts special properties for the two origins of replication of mtDNA. In particular, it predicts characteristic discontinuity of mutational pressure,(high pressure 5’ and low pressure 3’ to each of the two origins), especially on guanine nucleotides We used the “sliding subtractive window”, to test the presence of the origins: for each position of the genome we subtracted the G‐density 5’ to that position from the 3’ G‐density, using adjacent sliding windows of variable length. This “sliding subtractive window” creates a characteristic profile as it moves along the genome, which is expected to peak around the origins of replication if the mode of replication is mostly asymmetric and origin‐dependent. Indeed, we found the expected pattern with peaks in both origins, thus corroborating the classical asymmetric mode of mtDNA replication. We found that this pattern was highly conserved as far as among the entire chordata (Fleischmann et al., in preparation).In this study we asked whether this conservation is related to acute continuous mutational pressure (asymmetric replication is currently in use) or perhaps the pattern we observe is an ancient one, that's been created hundreds of millions years ago and was resistant to dissipation by random mutagenesis.To test this hypothesis, we performed numerical simulations where the actual nucleotide differences between human mtDNA and various chordata species' genomes were introduced into human mtDNA at randomized positions to create “randomized species”. We measured whether the distances between the “subtractive window” profiles of the randomized species and human mtDNA were larger than those between real species and human mtDNA. The significance of the difference in distance was estimated by counting the proportion of simulated randomized species which were further from human mtDNA than the corresponding real species.We demonstrate that the distances between subtractive window profiles of randomized species and human mtDNA are significantly larger than that from real species, i.e. chimpanzee. This implies that mutations are constrained is such a way that they change the subtractive window profile much less than random mutations would, and, because the profile reflects the mode of mtDNA replication, this confirms that keeping the profile stable is favored either by selection or by asymmetric mutational pressure. In either case this implies that the light origin of replication are currently of biological significance.There is a possibility though that the role of exceptional profile with discontinuity precisely at light strand origin of replication has some other function that it acquired over millions of years of evolution, however, this should have happened independently in several highly divergent groups like mammals and lampreys, which is unlikely. Furthermore, it is possible that other parts of the profile drive the similarity rather the origin. We will repeat the simulation concentrating on progressively narrower area around the origin to determine which part of the profile caries most importance for it invariability.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AbstractMutational spectrum of the mitochondrial genome (mtDNA) does not resemble signatures of any known mutagens and variation in mtDNA mutational spectra between different tissues and organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra may be affected by the oxidative damage which is increasing with cellular and organismal aging. However, the well-documented mutational signature of the oxidative damage, G>T substitutions, is typical only for the nuclear genome while it is extremely rare and age-independent in mtDNA. Thus it is still unclear if there is a mitochondria - specific mutational signature of the oxidative damage. Here, reconstructing mtDNA mutational spectra for human cancers originated from 21 tissues with various cell turnover rate, human oocytes fertilized at different ages, and 424 mammalian species with variable generation length which is a proxy for oocyte age, we observed that the frequency of AH>GHsubstitutions (H- heavy chain notation) is positively correlated with cellular and organismal longevity. Moreover, this mutational bias from AHto GHaffects nucleotide content at the fourfold degenerative synonymous positions leading to a deficit of AHand excess of GH, which is especially pronounced in long-lived mammals. Taking into account additionally, that AH>GHis sensitive to time being single stranded during mtDNA asynchronous replication and A>G is associated with oxidative damage of single-stranded DNA in recent bacterial experiments we propose that AH>GHis a mutational signature of oxidative damage in mtDNA.

A recent report (1) presents the long-awaited confirmation of paternal inheritance of mtDNA in humans (2). Surprisingly, paternal transmission of mtDNA (1) follows a bimodal pattern: About half of the offspring show fairly uniform paternal/maternal heteroplasmy levels, while the rest do not inherit paternal mtDNA at all. This pattern resembles the inheritance of a dominant nuclear gene. The authors explain this pattern as permissive inheritance resulting from a faulty “gatekeeper” gene (1). However, 3 groups (3⇓–5) instead suspect contamination with mtDNA nuclear pseudogenes (NUMTs), a notorious artifact (6). Based on our vast NUMT experience, we support the authors’ response (7), asserting that NUMT artifact is unlikely (further explanation is given in Supporting Notes [SN] sections SN1 and SN2, see ref. 8). However, we also demonstrate that the authors’ dominant gatekeeper explanation is incorrect, because spermatozoa are functionally diploid (SN3) … [↵][1]2To whom correspondence may be addressed. Email: p.j.i.ellis{at}kent.ac.uk or kkhrapko{at}gmail.com. [1]: #xref-corresp-1-1

Mitochondria are well-characterized regarding their function in both energy production and regulation of cell death; however, the heterogeneity that exists within mitochondrial populations is poorly understood. Typically analyzed as pooled samples comprised of millions of individual mitochondria, there is little information regarding potentially different functionality across subpopulations of mitochondria. Herein we present a new methodology to analyze mitochondria as individual components of a complex and heterogeneous network, using a nanoscale and multi-parametric flow cytometry-based platform. We validate the platform using multiple downstream assays, including electron microscopy, ATP generation, quantitative mass-spectrometry proteomic profiling, and mtDNA analysis at the level of single organelles. These strategies allow robust analysis and isolation of mitochondrial subpopulations to more broadly elucidate the underlying complexities of mitochondria as these organelles function collectively within a cell.

The mtDNA 'mutator' mouse, also called the 'POLG' mouse, is a well-characterized model frequently used for studies of progeroid aging. Harboring a mutation in the proofreading domain of the mitochondrial polymerase, polymerase-γ (Polg), POLG mice acquire mtDNA mutations at an accelerated rate. This results in premature mitochondrial dysfunction and a systemic aging phenotype. Previous work has demonstrated that the progeroid phenotype in POLG is attenuated following endurance exercise, the only reported intervention to extend health span and lifespan of these mice. Herein, oocyte quality was evaluated in sedentary and exercised POLG mice. In mice homozygous for the Polg mutation, litter size is dramatically reduced as compared to heterozygous Polg mice. Following ovarian hyper-stimulation, oocytes were retrieved until 9 months of age in exercised and sedentary groups, with no oocytes ovulated thereafter. Although ovulated oocyte numbers were not impacted by exercise, we did find a modest improvement in both the ovarian follicle reserve and in oocyte quality based on meiotic spindle assembly, chromosomal segregation and mitochondrial distribution at 7 months of age in exercised POLG mice as compared to sedentary counterparts. Of note, analysis of mtDNA mutational load revealed no differences between exercised and sedentary groups. Collectively, these data indicate that exercise differentially influences somatic tissues of the POLG mouse as compared to oocytes, highlighting important mechanistic differences between mitochondrial regulatory mechanisms in the soma and the germline.

The hypothesis that the evolution of humans involved hybridization between diverged species has been actively debated in recent years. We present novel evidence in support of this hypothesis: the analysis of nuclear pseudogenes of mtDNA (“NUMTs”). NUMTs are considered “mtDNA fossils”, as they preserve sequences of ancient mtDNA and thus carry unique information about ancestral populations. Our comparison of a NUMT sequence shared by humans, chimpanzees, and gorillas with their mtDNAs implies that, ca. 6 Ma, our evolutionary history involved the interbreeding of individuals whose mtDNA had diverged as much as ~4.5 Myr prior. This large divergence suggests a distant interspecies hybridization. Additionally, analysis of two other NUMTs suggests that such events occurred repeatedly. Our findings suggest a complex pattern of speciation in primate human ancestors and provide a potential explanation for the mosaic nature of fossil morphology found at the emergence of the hominin lineage.

The data and methods presented in this article are supplementing the research article "Integration of mtDNA pseudogenes into the nuclear genome coincides with speciation of the human genus. A hypothesis", DOI: 10.1016/j.mito.2016.12.001 (Gunbin et al., 2017) [1]. Mitochondrial DNA is known to get inserted into nuclear DNA to form NUMTs, i.e. nuclear DNA pseudogenes of the mtDNA. We present here the sequences of selected NUMTs, in which time of integration can be determined with sufficient precision. We report their chromosomal positions , their position within the great ape mtDNA phylogeny, and their times of integration into the nuclear genome. The methods used to generate the data and to control their quality are also presented. The dataset is made publicly available to enable critical or extended analyzes.

IntroductionOocytes contain the whole mitochondrial genomes of mammals, and the inheritance pattern of mitochondrial DNA in oocytes do not follow Mendel's rules of heredity. Mitochondrial DNA is maternally inherited in the oocytes, and only mother's mitochondrial genome can be passed to the next generation. The mitochondrial genome is a closed, circular piece of DNA which has a length of about 16kb, and there are hundreds to hundreds of thousands mitochondrial DNA in a single cell. Mitochondrial DNA mutation rate is generally difficult to detect and is often over‐estimated. This research is focusing on developing a sequencing strategy to accurately get mitochondrial DNA mutation rate of single, long molecules from a wild type mouse germline.Research ObjectivesThe research goal is to develop an efficient and accurate way to assess the mutation rate of mitochondrial DNA from wild type mouse oocytes.MethodsWe isolated and individually lysed single oocytes from a wild type female mouse. A serial dilution of the DNA sample was made to find its working concentration for generating single, long molecules, and prepared a PCR reaction by using the specific DNA sample concentration. A high‐fidelity DNA polymerase was used to generate 16kb PCR products originating from single template molecules. Sanger sequencing was used to sequence these DNA molecules.ResultsWe used single molecule PCR to amplify the mitochondrial DNA from three wild type mouse oocytes and sequenced all the PCR products, yielding a total coverage about 330,392bp. Twenty‐two mitochondrial genomes from three oocytes were sequenced, with an average length of about 15,017bp. Mouse mitochondrial DNA length is 16,299kb, so each of our mitochondrial DNA molecule has an average coverage about 92.13% of the whole mitochondrial DNA genome. Only two mutations were found in two separate mitochondrial genomes, and the resulting mutation rate of mitochondrial DNA in the oocytes was 6.05×10^‐6. This is a very low mutation rate across over 300,000bp.ConclusionThe mutation rate of mitochondrial DNA in wild type mouse germline is remarkable low in our research by using single, long molecule PCR. This PCR method allows for the accurate detection of mutations in the mouse mitochondrial DNA in oocytes. Our data indicates that, by using single, long molecule PCR combined with Sanger sequencing, we can accurately get informations from a molecule that has the most coverage of its genome. Additionally, we used a high‐fidelity enzyme to serve as DNA polymerase in the PCR reaction, thus preventing the chance of artificial mutations from the beginning of the PCR reaction. With the right procedure of our sequencing strategy, the rare mutations and the mutation rate in single germline cells can be accurately determined.Support or Funding InformationEllison Medical Foundation; NIH R37‐AG012279