Evolution in Reverse: Demystifying my Epilepsy
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If we are to have a complete understanding of origin and biological importance of mtDNA genetic diversity, we need to understand the role that selection plays in shaping mtDNA genetic diversity. To date, only a few mouse models have been generated and studied that provide insight into the importance of selection in sorting out mtDNA heteroplasmic mtDNA mutations. In one mouse model system, an mtDNA deletion was introduced into the mouse female germline via somatic cell cytoplasmic hybrid cybrid fusions Bunn et al.
The resulting synaptosome cybrids were screened for mtDNA mutations and one clone was identified that harbored a nt mtDNA deletion that removed six tRNAs and seven structural genes. Next, enucleated cell cytoplasts from this mtDNA nt deletion cell line were fused to single-cell mouse embryos, the embryos implanted into foster mothers, and the resulting mice found to be heteroplasmic for the deleted mtDNA.
Unlike human mtDNA deletion mutations, which are not generally maternally inherited, this deletion was transmitted through the female germline Inoue et al. In humans, it has been observed that mtDNA duplications can be maternally inherited and that these duplicated molecules generate deletions by intramolecular recombination within postmitotic tissues Wallace and Fan Hence, it is possible that the maternal transmission of the nt mtDNA deletion is the product of maternal transmission of a duplication, although this has not been determined.
Regardless of the molecular nature of the maternally transmitted rearranged mtDNA, analysis of the level of the deleted mtDNA in pups derived from heteroplasmic female mice revealed a striking directional loss of the rearranged mtDNA in successive litters. Interestingly, in two of the females, the mean mtDNA heteroplasmy of the pups of the first litter was higher than that of the tail of the mother.
The same striking decline in the percentage of deleted mtDNA was seen in the oocytes of two females that were superovulated at d 44 and In both cases, the oocyte heteroplasmy level in the first superovulation oocytes was less than that of the females and the reduction of heteroplasmy in the oocytes in the subsequent superovulation was even more marked. Apparently, then, oocytes with high mtDNA deletion levels can be ovulated while the mouse is young, but, as the female mouse ages, the high deletion oocytes are progressively lost.
Hence, there must be selection against the formation or ovulation of the oocytes with highest deletion levels throughout the female mouse's lifespan. When this cell line was enucleated and fused to a female mouse embryonic stem cell mfESC , one of the cybrid clones was found to harbor mtDNAs that had reversed the ND6 insC mutation by deletion of an adjacent T, thus restoring the reading frame.
Hence, there was a directional and concerted loss of the frameshift mtDNA over successive generations. Furthermore, the distribution of oocyte heteroplasmy levels was not Gaussian but was truncated such that there were oocytes with substantially less mutant mtDNA than the mother's tail genotype, but none had significantly more mutant mtDNAs than the mother.
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Therefore, germ cells or oocytes with a higher percentage of frameshift mtDNAs must have been selectively removed prior to ovulation. Thus, mammalian females have a prefertilization mtDNA selection that eliminates those oocytes harboring the most deleterious mtDNA mutations. This revealed that while deleterious tRNA mutations were introduced into the germline, there was a dearth of deleterious polypeptide gene mutations Stewart et al. The inheritance of the mtDNA harboring this mutation was monitored and found to be biased toward loss of the tRNA mutation. Mice harboring the tRNA Met delC mutation had relatively similar heteroplasmy levels across tissues and these did not change with age.
Hence, selection is clearly acting against this tRNA mutation at high levels of heteroplasmy. The mtDNA genotypes of 44 mothers and their offspring were analyzed. This conclusion was based on comparison of the offspring distribution of genotypes observed versus prediction made from the Kimura distribution that would be expected for a random genetic drift model.
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In contrast to the ND6 insC frameshift mutation and the nt deletion mtDNA mice in which the heteroplasmy levels of the pups of successive litters declined with age, the heteroplasmy distribution of the tRNA Met nt delC mutation mtDNAs did not significantly differ among generations Freyer et al. Thus, germline cells could be homoplasmic for the mutant even though no homoplasmic animals were generated. This indicated that early in female germ cell development there was a stochastic distribution of heteroplasmic levels, but that the highest levels of mutant mtDNAs were lost as the oogonia or oocytes matured.
Hence, the tRNA Met delC mutation levels seem to be maintained in the female germline by two opposing selective forces, one enriching for the mutant mtDNA at intermediate heteroplasmy levels and the other strongly selecting against the mutant at high levels of heteroplasmy. The enrichment of the mtDNAs was seen in the ovaries of the heteroplasmic females and to a lesser extent in the oocytes of the heteroplasmic females. This series of experiments unequivocally demonstrates that selection acts on mtDNA genotypes within the female germline, acting to weed out the most severe mtDNA mutations.
Furthermore, the bias toward the elimination of a deleterious mtDNA is correlated with the severity of the mitochondrial defect associated with the mtDNA mutation. The striking inconsistencies observed among different estimates of the mtDNA copy numbers in mouse female germline cells, the variation in estimates of heteroplasmy level variances in progeny cells and offspring of different studies and model systems, and the role of selection in defining the transmission of heteroplasmic mtDNA genotypes mean that it is currently impossible to define the factors that determine the origin and inheritance of pathogenic mtDNA mutations.
In an effort to more accurately compare different experiments and ultimately to better understand mtDNA mutant transmission, significant efforts have been made to model the transmission of heteroplasmic mtDNAs along the female germline. Unfortunately, three factors have limited the investigator's ability to generalize the results from different studies: 1 development of methods for comparing differences in heteroplasmy variance among samples and experiments and to calculate the statistical significance of these differences, 2 development of appropriate mathematical models to describe the origin and transmission of mtDNA heteroplasmy, and 3 development of theory and methods to determine the role of selection in mtDNA heteroplasmy transmission.
A major limitation for defining the principles covering heteroplasmy transmission has been difficulty in comparing the heteroplasmy variance levels observed across different experimental systems. Difficulties arise at two levels, correcting for the effects of parental allele frequencies on the potential range of heteroplasmy variances of their descendants and difficulties in calculating the standard error of heteroplasmy variance estimates for testing the statistical significance of observed differences.
The heteroplasmy variance of offspring or derived cells is a function of the heteroplasmy level of the mothers for offspring or of the parental cells for derived cells, the parental heteroplasmy designated as p 0. Under these conditions, the range of heteroplasmy values of the offspring or derived cells will be constrained by the frequency of the low parental allele, making the range of heteroplasmic variance levels aberrantly low. To correct for this systematic distortion in variance capacity, Samuels and collaborators have used Mendelian population genetics theory Wright to normalize the heteroplasmy variances in proportion to p 0.
This was accomplished by dividing the observed heteroplasmy variance by the term p 0 1- p 0. This term is at maximum 0. Hence, dividing the observed variance by p 0 1- p 0 proportionately increases population variance estimates for low values of p 0. With this correction, Samuels and associates discovered that a number of conclusions about changes in heteroplasmy variance drawn from various studies involving animals with low maternal p 0 could not be substantiated Samuels et al.
Once the effects of extreme values of p 0 on variance estimates were corrected, it became necessary to calculate the standard error SE of the variance so that the statistical significance of differences in variance among different sample determinations could be calculated.
At low levels of p 0 , the required sample size to obtain statistical significance rapidly increases. Their simulations indicate that these variance estimates have very large SEs. When Wonnapinij and colleagues estimated the SE of the variance for the original NZB-BALB mtDNA segregation of Jenuth and collaborators , they concluded that once variance normalization was applied and variance SEs were calculated, the reported differences in heteroplasmy levels between primary and mature oocytes were not statistically significant. However, the heteroplasmy variance of the PGCs was significantly lower than that of primary and mature oocytes.
This analysis supports the hypothesis that an mtDNA bottleneck occurs during the transition of PGCs to primary oocytes. Similarly, Wonnapinij and colleagues reanalyzed the mouse mtDNA heteroplasmy variation of the Wai and associates study. Once the heteroplasmy variances were normalized and SE was estimated, Wonnapinij and colleagues concluded that the heteroplasmy variance values determined on postnatal d 11 and after relative to those of d 8 and earlier were not significantly different.
Therefore, the conclusion of Wai and associates that an mtDNA heteroplasmy bottleneck occurs during postnatal folliculogenesis could not be substantiated Wonnapinij et al. Wonnapinij and colleagues also pointed out that the mtDNA heteroplasmy variance levels in the early postnatal period differed between the Jenuth study Jenuth et al. From these analyses, Wonnapinij and colleagues concluded that the major bottleneck responsible for mtDNA heteroplasmy variance occurs in the maternal germline between the PGCs and the oocytes. Application of these analytical approaches to mouse versus human mtDNA heteroplasmic variance data revealed that human heteroplasmy variance was greater than mouse.
To determine whether mtDNA heteroplasmy variance has been affected by nonrandom factors such as selection, it was necessary to define the expectation for heteroplasmy variance if only stochastic factors were contributing to mtDNA segregation. The calculation of the expectations of random heteroplasmy transmission required a theoretical model.
Despite its simplicity, this approach ignores much of the information that is present in the total heteroplasmy distribution, especially when this is not symmetric. Additionally, a common assumption when comparing heteroplasmy distributions between cells or individuals is that the population follows a normal distribution. However, Wonnapinij and colleagues have argued that the normal distribution is not the optimal choice for representing the potential distribution of mtDNA heteroplasmic genotypes for two main reasons. First, the normal or Gaussian distribution is defined over a range of infinite minus and plus tails, yet genetic variation is constrained to allele frequencies p 0 between zero and one.
Second, the normal distribution is always symmetric, whereas mtDNA heteroplasmy distribution is often skewed. In the s, Kimura developed a model of the possible allele frequency distribution for a finite population resulting from the random sampling of gametes. His continuous model includes a set of probability distribution functions that describe gene frequency distributions of populations under pure random genetic drift. This model has three parameters: the initial gene frequency, p 0 ; the effective population size, N e ; and the number of generations, t.
In the case of mtDNA heteroplasmy, p 0 is interpreted as the founder's heteroplasmy proportion or as a surrogate of the parental allele frequency calculated as the mean heteroplasmy in the derived cells or offspring of the progenitor. N e is interpreted as a parameter related to the number of segregating mtDNA units. Wonnapinij and colleagues refer to parameter b as the bottleneck parameter, which then determines the width of the heteroplasmy distribution in the offspring Wonnapinij et al.
To determine the statistical significance of the observed differences in the fit of experimental data with the Kimura distribution, the WCS-K tool uses the Kolmogorov-Smirmov KS test Wonnapinij et al. Using the Kimura model, Wonnapinij and colleagues reassessed the variance estimates and conclusions of a number of previous experimental studies on heteroplasmic segregation. Of course, the determination of whether an mtDNA is present or absent depends on the sensitivity of the method used to detect the minor allele. Analysis of two datasets on heteroplasmic Drosophila eggs Solignac et al.
In one D. However, in this case the founder female harbored Therefore, although this striking directional shift could not have occurred by chance, the WCS-K tool was unable to detect deviation from randomness. Finally, in a Drosophila dataset derived from eggs 30 generations away from the founder, the results did not fit the expectations of the Kimura distribution, suggesting that factor s in addition to genetic drift had acted on this mtDNA segregation pattern Wonnapinij et al. Thus, by applying the WCS-K tool to heteroplasmy distributions, Wonnapinij and colleagues concluded that many were consistent with stochastic processes.
Still, even this tool showed that some cases deviated significantly from the expectations of randomness. In contrast to the seemingly modest role played by selection in determining transmission of heteroplasmy when analyzed using the WCS-K tool, the mouse experiments that analyzed the transmission of deleterious mutations including the mtDNA deletion Sato et al.
One set of potential limitations of using the Kimura distribution to model a stochastic distribution of mtDNA genotypes resides in the three parameters that relate to the neutralist basis of the distribution. These include: 1 a large population of N diploid parents, 2 no mutation, selection, or migration, and 3 no overlapping generations Kimura Studies of mammalian embryonic development suggest that the number of mitochondria or mtDNA molecules may be drastically reduced Jansen and de Boer ; Krakauer and Mira By implementing the KS nonparametric test, the WCS-K tool avoids assuming that the data were sampled from Gaussian distributions, but there are drawbacks to using a nonparametric test.
Additionally, the KS test is most sensitive when the distribution functions differ in a global fashion near the center of the distribution. But if there are repeated deviations between the distribution functions or the distribution functions have or are adjusted to have the same mean values, then the distribution functions cross each other multiple times and the maximum deviation between the distributions is reduced Babu and Feigelson Because the predictive ability of the WCS-K tool is based on the assumption of random genetic drift in a finite large population, the strength of the predictions of the WCS-K tool is strongly influenced by sample size.
This makes it possible to change the statistical significance of a comparison simply by increasing the range of mtDNA heteroplasmy genotypes binned together.
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This limitation is compounded because the currently available program can only analyze transmissions through one generation, thus limiting detection of cumulative effects of selection. Moreover, the effects of population size fluctuations on selection are also not considered and therefore not modeled Wonnapinij et al. While these technical considerations are pertinent, perhaps a more important reason why the WCS-K tool is relatively insensitive in identifying the effects of selection on mtDNA mutations is that the Kimura model was developed for changes in the allele frequency of Mendelian genes within finite populations.
The Mendelian mode of inheritance was the only genetic model known in the s when Kimura developed his model. However, the Mendelian rules of inheritance derive from the behavior of chromosomal genes and the biological unit upon which selection acts on Mendelian genes is the diploid organism. This unit of selection is fundamentally different from the situation with the mtDNA in which selection acts on cells and organisms with several thousand-fold ploidy.
This does not adversely affect the mathematics of allelic transmission if all alleles are neutral. However, it has a profound effect on whether the Kimura model can detect the effects of purifying selection during the transmission of a deleterious heteroplasmy mtDNA mutation. For mtDNA genetics, selection acts on the cellular phenotype, which is a composite of the several thousand mtDNAs with different percentages of two or more allele systems.
Because cytokinesis divides the cell in half, low-frequency mtDNA alleles are much less likely to be lost in successive generations than nDNA alleles. Finally, the mitochondria and mtDNAs within the cell are not autonomous units. Rather, they function as a collective population through repeated fission and fusion and sharing of all their gene products. Also, low-frequency deleterious mtDNAs can be retained in a cell lineage for many generations without adversely affecting the cellular phenotype. An ND5 gene frameshift mutation, which was found to be homoplasmic in a human oncocytoma tumor, was present at low-heteroplasmy levels in the normal tissues of the patient and his two sisters.
Thus, this deleterious mutation was silently transmitted through the maternal lineage Gasparre et al. Hence, classical concepts of genotype—phenotype associations and their interaction with selection are violated by mtDNA genetics. In conclusion, the formulations of Wonnapinij and associates Wonnapinij et al. Heteroplasmic mtDNAs also segregate in somatic cells and tissues and this greatly complicates phenotypic predictions in patients.
Although it is standard practice to test for mutant nDNA genes in patient blood, the mtDNA heteroplasmy level in blood cells can be quite different from that of brain, heart, muscle, or kidney, the organs most susceptible to life-threatening mitochondrial disease. The first reports of directional segregation of mtDNA heteroplasmy in mouse and human somatic cells appeared in the s.
The directionality of mtDNA segregation was even more extreme when cells of different species were fused. This is the opposite of the well-established mouse—human hybrid segregation pattern Wallace et al. When clinically relevant mtDNA mutations became available, these mutants were also introduced into cultured cells. Shortly after it was discovered that mtDNA deletions accumulate in muscle of patients with mitochondrial myopathy Holt et al.
Moreover, mtDNA deletions in humans were rarely transmitted through the female germline Shoffner et al. The absence of mtDNA deletions in blood cells when they are prevalent in postmitotic tissues led to the hypothesis that severely deleterious mtDNA mutations might be inhibiting bone marrow stem cell replication.
Those stem cells that spontaneously segregated the deleted mtDNA would then have a proliferative advantage and repopulate the bone marrow. For the symptomatic subjects the heteroplasmy level declined 0. At the same time, the symptomatic individual's phenotypes became worse while the asymptomatic individuals remained asymptomatic Mehrazin et al. Hence, different mtDNA mutations show different tissue-specific stabilities indicating that a variety of physiological processes must modulate the fate of heteroplasmic variants in tissues.
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The original Jenuth et al. The heteroplasmy of the tail, cerebral cortex, gastrocnemius muscle, heart ventricle, and lung were reported to remain relatively stable Jenuth et al. In addition, it was found that seminal vesicles, ovaries, and pancreas also segregated toward mtDNAs. However, tail, brain, lung, and heart maintained relatively constant heteroplasmy Sharpley et al. Hence, striking differences exist between germline and somatic cell mtDNA heteroplasmy segregation.
Because the somatic tissue cells contain thousands of mtDNAs, this segregation is unlikely to be caused by a bottleneck involving a dramatic reduction in mtDNA copy number. Rather, this must involve a process by which either some mtDNAs are selectively replicated or eliminated or certain mitochondria are differentially propagated. This discovery provided the opportunity to map genetic loci involved in the mtDNA sorting process. Therefore, the authors surmised that direction and mechanism of segregation was not the result of differential mitochondrial physiology or mtDNA replication rate and thus might depend on mitochondrial maintenance or mitochondrial turnover Battersby and Shoubridge Absence of these genes should impair the presentation and recognition of mtDNA-coded peptides.
However, the kinetics of selection for the BALB mtDNA was unaltered indicating that segregation was not the result of presentation of mitochondrially encoded peptides by the major histocompatibility locus MHC Battersby et al. This results in the first AUG being used resulting in the addition of 58 amino acids to the amino terminus of the protein without affecting the carboxy-terminal transmembrane domain. It has been suggested that in hematopoietic tissues the selection is age-dependent and proportional to the initial heteroplasmy level.
This implies that selection occurs at the organelle level. Because Gimap3 is a mitochondrial outer membrane protein, it might participate in mitochondrial partitioning or sorting. Gimap3 is part of a vertebrate-specific gene family and the protein is anchored to the mitochondrial outer membrane by its carboxyl terminus. It has been reported to be important in T-cell survival and development. The discovery that Gimap3 regulates mtDNA segregation in hematopoietic cells does not seem to be the basis for the enrichment of the NZB mtDNAs in either liver or kidney, each of which is modulated by a different genetic locus.
This same variant has been found to be enriched in heteroplasmic cultured mouse L cells Fan et al. However, the similarity in respiration rate proved to be the result of a compensatory 1. When the increased ROS production was neutralized by antioxidants, the mtDNA copy number declined and mitochondrial respiratory chain function was reduced.
Because an alteration in a mitochondrial tRNA would be expected to inhibit mitochondrial protein synthesis, this could impede the synthesis of mitochondrial OXPHOS enzymes, inhibit electron transport, and result in increased ROS production. Assuming that the female germline cells have mitochondria with a limited number of mtDNAs and fusion and fission are inhibited, then each mitochondrion would act as a semi-autonomous unit.
In contrast to germline cells, somatic tissue cells and cultured cells have mitochondria that contain multiple nucleoids and mitochondria that actively fuse within a cell mixing their contents and gene products. In this case, the mitochondrial polysomes of a heteroplasmic somatic cell can translate both mtDNAs and mRNAs and can complement each other in trans. In heteroplasmic cells in which the mtDNA translation products were differentially labeled by growth in 35 S-methionine in the presence of cytosolic ribosome inhibitor, emetine, both MVI and MVII were equally labeled.
The high glucose would fully reduce the electron transport chain of the cells resulting in increased ROS production regardless of the haplotype combination. If mitochondrial ROS production is important in the replicative segregation of NZB versus BALB or mtDNA heteroplasmy animals, then one might anticipate that antioxidant defenses might also be relevant in regulating their segregation.
NADPH is also essential for the regulation of multiple enzymes and transcription factors via oxidation-reduction of thiol-disulfides Wallace Other mitochondrial quality assurance mechanisms such as mitochondrial dynamics and mitochondrial autophagy mitophagy might also be important in mtDNA heteroplasmy segregation Youle and van der Bliek ; Jokinen and Battersby The mitochondria in somatic tissues and cultured cells are highly dynamic undergoing a frequent fusion and fission cycle.
Perturbations of mitochondrial fission and fusion have been shown to affect the segregation of heteroplasmic mtDNAs. Mitochondrial fission is initiated by Drp1, a member of the dynamin protein family. Mitochondrial fusion is initiated by the Mfn1 and Mfn2 proteins that mediate the fusion of the outer mitochondrial membranes and OpaI, which mediates the fusion of the mitochondrial inner membranes Youle and van der Bliek Hence, in this replicating cell line, increased mitochondrial fusion was permissive for retention of mutant mtDNAs Malena et al. In mice in which the mitochondrial fusion genes, Mfn1 and Mfn2 , were knocked down in skeletal muscle via floxed Mfn1 and Mfn2 genes and the muscle-specific MLC1f promoter-driven Cre, profound mtDNA depletion ensued.
Mitochodnrial fission and fusion permit mtDNA mutant complementation within cells. The mtDNAs are contained in nucleoids and the distribution of mtDNAs within nucleoids and the partitioning of nucleoids into daughter mitochondria could be important in heteroplasmy segregation. The homoplasmic mtDNA deletion cells each had fragmented mitochondria and were deficient in mitochondrial protein synthesis and respiration.
However, in the hybrids containing the two deleted mtDNAs the mitochondrial were elongated and functional in both protein synthesis and respiration. By using hybridization probes that were internal to the two deletions and differentially fluorescently labeled, the presence of the two deleted mtDNAs were monitored.
This revealed that the respiring hybrids harbored both mtDNAs in elongated mitochondria and most of the nucleoids hybridized to either one or the other probe, though in some cases the two hybridization probes seemed to overlap. This indicated that nucleoids do not exchange mtDNAs, and that the instances where the deleted mtDNAs colocalized in the hybrids was the result of two nucleoids being adjacent to each other and thus not resolved by light microscopy Gilkerson and Schon ; Gilkerson et al.
By fragmenting the mitochondria, sorting the pico-green-stained mitochondria in a cell sorter, and performing differential PCR amplification for the deleted and normal mtDNA, it was again concluded that each nucleoid contained only one type of mtDNA Poe et al. Therefore, the current evidence favors the conclusion that individual nucleoids harbor only one type of mtDNA. The mtDNAs continually replicate within somatic cells including postmitotic cells, with the excess mtDNAs being removed by mitophagy.
Thus, mitophagy provides another mechanism by which heteroplasmic mtDNA mutations can be segregated. Mitophagy is preceded by mitochondrial fission, which is thought to be asymmetric such that one daughter mitochondrion is more energetically competent than the other. Mitophagy is then envisioned to preferentially degrade the more respiration-compromised mitochondrion. However, if the mitochondrial membrane potential is significantly reduced, PINK1 becomes stabilized in the mitochondrial outer membrane where it phosphorylates serines on target proteins. PINK1 phosphorylation results in the attraction of the cytosolic protein Parkin to the mitochondrial outer membrane.
Parkin is an E3 ubiquitin ligase that ubiquitinates mitochondrial outer membrane target proteins Narendra et al. Recent evidence has implied that Mfn1, Mfn2, and Miro1 a protein involved in mitochondrial transport along microtubules are important Parkin ubiquitination targets.
The relevance of mitophagy to mtDNA heteroplasmy segregation was first demonstrated in Parkin overexpression experiments. Partial inhibition of mitochondrial fusion with vMIA and of the electron transport chain with azide to reduce mitochondrial electron transport, increased the Parkin bound to the mitochondria. Hence, mitophagy is capable of modulating mtDNA heteroplasmy. Because of the multiplicity of cellular processes that can impinge on mtDNA heteroplasmy, it is not surprising that different mtDNA mutations show different directionality and kinetics of segregation in different tissues.
The absolute nature of maternal inheritance of the mtDNA creates an intractable dilemma for women that harbor high levels of a deleterious mtDNA mutation. They have a high probability that most if not all of their children will develop devastating diseases. Homoplasmic mtDNA mutations such as those commonly associated with LHON will be transmitted to all of a woman's offspring, though fortunately the penetrance of these milder mtDNA disease mutations is incomplete.
For more deleterious mtDNA mutations, the outcome can be much worse. Five of the conceptions were lost prenatally. One published example of a germline lethal mtDNA mutation. The two surviving term infants died within 2 to 3 d. The mother's twin sister had a similar reproductive history, yet both women were seemingly normal. From Yoon et al. Unfortunately, prenatal diagnostic procedures that have been effective in identifying fetuses with deleterious chromosomal gene mutations have proven to be relatively unreliable for diagnosing mtDNA diseases.
These difficulties arise from the exclusively maternal inheritance of the mtDNA and the unpredictable nature of heteroplasmic mtDNA mutation segregation. Obviously, all forms of prenatal diagnosis are useless for a woman harboring a homoplasmic mtDNA mutation because all of her fetuses will inherit her mutation. By contrast, heteroplasmic mtDNA mutations might be amenable to prenatal diagnosis if the woman was able to conceive a fetus with low levels of heteroplasmy.
However, this can also be problematic because it is not certain that the fetal brain, heart, muscle, or kidney will have the same level of mtDNA mutation as found in the chorionic villus or amniocentesis sample. Finally, some women might conceive multiple times and in every case the conceptus will have an unacceptably high level of mutant mtDNAs resulting in repeated fetal loss Thorburn and Dahl ; Poulton and Bredenoord The ambiguities associated with chorionic villus sampling and amniocentesis diagnosis of mtDNA disease have led to investigation of the potential value of preimplantation genetic diagnosis PGD.
By determining the mtDNA heteroplasmic genotype of oocytes or embryos before fertilization or implantation, only those embryos with a desirable mtDNA genotype could be retained for implantation into the mother. Hence, many potential embryos could be screened with only the rare embryo with the optimal genotype permitted to go through gestation.
It would be particularly attractive if it were possible to remove the first polar body from an oocyte and determine its mtDNA genotype. Only those oocytes with the lowest polar body heteroplasmy level would then be utilized. Another possibility could be to fertilize the oocytes in vitro and then collect and genotype blastomeres or trophoblast cells to identify the low-heteroplasmy embryos.
In these studies, it was found that the mtDNA heteroplasmy level of the first polar body was within 0. Analysis of the second polar body following fertilization revealed that the polar body mtDNA genotype correlated with the percentage of deletion in the embryo with a coefficient of 0. Because human mtDNA deletions have not been observed to be transmitted through the maternal lineage, the human female germ cells must have a lower tolerance for mitochondrial respiratory deficiency than the mouse Wallace et al. Based on the mouse polar body studies, sampling the mtDNA heteroplasmy levels of human meiosis I polar bodies would appear to be the most promising approach for PGD of heteroplasmic mtDNA diseases.
Unfortunately, when human studies were conducted it was found that the mtDNA genotype of the first polar body might be very different from that of the embryo. However, for half of the embryos the polar body genotype was significantly different from that of the embryo. In cases where the embryo's heteroplasmy levels were very high, the polar body genotype was significantly lower. Hence, it was concluded that in humans, polar body biopsy would not provide a reliable preimplantation test for human mtDNA diseases Gigarel et al.
The second possibility could be sampling one of the blastomeres of an early cleavage stage embryo. Studies of rhesus macaque oocytes having a heteroplasmy generated by karyoplast—cytoplast fusion, the heteroplasmy levels among primate blastomeres might be very different. In quantifying the divergence in percentage heteroplasmy, the coefficient of variance increased from Furthermore, the variance in heteroplasmy level among oocytes, blastomeres, and offspring was sufficiently great as to render absolute predictions on the transmission of heteroplasmy among cells and across generations unreliable Lee et al.
Fetal extraembryonic or embryonic tissues of 12 fetuses from seven carrier mothers were also found to be relatively consistent from gestational d to term. Thus, while there is marked variability in different embryos from the same woman, the tissues within an embryo can be relatively uniform even though the individual cells within a tissue can vary considerably Monnot et al.
Initial studies have suggested that preimplantation embryo blastomeres have relatively similar mtDNA heteroplasmy levels, in contrast to the macaque report. Comparison of blastomere genotypes from five cleavage-stage embryos with mean heteroplasmy levels of Twenty percent of the embryos lacked the mtDNA mutation. In contrast to the similarity of heteroplasmy levels of blastomeres within an embryo, the heteroplasmy levels among embryos varied considerably. Interestingly, the heteroplasmy levels of individual lymphocytes from a subject with a The woman then underwent two rounds of in vitro fertilization in association with PGD.
Fifty-nine cells from 10 embryos were analyzed and the mutation load found to be consistent within the blastomeres of the individual embryos. Therefore, in contrast to the macaque studies, the heteroplasmy levels of different blastomeres of human embryos appear to have relatively similar mtDNA heteroplasmy levels.
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Hence, blastomere biopsy and mtDNA heteroplasmy analysis from eight-cell embryos may provide a reasonable estimate of the heteroplasmy levels in the remaining blastomeres and reflect the heteroplasmy levels in the ensuing pregnancy amniocytes and cord blood samples Poulton and Bredenoord While mtDNA analysis of blastomere biopsies appears to be a promising approach for determining the heteroplasmy level of preimplantation embryos, blastomere biopsy removes a significant proportion of the embryo.
An alternative could be to biopsy extraembryonic cells in the trophectoderm of the blastocyst. Three to four trophectoderm cells were obtained and compared to one to three inner cell mass samples. The standard deviations of the independent samples from the various trophoblasts ranged from 2. Hence, there are still important ambiguities in the results on the effectiveness of human PGD in identifying and avoiding heteroplasmic mtDNA disease mutations. One recently recognized variable that might be relevant is that different pathogenic mtDNA mutations behave differently in both tissues and preimplantation embryos.
How such mutation-specific effects have affected the reliability of PGD remains to be determined. First, a woman may not produce oocytes that have low levels of mutant mtDNA, and, without the appropriate oocyte, her in vitro fertilization regime could be unproductive. Third, PGD is useless for homoplasmic women. Because of these limitations, there is increasing interest in developing methods to simply replace the mutant mtDNAs in preimplantation embryos by pronuclear or spindle transfer.
One early attempt at altering the mtDNA genotype in human oocytes was ooplasmic transfer. This technique was first used in an attempt to rejuvenate the oocytes of older women with the cytoplasm from the oocytes of younger women thus increasing their fertility. Multiple children have been born following application of this procedure and are alive today. Some of these children have been shown to be heteroplasmic for both the maternal oocyte mtDNA and the cytoplasmic donor mtDNA Barritt et al. Given that it has been reported that the mixing of two different mouse mtDNAs within the same female germline can lead to offspring with neuro-psychiatric defects Sharpley et al.
Rather than the transfer of mitochondria and mtDNAs, a more promising approach would be to isolate the nucleus from the oocyte or zygote of a woman harboring a deleterious mtDNA mutation and to transfer it into an enucleated oocyte or zygote from a woman with normal mtDNAs Wallace As a model for pronuclear transfer, in mouse embryos for the heteroplasmic mtDNA nt deletion, the zygote nuclei were removed by micropipette from the mtDNA mutant zygotes following disaggregation of the cytoskeleton and the resulting karyoplast plasma membrane bound pronuclei fused to an enucleated oocyte by electroshock.
Hence, zygote nuclear transfer resulted in a significant reduction in the transmission of the deleted mtDNAs. Pronuclear transfer technology has also been extended to human preimplantation embryos. Using uni-pronuclear or tri-pronuclear human embryos that otherwise would have been destroyed, the cytoskeleton was disrupted with cytochalasin B and nocodazole, the pronuclei pinched off into a karyoplast, the karyoplast placed under the zona pellucida of an enucleated recipient zygote, and the two cells fused together with inactivated viral envelope proteins.
Variation in the percentage of mutant mtDNAs in the different blastomeres within one eight-cell embryo was found to range from 5. Further refinement of the karyoplast isolation technique resulted in nine embryos with an average carryover of karyoplast mtDNAs of 1. Four of these embryos lacked detectable karyoplast mtDNA.
Of those embryos that harbored karyoplast mtDNA the maximum donor mtDNA was found in a nine-cell embryo in which seven blastomeres lacked detectable donor mtDNAs, while the remaining two blastomeres harbored 6. Interestingly, the spread in blastomere heteroplasmy levels in the first round pronuclear transfer embryos Craven et al. One possible explanation for this difference is that the oocyte cytoplasm may be relatively viscous resulting in the nonrandom partitioning of the mitochondria in different regions of the single-cell embryo.
Depending on the angles of the initial cleavages, the mtDNAs of the karyoplast and the cytoplast could be asymmetrically distributed into daughter blastomeres resulting in differing heteroplasmy levels. By contrast, oocytes and embryos derived from heteroplasmic germline cells of the two mtDNAs could be more evenly distributed throughout the cytoplasm such that cleavages would generate blastomeres with similar mtDNA heteroplasmy levels.
One concern with the pronuclear transfer procedure is that it destroys an embryo. This concern has been overcome by transfer of the chromosomes from the prefertilization mtDNA mutant oocyte to an enucleated oocyte with normal mtDNA. In this case, however, the chromosomes of the mature oocyte are arrested in meiotic metaphase II. Hence, this method involves the physical transfer of the meiotic II spindle. In the spindle transfer technique, the metaphase II MII spindle of the oocyte is visualized in the oocytes using the polarizing microscope and is removed by aspiration with a micropipette within a bleb of the ooycte cytoplasm and surrounded by the cell membrane.
Because the spindle is relatively free of surrounding cytoplasm and mitochondria very little mtDNA is transferred. The karyoplast is treated with a Sendai virus-derived fusigen and placed in the perivitelline space of the cytoplast opposite the first polar body where it fuses.
The resulting reconstituted oocyte can then be fertilized by intracytoplasmic sperm injection ICSI. This technology was first developed and applied to the oocytes of rhesus macaque monkeys. It resulted in preimplantation embryos that developed normally to generate pluripotent stem cells and when blastocysts of four- to eight-cell cleavage embryos were transferred into the reproductive tract of females, one pair of twins and two singleton infants were born.
These four monkeys had the maternal chromosomes of the spindle donor but the mtDNA of the cytoplast donor. Three years after birth, the four-spindle transfer monkeys were phenotypically like control monkeys and showed no significant change in mtDNA carryover in blood and skin samples Tachibana et al. Two female embryos generated from spindle transfer oocytes were permitted to develop in utero for d and then analyzed for mtDNA heteroplasmy.
One of the technological limitations of spindle transfer is that both the spindle donor karyoplast and the mtDNA donor cytoplast must be ready at the same time, requiring paired induction of ovulation of two women, which can be quite difficult. Studies on macaque oocytes have shown that the spindle donor oocyte can be frozen using a vitrification procedure. These oocytes can then be thawed, the spindle removed, and transferred to a fresh enucleated oocyte without diminution of fertilization. Vitrification of the cytoplasm donor oocytes, by contrast, blocked development Tachibana et al.
Hence, spindle transfer has the potential of eliminating most of the mutant mtDNAs from the maternal lineage, but low levels of heteroplasmy can still resurface during germline transmission. To assess the feasibility of moving this technology into the clinic, spindle transplantation has been applied to human oocytes.
The remainders were abnormal. The mean karyoplast derived mtDNA levels were very low, 0. In an independent study using parthenogenetically activated embryos that have undergone endoreduplication to establish a diploid karyotype, spindle transfer was also analyzed for its capacity to replace the mtDNA of the spindle donor mother. As before, the spindle was removed from the oocyte in a bleb of cytoplasm, the resulting karyoplast placed under the zona pellucida of a comparably enucleated oocyte, and the karyoplast fused to the cytoplast by either Sendai viral fusigen or electric shock.
Sendai fusion proved preferable because electroshock was prone to induce premature oocyte activation. Several embryos were permitted to develop into blastocysts and from these ESC lines with normal karyotypes were derived. These were capable of differentiating into pancreatic cells, neurons, fibroblasts, and cardiomyocytes.
Analysis of the mtDNA heteroplasmy level for all preimplantation embryos studied was 0. Fibroblasts derived from one of the spindle transfer stem cell lines were converted back to induced pluripotent stem iPS cells, yet the mtDNAs from the karyoplast did not reappear.
Finally, analysis of the mitochondrial respiratory complexes, respiration, and media acidification of one of the stem cell lines revealed that it was comparable to that of embryonic stem cells and iPS cells that had not undergone spindle transfer Paull et al. While both the pronuclear and spindle transfer techniques have been refined to transfer a minute amount of nuclear donor mtDNA, the possibility still remains that heteroplasmy can resurface in subsequent ESCs or maternal offspring Paull et al. While low-level heteroplasmy of a pathogenic mutation would probably be masked by the predominance of normal mtDNAs, minimizing the risk of a clinical phenotype, there is a risk of incompatibility between the two mtDNA haplogroup lineages Sharpley et al.
Then the mtDNA haplogroup of the woman harboring the deleterious mtDNA mutation could be matched with that of an oocyte donor; only matching mtDNA haplogroups would then be used. It has been argued that it may be unethical to manipulate human embryos in an effort to remove the risk of mtDNA disease. Certainly, all appropriate preclinical tests must be performed in an effort to reduce the risk for adverse outcomes in developing new human therapies.
Women harboring severely pathogenic mtDNA mutations want to have healthy children that are free of pain and can live productive lives. Currently children with high heteroplasmy levels of severely deleterious mtDNA mutations experience the progressive loss of mental and physical capabilities, often experiencing unremitting discomfort and pain and many will ultimately progress to premature death. For such families, this can mean repeated medical crises, numerous emergency room admissions, devastating medical bills, the loss of time, affection, and resources for other family members, ending only by the death of the child.
The sense of life, of self awareness, increased nearly tenfold in these moments, which flashed by like lightning. His mind, his heart were lit up with an extraordinary light; all his agitations, all his doubts, all his worries were as if placated at once, resolved in a sort of sublime tranquility, filled with serene, harmonious joy, and hope, filled with reason and ultimate cause. But these moments, these glimpses were still only a presentiment of that ultimate second never more than a second from which the fit itself began.
That second was, of course, unbearable. Reflecting on that moment afterwards, in a healthy state, he had often said to himself that all those flashes and glimpses of a higher self-sense and self-awareness, and therefore, of the "highest being" were nothing but an illness, a violation of the normal state, and if so, then this was not the highest being at all but, on the contrary, should be counted as the very lowest.
Was he dreaming some sort of abnormal and nonexistent visions of that moment, as from hashish, opium, or wine, which humiliate the reason and distort the soul? He could reason about it sensibly once his morbid state was over. Those moments were precisely only an extraordinary intensification of self-awareness - if there was a need to express this condition in a single word - self-awareness and at the same time a self-sense immediate in the highest degree. However, he did not insist on the dialectical part of his reasoning: dullness, darkness of soul, idiocy stood before him as the clear consequence of these "highest moments.
His reasoning, that is, his evaluation of this moment, undoubtedly contained an error, but all the same he was somewhat perplexed by the actuality of the sensation. Because it had happened, he had succeeded in saying to himself in that very second, that this second, in its boundless happiness, which he fully experienced, might perhaps be worth his whole life. It is interesting to read David Ingvar's biography of Dostoevsky in the book "Ten Brains" in which he emphasizes that there are few people with epilepsy symptoms that it has been written so much about.
Already at the turn of the eighteenth to the nineteenth century leading Russian and foreign neurologists published essays on Dostoevsky's epilepsy, although neither brain imaging nor electrophysiological methods EEC were available. David Ingvar mentions that Freud who dismissed the pathological Dostoyevsky obviously believed that emotional factors in childhood, especially youthful erotic escapades and bad relationship with his father could have had an impact on the disease. The father then had abused her sexually. It was this mental pain she constantly acted out.
When my father beat me up badly when I was 9 years, the most painful was not that he hit me blue and bloody, but that he tried raping my psyche and forced me to ask for forgiveness. To re-live this pain was a crucial experience to later re-live my birth trauma. The sum of these experiences of being raped made it later possible to live a life of self-development and to make my own journey through life.
Because I know her for several years, I reacted and pointed out to her that I did not think it was very funny. Then she changed her mind and her suffering welled up as well as her anger and anxiety towards the therapy center, where she was in treatment. This anger over poor treatment to unwarranted costs pursues her daily since a very long time. She feels destroyed, raped and abandoned. How raped she feels, in general, she showed when she said that I too had conceptually raped her when we at some point had discussed a visit to Spain, which she had interpreted would be at the price of sex.