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A Mouse Model of Mitochondrial Disease Reveals Germline Selection Against Severe mtDNA Mutations

Authors:
Weiwei Fan at Salk Institute for Biological Studies
Weiwei Fan
Katrina G Waymire
Katrina G Waymire
Katrina G Waymire
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Navneet Narula at Weill Cornell Medical College
Navneet Narula
Peng Li at University College London
Peng Li
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Abstract and Figures

The majority of mitochondrial DNA (mtDNA) mutations that cause human disease are mild to moderately deleterious, yet many random mtDNA mutations would be expected to be severe. To determine the fate of the more severe mtDNA mutations, we introduced mtDNAs containing two mutations that affect oxidative phosphorylation into the female mouse germ line. The severe ND6 mutation was selectively eliminated during oogenesis within four generations, whereas the milder COI mutation was retained throughout multiple generations even though the offspring consistently developed mitochondrial myopathy and cardiomyopathy. Thus, severe mtDNA mutations appear to be selectively eliminated from the female germ line, thereby minimizing their impact on population fitness.
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A Mouse Model of Mitochondrial Disease Reveals Germline
Selection Against Severe mtDNA Mutations
Weiwei Fan
1,2
, Katrina G. Waymire
1,2
, Navneet Narula
3
, Peng Li
4
, Christophe Rocher
1,2
,
Pinar E. Coskun
1,2
, Mani A. Vannan
4
, Jagat Narula
4
, Grant R. MacGregor
1,5,6
, and
Douglas C. Wallace
1,2,7,*
1
Center for Molecular and Mitochondrial Medicine and Genetics, University of California, Irvine,
CA 92697, USA.
2
Department of Biological Chemistry, University of California, Irvine, CA 92697, USA.
3
Department of Pathology, University of California, Irvine, CA 92697, USA.
4
Division of Cardiology, Department of Medicine, University of California, Irvine, CA 92697, USA.
5
Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA.
6
Developmental Biology Center, University of California, Irvine, CA 92697, USA.
7
Departments of Ecology and Evolutionary Biology and Pediatrics, University of California, Irvine,
CA 92697, USA.
Abstract
The majority of mitochondrial DNA (mtDNA) mutations that cause human disease are mild to
moderately deleterious, yet many random mtDNA mutations would be expected to be severe. To
determine the fate of the more severe mtDNA mutations, we introduced mtDNAs containing two
mutations that affect oxidative phosphorylation into the female mouse germ line. The severe ND6
mutation was selectively eliminated during oogenesis within four generations, whereas the milder
COI mutation was retained throughout multiple generations even though the offspring consistently
developed mitochondrial myopathy and cardiomyopathy. Thus, severe mtDNA mutations appear
to be selectively eliminated from the female germ line, thereby minimizing their impact on
population fitness.
The maternally inherited mitochondrial DNA (mtDNA) has a high mutation rate, and
mtDNA base substitution mutations have been implicated in a variety of inherited
degenerative diseases including myopathy, cardiomyopathy, and neurological and endocrine
disorders (1,2). Paradoxically, the frequency of mtDNA diseases is high, estimated at 1 in
5000 (3,4), yet only a few mtDNA mutations account for the majority of familial cases (2).
Because mutations would be expected to occur randomly in the mtDNA, the paucity of the
most severe mtDNA base substitutions in maternal pedigrees suggests that the severe
mutations may be selectively eliminated in the female germ line.
*
To whom correspondence should be addressed. dwallace@uci.edu.
Supporting Online Material
www.sciencemag.org/cgi/content/full/319/5865/958/DC1
Materials and Methods
Figs. S1 to S6
References
NIH Public Access
Author Manuscript
Science. Author manuscript; available in PMC 2011 March 7.
Published in final edited form as:
Science
. 2008 February 15; 319(5865): 958–962. doi:10.1126/science.1147786.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
To investigate this possibility, we have developed a mouse model in which the germline
transmission of mtDNA point mutations of different severity could be tested. An antimycin
A–resistant mouse LA9 cell line was cloned whose mtDNA harbored two homoplasmic
(pure mutant) protein-coding gene base change mutations: one severe and the other mild.
The severe mutation was a C insertion at nucleotide 13,885 (13885insC), which created a
frameshift mutation in the NADH dehydrogenase subunit 6 gene (ND6). This frameshift
mutation altered codon 63 and resulted in termination at codon 79 (Fig. 1A, top; fig. S1A,
bottom). When homoplasmic, this mutation inactivates oxidative phosphorylation complex I
(5). The mild mutation was a missense mutation at nucleotide 6589 (T6589C) in the
cytochrome c oxidase subunit I gene (COI) that converted the highly conserved valine at
codon 421 to alanine (V421A) (fig. S1A, top). When homoplasmic, this mutation reduces
the activity of oxidative phosphorylation complex IV by 50% (6,7).
LA9 cells homoplasmic for both the ND6 frameshift and COI missense mutations were
enucleated, and the mtDNAs were transferred by cytoplast fusion to the mtDNA-deficient
(ρ
o
) mouse cell line LMEB4, generating the LMJL8 transmitochondrial cybrid (8). LMJL8
mitochondria exhibited no detectable oxygen consumption when provided with NADH-
linked complex I substrates (fig. S1B) and no detectable complex I enzyme activity (fig.
S1C). However, the same mitochondria exhibited a 43% increase in succinate-linked
respiration and a 91% increase in complex II + III activity as well as a 62% increase in
complex IV activity (fig. S1, B and C), presumably as a compensatory response to the
severe complex I defect (9). Relative to LM(TK
−
) cells, mouse L cell lines homoplasmic for
the COI missense mutation also showed increased reactive oxygen species (ROS). Cells
homoplasmic for both the ND6 frameshift and COI missense mutations produced fewer
ROS than did the COI mutant cells. However, cells that were 50% heteroplasmic for both
the ND6 frameshift and the COI missense mutations had the highest ROS production (fig.
S1D).
To analyze the fates of the severe ND6 frameshift versus moderate COI missense mtDNA
mutations, we introduced these mutations into the mouse germ line. LMJL8 cybrids were
enucleated and the cytoplasts fused to the mouse female embryonic stem (ES) cell line
CC9.3.1 that had been cured of its resident mitochondria and mtDNAs by treatment with
rhodamine 6G (10–12). Of the 96 resulting ES cybrids, four (EC53, EC77, EC95, and EC96)
were found to be homoplasmic for the COI missense mutation. By quantitative primer
extension–denaturing high-performance liquid chromatography analysis, three of the ES cell
cybrids were also found to be homoplasmic for the ND6 frameshift mutation. However, one
ES cell cybrid, EC77, was heteroplasmic (mixture of mutant and normal mtDNAs); 96% of
the mtDNAs harbored the ND6 frameshift mutation (13885insC), whereas 4% of the
mtDNAs had sustained a secondary deletion of the adjacent T (13885insCdelT), restoring
the reading frame (Fig. 1A, bottom, and Fig. 1B). This ND6 revertant mutation encodes the
normal amino acid sequence but changes leucine codon 60 from TTA to TTG.
EC77 ES cells were injected into female C57BL/6NHsd blastocysts, and the chimeric
embryos were transferred into pseudo-pregnant females (12). Three chimeric females were
generated that contained varying proportions of three mtDNA genotypes: ND6 frameshift +
COI missense, ND6 revertant + COI missense, and wild type (fig. S2). The chimeras were
mated with C57BL/6J (B6) males and produced a total of 111 pups. Only one F
1
agouti
female pup, EC77-AG, was generated harboring the mutant mtDNAs. Analysis of tail
mtDNA by primer extension and by cloning and sequencing revealed that EC77-AG was
homoplasmic for the COI mutant allele but heteroplasmic for the ND6 frameshift (47%) and
ND6 revertant (53%) mtDNAs (fig. S3A). Post mortem analysis at 11 months revealed that
all analyzed tissues from EC77-AG had essentially the same genotype, with an average of
44 ± 3% (range 38% to 50%) of the mtDNAs harboring the ND6 frameshift plus COI
Fan et al. Page 2
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missense mutations (ND6 13885insC + COI T6589C) and 56% harboring the ND6 revertant
plus COI missense mutations (ND6 13885insCdelT + COI T6589C). The highest levels of
the frameshift mutant mtDNA were found in the brain and right oviduct; the lowest level
was found in the left ovary (Fig. 1C).
Throughout the 11 months of her life, EC77-AG displayed no overt phenotype. However,
post mortem mitochondrial enzymatic assays revealed a 10% to 33% decrease of complex I
activity in brain, heart, liver, and skeletal muscle (fig. S3B), a 56% and 46% decrease of
complex IV activity in brain and skeletal muscle, and a 19% and 39% increase of complex
IV activity in heart and liver (fig. S3C). This was associated with structures consistent with
lipid droplets in the heart mitochondria by ultrastructural analysis (compare fig. S3, D and
E).
To analyze transmission of the heteroplasmic, severe, ND6 frameshift (ND6 13885insC +
COI T6589C) mtDNA in successive maternal generations, we mated F
1
female EC77-AG,
which had 47% ND6 frameshift tail mtDNA, with B6 males. EC77-AG gave birth to six
litters totaling 56 pups (N
2
). The proportion of ND6 frameshift tail mtDNA, as assessed by
primer extension analysis, declined to 14% in the first litter of four pups (EC77 #1 to #4)
and the second litter of nine pups (EC77 #5 to #13), then to 6% in the third litter of 10 pups
(EC77 #14 to #23), and finally was lost (0%) in all subsequent litters (Fig. 2A).
To verify the reproducibility of the progressive loss of the ND6 frameshift mtDNA, we
mated N
2
female EC77 #4, which had 14% ND6 frameshift mtDNA, with B6 males. EC77
#4 gave birth to two litters totaling 12 pups (N
3
) (Fig. 2A). Three of the four pups of the first
litter had 6% ND6 frameshift mtDNA, whereas the remaining pup of the first litter and all
eight pups of the second litter had lost the ND6 frameshift mtDNA (0%). We also mated N
2
female EC77 #11, which had 14% ND6 frameshift mtDNA, with B6 males. EC77 #11 gave
birth to two litters totaling 21 pups. One of the 11 pups of the first litter had 6% ND6
frameshift mtDNA, whereas the remaining 10 pups of the first litter and all 10 pups of the
second litter had lost the frameshift mtDNA (0%). Mating of B6 males with N
3
females,
which had 6% ND6 frameshift mtDNA, only produced pups that lacked the ND6 frameshift
mtDNA. These data suggest that the mtDNA harboring the deleterious ND6 frameshift
mutation (13885insC) was selectively and directionally eliminated from the mouse female
germ line within four generations.
To determine whether the ND6 frameshift plus COI missense mtDNA was eliminated from
the female germ line in favor of the ND6 revertant plus COI missense mtDNA via selective
loss of those fetuses with the highest percentages of ND6 frameshift mtDNA, we compared
the litter sizes of females with different proportions of ND6 frameshift mtDNA. Females
with higher percentages of ND6 frameshift mtDNA would be predicted to generate pups
with higher proportions of the ND6 frameshift mtDNA and thus have higher fetal loss rates
and smaller litter sizes. We instead observed that the percentage of ND6 frameshift mtDNA
in the mother had no effect on litter size. The average litter size of F
1
female EC77-AG with
47% ND6 frameshift mtDNA was 9.3 pups per litter, whereas that of two of her daughters
with 14% ND6 frameshift mtDNA was 8.25 pups per litter, and that of her descendants with
6% ND6 frameshift mtDNAs was 8.75 pups per litter. Given that average litter size usually
decreases slightly when backcrossing onto a B6 strain background, the average litter size
appeared unaffected by the proportion of the mother’s mtDNA that harbored the ND6
frameshift mutation, thus arguing against preferential fetal loss as the segregation
mechanism.
To determine whether the ND6 frameshift mtDNA was lost before fertilization or ovulation,
we collected and genotyped individual oocytes from superovulated N
2
females containing
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14% ND6 frameshift plus COI missense and 86% ND6 revertant plus COI missense
mtDNA. Of the 12 oocytes that were successfully genotyped, four retained 10 to 16% of the
ND6 frameshift mtDNA, two retained 6% of the ND6 frameshift mtDNA, and five had lost
the frameshift mtDNA (Fig. 2B). Previous studies on mice heteroplasmic for the normal
NZB and Balb/c mtDNAs revealed that these mtDNAs segregated randomly when
transmitted through the female germ line (13). If this was the case for mice that were
heteroplasmic for the ND6 frameshift and ND6 revertant mtDNAs, we would expect that the
percentage of frameshift versus total mtDNAs would be normally distributed around the
mother’s genotype. In fact, none of the oocytes or progeny had a higher proportion of the
ND6 frameshift mutant mtDNA than the mother. This indicates that proto-oocytes with the
higher proportion of frameshift mutant mtDNA must have been eliminated by selection
before ovulation.
As seen with the F
1
female EC77-AG, the proportion of ND6 frameshift mtDNA in the
different tissues of three N
2
mice containing 14% ND6 frameshift and 86% ND6 revertant
mtDNA, all with the COI missense mutation, was relatively constant, ranging between 14%
and 16% (fig. S4A). Ultrastructural analysis of hearts taken from the 14% ND6 frameshift
plus 100% COI missense mice revealed mitochondrial proliferation, evidence of
mitochondrial autophagy, and myofibrillar degeneration (fig. S4, B to D). Biochemical
analysis of brain, heart, and liver of mice with 14% ND6 frameshift plus 100% COI
missense mutant mtDNAs revealed little reduction in complex I activity (fig. S4E);
however, complex IV activity in these tissues was reduced by 28%, 70%, and 59%,
respectively (fig. S4F).
Comparison of the complex I activity in mice harboring 0%, 6%, and 14% ND6 frameshift
and 100% COI missense mtDNAs confirmed that complex I was little affected, with the
possible exception of a modest reduction of complex I in muscle in animals with 14% ND6
frameshift mtDNAs (fig. S5A). The complex II + III activities were also relatively stable
(fig. S5B). However, complex IV was reduced about 50% in brain, liver, heart, and muscle
of the 14%, 6%, and 0% ND6 frameshift plus 100% COI missense mutant mice (Fig. 3A).
Hence, the predominant biochemical defect in animals with 14% or less ND6 frameshift
mtDNAs can be attributed to the homoplasmic COI missense mutation.
Mice that were homoplasmic for the COI missense mutation, linked to the ND6 revertant
mutation (13885insCdelT), transmitted this mtDNA to all of their offspring through multiple
backcrosses to B6 males. Although the phenotype of these mice was grossly normal, muscle
histology of 12-month-old animals revealed ragged red muscle fibers and abnormal
mitochondria characteristic of mitochondrial myopathy (Fig. 3, B to E).
In addition to the mitochondrial myopathy, echocardiographic analysis of 12-month-old COI
missense mice revealed that 100% of these animals (n = 7) had developed a striking
cardiomyopathy, as compared to age-matched B6 control mice (n = 5) (Fig. 4, A and B).
This cardiomyopathy was associated with a 35% increase in left ventricular wall thickness, a
23% reduction in left ventricular inner dimension at end-diastole (P < 0.001), and a 27%
increase in rotation in association with a 28% reduction in circumferential strain vectors (P
< 0.001) and a 42% reduction in radial stretch vectors (P < 0.001) (fig. S6, A and B).
Histology of the COI missense mutant hearts revealed the presence of myocyte hypertrophy,
myofibrillar lysis, binucleate cells, and interstitial fibrosis (Fig. 4, C and D). Focal
inflammation, interstitial edema, and increased blood vessel number and diameter were also
observed (fig. S6, C to E).However, no evidence of the myofiber disarray characteristic of
hypertrophic cardiomyopathy was seen. Cardiac ultrastructural analysis revealed loss of
myofilaments and mitochondrial abnormalities in mutant heart tissue, including
mitochondrial proliferation, reduction in mitochondrial matrix density, and cristolysis (Fig.
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4F), relative to age-matched controls (Fig. 4E). Hence, the milder mtDNA COI missense
mutation was successfully transmitted through repeated maternal generations, even though it
caused maternally inherited mitochondrial myopathy and cardiomyopathy.
Our studies suggest that the female germ line has the capacity for intraovarian selection
against highly deleterious mtDNA mutations such as the ND6 frameshift mutation, while
permitting transmission of more moderate mtDNA mutations such as the COI missense
mutation. This observation may explain why there is a dearth of severe mtDNA base
substitution pedigrees in humans, yet more moderate pathogenic mtDNA mutations are
repeatedly seen, such as those causing neurogenic muscle weakness, ataxia, and retinitis
pigmentosa (NARP) T8993G (ATP6 L156R) and Leber hereditary optic neuropathy
(LHON) G11778A (ND4 R340H) and T14484C (ND6 M64V) (2).
Although the mechanism by which the severe mtDNA mutations are recognized and
eliminated remains unclear, our observation that cells heteroplasmic for both the ND6
frameshift and the COI missense mutations have the highest ROS production provides one
possible explanation. It has been proposed that the primordial female germ cells have a
limited number of mtDNAs permitting rapid genetic drift toward pure mutant or wild-type
mtDNA during the approximately 20 female germline cell divisions (13). At birth, the
ovigerous cords reorganize to form single oogonia surrounded by granulosa cells. This
would lead to oogonia within fetal ovigerous cords with mtDNA genotypes symmetrically
distributed around the maternal mean percent heteroplasmy. Of these oogonia, only about
30% complete meiotic maturation; the remainder undergo apoptosis (14,15). Because
apoptosis in preovulatory follicles is thought to be induced by oxidative stress (16), it is
conceivable that the proto-oocytes with the highest percentage of severe mtDNA mutations
produce the most ROS and thus are preferentially eliminated by apoptosis. Such a process
would then lead to the progressive loss of the more deleterious mtDNA mutations over
successive female generations.
Among the pathogenic missense mutations that are observed, the more severe mtDNA
mutations such as NARP T8993G remain heteroplasmic through successive generations. By
contrast, the milder mtDNA mutations, such as LHON G11778A and T14484C, can
segregate to homoplasmic mutant (2). Because heteroplasmy would temper the biochemical
defect associated with the more severe mutations, this observation supports the concept that
the more severe mtDNA defects are eliminated within the maternal germ line.
The existence of a female germline filter for severely deleterious mtDNA mutations makes
evolutionary sense. Assuming that mtDNA variation is pivotal to species adaptation to
changing environments and that the uniparental mtDNA cannot generate diversity by
recombination, then mtDNA diversity must be generated through a high mutation rate (17–
19). However, a high mutation rate would generate many highly deleterious mutations that
could create an excessive genetic load and endanger species fitness. This dilemma can be
resolved by the addition of a graded filter in the female germ line that eliminates the most
severe mutations before conception. For such a filter to succeed, multiple cell divisions
resulting in a large population of proto-oocytes would be required to segregate out the new
mtDNA mutations. This may explain why the mammalian female generates millions of
primordial oogonia but ovulates only a few hundred mature oocytes.
References and Notes
1. Wallace DC. Annu. Rev. Genet 2005;39:359. [PubMed: 16285865]
2. Wallace, DC.; Lott, MT.; Procaccio, V. Emery and Rimoin’s Principles and Practice of Medical
Genetics. ed. 5. Rimoin, DL.; Connor, JM.; Pyeritz, RE.; Korf, BR., editors. Vol. vol. 1.
Philadelphia: Churchill Livingstone; 2007. p. 194-298.
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3. Schaefer AM, Taylor RW, Turnbull DM, Chinnery PF. Biochim. Biophys. Acta 2004;1659:115.
[PubMed: 15576042]
4. Schaefer AM, et al. Ann. Neurol. 2007 10.1002/ana.21217.
5. Bai Y, Attardi G. EMBO J 1998;17:4848. [PubMed: 9707444]
6. Acin-Perez R, et al. Hum. Mol. Genet 2003;12:329. [PubMed: 12554686]
7. Kasahara A, et al. Hum. Mol. Genet 2006;15:871. [PubMed: 16449238]
8. Trounce I, Wallace DC. Somat. Cell Mol. Genet 1996;22:81. [PubMed: 8643997]
9. Kokoszka JE, et al. Nature 2004;427:461. [PubMed: 14749836]
10. Levy SE, Waymire KG, Kim YL, MacGregor GR, Wallace DC. Transgen. Res 1999;8:137.
11. Sligh JE, et al. Proc. Natl. Acad. Sci. U.S.A 2000;97:14461. [PubMed: 11106380]
12. MacGregor, GR.; Fan, WW.; Waymire, KG.; Wallace, DC. Embryonic Stem Cells. Notarianni, E.;
Evans, MJ., editors. New York: Oxford Univ. Press; 2006. p. 72-104.
13. Jenuth JP, Peterson AC, Fu K, Shoubridge EA. Nat. Genet 1996;14:146. [PubMed: 8841183]
14. Hussein MR. Hum. Reprod. Update 2005;11:162. [PubMed: 15705959]
15. Tilly JL, Tilly KI. Endocrinology 1995;136:242. [PubMed: 7828537]
16. Tsai-Turton M, Luderer U. Endocrinology 2006;147:1224. [PubMed: 16339198]
17. Wallace DC. Annu. Rev. Biochem 2007;76:781. [PubMed: 17506638]
18. Ruiz-Pesini E, Mishmar D, Brandon M, Procaccio V, Wallace DC. Science 2004;303:223.
[PubMed: 14716012]
19. Ruiz-Pesini E, Wallace DC. Hum. Mutat 2006;27:1072. [PubMed: 16947981]
20. Supported by California Regenerative Medicine Predoctoral Fellowship TI-00008 (W.F.), NIH
grant HD45913 (G.R.M.), and NIH grants NS21328, AG13154, AG24373, DK73691, and
AG16573 (D.C.W.).
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Fig. 1.
Qualitative and quantitative analysis of the ND6 frameshift (13885insC) and ND6 revertant
(13885insCdelT) mutations in mouse ES cell cybrids and tissues of F
1
female EC77-AG.
(A) Sequence around nucleotide 13,885 of two clones of mtDNA from EC77 cells. Top
(13885insC), the single C insertion causing the frameshift (asterisk); bottom
(13885insCdelT), the T (arrow) deletion that restored the normal reading frame. (B)
Percentages of ND6 frameshift (13885insC, blue) versus revertant (13885insCdelT, purple)
in four independent mouse ES cybrids. (C) Proportions of ND6 frameshift (13885insC) and
revertant (13885insCdelT) mtDNAs in the tissues of F
1
female EC77-AG.
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Fig. 2.
Selective elimination of ND6 frameshift mtDNA (13885insC) from F
1
female EC77-AG and
her offspring. (A) Percentages of ND6 frameshift (13885insC) mtDNAs in EC77-AG and
her offspring, plus the pups of her daughters EC77 #4 and #11. Each offspring was analyzed
from multiple litters. Litter sizes are indicated in parentheses. (B) Percentages of the ND6
frameshift (13885insC, black) and revertant (13885insCdelT, gray) mtDNAs in 12 oocytes
isolated from EC77 progeny mice containing 14% of the ND6 frameshift mutation
(13885insC).
Fan et al. Page 8
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Fig. 3.
Decreased mitochondrial complex IV activity and mitochondrial myopathy in COI mutant
mice. (A) Complex IV activity was reduced to similar extents in various tissues of mutant
mice harboring 0%, 6%, or 14% ND6 frameshift mutations plus 100% COI missense.
Numbers of animals tested: six B6, seven 0% (0% ND6 frameshift plus 100% COI
missense), three 6% (6% ND6 frameshift plus 100% COI missense), and four 14% (14%
ND6 frameshift plus 100% COI missense), with three repeats performed for each test on
each animal. (B and C) Gomori trichrome staining shows increased ragged red fibers
(arrowheads) in skeletal muscle of 12-month-old COI mutant mice (C) compared to age-
matched control (B). (D and E) Electron microscopy (EM) shows altered mitochondrial
morphology (arrowheads) in skeletal muscle of 12-month-old COI mutant (E) mice
compared to age-matched control (D). Scale bars, 75 µm [(B) and (C)], 1 µm [(D) and (E)].
Fan et al. Page 9
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Fig. 4.
Mitochondrial cardiomyopathy in 12-month-old mice homoplasmic for COI missense
mutation. (A) Echocardiographic analysis of control heart. (B) Echocardiographic analysis
of mutant heart showing increased left ventricular wall thickness (LVWT) and decreased left
ventricular internal dimension in diastole (LVIDd). (C) Hematoxylin and eosin–stained
mutant heart showing myofibrolysis (black arrows), myocyte hypertrophy (long white
arrow), and binucleate cells (inset, white arrows). (D) Masson trichrome–stained mutant
heart showing interstitial replacement fibrosis (yellow arrows). (E) EM of mitochondria
(arrowheads) in normal heart. (F) EM of mutant heart showing mitochondrial proliferation,
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reduced matrix density, and cristolysis (arrowheads). Scale bars, 500 µm (C), 100 µm (D), 1
µm [(E) and (F)].
Fan et al. Page 11
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... In sexually reproducing species, mtDNA is exclusively maternally inherited while paternal mtDNA is eliminated by various mechanisms within the egg post-fertilization (Fan et al., 2008;Sato and Sato, 2013;Rojansky et al., 2016;Lee et al., 2023). Through poorly understood mechanisms, some maternal mtDNA is also selected against during fertilization (Latorre-Pellicer et al., 2019). ...
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  • Oct 2023
  • MITOCHONDRION
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 cross-specific 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. 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.
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... Collectively, these processes are thought to result in germline selection which has a major impact on the transmission of devastating mtDNAbased diseases, and, more generally, on the penetration of detrimental mtDNA mutations into populations and species. Germline selection has been actively discussed as a key means of eradicating of detrimental mutations since it was demonstrated that mtDNA mutations may be subject to negative germline selection (Fan et al. 2008;Stewart et al. 2008). On the other hand, the persistence of certain disease-causing mtDNA mutations in the human population hinted that detrimental mtDNA mutations may be, counterintuitively, under positive selection, as often proxied by the heteroplasmy shift (HS), i.e. the difference between the child's and the mother's heteroplasmy (See Suppl. ...
... As heteroplasmy levels exceed physiological threshold (Konstantin Khrapko and Turnbull 2014), the physiology of the entire cell may change, which, for example, may make the cell more prone to proliferation (Smith et al. 2020). Finally, at even higher mutant fractions, (as has been repeatedly proposed by many researchers in the field) detrimental mutations may become progressively more toxic to the cell and cause slowdown of proliferation or active removal of highly mutant germ cells cells (e.g. by attrition (Fan et al. 2008)), or by embryo demise. ...
A novel, wave-shaped profile of germline selection of pathogenic mtDNA mutations is discovered by bypassing a classical statistical bias.
Preprint
Full-text available
  • Nov 2023
The shift of the level of disease-causing mtDNA mutations (heteroplasmy) from mother to child is typically negatively correlated with the mother ′ s heteroplasmy (Hm). In other words, mothers with low Hm tend to have children with a higher mutation level (Hch) than their own. In contrast, mothers with high Hm typically see a decrease in heteroplasmy in their children. This peculiar trend has been commonly interpreted as a result of a descending germline selection profile, i.e., positive selection at low Hm, gradually turning negative at high Hm. Here we demonstrate, however, that the negative correlation is mostly driven by RTM, or ′ Regression To the Mean ′, a classical statistical bias. We further show that RTM can be nullified by using the average between the mother ′ s and child ′ s heteroplasmy, as a new variable, instead of the commonly used mother ′ s heteroplasmy in blood. Additionally, we demonstrate that mother/child average is a better proxy of the actual germline heteroplasmy. Moreover, the elimination of RTM revealed a previously hidden wave-shaped HS-profile (positive mother-to-child shift at intermediate average mother/child heteroplasmy, decreasing towards high and low average heteroplasmy). In confirmation of this finding, we show that simulations that involve both wave-shaped HS-profile and RTM, reproduce the observed patterns of inheritance of mtDNA mutations in unprecedented detail. From the health care perspective, the uncovering of the wave-shaped HS-profile (and the removal of the RTM bias) are crucial for millions of families affected by mtDNA disease. From the fundamental perspective, the wave-shaped profile offers a novel understanding of the germline dynamics of mtDNA and a novel potential mechanism that prevents the spread of detrimental mtDNA mutations in the population.
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... Therefore, most mutations arise as evolutionary 'dead ends' in somatic tissue that will not be inherited since only a small fraction of most animal cells are in the germ line (Surani et al., 2004) and most animals do not reproduce through fission (Howe et al., 2022); for a discussion on fission-inherited somatic mutations, see (Hanlon et al., 2019) Germ line mutations can be passed from parent to offspring and therefore are the basis for most genetic work to date. Germ line mutations are the fuel for evolutionary change via drift or selection (Fan et al., 2008;Lynch et al., 2016), but see Hanlon et al., 2019. Heritable diseases have in part been extensively studied due to their ease of detection: germ line mutations arise pre-fertilization and therefore proliferate into every cell of an organism (Campbell & Eichler, 2013). ...
On the distribution and diversity of tissue-specific somatic mutations in honey bee (Apis mellifera) drones
Article
Full-text available
  • Mar 2024
Somatic mutations originate from both exogenous (e.g. UV radiation, chemical agents) and endogenous (e.g., DNA replication, defective DNA repair) sources and can have significant impacts on an animal’s reproductive success. This may be especially true for haploid organisms that are susceptible to any deleterious alleles inherited from their parent and any that arise over their lifetime. Unfortunately, little is known about the rate of somatic mutation accumulation across individuals and tissues of haplodiploid animal populations, the functional processes through which they arise, and their distribution across tissues and the genome. Here, we generated short-read whole-genome sequencing data for four tissues of haploid honey bee males. We paired this with estimates of telomere length and tissue-specific DNA content to address three major questions: is there variance in somatic mutational load across haploid individuals and specific tissues therein, does increased DNA content in a tissue contribute to somatic mutational load, and does telomere length correlate with mutational load? Our results suggest that variance in somatic mutational load is better captured across individuals than across tissues, that tissue-specific DNA content is not associated with somatic mutation load, and that variance in telomere length does not correlate with somatic mutation loads across tissues. To our knowledge, this is the first observational study on somatic mutational load in Apoidea and likely Hymenoptera. It serves as a useful advent for additional studies understanding the processes through which haploids tolerate or repair somatic mutations.
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... Animals with a low aerobic requirement and a high rate of mtDNA replacements can tolerate suboptimal mitochondrial-nuclear genome matching and high free radical leakage. The elimination of harm from the rapid accumulation of heavy mutations in the genome occurs due to their selective removal from the female germ line, thereby minimizing their impact on population fitness (Fan et al., 2008;De Paula et al., 2013). ...
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... The D-loop (non-coding or control region) contains essential regulatory sequences for replication and transcription (34). Although the majority of harmful variants are removed by natural selection, some of these variants are introduced in certain populations and may influence the risk of developing certain disorders (35), whereas other variants such as m.152T>C may be selectively beneficial on some genetic backgrounds. Additionally, the silent m.15301G>A variant in the MT-CYB gene occurred in 30% of patients and 10% of controls (OR=3.8, ...
Comprehensive analysis of mitochondrial DNA variants, mitochondrial DNA copy number and oxidative damage in psoriatic arthritis
Article
Full-text available
  • Sep 2023
Growing evidence suggests that abnormalities in mitochondrial DNA (mtDNA) are involved in the pathogenesis of various inflammatory and immuno-mediated diseases. The present study analysed the entire mitochondrial genome by next-generation sequencing (NGS) in 23 patients with psoriatic arthritis (PsA) and 20 healthy controls to identify PsA-related variants. Changes in mtDNA copy number (mtDNAcn) were also evaluated by quantitative polymerase chain reaction (qPCR) and mtDNA oxidative damage was measured using an 8-hydroxy-2'-deoxyguanosine assay. NGS analysis revealed a total of 435 variants including 187 in patients with PsA only and 122 in controls only. Additionally, 126 common variants were found, of which 2 variants differed significantly in their frequencies among patients and controls (P<0.05), and may be associated with susceptibility to PsA. A total of 33 missense variants in mtDNA-encoded genes for complexes I, III, IV and V were identified only in patients with PsA. Of them, 25 variants were predicted to be deleterious by affecting the functions and structures of encoded proteins, and 13 variants were predicted to affect protein's stability. mtDNAcn analysis revealed decreased mtDNA content in patients with PsA compared with controls (P=0.0001) but the decrease in mtDNAcn was not correlated with patients' age or inflammatory biomarkers (P>0.05). Moreover, a higher level of oxidative damage was observed in patients with PsA compared with controls (P=0.03). The results of the present comprehensive analysis of mtDNA in PsA revealed that certain mtDNA variants may be implicated in the predisposition/pathogenesis of PsA, highlighting the importance of NGS in the identification of mtDNA variants in PsA. The current results also demonstrated that decreased mtDNAcn in PsA may be a consequence of increased oxidative stress. These data provide valuable insights into the contribution of mtDNA defects to the pathogenesis of PsA. Additional studies in larger cohorts are needed to elucidate the role of mtDNA defects in PsA.
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Influence of the Rate of Changes in the COX1 Gene on Body Size and Sexual Selection in Carp Hybridization
Article
  • Nov 2023
The influence of mtDNA cytochrome c-oxidase I gene fragment variability on body length was studied in twelve species of cyprinids, which may have hybrids with Rutilus rutilus L. and Abramis brama L., and in reciprocal hybrids (RA, AR) and alloplasmatic backcrosses (ARR, RAA) of roach (R) and bream (A). It has been established that the rate of nucleotide substitutions in COX1 is negatively related not only to body size but also to fish life span, which differentiates them into two groups: group I – species with a high rate of COX1 changes and a relatively small body size and group II – species with low sequence variability and relatively large body size. The boundary for the distinguished groups runs between species the same genus Leuciscus leuciscus and L. idus: with a twofold decrease in the rate of substitutions in ide, a twofold increase in body size and lifespan occurs, which indicates a decrease in the rate of cellular respiration and free radical leak, and the exact mitonuclear match respiratory complexes. Presumably, the decrease in the rate of COX1 changes in species of group II and in bleak Alburnus alburnus is associated with an increase in the size of genome, which provides additional protection of genes from chemical mutagens and, regardless of body size, reduces the rate of aerobic metabolism. It has been experimentally shown that mtDNA affects body length. When bream mtDNA is included in the roach nuclear genome, ARR backcrosses have the body length of a bream and high viability, while RAA backcrosses with roach mtDNA and the bream nuclear genome inherit the roach body length and reduce viability. Species of group II are not able to effectively use the highly polymorphic mtDNA of species of group I, which is also manifested by a violation of the inheritance of a longer bream body length in RA hybrids and leads to reproductive isolation. Group I species, such as Rutilus rutilus, can include mtDNA of both groups in their genome, which underlies sexual selection in hybridization. Accordingly, sexual size dimorphism has a genetic origin, and body size for a potential partner can be a signal for determining the mitonuclear compatibility of genomes in respiratory complexes.
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Germline ecology: Managed herds, tolerated flocks, and pest control
Article
  • Mar 2024
Multicopy sequences evolve adaptations for increasing their copy number within nuclei. The activities of multicopy sequences under constraints imposed by cellular and organismal selection result in a rich intranuclear ecology in germline cells. Mitochondrial and ribosomal DNA are managed as domestic herds subject to selective breeding by the genes of the single-copy genome. Transposable elements lead a peripatetic existence in which they must continually move to new sites to keep ahead of inactivating mutations at old sites and undergo exponential outbreaks when the production of new copies exceeds the rate of inactivation of old copies. Centromeres become populated by repeats that do little harm. Organisms with late sequestration of germ cells tend to evolve more “junk” in their genomes than organisms with early sequestration of germ cells.
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Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis
Article
Full-text available
  • Dec 2023
Hepatic steatosis is the result of imbalanced nutrient delivery and metabolism in the liver and is the first hallmark of Metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is the most common chronic liver disease and involves the accumulation of excess lipids in hepatocytes, inflammation, and cancer. Mitochondria play central roles in liver metabolism yet the specific mitochondrial functions causally linked to MASLD remain unclear. Here, we identify Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, liver-specific knockout mice are protected against high fat diet-induced steatosis and metabolic dysregulation. Additionally, Mtfp1 deletion inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers additional functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for MASLD.
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Jenuth, J.P. , Peterson, A.C. , Fu, K. & Shoubridge, E.A. Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA. Nat. Genet. 14, 146-151
Article
Full-text available
  • Nov 1996
Mitochondrial DNA (mtDNA) is maternally inherited in mammals. Despite the high genome copy number in mature oocytes (10(5)) and the relatively small number of cell divisions in the female germline, mtDNA sequence variants segregate rapidly between generations. To investigate the molecular basis for this apparent paradox we created lines of heteroplasmic mice carrying two mtDNA genotypes. We show that the pattern of segregation can be explained by random genetic drift occurring in early oogenesis, and that the effective number of segregating units for mtDNA is approximately 200 in mice. These results provide the basis for estimating recurrence risks for mitochondrial disease due to pathogenic mtDNA mutations and for predicting the rate of fixation of neutral mtDNA mutations in maternal lineages.
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The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme
Article
Full-text available
  • Sep 1998
Seven of the approximately 40 subunits of the mammalian respiratory NADH dehydrogenase (Complex I) are encoded in mitochondrial DNA (mtDNA). Their function is almost completely unknown. In this work, a novel selection scheme has led to the isolation of a mouse A9 cell derivative defective in NADH dehydrogenase activity. This cell line carries a near-homoplasmic frameshift mutation in the mtDNA gene for the ND6 subunit resulting in an almost complete absence of this polypeptide, while lacking any mutation in the other mtDNA-encoded subunits of the enzyme complex. Both the functional defect and the mutation were transferred with the mutant mitochondria into mtDNA-less (rho0) mouse LL/2-m21 cells, pointing to the pure mitochondrial genetic origin of the defect. A detailed biosynthetic and functional analysis of the original mutant and of the rho0 cell transformants revealed that the mutation causes a loss of assembly of the mtDNA-encoded subunits of the enzyme and, correspondingly, a reduction in malate/glutamate-dependent respiration in digitonin-permeabilized cells by approximately 90% and a decrease in NADH:Q1 oxidoreductase activity in mitochondrial extracts by approximately 99%. Furthermore, the ND6(-) cells, in contrast to the parental cells, completely fail to grow in a medium containing galactose instead of glucose, indicating a serious impairment in oxidative phosphorylation function. These observations provide the first evidence of the essential role of the ND6 subunit in the respiratory function of Complex I and give some insights into the pathogenic mechanism of the known disease-causing ND6 gene mutations.
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Transfer of chloramphenicol-resistant mitochondrial DNA into the chimeric mouse
Article
Full-text available
  • May 1999
The mitochondrial DNA (mtDNA) chloramphenicol (CAP)-resistance (CAPR) mutation has been introduced into the tissues of adult mice via female embryonic stem (ES) cells. The endogenous CAP-sensitive (CAPS) mtDNAs were eliminated by treatment of the ES cells with the lipophilic dye Rhodamine-6-G (R-6-G). The ES cells were then fused to enucleated cell cytoplasts prepared from the CAPR mouse cell line 501-1. This procedure converted the ES cell mtDNA from 100% wild-type to 100% mutant. The CAPR ES cells were then injected into blastocysts and viable chimeric mice were isolated. Molecular testing for the CAPR mutant mtDNAs revealed that the percentage of mutant mtDNAs varied from zero to approximately 50% in the tissues analyzed. The highest percentage of mutant mtDNA was found in the kidney in three of the chimeric animals tested. These data suggest that, with improved efficiency, it may be possible to transmit exogenous mtDNA mutants through the mouse germ-line.
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Maternal germ-line transmission of mutant mtDNAs from embryonic stem cell-derived chimeric mice
Article
Full-text available
  • Jan 2001
We report a method for introducing mtDNA mutations into the mouse female germ line by means of embryonic stem (ES) cell cybrids. Mitochondria were recovered from the brain of a NZB mouse by fusion of synaptosomes to a mtDNA-deficient (rho degrees ) cell line. These cybrids were enucleated and the cytoplasts were electrofused to rhodamine-6G (R-6G)-treated female ES cells. The resulting ES cell cybrids permitted transmission of the NZB mtDNAs through the mouse maternal lineage for three generations. Similarly, mtDNAs from a partially respiratory-deficient chloramphenicol-resistant (CAP(R)) cell line also were introduced into female chimeric mice and were transmitted to the progeny. CAP(R) chimeric mice developed a variety of ocular abnormalities, including congenital cataracts, decreased retinal function, and hamaratomas of the optic nerve. The germ-line transmission of the CAP(R) mutation resulted in animals with growth retardation, myopathy, dilated cardiomyopathy, and perinatal or in utero lethality. Skeletal and heart muscle mitochondria of the CAP(R) mice were enlarged and atypical with inclusions. This mouse ES cell-cybrid approach now provides the means to generate a wide variety of mouse models of mitochondrial disease.
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R EPORTS Effects of Purifying and Adaptive Selection on Regional Variation
Article
Full-text available
  • Feb 2004
  • SCIENCE
A phylogenetic analysis of 1125 global human mitochondrial DNA (mtDNA) sequences permitted positioning of all nucleotide substitutions according to their order of occurrence. The relative frequency and amino acid conservation of internal branch replacement mutations was found to increase from tropical Africa to temperate Europe and arctic northeastern Siberia. Particularly highly conserved amino acid substitutions were found at the roots of multiple mtDNA lineages from higher latitudes. These same lineages correlate with increased propensity for energy deficiency diseases as well as longevity. Thus, specific mtDNA replacement mutations permitted our ancestors to adapt to more northern climates, and these same variants are influencing our health today.
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An intragenic suppressor in the cytochrome c oxidase I gene of mouse mitochondrial DNA
Article
Full-text available
  • Mar 2003
We report here the identification of a cell line containing single and double missense mutations in cytochrome c oxidase (COX) subunit I gene of mouse mitochondrial DNA. When present in homoplasmy, the single mutant displays a normal complex IV assembly but a significantly reduced COX activity, while the double mutant almost completely compensates the functional defect of the first mutation. We discuss the potential structural consequences of those mutations based on the modeled structure of mouse complex IV. Based on genetic, biochemical and molecular analyses of cultured mouse cells we infer that: (i) deleterious mutations can arise and become predominant; (ii) cultured cells can maintain several mtDNA haplotypes at stable frequencies; (iii) the respiratory chain has little spare COX capacity; and (iv) the size of a cavity in the vicinity of Val421 in CO I of animal COX may affect the function of the enzyme.
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Inhibitors of oxidative stress mimic the ability of follicle-stimulating hormone to suppress apoptosis in cultured rat ovarian follicle
Article
  • Feb 1995
We have reported that members of the bcl-2 gene family are expressed and gonadotropin regulated in ovarian granulosa cells during follicular maturation and atresia. Because Bcl-2, a protein that prevents apoptosis in several cell types, is reported to function as an antioxidant or free radical scavenger, the present studies were designed to investigate if oxidative stress plays a role in granulosa cell apoptosis during follicular atresia in the immature rat ovary. In the first series of experiments, the role of oxidative stress in the induction of granulosa cell apoptosis was directly tested using a defined in vitro follicle culture system. Healthy antral follicles obtained from equine CG (eCG)-primed immature (27 day old) rats were incubated in serum-free medium for 24 h in the absence or presence of FSH (100 ng/ml; a control for inhibiting apoptosis), superoxide dismutase (SOD; 10-1000 U/ml), ascorbic acid (0.01-1 mM; a free radical scavenger), N-acetyl-L-cysteine (25-100 mM; a free radical scavenger and stimulator of endogenous glutathione peroxidase activity), or catalase (10-1000 U/ml). Granulosa cells within follicles incubated in medium alone exhibited extensive apoptosis after 24 h of incubation, and this onset of apoptosis was blocked by treatment with FSH (29 +/- 4% of controls; P < 0.001, n = 3). Moreover, apoptosis in follicles was also inhibited by treatment with SOD (44 +/- 4% of controls at 1000 U/ml; P < 0.01, n = 3), ascorbic acid (55 +/- 9% of controls at 1 mM; P < 0.05, n = 3), N-acetyl-L-cysteine (24 +/- 7% of controls at 100 mM; P < 0.001, n = 3), or catalase (35 +/- 6% of controls at 1000 U/ml; P < 0.001, n = 3). In the second series of experiments, complementary DNAs corresponding to secreted (SEC-SOD), copper/zinc-containing (Cu/Zn-SOD), and manganese-containing (Mn-SOD) forms of rat SOD, rat seleno-cysteine glutathione peroxidase (GSHPx), and rat catalase were isolated and used to synthesize antisense RNA probes for Northern and slot blot analysis of changes in SOD, GSHPx, and catalase gene expression during follicular maturation. In vivo priming of 25-day-old female rats for 2 days with 10 IU eCG, which promoted antral follicular growth and survival, increased levels of messenger RNA encoding SEC-SOD (216 +/- 9% of saline-treated controls, P < 0.05, n = 3) and Mn-SOD (222 +/- 14% of saline-treated controls, P < 0.05, n = 3) vs. saline-treated controls.(ABSTRACT TRUNCATED AT 400 WORDS)
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Production of transmitochondrial mouse cell lines by cybrid rescue of rhodamine-6G pre-treated L-cells
Article
  • Feb 1996
Treatment of mouse LMTK- cells with the toxic mitochondrial dye rhodamine 6G (R-6G) at 2.5 micrograms/ml for 7 days prevented cell growth while maintaining viability, with less than 10(-6) cells recovering to form colonies. Pre-treatment of LMTK- cells with R-6G was followed by fusion with enucleated mouse 501-1 cells harboring a homoplasmic point mutation in the mitochondrial DNA (mtDNA) 16S rRNA gene conferring chloramphenicol resistance (CAPR). Cybrids and any surviving unfused LMTK- cells were selected in BrdU with or without CAP and their mtDNAs screened for the presence of the CAPR marker. Approximately 1 colony per 2 x 10(5) LMTK- cells appeared in the fusion plates selected both with and without CAP. Most clones investigated were confirmed to be cybrids by showing the presence of the generally homoplasmic CAPR mutation, whether or not CAP selection was used. Hence, R-6G pre-treatment permits construction of transmitochondrial cybrid cell lines carrying a variety of mtDNAs, without the need for rho 0 cell lines.
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The epidemiology of mitochondrial disorders - Past, present and future
Article
  • Jan 2005
  • Biochim Biophys Acta
A number of epidemiological studies of mitochondrial disease have been carried out over the last decade, clearly demonstrating that mitochondrial disorders are far more common than was previously accepted. This review summarizes current knowledge of the prevalence of human mitochondrial disorders--data that has important implications for the provision of health care and adequate resources for research into the pathogenesis and treatment of these disorders.
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