Leber’s hereditary mitochondrial MICHAEL
optic neuropathy:
a model
neurodegenerative
U BROWN,
AND DOUGLAS
ALEXANDER
diseases
S. VOLJAVEC,’
MARIE
A number of human diseases have been attributed to defects in oxidative phosphorylation (OXPHOS) resulting from mutations in the mitochondrial DNA (mtDNA). One such disease is Leber’s hereditary optic neuropathy (LHON), a neurodegenerative disease of young adults that results in blindness due to atrophy of the optic nerve. The etiology of LHON is genetically heterogeneous and in some cases multifactorial. Eleven mtDNA mutations have been associated with LHON, all of which are missense mutations in the subunit genes for the subunits of the electron transport chain complexes I, III, and IV. Molecular, biochemical, and population genetic studies have categorized these mutations as high risk (class I), low risk (class II), or intermediate risk (class I/I!). Class I mutations appear to be primary genetic causes of LHON, while class II mutations are frequently found associated with class I genotypes and may serve as exacerbating genetic factors. Different LHON pedigrees can harbor different combinations of class I, II, or I/H mtDNA mutations, as shown by the complete sequence analysis of the mtDNAs of four LHON probands. The various mtDNA genotypes included an isolated class I mutation, combined class I + II mutations, and combined class I/H + II mutations. The occurrence of such genotypes supports the hypothesis that LHON may result from the additive effects of various genetic and environmental insults to OXPHOS, each of which increases the probability of blindness.Brown, M. D., Voljavec, A. S., Lott, M. T., MacDonald, I., Wallace, D. C. Leber’s hereditary optic neuropathy: a model for mitochondrial neurodegenerative diseases. FASEB J. 6: 2791-2799; 1992. ABSTRACT
DNA
Leber’s
mutations
LEBER’S
hereditary
oxidative
optic
phosphorylalion
HEREDITARY
OPTIC
neuropathy
mitochondrial
genotype
NEUROPATHY
Leber’s hereditary optic neuropathy (LHON)2 is a rare, maternally inherited, neurodegenerative disease of young adults that results in blindness due to optic nerve degeneration. Painless, progressive bilateral central vision boss between the ages of 15 and 35 is usually the only clinical manifestation of LHON, although cardiac conduction defects (preexcitation syndromes) and minor neurobogic abnormalities have been noted in some LHON pedigrees (1, 2). Variable penetrance is observed in almost all LHON pedigrees, and males are four times more likely to be affected than females. This inheritance pattern has previously been attributed to an X-lmked genetic factor; however, this interpretation is inadequate because males never transmit the disease. 0892-6638/92/0006-2791/$dl
.50.
IAN MACDONALD,t
©
FASEB
30322, University of
University School of Medicine, Atlanta, 20892, USA; and tFaculty of Medicine,
Georgia
The maternal transmission of LHON has also suggested cytoplasmic inheritance of the disease, as the cytoplasm of the zygote is derived almost exclusively from the ovum. The discovery that a point mutation in the mitochondrial DNA (mtDNA) at nucleotide pair (np) 11778 was a common feature of LHON patients confirmed the role of the mtDNA in the etiology of this disease (3). The 11778 mutation is a G to A transition in the NADH:ubiquinone oxidoreductase (NADH dehydrogenase) subunit 4 (ND4) gene and alters an evolutionanily conserved amino acid. It is found only in LHON patients, is associated with LHON regardless of race (mtDNA hapbotype), and accounts for roughly 50% of LHON cases worldwide (4, 5). The establishment of the np 11778 ND4 mutation as a causal genetic mutation in LHON was the first direct demonstration that a mtDNA point mutation can result in human disease. Subsequently, mtDNA mutations have been associated with other clinical phenotypes, including myocbonic epilepsy and ragged-red fiber (MERRF) disease, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like symptoms (MELAS), and maternally inherited diabetes (6).
GENETIC
CHARACTERISTICS
OF
mtDNA
Mitochondria provide the vast majority of cellular ATP through oxidative phosphorylation (OXPHOS). The 16,569 np mitochondrial genome is present in multiple copies per cell and codes for 13 polypeptides, 22 tRNAs, and 2 rRNAs. The 13 mitochondrial-encoded polypeptides are essential components of the five mitochondrial inner-membrane respiratory enzyme complexes involved in electron transport
(complexes Words:
T. LOT!’,
C. WALLACE’
Department of Genetics and Molecular Medicine, Emory USA; *National Institutes of Health, Bethesda, Maryland Ottawa, Ottawa, Ontario, Canada KIH 8M5
Key
for
multisubunit
I-IV)
and ATP synthesis
enzyme
complexes
are
(complex assembled
V). These from
both
nuclear and mitochondrial-encoded gene products. Complex I (NADH :ubiquinone oxidoreductase) contains seven polypeptides encoded by the mtDNA (NDI, ND2, ND3, ND4, ND4L, ND5, and ND6), complex III (ubiquinone:cytochrome c oxidoreductase) contains one (cytochrome b), complex IV
‘To
whom
University
correspondence
School
should
of Medicine,
be
addressed,
Department
Molecular Medicine, 1462 Clifton Road, Room 30322, USA. 2Abbreviations: LHON, Leber’s hereditary
at:
Emory
of Genetics
and
403,
GA
optic
Atlanta, neuropathy;
mtDNA, mitochondrial DNA; np, nucleotide pair; NADH dehydrogenase, NADH:ubiquinone oxidoreductase; ND4, NADH dehydrogenase subunit 4; MERRF, myocbonic epilepsy and raggedred fiber;
MELAS,
sis, and stroke-like tion;
nDNA,
nuclear
mitochondrial
symptoms; DNA;
encephalomyopathy,
lactic
acido-
OXPHOS,
class I/Il,
oxidative phosphorylaclass I and class II mutations. 2791
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c oxidase) contains three (COl, COIl, and and complex V (ATP synthase) contains two (ATPase 6 and 8). The cytoplasmic location of the mitochondria and the large number of mtDNA molecules per cell result in five distinctive characteristics of mitochondnial genetics (6). First, mtDNA is maternally inherited. Thus, pedigrees with deleterious mtDNA mutations frequently exhibit maternal
(13-16). These mutations create both conservative and nonconservative amino acid substitutions and can be found very rarely (np 5244 mutation) or frequently (np 4216 mutation) in the unaffected control population. In general, class II mutations are either found in higher frequencies (nps 4216 and 13708) in LHON pedigrees compared to control frequencies or are linked to a specific class I mutation, e.g., the nps 5244 and 15812 mutations are frequently found with the
transmission.
class
(cytochnome
COIII),
Second,
mtDNAs
undergo
replicative
segrega-
tion upon cell division. Because of the high copy number of mtDNA, a cell or tissue may harbor both mutant and normal mtDNAs (heteroplasmy). During cell division, the heteroplasmic mtDNAs are randomly distributed to daughter cells and thus the proportion of mutant mtDNAs can change over time, drifting toward predominantly mutant or wildtype (homoplasmy) mtDNA. Third, OXPHOS defects become associated with clinical manifestations when mitochondrial energy output falls below the minimum threshold level necessary for tissues to function normally. Because different tissues rely on OXPHOS ATP to a different extent, symptoms can vary widely among family members in pedigrees harboring heteroplasmic mtDNA mutations. The organ system most dependent on OXPHOS is the central nervous system (optic nerve, brain), followed by skeletal muscle, cardiac muscle, kidney, and liver. Thus clinical phenotype is the product of the interaction between the nature of the mtDNA mutation, the degree of heteroplasmy, and the dependence of an organ system on OXPHOS. Fourth, the efficiency of electron transport and OXPHOS declines with age. This is associated with a concomitant accumulation of mtDNA mutations with age, presumably due to damage caused by the high concentration of oxygen free radicals in the mitochondnion (7, 8). The secondary accumulation of somatic mtDNA mutations may explain why some mtDNA diseases are expressed in middle to late life and progress with age. Fifth, the mtDNA sequence evolves 10- to 20-fold more rapidly than nuclear DNA (nDNA). Hence, deleterious OXPHOS mutations are more likely to occur in mtDNA OXPHOS genes than in nDNA OXPHOS genes (9).
CLASS
I AND
CLASS
II LHON
MUTATIONS
In addition to the np 11778 mutation, 10 other mtDNA point mutations have been associated with LHON (Fig. 1). LHON mutations at nps 3460 (ND1 gene), 4160 (ND1 gene), 11778 (ND4 gene), and 15257 (cytb gene) appear to be highrisk (class I) mutations for LHON (Table 1). Generally, only one class I mutation occurs in each pedigree, and individuals harboring one of these mutations have a relatively high probability of going blind (3, 10-14). These mutations usually cause significant changes in evolutionarily conserved amino acids within functionally important polypeptide domains. Moreover, these mutations are found infrequently, if at all, in population surveys of unaffected individuals. Among LHON patients that lack the 11778 mutation, the np 3460 mutation has been found in 30% (9/30) of LHON families, the np 15257 mutation in 17% (4/23) of LHON families, and the np 4160 mutation has been found in only one large LHON pedigree, in which other severe neurological abnormalities are linked to LHON. Five LHON mutations (nps 4216, 4917, 5244, 13708, and 15812) are considered low-risk (class II) mutations for LHON (Table 1). When present in LHON patients, these mutations are usually found in combination with other class I or class II mutations, and the probability that an individual will go blind when
2792
Vol. 6
harboring
July 1992
only a class II mutation
is low
I np 15257 mutation.
Two mutations at np 7444 and np 3394 have features that appear to be intermediate between the class I and class II mutations
(class
I/TI)
(Table
1). Both
cause
significant
altera-
tions in electron transport chain polypeptides, but they are relatively uncommon in LHON patients. These mutations are found in about 1% of controls and in association with class
I as well
EXAMPLES Class
as class
OF
I LHON
II mutations.
LHON
GENOTYPES
Genotypes
Class I mutations account for the largest genetic subset of LHON patients and are the primary genetic causes of LHON. Presumably, these mutations significantly reduce the efficiency of the electron transport chain. The specific activity of complex I has been found to be reduced in patients harboring either the np 3460 mutation or the np 4160 mutation (11, 12, 17). A complex I enzymobogical defect has not been demonstrated for np 11778-positive patients; however, respiration studies using intact mitochondria revealed decreased oxidation of NADH-dependent substrates (17, 18). No biochemical data exist for the np 15257 (complex III) mutation. The initial studies of the np 11778 mutation demonstrated that a single class I mutation, by itself, was sufficient to cause the disease (3, 4). Eighty-five percent of the mtDNA coding region of a LHON proband was sequenced, and 25 base substitutions were detected upon comparison to the standard (wild-type) Cambridge sequence. Most of these could be ruled out as causal mutations because they did not change amino acids, altered nonconserved amino acids, were commonly seen mtDNA polymorphisms, or represented “errors” in the Cambridge mtDNA sequence. Only the 11778 mutation fulfilled the criteria for a disease-causing mutation and no other class I or class II LHON mutations were detected. Thus class I mutations can, in general, precipitate LHON as solitary pathogenic entities. For all four class I mutations, variable penetrance is observed among apparently homoplasmic LHON pedigrees. Therefore class I mutations appear to be necessary but not sufficient for the clinical manifestation of LHON. The reason some family members harboring class I mutations remain visually asymptomatic while others develop the disease is unclear and suggests that additional genetic (nuclear or mitochondrial), environmental, or physiological factors play a significant role in the expression of LHON. Class
I
+
II LHON
genotypes
Several LHON patients have mtDNA haplotypes that combine a class I mutation with one or more class II mutations. In these cases, the class II mutations may serve as additional predisposing or exacerbating genetic factors that increase the probability of expressing LHON. In other cases, where the role of a class II mutation in causing blindness is less clear, the mutation may simply be a LHON-associated mtDNA polymorphism of limited clinical significance.
The FASEB Journal
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ET AL.
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CLASS II LHON MUTATIONS
1158121
1137081
133941 -_
142161-’
149171 152441
ATPeseS
/
174441 [::::::.:.:.:l
Complex I genes
Complex HI genes
(NADH dehydrogenase)
:rh1.0me
____
Figure 1. Mitochondrial genome showing class I, II, and I/Il are located inside of the genome (circle) and class II mutations mediate (I/I!) between these two classes and are also shown
LHON
AND
MITOCHONDRIAL
NEURODEGENERATIVE
LHON
mutations. Class I (high are shown outside the genome. outside the genome.
DISEASE
TFafleT
C
____
Rlbosesnal
risk) LHON The np 3394
RNA genes
RNA genes
mutation and 7444
nucleotide mutations
positions are inter-
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TABLE
1. Class I and class II LHON
mutations
Class
Gene
Nucleotide change
3460
I
ND!
G-’A
A-FT
M
15
0
30
4160’ 11778 15257
I I I
ND1 ND4 Cytb
TC G-’A G-’A
L’P R-H D-N
H H H