J. tnher. Metab. Dis. 15 (1992) 472-479 ©~SSIEM and Kluwer Academic Publishers. Printed in the Netherlands

Diseases esulting fro Point Mutations

Mitochondrial DNA

D. C. WALLACE 1'2, M. T. LOTT 1, J. M. SHOFFNER 1'2 and M. D. BROWN 1

1Departments of Genetics and Molecular Medicine and 2Neurology, Emory University School of Medicine, 3031 Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322, USA Summary: A number of mitochondrial DNA (mtDNA) mutations have been identified which cause familial, late onset neuromuscular degenerative diseases. These include missense mutations in most of the mtDNA polypeptide genes as well as base substitutions in several tRNA genes. Missense mutations in the mitochondrial electron-transport genes cause Leber hereditary optic neuropathy. Ten mutations have been associated with this disease, but four at rips 11 178, 3460, 4160 and 15257 appear sufficient in themselves to cause the disease. One missense mutation in the ATPase 6 gene at np 8993 causes a second phenotype, neurogenic muscle weakness, ataxia and retinitis pigmentosum. Transfer RNA mutations have been identified for myoclonic epilepsy and ragged-red fibre disease in the tRNA Ly~gene at np 8344 and for the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome and for maternal mitochondrial myopathy and cardiomyopathy syndrome in the tRNA Leu~UUR) gene at nps 3234 and 3260, respectively. Deficiencies in mitochondrial oxidative phosphorylation enzymes have been observed in several common neurodegenerative diseases such as Alzheimer and Parkinson diseases. Perhaps mtDNA mutations play a role in these as well. M A T E R N A L L Y I N H E R I T E D DISEASES

A number of hereditary diseases have recently been found to result from point mutations in the mitochondrial DNA (mtDNA). These diseases show maternal transmission, consistent with the maternal inheritance of the mtDNA. However, in contrast to the mtDNA, the phenotypes of the diseases are not strictly maternally inherited, as phenotypes can appear sporadically and disease expression can vary significantly within pedigrees. It is now clear that this is the consequence of a number of other unique aspects of mtDNA genetics. mtDNA FUNCTION AND GENETICS

The mtDNA is a 16 569-base-pair (bp) closed circular molecule located within the mitochondrion (Anderson et al 1981). Each cell has hundreds of mitochondria and thousands of mtDNAs (Shuster et al 1988). The mtDNA codes for 13 potypeptides of the mitoehondriat energy-generating pathway, oxidative phosphorylation, as well 472

473

mtDNA Point Mutations

as the t2S and 16S rRNAs and 22 tRNAs involved in mitochondrial protein synthesis. Of the polypeptide genes, seven (ND1, ND2, ND3, ND4L, ND4, ND5, ND6) are subunits of complex I (NADH dehydrogenase); one (cytochrome b) is a subunit of complex III (ubiquinol:cytochrome c oxidoreductase); three (COl, COII, COIII) are subunits of complex IV (cytochrome c oxidase), and two (ATPase 6 and 8) are subunits of complex V (ATP synthase) (Anderson et al 1981; Shoffner and Wallace !990). Since all of the genes of the mtDNA are either subunits of oxidative phosphorylation enzymes or structural RNAs necessary for their expression, deleterious mtDNA mutations invariably result in deficiencies in mitochondrial energy metabolism. The genetics of the mtDNA are unique in five ways. First, the mtDNA is maternally inherited (Giles et al 1980), though some paternal input of mtDNA has recently been demonstrated in mice (Gyllensten et al t991). Second, heteroptasmic populations of mtDNAs (mixtures of mutant and normal) segregate during both mitotic and meiotic replication (Wallace 1986). Third, the phenotype resulting from mtDNA mutations is related to the severity of the oxidative phosphorylation defect and the differential reliance of different organs on mitochondrial ATP production. The severity of the oxidative phosphorylation defect is a product both of the nature of the mutation and, if it is heteroplasmic, the proportion of mutant mtDNAs in the cell. The relative reliance of tissues on oxidative phosphorylation energy decreases in the order: central nervous system, skeletal muscle, heart, kidney and liver (Shoffner and Wallace 1990). Fourth, the severity of mtDNA diseases increases with age. This is probably due to the natural decline of oxidative phosphorylation with age (Trounce et al 1989; Yen et al 1989), possibly because somatic mutations accumulate in the mtDNA with age (Piko et al 1988; Cortopassi and Arnheim 1990; Corral-Debrinski et al 1991). Finally, the mutation rate of mtDNA genes is much higher than that of the nuclear DNA genes (Brown et al 1979; Wallace et al 1986). Hence, mtDNA diseases are much more common than might be expected. DISEASE RESULTING FROM mtDNA POINT MUTATIONS

mtDNA point mutation diseases fall into two categories: missense mutations and structural RNA mutations. Missense mutations have been found in many of the mitochondrial open reading frames (Figure 1). However, structural RNA gene mutations have been confined to tRNA genes. Leber hereditary optic neuropathy is the most common phenotype resulting from mtDNA missense mutations. In this disease, individuals precipitously lose their central vision in mid to late life owing to death of the optic nerve. When familial, the disease appears sporadically along the maternal lineage, with males being preferentially affected. Ten mtDNA point mutations have now been proposed to be associated with Leber hereditary optic neuropathy. Four of these (np 11 778 in ND4, np 3460 in ND1, np 4160 in ND1 and np 15 257 in cytb) appear suflScient in themselves to cause the disease. All of these alter conserved amino acids. The np 11 778 mutation is most common, representing about 50% of cases, and converts an arginine to a histidine (Wallace et al 1988a). About half of all cases are familial, 14% are associated with d. lnher, Metab. Dis. 15 (1992)

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Wallace er al.

D-Loop Regton

/

PL

LHON (15257) '

~ MELAS (3243)

/ ~MMC LHON

~

(3260)

(3460)

LHON (4160)

L HS

LHON (11778) OL W

MERRF (8344)

NARP (8993)

\ ATPase8

Complex I genes (NADH dehydrogenase)

~

Complex IV genes (cytochrome c oxldase )

m

Complex H1 genes (ubiquinol : cytochrome c oxldoreductase) Complex V genes (ATP synthase)

- - - - - ] Transfer RNA genes

~

Ribosomal RNA genes

Figure 1 The human mtDNA genome, showing nucleotide positions (parenthesis) of mtDNA point mutations associated with disease. Only the four primary mutations of Leber hereditary optic neuropathy at nps 3460, 4t60 11778 and 15257 are indicated. On = heavy-strand replication origin; O e = light-strand DNA replication origin; Pn = promoter for heavy-strand transcription initiation; PL = promoter for light-strand transcription initiation

heteroplasmy in maternal relatives, and the median age of onset is 24 years but ranges from 8 to 60 years (Newman et al 1991a). For the remaining mutations, the np 3460 mutation changes an alanine to threonine in ND1 (Howell et al 1991, Houponen et al 1991); the np 15 257 mutation changes an aspartate to asparagine

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475

in cytb (Brown et al 1991, 1992); and the np 4160 mutation changes a leucine to proline in ND1 (Howell et al 1991b). The remaining six mutations in Leber hereditary optic neuropathy are generally found in conjunction with other mutations and appear to act synergistically to either increase or decrease the severity of the disease. These include mutations at np 4136 in ND1 which may ameliorate the effects of the np 4160 mutation (Howell et al 1991b); at np 4216 in ND1; at nps 4917 (Johns and Berman 1991); and 5244 in ND2 (Brown et al 1991, 1992); at np 13 708 in ND5 (Johns and Berman 1991; Brown et al 199t, 1992); and at np 15812 in cytb (Brown et al 1991, 1992). The synergistic interaction of the mtDNA mutations in Leber hereditary optic neuropathy is best demonstrated by one mtDNA lineage in which four mutations have accumulated sequentially, each increasing the probability that the individual will be affected. The first genotype was the np 13708 mutation alone. This was present in 8% of patients without the 11 778 mutation but also in 5% of controls. The second genotype included the npl3 708 + n p 15 257 mutations and was found in 8% of patients and 0.3% of controls. The third genotype encompassed the np 13708 + np 15257 + np 15812 mutations and was present in 4% of patients and 0.1% of controls, and the fourth included the np 13 708 + np 15257 + np 15 812 + a heteroplasmic np 5244 mutation and was present in 4% of patients and no controls. Thus as the number of deleterious mutations increased, the individual's respiratory capacity decreased. Consequently, the probability increased that an organ's energy production would fall below its energetic threshold, raising the chances for phenotypic expression (Brown et al 199t, 1992). mtDNA missense mutations have also been associated with a quite different phenotype: neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP). NARP results from a base substitution at np 8993 which changes a leucine to an arginine in the ATPase 6 gene. The mutation has been found to be heteroplasmic and to segregate rapidly, and variation in the genotype has been found to be associated with variation in the phenotype (Holt et al t990). Two pedigrees have now been found in which high proportions of the heteroplasmic NARP mutation are associated with Leigh disease, a lethal childhood disease associated with basal ganglial degeneration (Tatuch et al 1992; Shoffner et al, in press).

mtDNA PROTEIN SYNTHESIS MUTATIONS

A number of deleterious mtDNA point mutations in tRNA genes have also been identified. Each mutation is generally associated with a distinctive phenotype, though phenotypic overlap is common between pedigrees resulting from tRNA defects. Myoclonic epilepsy and ragged red fibre (MERRF) disease is associated with uncontrolled myoclonic jerks and a characteristic muscle pathology involving degeneration of the oxidative, type I, muscle fibres and accumulations of large numbers of abnormal mitochondria that stain red with Gomori modified trichrome. However, the phenotype of patients varies markedly along the maternal lineage, with only occasional individuals exhibiting the complete syndrome, and the severity of the phenotype is proportional to the degree of Complex I + IV deficiency in the J. Inher. Metab. Dis. 15 (1992)

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patient's skeletal muscle mitochondria (Wallace et al 1988b). MERRF results from a base substitution at np 8344 in the T¢C loop of tRNA TM (Shoffner et al 1990; Zeviani et al 1991a), causing inhibition of mitochondrial protein synthesis (Wallace et al 1986) (Figure: 2). The proportion of mutant mtDNAs varies among maternal relatives, and for individuals of the same age the percentage of mutant mtDNAs roughly correlates with the phenotype. However, individuals with similar genotypes develop progressively more severe biochemical defects and clinical symptoms as they age (Shoffner et al 1990), suggesting that the natural age-related decline in oxidative phosphorylation exacerbates the biochemical defect imparted by the mutation. If so, this concept could explain the late onset and progressive nature of many mtDNA diseases (Wallace 1992). Mitochondrial encephalomyopathy, lactic acidosis and stroke-like symptoms (MELAS) is also due to a mutation in a mtDNA tRNA gene. This disease is associated with transient stroke-like episodes and mitochondrial myopathy and is frequently caused by a heteroplasmic mutation at np 3243 in tRNA Leu(UuR)which also alters a transcriptional terminator element (Goto et al 1990; Hammans et al 1991; Hess et al 1991). A second mutation at np 3260 also alters tRNA Leu(uUR), but not the transcriptional terminator, and results in maternally inherited mitochondrial myopathy and hypertrophic cardiomyopathy (MMC) (Zeviani et al 1991b). Several additional tRNA mutations have also been attributed to disease phenotypes (Tanaka et al 1990; Yoon et al 1991; Lauber et al 1991), but the relationship between genotype and phenotype for many tRNA mutations remains to be clarified.

The np8344 MERRF mutation in tRNA Lys a'OH

A ~'p- C- G A-T C-G T-A G - C

T

T-A AAA

DHU LOOP

T TTCTC C

I

I

I

TTAGC

AC A A

,k.k&ka

AATCG C

• C LOOP

I

TT A T-A A GA T-A

c

AI-AI c

~, []

A-T A-T

cC-G A T A TTT ANTICODON

Figure 2 Human mitochondrial tRNALy~showing the A-to-G transition at np 8344 J. lnher. Metab. Dis,

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m t D N A Point Mutations

Table 1 Parkinson disease oxidative phosphorylation enzymoiogy Controls

Parkinson disease patie~lts

Complex

Assay ~

N

mean _+ SD

5%

1

2

3

4

I I+HI II]( II+III IV

NADH-DB NADH cyt c DBH2 cyt c Succ c y t c Cyt c oxidase FT Sonicated

11 11 11 11

194 + 60 229_+ 149 1952 + 700 605_+164

54 0 323 223

16 8 633 188

20 27 74 92 174 13 16 80 75 126 895 1206 2110 1164 2009 259 511 462 284 490

9 11

1426_+433 1615 +_ 309

373 896

1482 63

758 1772 t323 648 843 941

5

6

326 1026 677 1054

~ADH--DB = NADH-n-decyl coenzyme Q oxidoreductase; NADH-cyt c = NADH-cytochrome c oxidoreductase (rotenone-sensitive fraction); DBH2-cyt c = reduced n-decyl coenzyme Q-cytochrome c oxidoreductase; Cyt c oxidase = cytochrome c oxidase; FT = freeze-thaw bN, mean ± SD, 5% confidence level for older controls (age 45--69 years): complex I +III = 6, 135 _+66, 0 and complex II +III = 6, 606 ± 165, 148 Enzyme activity is reported in nanomoles of substrate per min per mg mitochondrial protein Reproduced from Wallace et al (in press), by permission

OXIDATIVE PHOSPHORYLATION D E G E N E R A T I V E DISEASES

DEFECTS IN C O M M O N

The analysis of the above rare m t D N A - b a s e d disorders indicate that a variety of late-onset neurological s y m p t o m s can result from m t D N A mutations associated with defects in the mitochondrial oxidative p h o s p h o r y l a t i o n enzymes. This has suggested that other neurodegenerative diseases m a y also result from oxidative phosphorylation dysfnnction. Oxidative phosphorylation defects have been observed in the platelet mitochondria of Alzheimer (Parker et al 1990) and Parkinson (Parker et al 1989) disease patients. In Parkinson patients, oxidative phosphorylation defects have also been reported in the substantia nigra of the brain (Mizuno et al 1989; Schapira et al 1989, 1990) and in isolated skeletal muscle mitochondria (Bindoff et al 1991; S hoffner e~: al t991). Interestingly, in one muscle mitochondrial study, four of the six patients had highly significant oxidative phosphorytation defects but the enzyme complexes affected varied (Shoffner et al t991; Wallace et al 1992) (Table 1). This suggests that the same disease can result from a variety of biochemical and presumably genetic defects, a portion of which might be m t D N A mutations. REFERENCES Anderson S, Banker AT, Barrell BG et al (1981) Sequence and organization of the human mitochondriM genome. Nature 290:457 465. Bindhoff LA, Birch-Machin, MA, Cartlidge NEF, Parker Jr WD, TurnbulI DM (1991) Respiratory chain abnormalities in skeletal muscle from patients with Parkinson's disease. J Neurol Sci 104: 203-208. Brown WM, George M, Wilson AC (1979) Rapid evolution of animal mitochondrial DNA. Proc Natl Acad Sci USA 76: 1967-1971. Brown MD, Lott MT, Voljavec AS, Torroni A, Wallace DC (1991) Mitochondrial DNA cytochrome b mutations associated with Leber's Hereditary Optic Neuropathy and evidence for deleterious interactions between mutations. Am J Hum Genet (SuppI) 49: 183.

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