Am. J. Hum. Genet. 49:939-950, 1991

Leber Hereditary Optic Neuropathy: Identification of the Same Mitochondrial NDI Mutation in Six Pedigrees Neil Howell,* L. A. Bindoff,t D. A. McCullough,* 1. Kubacka,* J. Poulton,$ D. Mackey,§ L. Taylor,t and D. M. Turnbullt *Biology Division, Department of Radiation Therapy, University of Texas Medical Branch, Galveston; tDivision of Clinical Neuroscience, University of Newcastle upon Tyne Medical School, Newcastle upon Tyne; $Department of Paediatrics, John Radcliffe Hospital, University of Oxford; and §The Murdoch Institute, Royal Children's Hospital, Melbourne

Summary Biochemical and molecular genetic evidence is presented that in six independent pedigrees the development of Leber hereditary optic neuropathy (LHON) is due to the same primary mutation in the mitochondrial ND1 gene. A LHON family from the Newcastle area of Great Britain was analyzed in depth to determine the mitochondrial genetic etiology of their disease. Biochemical assays of mitochondrial electron transport in organelles isolated from the platelet/white-blood-cell fraction have established that the members of this family have a substantial and specific lowering of flux through complex I (NADH-ubiquinone oxidoreductase). To determine the site of the primary mitochondrial gene mutation in this pedigree, all seven mitochondrial complex I genes were sequenced, in their entirety, from two family members. The primary mutation was identified as a homoplasmic transition at nucleotide 3460, which results in the substitution of threonine for alanine at position 52 of the ND1 protein. This residue occurs within a very highly conserved hydrophilic loop, is invariantly alanine or glycine in all ND1 proteins, and is adjacent to an invariant aspartic acid residue. This is only the second instance in which both a biochemical abnormality and a mitochondrial gene mutation have been identified in an LHON pedigree. The sequence analysis of the ND1 gene was extended to a further 11, unrelated LHON pedigrees that had been screened previously and found not to carry the mitochondrial ND4/R340H mutation. The ND1 /A52T mutation at nucleotide 3460 was found in five of these 11 pedigrees. In contrast, this sequence change was not found in any of the 47 non-LHON controls. The possible role of secondary complex I mutations in the etiology of LHON is also addressed in these studies.

Introduction

Leber hereditary optic neuropathy (LHON) is a mitochondrial genetic disease. In addition to strict maternal inheritance, the typical ophthalmological features include a severe loss of vision with centrocecal scotomata which occurs in early adulthood (Lundsgard 1944; van Senus 1963; Seedorff 1970; Nikoskelainen et al. 1987). A major advance in the understanding of the etiology of LHON came from the studies of WalReceived May 8, 1991; revision received June 20, 1991. Address for correspondence and reprints: Neil Howell, Biology Division, Department of Radiation Therapy, University of Texas Medical Branch, Galveston, TX 77550. © 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4905-0004$02.00

lace et al. (1988; Singh et al. 1989). They showed that in nine of 12 LHON families the disease was associated with a mitochondrial gene mutation at nucleotide 11778 which produces a substitution of histidine for arginine at position 340 of the ND4 protein, one of the seven mitochondrially encoded subunits of complex I (NADH-ubiquinone oxidoreductase). Subsequent screening studies by other laboratories have shown that about one-half of all LHON pedigrees carry this mutation (Holt et al. 1989; Vilkki et al. 1989; Poulton et al., in press). Further understanding of the etiology of LHON clearly requires a more complete elucidation of the mitochondrial gene mutations that have occurred in families which do not carry the ND4/R340H mutation (to maintain consistency, all mutations are desig939

Howell et al.

940

nated herein by their amino acid substitution). Toward that end, we have begun nucleotide sequencing analyses of the mitochondrial complex I genes in a series of LHON pedigrees from the United Kingdom and Australia. These studies have already demonstrated that the severe form of LHON in one Australian family is due to a mutation in the mitochondrial ND1 gene that results in the substitution of proline for the leucine at amino acid position 285 (Howell et al. 1991). The most important issue when analyzing nucleotide sequence data is judging whether a mutation is a causative factor in the etiology of LHON or an adventitious occurrence. This is a particularly acute problem with the rapidly evolving complex I genes, since numerous replacement substitutions are commonly observed (for an example, see Howell et al. 1991). The following are two criteria that have been applied in the present studies to differentiate etiologically important mutations from polymorphisms: 1. The mutation should be specific to LHON patients or family members and not found in non-LHON controls. The confidence concerning a particular mutation is increased if it is found to have occurred independently in more than one LHON family. 2. The mutation should result in a substitution which is likely to deleteriously affect function of complex I. Thus, nonconservative changes are particularly good candidates. Further support for causation would come from a demonstration that the amino acid substitution is predicted to alter the secondary or tertiary structure of the protein. An important corollary is that a defect in complex I activity should be demonstrable in the LHON patients. In an effort to adopt a consistent terminology, mutations which fulfill these two criteria are referred to as "primary" in our studies. The ND4/R340H (Wallace et al. 1988) and the ND1 /L285P (Howell et al. 1991) LHON mutations would be the primary genetic lesions which have been identified prior to the present report. In contrast to primary mutations, Johns and Berman (1991) have recently presented evidence that secondary mutations in the mitochondrial complex I genes may be involved in the etiology of LHON. The three substitution polymorphisms which they identify as secondary mutations occur in both LHON and non-LHON individuals but at a much higher frequency in the former. The studies presented here are part of our continuing efforts to identify the primary mitochondrial gene mutations in non-ND4/R340H LHON pedigrees.

The results obtained also bear on the question of secondary complex I mutations in the etiology of LHON. Experimental Procedures Isolation of Mitochondria and Biochemical Assays

Platelets were isolated from heparinized whole blood by centrifugation at 190 g for 1S min at room temperature. The supernatant of platelet-rich plasma was aspirated and recentrifuged at 2,800 g at room temperature to yield a platelet pellet. Mitochondrial fractions were then isolated by homogenization and differential centrifugation as described elsewhere (Singh Kler et al., in press). The activities of respiratory-chain complexes I (NADH-ubiquinone oxidoreductase), II (succinateubiquinone oxidoreductase), and IV (cytochrome c oxidase) were assayed as detailed elsewhere (BirchMachin et al. 1989). Citrate synthase was measured according to the method of Shepherd and Garland (1973). Each assay was performed in triplicate, with at least one reaction carried out at a different protein concentration to ensure linearity of rate with this variable. The variation among the three independent assays was always less than 10%. Protein was estimated using the modified Lowry procedure (Pedersen 1977). Cloning and Sequencing Strategy

The cloning and sequencing strategy of the mitochondrial complex I genes has been described in detail elsewhere (Howell and McCullough 1990; Howell et al. 1991). DNA was isolated, by standard procedures, from the white-blood-cell/platelet fraction of blood samples taken, with informed consent, from LHON and non-LHON subjects. The exception was that in two instances the DNA from non-LHON controls was isolated from muscle biopsy samples. All DNA samples were encoded with a four-digit laboratory sample number to ensure confidentiality. The seven mitochondrial complex I genes were PCR amplified as a series of 23 overlapping fragments. For each complex I gene fragment, a minimum of four independent PCR reactions were pooled; this was done to ensure that replication errors during amplification were diluted sufficiently to avoid being scored as mutations within the mitochondrial gene fragment. In several instances, nucleotide changes were heteroplasmic as evidenced by two types of M13 clones, those containing mtDNA inserts with the wild-type sequence and others containing mtDNA with the

941

Leber Hereditary Optic Neuropathy

basepair substitution. In these cases, PCR amplification and nucleotide sequencing were repeated, and the two sets of results were pooled: close correspondence between the two sets of results was uniformly observed. The PCR primers contained Sau3A or, in the case of one gene fragment, BglII restriction sites to facilitate cloning into M13 mpl 8 / 19 sequencing vectors. The recombinant M13 vectors were used for nucleotide sequencing using the modification of the dideoxy chain-termination method developed by Biggin et al. (1983). Data Analysis

Nucleotide sequencing data were stored and analyzed using the software developed in this laboratory (Howell and Howell 1988). Modeling of the ND1 protein was carried out using the PC GENE software package (Intelligenetics, Inc.). The programs used in these studies include the hydropathy analysis of Kyte and Doolittle (1982), with a window size of 11 residues; the Garnier et al. (1978) and GGBSM (Gascuel and Golmard 1988) methods for prediction of secondary structure; and three methods for prediction of hydrophobic transmembrane helices (Eisenberg et al. 1984; Klein et al. 1985; Rao and Argos 1986). LHON Pedigrees The LHON pedigrees from the United Kingdom (UK), Australia (AU), and the United States (US) that are analyzed in the present study are briefly described below. Standard diagnostic criteria were used (e.g., see Poulton et al., in press). All pedigrees were prescreened and shown not to carry the ND4/R340H LHON mutation (this was confirmed in this laboratory by nucleotide sequencing). Some of the UK LHON patients have been described elsewhere (Poulton et al., in press), and the identification numbers used in that study are shown below in parentheses. Pedigree A.-UK/0001-UK/0004 and UK/0037UK/0041 are members of a small LHON pedigree from the Newcastle area of Great Britain (figure 1).The disease in this family appears to be limited to the characteristic optic neuropathy. It should be noted

that family member 1-2 (UK/0039), husband of I-1, is an internal normal control. Pedigree B. - US /0006 is an affected male with no previous family history of LHON. Pedigree C. - UK/0007 (22 of Poulton et al., in press) is an affected male with no previous family history of LHON.

*1 P:2 451

5

6

Partial pedigree of Newcastle LHON family. The Figure I blackened figures indicate those family members who are afflicted with the optic neuropathy; III-1 is about 2 years old. The asterisks (*) denote individuals whose mitochondrial ND1 gene was sequenced.

Pedigree D.-UK/0009 (28 of Poulton et al., in press) is an affected female with a previous family history of LHON. UK/0008, UK/0087, UK/0088, and UK/0089 are her four sons, of which only the first is clinically affected. Pedigree E.-UK/0010 is the normal mother of an affected son, UK/0085 (19 of Poulton et al., in press), and an unaffected daughter, UK/0094. There is no previous history of LHON in this family. Pedigree F.-UK/0011 (9 of Poulton et al., in press) is an affected male with no previous family history of LHON. Pedigree G.-UK/0012 (21 of Poulton et al., in press) is an affected male from a family in which there is a history of LHON. Pedigree H.-UK/0013 (20 of Poulton et al., in press) is an affected male in which there is no previous family history of LHON. Pedigree 1. -AU/0014 is an affected male and AU! 0016 is an affected female from a large Tasmanian LHON family. Pedigree J. -AU /0017 is an affected male and AU! 0018 is an affected female from a small Australian LHON family. There are also minor neurological abnormalities of sensorineural hearing loss and epilepsy in this family (D. Mackey, unpublished data). Pedigree K. -AU/0019 is an affected female from a small Australian LHON family. Pedigree L. - UK/0022 (31 in Poulton et al., in press) is an affected male from an LHON family. Results The Newcastle LHON Family

This LHON family (pedigree A) was selected for detailed biochemical and molecular genetic analyses. As shown in figure 1, there are three affected members within generations I and II; family member 111-1 is 2

942

Howell et al.

Table I Specific Activities of Mitochondrial Respiratory-Chain Complexes SPECIFIC ACITIVITY

INDIVIDUAL(S) (age in years) I-1 (46) 1-3 (54) 1-5 (50) II-1 (23)

Complex I (nmol NADH oxidized/min/mg mitochondrial protein)

Complex II (nmol DCPIP reduced/min/mg mitochondrial protein)

Complex IV (apparent first order rate constant, per s/mg mitochondrial protein)

Citrate Synthase (nmol DTNB reduced/ min / mg mitochondrial protein)

3.4 2.4 2.8 5.4

3.7

33.6 28.9 76.5 78.9 66.2 56.8

.41 .48 .43 .34 .28 .39

420 296 309 273 197 299

16.0 8.4-27.9

47.9 21.9-84.6

.26 .16-0.37

176 166-219

.............

.............

.............

(30) II-5Mean

OF

............

............

4.5

............

Controls (n = 5; age range 30-62 years): Mean

Range

............

............

years old and too young to express the optic neuropathy. The maternal inheritance pattern is characteristic, as is the fact that not all maternally related family members are afflicted (also see Lundsgard 1944; van Senus 1963; Seedorf 1970; Nikoskelainen et al., 1987).

Assays of Mitochondrial Respiratory-Chain Complexes Platelet mitochondria were isolated from five members of the Newcastle LHON pedigree and assayed for flux through mitochondrial respiratory-chain complexes I, II, and IV. The values shown in table 1 are activities expressed on the basis of mitochondrial pro-

tein concentration. It is clear that in the LHON family members the specific activity for complex I is lowered compared with the values found for five age-matched normal controls. The mean and range of values for complex I specific activities for mitochondria from the normal controls were very similar to those obtained by Parker et al. (1989) in their assays with platelet mitochondria. In contrast, the specific activities of complexes II and IV and of citrate synthase were in the normal range or elevated. Since mitochondrial fractions are invariably contaminated with nonmitochondrial proteins, it is important to relate enzyme activity to a mitochondrial marker enzyme. In these

Table 2 Activities of Respiratory-Chain Complexes, Normalized to Activity of Citrate Synthase ACTIVITY

INDIVIDUAL(S) (age in years) I-1 (46) 1-3 (54)

I-5 (50) Il-1 (23) II-5 (30) Mean

..............

..............

.............. ..............

.............. ..............

OF

Complex I (nmol NADH oxidized/ citrate synthase [nmol DTNB reduced] x 103)

Complex II (nmol DCPIP reduced/ citrate synthase [nmol DTNB reduced]

Complex IV (apparent first-order rate constant, per s/citrate synthase [nmol DTNB reduced] x 103)

8.3 8.1 9.1 19.7 22.6 13.6

.08 .26 .09 .29 .34 .21

.98 1.61 1.41 1.24 1.44 1.34

87.6 57.4-127.3

.27 .13-.47

1.51 .94-2.20

Controls (n = 5): Mean

Range

..............

..............

Leber Hereditary Optic Neuropathy

943

Table 3 Nucleotide Sequence Changes in Mitochondrial Complex I Genes of Newcastle LHON Family Members

Gene and Nucleotide

Sequence

Change

Amino Acid Residue

ND1: 3423 3460

.........

.........

GC-TAa GC-AT

VAL39 unchanged ALA52-THR

TA-CG AT-GCa GC-ATa

ASN78 unchanged MET100 unchanged GLN172 unchanged

AT-GC TA-bCGa AT- GC GC -ATa

THR13- ALA ASN192 unchanged LEU233 unchanged

GC-AT GC- CGa CG- TA AT-GC

LEU12 unchanged GLY456-aARG THR533- MET

CG-ATa CG-GCa CG-GCa CG-GCa GC- AT

PRO159- THR PHE134- LEU VAL103 unchanged PHE102- LEU VAL38 unchanged

ND2:

4703 4769 4985 ND4/ND4L:

.........

.........

.........

10,506 11,335

.........

.........

11,467 11,719 ND5: 12,372 ......... 13,702 ......... 13,934 ......... 14,139 ......... ND6: -14,199 - 14,272 - 14,365 .........

.........

.........

.........

.........

-

14,368

-

14,560

.........

.........

GLY320 unchanged

LEU601 unchanged

a Also found in mitochondrial ND genes of normal control subjects (data not shown).

experiments, citrate synthase is used for this purpose, and the results (table 2) once again show a marked lowering in complex I activity for the LHON family members. It is interesting that complexes II and IV show greater concordance between LHON family members and normal controls when the activities are expressed on the basis of citrate synthase activity. These results suggest that the specific activities of complexes II and IV and of citrate synthase are increased in the LHON patients. A Mutation in the ND I Gene

To determine the sites of mitochondrial gene mutations in the Newcastle LHON family, the seven complex I genes from two family members (I-1 /UK0001 and II-1 /UK0002 in the lineage shown in fig. 1) were sequenced in their entirety. This represents the analysis of 506 M13 clones and about 150,000 bp of DNA sequence. The results obtained were identical for both individuals. As shown in table 3, there were 18 nucleotide changes from the standard Cambridge sequence (Anderson et al. 1981). Ten of these have also been

found in non-LHON control subjects and are not considered further (seven have been found in all LHON and non-LHON individuals analyzed thus far in this laboratory and may represent errors in the Cambridge sequence (for additional discussion, see Howell et al. 1991). Of the remaining eight nucleotide changes, five produce silent polymorphisms and, as a result, are also eliminated from further consideration as primary LHON mutations. It was found that the sequence change producing the ND6/VAL38 silent polymorphism was heteroplasmic in both family members (data not shown). The three remaining nucleotide changes produce alterations in the amino acid sequence of three complex I subunits (table 3) and are designated as follows: ND1/A52T, ND4L/T12A, and ND5/T533M. Of these three, the ND1 /A52T mutation was the clear choice as the primary mitochondrial genetic cause of the disease in this family, despite the fact that this amino acid substitution is classed as highly conservative in mutation substitution matrices (Schwartz and Doyhoff 1978) and in comparative three-dimensional structure studies (Bordo and Argos 1991). However, within the specific context of the ND1 amino acid sequence bracketing the ALA52 residue, the threonine substitution is clearly nonconservative (table 4). At the position equivalent to the ALA52 residue of the human ND1 protein, only alanine and glycine are found among the proteins from evolutionarily diverse species. Furthermore, this entire region of the protein shows a remarkable degree of sequence conservation, particularly with regard to acidic and basic residues. The alanine or glycine residue at position 52 is adjacent to an invariant aspartic acid at position 51, while there are invariant lysine residues at positions 54 and 58. With the exception of the ND1 proteins from Paramecium and trypanosomes, there is a glutamic acid residue at position 59. In addition to the conservation of these highly polar amino acid residues, there is a hydrophobic residue at position 53, while there are consensus hydrophobic or aromatic residues at positions 55-57. This stringent amino acid sequence conservation is striking in view of the low overall sequence conservation (about 20%) of the ND1 proteins from liverwort chloroplasts and Paramecium. The ND1 protein is a highly hydrophobic protein, as shown by its Kyte-Doolittle hydropathy profile (fig. 2). The conserved segment containing the ALA52 residue and spanning residues 50-59 is within one of the four major hydrophilic regions and is predicted to have alpha-helical secondary structure. All methods

Howell et al.

944 Table 4 Sequence Comparison of ND I Proteins Amino Acid Sequencea

Reference

58 52 A -D-A-M-K-L-F-T -K - E A - D -A- I - K-L-F- I -K - E -M-K-L-F-M-K-E A -D A -D-A-M-K-L-F-M-K-E A -D-G-V-K-L-F- I -K-E A -D-G-V-K-Y-F- I -K -E A -D-G-M-K-V-FI -K - E C -D-A- I -K-L-F-T - K - E A -D-A- L -K-L-L-L - K - E -K - E A -D-A- L -K-L-L-L A -D-A- L -K-L-L-L - K - E W-D-G-L-K-L-G-V - K - E T - D - G - V - K - L - F -V - K - F A - D - G - I - K L F- L- K E A -D-A- L-K-L-F-L - K - G

Anderson et al. 1981 Anderson et al. 1982 Gadaleta et al. 1989 Bibb et al. 1981 Desjardins and Morais 1990 Roeet al. 1985 Jacobs et al. 1988 Clary and Wolstenholme 1985 Burger and Werner 1983 Brown et al. 1983 Cummings et al. 1988 Boer and Gray 1988 Hensgens et al. 1984 Kohchi et al. 1988 Pritchard et al. 1990

Species Human ................... Bovine ................... Rat ......... .......... Mouse ................... Chicken ................... Xenopus ................... Sea urchin ................... Drosophila ................... Neurospora ...................

Aspergillus ................... Podospora ................... Chlamydomonas Trypanosome .................. Chloroplast ................... ..............

-

Paramecium ...................

a Numbered according to human ND1 protein.

used predict that the ND1 protein has five hydrophobic transmembrane helices, with one spanning residues 68-88. Thus, the conserved hydrophilic loop lies just proximal to this transmembrane helix. The region proximal to this hydrophilic loop lacks a clearly predicted secondary structure and may be random coil or extended.. * this n -I.... . .role . ...... In view of the likely mutation as the of primary etiologic event, the relevantt region of the ND1 gene has been cloned and sequienced from six -

.1

Is

**

................*

x 0 D 0

0

I

1

60

120

180

240

300

Amino Acid Position

Figure 2

Hydropathy profile of mitochon drial ND1 protein.

The larger arrow indicates the hydrophilic loop in which the A52T substitution is found. The smaller arrow indica the location of the L285P substitution which occurs in the C family (Howell et al. 1991). It should be noted th atneithermutation changes the ND1 protein hydropathy profile.

tes

additional maternally related family members (fig. 1). All were found to carry the mutation at nucleotide 3460. There is no evidence for heteroplasmy at this site in any of these eight individuals, as the mutation occurs among all 88 independent clones used for nucleotide sequencing. The ND I Mutation Occurs in Five Other LHON Pedigrees It was reasoned that, if the ND1 /A52T mutation is the primary genetic cause of the disease in the Newcastle LHON family, then it might occur also in other, unrelated LHON pedigrees. Conversely, the mutation should not be found in non-LHON controls. Therefore, this region of the ND1 gene was sequenced from a number of LHON and non-LHON individuals. Thus far, this mutation has not been observed within any of the 47 control subjects (a total of 434 independent M13 clones were sequenced); these controls included clinically normal individuals as well as those afflicted with non-LHON neurological or mitochondrial diseases (data not shown). The mitochondrial ND1 gene region spanning nucleotide 3460 has been sequenced for 11 additional LHON pedigrees. The ND1/A52T mutation was found in 12 members of five of these pedigrees (the nucleotide sequence of 185 M13 clones was determined). In contrast, this mutation was not found among seven members of the other six LHON pedigrees (a total of 53 clones analyzed). It should also be noted that none of the 12 LHON pedigrees carries the

Leber Hereditary Optic Neuropathy

94S

15/21

14/18

12/16

16/17

26/26

LHON pedigree D. For each family member, the Figure 3 fractions indicate the number of independent clones carrying the ND1 /A52T mutation, per total number of clones sequenced.

ND1 /L285P mutation identified in the Queensland LHON family. The results for the LHON pedigrees carrying the ND1 /A52T mutation are summarized briefly in the following paragraphs. I. Pedigree D.-The affected mother (UK/0009) was heteroplasmic for the mutation (15/21 clones contained the mutation), while her affected son (UK/ 0008) was homoplasmic (26/26 clones). The unaffected sons UK/0087, UK/0088, and UK/0089 were heteroplasmic. These results are shown in figure 3. 2. Pedigree E. -The unaffected mother (UK/0010) was apparently homoplasic for the mutation (15/15 clones), as were the affected son (UK/0085; 7/7 clones) and unaffected daughter (UK/0094; 9/9 clones). 3. Pedigree H.-This affected male was heteroplasmic for the mutation (9/23 clones). 4. Pedigree J.-Both the affected male (AU/0017) and the affected female (AU/0018) were apparently homoplasmic for the mutation (10 / 10 and 7/7 clones, respectively), although the total number of clones analyzed is small. 5. Pedigree 1.-This affected male (UK/0022) was homoplasmic for the mutation (16/16 clones). Screening of additional LHON patients for the ND1 /AS2T mutation should be facilitated by the fact that the transition at nucleotide 3460 results in the loss of AcyI and HgaI sites. In a preliminary survey of nine sporadic LHON patients from Australia, one further occurrence of the ND1 /A52T mutation has been found with this approach (D. Mackey, unpublished data) and has been confirmed in this laboratory by DNA sequencing (data not shown). The six LHON pedigrees carrying the ND1 /A52T mutation are unrelated, on the basis of the best available genealogical information. Two of these, pedigrees E and H, are affected males with no previous family history of LHON, although in the former instance the unaffected mother is known to carry the mutation. Although the genealogical data suggest that

the mutation has occurred independently in these six pedigrees, the optic neuropathy commonly skips generations, and such data therefore are not conclusive. To obtain more robust results, the distribution of mtDNA polymorphisms among these LHON pedigrees is being ascertained. The results obtained thus far are presented in table 5. While the data are limited to portions of the mitochondrial ND1, ND2, ND4L/ 4, and ND5 genes, there are clear-cut differences among these LHON pedigrees: they differ from each other at a minimum of three sites. These results confirm that these families are not closely related and thus support the independent origin of the ND1 /AS2T mutation. Assessing the Role of Secondary Complex I Mutations

Johns and Berman (1991) observed that three substitution polymorphisms in the mitochondrial complex I genes were found at a higher frequency in LHON patients (both ND4/R340H and non-ND4 LHON cases were studied) than in normal controls (ND1/Y304H, ND2/D15ON, and ND5/A458T). Representatives of the six ND1 /A52T LHON pedigrees have been analyzed for the occurrence for these substitutions, and the results are included in table 5. Johns and Berman (1991) reported that 19/28 (70%) of their non-ND4 LHON patients carried the ND1 /Y304H substitution, versus 4/49 (8%) normal controls. In contrast, this mutation was found in only 1 /6 (17%) of those carrying the ND1 /A52T mutation and in 5/20 (25%) of our non-LHON controls (these included both normals and non-LHON diseases). The ND2 / D15ON mutation occurs in one (17% ) of the six ND1 /A52T LHON pedigrees tested but was found in 10/28 (36%) non-ND4 LHON patients and 2/ 49 (4%) controls by Johns and Berman. Finally, they detected the ND5/A458T mutation in 12/28 (43%) non-ND4 LHON patients but in only 2/42 (5%) controls. This polymorphism was not found in the six ND1 /A52T LHON pedigrees analyzed. None of these three polymorphisms was found in the Queensland LHON family described elsewhere (Howell et al. 1991). The sequence analysis of the mitochondrial complex I genes in the Newcastle LHON family indicated that, in addition to the ND1 /AS2T mutation, there were two other sequence changes which altered the amino acid sequence of the protein: the ND4L/T13A and ND5/T533M mutations. The possibility has been tested that these sequence changes are associated at high frequency with LHON pedigrees and, specifi-

Howell et al.

946 Table 5 Mitochondrial DNA Polymorphisms among NDI/A52T LHON Pedigrees NUCLEOTIDE COORDINATE

ND1 /3460 ND1/3360 ND1 /4026 ND2/4580 ND2/4703 ND2/5263 ND2/5378 ND2/5472

ND2/5492

......

...... ...... ......

......

......

......

......

......

ND4L/ 10506 .. ND4L/10658 ND4L/10754 ND4/10825 ND4/11719 ND5/13711 ND1 /4216C ..... ND2/4917C NDS/13708C ....

...

...

.....

.....

.....

.....

AMINO ACID SEQUENCEa

AS2T A18A T240T M37M D78D A265V T303A L335L P341P T13A M63M L9SL M22M G320G A459T Y304H N1SOD A458T

NUCLEOTIDE CHANGE IN PEDIGREE

(family member)b

A (UK/0001)

D (UK/0008)

E (UK/0085)

H (UK/0013)

J (AU/0017)

L (UK/0022)

+ -

+ + +

+ -

-

-

-

+ NT -

-

+

-

+

-

-

-

-

-

+

-

+

-

-

+ -

-

+ -

+ -

+

+ -

+

+

-

-

-

+

-

-

+ -

+

+

-

+

+

-

The first letter indicates the amino acid residue present in the predicted wild-type protein sequence (Anderson et al. 1981) at that position, and the second letter is the amino acid residue in the protein from the LHON pedigree. Note that many of the nucleotide changes are silent polymorphisms which do not change the predicted amino acid sequence. b A plus sign ( + ) denotes presence of the nucleotide change; a minus sign (-) denotes absence of the nucleotide change. NT = not yet tested. c Substitution polymorphisms described by Johns and Berman (1991). a

cally, with those carrying the ND1 /A52T mutation. The ND4L gene has been sequenced for members of the other five ND1 /A52T LHON pedigrees (table 5) and in nine non-LHON controls; the ND4L/T13A substitution does not occur in any of these individuals. These results thus indicate that this substitution is most likely an infrequent polymorphism. The NDS/T533M mutation presents a more complicated picture. Nucleotide sequencing studies have revealed that five of the 12 LHON pedigrees analyzed here have five different replacement substitutions within a small segment of the C-terminal region of the NDS protein (data not shown). The situation is complicated by the fact that these NDS mutations occur both in LHON pedigrees that carry the ND1 / AS2T mutation and in those that lack it. Additional LHON pedigrees and controls are being analyzed to test comprehensively the possibility that LHON pedigrees frequently and selectively carry a secondary mutation in the NDS gene. The results of this study will be published separately.

Discussion

The results presented here provide only the second instance of a LHON pedigree in which an association has been found for maternal transmission of the optic neuropathy, a biochemical defect in mitochondrial complex I specific activity, and a nonconservative mutation (ND1 /A52T) in a mitochondrial gene encoding a complex I subunit. This mutation was also found in five other, unrelated LHON pedigrees. The ND1 / A52T mutation, therefore, is the primary mitochondrial genetic etiologic event in these LHON pedigrees, according to the criteria listed earlier. Since this mutation was found in about 50 % ofthe non-ND4/R340H pedigrees that were analyzed, and since 30%-50% of all LHON pedigrees do not carry the ND4/R340H mutation (Holt et al. 1989; Vilkki et al. 1989; Poulton et al., in press), it can be suggested by extrapolation that this genetic lesion is the primary mutation in 15%-25% of all LHON pedigrees. After the completion of these studies, it has been reported that three of 21 Finnish LHON pedigrees carry the ND1 /A52T

Leber Hereditary Optic Neuropathy mutation (Huoponen et al. 1991). Thus, the frequency of this mutation among the Finnish pedigrees is in agreement with our estimate. However, since the LHON pedigrees of both studies have been limited to Caucasians of northern-European descent, analysis of LHON patients of different ethnic backgrounds will be necessary to generalize this preliminary estimate. Conversely, the present results and those of Huoponen et al. (1991) both suggest that the disease in a substantial proportion of LHON patients is caused by mutations other than those encoding the ND4/R340H or ND1 /A52T substitutions. The ND1 /A52T substitution occurs within a highly conserved hydrophilic loop with a predicted alphahelical secondary structure. This loop is striking in the invariance of both acidic and basic residues, and it is likely that these sidechains are involved in the formation of a surface charge network or multivalent salt bridge. Such electrostatic interactions are often involved in the stabilization of protein tertiary structure (Barlow and Thornton 1983; Akke and Forsen 1990). The threonine substitution is adjacent to an invariant aspartic acid residue, and the additional electronegativity of the threonine hydroxyl group - and/or its greater sidechain volume - might distort or weaken the electrostatic interactions among the charged residues, thereby altering the tertiary structure of this loop and/or its dielectric environment (Saqi and Goodfellow 1990; Serrano et al. 1990). There are now two mutations in the mitochondrial ND1 gene which have been shown to be the primary genetic causes of LHON. In the large Queensland LHON family (Wallace 1970), the disease is caused by the ND1/L285P mutation (Howell et al. 1991). The ND1 subunit is the site of rotenone binding and is located within the hydrophobic core of complex I, where it probably plays an important role in the quinone-reduction portion of the catalytic pathway (reviewed in Ragan 1987). These two mutations present some interesting similarities. Both the ND1 / L285P and ND1 /A52T amino acid replacements occur within evolutionarily conserved hydrophilic alphahelical loops. The similar location of the two mutations is particularly striking, as the ND1 protein is highly hydrophobic and contains relatively few hydrophilic regions (fig. 2). Moreover, neither of these mutations occurs within the protein's central region, which has been postulated to form the quinone reductase site (Freidrich et al. 1990). The second similarity is that the disease in both the

947

ND1 /L285P Queensland and ND1-/A52T Newcastle LHON families is associated with a substantial lowering in flux through complex I (for the biochemical analysis of the Queensland LHON family, see Parker et al. 1989). The biochemical effects that these two mutations have on complex I structure and function remains an important area of investigation, particularly in light of the fact that the disease in the Queensland LHON family is accompanied by severe neurological abnormalities (Wallace 1970). In contrast, the disease in the Newcastle LHON family (pedigree A) appears to be limited to the optic neuropathy. One possible clue is that in the Newcastle LHON pedigree there may be a compensatory increase in mitochondrial biogenesis (table 1 and 2), to ameliorate the reduced flux through complex I. The demonstration of lowered complex I activity in asymptomatic members of the Newcastle LHON family is both intriguing and difficult to explain. Age at onset varies greatly in LHON patients, and one of the affected members of this (1-3) family first developed symptoms at age 38 years. Nevertheless, family members aged 46 years (I-1) and 50 years (I-5) remain asymptomatic, despite the fact that both carry the ND1 /A52T mutation and have lower complex I activity. These results, therefore, support the hypothesis that secondary factors, genetic or environmental, are involved in the etiology of LHON. Recent evidence has indicated that an X-chromosome locus influences expression of the optic neuropathy in LHON pedigrees (Vilkki et al. 1991). In view of the localization of both ND1 LHON mutations to hydrophilic loops (rather than in membrane-buried regions), one possible explanation as to why these particular amino acid replacements are etiologically important is that they generate autoantigens. That is, there may be an autoimmune component to the pathogenesis of LHON. Two recent observations are relevant in this regard. The first concerns the maternally transmitted antigen of mice, for which it has now been shown that the epitopes involved map to the N-terminal transmembrane domain of the ND1 protein (Loveland et al. 1990). Changes in the structure of the ND1 subunit therefore can be recognized by the immune system. Second, there is at least one example of a mitochondrial encephalopathy in which autoantibodies have been detected, although it is not known whether these are directly involved in the disease (Schapira et al. 1990). The work of Johns and Berman (1991) has raised

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the important possibility that LHON may have a multistep mitochondrial genetic etiology and that secondary complex I mutations may play a role in the clinical course of the disease. On the one hand, the results presented here do not provide clear-cut support for Johns and Berman's conclusions about the three mutations that they analyzed. There was no indication that any one of the three was frequently associated with the ND1 /A52T LHON pedigrees, although two (33%) of the pedigrees carried one of the three. On the other hand, the preliminary results did suggest that mutations in the 3' end of the NDS gene may occur at relatively high frequency in non-ND4 LHON pedigrees. It may be that etiology of LHON involves, at least in some pedigrees, a primary complex I mutation and any one of several secondary mutations, but much further work is necessary before rigorous conclusions can be drawn. That secondary mitochondrial gene mutations, specifically ones occurring within genes encoding complex I subunits, may influence the development of this disease is in accord with the evidence for an ND1 intragenic suppressor mutation in the Queensland LHON family (Howell et al. 1991). The mitochondrial genetic etiology of LHON is proving to be surprisingly complex both in terms of the number of primary complex I mutations identified thus far and in the likelihood that secondary mutations, both nuclear and mitochondrial, have an etiologic role. There is a corresponding complexity in the neurological abnormalities which can accompany the characteristic optic neuropathy (reviewed in Howell et al. 1991). It is tempting to speculate that the genetic and pathological complexities are mechanistically related. The continuing molecular genetic and biochemical analyses will clarify the etiology of LHON, but further surprises can also be anticipated.

Acknowledgments This research was supported by grants P01 HD 08315 from the National Institutes of Health and 2505-88 from the John Sealy Memorial Endowment Fund (both to N.H.) and by grants from the Muscular Dystrophy Group of Great Britain and from the Newcastle University Research Committee (both to L.A.B. and D.M.T.). D. M. was supported by a Clinical Research Fellowship of the Royal Children's Hospital. J. P. was supported by a grant from the Medical Research Council of the United Kingdom. This research was aided enormously by the efforts of Dr. J. Bronte-Stewart and Professor W. S. Foulds (Tennant Institute of Ophthalmology, University of Glasgow). We also acknowledge the advice and cooperation of our colleagues Drs. Davis Parker,

Howell et al. Jr., Frank Frerman, and Stephen I. Goodman at the University of Colorado Health Sciences Center. Technical assistance was provided by Mian Xu and Steven Halvorson; particular acknowledgment is made of the assistance of Barbara Howell in data analysis and presentation.

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