Editor—Mitochondrial DNA (MtDNA) is highly polymorphic. Each person is estimated to differ from another on average at about 25 base pairs among the 16 569 that comprise the mitochondrial genome.1 Thus, only a small fraction of mtDNA variants are likely to be of pathogenic significance. Criteria currently used for determining the likelihood that a missense mutation is pathogenic include heteroplasmy (the percentage of mtDNA molecules within a cell or tissue harbouring a mutation), evolutionary conservation of the altered amino acid, a maternal inheritance pattern, absence of the mutation in controls, clinical features commonly linked to known pathogenic mtDNA mutations, and defects in mitochondrial morphologies and enzyme activities.1 However, these criteria are inadequate for several reasons. Many mitochondrial missense mutations are homoplasmic. Pathogenic mtDNA mutations are typically characterised by incomplete penetrance, even when homoplasmic, presumably reflecting interactions with environmental and genetic factors.2 As a result, inherited mtDNA mutations may manifest as “sporadic” disorders rather than with the classical maternal inheritance pattern. Biochemical assays may also be inconclusive, as the expression of a defect in mitochondrial function depends on the nuclear background and tissue type in which the mutation is studied.3 ,4 As a result, mtDNA mutations identified in rare families or subjects with a putative mitochondrial genetic disorder are often of uncertain pathogenic significance.

Over 100 point mutations have been identified in mitochondrial genes in association with human disease, at least 45 of which are missense mutations in protein encoding genes.5 However, frameshift mtDNA mutations are exceedingly rare. An acquired frameshift 4 bp deletion mutation was identified in the cytochrome b gene at nucleotide position (np) 14 787-14 790 in a patient with parkinsonism-MELAS overlap syndrome6 and somatic mutations including frameshift mutations have been found in human cancers.7 ,8In contrast, inherited frameshift mutations in mtDNA have not previously been reported. We now report the identification of an inherited frameshift mutation in a patient with dystonia and maternally inherited cataracts. The normal base pair (T) is replaced by AC at np 3308 (T3308AC) in the mitochondrial gene encoding the ND1 subunit of complex I. Dystonia9 ,10 and cataracts11-13 have each been linked previously to complex I dysfunction and to mtDNA mutations but, for the reasons outlined above, the pathogenicity of the T3308AC mutation remains uncertain.

DNA was isolated by standard proteinase K and SDS digestion followed by phenol and chloroform extractions. DNA was isolated from muscle (III.4), fibroblasts (II.8), or blood (I.1, III.1, IV.1, IV.5, and IV.6). Each of these subjects (except IV.1) underwent neurological and ophthalmological examinations. Clinical and molecular data were unavailable from other family members. Polymerase chain reaction (PCR) amplification of mtDNA and sequencing on an ABI 377 automated sequencer (Perkin-Elmer) were performed as previously described.14PCR reactions for restriction digests were performed with primers at np 3207-3223 (upper) and 3414-3401 (lower). Restriction digests were performed with MsiI (New England Biolabs) and analysed by ultraviolet illumination of a 2% agarose gel permeated with ethidium bromide. The mutation eliminates the single restriction site for this enzyme. The normal 208 base pair (bp) PCR product is cut into two 104 bp fragments, but in the presence of the T3308AC mutation, a single 208 bp fragment remains. A normal control DNA sample was included in each assay to confirm complete digestion by the enzyme. Other PCR and sequencing primers have been published previously.14

Immunoblotting of the ND1 subunit of complex I was performed using lysates of fibroblasts obtained from three affected family members and one control. Samples (10 μg) were loaded and run on a 12% acrylamide minigel, rinsed, transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MD), and incubated in blocker containing primary antibody (1:500), as described previously.15 Membranes were thoroughly rinsed, then incubated with horseradish peroxidase conjugated secondary antibody. Secondary antibodies were detected by chemiluminescence (Amersham ECL, UK).

The proband (III.4, fig 1) developed a unilateral (right) cataract in her late teens. At the age of 30 she experienced several episodes lasting 30 minutes each of bilateral paraesthesias in her arms and legs with right hemifacial paraesthesias and paresis. Neurological examination showed sensory loss and weakness in the right face, as well as right arm weakness. The following year, examination showed a right facial dystonia and diffuse hyperreflexia with a positive Hoffman's sign on the left, but full strength and normal sensation. Plantar responses were flexor. Her sister (III.1) developed cataracts in her early teens with a severe left and mild right cataract. This sister's daughter (IV.1) died a few months after birth with a hypoplastic left heart, polycystic kidneys, and an ectopic pancreas. The maternal grandmother (I.1) developed a right cataract by the age of 40 years. There was no history of ocular trauma in any of the family members with cataracts.

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Figure 1

Pedigree. The darkened circle (III.4) represents the proband with early onset cataracts, focal dystonia, and episodes of paraesthesias. Cross hatched circles represent maternal relatives with early onset cataracts. Subject IV.1 died in infancy of multiple congenital defects. Clinical examinations and DNA analyses were available for I.1, II.8, III.1, III.4, IV.1, IV.5, and IV.6. See text for further clinical details.

An extensive evaluation of III.4 included normal brain computed tomography and magnetic resonance imaging. Electromyography and nerve conduction studies were normal. Muscle biopsy (right vastus lateralis) showed normal light and electron microscopic results. Cytochrome oxidase c staining was normal. No ragged red fibres were seen. Complex I activity, measured as rotenone sensitive NADH cytochrome c reductase activity normalised to citrate synthase, was normal. Cytochrome oxidase activity was normal. Citrate synthase activity was raised (11.08 μmol/min/g compared to 3.35 ± 1.1 for controls). Serum and cerebrospinal fluid lactates were normal. Serum ammonia was raised at 45 μmol/l (compared to normal of 9-33 μmol/l). Cerebrospinal fluid glucose and protein were normal with no cells or oligoclonal bands. Serum creatine kinase levels were normal.

Sequencing both the H and L strands of PCR amplified muscle derived DNA in III.4 showed a T to AC insertion/deletion at np 3308 (T3308AC) (fig2). This converts an ATA (methionine) codon to AACA, creating a frameshift in the initiating methionine codon of the mtDNA gene encoding the ND1 subunit of complex I. The presence of a homoplasmic mutation was confirmed by the elimination of anMsiI (New England Biolabs) restriction site in III.4 as well as in each of her maternal relatives from whom DNA was available for analysis (I.1, II.8, III.1, IV.1, IV.5, and IV.6). The mutation was absent in 108 control subjects including 29 with Parkinson's disease and 23 with adult onset focal dystonia. An initiating methionine at this site is highly conserved evolutionarily.16

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Figure 2

Electrophoretograms of subject III.4 (top) and a control showing a frameshift mutation at position 3308 where a T is replaced by an AC in subject III.4.

Sequencing of the entire mitochondrial genome in subject III.4 showed 17 known errors or consensus changes in the Cambridge sequence.17 Additional changes were observed as follows. Synonymous base pair changes: T6620C, C7028T, G11719A, G12007A, C12705T, and A14470G. Known polymorphisms in the non-coding D loop: C16223T, C16290T, G16319A, T16325C, T16362C, C64T, A73G, T146C, A153G, A235G, T310C, and C514CAC. Known polymorphisms in rRNA genes: A663G, A1736G, and A2706G. Known polymorphisms in protein coding genes associated with an altered amino acid: A4824G (ND1), G8027A (COX II), and C8794T (ATP6). Mutations at non-conserved sites within the non-coding D loop (but not known polymorphisms): C461T, C505T, and a TT insertion at np 311. The TT insertion at 311 is not a known polymorphism, but a CC insertion at this site is a known polymorphism. An insertion of a C at 956 occurs in a non-conserved region of the 12S rRNA gene. A mutation identified at 14 280 (A to G) in theND6 gene alters a non-conserved amino acid.

Immunoblotting detected the ND1 protein (apparent MW ∼33 kDa) in all fibroblast samples (fig 3). Thus, the ND1 protein is expressed in subjects with the frameshift mutation. An additional, minor, cross reacting band of unknown identity and significance was also seen.

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Figure 3

Western blot analysis of ND1 expression using a polyclonal ND1 specific antibody. Lanes 1, 2, and 4 are from subjects with homoplasmic mutations (lane 4 is the proband); lane 3 is a control subject. ND1 immunoreactivity (arrow) was clearly detected in all subjects. A minor cross reacting band was also seen (upper band).

Suspicion of a mitochondrial complex I gene mutation in this family was raised by the presence of dystonia and cataracts, each of which has been associated with complex I dysfunction and mtDNA mutations.9-13 The origin of the complex I deficiency in dystonia is unknown. In one study, lung carcinoma derived cell lines expressing mtDNA from nine patients with focal dystonia did not manifest complex I deficiency.18 However, the biochemical expression of mtDNA mutations is influenced by tissue type as well as nuclear background.3 ,4 Thus, the role of mtDNA mutations in the complex I defect of patients with focal dystonia remains uncertain. Dystonia clearly can be a prominent manifestation of mtDNA mutations, as shown by missense mutations in a mitochondrial complex I gene in several families with dystonia plus Leber's hereditary optic neuropathy (LHON).19-22 Dystonia is commonly a prominent component of Leigh's syndrome, which can be associated with mtDNA point mutations or deletions.23 ,24 Dystonia, along with other neurological deficits, was reported in a subset of affected members of a family harbouring the LHON associated 11 778 complex I mutation,14 and can be the presenting feature of the 3243 “MELAS” mutation.25 Cataracts have also been reported as a clinical correlate of mitochondrial complex I dysfunction11-13 and in patients with mtDNA mutations, including large deletions.13 The occurrence of both dystonia and maternally inherited early onset cataracts in our patient suggested the possibility of a mtDNA mutation.

Though a mtDNA mutation was suspected in this family, the identification of a frameshift mutation was surprising given the mild phenotype and the lack of any previous reports of inherited frameshift mutations in human mitochondrial genes. Translation of the ND1 subunit normally begins at the initiating methionine codon at np 3307-9. An initiating methionine is highly conserved evolutionarily.16 If translation begins at the 3307-9 codon for the mutant sequence, then the frameshift at 3308 would result in an asparagine (rather than methionine) as the initial amino acid and a premature stop codon after the 28th amino acid, whereas the full length ND1 subunit normally consists of 318 amino acids. Such an abnormality seems unlikely given the relatively mild clinical and biochemical features associated with the mutation. Alternatively, translation may begin in frame at the next methionine codon, which occurs at np 3313-5, resulting in a truncation of the first two amino acids (methionine and proline) at the amino terminal end of the ND1 subunit, with preservation of a methionine at the amino terminal end. Except for the initiating methionine, amino acids at the amino terminal end are not highly conserved evolutionarily.26

Mutations involving np 3308 have been reported in several patients with neurological abnormalities including two with dystonia. Camposet al 16 reported a woman with transient ataxia and later seizures and marked generalised dystonia who harboured a missense point mutation at np 3308. Two other unrelated patients, each with multiple neurological deficits, also were found to harbour a missense point mutation at 3308.27 One additional family with maternally inherited hearing loss attributed to the T7511C mtDNA mutation also harboured a T3308C point mutation28 but it was not thought likely to be pathogenic as it was homoplasmic and had been reported previously in controls.27 MtDNA mutations at np 3308 were not present in any of our 108 controls. Campos et al 16 also found no mutations at this site in 130 controls. In contrast, Vilarinho et al 27 reported that four of 150 controls harboured a point mutation at 3308. This may reflect the ethnic differences between these control groups. None of the combined 388 controls in these studies carried a frameshift mutation as found in the family reported here. However, the molecular consequences of a point mutation at this site may be identical to that of the T3308AC frameshift mutation, resulting in a truncation of the first two amino acids. The cellular impact of the T3308AC mutation is uncertain. A missense mutation in the initiating codon of the cytochrome c oxidase subunit II gene has been shown to result in lower levels of protein synthesis for this subunit.29 In contrast, we found no evidence of altered ND1 expression in association with the T3308AC mutation, and muscle complex I activity was normal. The possibility remains that complex I activity could be altered in the basal ganglia, the primary site of pathology in many dystonia patients.30 Furthermore, our complex I assay measures maximal enzyme activity (“Vmax”), and thus an effect of the mutation on enzyme kinetics cannot be excluded.

This is the first identification of an inherited frameshift mutation in a human mitochondrial protein coding gene. However, the pathogenic significance of this mutation remains uncertain. This case highlights the difficulties often encountered when interpreting associations between rare mtDNA variants and putative mitochondrial disorders.