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Since the identification of the first mtDNA point mutation in the ND4 gene of LHON patients (1), more than 100 mtDNA mutations in transfer RNA genes have been identified, but, over the past few years, increasing attention has been focused on mtDNA mutations in protein-coding genes. Point mutation in the ND1, ND3, ND4, ND5, and ND6 subunit genes of complex I have been found in various mitochondrial disorders (2–5). ND3 mutations were identified in patients with severe early-onset encephalopathy, Leigh syndrome, and progressive neurologic deterioration (6–8).

Here we report a novel heteroplasmic m.10197G>A mutation in the ND3 gene in three Korean children with bilateral basal ganglia lesions and partial deficiencies of respiratory chain complex I activity. These findings reinforce the concept that the protein-coding mtDNA genes are important causes of complex I deficiency in diverse mitochondrial syndromes.

PATIENTS AND METHODS

Informed consent was obtained from the parents of each patient for the clinical studies including tissue and blood sampling. The patient's tissues were studied under a Columbia University Institutional Review Board protocol.

Patient 1.

This 9-y-old girl was born at full term without complications. She was healthy until age 7 y when she developed right-hand weakness and emotional lability followed by progressive gait ataxia, dystonia, poor coordination, and dysarthria. On examination, she had a normal mental status, hyperactive tendon reflexes with ankle clonus, and cerebellar ataxia. Her venous lactate level was normal (1.5 mM, normal <2.5). There were no abnormalities in copper metabolism including serum and urine copper levels and serum ceruloplasmin level. Urine and serum amino acid and organic acid analysis were also normal. Her parents are healthy, but her older brother (patient 2) had similar symptoms. Magnetic resonance imaging (MRI) of her brain revealed increased signal intensities in both globus pallidi (Fig. 1 A). A muscle biopsy specimen showed moderate subsarcolemmal proliferation of mitochondria by succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) histochemical stains and strongly SDH-positive blood vessels (Fig. 2 A). No COX-deficient fibers were seen (Fig. 3).

Figure 1
figure 1

Brain MRI findings of three patients. Patient 1 (A) and patient 2 (B) showed similar abnormalities in both basal ganglia. (C) Patient 3 showed abnormal signal intensities in both basal ganglia and midbrain more prominent on right side.

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Figure 2
figure 2

SDH histochemistry of muscle biopsy specimens. (A) Muscle biopsy specimen of patient 1 shows strongly SDH-positive blood vessels (SSVs) (arrows). (B) Muscle biopsy specimen of patient 3 shows focal collections of subsarcolemmal mitochondria in some muscle fibers (arrows). Bar = 50 μM.

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Figure 3
figure 3

(A) Sequences of ND3 gene showing heteroplasmic change in patient 1 and a control. (B) Mismatch polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of mtDNA in the three probands, the parents of patient 3, and a control. Lane 1, uncut 245-bp PCR product; lane 2, normal control muscle DNA; lane 3, patient 1 muscle DNA; lane 4, patient 2 muscle DNA; lane 5, patient 3 muscle DNA; lane 6, blood DNA of the mother of patient 3; lane 7, blood DNA of the father of patient 3; and lane M, 100-bp DNA ladder.

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Patient 2. The older brother of patient 1, patient 2 was normal until age 4 y when he began to walk on his toes. His gait abnormality worsened, and he developed dysarthria, impaired fine motor coordination, and dystonia. He became increasingly ataxic and fell frequently. His tone was increased in all four limbs, with hyperactive tendon reflexes. His cognitive function was unaffected, and he was able to describe his symptoms well. His lactate level was borderline high (2.5 mM, normal <2.5). Serum and urine copper levels and serum ceruloplasmin level were normal, and there were no abnormalities on other metabolic screenings. His brain MRI was similar to that of his sister (Fig. 1B). On muscle histology, ragged-red fibers and COX-deficient fibers were absent, but SDH and COX histochemistry showed mild subsarcolemmal proliferation of mitochondria.

Patient 3.

He was born at full term with normal spontaneous uneventful delivery. At age 3 mo, he developed sudden left-sided weakness without cognitive changes. The left-sided weakness progressed slowly. At age 6 mo, he was evaluated at the Seoul National University Children's Hospital. Lactate levels were elevated in venous blood (22.3 mg/dL, normal <12.5) and normal in cerebrospinal fluid (2.3 mM, normal <2.5). There were no abnormalities in other metabolic screenings. His brain MRI showed abnormal high signal intensities in both basal ganglia and midbrain, more prominent on right side (Fig. 1C). Muscle histology demonstrated mild mitochondrial proliferations in some fibers on SDH (Fig. 2B) and COX staining without COX-deficient fibers. With physical therapy for 1 y, his left-sided weakness improved slowly.

Biochemistry.

Activities of respiratory chain enzyme complexes were measured in a supernatant from muscle homogenates and expressed relative to the activity of the citrate synthase, a marker of mitochondrial mass as described (9).

Mitochondrial DNA analyses.

Total DNA was extracted from muscle homogenates (PUREGENE, Gentra Systems, Inc., Minneapolis, MN). Direct sequencing of the ND genes was performed with the BigDye terminator cycle sequencing methodology on an ABI PRISM 310 Genetic Analysis (Applied Biosystems, Foster City, CA). The m.10197G>A mutation was further investigated by PCR-RFLP analysis, using mismatch primers to introduce recognition sites for the restriction endonuclease PmI I. Oligonucleotide primers forward 5′-GAGGGTTCTACTCTTTTAGT and reverse 5-AGAAGAATTTTATGGAGAAAGGGACGCACGAGG (mismatch nucleotide shown in bold italics) amplified a 245-bp DNA fragment. The relative proportion of wild-type and mutant mtDNA was determined by last-cycle hot PCR-RFLP analysis (6). The m.10197G>A transition creates a PmI I site, cutting the 245-bp fragments into 215-bp and 30-bp segments.

RESULTS

Biochemical assays of mitochondrial respiratory chain activities in muscle tissue homogenates from these three patients showed partial reductions of complex I activities (Table 1). Genetic screening for mtDNA rearrangements and tRNA mutations were negative. We sequenced ND coding regions of the patients' mtDNA and identified several nucleotide variants, which were previously reported as polymorphisms (patients 1 and 2: m.3618T>C, m.10873T>C, m.11864T>C, m.12810A>G, m.13359G>C, and m13620T>C; and patient 3: m.11719G>A, 12705C>T, m.14062A>G, and m.14067C>T), but one nucleotide transition, m.10197G>A in ND3 has not been described. This change changes amino acid 47 from alanine to theonine and was heteroplasmic with a level of 98% in patient 1, 86% in patient 2, and 80% in patient 3 by PCR-RFLP analysis (Fig. 2). The parents of patients 1 and 2 declined further investigation. The mutation was not detected in the blood of the mother of patient 3. This amino acid is highly conserved across different species (Table 2). This mutation was not found in 100 control muscle DNA samples (59 healthy and 41 neuromuscular patients).

Table 1 Respiratory chain enzyme activities in muscle homogenates of patients normalized to citrate synthase
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Table 2 Multiple sequence alignment of the ND3 protein segment containing the mutation
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DISCUSSION

Interest in mitochondrial diseases has been shifted from mtDNA to nuclear DNA, particularly in children, in part because a larger number of nuclear than mitochondrial genes are required for mitochondrial functions (10). Nevertheless, since the identification of the first point mutation of mtDNA in a protein-coding gene (1), the number of mutations in mtDNA genes encoding structural respiratory chain subunit has gradually increased.

We identified partial deficiencies of complex I in two siblings with childhood-onset dystonia, and another child with a strokelike episode in early infancy and bilateral basal ganglia lesions. Isolated complex I deficiency, the most frequent respiratory chain disorder in infants and children, is genetically and clinically heterogeneous. Mutations in seven mtDNA and nine nuclear genes encoding structural subunits have been associated with complex I deficiency and their clinical phenotypes have varied from severe encephalopathies such as Leigh syndrome to neonatal cardiomyopathy (11). The clinical phenotype of our two families differed; one had childhood-onset dystonia without cognitive impairment and the other had a solitary stroke; both are unusual clinical presentation of isolated complex I deficiency. There have been a few reports of patients with dystonia due to mitochondrial disorders, especially associated with complex I deficiency (12–14), but most of the patients also had deafness, cognitive impairment, or optic neuropathy. Generalized dystonia without cognitive impairment, as in our patients 1 and 2, was an uncommon clinical presentation. Considering the frequency of multiorgan involvement in mitochondrial disorders, we need to monitor carefully our patients for other organ involvement.

Despite the mild muscle morphologic findings and normal venous lactates, biochemical assay of respiratory chain enzyme revealed decreased activity of complex I. Based on the biochemical abnormalities, we performed direct sequencing of mtDNA-encoding complex I subunits and identified a novel m.10197G>A heteroplasmic mutation in ND3, which is probably a pathogenic mutation based on the following lines of evidence: (1) the transition is heteroplasmic at high levels in postmitotic tissue (skeletal muscle); (2) it presented with similar encephalopathies in siblings; (3) it causes a significant amino acid change (nonpolar to polar R group), at a site that is evolutionally highly conserved (Table 2); (4) this mutation is associated with reduced activity of respiratory chain enzyme complex I; and (5) it was not found in 100 normal and disease controls.

Interestingly, another missense mutation in ND3, m.10191T>C, causes an S45P change two amino acid upstream from our patients' mutation and was reported in patients with early-onset and progressive encephalopathy (6,15). This mutation in the hydrophilic loop situated between two of the three membrane-spanning α-helices of ND3 was confirmed to be pathogenic by cybrid cell analysis demonstrating that the m.10191T>C genotype caused severe complex I deficiency (15). The A47T mutation is likely to affect folding of ND3 and hence enzyme function via interaction with other complex I subunits (6).

Despite the nearly normal muscle pathology, mutation loads in skeletal muscle of our three patients were ≥80% and were associated with significant defects of complex I activity. This dissociation between morphologic and biochemical findings is not uncommon among mutations of mtDNA-coding structural respiratory chain subunits, particularly those affecting complex I and is clearly different from tRNA mutations which typically cause ragged-red and COX-deficient fibers (16,17).

Defects of complex I due to mtDNA or nuclear DNA mutations may comprise the most common cause of mitochondrial disease in infants and children who present with diverse severe progressive encephalopathies including Leigh syndrome (18). In particular, there is a growing number of reports describing ND5 mutations; therefore, this gene appears to be a pathogenic hotspot in these diseases (19–21). In light of recent reports describing mutations in ND3 (6,7,15), this gene could be another mtDNA hotspot for mutations in pediatric patients with diverse encephalopathies.

Classic mitochondrial syndromes are seen in only a small proportion of children investigated for mitochondrial disorders. Instead, the vast majority of children suspected of having a mitochondrial disease present like our three cases with nonspecific or unusual encephalopathies. This novel mutation in the ND3 gene also reinforces the concept that protein-coding genes in mtDNA are still significant and diagnostically challenging causes of disease in children and extensive sequencing of the mtDNA-coding region might be informative in patients with normal muscle histochemical studies.