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Molecular Epidemiology of Childhood Mitochondrial Encephalomyopathies in a Finnish Population: Sequence Analysis of Entire mtDNA of 17 Children Reveals Heteroplasmic Mutations in tRNA^Arg, tRNA^Glu, and tRNA^Leu(UUR) Genes

Johanna Uusimaa, MD, PhD^*,,, Saara Finnilä, MD, PhD^,,||, Anne M. Remes, MD, PhD^,,||, Heikki Rantala, MD, PhD^*, Leena Vainionpää, MD, PhD^*, Ilmo E. Hassinen, MD, PhD and Kari Majamaa, MD, PhD^,,||

^* Departments of Pediatrics
Medical Biochemistry and Molecular Biology
^|| Neurology
Biocenter, University of Oulu, Oulu, Finland

	ABSTRACT

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Objectives. Many heteroplasmic point mutations in tRNA genes of mitochondrial DNA (mtDNA) have been associated with human diseases. We recently reported on a prospective 7-year study in which we enrolled 116 consecutive children with undefined encephalomyopathy. Seventeen of them were found to have both a defect in the mitochondrial respiratory chain and abnormal ultrastructure of muscle mitochondria, suggesting a clinically probable mitochondrial encephalopathy.

Methods. We determined the frequency of mtDNA mutations in these 17 children by analyzing the entire sequence of mtDNA by conformation-sensitive gel electrophoresis and sequencing.

Results. Three heteroplasmic tRNA mutations that were considered to be pathogenic were detected. Two of the mutations were novel transitions, 10438A>G in the tRNA^Arg gene and 14696A>G in the tRNA^Glu gene, whereas the third one was 3243A>G, the common MELAS mutation. The mutant load was very high in the blood and skeletal muscle of the patients and markedly lower in the blood of asymptomatic maternal relatives. The 10438A>G mutation changes the nucleotide flanking the anticodon, whereas 14696A>G changes a nucleotide in the stem of the pseudouridine loop, creating a novel base pair and reducing the wobble.

Conclusions. Our results emphasize that the analysis of the entire sequence of mtDNA is worthwhile in the diagnostic evaluation of patients with clinically probable mitochondrial encephalomyopathy. The frequency of pathogenic mtDNA mutations was found to be 18% among children with biochemically and histologically defined mitochondrial disease, suggesting that the likelihood of nuclear DNA mutations in such a group is several times higher than that of mtDNA mutations.

Key Words: encephalomyopathy • mitochondrial DNA • tRNA mutation • conformation sensitive gel electrophoresis

Abbreviations: mtDNA, mitochondrial DNA • RRF, ragged red fiber • OXPHOS, mitochondrial oxidative phosphorylation system • CSGE, conformation-sensitive gel electrophoresis • PCR, polymerase chain reaction • EEG, electroencephalography • RFLP, restriction fragment length polymorphism • MRI, magnetic resonance imaging • COX, cytochrome-c oxidase

Encephalomyopathies caused by mutations in mitochondrial DNA (mtDNA) are a genetically and phenotypically variable group of disorders. Biochemically, they are characterized by defects in the mitochondrial respiratory chain, and the most ominous histologic finding is the accumulation of mitochondria in muscle, leading to ragged red fibers (RRFs). Brain, cardiac muscle, and skeletal muscle have the highest requirements for mitochondrial oxidative phosphorylation; therefore, they are the most sensitive tissues to the deleterious effects of mtDNA mutations. More than 100 point mutations in mtDNA have been reported so far.¹ Because the polypeptides encoded by the 13 mtDNA genes interact with 70 nuclear-encoded polypeptides to form 5 multisubunit enzyme complexes (I to V) within the mitochondrial oxidative phosphorylation system (OXPHOS), a mitochondrial disease may be caused by abnormalities in either nuclear or mitochondrial genes.

Conformation-sensitive gel electrophoresis (CSGE) has been proved to be an efficient method in screening for sequence differences in polymerase chain reaction (PCR) amplified fragments. It is based on the separation of heteroduplexes that contain single base-pair mismatches from homoduplexes composed of perfectly base-paired DNA strands in a polyacrylamide gel,² is simple, has a large capacity, and does not need radioactivity for detection purposes.³ The heteroduplexes are generated in a denaturation-renaturation cycle with a homologous reference fragment, allowing homozygous nuclear DNA variants or homoplasmic mtDNA variants to be detected.^3,4 This technique is rapid and cost-effective compared with direct sequencing when searching for mutations among a large number of samples, because sequencing is required only when the results of a CSGE analysis suggest a mutation.

Mitochondrial dysfunction may be one of the most common causes of undefined encephalomyopathies. We recently assessed the frequency of mitochondrial disorders among 116 children with undefined encephalomyopathies in a 7-year prospective study.⁵ Seventeen of the 116 patients were found to have both defects in oxidative phosphorylation and abnormalities in muscle mitochondria, suggesting a clinically probable mitochondrial disease.⁵ We are concerned here with the molecular cause of encephalomyopathies in these 17 children. Sequence analysis of the entire mtDNA by CSGE and sequencing revealed pathogenic mutations in 3 of the 17 patients. Two of them harbored novel heteroplasmic mutations in tRNA^Arg and tRNA^Glu genes, respectively, whereas the third child had the common MELAS mutation in the tRNA^Leu(UUR) gene.

	METHODS

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The patient group comprised 17 children who had a defect in oxidative phosphorylation and abnormal ultrastructure of muscle mitochondria and had been identified in our prospective 7-year study that included 116 children with unknown encephalomyopathy.⁵ The pediatric population in the catchment area numbered 97 609 on the last day of patient inclusion. The patients are identified here according to Table 5 of our previous article.⁵ The most common clinical features among the 17 children were mental and motor retardation (n = 16), seizures (n = 8), muscle hypotony (n = 8), progressive course (n = 6), and short stature (n = 5). Electroencephalography (EEG) was abnormal in 13 children, brain imaging was abnormal in 11 children, blood or cerebrospinal fluid lactate was increased in 8 children, and RRFs in muscle were found in 3 children. The samples were examined after obtaining informed consent from the parents of the children. The research protocol was approved by the Ethics Committee of the Medical Faculty, University of Oulu.

Control blood samples were obtained from 403 volunteers, who reported that they and their mothers were healthy with respect to diabetes, sensorineural hearing impairment, and neurologic ailments.⁶ Furthermore, the complete mtDNA sequence has recently been determined in 192 Finnish samples.⁷ In addition to the analysis of the entire mtDNA sequence of 17 patients, the skeletal muscle mtDNA samples of the remaining of 99 patients⁵ were tested for novel tRNA^Arg and tRNA^Glu mutations by restriction fragment length polymorphism (RFLP) analysis.

Case Reports
Patient 3 (patient identification according to Table 5 of the previous article⁵) is a boy who is the first child of nonconsanguineous parents. He was born after an uncomplicated full-term pregnancy and normal delivery. Early psychomotor development was not delayed (Table 1), but at the age of 10 years, he was evaluated for poor school performance and delayed speech development. He has had migraine attacks, exercise intolerance, and attacks of loss of tone in the legs lasting for 5 to 10 minutes. Ophthalmologic examination showed prominent scleral veins, anomalous chamber corners, and elevated ocular pressure. He has progressive sensorineural hearing loss. At the last follow-up visit, at the age of 16 years, his muscles were hypotonic and he had predominantly right-sided dysmetria. He had dyslalia and dysarthria, and he was found to have moderate retardation according to International Classification of Diseases, Tenth Revision criteria.

Patient 5 is a boy who is the second child of nonconsanguineous parents. He was born at term after an uncomplicated pregnancy and normal delivery. During the first few months of life, he had jitteriness in his hands, a poor gaze contact, and a continuous coarse horizontal nystagmus. Computed tomography of the head, EEG, a flash electroretinogram, and visual evoked potentials were normal. His psychomotor development was only mildly delayed in early childhood (see Table 1). However, by the age of 6 years, he had mild retardation, and at the age of 8 years 8 months, he had moderate retardation. He was clumsy, his gait was broad based, his facial muscles were weak, and he was drooling, in addition to which his speech was slow and he had some phonologic difficulties. He had a continuous coarse horizontal nystagmus, and his visual acuity was decreased, but the fundi were normal. In other respects, his neurologic and physical examinations were normal.

The blood lactate/pyruvate ratio was increased (Table 1), but other routine laboratory measurements were normal (for the complete list of tests, see case history of patient 3). Histologic examination of the muscle at the age of 1 year had revealed nonspecific myopathy with a predominance of type 1 fibers but no RRFs. An increased amount of fat and giant mitochondria were seen in electron microscopy. The activity of complex IV was decreased, whereas complexes I, I+III, and II+III activities were normal (Table 1). T2-weighted MRI at the age of 3 years suggested dysmatured myelin formation. Two years later, myelinization was normal, but increased signal intensity was noted in both thalami, and the white matter was unusually sparse at the level of the trigonum. Other family members were asymptomatic, including the proband's elder sister, and a neurologic examination of the mother of the proband was normal.

Patient 17 is a girl who is the second child of nonconsanguineous healthy parents. Her elder brother is healthy. She was born at term after an uncomplicated pregnancy and normal delivery. Her psychomotor development was delayed (Table 1), she had muscle hypotonia and severe dysphasia, and at the last follow-up visit at the age of 6 years 1 month, her muscles were hypotonic and she could not walk on her toes or heels. Her neurologic and physical examinations were otherwise normal. She was judged to have mild retardation.

Blood lactate and pyruvate levels have been within normal limits (Table 1), and although serum-free carnitine was measured once to be 18 mmol/L, subsequent measurements were within the normal values for the laboratory (35–70 mmol/L). Other routine laboratory measurements were normal (for the complete list of tests, see case history of patient 3). Histologic examination of muscle samples revealed RRFs, and increased amounts of fat and mitochondria were seen in electron microscopy. The activities of complexes I and I+III of the respiratory chain were decreased, whereas complexes II+III and IV activities were normal (Table 1). Brain MRI at the age of 1 year 10 months showed slightly enlarged ventricles, wide cerebral sulci, and a thin corpus callosum. EEG was normal.

Molecular Methods
DNA Extraction
Total DNA was isolated from blood cells, buccal epithelial cells, and skin fibroblasts using a QIAamp Blood Kit (Qiagen, Hilden, Germany) and from skeletal muscle biopsies by standard extraction with phenol and chloroform. mtDNA haplogroups were determined by analysis of RFLP⁸ and by sequencing the hypervariable segment I.

CSGE and Sequencing
MtDNA from the patients was screened for polymorphisms and mutations by CSGE and subsequent sequencing.^2,4 Muscle DNA was used as the template in 14 patients, and in the remaining 3 cases, some of the amplifications were performed with blood DNA as the template because of an insufficient amount of muscle DNA. The heteroduplexes that differed in mobility on CSGE from wild-type homoduplexes were analyzed by automated sequencing (ABI PRISM 377 Sequencer using the Dye Terminator Cycle Sequencing Ready Kit; Perkin Elmer, Foster City, CA) after treatment with exonuclease I and shrimp alkaline phosphatase.⁹ The primers used for sequencing were the same as those used in the amplification reactions for CSGE. The D loop was amplified in a fragment spanning nts 15975 to 00725, and the sequence was determined in the interval nts 16024 to 16400.

Detection of Heteroplasmy
Heteroplasmy of the putative pathogenic mtDNA mutations 5452C>T, 7859G>A, 10438A>G, and 14696A>G was examined by RFLP analysis of amplified DNA from the study subjects or by screening bacterial clones harboring the mutation. All the enzymes used were from New England Biolabs (Beverly, MA).

mtDNA contains a Taq I restriction site at nt 10438, but the 10438A>G transition abolishes this restriction site, thus providing a basis for restriction fragment analysis of the mutation. A 413-bp fragment spanning nts 10288 to 10700 was amplified by PCR. After digestion with Taq I, the fragments were electrophoresed through a 1.5% agarose gel. Restriction fragment analysis was used to detect the 10438A>G mutation in 403 control subjects and in 99 children with unexplained encephalomyopathy. The proportions of wild-type and mutant mtDNA in the proband and the asymptomatic mother were determined using subcloning and RFLP analysis.¹⁰ First, amplified 413-bp fragments were subcloned into a pCR 2.1-TOPO vector (TOPO TA Cloning Kit; Invitrogen, Leek, Netherlands). Positive clones were cultured overnight in 2 mL of LB medium that contained 50 µg/mL ampicillin. Portions of 1 µL from the cultures were then incubated for 10 minutes at 94°C to lyse the cells and inactivate the nucleases, and DNA was amplified in the presence of a vector-specific pair of primers. A nested PCR was then conducted using the same pair of primers as in the RFLP analysis of 10438A>G, and digestion with Taq I was used to detect 10438A>G. The proportions of mutant and wild-type mtDNA were calculated. Second, the proportion of the 10438A>G mutation in mtDNA was determined by RFLP analysis of a fragment synthesized in the presence of 35S-dATP. The digested product was electrophoresed through a 10% nondenaturing polyacrylamide gel, which was dried and autoradiographed at –72°C overnight using Kodak XAR film with an intensifying screen. The intensities of the bands on the autoradiography film were quantified using a GS-710 Calibrated Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA).

The proportion of the 14696A>G mutation was determined by RFLP analysis of a fragment synthesized in the presence of 35S-dATP as described above. 14696A>G creates a novel Aci I restriction site. Thus, the amplified DNA fragment (spanning nts 14560–14820) of 261 bp harboring 14696A>G was digested into fragments of 136 bp and 125 bp. The heteroplasmy of the 14696A>G mutation in skeletal muscle was also determined by subcloning as described above. The amplified DNA fragments were digested with Aci I, and the cleavage was verified on 3% MetaPhor agarose (FMC BioProducts, Rockland, ME). This RFLP analysis was also used to detect the 14696A>G mutation in 403 control subjects and in 99 children with unexplained encephalomyopathy.

The common MELAS mutation 3243A>G was detected by Apa I digestion of the appropriate mtDNA fragment amplified in the presence of 35S-dATP.¹¹ The possible heteroplasmic state of nucleotide positions 5452 and 7859 was determined by cloning. The amplified fragment between nts 7813 and 8203 was cloned, and 7859G>A was verified by loss of the Dpn II site. Similarly, amplified DNA fragment spanning nts 5287 to 5697 was subcloned, and, thereafter, 120 clones were studied by CSGE to detect the mutant clones harboring 5452C>T. For sequencing, M13 forward and M13 reverse primers were used to amplify the template, and the sequencing was conducted using plasmid-specific and insert-specific primers.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Nucleotide Sequence of the Entire mtDNA in 17 Children With Encephalomyopathy
We analyzed the entire mitochondrial genome in 17 patients with defects in oxidative phosphorylation and abnormal mitochondrial ultrastructure by CSGE and sequencing. We identified several mtDNA polymorphisms that have been reported in MITOMAP¹² or in a recent compilation of 435 European mtDNA sequences¹³ or have been found in Finnish controls.⁷ Furthermore, we found 4 substitutions—5452C>T, 7859G>A, 10438A>G, and 14696A>G—that have not been described earlier and the common MELAS mutation 3243A>G (Fig 1). We found that 5452C>T (Thr328Met in mtND2 gene) and 7859G>A (Asp92Asn in mtCO2 gene) were homoplasmic substitutions, whereas 10438A>G and 14696A>G were heteroplasmic and, therefore, were studied further.

View larger version (115K): [in this window] [in a new window]   Fig 1. Sequence variation in the mtDNA of the 17 children with mitochondrial encephalomyopathy. Nucleotide changes are shown relative to the revised Cambridge reference sequence.29,30 Grey, polymorphism reported in MITOMAP,12 in a recent compilation of European mtDNA sequences,13 or found in Finnish control subjects7; black, putative pathogenic mutation.

View larger version (62K): [in this window] [in a new window]   Fig 2. Restriction fragment analysis of tRNAArg, tRNAGlu, and tRNALeu(UUR) mutations. A, The heteroplasmy of the 10438A>G mutation was determined by Taq I digestion. Wild-type mtDNA fragments are cleaved into 2 fragments of 148 bp and 265 bp (arrows), whereas the 10438A>G mutation in 10438 abolishes the Taq I restriction site. Lane 1, undigested wild-type control; lane 2, digested wild-type control; lanes 3 to 4, mtDNAs of the proband's grandmother: lane 3, blood; lane 4: buccal epithelial cells; lanes 5 to 8, mtDNAs of the proband's mother: lane 5, blood; lane 6, buccal epithelial cells; lane 7, skin fibroblasts; lane 8, skeletal muscle; lanes 9 to 10, mtDNAs of the proband: lane 9, blood; lane 10, skeletal muscle. B, The heteroplasmy of the 14696A>G mtDNA mutation was detected by Aci I digestion. The 14696A>G mutation leads to the gain of an Aci I cleavage site, and the amplified DNA fragment of 261 bp harboring this mutation will be digested into fragments of 136 bp and 125 bp. Lane 1, blood mtDNA from a healthy wild-type control; lane 2, blood mtDNA from the proband's grandmother; lane 3, blood mtDNA from the proband's mother; lanes 4 to 5, mtDNAs of the proband: lane 4, blood; lane 5, skeletal muscle; lane 6, blood sample from an anonymous control belonging to haplogroup W. The fragments of 136 bp and 125 bp co-migrate on the polyacrylamide gel (arrow). C, The heteroplasmy of the 3243A>G mutation was determined by Apa I digestion. The 3243A>G mutation leads to the gain of an Apa I cleavage site, and the amplified DNA fragment of 410 bp harboring this mutation will be digested into fragments of 307 bp and 103 bp (arrows). Lane 1, blood mtDNA from a healthy control; lane 2, blood from the proband's grandmother; lane 3, blood mtDNA from the proband's mother; lanes 4 to 5, mtDNAs of the proband: lane 4, blood at the age of 10 years; lane 5, blood at the age of 15 years; lane 6, skeletal muscle at the age of 10 years.

Frequency of Pathogenic mtDNA Mutations
In addition, we found the common MELAS mutation 3243A>G in patient 3 and in his mother and maternal grandmother (Fig 2C, Table 2). Altogether, 3 heteroplasmic tRNA mutations were detected in 3 of 17 children with mitochondrial encephalomyopathy, giving a frequency of 18% for definitely pathogenic mtDNA mutations (95% confidence interval: 4%–43%). These 17 patients belonged to a cohort of 116 children with unknown encephalomyopathy, suggesting that the frequency of pathogenic mtDNA in this group was 2.6% (95% confidence interval: 0.5%–7.4%).

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
We determined the nucleotide sequence of the entire mtDNA in 17 children with probable mitochondrial encephalomyopathy. These children were identified previously in a prospective survey of a defined population on the basis of a defect in mitochondrial oxidative phosphorylation and abnormal ultrastructure of the muscle mitochondria.⁵ We found 3 children with heteroplasmic tRNA mutations, 2 of which were novel transitions 10438A>G in tRNA^Arg and 14696A>G in tRNA^Glu, whereas the third one was the common MELAS mutation 3243A>G. In addition, we found several known polymorphisms that co-segregated with mtDNA haplogroups (Fig 1) and 2 homoplasmic substitutions 5452C>T and 7859G>A that had not been described earlier^12,13 or were not found among the control subjects.

We believe that the 10438A>G mtDNA mutation is the cause of encephalomyopathy in patient 5. This mutation was absent in 403 healthy unrelated control subjects; furthermore, the mutation was heteroplasmic, which is typical of pathogenic tRNA mutations in mtDNA. The 10438A>G mutation changes the nucleotide flanking the anticodon (Fig 3A). Three other heteroplasmic mutations have been reported at a position homologous to nt 10438. The 14709A>G mutation in the tRNA^Glu gene causes encephalomyopathy and diabetes,^14–16 4295A>G in tRNA^Ile is associated with hypertrophic cardiomyopathy,¹⁷ and 12301A>G in the tRNA^Leu(CUN) gene has been found in a patient with acquired idiopathic sideroblastic anemia.¹⁸ Alignment of tRNA^Arg sequences from various species revealed that adenine at position 10438 is highly conserved in all mammals and in various eukaryotic species, the exceptions being some reptiles, amphibians, fish, and nematodes. In a similar manner, 14709A>G in tRNA^Glu occurs at a position that is highly conserved except in nematodes.¹⁵ Such point mutations flanking the anticodon triplet affect codon matching and possibly tRNA synthetase recognition, with a decrease in the accuracy and rate of translation.¹⁹ The position 10438 in human mtDNA corresponds to position 37 in the canonical tRNA molecule.²⁰ It is interesting that this nucleotide is subject to posttranscriptional modification in 2 of the 3 human mitochondrial tRNAs that have been sequenced so far, suggesting functional importance.^21,22 Mitochondrial tRNA^Lys is modified at position 37 in 4 mammalian tRNA^Lys molecules.²³ Furthermore, the nucleotide at this position is methylated in the tRNA^Arg of Escherichia coli,²⁴ suggesting that this modification may also be important in human tRNA^Arg molecules. Unfortunately, in the lack of fresh muscle, it was not possible to verify the pathogenicity of 10438A>G by the use of, eg, enzyme histochemistry of cytochrome-c oxidase (COX) and subsequent analysis of mutation heteroplasmy in single COX-negative and COX-positive fibers.

View larger version (14K): [in this window] [in a new window]   Fig 3. The cloverleaf structures of human mitochondrial tRNAArg and tRNAGlu and sites of the mutations. The arrows denote the location of A>G transitions (U>C in tRNAGlu) at mtDNA positions 10438 and 14696.

The clinical phenotypes of our patients were variable, although they shared some features with patients who harbor other mitochondrial tRNA mutations. The main features were progressive encephalomyopathy, an OXPHOS defect, abnormal ultrastructure of the skeletal muscle mitochondria, and lactic acidosis or RRFs. The mutant load was very high in the blood of the patients but markedly lower in the blood of asymptomatic maternal relatives. The higher percentage of the mutant DNA in both blood cells and muscle observed in the proband may explain the severity of the clinical phenotype in these patients. The families thus resembled many MELAS pedigrees,²⁶ whereby oligosymptomatic and asymptomatic carriers of the mutation co-occur with affected family members. It has been shown that tRNA point mutations seem to act as recessive mutations so that a clinical phenotype is expressed only when the proportion of mutant mtDNA is high.²⁷ In the cases described here, the degree of mutant heteroplasmy was relatively high in 2 of the probands. It is interesting that segregation to near homoplasmy was detected in the proband with 14696A>G, suggesting that the threshold for this mutation is high, whereas those for the common MELAS mutation and the 10438A>G mutation are lower.

Because the prevalence of the common MELAS mutation has been found to be 16/100 000 in the adult population of northern Finland,²⁶ it was somewhat surprising that we found only 1 child with this mutation and 2 children with a mutation in other tRNA genes. Thus, the frequency of definitely pathogenic mtDNA mutations was calculated to be 2.6% among children with encephalomyopathy of unknown cause. Furthermore, the 3 children belonged to a group of 17 patients with a clinically probable mitochondrial disease, making the frequency of pathogenic mtDNA mutations in this cohort 18% and giving a minimum prevalence of 3/100 000 among the children in the general population. A fairly similar prevalence of mitochondrial encephalomyopathies, 5/100 000, has been reported in children in western Sweden.²⁸

The diagnosis of mitochondrial diseases is based on clinical, histologic, biochemical, and molecular genetic studies. The 17 patients of the present study had received diagnoses of progressive encephalomyopathy, an OXPHOS defect, and abnormal ultrastructure of the skeletal muscle mitochondria. Three children were found to harbor a pathogenic mtDNA mutation, emphasizing that the analysis of the entire sequence of mtDNA is worthwhile in the diagnostic evaluation of such patients. We conclude that children who present with an unexplained encephalopathy and combination of neuromuscular or nonneuromuscular symptoms, with OXPHOS defects and with abnormalities in muscle mitochondria, and lacking the most common known mtDNA mutations should be subjected to molecular analysis of the entire mtDNA. However, we were left with 14 children who did not have pathogenic mutations in mtDNA, suggesting that the likelihood of nuclear DNA mutations in children with a mitochondrial encephalopathy may be even higher than that of mtDNA mutations.

	ACKNOWLEDGMENTS

 
This work was supported by grants from the Medical Research Council of the Academy of Finland, the Arvo and Lea Ylppö Foundation, the Foundation for Pediatric Research, and the Sigrid Juselius Foundation.

The expert technical assistance of Irma Vuoti and Kaisu Korhonen is gratefully acknowledged.

	FOOTNOTES

 
Received for publication Nov 21, 2002; Accepted Dec 19, 2003.

Reprint requests to (K.M.) Department of Neurology, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland. E-mail: kari.majamaa{at}oulu.fi

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 

1. Servidei S. Mitochondrial encephalomyopathies: gene mutation. Neuromuscul Disord.2000; 10 :X –XVI
2. Ganguly A, Rock MJ, Procop DJ. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A.1993; 90 :10325 –10329[Abstract/Free Full Text]
3. Körkkö J, Annunen A, Pihlajamaa T, Prockop DJ, Ala-Kokko L. Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing. Proc Natl Acad Sci U S A.1998; 95 :1681 –1685[Abstract/Free Full Text]
4. Finnilä S, Hassinen IE, Ala-Kokko L, Majamaa K. Phylogenetic network of mitochondrial DNA haplogroup U in Northern Finland based on sequence analysis of the complete coding region by conformation sensitive gel electrophoresis. Am J Hum Genet.2000; 66 :1017 –1026[CrossRef][ISI][Medline]
5. Uusimaa J, Remes A, Rantala H, et al. Childhood encephalopathies and myopathies: a prospective study in a defined population to assess the frequency of mitochondrial disorders. Pediatrics.2000; 105 :598 –603[Abstract/Free Full Text]
6. Meinilä M, Finnilä S, Majamaa K. Evidence for mtDNA admixture between the Finns and the Saami. Hum Hered.2001; 52 :160 –170[CrossRef][ISI][Medline]
7. Finnilä S, Lehtonen MS, Majamaa K. Phylogenetic network for European mitochondrial DNA. Am J Hum Genet.2001; 68 :1475 –1484[CrossRef][ISI][Medline]
8. Torroni A, Huoponen K, Francalacci P, et al. Classification of European mtDNAs from an analysis of three European populations. Genetics.1996; 144 :1835 –1850[Abstract]
9. Werle E, Schneider C, Renner M, Volker M, Fiehn W. Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Res.1994; 22 :4354 –4355[Free Full Text]
10. Finnilä S, Hassinen IE, Majamaa K. Restriction fragment analysis as a source of error in detection of heteroplasmic mtDNA mutations. Mutat Res.1999; 406 :109 –114[ISI][Medline]
11. Kobayashi Y, Momoi MY, Tominaga K, et al. A point mutation in the mitochondrial tRNALeu (UUR) gene in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). Biochem Biophys Res Commun.1990; 173 :816 –882[CrossRef][ISI][Medline]
12. Kogelnik AM, Lott MT, Brown MD, Navathe SB, Wallace DC. MITOMAP: a human mitochondrial genome database-1998 update. Nucleic Acids Res.1998; 26 :112 –115[Abstract/Free Full Text]
13. Herrnstadt C, Elson JL, Fahy E, et al. Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups. Am J Hum Genet.2002; 70 :1152 –1171[CrossRef][ISI][Medline]
14. Hanna MG, Nelson I, Sweeney MG, et al. Congenital encephalomyopathy and adult-onset myopathy and diabetes mellitus: different phenotypic associations as a new heteroplasmic mtDNA tRNA glutamic acid mutation. Am J Hum Genet.1995; 56 :1026 –1033[ISI][Medline]
15. Hao H, Bonilla E, Manfredi G, DiMauro S, Moraes CT. Segregation patterns of a novel mutation in the mitochondrial tRNA glutamic acid gene associated with myopathy and diabetes mellitus. Am J Hum Genet.1995; 56 :1017 –1025[ISI][Medline]
16. Vialettes BH, Paquis-Flucklinger V, Pelissier JF, et al. Phenotypic expression of diabetes secondary to a T14709C mutation of mitochondrial DNA. Comparison with MIDD syndrome (A3243G mutation): a case report. Diabetes Care.1997; 20 :1731 –1737[Abstract]
17. Merante F, Myint T, Tein I, Benson L, Robinson BH. An additional mitochondrial tRNA^Ile point mutation (A-to-G at nucleotide 4295) causing hypertrophic cardiomyopathy. Hum Mutat.1996; 8 :216 –222[CrossRef][ISI][Medline]
18. Gattermann N, Retzlaff S, Wang Y-L, et al. A heteroplasmic point mutation of mitochondrial tRNALeu(CUN) in non-lymphoid haemopoietic cell lineages from a patient with acquired idiopathic sideroblastic anemia. Br J Haematol.1996; 93 :845 –855[CrossRef][Medline]
19. Yoon KL, Aprille JR, Ernst SG. Mitochondrial tRNA^Thr mutation in fatal infantile respiratory enzyme deficiency. Biochem Biophys Res Commun.1991; 176 :1112 –1115[CrossRef][Medline]
20. Sprinzl M, Horn C, Brown M, Ioudovitch A, Steinberg S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleid Acids Res.1998; 26 :148 –153
21. Brule H, Holmes WM, Leith G, Giege R, Florentz C. Effect of a mutation in the anticodon of human mitochondrial tRNAPro on its post-transcriptional modification pattern. Nucleic Acids Res.1998; 26 :537 –543[Abstract/Free Full Text]
22. Helm M, Giege R, Floretz C. A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys. Biochemistry.1999; 38 :13338 –13346[CrossRef][Medline]
23. Helm M, Brule H, Degoul F, et al. The presence of modified nucleotides is required for cloverleaf folding of a human mitochondrial tRNA. Nucleic Acids Res.1998; 26 :1636 –1643[Abstract/Free Full Text]
24. McClain WH, Foss K, Jenkins RA, Schneider J. Nucleotides that determine Escherichia coli tRNAArg and tRNALys acceptor identities revealed by analyses of mutant opal and amber suppressor tRNAs. Proc Natl Acad Sci U S A.1990; 87 :9260 –9264[Abstract/Free Full Text]
25. Brown MD, Wallace DC. Molecular basis of mitochondrial DNA disease. J Bioenerg Biomembr.1994; 26 :273 –289[CrossRef][ISI][Medline]
26. Majamaa K, Moilanen JS, Uimonen S, et al. Prevalence, in an adult population, of A3243G, the mutation for mitochondrial encephalomyopathy, lactic acidosis, and stroke like episodes. Am J Hum Genet.1998; 63 :447 –454[CrossRef][ISI][Medline]
27. Shoubridge EA. Mitochondrial DNA disease: histological and cellular studies. J Bioenerg Biomembr.1994; 26 :301 –310[CrossRef][ISI][Medline]
28. Darin N, Oldfors A, Moslemi AR, Holme E, Tulinius M. The incidence of mitochondrial encephalomyopathies in childhood: clinical features and morphological, biochemical, and DNA abnormalities. Ann Neurol.2001; 49 :377 –383[CrossRef][ISI][Medline]
29. Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature.1981; 290 :457 –465[CrossRef][Medline]
30. Andrews RM, Kubacka I, Chinnery PF, et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet.1999; 23 :147[CrossRef][ISI][Medline]

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