Clinical and Genetic Heterogeneity in Progressive External Ophthalmoplegia Due to Mutations in Polymerase ␥

ORIGINAL CONTRIBUTION Clinical and Genetic Heterogeneity in Progressive External Ophthalmoplegia Due to Mutations in Polymerase ␥

Massimiliano Filosto, MD; Michelangelo Mancuso, MD; Yutaka Nishigaki, MD; Jacklyn Pancrudo, BS; Yadollah Harati, MD; Clifton Gooch, MD; Ami Mankodi, MD; Lydia Bayne, MD; Eduardo Bonilla, MD; Sara Shanske, PhD; Michio Hirano, MD; Salvatore DiMauro, MD

Background: The mendelian forms of progressive ex- Results: Four unrelated patients had novel POLG mu- ternal ophthalmoplegia (PEO) associated with multiple mi- tations. A woman with PEO and mental retardation had tochondrial DNA deletions are clinically heterogeneous a heterozygous Gly1076Val mutation. Two patients, one disorders transmitted as dominant or recessive traits. Au- with PEO, exercise intolerance, and gastrointestinal dys- tosomal dominant PEO is caused by mutations in at least motility and the other with PEO, neuropathy, deafness, 3 genes: adenine nucleotide translocator-1 (ANT1), en- and hypogonadism, both had a Pro587Leu change. The coding the muscle-specific adenine nucleotide transloca- fourth patient, who was compound heterozygous for tor; chromosome 10 open reading frame 2 (C10orf2), en- Ala889Thr and Arg579Trp mutations, had PEO, gastro- coding Twinkle helicase; and polymerase ␥ (POLG), intestinal dysmotility, and neuropathy. These muta- encoding the ␣ subunit of polymerase ␥. Mutations in tions were not detected in 120 healthy control alleles. POLG can also cause autosomal recessive PEO, which is often associated with multisystemic disorders. Conclusions: Our results demonstrate that POLG mu- Objective and Methods: To further investigate the tations account for a substantial proportion of patients frequency and genotype-phenotype correlations of mu- (13%) with PEO and multiple mitochondrial DNA de- tations in the POLG gene, we used single-stranded con- letions and cause both clinically and genetically heter- formational polymorphism analysis and direct sequenc- ogeneous disorders. ing to screen 30 patients with familial or sporadic PEO and multiple mitochondrial DNA deletions in muscle but without mutations in ANT1 and C10orf2. Arch Neurol. 2003;60:1279-1284

ROGRESSIVE EXTERNAL oph- identified in patients with either adPEO thalmoplegia (PEO) with or autosomal recessive PEO (arPEO), multiple mitochondrial DNA which is often associated with multisys- (mtDNA) deletions in muscle temic disorders.6-8 In this study, we can be inherited as a domi- screened the coding region of POLG in 30 nantP or a recessive trait.1,2 Clinical manifes- patients with PEO and multiple mtDNA tations are heterogeneous and are usually deletions to identify mutations, investi- more variable and severe in the recessive gate their frequency, and assess phenotype- forms.2 Besides PEO, clinical features in- genotype correlations. clude deafness, cataracts, depression, dys- From the Department of phagia, hypogonadism, neuropathy, and Neurology, Columbia 3 METHODS University College of sensory ataxia. Muscle biopsy results typi- Physicians and Surgeons, New cally show ragged red fibers and cyto- PATIENTS York, NY (Drs Filosto, chrome c oxidase–negative fibers.3 Mancuso, Nishigaki, Gooch, Autosomal dominant PEO (adPEO) Thirty patients (16 men and 14 women) with Bonilla, Shanske, Hirano, and has been linked to mutations in genes en- PEO and multiple mtDNA deletions in muscle DiMauro and Ms Pancrudo); coding adenine nucleotide transloca- were included in this study. Twenty-one pa- Baylor College of Medicine, tor-1 (ANT1) and Twinkle, a putative tients had complex clinical diagnoses, includ- Houston, Tex (Dr Harati); mtDNA helicase, or chromosome 10 open ing neuropathy, ataxia, psychiatric disorders, School of Medicine and deafness, and cataracts. Twenty-three pa- Dentistry, University of reading frame 2 (C10orf2). Both protein tients (77%) had affected relatives, and the other Rochester, Rochester, NY products are involved in mtDNA mainte- cases were sporadic. None had mutations in 4,5 (Dr Mankodi); and Good nance. Mutations in a third gene, poly- ANT1 and C10orf2. In this article we describe Samaritan Clinic, Portland, Ore merase ␥ (POLG), encoding the ␣ sub- the clinical features in only the 4 patients with (Dr Bayne). unit of mtDNA polymerase ␥, were POLG mutations.

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 because of severe deafness and oropharyngeal muscle weakness. A B In addition, he had bilateral ptosis and ophthalmoparesis, with restriction of eye movement in all directions to 60% of normal. There was also proximal and distal symmetrical limb weakness, more severe in the legs than in the arms, and impaired vibration sensation in the feet. Electromyograms showed mixed neuro- pathic and myogenic features. His 35-year-old brother has similar but more severe symptoms and is wheelchair-bound. An- +/– –/– +/– +/– other brother, 2 sisters, their children, and both parents are healthy. Patient 4 (Figure 1D), a 58-year-old woman, at age 30 years noted slowly progressive ptosis and limitation of eye movements. Later, she developed ataxia, orthostatic dizziness, cataracts, and gastrointestinal dysmotility with diarrhea and constipation. Neurological examination results showed normal C D mental status, ptosis with almost complete PEO, vibration sensory loss, sensory ataxia, and areflexia. Her sister, who had died in a car accident, reportedly had PEO. Her parents and her 4 children are healthy. +/+ BIOCHEMICAL +/– AND DNA ANALYSIS

Figure 1. Pedigrees of 4 families with polymerase ␥ (POLG) mutations. Routine histological studies and histochemical staining for cy- Affected individuals are indicated by solid symbols. Stippled symbols tochrome c oxidase and succinate dehydrogenase were per- indicate subjects without progressive external ophthalmoplegia (PEO) but formed as previously described.9 Respiratory chain enzyme ac- with other symptoms. Asymptomatic individuals are indicated by open tivities in muscle in patients 2, 3, and 4 were measured as symbols. Triangles indicate individuals whose sex was unknown. Arrows 10 indicate index cases. Slashed symbols indicate deceased subjects. previously described. Heterozygotes are indicated by +/-, healthy individuals by -/-, and compound Total DNA was extracted from muscle by using a DNA iso- heterozygotes by +/+. A, patient 1; B, patient 2; C, patient 3; D, patient 4. lation kit (Puregene; Gentra, Minneapolis, Minn), and mtDNA was analyzed with Southern blotting as previously described to 11 Patient 1 (Figure 1A), a 51-year-old Ukrainian woman, check for multiple deletions. Mutations were detected with was born to nonconsanguineous parents. She was healthy un- single-stranded conformational polymorphism analysis. We used til age 4 years, when she was noted to have a learning disabil- a set of primers that amplified all exons with flanking intronic ity. Mental retardation was later diagnosed, and she required sequences of POLG, except exon 1, which is not translated special schooling throughout childhood. At age 41 years, she (Table 1). Reactions were performed in 25 µL of 10mM Tris had difficulty moving her eyes, and she had to turn her head hydrochloride (pH 8.9) containing 0.4µM each of the forward to fixate on objects. She also developed progressive bilateral and reverse oligonucleotide primers; 1.5 mM of magnesium chlo- ptosis. She did not have dysarthria, dysphagia, dyspnea, or limb ride; 0.2mM each of dATP, dGTP, and dTTP; 0.02mM of dCTP; ␣ 32 weakness. Both her father, who died at age 72 years, and her 1 µCi (37 kBq) of - P dCTP; and 1.25 units of Taq DNA poly- paternal grandmother, who died at age 93 years, had long- merase (Roche, Indianapolis, Ind). Polymerase chain reaction standing histories of bilaterally impaired eye movements. The (PCR) conditions for exons 4, 5, 6, 7, 8, 9, 10, 22, and 23 were patient’s brother and his 7-year-old daughter are healthy. Neu- 94°C for 5 minutes, followed by 35 cycles at 94°C for 1 minute, rological examination results showed bilateral ptosis, despite 56°C for 1 minute, 72°C for 1 minute, and a final extension step previous blepharoplasties, and minimal lateral and conver- at 72°C for 7 minutes. For exons 2, 17, and 18, the annealing gence eye movements. No other neurological abnormalities were temperature was 60°C, whereas it was 64°C for exons 11, 12, noted. Endocrinological study results, including parathyroid 13, 14, 15, 16, 19, 20, and 21. Samples were denatured and sepa- and thyroid function test results, were normal. Magnetic reso- rated on 6% mutation detection enhancement polyacrylamide nance imaging of the brain demonstrated mild dilatation of the gel (Cambrex BioScience Rockland Inc, Rockland, Me) with 5% subarachnoid spaces, prominence of the cerebellar folia, and a glycerol, according to the manufacturer’s protocol. retention cyst along the posterior wall of the sphenoid sinus. Conformations of single-stranded DNA were visualized Patient 2 (Figure 1B), a 61-year-old man, noted numb- with autoradiography by using BioMax film (Kodak, Roches- ness in his legs at age 47 years. Later, he developed PEO, gen- ter, NY). Samples with abnormal patterns were directly se- eralized muscle weakness, exercise intolerance, unsteadiness quenced by using the ABI PRISM BigDye Terminators v3.0 Cycle (especially in the dark or with eyes closed), abdominal cramp- Sequencing Kit and a 310 Genetic Analyzer (Applied Biosys- ing, and gastrointestinal dysmotility with alternating diarrhea tems, Foster City, Calif). The presence of the mutations was and constipation. A 63-year-old sister had an 18-year history confirmed by means of PCR–restriction fragment length poly- of PEO, exercise intolerance, distal limb weakness, and diabe- morphism analysis. PCR conditions were 94°C for 5 minutes, tes mellitus. She also had peripheral neuropathy with stocking- followed by 35 cycles of 94°C for 1 minute, annealing for 1 glove numbness. Her 44-year-old son and a 42-year-old daugh- minute, 72°C for 1 minute, and a final extension step at 72°C ter have diabetes mellitus. The proband’s parents are described for 7 minutes. Primers, annealing temperatures, and restric- as healthy, although the mother apparently had mood swings. tion endonucleases used to perform PCR–restriction fragment A maternal aunt had PEO. length polymorphism analysis are listed in Table 2. Patient 3 (Figure 1C) was a 43-year-old man, who, at age 7 years, developed progressive hearing loss. At age 10 years, he had difficulty with his balance, and at age 25 years, he had dyscon- RESULTS jugate gaze. At age 31 years, he had hypogonadism with low tes- tosterone levels and normal pituitary function. Neurological ex- Muscle biopsy results in all 30 patients revealed ragged amination results showed impaired communication and speech red fibers and cytochrome c oxidase–negative fibers (data

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 not shown). Biochemical analysis of muscle extracts showed decreased activities of multiple respiratory chain en- Table 1. Primer Sequences zymes; values in patients with POLG mutations are listed in Table 3). All patients had multiple mtDNA deletions Annealing Base Pair Primer Sequence Temperature, detected with Southern blot analysis (data not shown). Fragment (5؅ to 3؅) Exon °C We found POLG mutations in 4 patients (13% of all cases; 17% of familial cases). In agreement with results from 846 7 F TCCAC, GTCTT, CCAGC, CAGTA 2 60 a previous report, we considered the A of the AUG chain- F GTCCC, TGCAC, CAACC, ATGAG initiation codon of the POLG messenger RNA as nucleo- R ACCTT, CCTCC, AGAGC, AGGCG tide position +1 and the corresponding methionine as the F GCAAG, TGCTA, TCCTC, GGAGG first amino acid residue of the protein sequence. R TCCTT, GCCCG, AAGAT, TTGCT Patient 1 was heterozygous for a G3227T transition F GTCGA, GCACC, TGCAG, AAGCA R TAGGG, CAGGC, TCTGC, TTCTG in exon 20 of the POLG gene, which resulted in a Gly1076Val F AACTT, GCTGT, TGCAG, GCCCA amino acid change. Analysis of DNA in her healthy brother R ACCTC, CACGT, CGAAC, ACCAG did not show any POLG mutation (Figure 2A). R AGCAC, GTAAC, AGGAC, CTCAG Patient 2 and his affected sister were both hetero- 330 zygous for a C1760T transition in exon 10, which re- F AGTGG, TTGTT, GTGGA, GTGGA 3 62 sulted in a Pro587Leu amino acid change (Figure 2B). R AACCA, CTGAG, ATTAG, GGCTC 819 Patient 3 was heterozygous for the same C1760T muta- F AGGTC, CACAC, CACCA, AGCAG 4 56 tion identified in patient 2 (Figure 2C). F ATAGT, GCTTG, GGACC, CCCTC 5 Patient 4 was compound heterozygous for a G2665A R AGCAA, GGGAC, ATGGC, AGATC 4 mutation in exon 17, which resulted in an Ala889Thr F TAGCT, GCACT, TGGGG, AGATG 6 amino acid change (Figure 2D), and for a C1735T tran- R ATTAC, CACCA, CCAGC, TCTCA 5 sition in exon 10, which resulted in an Arg579Trp amino R CAGCG, CCACC, TGATT, ACAGT 6 851 acid change (Figure 2E). None of these mutations was F AGTCT, TGCCT, CCTGT, GGTCA 7 56 found in 120 control alleles from patients who under- F CTTGG, GTGGG, CAGGA, TCTAG 8 went diagnostic muscle biopsy but were ultimately R CTAAG, GCTAA, GCCGA, AGGCT 7 deemed free of neuromuscular disease. F TGTCT, CTGGG, AAGGT, CCTGT 9 In these 4 patients with POLG mutations, we also found R CACCC, GCTCA, TCTCC, AGAAG 8 a Gln1236His change already described as a polymor- R CTGTC, CTGAG, AATGG, AGCAA 9 7 384 phism and 2 heterozygous nucleotide transitions, G2178A F TGTGA, GAGAG, AGAAC, CTTCC 10 56 and C2254T, that do not alter amino acid residues. R TAGCC, TGAGC, TGACC, AGCCA 921 F GTGGG, CATCT, GGTAA, TCAGC 11 64 COMMENT R CCTGG, GTGGA, ATACA, GAGAC F TACCC, AGTCC, CCTGC, TTCAC 12 R ACTCT, GGCTG, GGAAG, AACTA Mutations in 3 nuclear genes, ANT1, C10orf2, and POLG, F TGAAT, GCAGG, TGCTG, GAGCA 13 have been associated with adPEO.4-7,12-14 Adenine nucleo- R GAGCA, GAATG, AGGAA, ACACC tide translocator-1, the gene responsible for the chro- 285 mosome 4q35–linked form of adPEO,15 encodes an iso- F GAGTG, CAGGT, ACTCA, CGTTG 14 64 R AAGAG, AGGGG, AAAGG, CATCC form of the adenosine triphosphate–adenosine 382 diphosphate translocator common to muscle, heart, and F TAGAT, TCTGC, TTCCC, ATGGC 15 64 brain. Adenine nucleotide translocator-1 regulates the ad- F GAACT, GAGGT, GAGGT, GGTAG 16 enine nucleotide pool within mitochondria and is a struc- R AGCAG, GGTGC, TTTGG, CCTTA 15 tural element of the mitochondrial permeability transi- R ACAGA, CCTGG, GAGAG, GAAGA 16 tion pore, thus playing an important role in apoptosis 615 5 F TCAAC, TCTGG, CTCCA, GGAAT 17 60 mediated by mitochondria. Twinkle, the product of the R ATATG, CTACC, TGAGG, GGCTG C10orf2 gene, appears to be an adenine nucleotide– F CATTG, GGCAG, AATGT, TCCTG 18 dependent mtDNA helicase.4 Twinkle alterations may en- R TAAAG, CAGGC, CTCGG, GTCCT hance dNTP breakdown and impair mtDNA replication 914 through nucleotide pool imbalances.4 F AGCGT, GGCAC, AGGAA, GCACT 19 64 The ␣ subunit of polymerase ␥, the product of the R ATCCA, AGCTC, TTCTG, GGGCA F GCTTT, GAGAT, GCTGG, AAGAC 20 POLG gene, is responsible for the chromosome 15q22- R TAAGG, TCCAC, AGGGA, GCTCT 6 26–linked form of adPEO. Polymerase ␥ is a het- F TCTCT, TGGGG, CTTCT, ACCCT 21 erodimer composed of a 140-kDa ␣ subunit and an ac- R AGCCC, CACAT, AGGAG, CACAT cessory protein of about 41 kDa (␤ subunit).16 It is part 267 of a multienzymatic complex located within the inner mi- F GTGAT, GCATC, TGTTC, ACAGG 22 56 tochondrial membrane and required for mtDNA repli- R TCCAC, CTCAG, ATCCT, ATGTG 269 17 ␣ cation. The subunit is catalytic and contains both poly- F AGCTC, CTTTG, CTCAC, TTCTG 23 56 merase and exonuclease activities, whereas the ␤ subunit R ATGGC, TGGCC, TTAGG, CAAGC seems to enhance DNA binding and promote DNA synthesis.18 Mutations in the ␤ subunit have not been de- Abbreviations: F, forward; R, reverse.

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Mutation Oligonucleotide Sequences Annealing Temperature, °C Restriction Endonuclease G3227T F 5Ј-GCGTGGCACAGGAAGCACTCC 64 BbsI R5Ј-AGGGCTCGGCTGATGCAGAA mm C1760T F 5Ј-TGTGAGAGAGAGAACCTTCC 56 SmaI R5Ј-TAGCCTGAGCTGACCAGCCA G2665A F 5Ј-TCAACTCTGGCTCCAGGAAT 60 Csp6I R5Ј-ATATGCTACCTGAGGGGCTG C1735T F 5Ј-CCCTGGGTCAGGGTATGCTT 55 NciI R5Ј-TCCATGCAGGGTCGTCTAGC

Abbreviations: F, forward; mm, mismatched; R, reverse.

Table 3. Enzyme Values*in Patients With Polymerase ␥ Mutations

Enzyme Patient 2 Patient 3 Patient 4 Control Alleles† Cytochrome c oxidase 0.098 (35) 0.196 (69) 0.110 (39) 0.283 ± 0.052 Succinate cytochrome c reductase 0.051 (73) 0.033 (47) 0.018 (26) 0.070 ± 0.023 NADH−cyctochrome c reductase 0.033 (29) 0.040 (35) 0.044 (39) 0.115 ± 0.038 NADH dehydrogenase 0.910 (24) 3.321 (56) 1.261 (34) 3.760 ± 0.715 Succinate dehydrogenase 0.066 (66) 0.096 (95) 0.041 (40) 0.101 ± 0.053

Abbreviation: NADH, reduced nicotinamide adenine dinucleotide. *Data are expressed as micromoles per minute or fresh tissue; data in parentheses are the percentage of normal. †Data are the mean plus or minus SD.

scribed in humans, but a null mutation of this subunit actly in the middle of motif A of the polymerase do- causes early death in Drosophila melanogaster.19 main.20 Although the alanine-to-threonine amino acid The human enzyme has weak homology with fam- change is mild, its pathogenicity is probably enhanced ily A of DNA polymerases.20-22 Sequences in higher eu- by the association with the Arg579Trp change. Arginine karyotes differ from those in lower eukaryotes, and simi- 579 is relatively well conserved through evolution, al- larities are confined mainly to the the amino-terminal though in some species it has been replaced by a lysine exonuclease domain involved in proofreading and to the residue. Both arginine and lysine contain positively COOH-terminal polymerase domain.22 A unique char- charged lateral groups, while tryptophan has an aro- acteristic of human POLG is the presence of 10 gluta- matic side group. Neither mutation produces clinical ef- mine repeats near the amino-terminal.20 The identifica- fects in heterozygosity, but when both mutations coex- tion of novel POLG mutations in 4 families confirms that ist in compound heterozygotes, they appear to have defects of POLG are common autosomal causes of PEO.6,7 deleterious effects on POLG function. Patient 1 had a heterozygous Gly1076Val substitu- Most mutations described in POLG affect function (Figure 2A). Glycine in residue 1076 is well con- tional exonuclease or polymerase domains.6,7 Two of the served throughout evolution and is located near the 4 novel mutations described here are located in exon 10, COOH-terminal polymerase domain, between motifs B outside functional domains, which shows that the pro- and C.20 Glycine and valine have different structural char- tein region encoded by exon 10 and adjacent to the exo- acteristics. The simpler structure of glycine and the mini- nuclease domain is important for the stability and cor- mum steric impediment of its lateral chains allow greater rect conformation of the human protein. flexibility of protein structures than other amino acid resi- Authors of in vitro studies have demonstrated that dues have; therefore, a glycine-to-valine amino acid change mutations in POLG can promote accumulation of mtDNA is likely to alter the stability of the secondary structure mutations, deletions, and depletion,20,23 but the under- in this important region. The heterozygous Pro587Leu lying mechanisms are unknown. Because Y955C mu- mutation identified in patients 2 and 3 (Figure 2B and tated POLG has an enhanced base substitution error rate, C) may produce a similar effect on the protein second- Ponamarev et al23 proposed that the mutated protein could ary structure because proline is structurally different from promote deletions between direct repeats of mtDNA. A leucine and has a secondary amino group that main- misinsertion event after correct synthesis of a repeat se- tains a rigid conformation and reduces the structural flex- quence of mtDNA could be followed by strand slippage ibility of polypeptidic chains. Proline 587 is located im- between the direct repeats, thereby creating a matched mediately adjacent to motif III of the exonuclease DNA terminus at the downstream template sequence.23 domain.20 In some species, proline is substituted by glu- The results of this study can be used to confirm the tamic acid or threonine but never by leucine. genetic and clinical heterogeneity of diseases caused by Patient 4 was compound heterozygous for Ala889Thr POLG mutations. While mutations in ANT1 and C10orf2 and Arg579Trp substitutions (Figure 2D and E). The ala- have been associated with dominant or sporadic cases of nine at residue 889 was also conserved and located ex- PEO,3-5,12-14 mutations in POLG can cause either adPEO

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 A B A T A C C A C G T A CCCCGGT G C T GG C T G C T GCA A G A C G A CCCT G CA T GG A CCCT GG G CCCCA G C 30 140 150 160 90 100 110 120

MPUB M P AS C 384 bp 500 bp

449 bp 300 bp 264 bp 429 bp

120 bp 300 bp 100 bp

C D G AC G A CCCT G C AT GG A CCCT GGG CCCCA G CC AG C T C TTGGG A GT CCA C A T C A G A CCCA C AA 30 90 100 110 80 90 100 110

MPPCC MP CC

384 bp 300 bp 247 bp 300 bp 264 bp

128 bp 120 bp 100 bp 119 bp 100 bp

E C GG AAG C T C T G CCCC GG C T A G A C G A CCCT 60 70 80 90

MPCU

400 bp

300 bp 312 bp 290 bp

Figure 2. Electropherograms (top) and polymerase chain reaction–restriction fragment length polymorphism analyses (bottom) of the mutations. A, Patient 1. Heterozygous mutation G3227T in exon 20 (arrow). Restriction endonuclease BbsI cuts the 449–base pair (bp) mutant DNA into 2 fragments of 429 and 20 bp (smaller fragment not shown). Whereas the patient (P) shows the abnormal 429-bp fragment, her unaffected brother (UB) does not. M indicates marker. B, Patient 2. Heterozygous mutation C1760T in exon 10 (arrow). Restriction endonuclease SmaI cuts the 384-bp wild-type DNA into 2 fragments of 264 and 120 bp. Both the patient and his affected sister (AS) show an uncut 384-bp band. C indicates control. C, Patient 3. Heterozygous mutation C1760T in exon 10 (arrow). Restriction endonuclease SmaI cuts the wild-type DNA into 2 fragments of 264 and 120 bp. The patient shows an uncut band of 384 bp. D, Patient 4. Heterozygous mutation G2665A in exon 17 (arrow) in a reverse primer sequence. Restriction endonuclease Csp6I cuts the patient’s 247-bp DNA into 2 fragments of 128 and 119 bp. E, Patient 4. Heterozygous mutation C1735T in exon 10 (arrow). Restriction endonuclease NciI cuts the wild-type DNA into 2 fragments of 290 and 22 bp (smaller fragment not shown). The patient shows an uncut band of 312 bp. U indicates uncut.

or arPEO.6-8 The clinical phenotypes of patients with POLG more severe than those of patients with ANT1 and C10orf2 mutations appeared to be both more heterogeneous and mutations.6-8,24 In addition to PEO, symptoms included

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 psychiatric disorders, dysphagia, dysphonia, facial diple- versity College of Physicians and Surgeons, 630 W 168th gia, neuropathy, ataxia, extrapyramidal syndromes, and St, New York, NY 10032 (e-mail: [email protected]). profound muscle weakness.7,24 In contrast, psychiatric disorders, neuropathy, and a REFERENCES motor neuron disease–like scenario were described in some 4,14,25 cases of C10orf2 mutations, whereas mutations in ANT1 1. Zeviani M, Servidei S, Gellera C, Bertini E, DiMauro S, DiDonato S. An autoso- caused a relatively homogeneous phenotype, usually lim- mal dominant disorder with multiple deletions of mitochondrial DNA starting at 3,5,12,13 the D-loop region. Nature. 1989;339:309-311. ited to adPEO, ptosis, and muscle weakness ; only 1 2. Bohlega S, Tanji K, Santorelli FM, Hirano M, al-Jishi A, DiMauro S. 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Recessive POLG mutations present- ing with sensory and ataxic neuropathy in compound heterozygote patients with ure 1B and C), but a maternal aunt was affected in family progressive external ophthalmoplegia. Neuromuscul Disord. 2003;13:133-142. 2 (Figure 1B), which suggests that the Pro587Leu muta- 9. Sciacco M, Bonilla E. Cytochemistry and immunocytochemistry of mitochon- tion has variable penetrance. Our data agree with those of dria in tissue sections. In: Attardi G, Chomyn A, eds. Methods in Enzymology. Vol 264. New York, NY: Academic Press; 1996:509-521. a previous report that also showed variable penetrance, evi- 10. DiMauro S, Servidei S, Zeviani M, et al. Cytochrome c oxidase deficiency in Leigh dence of anticipation, and variable clinical expression of syndrome. Ann Neurol. 1987;22:498-506. POLG mutations.7 11. Andreu AL, Marti R, Hirano M. Analysis of human mitochondrial DNA mutations. In: Potter NT, ed. Methods in Molecular Biology. Vol 217. Totowa, NJ: Hu- We show that POLG mutations account for a sub- mana Press; 2002:185-197. stantial proportion of patients with PEO and multiple 12. Komaki H, Fukazawa T, Houzen H, Yoshida K, Nonaka I, Goto Y. A novel D104G mutation in the adenine nucleotide translocator 1 gene in autosomal dominant mtDNA deletions (13% of all cases; 17% of familial cases). progressive external ophthalmoplegia patients with mitochondrial DNA with mul- However, the proportion of patients with familial autoso- tiple deletions. Ann Neurol. 2002;51:645-648. mal PEO with POLG mutations in our series is lower than 13. Napoli L, Bordoni BS, Zeviani M, et al. A novel missense adenine nucleotide trans- 7 locator-1 gene mutation in a Greek adPEO family. Neurology. 2001;57:2295-2298. that in a previous study from Europe (46% [13 of 28]). 14. Lewis S, Hutchison W, Thyagarajan D, Dahl HH. Clinical and molecular features This smaller number may be because of differences in the of adPEO due to mutations in the Twinkle gene. J Neurol Sci. 2002;201:39-44. ethnic backgrounds of the 2 series of patients. 15. Fan YS, Yang HM, Lin CC. Assignment of the human muscle adenine nucleotide translocator gene (ANT1) to 4q35 by fluorescence in situ hybridization. Cyto- We suggest screening of POLG in patients with famil- genet Cell Genet. 1992;60:29-30. ial PEO with multiple mtDNA deletions, especially in pa- 16. Longley MJ, Ropp PA, Lim SE, Copeland WC. Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma. Biochemis- tients with multisystemic involvement. However, it is also try. 1998;37:10529-10539. important to note that the genetic abnormalities underly- 17. Shadel GS, Clayton DA. Mitochondrial DNA maintenance in vertebrates. Annu ing many cases of adPEO and arPEO remain unknown, Rev Biochem. 1997;66:409-435. 18. Lim SE, Longley MJ, Copeland WC. The mitochondrial p55 accessory subunit of which indicates that other genes are involved in the cause human DNA polymerase gamma enhances DNA binding, promotes processive of these syndromes and remain to be identified. DNA synthesis, and confers N-ethylmaleimide resistance. J Biol Chem. 1999; 274:38197-38203. 19. Iyengar B, Luo N, Farr CL, Kaguni LS, Campos AR. The accessory subunit of DNA Accepted for publication February 13, 2003. polymerase gamma is essential for mitochondrial DNA maintenance and develop- Author contributions: Study concept and design (Drs ment in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2002;99:4483-4488. 20. Spelbrink JN, Toivonen JM, Hakkaart GA, et al. In vivo functional analysis of the Filosto, Mancuso, Shanske, Hirano, and DiMauro); acqui- human mitochondrial DNA polymerase POLG expressed in cultured human cells. sition of data (Drs Filosto, Mancuso, Nishigaki, Harati, J Biol Chem. 2000;275:24818-24828. Gooch, Mankodi, Bayne, and Bonilla and Ms Pancrudo); 21. Braithwaite D, Ito J. Compilation, alignment and phylogenetic relationship of DNA polymerases. Nucleic Acids Res. 1993;21:787-802. analysis and interpretation of data (Drs Filosto, Mancuso, 22. Ropp PA, Copeland WC. Cloning and characterization of the human mitochon- Shanske, Hirano, and DiMauro); drafting of the manu- drial DNA polymerase, DNA polymerase gamma. Genomics. 1996;36:449-458. 23. Ponamarev MV, Longley MJ, Nguyen D, Kunkel TA, Copeland WC. Active site mu- script (Drs Filosto and DiMauro and Ms Pancrudo ); criti- tation in DNA polymerase gamma associated with progressive external ophthal- cal revision of the manuscript for important intellectual con- moplegia causes error-prone DNA synthesis. J Biol Chem. 2002;277:15225- tent (Drs Mancuso, Nishigaki, Harati, Gooch, Mankodi, 15228. 24. Van Goethem G, Martin JJ, Lofgren A, et al. Unusual presentation and clinical Bayne, Bonilla, Shanske, Hirano, and DiMauro); study su- variability in Belgian pedigrees with progressive external ophthalmoplegia and pervision (Drs Filosto, Hirano, and DiMauro). multiple deletions of mitochondrial DNA. Eur J Neurol. 1997;4:476-484. This study was supported by National Institutes of 25. Suomalainen A, Majander A, Wallin M, et al. Autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Neurology. 1997; Health grants NS11766 and PO1HD 32062 and by a grant 48:1244-1253. from the Muscular Dystrophy Association; Department of 26. Siciliano G, Tessa A, Petrini S, et al. Autosomal dominant external ophthalmoplegia and bipolar affective disorder associated with a mutation in the ANT1 gene. Neurological Sciences and Vision, University of Verona, Neuromuscul Disord. 2003;13:162-165. Italy (Dr Filosto); and Department of Neurosciences, Uni- 27. Nishino I, Spinazzola A, Hirano M. Thymidine phosphorylase gene mutations in versity of Pisa, Italy (Dr Mancuso). MNGIE, a human mitchondrial disorder. Science. 1999;283:689-692. 28. Rovio AT, Marchington DR, Donat S, et al. Mutations at the mitochondrial DNA Corresponding author and reprints: Salvatore Di- polymerase (POLG) locus associated with male infertility. Nat Genet. 2001;29: Mauro, MD, Department of Neurology, 4-420 Columbia Uni- 261-262.

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