A late-onset mitochondrial myopathy is associated with a novel mitochondrial DNA (mtDNA) point mutation in the tRNATrp gene

Neuromuscular Disorders 8 (1998) 291–295

A late-onset mitochondrial myopathy is associated with a novel mitochondrial DNA (mtDNA) point mutation in the tRNATrp gene G. Silvestri a, M. Rana a, A. DiMuzio b, A. Uncini b, P. Tonali a, S. Servidei a ,* a

Neurological Institute, Catholic University, UILDM, L. go A. Gemelli 8, 00168 Rome, Italy b Neurological Institute, University of Chieti, Chieti, Italy

Received 27 October 1997; revised version received 6 January 1998; accepted 29 January 1998

Abstract We detected a novel pathogenic mutation, a G → A transition at position 5521 of mitochondrial tRNATrp gene, in association with familial late-onset mitochondrial myopathy. The mutation was detected in muscle but not in leukocytes from the family’s proband. Morphological and biochemical studies documented a severe defect of muscle cytochrome c oxidase (COX) activity. RFLP analysis of single muscle fibers demonstrated segregation of higher percentages of mutated genomes in COX-negative ragged red fibres compared with normal fibers. A predominant impairment in synthesis of subunits I and III of complex IV due to their highest relative content of tryptophane might explain the greater susceptibility of complex IV to the pathogenic effect of this mutation. A progressive accumulation of mutated genomes in muscle can account for the late onset of symptoms observed in affected members.  1998 Published by Elsevier Science B.V. All rights reserved Keywords: Late-onset mitochondrial myopathy; Point mutation; tRNATrp gene

1. Introduction Mitochondrial diseases due to mitochondrial tRNA gene mutations are usually multisystem disorders with infantile or juvenile onset of symptoms. Late onset rarely exceeds middle-age and is seldom reported in full-syndrome patients. It can, however, be more commonly observed in oligosymptomatic relatives. The biochemical phenotype expressed by a tRNA mutation is usually characterized by a partial generalized decrease of all respiratory chain enzymes containing mtDNA-encoded subunits, which reflects the generalized impairment of mtDNA translation caused by tRNA malfunction. We now report a novel mtDNA point mutation located in the tRNATrp gene associated with a familial mitochondrial myopathy characterized by peculiar clinical and biochemical features. All affected members had a late onset of symptoms; biochemical studies on the proband’s muscle showed a selective decrease of cytochrome c oxidase (COX) activ-

* Corresponding author. Tel.: +39 6 30154435; fax: +39 6 35501909.

ity, while other respiratory chain activities were in the normal range.

2. Materials and methods Histochemistry and biochemistry on muscle, DNA extraction, Southern blot and analysis for MELAS 3243/ 3271, MERRF 8344/8356 and PEO 4285/5703/5692 mutations and the sequence of tRNA genes were done as described [1]. Single-fiber PCR was performed according to Moraes and Schon [2]. Screening for the G → A at nt. 5521 of mtDNA was done by RFLP analysis of a 91 bp PCR fragment amplified by modified primers. We used a reverse oligonucleotide (nt. 5550–5580 according to Anderson’s sequence [3]) previously designed for screening for the G → A at nt. 5549, which contains two mismatches as reported in the original paper [4]; one of them, a G → A at nt. 5552, eliminates the DdeI site (C/TNAG) present in the reference sequence. The forward primer (nt. 5490–5519) contains a C → T mismatch at nt. 5517 that introduces a novel DdeI site in the amplified fragment (nt. 5517 -C/TTAG- nt. 5521), i.e. DdeI

0960-8966/98/$19.00  1998 Published by Elsevier Science B.V. All rights reserved PII S0960-8966 (98 )0 0037-6

292

G. Silvestri et al. / Neuromuscular Disorders 8 (1998) 291–295

Fig. 1. Cryostat serial muscle sections stained for COX (a) and SDH (b) reactions. There is a focal decrease or absence of COX activity in the majority of fibers, which appear pale in (a) accompanied by a marked mitochondrial proliferation, as shown by their strong SDH reaction in (b).

cuts normal mtDNA into two fragments of 63 and 28 bp; the G → A at nt. 5521 eliminates this site (nt. 5517 -CTTAAnt. 5521) and the mutant mtDNA remains uncut. The amplification conditions were 1 min at 94°C, 1 min at 50°C and

42 s at 72°C for 25 cycles. PCR (30 ml) was digested in 50 ml by 10 U of DdeI for 2 hours at 37°C. Digestion products were separated on a 12% polyacrylamide gel. For quantification of the percentage of mutation in tissues and in single

Fig. 2. (Left) tRNATrp structure and location of the mutation in the D-stem. Sequence analysis of the tRNATrp gene, showing the G → A transition at nt. 5521 in the patient. (Right) Comparison of tRNATrp sequences through evolution, revealing a high degree of conservation of the site in all species.

G. Silvestri et al. / Neuromuscular Disorders 8 (1998) 291–295

fibers 50 pmol of [g-32P]ATP end-labeled backward primer was added to each PCR reaction before the last cycle. Densitometric analysis of bands from X-ray films exposed to gels was performed using a BioRad densitometer GS670. Statistical analysis (mean, standard deviation, Student’s ttest) was performed using Microsoft Excel software version 5.0.

3. Case report and results A 68-year-old male noticed progressive bilateral ptosis and fatigue since the age of 50. His mother, deceased at age 90, and one of his brothers were reported to be similarly affected (the pedigree is represented in Fig. 3). Clinical examination documented bilateral ptosis without ophthalmoplegia, dysphonia and mild proximal muscle wasting and weakness (F = 4, MRC scale). CK and LDH were normal. EMG was myopathic. Muscle biopsy showed a mitochondrial myopathy with many COXnegative ragged red fibers (COX− RRF) (Fig. 1). Biochem-

293

istry documented a severe COX deficiency in muscle (Table 1). Routine mtDNA studies, including an extensive search for known mtDNA mutations (see Section 2), were negative. The sequence of mitochondrial tRNA genes revealed a G → A transition at position 5521 of the tRNATrp gene (Fig. 2). The mutation, located in a highly conserved position of the D-stem (Fig. 2), has never been reported in the literature as neutral polymorphism and it was not found in any of 110 muscle control DNAs. It was heteroplasmic in muscle (98%) while it was absent in leukocytes (Fig. 3). Singlefiber PCR (Fig. 3) documented a significantly higher amount of mutated DNAs in COX− RRFs compared with normal COX-positive (COX+) fibers (COX− RRFs (n = 23), mean % of mutant mtDNA 89.90 ± 11.76; COX+ fibers (n = 22), mean % of mutant mtDNA 69.35 ± 26.22, P , 0.005).

4. Discussion The G → A transition at nt. 5521 of mitochondrial

Fig. 3. (Upper) ‘Last cycle hot’ PCR and RFLP analysis for the G → A transition at nt. 5521. A 91 bp fragment encompassing the tRNATrp gene is amplified by modified primers and end-labeled before the last cycle (see Section 2); the wild-type mtDNA is cut by DdeI at nt. 5521 into two fragments of 63 and 28 bp (the 28 bp fragment is unlabeled and is not detectable on the gel). The G → A at nt. 5521 eliminates this site and the mutant DNA remains uncut. (Left) Analysis of DNA extracted from the patient’s tissues (II 7 in the pedigree) and from a control; U, uncut; M, muscle; B, blood; C, control. (Right) Analysis of single muscle fibers; N, COX+ fibers; R, COX− RRFs; numbers below each line indicate the percentage of mutant mtDNA. (Lower) Scheme of the pedigree; black symbols indicate affected members, while open symbols indicate healthy members. The arrow indicates the proband (II 7).

294

G. Silvestri et al. / Neuromuscular Disorders 8 (1998) 291–295

Table 1 Mitochondrial enzymes activities on muscle homogenate

Cytochrome c oxidase NADH-Cyt c reductase Succinate-Cyt c reductase NADH dehydrogenase SDH Citrate synthase

Patient

Controls

0.49 0.65 0.52 37.50 1.35 19.66

3.20 1.23 1.16 39.62 1.73 13.43

± ± ± ± ± ±

0.99 0.58 0.52 9.44 0.431 3.10

Activities are expressed as mmol substrate utilized/min per g muscle tissue.

tRNATrp gene fulfills all diagnostic criteria for pathogenic mutations, i.e. (i) it is located in a conserved region of the gene, (ii) it has never been reported as neutral polymorphism, nor found in 110 controls and (iii) RFLP analysis demonstrated a condition of heteroplasmy and single-fiber PCR documented higher amounts of mutated mtDNAs in COX− RRFs compared with normal fibers. There was a good correlation between clinical phenotype, characterized by a pure myopathy, and genetic findings. In fact, the mutation segregated in a high percentage (98%) in the patient’s muscle. Accordingly, morphology showed many COX− RRFs and biochemistry documented a severe decrease of COX activity up to 16% of normal mean values. Other respiratory chain enzyme activities were normal. The biochemical phenotype expressed by our patient, characterized by a selective decrease of COX activity, is not typical for a mitochondrial tRNA gene mutation; such a defect more frequently produces a global impairment of mtDNA translation and, consequently, is associated with a partial generalized decrease of all respiratory chain enzymes containing mtDNA-encoded subunits. Nevertheless, a marked and selective COX deficiency has already been reported in association with other tRNA mutations, i.e. the A → G at nt. 8344 in the tRNALys gene [5,6]. The greater susceptibility of cytochrome c oxidase to the pathogenic effect of some tRNA mutations needs to be elucidated. The occurrence of a higher rate of synthesis of complex IV subunits compared with other mtDNA-encoded respiratory chain subunits recently documented in human myoblast clones [7] might explain why this enzyme can be primarily affected by the pathogenic effect of tRNA mutation which interferes with translational efficiency. We believe that in the case of the G → A transition at nt. 5521, two pathogenic mechanisms can synergistically act to decrease the rate of synthesis of COX subunits leading to the severe biochemical defect, i.e. (a) the site of mutation, located in the D-stem, can specifically affect tRNA–ribosome interaction with a consequent decrease in the rate of synthesis of mitochondrial proteins and (b) the higher relative content of tryptophane in two catalytic subunits of COX (COI and COIII) compared to other mtDNA-encoded respiratory chain subunits (Table 2) can further affect the translational efficiency of these subunits. Another potential pathogenic mechanism of mutations in

the tRNATrp gene, such as the G → A at nt. 5521, could be related to the position of this gene in the mitochondrial genome. In fact, it is located close to the origin of mtDNA light-strand replication (OL). Therefore, mutations affecting such a gene could interfere with the regulation of mtDNA light-strand (L-strand) replication, perhaps altering the L-strand secondary structure formed after the H-strand displacement. Southern blot analysis, however, failed to show gross quantitative mtDNA alterations both in our case (data not shown) and in the two other reported tRNATrp gene mutations [4,8]. Further studies, in particular analysis of genotype and phenotype expression of the mutation in r° systems, are needed to clarify this issue. The clinical phenotype expressed in our patient, affected by a pure myopathy, is distinct from the other two tRNATrp mutations reported in literature. The G → A transition at nt. 5549 [4] and the single thymidine insertion at nt. 5537 [8] are in fact both associated with a severe multisystem mitochondrial disorder dominated by central nervous system involvement and psychiatric features. In these cases mtDNA studies documented a widespread distribution of the mutation in all tissues, including leukocytes, with the highest percentage segregating in the brain. Conversely, our patient apparently segregated a very high percentage of mutated genomes (98%) in the muscle only. According to the very high amount of mutation, muscle biopsy showed marked mitochondrial proliferation and a severe COX defect (Fig. 1). A marked decrease in COX activity was detected also in muscle from the patient carrying the single-base insertion at nt. 5537, who in fact segregated an elevated proportion of mutated genomes (.92%) in the tissue. By contrast, the patient with the 5549 mutation, who did not have a clinical myopathy, had a lower amount of mutation (85%) in muscle, fewer COX− RRFs and normal COX activity. Therefore, the occurrence of a Table 2 Absolute and relative amounts of Trp residues in mtDNA-encoded respiratory chain subunits Subunit

Ratio (%)a

CO1 CO2 CO3 ND1 ND2 ND3 ND4 ND4L ND5 ND6 ATPase6-8 apoCytb

16/513 (3.1) 4/227 (1.7) 12/261 (4.5) 9/318 (2.5) 11/347 (3.1) 4/115 (3.5) 13/459 (2.8) 0/98 (0) 12/603 (1.9) 5/174 (2.8) 4/226 (1.7) 12/380 (3.1)

CO, COX; ND, NADH dehydrogenase; apoCytb, apocytochrome b. Arabic numbers (i.e. CO1) indicate the corresponding subunits. a The ratio of the absolute amount of Trp residues versus the total number of amino acids and the relative percentage are indicated for each subunit.

G. Silvestri et al. / Neuromuscular Disorders 8 (1998) 291–295

variable pattern of mitotic segregation may explain why different mutations in the same gene can display distinct clinical and biochemical phenotypes. Besides multiple mtDNA deletions and the A → G transition at nt. 3243 in the tRNALeu(UUR) gene [9,10], the G → A at nt. 5521 represents another mtDNA defect associated with a late-onset myopathy. The late onset and the progression of symptoms observed in all affected members from our family could be explained by a gradual accumulation of mutated genomes in muscle with time. According to this hypothesis, Weber et al. [11] have recently documented an increase in the level of mutated genomes in three biopsies taken over a 12-year period from a patient with a progressive mitochondrial myopathy associated with a novel mitochondrial tRNA Leu(CUN) mutation. In conclusion, we described a novel pathogenic mtDNA mutation in the tRNATrp gene. We found a significant correlation between clinical phenotype, characterized by a pure myopathy, and genetic data, showing the presence of mutation in muscle, but not in leukocytes. This pattern of segregation, although uncommon for mtDNA point mutations, has been reported in other cases [1,11–13] and confirms in patients with pure muscle involvement the importance of performing mtDNA studies on muscle rather than blood, as the latter may give false negative results.

[2]

[3] [4]

[5]

[6]

[7]

[8]

[9] [10]

[11]

Acknowledgements [12]

We thank Enrico Paris and Manuela Papacci for their precious technical assistance. This work was supported by UILDM sez. Laziale, Rome and by Telethon Italy (grant n.52).

References [1] Silvestri G, Servidei S, Rana M, et al. A novel mitochondrial DNA

[13]

295

point mutation in the tRNAIle gene is associated with progressive external ophthalmoplegia. Biochem Biophys Res Commun 1996; 220:623–627. Moraes CT, Schon EA. Detection and analysis of mitochondrial DNA and RNA in muscle by in situ hybridization and single-fiber PCR. Methods Enzymol 1996;264:522–540. Anderson S, Bankier AT, Barrel BG, et al. Sequence and organization of human mitochondrial genome. Nature 1981;290:457–465. Nelson I, Hanna MG, Alsanjari N, Scaravilli F, Morgan Hughes JA, Harding AE. A new mitochondrial DNA mutation associated with progressive dementia and chorea: a clinical pathological, and molecular genetic study. Ann Neurol 1995;37:400–403. Lombes A, Mendell JR, Nakase H, et al. Myoclonic epilepsy and ragged red fibers with cytochrome oxidase deficiency: neuropathology, biochemistry and molecular genetics. Ann Neurol 1989;26:20– 29. Silvestri G, Ciafaloni E, Santorelli FM, et al. Clinical features associated with the A → G transition at nucleotide 8344 of mtDNA (‘MERRF mutation’). Neurology 1993;43:1200–1206. Hanna MG, Nelson IP, Morgan-Hughes JA, et al. Impaired mitochondrial translation in human myoblasts harbouring the mitochondrial DNA tRNA lysine 8344 A → G (MERRF) mutation: relationship to proportion of mutant mitochondrial DNA. J Neurol Sci 1995;130:154–160. Santorelli FM, Tanji K, Sano M, et al. Maternally inherited encephalopathy associated with a single base insertion in the mitochondrial tRNATrp gene. Ann Neurol 1997;42:256–260. Johnston W, Karpati G, Carpenter S, Arnold D, Shoubridge EA. Late onset mitochondrial myopathy. Ann Neurol 1995;37:16–23. Moraes CT, Ciacci F, Silvestri G, et al. Atypical clinical presentations associated with the MELAS mutation at position 3243 of human mitochondrial DNA. Neuromusc Disord 1992;3:43–50. Weber K, Wilson JN, Taylor L, et al. A new mtDNA mutation showing accumulation with time and restriction to skeletal muscle. Am J Hum Genet 1997;60:373–380. Dumoulin R, Sagnol I, Ferlin T, Bozon D, Stepien G, Mousson B. A novel Gly290Asp mitochondrial cytochrome b mutation linked to complex III deficiency in progressive exercise intolerance. Mol Cell Probes 1996;10:389–391. Fu K, Hartlen R, Johns T, et al. A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggest an approach to therapy. Hum Mol Genet 1996;5:1835–1840.