The novel mitochondrial trnaasn gene mutation m. 5709t>c produces ophthalmoparesis and respiratory impairment

ABSTRACT Although mutations in mitochondrial tRNAs constitute the most common mtDNA defect, the presence of pathological variants in mitochondrial tRNAAsn is extremely rare. We were able to

identify a novel mtDNA _tRNA__Asn_ gene pathogenic mutation associated with a myopathic phenotype and a previously unreported respiratory impairment. Our proband is an adult woman with

ophthalmoparesis and respiratory impairment. Her muscle biopsy presented several cytochrome _c_ oxidase-negative (COX−) fibres and signs of mitochondrial proliferation (ragged red fibres).

Sequence analysis of the muscle-derived mtDNA revealed an m.5709T>C substitution, affecting mitochondrial _tRNA__Asn_ gene. Restriction-fragment length polymorphism analysis of the

mutation in isolated muscle fibres showed that a threshold of at least 91.9% mutated mtDNA results in the COX deficiency phenotype. The new phenotype further increases the clinical spectrum

of mitochondrial diseases caused by mutations in the _tRNA__Asn_ gene. SIMILAR CONTENT BEING VIEWED BY OTHERS IDENTIFICATION OF TWO NOVEL _RRM2B_ VARIANTS ASSOCIATED WITH AUTOSOMAL RECESSIVE

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X-LINKED MYOTUBULAR MYOPATHY Article 29 August 2020 INTRODUCTION Many mitochondrial disorders are associated with mutations in mitochondrial tRNAs. To date, over 200 point mutations

affecting mitochondrial _tRNA_ genes have been described.1 Nevertheless, mutations in the _tRNA__Asn_ gene are very rare; until now, only five pathogenic variants have been associated with

clinical phenotypes ranging from chronic external ophthalmoplegia (cPEO), with or without mitochondrial myopathy, to lethal early-onset encephalomyopathy.2 Here we present clinical and

molecular features of a patient with ophthalmoparesis and respiratory impairment associated with a novel heteroplasmic mutation (m.5709T>C) disclosed in the mitochondrial _tRNA__Asn_

gene. CASE REPORT The proband is a 51-year-old woman with a several year history of progressive external ophthalmoparesis (PEO) and bilateral eyelid ptosis, which was surgically corrected at

age 29, but which gradually worsened again in the following years. At age 47 years, she was admitted to hospital for subacute onset of a severe respiratory insufficiency and she was

diagnosed with a restrictive syndrome with indication for non-invasive ventilation (B-PAP) during the night. Her clinical history is complicated by hypothyroidism (she is taking replacement

therapy) and anxious–depressive syndrome. Neurological examination showed bilateral eyelid ptosis and bilateral, both vertical and horizontal, ophthalmoparesis without diplopia. She had mild

axial and proximal upper limb weakness (bilateral sternocleidomastoid and deltoid muscles) with brisk tendon reflexes and no sensitive alterations. Neither cerebellar nor gait dysfunction

were observed. Blood tests were normal, except for mildly elevated CK (between 290 and 450 U/L, nv <185 U/L), LDH (872 U/L with nv 125–243 U/L) and basal lactate (3.9 mmol/l venous, with

nv 0.90–1.70 mmol/l and 3.1 mmol/l arterial with nv 0.36–1.25 mmol/l). EMG examination disclosed mild non-specific abnormalities in both deltoid and quadriceps muscles. Cardiological

evaluation and tests were normal. Respiratory functional tests confirmed a chronic respiratory insufficiency with restrictive syndrome. Her younger sister, aged 42 years, has mild mental

retardation and unspecified psychiatric disorders, but no eyelid ptosis or ophthalmoparesis. Her serum CK levels are normal. Both the mother and the maternal grandmother are reported

affected with sarcoidosis and ophthalmoparesis (no clinical reports available). The father is healthy and neither the proband nor the sister has children. At age 49 years, the patient

underwent left deltoid muscle biopsy, which was consistent with a mitochondrial disorder. MATERIALS AND METHODS Histological and histochemical analysis of the muscle biopsy, Southern blot

analysis of muscle mtDNA and PCR assay for multiple deletions were performed as described.3, 4, 5 Fragments encompassing the 22 _tRNA_ genes were PCR-amplified, using a set of primers

contained in the MitoSEQ Resequencing System (Applied Biosystem, Foster City, CA, USA) and sequenced on an Applied Biosystem 3100 Genetic Analyzer. A 750-bp fragment encompassing

m.5709T>C mutation was PCR-amplified. The mutated nucleotide C introduces a BstXI-restriction site. BstXI cuts the mutated molecules into two fragments of 500 and 250 bp, whereas the

wild-type PCR fragment remains uncut. Single fibres were microdissected from 30-μm thick muscle sections using the Leica Laser Microdissection Microscope ASLMD (Leica Microsystems, Wetzlar,

Germany) and were processed as described.6 Mutational load assessment in single fibres, muscle biopsy and blood was performed by last-cycle hot PCR7 followed by restriction-fragment length

polymorphism (RFLP) analysis. Aliquots (20 _μ_l) of PCR products were digested and electrophoresed in 5% non-denaturing acrylamide gel. The proportion of mutant mtDNA was evaluated by

densitometry using the NIH ImageJ software (http://rsbweb.nih.gov/ij/). RESULTS MORPHOLOGICAL AND HISTOCHEMICAL INVESTIGATIONS Left deltoid muscle biopsy showed well-preserved muscle

architecture with evidence of ragged red fibres (RRFs; Figures 1a and c). Histochemical analysis revealed several cytochrome _c_ oxidase (COX)-negative fibres (Figure 1b), many of them with

increased SDH activity (Figure 1d) indicating mitochondrial proliferation. MOLECULAR GENETIC ANALYSIS Multiple deletions of mtDNA were ruled out by Southern blot and PCR analyses. Direct

sequencing of PCR-amplified fragments encompassing the 22 _tRNA_ genes of muscle mtDNA revealed a T-to-C transition at nucleotide position 5709. This substitution results in the replacement

of a well-conserved A with a G in the cloverleaf secondary structure of the _tRNA__Asn_ gene, whose transcription proceeds on the opposite strand (Figure 1e). The A-to-G transition seems to

result in a shift in the nucleotides involved in the D-stem formation, leading to an impaired D-stem and loop structure (Figure 1f). The mutation was not found in 100 Caucasian controls.

PCR–RFLP analysis showed that the mutation was heteroplasmic in skeletal muscle and in white blood cells, percentage of mutated genomes being 89.0% and 21.7%, respectively. The mutation was

not detected in blood from the proband's sister (Figure 1g). Single fibre analysis showed a higher degree of heteroplasmy in COX-deficient fibres (93.1±1.5%, _n_=5) compared with

COX-positive muscle fibres (78.6±5.5%, _n_=4, Figures 1g and h). The difference between COX-positive and COX-negative fibres was statistically significant (_P_<0.01). DISCUSSION More than

half of the pathogenic mtDNA mutations are concentrated in _tRNA_ genes.2 So far, five pathogenic point mutations within _tRNA__Asn_ gene have been described in seven probands: m.5703G>A

associated with bilateral ptosis, cPEO and mitochondrial myopathy,8, 9 m.5692T>C associated with cPEO,10 m.5698G>A causing an isolated myopathy with cPEO in two sporadic patients,11,

12 m.5693T>C associated with a lethal early-onset encephalomyopathy13 and m.5728T>C leading to severe multiorgan failure.14 Here we report the novel substitution m.5709T>C in

muscle-derived mtDNA from a woman affected with ophthalmoparesis and respiratory impairment. Our study meets most of the consensus criteria used to assign pathogenicity to mitochondrial tRNA

variants:15 (i) m.5709T>C was found in heteroplasmic state; (ii) the proportion of mutated mtDNA was higher in the affected tissue (skeletal muscle) than in an unaffected tissue with

rapid turnover (blood); (iii) the mutation occurs at an evolutionary conserved site in the mitochondrial genome and it seems to cause a significant structural impairment in the tRNAAsn

D-stem and loop; (iv) m.5709T>C was absent in a cohort of ethnically matched healthy controls; (v) mutation load was significantly higher in isolated COX-negative fibres than in

COX-positive single muscle fibres. We could not prove the matrilineal transmission, suggested by both the kind of mutation and the family history, as maternal mtDNA was unavailable.

Muscle-derived mtDNA from the sister was not available to ascertain the presence of the mutation and, however, she does not have a myopathic phenotype. Because we did not detect mutated

molecules in her leukocytes, we are currently unable to say whether the sister's clinical condition, mainly affecting the central nervous system, is another possible manifestation of

the mutation-related clinical picture. The small number of tRNAAsn mutations reported so far makes a genotype–phenotype correlation a hazardous task. Heterogeneous clinical presentations can

be divided into two groups: an early onset syndromic disease leading to lethal mitochondrial encephalomyopathy13 or multi-organ failure,14 and an adult onset phenotype characterized by

cPEO, myopathy and a neurological syndrome resembling MERRF (myoclonus epilepsy and RRF).8, 9, 10, 11, 12 As expected, mutations resulting in severe phenotypes or fatal outcome were present

at almost homoplasmic levels in blood and skeletal muscle (>97%), whereas mutation load appeared lower in adult-onset patients, ranging from 46 to 80%. In our proband, the level of

heteroplasmy in leukocytes was moderately low, whereas the mutation appeared largely heteroplasmic in skeletal muscle tissue. A narrow, although significant, mutational threshold could be

still identified analysing isolated muscle fibres. Our patient shared the myopathic phenotype and developed cPEO, followed by a previously unreported respiratory impairment. Considering her

sister's clinical features, an involvement of the central nervous system by the same mutation cannot be excluded. Ventilatory failure is frequently described in both acute and

chronic-progressive neuromuscular disorders.16 In the latter, it usually appears late in the course of the disease, but occasional presentation with respiratory failure has been reported,

including restrictive syndrome in mitochondrial disorders.17 Two pathophysiological mechanisms leading to respiratory failure in patients with mitochondrial disorders have been proposed: (i)

abnormal respiratory drive caused by dysfunction in the central nervous system (brainstem) respiratory centers; (ii) weakness and/or fatigue of the inspiratory muscles. In our patient, the

first mechanism is unlikely to be involved, because other clinical signs of brainstem dysfunction were absent. We therefore hypothesise an inspiratory muscle dysfunction due to both the

fatigue from inappropriate energetic supply and from primary respiratory muscle weakness. Pulmonary function tests in our patient (single parameters not available) confirmed respiratory

muscle weakness with evidence of a restrictive syndrome. In conclusion, our data provide strong elements supporting the pathogenic role of the m.5709T>C mutation, and help define its

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ACKNOWLEDGEMENTS Gratitude has to be expressed to the patient for participating in this research. We wish to thank especially the ‘Associazione Amici del Centro Dino Ferrari’ for their

support. The financial support of the following research grant is gratefully acknowledged: Telethon – UILDM Project GUP09004 ‘Construction of a database for a nation-wide Italian

collaborative network of mitochondrial diseases’, Associazione Amici del Centro Dino Ferrari, University of Milan, the Telethon project GTB07001, the Eurobiobank project QLTR-2001-02769 and

RF 02.187 Criobanca Automatizzata di Materiale Biologico. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Neurological Sciences, Dino Ferrari Centre, University of Milan, IRCCS

Foundation Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Dario Ronchi, Monica Sciacco, Andreina Bordoni, Michela Ripolone, Elisa Fassone, Alessio Di Fonzo, Mafalda Rizzuti, Patrizia

Ciscato, Alessandra Cosi, Maura Servida, Maurizio Moggio, Stefania Corti, Nereo Bresolin & Giacomo P Comi * Department of Neurology, Neurocenter of Southern Switzerland, Lugano,

Switzerland Monika Raimondi * Centre of Excellence on Neurodegenerative Diseases, University of Milan, Milan, Italy Stefania Corti, Nereo Bresolin & Giacomo P Comi * IRCCS Eugenio Medea,

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authors declare no conflict of interest. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ronchi, D., Sciacco, M., Bordoni, A. _et al._ The novel

mitochondrial _tRNA__Asn_ gene mutation m.5709T>C produces ophthalmoparesis and respiratory impairment. _Eur J Hum Genet_ 20, 357–360 (2012). https://doi.org/10.1038/ejhg.2011.238

Download citation * Received: 29 April 2011 * Revised: 28 October 2011 * Accepted: 11 November 2011 * Published: 21 December 2011 * Issue Date: March 2012 * DOI:

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currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * progressive external ophthalmoplegia * tRNA(Asn) *

mitochondrial myopathy