Mitochondrial m.13513G>A Point Mutation in ND5 in a 16-Year-Old Man with Leber Hereditary Optic Neuropathy Detected by Next-Generation Sequencing

Abstract

This article reports a Leber hereditary optic neuropathy (LHON) case associated for the first time with mitochondrial m.13513G>A mutation. We present a 16-year-old man who complained of subacute, painless, visual loss. Ocular examination showed optic nerve atrophy, papillary pseudoedema, and optic disc pallor. Extraocular manifestations included hypertrophic myocardiopathy and myopathy. Initial genetic analysis excluded the three most common LHON mutations. Sanger sequencing of the whole mitochondrial deoxyribonucleic acid showed no mutation. Next-generation sequencing (NGS) revealed m.13513G>A mutation in the NADH dehydrogenase (ND5) subunit gene ( MT-ND5 ). The m.13513G>A mutation has never been associated with LHON phenotype without Leigh/mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes features. NGS techniques should be considered when this diagnosis is strongly suspected.

Introduction

Leber hereditary optic neuropathy (LHON) is the most common optic neuropathy caused by a primary mutation in mitochondrial deoxyribonucleic acid (mtDNA). It is characterized by chronic or subacute painless visual failure, which typically starts in the second decade of life. Ophthalmologic examinations, such as fundoscopy, fluorescein angiography, or optical coherence tomography (OCT) often reveal papillary thickness, hyperemia, and edema in the acute phase or atrophy of retinal nerve fiber layer (RNFL) in the chronic phase. Extraocular manifestations can happen, including peripheral neuropathy, nonspecific myopathy, or cardiac conduction abnormality, like Wolff–Parkinson–White (WPW) syndrome.

LHON is caused in 95% of cases by one of the three common mtDNA variants (m.3460G>A, m.11778G>A, or m.14484T>C). However, the presence of other less frequent mutations in mtDNA should be investigated, if clinician suspicion remains high, since other rare mutations have been described to be associated with LHON.

Although mitochondrial diseases lack curative treatment, genetic diagnoses should be pursued, since families can benefit from genetic counseling ¹ and other valuable information can be obtained. Furthermore, nowadays, there are clinical trials whose participants are enrolled based on molecular criteria, ² and therefore, fast diagnosis of LHON patients is crucial to enable enrollment.

Methods

Case Report

A 16-year-old man was seen in the neurology department complaining of progressive painless visual loss of the right eye which had started about 5 months ago. Past medical history was noncontributory. There was neither psychomotor regression, nor episodes suggestive of seizures, weakness, muscle pain, or cramps. There was no family history of visual impairment or any other feature suggestive of mitochondrial disease in any first-degree relative.

At the initial examination, the patient was unable to count fingers with the right eye. Visual acuity was normal in the left eye and no signs suggestive of external ophthalmoplegia were detected. No other abnormalities were detected. Two months later, the patient had worsened, with progression to bilateral blindness. Corticoid therapy was initiated but no benefit was noted.

Fundoscopy showed papillary pseudoedema in the left eye and optic disc pallor in the right eye. Fluorescein angiography showed bilateral peripapillary telangiectatic blood vessels. Papillary OCT showed thickened in the left eye and was normal in the right eye. OCT showed thinner RNFL in the macula of both eyes. Brain magnetic resonance imaging (MRI) showed pituitary macroadenoma, diagnosed as a nonfunctioning pituitary macroadenoma when further studied. Cerebrospinal fluid analysis was normal. Serum aquaporin-4 antibodies were negative. No hyperlactacidemia was present.

Electromyography showed decreased duration of motor unit potentials in the right biceps brachii and tibialis anterior muscles, suggesting myopathy. Visual evoked potentials exhibited increased latencies, with decreased amplitude in the left side and absent response in the right side.

Transthoracic echocardiogram showed moderate concentric left ventricular hypertrophy, global hypercontractility with ejection fraction of 75%, with mild dilated left atria (36 mL/m ² ) and without obstruction to left ventricle outflow. There was no valvular disease. All these features were suggestive of nonobstructive hypertrophic cardiomyopathy. Electrocardiogram and Holter registry over 24 hours did not show abnormalities of cardiac rhythm.

Mitochondrial DNA Genetic Analysis

Initial genetic analysis of blood sample showed none of the three most common mtDNA pathogenic variants of LHON. Therefore, and taking into account the clear clinical diagnosis of LHON, we decided to sequence the entire mtDNA from the patient's blood, by the classical Sanger's method commonly used in different laboratories, but no mutations were found. Since lower levels of mutation are not clearly detected by this methodology, we finally decided to reanalyze the whole mtDNA genome by next-generation sequencing (NGS) and a mutation at position m.13513G>A was identified. This mutation, localized in the MT-ND5 of complex I of the oxidative phosphorylation system, was present at 29% heteroplasmy. Sanger's sequencing of a fragment of mtDNA containing this mutation was performed to confirm the result ( Fig. 1 ).

Fig. 1.

Sanger confirmation of the m.13513G>A.

Discussion

In this work, we present a case of progressive visual failure, hypertrophic cardiomyopathy, and myopathy. Ophthalmologic examination strongly suggested the diagnosis of LHON. Brain MRI showed nonfunctioning pituitary macroadenoma, but was otherwise unremarkable. This MRI finding was unrelated to visual failure. After negative preliminary genetic analysis, NGS detected a m.13513G>A transition with a mutation load in blood of 29% that was later confirmed by Sanger' sequencing. The pathophysiology of the mutation results in a substitution of an evolutionarily conserved amino acid of the ND5 subunit, causing decrease in complex I activity. We considered this mutation to be likely the cause of the clinical picture of the patient, given its well-known pathogenicity. The mutation has been reported to have a pathogenicity score of 39. ³ Efforts to describe genotype–phenotype correlations have been attempted, without significant results. The m.13513G>A mutation was first described as a cause of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), ⁴ and it is also known to be a frequent cause of Leigh disease, even with low mutation load. ⁵ Patients with Leigh disease and m.13513G>A mutation can develop visual disturbances with Leber features. ⁶ As many other mtDNA mutations, the spectrum of clinical phenotypes related to this mutation have been recently expanded, ^{7 8 9} from catastrophic neonatal forms to asymptomatic cases. But, as far as we are aware, the mutation has never been reported as a cause of LHON without Leigh or MELAS features.

WPW syndrome has been previously described to be related to m.13513G>A mutation in the literature. ^{8 10 11 12} However, WPW syndrome has always been described in association with Leigh syndrome, and never with LHON or MELAS phenotypes. ⁷ We neither reported conduction abnormalities in our patient nor in his mother. However, hypertrophic myocardiopathy was found in our patient, which was asymptomatic and was detected in the setting of mitochondrial disease study.

For a better comparison with previous published results, we provide a table with prior cases summarizing their symptoms, age of onset, and heteroplasmy levels in different tissues ( Table 1 ).

Table 1. Characteristics of patients with m.13513G>A mutation previously published.

Number	Age of onset	Syndrome	Visual phenotype	Cardiac conduction abnormalities	Heteroplasmy levels	Reference
2	Patient 1 : 18 mo Patient 2 : 9 y	Leigh	Patient 1 : Optic atrophy Patient 2 : Optic atrophy	WPW	Patient 1: Muscle: 63% Blood: 42% Patient 2 : Muscle: 64% Blood 26%	11
2	Patient 1 : 4 mo Patient 2 : 11 y	Patient 1 : MELAS/Leigh Patient 2 : MELAS	Patient 1 : Retinitis Pigmentosa Patient 2 : Chronic progressive external ophthalmoplegia	–	Patient 1: Muscle: 16% Blood: 11–17% Patient 2 : Muscle 13–15% Blood: 4–6%	9
3	Patient 1 : 11 y Patient 2 : 12 y Patient 3 : 19 mo	Patient 1 : MELAS/Leigh Patient 2 : Leigh patient Patient 3 : MELAS/Leigh	Patient 1 : Ophthalmoplegia Patient 2 : – Patient 3 : Optic atrophy	Patient 1 :– Patient 2 : WPW Patient 3 : WPW	Patient 1: Brain: 46% Patient 2: Blood: 52% Patient 3: Blood: 61%	8
3	Patient 1 : 8 y Patient 2 : Intrauterine growth retardation Patient 3 (mother): Asymptomatic	Patient 1 : MELAS Patient 2 : Pneumonia with sepsis and chorioamnionitis	Patient 1 : Optic neuropathy Diplopia Patient 2 : – Patient 3 : –	Patient 1 : – Patient 2: Enlarged heart Patient 3 : –	Patient 1: Urine: 42% Muscle: 30% Patient 2: Liver: 66% Heart: 44% Patient 3: Urine: 7% Blood: Undetectable	10
2	Patient 1 : 3 mo Patient 2 (mother):	Patient 1 : Leigh Patient 2 : Asymptomatic	Patient 1 : Normal fundoscopy Patient 2 : –	Patient 1: Mild left ventricular hypertrophy without outflow tract obstruction and a patent foramen ovale Patient 2: WPW	Patient 1: – Patient 2: Blood 14% Urine 54%	7
1	12 mo	Tachyarrhythmia Hyponatremia inappropriate ADH secretion	Exotropia and a new–onset downbeat nystagmus with unremarkable retinae and optic nerve discs	WPW-like conduction defect	Muscle and lymphocytes: 50%	12

Abbreviations: ADH, antidiuretic hormone; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; WPW, Wolff–Parkinson–White.

As can be observed in Table 1 , in general, heteroplasmy levels are not well correlated with illness severity. In our patient, we found a mutational load of 29% in blood sample as it is usually performed in LHON genetic studies.

Electromyographic features of myopathy were also found, which were not symptomatic, as it is known can occur in LHON. ¹³

One aspect of interest of our study is the fact that the initial genetic analysis by Sanger sequencing was negative. Even though Sanger has been reported to detect levels as low heteroplasmy as 15%, ¹⁴ it is known that low levels of heteroplasmy can result in false negatives. NGS allows to detect very low levels of heteroplasmy. ¹⁵ Therefore, NGS should be considered when there is strong evidence of suspicion of LHON.

Our study has many limitations. For example, we could not test the presence of mutation in the mother or any other family members. Anyway, the mother was not on fertile age and, thus, we considered she would not have obtained any benefit of genetic counseling. Moreover, she was asymptomatic and electrocardiographic evaluation did not show any features suggestive of WPW. Therefore, we do not know whether our patient carries a de novo mutation. Though the patient had LHON phenotype at the time of diagnosis, we cannot exclude that he can develop a mixed phenotype with Leigh or MELAS features in the future. Finally, it would have been interesting to study this mutation with a muscle biopsy to investigate if the level of heteroplasmy could be higher. However, since the molecular diagnosis was made with a noninvasive test, we rejected this option.

Conclusion

LHON can be caused by the m.13513G>A transition. Phenotypes associated with this mutation are widely variable. We think that this mutation should be tested for in suspected cases of LHON, even when no association to features of Leigh or MELAS syndromes is present. We also think that negative initial results of whole mtDNA sequencing by classic methods do not exclude LHON or other mtDNA diseases when mutations are present at low level of heteroplasmy. NGS is suitable for diagnosis if clinician suspicion remains high.

Funding Statement

Funding Dr. Garcia Arumi reports grants from Instituto de Salud Carlos III, during the conduct of the study. Dr. Montoya reports grants from Instituto de Salud Carlos III, grants from Departamento de Ciencia, Tecnología y Universidad del Gobierno de Aragón (Grupos Consolidados B33), and FEDER Funding Program from the European Union, during the conduct of the study.