- Email: [email protected]
Leber hereditary optic neuropathy in the population of Serbia
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
Official Journal of the European Paediatric Neurology Society
Original article
Leber hereditary optic neuropathy in the population of Serbia Jasna Jancic a,*, Ivana Dejanovic a, Janko Samard zic d, Sasa Radovanovic e, c f, Vladimir Kostic b Ana Pepic a, Natalija Kosanovic-Jakovic c, Mila Cetkovi a
Clinic of Neurology and Psychiatry for Children and Youth, Medical Faculty, University of Belgrade, Serbia Clinic of Neurology, Medical Faculty, University of Belgrade, Serbia c Institute of Ophthalmology, Medical Faculty, University of Belgrade, Serbia d Institute of Pharmacology and Toxicology, Medical Faculty, University of Belgrade, Serbia e Institute for Medical Research, University of Belgrade, Serbia f Institute of Histology and Embryology, Medical Faculty, University of Belgrade, Serbia b
article info
abstract
Article history:
Background: Leber hereditary optic neuropathy (LHON) is the most common mitochondrial
Received 19 June 2013
disorder. However, few countries have published their population-based findings related to
Received in revised form
this multisystemic disease.
13 January 2014
The aim: In order to get a better insight into the epidemiological and clinical picture of this
Accepted 19 January 2014
maternally inherited disorder, we performed the first population-based clinical and molecular-genetic study of LHON in the Serbian population.
Keywords:
Methods: Prospective study included patients who were diagnosed with LHON after detailed
Mitochondrial disease
medical examination and molecular-genetic confirmation.
Prevalence
Results: We identified 41 individuals from 12 genealogically unrelated families, carrying one
Primary mutations
of the three “primary” mitochondrial (mt) DNA point mutations associated with LHON.
Clinical picture
Fourteen of them were clinically affected, giving a minimum point prevalence of 1.9 per 1 000 000. The minimum point prevalence for mtDNA LHON mutations was 5.2 per 1 000 000. Male to female ratio was 6:1. Only one affected patient harboured mutant mtDNA in heteroplasmic condition. All patients were presented with common clinical findings. Conclusion: We observed significantly lower prevalence and higher gender ratio than expected. However, frequencies of primary mutations, incidence of heteroplasmy and clinical findings are in accordance with other studies in Caucasoid populations. Our results might be a consequence of poor recognition and misdiagnosis due to lack of diagnostic possibilities of the entity in different region of our country or less likely be in part due to specific haplotype background of Serbian population which should be further investigated. ª 2014 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
a 6a, 11000 Belgrade, Serbia. Tel.: þ381 65 * Corresponding author. Clinic of Neurology and Psychiatry for Children and Youth, Dr Subotic 666 6733; fax: þ381 11 2645 064. ic ). E-mail address: [email protected] (J. Janc 1090-3798/$ e see front matter ª 2014 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejpn.2014.01.005
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
1.
Introduction
Leber hereditary optic neuropathy (LHON, MIM 535000), the most common mitochondrial disorder, is characterized by acute or subacute painless loss of central vision, usually in young adults, predominantly male.1,2 Apart from visual loss as its distinctive feature, LHON may be associated with other neurological, cardiac and skeletal abnormalities, characterizing the “LHON plus” phenotype.2 This maternally inherited disorder is mainly associated with mitochondrial DNA (mtDNA) mutations 11778G>A, 3460G>A and 14484T>C, all involving genes encoding complex I subunits of mitochondrial respiratory chain.3 The median age of onset in LHON is between 15 and 35 years, but an individual can become affected at any age between 2 and 80.4e7 A few reports found a bit later age of onset in females.8,9 The disease usually presents with a loss of central vision involving first one eye and then the other with a mean interval of 2 months (75% of cases).10 The fellow eye can also be affected simultaneously (25%) or very rarely the disease can remain monocular.8,10,11 LHON is acute or subacute, but may also manifest as slowly progressive visual loss. Visual impairment is usually severe with visual acuity ranging from 20/200 to counting fingers, in association with profound dyschromatopsia, centrocaecal scotomas and relative preservation of peripheral visual field and pupillary reflexes.12,13 Following the acute phase, on average within six weeks from the disease onset, the optic disc becomes atrophic. Significant improvements in visual acuity are rare and in most individuals vision remains severely impaired. The clinical diagnosis is based on the ophthalmologic features and molecular-genetic testing. Relevant testing may include fundus examination (to identify the characteristic optic disc and vascular changes), kinetic (Goldman) or static perimetry (for central or centrocaecal scotoma), electrophysiological studies in selected cases (visual evoked potentials and pattern electroretinogram), optical coherence tomography (to assess the retinal nerve fibre layer thickness and the optic nerve anatomy) and neuroimaging (to exclude compressive, infiltrative and inflammatory causes of bilateral optic neuropathy).3 In order to get a better insight into the epidemiological and clinical picture of LHON, we present the first population-based clinical and molecular study of LHON in the population of Serbia as region of Europe, in particular as part of the West Balkan area.
2.
Neurology and Psychiatry for Children and Youth in Belgrade is the only referent institution in Serbia where the diagnosis of LHON could be established, therefore it poses the unique database of LHON patients for this region. Prior to inclusion in the study, thorough history was obtained and pedigree analysis performed and all participants underwent physical, neurological and neuro-ophthalmologic examinations. In addition, we have performed fluorescent angiography (FSA), visual electrophysiology, serum lactate and pyruvate levels, blood levels of thyroid hormones, cerebrospinal fluid analysis and magnetic resonance imaging (MRI) of the brain and optic nerves in order to exclude the other ocular or systemic diseases. Probands and their family members were thoroughly informed about the clinical findings. Those with a clinical diagnosis of LHON and their at-risk asymptomatic family members were advised to have a molecular-genetic testing to rule in or rule out the diagnosis. Having obtained their informed written consent, moleculargenetic tests were performed from venous blood samples (5 ml) in the Laboratory of Neurogenetics, Department of Neurological Sciences, Bologna, Italy and the Laboratory of Neurogenetics in the Clinic of Neurology, Clinical Centre of Serbia, Belgrade. The inclusion criteria were having primary (11778, 3460 and 14484) or secondary (14495, 14482, 10663) mtDNA LHON-causing mutations. Local ethical committee approved the research protocol according to the national regulations and study was performed in accordance to ethical standards of the Declaration of Helsinki.
3.
Results
During a 12 year follow-up period we identified 41 individuals, from 12 genealogically unrelated families, carrying mitochondrial DNA mutations associated with LHON. Fourteen of them were clinically affected (probands), giving a minimum point prevalence of 1.9 per 1 000 000. All of the 12 Serbian LHON families harboured a primary mutation. This makes the minimum point prevalence of LHON mutations 5.2 per 1 000 000. Mutations were distributed as follows: MT-11778/ND4 e 58.5% (9/14), MT-3460/ND1 e 34.2% (4/14) and MT-14484/ND6 e 7.3% (1/14) (Fig. 1). In the affected group there were 12 (85.7%) male and 2 (14.3%) female subjects. The gender ratio in this group was different depending on the mutation type. The 11778/ND4 mutation was associated with a less prominent gender ratio (7 males and 2 females), while marked male predominance was
Materials and methods
This prospective study included patients with clinical signs of LHON who were examined and ambulatory monitored in the Clinic of Neurology and Psychiatry for Children and Youth, Clinic of Neurology and Institute of Ophthalmology, Clinical Centre of Serbia, Belgrade in the period of 2000 until February 2013. The study also included family members at risk of developing LHON, being unaffected mutation carriers. The follow-up period was approximately 12 years. The Clinic of
355
Fig. 1 e The distribution of primary mutations.
356
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
Fig. 2 e Gender ratios among primary mutation groups in the proband group and in the group of subclinical affected patients.
found in patients with 3460/ND1 mutation (4 males). In the 14484/ND6 family, there was only one affected male (Fig. 2). The mean age at the time of evaluation was 36.5 years (range 19e66 years). The age at onset of visual loss ranged from the age of 11 to the age of 47 (mean 23 years). The mean age of onset was 21 years for males and 32 years for females, although there were only two affected female subjects. The vast majority of probands (10/11) harboured only mutant mtDNA (homoplasmy). Heteroplasmy was detected only in one proband whose family was the only one harbouring 14484/ND6 point mutation. All patients presented with blurred vision. Bilateral simultaneous onset with monophasic progression was reported in 8 out of 14 cases; there were 5 patients with monocular onset, a biphasic course and involvement of the other eye after 2 weeks, 7 months and 3 years; while in one patient the disease remained monocular. In most cases, the disease started with painless loss of central vision, followed by impairment of visual acuity. In addition, one patient complained of persistent headache and the other of instability and occasional vertigo. Decreased visual acuity was detected in all probands, with an average value between 1/60 and 3/60. The main ophthalmoscopic findings of this group are summarized in Table 1. All 14 patients had a severe impairment of central vision function, a central scotoma in their visual fields and colour vision deficiency. Peripheral visual field constriction was detected in 4 cases. In three patients pupillary light responses were marked as slow. Afferent pupillary defect was found in 2 members of this group. All affected individuals had preserved eye movements. Absence of leakage from the optic disc (typical for LHON) during FSA was observed in 6 affected patients, while in 8 cases it was not performed. Neurological exams were completely normal for almost all probands (13 out of 14), except for one 29-year-old male patient with 3460/ND1 point mutation who had a postural tremor of hands that occurred at the onset (at the age of 17) and remained unchanged in amplitude and frequency to date. In the group of clinically asymptomatic mutation carriers we identified 10 individuals with subclinical form of the
disease. There were 2 (20%) male and 8 (80%) female subjects, which is in contrast to the ratio observed in the group of clinically affected patients. 3460/ND1-positive patients had a lower sex ratio (4 females vs. 1 male). Among 11778/ND4positive subclinical affected there were no males, while the group of 14484/ND6 positive consisted of one male patient (Fig. 2). The median age at the time of evaluation was 48.4 years (range 26e64 years). Two members of this group from 2 genealogically unrelated families had LHON, causing mutation in heteroplasmic condition - one harboured 3460/ND1 and the other 14484/ND6 point mutation. It was revealed by examination that eight out of 10 clinically unaffected patients had normal vision. One patient had visual acuity 0.2e0.3 and another 0.6e0.7 with no potential for improvement. The examination of the ocular fundus showed several subclinical abnormalities (described in Table 1). Impairment of central visual function was not detected in this group. Only one patient had a peripheral visual field constriction (1/10), two showed a decrease in colour perception (2/10), and one exhibited colour vision deficiency (1/10). All unaffected (10/10) individuals had preserved eye movements. Only one family member had afferent pupillary defect.
Table 1 e Ophtalmoscopic findings in affected and subclinical affected patients. Ophtalmologic findings Normal Pallor of disc Optic atrophy Turtiosity of the peripapillary retinal vessels Optic disc hyperemia Optic disc pseudoedema Narrow blood vessels Pigment irregularity Circumpapillary telangiectasia
Affected Subclinical affected subjects e no. subjects e no. / 8/14 8/14 6/14
4/10 4/10 2/10 8/10
4/14 4/14 3/14 / /
2/10 2/10 3/10 2/10 1/10
357
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
The neurological findings were unremarkable in the unaffected mutation carriers. Group of asymptomatic mutation carriers consisted of 17 patients (3 males vs. 14 females). The mean age at the time of evaluation was 48 years. Gender ratios among the 3460/ND1 and 11778/ND4 e positive mutation carriers were similar (4 females vs. 1 male and 9 females vs. 2 males, respectively). The group of 14484/ND6 - positive mutation carriers consisted of one female. Two members of this group from two genealogically unrelated families were harbouring mutation in the heteroplasmic condition (one was harbouring 11778/ND4 and the other 14484/ND6 point mutation).
4.
Discussion
LHON has been receiving much attention from the scientific community for more than two decades. However, few countries have published their population-based findings related to this multisystemic disease. We performed the first population-based and moleculargenetic study of LHON in the population of Serbia, as region of Europe, in particular region of the West Balkan. At the time of evaluation, based on census data from year 2012, 7 411 569 individuals were living in Serbia. Furthermore, our Clinics have patients coming from border regions of neighbouring countries, namely Montenegro and partly from Bosnia-Herzegovina, making population number data higher. Therefore, it has been assumed that around 8 million people are referring to our Clinics for this particular pathology. According to the previous studies from the literature, LHON is the most common mtDNA disorder.14 However, the prevalence of this maternally inherited disease in most populations is unknown. Known prevalences of LHON and LHON mutations in various countries of the world are shown in Table 2.14e17 The well-known male predominance among affected patients, which varies significantly worldwide, was also found in this study (85.7% of patients were male and 14.3% were female) (Table 3).15,16,18e24 As expected, in the groups of subclinical affected and asymptomatic mutation carriers gender ratios were reversed (approximately 20% were males and 80% were females in these groups). This male predominance among the affected patients could be explained by the fact that additional environmental and genetic factors interact
Table 2 e Prevalences of LHON and LHON mutations in various countries of the world. Country Netherlands Finland North East of England Australia Croatia Serbia
Prevalence of LHON
Prevalence of LHON mutations
1: 39 000 1: 50 000 1: 31 054
1: 9000 1: 9000 1: 9000
1: 113 300 1: 400 000 1: 520 000
1: 8500 unknown 1: 180 000
Table 3 e Gender ratios in various populations. Population Serbian Finnish Dutch American Chinese Japanese Iranians Turkish Russian Thai
Male: female ratio 6: 1 3.4: 1 5.4: 1 4.6: 1 4.6: 1 8: 1 13: 1 2.2: 1 2.25: 1 2.6: 1
with the primary mtDNA defect and determine whether an individual will ultimately develop optic nerve dysfunction and visual failure.10 The distribution of primary mutations (11778/ND4, 3460/ ND1 and 14484/ND6) was similar to those in the other Caucasoid populations studied where approximately 65% of the families carried 11778/ND4 mutation, 15%e20% the 3460/ ND1 mutation and approximately 14% the 14484/ND6 mutation.14e16,25 When considering primary mutations, marked male predominance was observed among the patients with the 14484/ ND6 mutation but not with the 11778/ND4 and 3460/ND1 mutations.1 In our study population 11778/ND4 mutation was also associated with a less prominent gender ratio, while other patients (3460/ND1 and 11448/ND6 mutation carriers) were male. Incidence of heteroplasmy in our groups of affected (one proband) and unaffected patients (in whom it was performed) were similar to those in other studies published to date.14,26 Previous studies have shown a variable prevalence of heteroplasmy based on the type of mutation.1 Individuals with 11778/ND4, 3460/ND1 and 14484/ND6 mutations showed levels of heteroplasmy equal to 5.6%, 40% and 36.4%.27 Since we had only one proband with heteroplasmic status (14484/ND6 mutation), we could not estimate prevalence of heteroplasmy among different mutation groups in our study. The median age of onset (23 years - 21 years in males, 32 years in females) and the percentage of patients with positive family history (44.4%) are in accordance with the previous studies published.4,5,8e10 As it is known, apart from bilateral loss of central vision, in most LHON patients there are no other neurological manifestations. However, some rare cases of ‘‘LHON plus’’ have been described. One of theme is a case of 12-year-old girl who presented with reduced visual acuity secondary to optic atrophy at 8 months of age. This led to a clinical diagnosis of LHON, which have been confirmed at the molecular-genetic level (11778G>A mtDNA point mutation was identified). Psychomotor regression, refractory epilepsy and progressive neurological abnormalities (due to neurodegeneration and corticospinal tract dysfunction) developed subsequently. Except for age of onset and gender, this case is unusual for neurological findings and evolution as well, indicating a wider central nervous system involvement.28 This is supported by the fact that although the most of the patients in our study
358
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
presented with common clinical findings (blurred vision, central scotoma, colour vision deficiency and characteristic fundus abnormalities, as well as peripheral visual field constriction), two recalled having other symptoms at the time of visual loss: one had persistent headaches and the other had walking instability and occasional vertigo. Moreover, one affected male patient with 3460/ND1 point mutation had a postural tremor of hands as associated neurological changes. Relative preservation of pupillary reflexes and eye movements are also seen in our study group. Considering that fluorescein angiogram was typical for LHON in all affected patients (in whom it was performed) (6/6), high specificity of FSA in differential diagnosis of this condition in the acute phase are confirmed. Furthermore, while still clinically asymptomatic, some of the unaffected mDNA mutation carriers may exhibit subtle impairment of the optic nerve function, such as colour vision impairment.29e31 In our study group of clinical unaffected individuals (27) only 2 showed a decrease in colour perception and one manifested colour vision deficiency, which is less than in another study published where 49% (27 out of 55) of unaffected individuals manifested colour vision impairment.30 However, during examination, we might have used the test with lower sensitivity (Ishihara colour vision test), which may explain the difference. It is important to emphasize that as much as 37% of unaffected patients had some abnormal ophthalmoscopic findings (described in Table 1) constituting the group of subclinical affected patients. One of them was found to have afferent pupillary defect, which is the first such case described in literature. In conclusion, we have shown that even though the frequencies of primary LHON mutations, incidence of heteroplasmy and clinical findings are largely consistent with those in other Caucasoid populations studied, the gender ratio appears to be higher and prevalence of this maternally inherited disorder significantly lower (1 per 530 000) than expected (1 per 45 000, reported in the studies from the European regions). The possible rationale behind this might be the poor recognition and misdiagnosis of the disease, mostly due to lack of the diagnostic tools for this disease in different regions of the Western Balkan. The other explanation might be a specific haplotype background of population in this region of Europe. We believe that the first reason is more likely. This also means that a number of cases in this region is yet to be examined and diagnosed. It is clear that further investigations and molecular-genetic testing of the entity are needed, but also efforts should be made to raise the awareness of LHON disease in the region.
Acknowledgment Our thanks are due to Dr Valerio Carelli, Laboratory of Neurogenetics, Department of Neurological Sciences, University of Bologna, Italy, for his help in molecular-genetic tests of -Jovic ic , PhD, patients. Also, we are grateful to Milica Ðuric School of Electrical Engineering, University of Belgrade, for valuable technical support in preparation of manuscript.
references
1. Carelli V, Ross-Cisneros FN, Sadun AA. Mitochondrial dysfunction as a cause of optic neuropathies. Prog Retin Eye Res 2004;23:53e89. 2. Yu-Wai-Man P, Griffiths PG, Chinnery PF. Mitochondrial optic neuropathies e disease mechanisms and therapeutic strategies. Prog Retin Eye Res 2011;30:81e114. 3. Fraser JA, Biousse V, Newman NJ. The neuroophthalmology of mitochondrial disease. Surv Ophthalmol 2010;55:299e334. 4. Newman NJ, Lott MT, Wallace DC. The clinical characteristics of pedigrees of Leber’s hereditary optic neuropathy with the 11778 mutation. Am J Ophthalmol 1991;111:750e62. 5. Riordan-Eva P, Sanders MD, Govan GG, Sweeney MG, Da Costa J, Harding AE. The clinical features of Leber’s hereditary optic neuropathy defined by the presence of a pathogenic mitochondrial DNA mutation. Brain 1995;118:319e37. 6. Brenner L, Bynke G, Bynke H. Leber’s hereditary optic neuropathy. A report of two unusual cases. Neuro Ophthalmol 1999;22:239e44. 7. Buchan JC, Onq C, Dabbs TR. Acute Leber hereditary optic neuropathy in a 73-year-old man. Eye 2007;21:859e60. 8. Nikoskelainen EK, Huoponen K, Juvonen V, Lamminen T, Nummelin K, Savontaus ML. Ophthalmologic findings in Leber hereditary optic neuropathy, with special reference to mtDNA mutations. Ophthalmology 1996;103:504e14. 9. Leo-Kottler B, Christ-Adler M. Leber optic neuropathy in women and children. Ophthalmology 1999;96:698e701. 10. Yu-Wai-Man P, Turnbull DM, Chinnery PF. Leber hereditary optic neuropathy. J Med Genet 2002;39:162e9. 11. Biousse V, Newman NJ. Neuro-ophthalmology of mitochondrial diseases. Semin Neurol 2001;21:275e91. 12. Nikoskelainen EK. Clinical picture of LHON. Clin Neurosci 1994;2:115e20. 13. Sadun F, DeNegri A-M, Carelli V, et al. Ophthalmologic findings in a large pedigree of 11778/haplogroup J Leber hereditary optic neuropathy. Am J Ophthalmol 2004;137:271e7. 14. Yu-Wai-Man P, Griffiths PG, Brown DT, Howell N, Turnbull DM, Chinnery PF. The epidemiology of Leber hereditary optic neuropathy in the North East of England. Am J Hum Genet 2003;72:333e9. 15. Spruijt L, Kolbach DN, de Coo RF, et al. Influence of mutation type on clinical expression of Leber hereditary optic neuropathy. Am J Ophthalmol 2006;141:676e82. 16. Puomila A, Ha¨ma¨la¨inen P, Kivioja S, et al. Epidemiology and penetrance of Leber hereditary optic neuropathy in Finland. Eur J Hum Genet 2007;15:1079e89. 17. Martin-Kleiner I, Gabrilovac J, Bradvica M, et al. Leber’s hereditary optic neuroretinopathy (LHON) associated with mitochondrial DNA point mutation G11778A i two Croatian families. Coll Antropol 2006;30:171e4. 18. Mashima Y, Yamada K, Wakakura M, et al. Spectrum of pathogenic mitochondrial DNA mutations and clinical features in Japanese families with Leber’s hereditary optic neuropathy. Curr Eye Res 1998;17:403e8. 19. Jia X, Li S, Xiao X, Guo X, Zhanq Q. Molecular epidemiology of mtDNA mutations in 903 Chinese families suspected with Leber hereditary optic neuropathy. J Hum Genet 2006;51:851e6. 20. Smith KH, Johns DR, Heher KL, et al. Heteroplasmy in Leber’s hereditary optic neuropathy. Arch Ophthalmol 1993;111:1486e90. 21. Houshmand M, Sharifpanah F, Tabasi A, et al. Leber’s hereditary optic neuropathy: the spectrum of mitochondrial DNA mutations in Iranian patients. Ann N Y Acad Sci 2004;1011:345e9.
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
22. Dogulu CF, Kansu T, Seyrantepe V, Ozguc M, Topaloglu H, Johns DR. Mitochondrial DNA analysis in the Turkish Leber’s hereditary optic neuropathy population. Eye 2001;15:183e8. 23. Volod’ko NV, L’vova MA, Starikovskaia EB, et al. Spectrum of pathogenic mtDNA mutations in Leber hereditary optic neuropathy families from Siberia. Genetika 2006;42:89e97. 24. Phasukkijwatana N, Chuenkongkaew WL, Suphavilai R, et al. The unique characteristics of Thai Leber hereditary optic neuropathy: analysis of 30 G11778A pedigrees. J Hum Genet 2006;51:298e304. 25. Yu-Wai-Man P, Morris CM, Zeviani M, Carrara F, Turnbull DM, Chinnery PF. The role of APOE in the phenotypic expression of Leber hereditary optic neuropathy. J Med Genet 2003a;40:41e3. 26. Chinnery PF, Andrews RM, Turnbull DM, Howell N. Leber hereditary optic neuropathy: does heteroplasmy influence the inheritance and expression of the G11778A mitochondrial DNA mutation? Am J Med Genet 2001a;98:235e43.
359
27. Jacobi FK, Leo-Kottler B, Mittelviefhaus K, et al. Segregation patterns and heteroplasmy prevalence in Leber’s hereditary optic neuropathy. Invest Ophthalmol Vis Sci 2001;42:1208e14. 28. Grazina MM, Diogo LM, Garcia PC, et al. Atypical presentation of Leber’s hereditary optic neuropathy associated to mtDNA 11778G>A point mutation - a case report. Eur J Paediatr Neurol 2007;11:115e8. 29. Carroll WM, Mastaglia FL. Leber’s optic neuropathy. A clinical and visual evoked potentials study of affected and asymptomatic members of a six generation family. Brain 1979;102:559e80. 30. Quiros PA, Torres RJ, Salomao S, et al. Colour vision defects in asymptomatic carriers of the Leber’s hereditary optic neuropathy (LHON) mtDNA 11778 mutation from a large Brazilian LHON pedigree: a case-control study. Br J Ophthalmol 2006;90:150e3. 31. Yu-Wai-Man P, Griffiths PG, Hudson G, Chinnery PF. Inherited mitochondrial optic neuropathies. J Med Genet 2009;46:145e58.
Official Journal of the European Paediatric Neurology Society
Original article
Leber hereditary optic neuropathy in the population of Serbia Jasna Jancic a,*, Ivana Dejanovic a, Janko Samard zic d, Sasa Radovanovic e, c f, Vladimir Kostic b Ana Pepic a, Natalija Kosanovic-Jakovic c, Mila Cetkovi a
Clinic of Neurology and Psychiatry for Children and Youth, Medical Faculty, University of Belgrade, Serbia Clinic of Neurology, Medical Faculty, University of Belgrade, Serbia c Institute of Ophthalmology, Medical Faculty, University of Belgrade, Serbia d Institute of Pharmacology and Toxicology, Medical Faculty, University of Belgrade, Serbia e Institute for Medical Research, University of Belgrade, Serbia f Institute of Histology and Embryology, Medical Faculty, University of Belgrade, Serbia b
article info
abstract
Article history:
Background: Leber hereditary optic neuropathy (LHON) is the most common mitochondrial
Received 19 June 2013
disorder. However, few countries have published their population-based findings related to
Received in revised form
this multisystemic disease.
13 January 2014
The aim: In order to get a better insight into the epidemiological and clinical picture of this
Accepted 19 January 2014
maternally inherited disorder, we performed the first population-based clinical and molecular-genetic study of LHON in the Serbian population.
Keywords:
Methods: Prospective study included patients who were diagnosed with LHON after detailed
Mitochondrial disease
medical examination and molecular-genetic confirmation.
Prevalence
Results: We identified 41 individuals from 12 genealogically unrelated families, carrying one
Primary mutations
of the three “primary” mitochondrial (mt) DNA point mutations associated with LHON.
Clinical picture
Fourteen of them were clinically affected, giving a minimum point prevalence of 1.9 per 1 000 000. The minimum point prevalence for mtDNA LHON mutations was 5.2 per 1 000 000. Male to female ratio was 6:1. Only one affected patient harboured mutant mtDNA in heteroplasmic condition. All patients were presented with common clinical findings. Conclusion: We observed significantly lower prevalence and higher gender ratio than expected. However, frequencies of primary mutations, incidence of heteroplasmy and clinical findings are in accordance with other studies in Caucasoid populations. Our results might be a consequence of poor recognition and misdiagnosis due to lack of diagnostic possibilities of the entity in different region of our country or less likely be in part due to specific haplotype background of Serbian population which should be further investigated. ª 2014 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
a 6a, 11000 Belgrade, Serbia. Tel.: þ381 65 * Corresponding author. Clinic of Neurology and Psychiatry for Children and Youth, Dr Subotic 666 6733; fax: þ381 11 2645 064. ic ). E-mail address: [email protected] (J. Janc 1090-3798/$ e see front matter ª 2014 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejpn.2014.01.005
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
1.
Introduction
Leber hereditary optic neuropathy (LHON, MIM 535000), the most common mitochondrial disorder, is characterized by acute or subacute painless loss of central vision, usually in young adults, predominantly male.1,2 Apart from visual loss as its distinctive feature, LHON may be associated with other neurological, cardiac and skeletal abnormalities, characterizing the “LHON plus” phenotype.2 This maternally inherited disorder is mainly associated with mitochondrial DNA (mtDNA) mutations 11778G>A, 3460G>A and 14484T>C, all involving genes encoding complex I subunits of mitochondrial respiratory chain.3 The median age of onset in LHON is between 15 and 35 years, but an individual can become affected at any age between 2 and 80.4e7 A few reports found a bit later age of onset in females.8,9 The disease usually presents with a loss of central vision involving first one eye and then the other with a mean interval of 2 months (75% of cases).10 The fellow eye can also be affected simultaneously (25%) or very rarely the disease can remain monocular.8,10,11 LHON is acute or subacute, but may also manifest as slowly progressive visual loss. Visual impairment is usually severe with visual acuity ranging from 20/200 to counting fingers, in association with profound dyschromatopsia, centrocaecal scotomas and relative preservation of peripheral visual field and pupillary reflexes.12,13 Following the acute phase, on average within six weeks from the disease onset, the optic disc becomes atrophic. Significant improvements in visual acuity are rare and in most individuals vision remains severely impaired. The clinical diagnosis is based on the ophthalmologic features and molecular-genetic testing. Relevant testing may include fundus examination (to identify the characteristic optic disc and vascular changes), kinetic (Goldman) or static perimetry (for central or centrocaecal scotoma), electrophysiological studies in selected cases (visual evoked potentials and pattern electroretinogram), optical coherence tomography (to assess the retinal nerve fibre layer thickness and the optic nerve anatomy) and neuroimaging (to exclude compressive, infiltrative and inflammatory causes of bilateral optic neuropathy).3 In order to get a better insight into the epidemiological and clinical picture of LHON, we present the first population-based clinical and molecular study of LHON in the population of Serbia as region of Europe, in particular as part of the West Balkan area.
2.
Neurology and Psychiatry for Children and Youth in Belgrade is the only referent institution in Serbia where the diagnosis of LHON could be established, therefore it poses the unique database of LHON patients for this region. Prior to inclusion in the study, thorough history was obtained and pedigree analysis performed and all participants underwent physical, neurological and neuro-ophthalmologic examinations. In addition, we have performed fluorescent angiography (FSA), visual electrophysiology, serum lactate and pyruvate levels, blood levels of thyroid hormones, cerebrospinal fluid analysis and magnetic resonance imaging (MRI) of the brain and optic nerves in order to exclude the other ocular or systemic diseases. Probands and their family members were thoroughly informed about the clinical findings. Those with a clinical diagnosis of LHON and their at-risk asymptomatic family members were advised to have a molecular-genetic testing to rule in or rule out the diagnosis. Having obtained their informed written consent, moleculargenetic tests were performed from venous blood samples (5 ml) in the Laboratory of Neurogenetics, Department of Neurological Sciences, Bologna, Italy and the Laboratory of Neurogenetics in the Clinic of Neurology, Clinical Centre of Serbia, Belgrade. The inclusion criteria were having primary (11778, 3460 and 14484) or secondary (14495, 14482, 10663) mtDNA LHON-causing mutations. Local ethical committee approved the research protocol according to the national regulations and study was performed in accordance to ethical standards of the Declaration of Helsinki.
3.
Results
During a 12 year follow-up period we identified 41 individuals, from 12 genealogically unrelated families, carrying mitochondrial DNA mutations associated with LHON. Fourteen of them were clinically affected (probands), giving a minimum point prevalence of 1.9 per 1 000 000. All of the 12 Serbian LHON families harboured a primary mutation. This makes the minimum point prevalence of LHON mutations 5.2 per 1 000 000. Mutations were distributed as follows: MT-11778/ND4 e 58.5% (9/14), MT-3460/ND1 e 34.2% (4/14) and MT-14484/ND6 e 7.3% (1/14) (Fig. 1). In the affected group there were 12 (85.7%) male and 2 (14.3%) female subjects. The gender ratio in this group was different depending on the mutation type. The 11778/ND4 mutation was associated with a less prominent gender ratio (7 males and 2 females), while marked male predominance was
Materials and methods
This prospective study included patients with clinical signs of LHON who were examined and ambulatory monitored in the Clinic of Neurology and Psychiatry for Children and Youth, Clinic of Neurology and Institute of Ophthalmology, Clinical Centre of Serbia, Belgrade in the period of 2000 until February 2013. The study also included family members at risk of developing LHON, being unaffected mutation carriers. The follow-up period was approximately 12 years. The Clinic of
355
Fig. 1 e The distribution of primary mutations.
356
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
Fig. 2 e Gender ratios among primary mutation groups in the proband group and in the group of subclinical affected patients.
found in patients with 3460/ND1 mutation (4 males). In the 14484/ND6 family, there was only one affected male (Fig. 2). The mean age at the time of evaluation was 36.5 years (range 19e66 years). The age at onset of visual loss ranged from the age of 11 to the age of 47 (mean 23 years). The mean age of onset was 21 years for males and 32 years for females, although there were only two affected female subjects. The vast majority of probands (10/11) harboured only mutant mtDNA (homoplasmy). Heteroplasmy was detected only in one proband whose family was the only one harbouring 14484/ND6 point mutation. All patients presented with blurred vision. Bilateral simultaneous onset with monophasic progression was reported in 8 out of 14 cases; there were 5 patients with monocular onset, a biphasic course and involvement of the other eye after 2 weeks, 7 months and 3 years; while in one patient the disease remained monocular. In most cases, the disease started with painless loss of central vision, followed by impairment of visual acuity. In addition, one patient complained of persistent headache and the other of instability and occasional vertigo. Decreased visual acuity was detected in all probands, with an average value between 1/60 and 3/60. The main ophthalmoscopic findings of this group are summarized in Table 1. All 14 patients had a severe impairment of central vision function, a central scotoma in their visual fields and colour vision deficiency. Peripheral visual field constriction was detected in 4 cases. In three patients pupillary light responses were marked as slow. Afferent pupillary defect was found in 2 members of this group. All affected individuals had preserved eye movements. Absence of leakage from the optic disc (typical for LHON) during FSA was observed in 6 affected patients, while in 8 cases it was not performed. Neurological exams were completely normal for almost all probands (13 out of 14), except for one 29-year-old male patient with 3460/ND1 point mutation who had a postural tremor of hands that occurred at the onset (at the age of 17) and remained unchanged in amplitude and frequency to date. In the group of clinically asymptomatic mutation carriers we identified 10 individuals with subclinical form of the
disease. There were 2 (20%) male and 8 (80%) female subjects, which is in contrast to the ratio observed in the group of clinically affected patients. 3460/ND1-positive patients had a lower sex ratio (4 females vs. 1 male). Among 11778/ND4positive subclinical affected there were no males, while the group of 14484/ND6 positive consisted of one male patient (Fig. 2). The median age at the time of evaluation was 48.4 years (range 26e64 years). Two members of this group from 2 genealogically unrelated families had LHON, causing mutation in heteroplasmic condition - one harboured 3460/ND1 and the other 14484/ND6 point mutation. It was revealed by examination that eight out of 10 clinically unaffected patients had normal vision. One patient had visual acuity 0.2e0.3 and another 0.6e0.7 with no potential for improvement. The examination of the ocular fundus showed several subclinical abnormalities (described in Table 1). Impairment of central visual function was not detected in this group. Only one patient had a peripheral visual field constriction (1/10), two showed a decrease in colour perception (2/10), and one exhibited colour vision deficiency (1/10). All unaffected (10/10) individuals had preserved eye movements. Only one family member had afferent pupillary defect.
Table 1 e Ophtalmoscopic findings in affected and subclinical affected patients. Ophtalmologic findings Normal Pallor of disc Optic atrophy Turtiosity of the peripapillary retinal vessels Optic disc hyperemia Optic disc pseudoedema Narrow blood vessels Pigment irregularity Circumpapillary telangiectasia
Affected Subclinical affected subjects e no. subjects e no. / 8/14 8/14 6/14
4/10 4/10 2/10 8/10
4/14 4/14 3/14 / /
2/10 2/10 3/10 2/10 1/10
357
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
The neurological findings were unremarkable in the unaffected mutation carriers. Group of asymptomatic mutation carriers consisted of 17 patients (3 males vs. 14 females). The mean age at the time of evaluation was 48 years. Gender ratios among the 3460/ND1 and 11778/ND4 e positive mutation carriers were similar (4 females vs. 1 male and 9 females vs. 2 males, respectively). The group of 14484/ND6 - positive mutation carriers consisted of one female. Two members of this group from two genealogically unrelated families were harbouring mutation in the heteroplasmic condition (one was harbouring 11778/ND4 and the other 14484/ND6 point mutation).
4.
Discussion
LHON has been receiving much attention from the scientific community for more than two decades. However, few countries have published their population-based findings related to this multisystemic disease. We performed the first population-based and moleculargenetic study of LHON in the population of Serbia, as region of Europe, in particular region of the West Balkan. At the time of evaluation, based on census data from year 2012, 7 411 569 individuals were living in Serbia. Furthermore, our Clinics have patients coming from border regions of neighbouring countries, namely Montenegro and partly from Bosnia-Herzegovina, making population number data higher. Therefore, it has been assumed that around 8 million people are referring to our Clinics for this particular pathology. According to the previous studies from the literature, LHON is the most common mtDNA disorder.14 However, the prevalence of this maternally inherited disease in most populations is unknown. Known prevalences of LHON and LHON mutations in various countries of the world are shown in Table 2.14e17 The well-known male predominance among affected patients, which varies significantly worldwide, was also found in this study (85.7% of patients were male and 14.3% were female) (Table 3).15,16,18e24 As expected, in the groups of subclinical affected and asymptomatic mutation carriers gender ratios were reversed (approximately 20% were males and 80% were females in these groups). This male predominance among the affected patients could be explained by the fact that additional environmental and genetic factors interact
Table 2 e Prevalences of LHON and LHON mutations in various countries of the world. Country Netherlands Finland North East of England Australia Croatia Serbia
Prevalence of LHON
Prevalence of LHON mutations
1: 39 000 1: 50 000 1: 31 054
1: 9000 1: 9000 1: 9000
1: 113 300 1: 400 000 1: 520 000
1: 8500 unknown 1: 180 000
Table 3 e Gender ratios in various populations. Population Serbian Finnish Dutch American Chinese Japanese Iranians Turkish Russian Thai
Male: female ratio 6: 1 3.4: 1 5.4: 1 4.6: 1 4.6: 1 8: 1 13: 1 2.2: 1 2.25: 1 2.6: 1
with the primary mtDNA defect and determine whether an individual will ultimately develop optic nerve dysfunction and visual failure.10 The distribution of primary mutations (11778/ND4, 3460/ ND1 and 14484/ND6) was similar to those in the other Caucasoid populations studied where approximately 65% of the families carried 11778/ND4 mutation, 15%e20% the 3460/ ND1 mutation and approximately 14% the 14484/ND6 mutation.14e16,25 When considering primary mutations, marked male predominance was observed among the patients with the 14484/ ND6 mutation but not with the 11778/ND4 and 3460/ND1 mutations.1 In our study population 11778/ND4 mutation was also associated with a less prominent gender ratio, while other patients (3460/ND1 and 11448/ND6 mutation carriers) were male. Incidence of heteroplasmy in our groups of affected (one proband) and unaffected patients (in whom it was performed) were similar to those in other studies published to date.14,26 Previous studies have shown a variable prevalence of heteroplasmy based on the type of mutation.1 Individuals with 11778/ND4, 3460/ND1 and 14484/ND6 mutations showed levels of heteroplasmy equal to 5.6%, 40% and 36.4%.27 Since we had only one proband with heteroplasmic status (14484/ND6 mutation), we could not estimate prevalence of heteroplasmy among different mutation groups in our study. The median age of onset (23 years - 21 years in males, 32 years in females) and the percentage of patients with positive family history (44.4%) are in accordance with the previous studies published.4,5,8e10 As it is known, apart from bilateral loss of central vision, in most LHON patients there are no other neurological manifestations. However, some rare cases of ‘‘LHON plus’’ have been described. One of theme is a case of 12-year-old girl who presented with reduced visual acuity secondary to optic atrophy at 8 months of age. This led to a clinical diagnosis of LHON, which have been confirmed at the molecular-genetic level (11778G>A mtDNA point mutation was identified). Psychomotor regression, refractory epilepsy and progressive neurological abnormalities (due to neurodegeneration and corticospinal tract dysfunction) developed subsequently. Except for age of onset and gender, this case is unusual for neurological findings and evolution as well, indicating a wider central nervous system involvement.28 This is supported by the fact that although the most of the patients in our study
358
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
presented with common clinical findings (blurred vision, central scotoma, colour vision deficiency and characteristic fundus abnormalities, as well as peripheral visual field constriction), two recalled having other symptoms at the time of visual loss: one had persistent headaches and the other had walking instability and occasional vertigo. Moreover, one affected male patient with 3460/ND1 point mutation had a postural tremor of hands as associated neurological changes. Relative preservation of pupillary reflexes and eye movements are also seen in our study group. Considering that fluorescein angiogram was typical for LHON in all affected patients (in whom it was performed) (6/6), high specificity of FSA in differential diagnosis of this condition in the acute phase are confirmed. Furthermore, while still clinically asymptomatic, some of the unaffected mDNA mutation carriers may exhibit subtle impairment of the optic nerve function, such as colour vision impairment.29e31 In our study group of clinical unaffected individuals (27) only 2 showed a decrease in colour perception and one manifested colour vision deficiency, which is less than in another study published where 49% (27 out of 55) of unaffected individuals manifested colour vision impairment.30 However, during examination, we might have used the test with lower sensitivity (Ishihara colour vision test), which may explain the difference. It is important to emphasize that as much as 37% of unaffected patients had some abnormal ophthalmoscopic findings (described in Table 1) constituting the group of subclinical affected patients. One of them was found to have afferent pupillary defect, which is the first such case described in literature. In conclusion, we have shown that even though the frequencies of primary LHON mutations, incidence of heteroplasmy and clinical findings are largely consistent with those in other Caucasoid populations studied, the gender ratio appears to be higher and prevalence of this maternally inherited disorder significantly lower (1 per 530 000) than expected (1 per 45 000, reported in the studies from the European regions). The possible rationale behind this might be the poor recognition and misdiagnosis of the disease, mostly due to lack of the diagnostic tools for this disease in different regions of the Western Balkan. The other explanation might be a specific haplotype background of population in this region of Europe. We believe that the first reason is more likely. This also means that a number of cases in this region is yet to be examined and diagnosed. It is clear that further investigations and molecular-genetic testing of the entity are needed, but also efforts should be made to raise the awareness of LHON disease in the region.
Acknowledgment Our thanks are due to Dr Valerio Carelli, Laboratory of Neurogenetics, Department of Neurological Sciences, University of Bologna, Italy, for his help in molecular-genetic tests of -Jovic ic , PhD, patients. Also, we are grateful to Milica Ðuric School of Electrical Engineering, University of Belgrade, for valuable technical support in preparation of manuscript.
references
1. Carelli V, Ross-Cisneros FN, Sadun AA. Mitochondrial dysfunction as a cause of optic neuropathies. Prog Retin Eye Res 2004;23:53e89. 2. Yu-Wai-Man P, Griffiths PG, Chinnery PF. Mitochondrial optic neuropathies e disease mechanisms and therapeutic strategies. Prog Retin Eye Res 2011;30:81e114. 3. Fraser JA, Biousse V, Newman NJ. The neuroophthalmology of mitochondrial disease. Surv Ophthalmol 2010;55:299e334. 4. Newman NJ, Lott MT, Wallace DC. The clinical characteristics of pedigrees of Leber’s hereditary optic neuropathy with the 11778 mutation. Am J Ophthalmol 1991;111:750e62. 5. Riordan-Eva P, Sanders MD, Govan GG, Sweeney MG, Da Costa J, Harding AE. The clinical features of Leber’s hereditary optic neuropathy defined by the presence of a pathogenic mitochondrial DNA mutation. Brain 1995;118:319e37. 6. Brenner L, Bynke G, Bynke H. Leber’s hereditary optic neuropathy. A report of two unusual cases. Neuro Ophthalmol 1999;22:239e44. 7. Buchan JC, Onq C, Dabbs TR. Acute Leber hereditary optic neuropathy in a 73-year-old man. Eye 2007;21:859e60. 8. Nikoskelainen EK, Huoponen K, Juvonen V, Lamminen T, Nummelin K, Savontaus ML. Ophthalmologic findings in Leber hereditary optic neuropathy, with special reference to mtDNA mutations. Ophthalmology 1996;103:504e14. 9. Leo-Kottler B, Christ-Adler M. Leber optic neuropathy in women and children. Ophthalmology 1999;96:698e701. 10. Yu-Wai-Man P, Turnbull DM, Chinnery PF. Leber hereditary optic neuropathy. J Med Genet 2002;39:162e9. 11. Biousse V, Newman NJ. Neuro-ophthalmology of mitochondrial diseases. Semin Neurol 2001;21:275e91. 12. Nikoskelainen EK. Clinical picture of LHON. Clin Neurosci 1994;2:115e20. 13. Sadun F, DeNegri A-M, Carelli V, et al. Ophthalmologic findings in a large pedigree of 11778/haplogroup J Leber hereditary optic neuropathy. Am J Ophthalmol 2004;137:271e7. 14. Yu-Wai-Man P, Griffiths PG, Brown DT, Howell N, Turnbull DM, Chinnery PF. The epidemiology of Leber hereditary optic neuropathy in the North East of England. Am J Hum Genet 2003;72:333e9. 15. Spruijt L, Kolbach DN, de Coo RF, et al. Influence of mutation type on clinical expression of Leber hereditary optic neuropathy. Am J Ophthalmol 2006;141:676e82. 16. Puomila A, Ha¨ma¨la¨inen P, Kivioja S, et al. Epidemiology and penetrance of Leber hereditary optic neuropathy in Finland. Eur J Hum Genet 2007;15:1079e89. 17. Martin-Kleiner I, Gabrilovac J, Bradvica M, et al. Leber’s hereditary optic neuroretinopathy (LHON) associated with mitochondrial DNA point mutation G11778A i two Croatian families. Coll Antropol 2006;30:171e4. 18. Mashima Y, Yamada K, Wakakura M, et al. Spectrum of pathogenic mitochondrial DNA mutations and clinical features in Japanese families with Leber’s hereditary optic neuropathy. Curr Eye Res 1998;17:403e8. 19. Jia X, Li S, Xiao X, Guo X, Zhanq Q. Molecular epidemiology of mtDNA mutations in 903 Chinese families suspected with Leber hereditary optic neuropathy. J Hum Genet 2006;51:851e6. 20. Smith KH, Johns DR, Heher KL, et al. Heteroplasmy in Leber’s hereditary optic neuropathy. Arch Ophthalmol 1993;111:1486e90. 21. Houshmand M, Sharifpanah F, Tabasi A, et al. Leber’s hereditary optic neuropathy: the spectrum of mitochondrial DNA mutations in Iranian patients. Ann N Y Acad Sci 2004;1011:345e9.
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y 1 8 ( 2 0 1 4 ) 3 5 4 e3 5 9
22. Dogulu CF, Kansu T, Seyrantepe V, Ozguc M, Topaloglu H, Johns DR. Mitochondrial DNA analysis in the Turkish Leber’s hereditary optic neuropathy population. Eye 2001;15:183e8. 23. Volod’ko NV, L’vova MA, Starikovskaia EB, et al. Spectrum of pathogenic mtDNA mutations in Leber hereditary optic neuropathy families from Siberia. Genetika 2006;42:89e97. 24. Phasukkijwatana N, Chuenkongkaew WL, Suphavilai R, et al. The unique characteristics of Thai Leber hereditary optic neuropathy: analysis of 30 G11778A pedigrees. J Hum Genet 2006;51:298e304. 25. Yu-Wai-Man P, Morris CM, Zeviani M, Carrara F, Turnbull DM, Chinnery PF. The role of APOE in the phenotypic expression of Leber hereditary optic neuropathy. J Med Genet 2003a;40:41e3. 26. Chinnery PF, Andrews RM, Turnbull DM, Howell N. Leber hereditary optic neuropathy: does heteroplasmy influence the inheritance and expression of the G11778A mitochondrial DNA mutation? Am J Med Genet 2001a;98:235e43.
359
27. Jacobi FK, Leo-Kottler B, Mittelviefhaus K, et al. Segregation patterns and heteroplasmy prevalence in Leber’s hereditary optic neuropathy. Invest Ophthalmol Vis Sci 2001;42:1208e14. 28. Grazina MM, Diogo LM, Garcia PC, et al. Atypical presentation of Leber’s hereditary optic neuropathy associated to mtDNA 11778G>A point mutation - a case report. Eur J Paediatr Neurol 2007;11:115e8. 29. Carroll WM, Mastaglia FL. Leber’s optic neuropathy. A clinical and visual evoked potentials study of affected and asymptomatic members of a six generation family. Brain 1979;102:559e80. 30. Quiros PA, Torres RJ, Salomao S, et al. Colour vision defects in asymptomatic carriers of the Leber’s hereditary optic neuropathy (LHON) mtDNA 11778 mutation from a large Brazilian LHON pedigree: a case-control study. Br J Ophthalmol 2006;90:150e3. 31. Yu-Wai-Man P, Griffiths PG, Hudson G, Chinnery PF. Inherited mitochondrial optic neuropathies. J Med Genet 2009;46:145e58.