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Abstract
Over 90% of Leber's hereditary optic neuropathy (LHON) is caused by one of three mtDNA mutations (m.11778A>G, m.3460G>A, m.14484T>C). The remaining cases are due to rare mutations in different genes encoding subunits of the respiratory chain. The proband is a 17-year-old male with symptoms of optic nerve atrophy. No common LHON mutations were found, but detailed sequencing identified a rare, homoplasmic mutation m.3635G>A in the ND1 gene.
- Molecular Genetics
- Ophthalmology
- Genetics
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Leber's hereditary optic neuropathy (LHON) is a progressive optic nerve atrophy due to the degeneration of the retinal ganglion cells. Over 90% of LHON cases are caused by one of three mutations in mitochondrial DNA (m.11778A>G, m.3460G>A or m.14484T>C), all affecting components of complex I. Vision loss is usually permanent, but spontaneous recovery can occur. Only approximately 50% of men and 10% of women harbouring one of the three primary pathogenic mutations develop visual failure. The age of onset is usually between 15 years and 35 years. Both eyes are involved, and vision loss is usually sequential with the time interval between the onsets in the two eyes varying from some weeks to a few months.1
Fourteen rare LHON mutations are known, most of them localised in the ND1 or the ND6 genes. Some cause a variable clinical manifestation, from vision loss to complex neurological disorders called LHON+.2 There are also 18 candidate primary LHON mutations found only in a single family or in a single case (MITOMAP, http://www.mitomap.org).
Even severe mutations like m.11778G>A in most cases are present in a homoplasmic or almost homoplasmic state. In heteroplasmic families (about 15%), the level of heteroplasmy can vary extensively between generations, between offspring in the same family, as well as between different tissues in an affected individual.1 ,3
In this study, we report the identification of a Polish LHON family with a rare m.3635G>A mutation in the ND1 gene.
Clinical findings
A 17-year-old male of Polish origin was referred to a medical genetics clinic due to hereditary optic nerve atrophy of unknown aetiology. He has three younger asymptomatic siblings: two brothers and one sister. However, optic nerve atrophy was diagnosed in his maternal uncle, maternal first cousin, and two brothers and two nephews of his maternal grandmother (pedigree, figure 1)
Pedigree of the family with Leber hereditary optic neuropathy caused by m.3635 G>A mutation.
The proband was born from the first uneventful pregnancy, during the 38th week of gestation, with a birth weight 2870 g and an Apgar score of 9. His psychomotor development was normal. During childhood he was healthy and his vision was normal. Aged 16 years, he noted a dramatic decrease in visual acuity of his left eye and 2 months later also of his right eye. The typical swelling and hyperaemia of the optic nerve discs were found, and then on the basis of funduscopy, abnormal results of visual evoked responses and optical coherence tomography of the retinal nerve fibre layer, the diagnosis of bilateral optic nerve atrophy was made. His actual visual acuity is counting fingers from 15 to 30 cm. All the above-mentioned data allowed a diagnosis of LHON.
Materials and methods
Three members of the family were investigated: the proband (IV:11, figure 1), his mother (III:12, figure 1) and his 24-year-old maternal cousin (IV:8, figure 1). Total genomic DNA was isolated from blood leukocytes using a standard phenol/chloroform method.
PCR:RFLP test for m.11778G>A, m.3460G>A and m.14484T>C mutations was performed on total DNA from the peripheral blood of the patient. Then the sequencing of four mtDNA genes encoding subunits of complex I (subunits ND2, ND4, ND5, ND6) was performed using previously described primers.4
Heteroplasmy of the m.3635G>A mutation was determined using last cycle hot PCR-RFLP with α-32P-dATP.5 The region spanning this mutation was PCR-amplified using primers: forward, 3608–3633 (C/T mismatch at position 3632), reverse 4239–4219 in standard PCR conditions. Radiolabelled 632 bp PCR product was cut with Tru1I (Fermentas) and separated on a 12% non-denaturing polyacrylamide gel. Two fragments (454, 178 bp) for wild type, and three (454, 153 and 25 bp) for 3635G>A mutation were obtained.
Results
The test for common mtDNA LHON mutations gave negative results. Sequencing of the four genes encoding subunits of complex I of the respiratory chain revealed a homoplasmic m.3635G>A mutation. The mutation substitutes an asparagine for a highly conserved serine at ND1 amino acid 110 (S110N). The same mutation was found in DNA isolated from the blood of the patient's mother and the patient's maternal cousin, however, in contrast with the proband, the mutation in his mother and cousin was heteroplasmic (figures 2 and 3), with heteroplasmy of 71.8% ± 0.13% and 67% ± 0.47%, respectively. The patient's mtDNA was found to belong to haplogroup J.
Electrophoregrams of the region encompassing the m.3635 G>A mutation. The presence of the 3635 G>A variant in the heteroplasmic state was confirmed in blood of the patient's mother (A) and in blood of the patient's maternal cousin (B), but in the patient's blood, it was in the homoplasmic state (P). C- control (wild type) sequence.
Heteroplasmy analysis of m.3635G>A transition using last cycle hot PCR-RFLP. The wild type sequence is cut into two fragments of 454 and 178 bp. The 3688G>A transition introduces a new restriction site for Tru1I, and after digestion, three fragments of length 454, 153 and 25 bp (not shown) were obtained. A—blood of patient mother; B—blood of patient maternal cousin; P—blood of patient; C—control (wild type) DNA; U—uncut PCR product.
Discussion
In this study, we identified the m.3635G>A mutation in a Polish family. This mutation was initially identified in a single Russian LHON family and in 10 Chinese LHON patients.6–9 In six previously described cases, probands had a family history of LHON, and five cases were sporadic. Our patient had the m.3635G>A mutation in a homoplasmic state, but his symptomatic cousin had this mutation in a heteroplasmic state, and the heteroplasmy level exceeded 67%. Only one previously described patient had the m.3635G>A mutation in the heteroplasmic state, however, the level of heteroplasmy in this patient was not determined. Our patient's mother, despite having the m.3635G>A mutation at a higher level than the patient's cousin, was asymptomatic.
mtDNA of our patient belongs to haplogroup J, known to modify (worsen) the expression of LHON in case of m.14484T>C and m.11778G>A mutation.10
Our observations support a pathogenic role of the m.3635G>A mutation in patients with LHON.
Take home message
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m.3635G>A mutation confirmed as pathogenic in Leber's hereditary optic neuropathy.
Footnotes
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Contributors AK DNA analysis, manuscript preparation, MJK clinical diagnosis, manuscript preparation, KT coordination, DNA study design, EB study design, manuscript preparation.
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Funding This work was supported by grant 2011/03/N/NZ3/00655 from the National Science Centre.
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Competing interests None.
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Ethics approval Bioethics Committee of Warsaw Medical University.
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Provenance and peer review Not commissioned; internally peer reviewed.