Optic atrophy accounts for about 11% of all cases of blindness in India, according to a population-based study in Hyderabad.1 Its aetiology often remains unidentified. Leber's hereditary optic neuropathy (LHON) comprises a small but specific subgroup of optic atrophy. It is characterised by acute or subacute loss of vision, usually in males in the second or third decade of life. It is a common cause of familial optic atrophy. The exact prevalence of this disease in India is not known. In the United Kingdom, Man et al2 estimated it to be about 3.22 per 100,000 of the population. In Australia, LHON contributes about 2% of all invalid blind persons.3 The disease is transmitted through mitochondrial genes which are usually inherited from the mother. The mitochondrial origin of this disorder was established by Wallace in 1988, by demonstrating point mutations in the mitochondrial gene ND4 (MTND4), a subunit of NADH dehydrogenase complex 1.4 Since then an increase in number of cases have been reported from all over the world including Europe, America, Japan, China and Australia.235 The mutations in the mitochondrial genes MTND1, MTND4, MTND5 and MTND6 are now known to be associated with LHON.5 Cases of LHON occur in India, but no studies on molecular mutations in LHON could be identified even after an extensive search of indexed medical journals. This study reports two families with cases of blindness due to optic atrophy in young individuals in whom diagnosis of LHON was established through molecular studies. Molecular studies were performed after extracting DNA from individuals' blood by polymerase chain reaction (PCR) using mutation specific primers.
Materials and Methods
Two index cases from different families were chosen for study; clinical data from these two cases were recorded to evaluate the mode of inheritance and to identify other relatives at risk. A detailed ophthalmological examination was carried out including fundus examination, perimetry, electroretinogram, neuroimaging and laboratory tests such as blood lactate and so on. Molecular studies were carried out to confirm the diagnosis after obtaining informed consent from each individual tested.
Molecular genetic analysis: Peripheral blood samples obtained from subjects were anticoagulated with EDTA and the total genomic as well as mitochondrial DNAs were extracted by the salt precipitation method.6 Mutation analysis was carried out for three primary LHON mutations namely, G3460A, G11778A and T14484C (Table 1). ARMS (Amplified refractory mutation detection system) technique was used for detection of mutations G3460A and G11778A, and restriction enzyme digest method for LHON mutation T14484C.78 Briefly for ARMS test, PCR amplification was carried out with 50 pmol of each primer (sequences provided in Table 1, 10mM Tris HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 125 microM dNTPs and one unit Taq DNA polymerase (Amersham Pharmacia) in a volume of 25 microL. PCR cycles included 95°C for 5 minutes, followed by 30 cycles of 94°C for 1 minute, 60°C for 1 minute and 72°C for 1 minute followed by 72°C for 10 minutes in a thermal cycler (model number PTC-100 MJ Research Inc. Waltham, MA). The PCR product was run directly on 2% Agarose gel and stained by Ethidium bromide and observed under ultraviolet transillumination. The PCR conditions remained the same for detection of both mutations - G3460A and G11778A in the mitochondrial genes MTND1 and MTND4.
Mutation LHON T14484C was analysed by amplifying mitochondrial DNA with primers provided in Table 1, followed by digestion with restriction enzyme Ban 1 (NEB) at 370subC for three hours. In the presence of mutation, the 125 bp fragment cuts to 108+ 17 bp. The digested PCR product was analysed by 6% polyacrylamide gel electrophoresis (PAGE) and visualised by silver staining.8
The clinical details of the two patients were as follows:
Case 1: A fourteen-year-old boy, R (DOB 19.02.1987) presented to the hospital in May, 2001 with complaints of diminution of vision in both eyes for one month. The visual loss was gradual and painless. There were no associated symptoms of headache, diplopia, altered colour perception, seizures, abnormal movements or loss of consciousness. The patient did not have any history head injury, ocular injury and other neurological complaints.
The patient's eldest sister had suffered from a similar illness 14 years ago, at the age of 11 years. She had suffered a gradual onset of painless diminution of vision from both eyes, which progressed for over two months, and became static after 1.5 to 2 years. Her clinical records revealed visual acuity of finger counting from 6 meters and 2 meters from right and left eyes, respectively. There was altered macular reflex, macular oedema, temporal pallor, reduced amplitudes and normal latencies in visual evoked potential in both eyes. Fundus examination at a later date showed bilateral early optic atrophy. No other family member had clinical manifestations related to vision at that time (Figure 1).
The patient was evaluated in May 2001, and found to have visual acuity of 6/60 in both eyes. Pupillary reactions (direct and consensual) were present. Fundus examination showed nasal blurring of disc margins; fundal reflex was present and found to be normal. Other systemic examinations including examination of the cardiovascular and central nervous system did not reveal any abnormality apart from reduced visual acuity. A flash visually evoked potential (VEP) revealed delayed latencies but normal amplitudes in both eyes. Initially bilateral optic neuritis was diagnosed and oral prednisolone was administered by the ophthalmologist. The dosage was 30 mgs per day for two weeks. The visual acuity after two weeks remained the same. Repeat flash VEP (July, 2001) eight weeks after the initial evaluation was similar to the previous one, and fundus examination showed disc hyperaemia in both eyes, the left more than the right, with venous engorgement. Papilloedema was suspected and pulse methyl prednisolone was administered for three days (1 gm per day by intravenous infusion), but no improvement was observed. Visual acuity reduced to finger counting to 1 metre in both eyes by the end of August 2001. Pupillary reaction was ill sustained and sluggish. Optic disc showed bilateral temporal pallor. The possibility of LHON was then considered and appropriate investigations were carried out. Blood lactate was 27.3 mg/dl (normal upto 19 mg/dl), brainstem auditory evoked potentials (BAEP) and somatosensory evoked potentials (SSEP) in bilateral median nerves showed no abnormality. MRI scan of brain and orbit was performed and no significant abnormality was noted.
Molecular studies for LHON were carried out in the patient (proband), in all his siblings and in the parents. DNA was analysed for the three primary mutations. The PCR product run on agarose gel showed the presence of two bands, one mutation specific band of 318 bp and another control band of 500 bp, in all subjects studied. Thus, the mutation G3460A in the mitochondrial gene MTND1 was confirmed in the proband, all his siblings and the mother (see pedigree in Figure 1 for details). The family members were tested for the presence of the other two mutations also and it was found to be negative.
Case 2: Patient PV, a fourteen year-old boy was evaluated in March 2000 with main complaints of gradual and painless diminution of vision in both eyes of one year's duration. The diminution of vision was initially associated with headache, which was severe, bilateral and episodic in nature. There were no associated neurological or systemic symptoms.
There were four other family members affected with loss of vision (Figure 2). The elder brother, a maternal aunt and an uncle, and a second cousin, that is, a son of the mother's maternal first cousin had loss of vision in a similar manner. Only the 26-year-old maternal uncle was available for examination. He had a history of painless, gradual and sequential diminution of vision from the age of seventeen. Visual loss started in the left eye and six months later, it started in the right eye. There were no associated symptoms. Fundus examination revealed bilateral optic atrophy. The maternal aunt also had optic atrophy of both eyes and was unable to see even near objects. She was not available for examination.
In the child (PV), the systemic examination was unremarkable. Visual acuity was finger counting at 1 meter in both eyes. Pupillary reaction was very sluggish and ill sustained bilaterally. Fundus examination showed blurred disc margin nasally along with temporal pallor bilaterally. Study of fundus one year earlier had shown blurred disc margins and papilloedema. CT scan of the brain was performed and found to be unremarkable. VEP showed delayed latencies bilaterally. He was suspected to have LHON in view of family history of similar affliction on the maternal side and optic atrophy on fundus examination.
Molecular studies to confirm diagnosis of LHON were carried out in the affected child and maternal uncle (SP). Both ARMS PCR and restriction enzyme digest methods were employed in order to detect the three primary LHON mutations. The mutation G11778A in the mitochondrial gene MTND4 was identified in the patient and his maternal uncle, who were the only family members available for testing (Figure 3). The Ethidium bromide stained agarose gel showed presence of both mutation specific band (222 bp) and control band (500 bp). The patient and his uncle were negative for mutations G3460A and T14484C.
LHON is a distinct cause of optic atrophy in adults, with a unique pattern of mitochondrial inheritance. It is a major cause of hereditary blindness in young adult males. Because of its rarity and varied presentation, the disease often remains undiagnosed. In fact, in both the cases under study, LHON was not considered the initial diagnosis. Optic neuritis was first diagnosed in Case 1, which led to treatment with steroids, while Case 2 was diagnosed as papilloedema and referred to a neurosurgeon for a possible intra-cranial space occupying lesion. Only after the above two conditions were ruled out, was the diagnosis of LHON considered. Molecular studies enabled a definitive diagnosis of this disorder in these families.
Mackey et al9 reported that about 95% cases of LHON harbour one of the three 'primary' mutations - G11778A in MTND4 gene (69% of cases) , T14484 in MTND6 gene (14%) and G3460A in MTND1 gene (13% of cases).9 These mutations act as major predisposing factors for disease.5 The primary LHON mutations characteristically do not occur together and are absent in normal subjects. Identification of one of the three major primary pathogenic mutations establishes the diagnosis even in the absence of a family history.10 All the primary mutations occurring in LHON are missense mutations leading to change in amino acids in the protein.5 The worldwide occurrence of the most common mutation G11778A varies from 50% to 82% in different studies.511 Mutations G11778A and G3460A were observed in the two families under study In Case 1, the affected boy, his mother and all the siblings (3 sisters) were tested for mitochondrial mutations. All five members of the family were found to harbour the primary mutation G3460A, though only two of them, the boy and eldest sister, manifested blindness (pedigree 1). In Case 2, only the proband and his blind maternal uncle were tested and both were found to be positive for the most common primary LHON mutation G11778A.
The 'secondary' mutations known to be associated with LHON usually co-occur with the 'primary' mutations and are also prevalent in the general population.12 The mitochondrial pattern of inheritance makes this disease unique, as it is inherited almost exclusively from the egg of the mother rendering all the offspring, irrespective of sex to be at risk of disease, unlike X linked recessive inheritance where only males are at risk of disease. For reasons not established, there is a striking male preponderance, although an X-linked factor predisposing to the disease has been suggested.13
The occurrence of disease and its severity differs depending upon the percentage of mutated mitochondria inherited by the offspring (genetic heteroplasmy), the sex (males predominate as mentioned above) and additional environmental factors such as smoking, alcohol intake etc.14 In the first family in our case series, from among the five members who tested positive for LHON mutation, only two manifested with blindness typical of LHON. The other three sisters were spared of the disease in spite of testing positive for the mutation, thereby suggesting the presence of low level of mutant mitochondrial population or heteroplasmy.
Genetic counselling forms an integral part of the management of patients and families with LHON. Once the diagnosis is established by mutation detection in the proband, other at risk family members should be tested. In about 60% of the cases, a history of visual loss affecting maternal relatives at a young age is present, while nearly 40% of the cases appear to be sporadic. The risk to siblings and other maternal relatives depends upon the genetic status of the mother. If the mother has the mitochondrial LHON mutation, all siblings are at risk of inheriting, as in the first family in this series14.
Over 50% of males and 85% females who harbour one of the 'primary' LHON mitochondrial DNA mutations homoplasmically do not develop blindness suggesting reduced penetrance of the LHON mutations. The lifetime risk of visual loss in individuals with a homoplasmic 'primary' LHON mitochondrial DNA mutation is about 40% for males and 10% for females.14 The specific risks vary with the mutation. Some mutations show distinct phenotypes, for example, G11778A generally causes the most severe visual failure with slim chance of recovery, whereas T14484C has the best long-term prognosis with upto 50% remission rate as compared to G11778A mutation where it is only 7%15.
Regarding the management, those at risk (tested to have mitochondrial mutations for LHON) can abstain from smoking and moderate their intake of alcohol14. Some studies have reported benefit from using quinine analogues (ubiquinone and idebenone) although the mechanism is unclear.16
In conclusion, this paper reports the identification of mutations in Indian patients with LHON, and describes the implications of this diagnosis for the individual and the other family members.
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