Phenotypic expressions of chronic leber's hereditary optic neuropathy patients with primary and secondary mitochondrial mutations presenting at tertiary eye care center, South India

Naveen Kumar Challa; Hima Bindu Narsini; Sulaiman Aldakhil

doi:10.4103/erj.erj_7_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 9 | Issue : 1 | Page : 8-13

Phenotypic expressions of chronic leber's hereditary optic neuropathy patients with primary and secondary mitochondrial mutations presenting at tertiary eye care center, South India

Naveen Kumar Challa¹, Hima Bindu Narsini², Sulaiman Aldakhil³
¹ Department of Optometry, College of Applied Medical Sciences, Qassim University, Buraidah, Saudi Arabia; Department of Vitreo-retinal Services, L V Prasad Eye Institute, Hyderabad, Telangana, India
² Department of Vitreo-retinal Services, L V Prasad Eye Institute, Hyderabad, Telangana, India
³ Department of Optometry, College of Applied Medical Sciences, Qassim University, Buraidah, Saudi Arabia

Date of Submission	13-Oct-2022
Date of Acceptance	09-Jan-2023
Date of Web Publication	11-May-2023

Correspondence Address:
Dr. Naveen Kumar Challa
Department of Optometry, College of Applied Medical Sciences, Qassim University, Buraidah

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/erj.erj_7_22

Abstract

Aim: The aim was to report the phenotypic expression of chronic Leber's hereditary optic neuropathy (LHON) patients with primary and secondary mitochondrial mutations presenting at a tertiary eye care center in South India. Materials and Methods: Of 51 patients suspected to have LHON, 15 patients turned out to be positive for mitochondrial mutations. All 15 patients with LHON are in the chronic phase of the disease and have undergone clinical examination that included visual acuity, fundus examination, visual fields, and optical coherence tomography. Clinical data were compared among the LHON patients with primary mutations of G11778A and T14484C and grouped secondary mutations. Descriptive analysis was reported. Results: There was no significant difference found in visual acuities in both the eyes of the three genotypic groups (G11778A, T14484C, and secondary mutations) (P > 0.05). Retinal nerve fiber layer (RNFL) analysis showed that nine patients carrying G11778A and three patients carrying secondary mutations were similar, while two patients with T14484C showed severe thinning of RNFL in all four quadrants. Conclusions: Among a small percentage of patients who were positive for LHON mutations, severe RNFL thinning was the most prominent finding in the T14484C mutation that differentiates from G11778A and secondary mutations. Other clinical features seem to be similar among all genotypic groups examined.

Keywords: Genotyping, Leber's hereditary optic neuropathy, optical coherence tomography, retinal nerve fiber layer, South India

How to cite this article:
Challa NK, Narsini HB, Aldakhil S. Phenotypic expressions of chronic leber's hereditary optic neuropathy patients with primary and secondary mitochondrial mutations presenting at tertiary eye care center, South India. Egypt Retina J 2022;9:8-13

How to cite this URL:
Challa NK, Narsini HB, Aldakhil S. Phenotypic expressions of chronic leber's hereditary optic neuropathy patients with primary and secondary mitochondrial mutations presenting at tertiary eye care center, South India. Egypt Retina J [serial online] 2022 [cited 2023 Jun 7];9:8-13. Available from: https://www.egyptretinaj.com/text.asp?2022/9/1/8/376075

Introduction

Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disorder that results from mitochondrial deoxyribonucleic acid (DNA) mutations with the clinical manifestation of optic nerve degeneration leading to progressive bilateral vision loss.^[1],[2],[3] LHON seems to affect all age groups, but the most commonly affected individuals were men between the second and third decades, and the estimated prevalence in various populations has ranged from 1 in 30,000 to 1 in 53,000.^[4],[5] The vision loss is usually sequential in 75% of the cases, where one eye is affected, and weeks to months later, other eye gets involved, and 25% of the cases are also reported to have simultaneous vision loss.^[6],[7] Visual recovery among LHON patients usually occurs within a year from the onset of the disease, and the chances of recovery seem to be more in the individuals who manifest the disease in the 1^st or 2^nd decades of life and also based on the type of mutation present.^[8]

LHON is known to be caused by three common pathogenic mitochondrial mutations, m.3460G>A, m.11778G>A, and m.14484T>C, which takes place in genes encoding complex 1 subunits of the respiratory chain. Over 95% of the patients diagnosed as LHON harbor at least one of these primary mitochondrial DNA mutations.^[9] The occurrence of m.11778G>A is higher in East Asia than in European-derived populations,^[2],[10] and m.14484T>C is more common in French Canadians.^[11] The frequency of m.11778G>A is higher in Indian patients compared to other primary mutations causing LHON.^[2],[12] In addition to the primary mutations, secondary mutations were also reported in LHON patients from the Indian population.^[13] These novel secondary mutations were reported in MT-CO3, MT-CYB, and MT-ATP6 genes.

Studies have reported that it is important to know the spectrum of mitochondrial mutations in understanding the pathogenesis of LHON and phenotypic expression of LHON as each type of mutation has a different type of pathogenesis and clinical expression. The type of mutation has different penetrance among haplogroups as certain alleles in particular haplogroups have protective or deleterious effects.^[13]

There are studies that have reported the relation between genotype and clinical expression of LHON patients; however, they were limited to a few clinical features specific to mutation.^{[1],[6],[13],[14],[15],[16],[17],[18]} To the best of our knowledge, there are only a few studies^[12],[19] which reported the mitochondrial mutations and corresponding detailed clinical features in LHON patients from India. Considering the differences in population diversity and environmental variation, between north and south India, this study aimed to evaluate all the known mitochondrial mutations and corresponding clinical features in LHON patients presenting at a tertiary eye care center in south India.

Materials and Methods

All the patients with LHON suspect, who visited Neuro Ophthalmology Services at L V Prasad Eye Institute, Hyderabad, between July 2017 and February 2018, were prospectively screened for LHON confirmation. Patients with LHON were diagnosed by an experienced ophthalmologist after detailed testing, including visual acuity with a LogMAR chart, visual field testing using automated perimetry, evaluation of pupillary reactions, fundus examination, peripapillary and macular optical coherence tomography (OCT), and OCT angiography. Neuroimaging tools were also used to rule out differential diagnosis where required. Patients suspected to have LHON based on their clinical history were subjected to mutational analysis after informed consent.

Approval of the study was obtained from the Institutional Review Board of LV Prasad Eye Institute, Hyderabad, India, and the Institutional Ethics committee of the University of Hyderabad, India. Its conduct followed the tenets of the Declaration of Helsinki. Informed oral consent was obtained from the study participants who were above 18 years. For minors (who were less than 18 years), parents or legal guardians of the patients provided the required verbal informed consent.

Sample collection

Blood was collected through venipuncture (from the forearm) from clinically suspected LHON patients. The blood sample was stored at 0°C–4°C in ethylenediaminetetraacetic acid (EDTA) vacutainer until further use.

DNA isolation and processing

The method adopted for DNA isolation was the salting out method. The procedure is carried out under all aseptic conditions in the laboratory. One microliter of blood is drawn in an EDTA vacutainer and mixed well by inverting the tube and is stored at 4°C till use. The blood sample is brought to room temperature; 300 μl is transferred into 1.5 μl of Eppendorf tube and 900 μl of TKM buffer. Then, a 90 μl of triton X is added to lyse the red blood cells and mixed well inverting the tube several times; then, the sample is centrifuged at 8000 rpm for 5 min at room temperature. Slowly, the supernatant is poured off saving the nuclear pellet settled at the bottom of the tube. The pellets are washed in 900 μl of TKM1 buffer and centrifuged. This step is repeated if lysis is incomplete. Then, 260 μl of TKM 2 is added to the pellet, and 40 μl of SDS is used to lyse the white blood cells, and then, the whole suspension is mixed thoroughly and is incubated for 25 min at room temperature. Then, 600 μl of NaCl is added and mixed well so that protein gets precipitated by inversion and is centrifuged at 8000 rotations per minute for 5 min in a microcentrifuge. The supernatant containing DNA is transferred into a prechilled Eppendorf with 300 μl of isopropanol, and the pellet containing precipitated proteins is discarded. The tube is inverted several times to precipitate the DNA. The precipitated DNA is then centrifuged and washed with 300 μl of 70% ethanol and again centrifuged for 5 min at 8000 rpm. The pellets are allowed to air dry for a minimum of 1 h and resuspended into 50 μl of Tris-EDTA buffer. The isolated DNA is stored at − 80°C till further use.

The isolated DNA sample is subjected to polymerase chain reaction (PCR) for amplification. We recommend assembling all reaction components on ice and quickly transferring 2.5 μl 10 × Standard Taq Reaction Buffer, 0.50 μl of dNTPs, 0.50 μl of 10 μM Forward Primer, 0.50 μl of 10 μM Reverse Primer, Template DNA, 0.125 μl Taq DNA Polymerase, and 25 μl Nuclease-free water reactions to PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling. Initial denaturation is done at 95°C for 30 s. The annealing step is typically 15–60 s. The annealing temperature is based on the T_m of the primer pair and is typically 45°C–68°C. The recommended extension temperature was 68°C for 5 min. This cycle repeats 25–35 times in a typical PCR reaction, which generally takes 2–4 h, depending on the length of the DNA region being copied. The results of the PCR reaction are visualized (made visible) using gel electrophoresis. Fragments of DNA are pulled through a gel matrix by an electric current, separating DNA fragments according to size. A standard, or DNA ladder, is typically included to determine the size of the fragments attained in PCR.

The processed PCR product was initially sequenced by capillary electrophoresis (Sanger sequencing) for the three primary LHON mutations at Genome foundation (Hyderabad, India). Those samples were negative for all three known primary mitochondrial mutations; the whole mitochondrial genome sequencing was carried out to find out other secondary and novel mutations that may be involved in the pathogenesis of the disease.

Clinical investigations

A total of 15 patients were stable and in the chronic phase of the disease while performing the investigations. All patients underwent a complete clinical examination, including visual acuity testing, complete ophthalmic examination, visual fields (where visual acuity allowed the performance of a reliable visual field), and OCT of the retinal nerve fiber layer. Visual acuity was recorded using a LogMAR chart. If the patient is unable to read the letters from 4 m, the chart is placed at 2 m and 1 m from the patient until he sees the first line of the chart, and visual acuity is converted to the standard LogMAR value according to distance. A swept-source OCT-Topcon DRI OCT Triton plus (Topcon Corporation, Tokyo, Japan) coupled with the noninvasive OCT angiography technology (SS-OCT Angio™) was used to obtain the images. 6.0 mm × 6.0 mm scans with a resolution of 320 × 320 were obtained at a speed of 100,000 A scan/s. Repeated scans were taken till a good-quality image was obtained for both eyes. OCT images were analyzed by a single investigator. Images of poor quality with signal strength <40 or with residual motion artifacts like discontinuous vessels or disc boundaries were excluded from further analysis.

Results

Of 51 patients suspected to have LHON, 15 patients turned out to be positive for mitochondrial mutations which may be implicated in the pathogenesis of LHON. All the LHON patients were in the chronic phase of the disease.

The data of these 15 unrelated patients were collected and used for the study. All 15 patients who were positive for a mutation that led to LHON were males with the mean age of onset of the disease being 16.6 ± 5.2 years. Twelve of 15 patients reported that they were experiencing simultaneous visual loss, while the remaining three patients experienced sequential vision loss. Of the 15 patients, 11 patients were positive for primary mitochondrial mutations, of which nine patients were positive for G11778A mutation and two were positive for T14484C mutation. Four out of fifteen patients had secondary mitochondrial mutations, according to the current study. The first patient out of four tested positive for the A4136G mutation, the second tested positive for the T14864C mutation, and the third tested positive for the T15908C mutation. The fourth patient had four disease-associated variations, including T4216C, A4917G, as well as G5556A (indicating combined OXPHOS insufficiency), and G15928A (indicating multiple sclerosis) variants which were suggestive of having a potential LHON + syndrome. The mutant DNA and clinical features of all LHON patients are presented in [Table 1].

Table 1: The clinical descriptive data and corresponding deoxyribonucleic acid mutations of all Leber's hereditary optic neuropathy patients participated in the current study

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Visual acuity

Visual acuity of the patients with primary and secondary mutations is presented in [Figure 1]. The mean LogMAR visual acuity in patients with G11778A mitochondrial mutation was 1.05 ± 0.36 in the right eye and 1.15 ± 0.63 in the left eye, and patients with T14484C mitochondrial mutation had a mean visual acuity of 1.15 ± 0.15 in the right eye and 0.8 ± 0.5 in the left eye. Patients with secondary mitochondrial mutations showed a mean visual acuity of 0.8 ± 0.49 in the right eye and 1.50 ± 0.74 in the left eye. Analysis of variance (ANOVA) shows that there is no significant difference in visual acuity of the right eye (P = 0.1) among the three genotypic groups. Similarly, there is no significant difference in visual acuity of the left eye (P = 0.6) among the three genotypic groups.

Figure 1: The mean LogMAR visual acuity in the right eye and left eye in three different genotypic groups

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Fundus examination

The common optic nerve head appearance in LHON patients was either temporal pallor or diffuse pallor.

The optic disc findings in all three mutations are presented in [Figure 2]. There was no specific pattern observed in G11778A and secondary mutation groups; however, patients carrying T14484C showed a hallmark of diffuse pallor of the disc. Of 30 eyes of 15 patients, nine eyes of five patients carrying G11778A mutation, four eyes of two patients carrying T14484C mutation, and six eyes of three patients carrying secondary mitochondrial mutation showed diffuse pallor of the optic disc. While nine eyes of five patients carrying G11778A mutation and two eyes of one patient carrying secondary mutation showed temporal pallor on the optic disc.

Figure 2: Bar graph showing the optic disc findings in LHON patients with primary and secondary mutations. The vertical axis shows the number of patients, and the horizontal axis shows the type of mutation present. LHON: Leber's hereditary optic neuropathy

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Visual fields

The Humphrey visual field analyzer was used to record the visual fields. Visual field examination was possible in 23 eyes of 13 patients. Due to poor visual acuity, three eyes of two patients' visual fields testing were not possible. Visual field loss in LHON patients was mainly of two types. The first type of field loss was generalized, and the second type of field loss was central. There was no specific pattern observed in any of the three genotypic groups. Of nine patients carrying G11778A mutations, six patients showed central scotoma and two patients showed generalized field loss on visual fields. Of two patients carrying T14484C mutations, two patients showed central scotoma, while one patient showed a generalized total loss in one eye and could not perform a visual field test for the other eye due to poor visual acuity. Of four patients carrying secondary mitochondrial mutation, three patients showed central scotoma, and one patient showed a generalized total loss.

Retinal nerve fiber layer thickness

Retinal nerve fiber layer (RNFL) analysis was possible in 24 eyes of 12 patients. RNFL thickness was studied in all four quadrants (inferior, superior, nasal, and temporal) and was measured using a swept-source OCT-Topcon DRI OCT Triton plus (Topcon Corporation, Tokyo, Japan). The right and left eye mean RNFL thickness in all four quadrants of the genotypic groups is presented in [Figure 3]a and [Figure 3]b, respectively. Patients carrying G11778A mutation and those harboring a secondary mitochondrial mutation showed a similar loss of RNFL in all the quadrants of both eyes indicating a loss of retinal ganglion cells in both groups; two patients carrying T14484C mutation showed a clinically significant RNFL loss compared to the other two groups although it was established that the patients with T14484C mutation have a RNFL loss less compared to G11778A mutation group; we require more sample size to confirm if this finding holds true for our ethnic group.

Figure 3: RNFL thickness along four quadrants of (a) right eye and (b) left eye of LHON patients with different mutations. The vertical axis shows RNFL thickness in μM, and the horizontal axis shows the type of mutation. RNFL: Retinal nerve fiber layer

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Discussion

Although there were studies that looked at the genotype and phenotype correlations in LHON patients, they were limited to evaluate one or a few of the clinical investigations such as visual acuity, disc appearance, visual fields, and RNFL thickness.^{[1],[6],[13],[14],[15],[16],[18]} This is the first study from South India which reported the differences in the clinical features that included RNFL thickness of LHON patients with different mutations. It is important to note that LHON disease affects one in 50,000 patients, so even analysis of small sample data would be very useful in understanding the disease clinical presentation in various mutations.

The present study has explored the genotyping and phenotyping associated with LHON patients from India. The mean age of onset of LHON in the current study was 16.6 ± 5.2 years. The age of onset of LHON seems to vary in different studies ranging from 8 to 62 years, but the mean age of onset is approximately 20 years.^{[7],[19],[20],[21]} In a recent study with a large cohort of 189 LHON patients, only 17.5% of them showed primary mutations and 25% showed other variants. The current study also shows that 21% of the suspected LHON patients only were positive for primary mitochondrial mutations and 7% of them were positive for secondary mitochondrial mutations. Hence, it is important to do complete mtDNA sequencing to diagnose LHON as there are other disease variants throughout the genome.^[21] Unlike reports from other studies,^[6],[7] most of the current study patients reported having a simultaneous loss of vision in both eyes. This may be attributed to the poor history recalling by some of our patients, where the loss of vision in one eye may have gone unnoticed. All the patients who were positive for LHON in this study from July 2017 to February 2018 were male. Previous studies from India also reported similar results that LHON disease is mainly affecting males.^[12],[19] From these studies, it may be observed that the female carriers with the mutation are less likely to express the phenotypic characteristics of Indian ethnicity.

In the chronic phase of LHON disease, there is no significant difference in the LogMAR visual acuity of the patients carrying primary or secondary LHON mitochondrial DNA mutations. Previous reports from Korea and the United Kingdom have also reported that there is no difference in the mean visual acuities of the LHON patients carrying different mutations.^[6],[16] The qualitative assessment of visual fields was done, on which 60% of the patients showed central scotoma, while 40% of the patients showed generalized total field loss or visual acuity very poor to perform HVF. All of our patients who were diagnosed to have LHON are in the chronic phase of the disease; hence, the fundus examination in our study largely showed diffuse or temporal pallor of the optic disc.

Seo et al.,^[16] in 2009, reported that the average RNFL thickness in the chronic phase of LHON patients carrying G11778A mutation was significantly less than that of T14484C group. On the contrary, the current study shows that T14484C group patients show severe NFL thinning in the chronic phase of the LHON compared to the G11778A mutation group in south Indian patients. In addition to this, the RNFL thickness of the patients harboring G11778A mutation was overlapping with the results of patients carrying secondary mitochondrial mutations suggesting that secondary mutations presented here show similar RNFL changes as G11778A. Future studies with a larger sample size are needed to validate these initial data.

The limitations of the study were as follows: (a) all patients, who were diagnosed to have LHON, were in the chronic stage of the disease; hence, acute phase clinical features were missed; (b) all secondary mutations were grouped together as the sample size is very small. Applying statistical analysis of RNFL thickness among the three groups was difficult as the sample size in each group is too small.

Conclusions

Based on a small sample of our LHON patients, no significant differences seem to present among the clinical features in various genotypic groups except severe RNFL thinning in patients with T14484C mutation. However, a detailed study with larger samples would be needed to validate these initial data.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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