Comparison of thickness changes in retinal nerve fibre layer in Leber’s hereditary optic neuropathy patients with 11778, 14484 and 3460 mutations

Introduction

Leber’s hereditary optic neuropathy (LHON) is the most prevalent maternally inherited disorder causing visual impairment (1). The typical clinical manifestation of LHON involves pain-free central vision loss, with sequential involvement in both eyes occurring in 75% of cases, while 25% experienced simultaneous occurrence (2). The pathogenesis of LHON stems from mutations in mitochondrial DNA (mtDNA), impairing the function of respiratory chain complex I (3). This leads to energy deficiency and heightened production of reactive oxygen species. Then ganglion cells and their axons are damaged, ultimately leading to optic nerve atrophy (3). Research advancements have led to the discovery of over 50 distinct mtDNA variations associated with LHON, with over 95% of LHON cases attributed to three primary mtDNA changes. These primary mutations include the m.11778G>A mutation in the MT-ND4 gene, the m.3460G>A mutation in the MT-ND1 gene, and the m.14484T>C mutation in the MT-ND6 gene (4). Generally, visual outcomes of LHON patents are poor, whereas some were observed to have certain degree of visual recovery. Notably, the LHON patients with T14484C mtDNA mutations have the best prognosis with partial visual recovery rates from 37% to 65% (5-7).

Optical coherence tomography (OCT) can demonstrate the swelling and atrophy of the retinal nerve fibre layer (RNFL) in LHON with in vivo real-time imaging (8), which can help us detect axonal damage at an early stage (9). Previous studies have suggested that patients with thicker RNFL might have better visual prognosis (8). Our previous research evaluated RNFL thickness changes in LHON patients with the m.11778G>A mutation in MT-ND4 (10). However, there was limited research comparing RNFL involvement among different mtDNA mutations associated with varying visual prognoses (11).

In this study, we aimed to compare RNFL involvement in LHON patients with three specific mtDNA mutations (11778, 14484, and 3460) using OCT. We present this article in accordance with the STROBE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-589/rc).

Methods

Study participants

A total of 121 LHON patients (189 eyes) with G11778A mtDNA mutations, 10 patients (20 eyes) with T14484C mtDNA mutations, 15 patients (28 eyes) with G3460A mtDNA mutations, and 26 healthy controls (52 eyes) were enrolled in the study from July 2017 to December 2020. Patients were divided into three groups based on the duration of the disease (<6, 6–12, and >12 months) (12). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College (No. TJ-IRB20180316). Written informed consent was obtained from all individual participants, and from the parents or legal guardians of those under 18 years old.

Inclusion criteria

Patients between 12 and 45 years of age who were clinically confirmed with LHON based on characteristic symptoms and examination findings, harbored either the m.11778G>A mutation in the MT-ND4 gene, the m.14484T>C mutation in the MT-ND6 gene, or the m.3460G>A mutation in the MT-ND1 gene, and provided written informed consent were eligible for inclusion in this study.

Exclusion criteria

In this study, individuals with glaucoma, high myopia, or other retinal and optic nerve disorders unrelated to LHON were excluded from participation. For the purpose of documenting the disease progression, the onset of uncorrectable vision loss or visual dysfunction, as previously defined in our earlier research (10), was taken as the starting point for tracking the clinical course. Childhood-onset patients with an age of onset of less than 12 years and late-onset patients with an age of onset greater than 45 years were excluded. Additional exclusion criteria were previous treatment with gene therapy product, smokers and heavy drinkers as previously described (13). We have excluded patients who have received any drug (such as idebenone, vitamins, or traditional Chinese medicine) or therapy within 6 months or since the onset of the disease.

Study grouping

LHON patients were divided into groups based on the following criteria: the acute phase (onset within 6 months), the dynamic phase (onset between 6–12 months), and the chronic phase (onset over 12 months) (12). We recruited age-matched healthy individuals as the control group (both eyes of 26 healthy individuals, totaling 52 eyes, were included in this study). The inclusion criteria for the control group were as follows: best-corrected visual acuity (BCVA) greater than 20/25, refractive error less than 6 diopters (sphere) and 2 diopters (cylinder), intraocular pressure less than 21 mmHg, and no systemic or central nervous system diseases, as previously described (10).

Instrumentation and procedures

The RNFL thickness of LHON patients and healthy controls was assessed using the Spectralis^® HRA + OCT system (Heidelberg Engineering GmbH, Heidelberg, Germany). The thickness measurements were obtained from the superior, inferior, temporal, and nasal regions, and the 360° average values were also calculated. BCVA was evaluated using a standard 2.5-meter logMAR visual acuity chart (produced by Star Kang Medical Technology Co., Ltd., Wenzhou, China).

Statistical analysis

The data are presented as mean ± standard deviation. Statistical analysis was conducted using SPSS (version 21.0; IBM Corp., Armonk, NY, USA). A Kruskal-Wallis test was performed to evaluate differences in RNFL thickness among the groups. For all analyses, the significance level was set at P<0.05.

Results

Patient demographic data

Table 1 showed that the disease duration and the age of onset did not show any significant differences among LHON patients with G11778A, T14484C, and G3460A mtDNA mutations. Visual acuity was significantly better in LHON patients with T14484C mtDNA mutations than in patients with G11778A mtDNA mutations (P<0.01) or G3460A mtDNA mutations (P<0.01). No statistically significant differences in age were demonstrated among the three groups of LHON patients with different mutations.

RNFL thickness in all quadrants in healthy controls and LHON patients with G11778A, T14484C and G3460A mtDNA mutations with different disease durations

In the subacute phase, compared to healthy controls, the RNFL thickness of the nasal quadrant in LHON patients with G11778A mtDNA mutations increased, whereas it decreased in the remaining quadrants, and the decrease in temporal RNFL thickness reached significant differences (P<0.01). We further divided the LHON patients with G11778 mtDNA mutations in the subacute phase into 2 groups according to the duration of the disease (<3 and 3–6 months). Within 3 months of disease onset, all quadrants and the average RNFL thickness thickened, except for the temporal quadrant, however, no significant difference was reached. By 3–6 months from onset, all quadrants and the average RNFL thickness thinned. A notable decrease in RNFL thickness was detected in LHON patients with the G11778A mutation during both the dynamic and chronic phases, affecting all quadrants and the overall average, with all reductions achieving statistical significance (P<0.01) (Figure 1A; Table 2).

Figure 1 RNFL thickness in all quadrants in healthy controls and LHON patients with G11778A, T14484C and G3460A mutations with different disease durations. (A) Line diagram showed that the RNFL involvement in LHON patients with different mutation types showed similar pattern: firstly, the temporal RNFL showed earlier and more prominent decrease, followed by RNFL in the inferior and superior quadrants, and finally, the nasal RNFL. (B) Bar diagram showed that in the subacute phase, the nasal RNFL thickness was significantly thinner in LHON patients with G3460A mutations than in LHON patients with T14484C mutations (P=0.03). In the dynamic phase, the RNFL thickness in all quadrants of LHON patients with G11778A mutations was thinner than that of LHON patients with T14484C mutations; the decrease in the nasal quadrant (P<0.01), superior quadrant (P<0.01), inferior quadrant (P<0.01), and the average (P<0.01) RNFL reached significant differences. The RNFL thickness in all quadrants of LHON patients with G3460A mutations was thinner than that of LHON patients with T14484C mutations; the decrease in RNFL thickness in the nasal (P=0.01) quadrant reached significant differences. In the chronic phase, there were no significant differences in the RNFL thickness of each quadrant and the average RNFL thickness among LHON patients with G11778A, T14484C, and G3460A mutations. *, P<0.05 when compared among LHON patients with G11778A, T14484C and G3460A mutations with different disease durations. LHON, Leber’s hereditary optic neuropathy; RNFL, retinal nerve fibre layer.

In the subacute and dynamic phases, the RNFL thickness in the remaining quadrants in LHON patients with T14484C mtDNA mutations increased except that in the temporal quadrant, which had decreased (Table 2). Excluding the nasal quadrant (P=0.10), the RNFL thickness was significantly reduced in all remaining quadrants and on average in the chronic phase for LHON patients with the T14484C mutation (Figure 1A).

In the subacute phase, the RNFL thickness of LHON patients with G3460A mtDNA mutations in all quadrants and the average thickness are significantly reduced, with the most significant reduction observed in the temporal quadrant (from 87.67 to 47.20 µm). In the dynamic phase, the RNFL thickness in all quadrants of G3460A were significantly thinner than those in the healthy controls except for the nasal quadrant (P=0.29). In the chronic phase, the RNFL thickness of each quadrant and the average RNFL thickness of G3460A LHON patients were significantly thinner than those of the healthy control group (P<0.01) (Figure 1A; Table 2).

Comparison of RNFL thickness in all quadrants among LHON patients with G11778A, T14484C and G3460A mtDNA mutations with different disease durations

In the subacute and dynamic phases, the RNFL thickness in all quadrants of LHON patients with G11778A mtDNA mutations or G3460A mtDNA mutations was thinner than that of LHON patients with T14484C mtDNA mutations (Figure 1B; Table 2).

In the chronic stage, the RNFL thickness measurements, both quadrant-specific and average, were comparable among LHON patients harboring G11778A, T14484C, or G3460A mtDNA mutations (Figure 1B; Table 2).

There was no significant difference in visual acuity among LHON patients with G11778A, T14484C, and G3460A mtDNA mutations during the subacute and dynamic phases. In the chronic phase, individuals with the G11778A mtDNA mutation experienced markedly poorer vision compared to those with the T14484C mutation, with a statistically significant difference (P<0.01) as detailed in Table 2.

Discussion

In this study, we measured and compared the changes in visual acuity and RNFL thickness among LHON patients with G11778A mtDNA mutations, T14484C mtDNA mutations, and G3460A mtDNA mutations across different disease durations. In patients with LHON carrying different mutations, the progression of RNFL thinning exhibited a consistent pattern across all cases. Initially, the temporal region demonstrated the earliest and most significant reduction in RNFL thickness. Subsequently, thinning became apparent in the inferior and superior quadrants. Finally, the nasal region was affected. These findings align with previous observations regarding the natural progression of LHON (14). Notably, LHON patients with G11778A mtDNA mutations and G3460A mtDNA mutations demonstrated earlier and more prominent RNFL atrophy than patients with T14484C mtDNA mutations. Also, in the chronic phase, the visual acuity of LHON patients with G11778A mtDNA mutations was markedly poorer compared to those with the T14484C mtDNA mutations.

In the subacute phase, the RNFL thickness in LHON patients with G11778A mtDNA mutations or T14484C mtDNA mutations showed a tendency to first thicken and then thinned, consistent with previous studies (14). The mechanisms of RNFL thickening remains to be elucidated (14). One hypothesis postulates that energy deficiency at onset triggers a compensatory engorgement of peripapillary vessels, resulting in peripapillary RNFL (pRNFL) thickening (15). An alternative theory attributes the thickening to impaired axoplasmic transport and a compensatory rise in mitochondrial biogenesis, both consequences of the energy crisis and elevated oxidative stress (16). And as the disease progressed, we found that LHON patients with different mutation types showed earliest and most severe RNFL atrophy in the temporal quadrant (mainly composed of papillary macular bundles). This might be related to the natural pathological process of LHON, independent of the mutation type. The smaller-caliber fibers anatomically restricted mitochondrial axoplasmic transport and were characterised by low energy production and high energy demand (8,17). Therefore, the papillary macular bundle is more sensitive to this degenerative process and characteristically involved in the early stages of the disease (18).

In the subacute and dynamic phases, the RNFL thickness in all quadrants of LHON patients with G11778A mtDNA mutations or G3460A mtDNA mutations was thinner than that of LHON patients with T14484C mtDNA mutations (Figure 1B; Table 2). In our study, we found that LHON patients with G11778A mtDNA mutations and G3460A mtDNA mutations showed earlier and more prominent RNFL atrophy than patients with T14484C mtDNA mutations. A T-to-C transition at nucleotide 14484 in the ND6-gene converts a methionine to a valine in one of the most conserved domains of the ND6-gene. However, the conservation of the mutant residue and the nature of the amino acid substitution are less noteworthy than those seen in other primary mutations (19). An extended respiration study revealed that the 3460 mutation reduced the maximal respiration rate at 20–28%, the 11778 mutation 30–36% and the 14484 mutation 10–15% (20). That might imply less dysfunction of mitochondrial respiratory chain complex I, which might partly explain the better prognosis of LHON patients with 14484 mtDNA mutations.

As the disease progressed further, with further ganglion cell death and axonal loss, the RNFL thickness of all quadrants and the average RNFL thicknesses of LHON patients with the three primary mutation types were significantly thinner than that in the healthy controls. In summary, the thinning pattern of the RNFL in LHON patients with the three primary mutation types exhibits a consistent trend (Figure 1A). The process begins with thinning in the temporal region, followed by changes in the inferior and superior regions, and concludes with thinning in the nasal region, which is consistent with our previous studies (10), and this feature may help us to identify it with other optic nerve diseases. Our study also observed that LHON patients with G11778A mtDNA mutations had significantly worse visual acuity than in LHON patients with T14484C mtDNA mutations in the chronic phase, which is consistent with previous studies (11). Previous studies showed that LHON patients with thicker nerve fibre layer thickness might have a better prognosis (8). In our study, even in the chronic phase, the RNFL thickness of the average, superior, inferior and nasal quadrants in LHON patients with T14484C mtDNA mutation were discreetly thicker than those with the other two primary mutations and that only the temporal quadrant was comparable. However, the RNFL thicknesses changes were not statistically significant. Research has revealed a correlation between retinal structural alterations and visual impairment (21). Furthermore, studies have indicated that a reduction in the thickness of the RNFL, ganglion cell layer, and inner plexiform layer may be linked to partial vision loss in individuals with LHON (21). This might indicate that even in the chronic phase, LHON patients with T14484C mtDNA mutation retain relatively more ganglion cells and their axons. However, it might attributable to the small sample size of LHON patients with T14484C mtDNA mutation. To validate this result, supplementary evidence is essential.

Our study has some limitations. This study solely conducted a cross-sectional analysis to examine the variations in RNFL thickness across patients harboring distinct mutation types. Nevertheless, validation in larger prospective studies is required. Due to the rarity of LHON patients, binocular data were included in the statistics, and thus, potential confounding factors from binocular data were not accounted for. Further support from prospective evidence is needed for this part of our study. The number of patients with LHON types T14484C or G3460A was limited. However, the rare incidence of LHON with the T14484C or G3460A mtDNA mutation should also be considered. Our results need to be validated by further studies of larger samples. An acknowledged limitation is the omission of heteroplasmy and mtDNA copy number, which will be included in future studies to strengthen the rigor of our approach.

Conclusions

Our findings demonstrated that the RNFL involvement patterns in LHON patients with the three primary mutations were consistent, indicating that such involvement is unrelated to the specific mutation type. This observation offers insights into distinguishing LHON from other forms of optic neuropathy. In addition, our study also revealed that the visual acuity and RNFL thickness change of the three subtypes of LHON patients still have their own characteristics, and LHON patients with G11778A mutations and G3460A mtDNA mutations showed earlier and more prominent RNFL atrophy than patients with T14484C mtDNA mutations. In conclusion, OCT can play a great role in studying nerve fibre layer thickness changes in patients with different mutations of LHON.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-589/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-589/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-589/prf

Funding: This study was supported by the Hubei Province Natural Science Foundation of China (Nos. 2022CFB893 and 2025AFB047), the Scientific Research Foundation Program of Wuhan Children’s Hospital (No. 2023TFJH04), the Guangdong Provincial Natural Science Foundation of China (No. 2023A1515010382), and the Scientific Research Foundation Program of Tongji Hospital (No. 2024B23).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-589/coif). B.L. is from Wuhan Neurophth Biological Technology Limited Company. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College (No. TJ-IRB20180316). Written informed consent was obtained from all individual participants, and from the parents or legal guardians of those under 18 years old.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Martin-Kleiner I, Gabrilovac J, Bradvica M, et al. Leber's hereditary optic neuroretinopathy (LHON) associated with mitochondrial DNA point mutation G11778A in two Croatian families. Coll Antropol 2006;30:171-4.
2. Meunier I, Lenaers G, Hamel C, et al. Hereditary optic neuropathies: from clinical signs to diagnosis. J Fr Ophtalmol 2013;36:886-900. [Crossref] [PubMed]
3. Coussa RG, Merat P, Levin LA. Propagation and Selectivity of Axonal Loss in Leber Hereditary Optic Neuropathy. Sci Rep 2019;9:6720. [Crossref] [PubMed]
4. Shamsnajafabadi H, MacLaren RE, Cehajic-Kapetanovic J. Current and Future Landscape in Genetic Therapies for Leber Hereditary Optic Neuropathy. Cells 2023;12:2013. [Crossref] [PubMed]
5. 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;141:676-82. [Crossref] [PubMed]
6. Mackey D, Howell N. A variant of Leber hereditary optic neuropathy characterized by recovery of vision and by an unusual mitochondrial genetic etiology. Am J Hum Genet 1992;51:1218-28.
7. Johns DR, Heher KL, Miller NR, et al. Leber's hereditary optic neuropathy. Clinical manifestations of the 14484 mutation. Arch Ophthalmol 1993;111:495-8. [Crossref] [PubMed]
8. Barboni P, Savini G, Valentino ML, et al. Retinal nerve fiber layer evaluation by optical coherence tomography in Leber's hereditary optic neuropathy. Ophthalmology 2005;112:120-6. [Crossref] [PubMed]
9. Manogaran P, Hanson JV, Olbert ED, et al. Optical Coherence Tomography and Magnetic Resonance Imaging in Multiple Sclerosis and Neuromyelitis Optica Spectrum Disorder. Int J Mol Sci 2016;17:1894. [Crossref] [PubMed]
10. Wang D, Liu HL, Du YY, et al. Characterisation of thickness changes in the peripapillary retinal nerve fibre layer in patients with Leber's hereditary optic neuropathy. Br J Ophthalmol 2021;105:1166-71. [Crossref] [PubMed]
11. Seo JH, Hwang JM, Park SS. Comparison of retinal nerve fibre layers between 11778 and 14484 mutations in Leber's hereditary optic neuropathy. Eye (Lond) 2010;24:107-11. [Crossref] [PubMed]
12. Carelli V, Carbonelli M, de Coo IF, et al. International Consensus Statement on the Clinical and Therapeutic Management of Leber Hereditary Optic Neuropathy. J Neuroophthalmol 2017;37:371-81. [Crossref] [PubMed]
13. Zhang Y, Li X, Yuan J, et al. Prognostic factors for visual acuity in patients with Leber's hereditary optic neuropathy after rAAV2-ND4 gene therapy. Clin Exp Ophthalmol 2019;47:774-8. [Crossref] [PubMed]
14. Barboni P, Carbonelli M, Savini G, et al. Natural history of Leber's hereditary optic neuropathy: longitudinal analysis of the retinal nerve fiber layer by optical coherence tomography. Ophthalmology 2010;117:623-7. [Crossref] [PubMed]
15. Teng D, Peng CX, Qian HY, et al. Structural impairment patterns in peripapillary retinal fiber layer and retinal ganglion cell layer in mitochondrial optic neuropathies. Int J Ophthalmol 2018;11:1643-8. [Crossref] [PubMed]
16. Borrelli E, Triolo G, Cascavilla ML, et al. Changes in Choroidal Thickness follow the RNFL Changes in Leber's Hereditary Optic Neuropathy. Sci Rep 2016;6:37332. [Crossref] [PubMed]
17. Sadun AA, Win PH, Ross-Cisneros FN, et al. Leber's hereditary optic neuropathy differentially affects smaller axons in the optic nerve. Trans Am Ophthalmol Soc 2000;98:223-32; discussion 232-5.
18. Carelli V, Ross-Cisneros FN, Sadun AA. Mitochondrial dysfunction as a cause of optic neuropathies. Prog Retin Eye Res 2004;23:53-89. [Crossref] [PubMed]
19. Savontaus ML. mtDNA mutations in Leber's hereditary optic neuropathy. Biochim Biophys Acta 1995;1271:261-3. [Crossref] [PubMed]
20. Brown MD, Trounce IA, Jun AS, et al. Functional analysis of lymphoblast and cybrid mitochondria containing the 3460, 11778, or 14484 Leber's hereditary optic neuropathy mitochondrial DNA mutation. J Biol Chem 2000;275:39831-6. [Crossref] [PubMed]
21. Moster SJ, Moster ML, Scannell Bryan M, et al. Retinal Ganglion Cell and Inner Plexiform Layer Loss Correlate with Visual Acuity Loss in LHON: A Longitudinal, Segmentation OCT Analysis. Invest Ophthalmol Vis Sci 2016;57:3872-83. [Crossref] [PubMed]