ABSTRACT
In numerous neurodegenerative disorders, neurofilaments, especially their subunits such as the Neurofilament light chain (NfL), are recognized as significant biomarkers of axonal injury when increased in blood or cerebrospinal fluid. Dominant optic atrophy (DOA) is characterized by a degeneration of retinal ganglion cells leading to axonal injury. Aim of this study was the evaluation of serum NfL (sNfL) levels in patients with DOA. sNfL concentration was quantified by a Single Molecule Array (Simoa) SR-X analyzer. Primary aim was the comparison of sNfL between patients with OPA1-DOA confirmed by genetic testing and controls. We further investigated associations between sNfL and age, visual acuity, peripapillary retinal nerve fiber layer thickness (pRNFLT) and disease duration. 22 DOA patients and 22 controls were included in this study. sNfL concentration was higher in DOA patients but did not differ significantly between the DOA group (Median (IQR) = 7.39 (5.25, 11.26) and controls (Median (IQR) = 5.86 (4.50, 9.88); p = .405). We found significant correlations between sNfL and age in both groups (DOA group: rho = 0.77, p < .001; control group: rho = 0.79, p < .001). Correlations between sNfL and visual acuity, pRNFLT and disease duration were not significant. Although elevated sNfL values were found in patients with DOA, we did not observe a significant difference between DOA patients and healthy controls.
KEYWORDS: Blood biomarker, serum neurofilament light chain, dominant optic atrophy, biomarker, OPA1
Introduction
Dominant optic atrophy (DOA) is the most common form of hereditary optic atrophies.1 Between 1:10,000 and 1:50,000 individuals are affected by this disease.2 In 60–70% of patients, a mutation in the OPA1 gene can be found.3 This leads to a bilateral degeneration of the optic nerves, primarily affecting the retinal ganglion cells and their axons. In most cases, patients experience visual loss accompanied by central or paracentral visual field defects and color vision disturbances. Extraocular symptoms such as neurosensory hearing impairment, myopathy, peripheral neuropathy, spastic paraplegia, and the rare chronic progressive external ophthalmoplegia occur in approximately 20% of DOA patients.4
The axonal cytoskeleton is primarily composed of neurofilaments, which consist of three major protein subunits known as neurofilament light chain (NfL), neurofilament medium chain (NfM), and neurofilament heavy chain (NfH). Through axonal damage, neurofilaments enter the interstitial fluid and then diffuse into the blood and cerebrospinal fluid.5,6
Serum NfL (sNfL) is a valid biomarker for neuro-axonal injury and increased levels of this marker has been demonstrated in a variety of acute and chronic neurological disorders like prion diseases, dementia, amyotrophic lateral sclerosis, Parkinson disease and multiple sclerosis.5
With the development of fourth-generation assays such as Single Molecule Array (Simoa) highly sensitive detection of NfL across its full concentration range in blood serum and cerebrospinal fluid has become possible, outperforming both the second-generation Enzyme-Linked Immunosorbent Assay (ELISA) and the third generation Electrochemiluminescence (ECL).7
Building upon these findings, we investigated if sNfL quantified by the Simoa technique would be sensitive enough to capture neuro-axonal injury related to DOA.
In this preliminary study, we aimed to investigate sNfL concentration in DOA patients compared to healthy controls.
Materials and methods
In this prospective case-control study patients with DOA and healthy controls were enrolled between July 2020 and December 2021 at the Department of Ophthalmology, Medical University of Graz (Austria). The study was approved by the local Ethics Committee (Medical University of Graz, EC number: 31–504 ex 18/19) and adhered to the Declaration of Helsinki. Written informed consent was obtained from all patients.
Inclusion criterion of the DOA group was a genetically confirmed OPA1-mutation, which was analyzed at the Institute of Human Genetics. Mutation status was assessed using next-generation sequencing (NGS), polymerase chain reaction (PCR) amplification, and direct DNA sequencing. DOA plus was not included in this study.
For the control group healthy participants especially without any the following diseases were selected: optic atrophy, glaucoma, neurodegenerative diseases (for example Multiple Sclerosis, Dementia, Parkinson disease, Huntington disease) history of stroke or traumatic brain injury.
Blood volume, assessed through a blood sample, was within normal limits, severe renal insufficiency and mental illness were excluded, and there was no female participant pregnant at that time. Controls were age- and gender-matched.
Clinical examination of all patients included best-corrected visual acuity (BCVA, converted in Logarithm of the Minimum Angle of Resolution (logMAR)) and a slit lamp examination. Furthermore, an optical coherence tomography (OCT, Heidelberg Engineering GmbH, Germany) of the peripapillary optic nerve head was performed. Therefore, a 12° scan was focused automatically on the optic nerve head. The built-in segmentation software segmented the retinal nerve fiber layer. Segmentation was checked by a single reviewer and corrected manually. The global peripapillary retinal nerve fiber layer thickness (pRNFLT) was generated automatically (Spectralis Family Acquisition Module Software Version 6.16.8.0). Duration of the disease was defined as difference between biological age and the age of anamnestic first presentation of DOA symptoms.
Primary aim of our study was the comparison of sNfL between DOA patients and healthy controls. To determine sNfL, a venipuncture was performed. Samples were stored at −80°C and analyzed at the Department of Neurology (Medical University of Graz, Austria) through Simoa SR-X Analyzer. We further investigated associations between sNfL and age, visual acuity, peripapillary retinal nerve fiber layer thickness (pRNFLT) and disease duration. In a subgroup analysis including only DOA patients, sNfL concentration was compared between individuals with high vs. low levels of BCVA and pRNFLT.
Categorical data are presented as relative and absolute frequencies. Continuous data are presented as median and interquartile range. To compare patient characteristics between the DOA and control groups, categorical data were compared with Fisher’s exact test and continuous data with Wilcoxon rank-sum test. Correlations were investigated using Spearman’s correlations. In both groups, correlations were also computed with Spearman partial correlation while controlling for age. Subgroups were built by median split of BCVA and pRNFLT within the group of DOA patients. The sample-size determination was calculated based on a mean of serum NfL of 34.7 pg/mL and a SD of 13.1 pg/mL.8 Thus, we would have a power of more than 80% to detect a difference of 12 pg/mL with a sample size of 22 participants. P-values <0.05 were considered statistically significant. Statistical analysis was performed with R software (version 4.1.0).9
This manuscript was written according to the Strobe guidelines for case-control studies.10
Results
22 patients with a genetic confirmed OPA1-DOA and 22 controls were included in this study. The distribution of age and sex between groups was equal. Patients of the DOA group had significantly worse BCVA and lower pRNFLT than the control group (p < .001). Baseline characteristics are depicted in Table 1.
Table 1.
Baseline characteristics.
| DOA group |
Controls |
||||
|---|---|---|---|---|---|
| N | Median (IQR) | N | Median (IQR) | P | |
| Age (years) | 22 | 41.50 (37.00, 56.75) | 22 | 41.50 (35.00, 57.00) | .9441 |
| Sex | 22 | 22 | >.9992 | ||
| Male (N, %) | 14 (63.6) | 14 (63.6) | |||
| Female (N, %) | 8 (36.4) | 8 (36.4) | |||
| BCVA (logMAR) | 22 | 0.50 (0.26, 0.65) | 18 | 0.00 (−0.10, 0.04) | <.0011 |
| pRNFLT (µm) | 22 | 59.75 (51.50, 65.25) | 20 | 102.00 (93.88, 107.75) | <.0011 |
IQR = Interquartile range; BCVA = best corrected visual acuityp; RNFLT= peripapillary retinal nerve fiber layer thickness; 1: Wilcoxon Rank Sum Test; 2: Fisher’s Exact Test; *p- value < 0.05.
The DOA group appeared to show a trend toward a higher sNfL concentration (median (IQR) = 7.39 (5.25, 11.26)) than the control group (median (IQR) = 5.86 (4.50, 9.88)), but this difference was not significant (p = .405, Figure 1).
Figure 1.
sNfL concentration in the DOA group and controls.
We found a positive correlation between sNfL and age in both groups (DOA group: rho = 0.77, p < .001, control group: rho = 0.79, p < .001, Figure 2).
Figure 2.
Correlation between sNfL concentration and age in DOA group and control group.
In both groups, we did not observe a significant correlation between sNfL concentration and BCVA (DOA group: rho = −0.28, p = .201, control group: rho = 0.37, p = .134, Figure 3) or pRNFLT (DOA group: rho = −0.14, p = .522, control group: rho = −0.01, p = .975, Figure 4). In the DOA group we also analyzed the RNFL in the temporal quadrant and here we did not find a correlation either (rho = −0.14, p = .522). Spearman partial correlations, controlling for age, did not change results. There was no significant correlation between BCVA and sNfL levels (DOA group: rho= −0.09, p = .70, control group: rho = 0.15, p = .55). Furthermore, no significant differences were found between OCT and sNfL concentration in both the DOA group (rho = 0.00, p = .99) and control group (rho = 0.08, p = .76).
Figure 3.
Correlation between sNfL concentration and BCVA in DOA and control group.
Figure 4.
Correlation between sNfL concentration and pRNFLT in DOA and control group.
To investigate the correlation between sNfL and disease duration three DOA patients were excluded, because they did not perceive any symptoms. We could not find a correlation between sNfL concentration and disease duration (median (IQR) of disease duration = 23.00 (14.00, 36.50), rho = 0.29, p = .226).
We performed a subgroup analysis to examine a possible difference in sNfL concentration in DOA patients depending on BCVA. Therefore, the DOA cohort was divided into two groups: better BCVA (≤0.50 logMAR, n = 11) and worse BCVA (>0.50 logMAR, n = 11). Patients with better BCVA had higher sNfL concentration (median (IQR) = 10.41 (6.07, 12.51)) than patients with worse BCVA (median (IQR) = 7.03 (4.36, 8.10)), but this difference was not significant (p = .171).
For the subgroup analysis sNfL levels depending on pRNFLT, the DOA cohort was divided into a group with thinner (≤59.75 µm, n = 11) and thicker (>59.75 µm, n = 11) pRNFLT. Also, in this subgroup analysis we could not observe a significant difference in sNfL concentration (thinner pRNFL: median (IQR) = 7.14 (6.09, 10.67); thicker pRNFL: median (IQR) = 7.64 (4.09, 10.99); p = .652).
Discussion
This is the first study to investigate sNfL concentration in DOA patients. Our results showed a trend toward elevated sNfL values in DOA patients compared to controls, but the difference was not statistically significant. A larger sample size would be useful to determine this difference more precisely.
Previous studies have already shown elevated sNfL levels in various neurodegenerative disorders.5 Until now, there has been a limited amount of research investigating NfL levels in neurodegenerative ocular disorders. Recently, both animal models and human studies have shown an elevation in retinal neurofilament proteins, especially NfHSMI35, during optic neuritis.11,12 Comacchio et al.13 conducted a study that demonstrated elevated sNfL levels in patients with glaucoma. However, after adjusting for age, this difference lost its statistical significance, likely because both the frequency of glaucoma and NfL values increased with biological age in the studied cohort. In 2022, Woltsche et al.14 were the first to demonstrate increased NfL levels in the anterior chamber fluid of glaucoma patients. However, they did not find elevated sNfL levels.
In 2008, Guy et al.15 evaluated serum phosphorylated Neurofilament Heavy Chain (pNfH) levels in Leber hereditary optic neuropathy (LHON) patients and carriers using the ELISA method. Their findings suggested that elevated pNfH levels in the serum of LHON patients may indicate axonal degeneration. This elevation tends to occur after a certain point following the loss of visual function. However, the precise onset and irreversibility of degeneration in retinal ganglion cells and optic nerve axons remain undetermined.
Similar to the findings reported in the studies conducted by Khalil et al.8 and Nielsen et al.,16 our study also identified a significant association between sNfL levels and age in both the DOA and control groups.
We did not find any significant correlation, even after adjusting for age, between sNfL concentration and BCVA or pRNFLT in either of the two groups. Unexpectedly, subgroup analysis showed that DOA patients with better BCVA had higher sNfL concentrations than those with worse BCVA, although the difference was not statistically significant. Similarly, DOA patients with thicker pRNFLT had higher sNfL levels than those with thinner pRNFLT. This may be due to the fact, that patients may have more neurons left as a possible source for sNfL in the early course of the disease.
Furthermore, in the control group one of the patients showed a sNfL value exceeding 30 pg/mL, yet no apparent explanation could be discerned for this observation.
Our findings suggest that although sNfL may be a marker of neurodegeneration, its concentration in serum does not necessarily reflect the severity of visual impairment or the thinning of the retinal nerve fiber layer in DOA. A possible explanation could be that either the axonal damage is too small to cause a systemic change in NfL levels or that we still need more sensitive examination techniques to detect these changes.
Our study has some limitations, which need to be acknowledged: The sample size of the investigated cohort in this preliminary study was rather small, limiting statistical power. Another aspect to be taken into consideration is that our statistical analysis did not consider body mass index (BMI). According to Benkert et al.,17 NfL values are not consistent as they tend to increase with age and decrease with BMI. Further it would be informative to perform a longitudinal study to track the evolution of NfL concentration over time and to include extraocular symptoms to obtain a more comprehensive picture of neurodegenerative changes.
In conclusion, we observed a trend of elevated sNfL levels in DOA patients compared to controls in our study, but the difference was not statistically significant. Further research with a larger sample size is needed to clarify the potential role of NfL as a biomarker in DOA and other neurodegenerative eye diseases.
Acknowledgments
The authors thank all patients for the participation in this study.
Funding Statement
This study was funded by the Adele-Rabensteiner-Foundation
Disclosure statement
No potential conflict of interest was reported by the author(s).
References
- 1.Eliott D, Traboulsi EI, Maumenee IH.. Visual prognosis in autosomal dominant optic atrophy (Kjer Type). Am J Ophthalmol. 1993;115(3):360–367. doi: 10.1016/S0002-9394(14)73589-5. [DOI] [PubMed] [Google Scholar]
- 2.Puomila A, Huoponen K, Mäntyjärvi M, et al. Dominant optic atrophy: correlation between clinical and molecular genetic studies. Acta Ophthalmol Scand. 2005;83(3):337–346. doi: 10.1111/J.1600-0420.2005.00448.X. [DOI] [PubMed] [Google Scholar]
- 3.Amati-Bonneau P, Maria Lucia Valentino Ã, Pascal Reynier Ã, et al. OPA1 mutations induce mitochondrial DNA instability and optic atrophy “Plus” Henotypes Brain. doi: 10.1093/brain/awm298. [DOI] [PubMed] [Google Scholar]
- 4.Lenaers G, Hamel C, Delettre C, et al. Dominant optic atrophy. Orphanet J Rare Dis. 2012;7(1):1–12. doi: 10.1186/1750-1172-7-46/TABLES/2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Gordon BA. Neurofilaments in disease: what do we know? Curr Opin Neurobiol. 2020;61:105. doi: 10.1016/J.CONB.2020.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Petzold A. Neurofilament phosphoforms: surrogate markers for axonal injury, degeneration and loss. J Neurol Sci. 2005;233(1–2):183–198. doi: 10.1016/J.JNS.2005.03.015. [DOI] [PubMed] [Google Scholar]
- 7.Kuhle J, Barro C, Andreasson U, et al. Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA, electrochemiluminescence immunoassay and Simoa. Clin Chem Lab Med. 2016;54(10):1655–1661. doi: 10.1515/cclm-2015-1195. [DOI] [PubMed] [Google Scholar]
- 8.Khalil M, Pirpamer L, Hofer E, et al. Serum neurofilament light levels in normal aging and their association with morphologic brain changes. Nat Commun. 2020;11(1). 10.1038/S41467-020-14612-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.R Core Team . R: a language and environment for statistical computing, Vienna, Austria. R Foundation for Statistical Computing. https://www.r-project.org/. 2021. Accessed May 4, 2024. [Google Scholar]
- 10.von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol. 2008;61(4):344–349. doi: 10.1016/J.JCLINEPI.2007.11.008. [DOI] [PubMed] [Google Scholar]
- 11.Petzold A. Axonal degeneration and inflammation in acute optic neuritis. J Neurol Neurosurg Psychiatry. 2004;75(8):1178. doi: 10.1136/JNNP.2003.017236. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Weissert R, Hugosson T, Petzold A. Upregulated retinal neurofilament expression in experimental optic neuritis. Neuro-Ophthalmol. 2022;46(4):215. doi: 10.1080/01658107.2022.2025852. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Chomacchio F, Mariotto SN. Neurofilament light chain: a novel biomarker in patients with glaucoma | IOVS | ARVO journals. ARVO Annual Meeting Abstract Published 2020. https://iovs.arvojournals.org/article.aspx?articleid=2766416. Accessed October 14, 2020.
- 14.Woltsche N, Valentin K, Hoeflechner L, et al. Clinical science neurofilament light chain: a new marker for neuronal decay in the anterior chamber fluid of patients with glaucoma. Br J Ophthalmol. 2022:1–6. doi: 10.1136/bjophthalmol-2021-320828. [DOI] [PubMed] [Google Scholar]
- 15.Guy J, Shaw G, Ross-Cisneros FN, et al. Phosphorylated neurofilament heavy chain is a marker of neurodegeneration in Leber Hereditary Optic Neuropathy (LHON). Mol Vis. 2008;14:2443–2450. http://www.molvis.org/molvis/v14/a281. Accessed October 17, 2020. [PMC free article] [PubMed] [Google Scholar]
- 16.Krogh Nielsen M, Subhi Y, Rue Molbech C, Sellebjerg F, Sørensen TL. Serum neurofilament light chain in healthy elderly and in patients with age-related macular degeneration. Acta Ophthalmol. 2020;98(3):e393–e394. doi: 10.1111/AOS.14270. [DOI] [PubMed] [Google Scholar]
- 17.Benkert P, Meier S, Schaedelin S, et al. Serum neurofilament light chain for individual prognostication of disease activity in people with multiple sclerosis: a retrospective modelling and validation study. Lancet Neurol. 2022;21(3):246–257. doi: 10.1016/S1474-4422(22)00009-6. [DOI] [PubMed] [Google Scholar]