Leber Hereditary Optic Neuropathy in a Family of Carriers of MT-ND5 m.13042G>T (A236S) Novel Variant

Background: A Slovenian three-generation family with 3 individuals with bilateral optic neuropathy and 2 unaffected relatives with a novel homoplasmic missense variant m.13042G > T (A236S) in the ND5 gene is described. A detailed phenotype at initial diagnosis and a follow-up of bilateral optic neuropathy progression is presented for 2 affected individuals. Methods: A detailed phenotype analysis with clinical examination in the early and chronic phase with electrophysiology and OCT segmentation is presented. Genotype analysis with full mitochondrial genome sequencing was performed. Results: Two affected male individuals (maternal cousins) had a profound visual loss at an early age (11 and 20 years) with no recovery. The maternal grandmother exhibited bilateral optic atrophy with a history of visual loss at the age 58 years. The visual loss of both affected male individuals was characterized by centrocecal scotoma, abnormal color vision, abnormal PERG N95, and VEP. Later with disease progression, retinal nerve fiber layer thinning was observed on OCT. We observed no other extraocular clinical features. Mitochondrial sequencing identified a homoplasmic novel variant m.13042G > T (A236S) in the MT-ND5 gene, belonging to a haplogroup K1a. Conclusions: Novel homoplasmic variant m.13042G > T (A236S) in the ND5 gene in our family was associated with Leber hereditary optic neuropathy-like phenotype. However, predicting the pathogenicity of a novel ultra-rare missense variant in the mitochondrial ND5 gene is challenging. Genetic counseling should consider genotypic and phenotypic heterogeneity, incomplete penetrance, haplogroup type, and tissue-specific thresholds.

Original Contribution Section Editors: Clare Fraser, MD Susan Mollan, MD Leber Hereditary Optic Neuropathy in a Family of Carriers of MT-ND5 m.13042G.T (A236S) Novel Variant tar Habjan, PhD, Jelka Brecelj, PhD, Sanja Petrovi c Paji c, MD, MSc, Maja Sus Maver, MD, PhD, Ana Fakin, MD, PhD, Marija Volk, MD, PhD, Ales Gregor Jezernik, PhD, Borut Peterlin, MD, PhD, Damjan Glava c, PhD, Marko Hawlina, MD, PhD, Martina Jarc-Vidmar, MD, PhD Background: A Slovenian three-generation family with 3 individuals with bilateral optic neuropathy and 2 unaffected relatives with a novel homoplasmic missense variant m.13042G . T (A236S) in the ND5 gene is described. A detailed phenotype at initial diagnosis and a follow-up of bilateral optic neuropathy progression is presented for 2 affected individuals. Methods: A detailed phenotype analysis with clinical examination in the early and chronic phase with electrophysiology and OCT segmentation is presented. Genotype analysis with full mitochondrial genome sequencing was performed. Results: Two affected male individuals (maternal cousins) had a profound visual loss at an early age (11 and 20 years) with no recovery. The maternal grandmother exhibited bilateral optic atrophy with a history of visual loss at the age 58 years. The visual loss of both affected male individuals was characterized by centrocecal scotoma, abnormal color vision, abnormal PERG N95, and VEP. Later Eye Hospital (SPP, MJV, BSK, MS, JB, AF, MSH), University Medical Centre Ljubljana, Ljubljana, Slovenia; Clinic for Eye Diseases (SPP), Clinical Centre of Serbia, Belgrade, Serbia; Department of Molecular Genetics (DG), Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; Center for Human Genetics and Pharmacogenomics (GJ, DG), Faculty of Medicine, University of Maribor, Maribor, Slovenia; Clinical Institute of Genomic Medicine (MV, AM, BP), University Medical Centre Ljubljana, Ljubljana, Slovenia. The authors report no conflicts of interest. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www. jneuro-ophthalmology.com). This research was funded by Slovenian Research Agency (ARRS Program P3-0333 and P3-0427) Patients provided written informed consent according to regulations of University Medical Centre Ljubljana; the use of clinical data was approved by the National committee for Medical Ethics (No:0120– 626/2019/5, date: 17/3/2020). Address correspondence to Damjan Glava c, PhD, Department of Molecular Genetics, Faculty of Medicine, University of Ljubljana, Korytkova 2, SI-1000 Ljubljana Slovenia; E-mail: damjan.glavac@mf. uni-lj.si Petrovi c Paji c et al: J Neuro-Ophthalmol 2023; 43: 341-347 with disease progression, retinal nerve fiber layer thinning was observed on OCT. We observed no other extraocular clinical features. Mitochondrial sequencing identified a homoplasmic novel variant m.13042G . T (A236S) in the MT-ND5 gene, belonging to a haplogroup K1a. Conclusions: Novel homoplasmic variant m.13042G . T (A236S) in the ND5 gene in our family was associated with Leber hereditary optic neuropathy-like phenotype. However, predicting the pathogenicity of a novel ultra-rare missense variant in the mitochondrial ND5 gene is challenging. Genetic counseling should consider genotypic and phenotypic heterogeneity, incomplete penetrance, haplogroup type, and tissue-specific thresholds. Journal of Neuro-Ophthalmology 2023;43:341–347 doi: 10.1097/WNO.0000000000001820 © 2023 by North American Neuro-Ophthalmology Society L eber hereditary optic neuropathy (LHON) is a mitochondrial neurodegenerative disease presented as a painless, acute, or subacute visual loss. More than 90% of all LHON cases occur due to pathogenic, usually homoplasmic, mtDNA variants located in nucleotides 11778 (70%), 14484 (14%), and 3460 (13%) which affect genes encoding the NADH-ubiquinone oxidoreductase chain ND1, ND4, and ND6 subunits of complex I of the mitochondrial respiratory chain (1). The rare, atypical, usually heteroplasmic, pathogenic variants identified in mtDNA usually occur within individual families and are also considered pathogenic if functional studies confirm reduced mitochondrial function (2). We report a novel homoplasmic mtDNA MT-ND5 gene missense variant m.13042G . T (A236S) identified in a Slovenian family. Alternative variant m.13042G . A resulting in a different amino acid change (A236T) in heteroplasmy has been reported as pathogenic in several sporadic cases with overlapping phenotypes including LHON, MERRF/ MELAS, and Leigh syndrome (3–6). Only the Italian report 341 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution showed a familial occurrence where affected individuals had predominantly ocular phenotype (3). The aim of this study was to present a detailed phenotype course of bilateral optic neuropathy in a Slovenian family with a novel MT-ND5 variant m.13042G . T (A236S) and compare the phenotype with an Italian family harboring an alternative MT-ND5 pathogenic variant m.13042G . A (A236T) (3). METHODS A Slovenian family with 3 affected individuals (grandmother and 2 grandsons) who suffered from LHON-like optic neuropathy and 2 unaffected first-degree relatives is presented. The family pedigree is shown in Figure 1. The affected patients experienced bilateral simultaneous (n = 2) or sequential (n = 1) VA loss. Two patients (proband and patient III-5) underwent a detailed diagnostic and genetic workup. Detailed methodology is presented in Supplemental Digital Content 1 (see File 1, http://links.lww.com/ WNO/A676). RESULTS Proband A 20-year-old man suffered profound, bilateral, sequential, central vision loss within a month-and-a-half interval, first on the left eye (LE). During examination, the Snellen visual acuity (VA) on his better right eye (RE) was 0.2 and hand movement (HM) at 30 cm (Approx. 0.005 Snellen) in LE. Bilateral central scotomas were recorded. The patient reported having strong headaches and flashes before VA loss. Fundus examination revealed a right optic nerve head (ONH) hyperemia with peripapillary telangiectatic blood vessels, vascular tortuosity, and swelling of the retinal nerve fiber layer (RNFL) around the optic disc. The left ONH was paler with tortuous vessels (Fig. 2); no leakage on fluorescein angiography (FA) was noted in either eye. VEP showed prolonged peak times bilaterally and a reduced amplitude on the LE. The RE PERG N95 amplitude was within the normal range, while LE was significantly lower (Fig. 2), corresponding to asymmetry in VA. Patient III-5 An 11-year-old boy, the maternal cousin of the proband, presented 2 years later with a two-week history of simultaneous asymmetrical bilateral visual loss. VA at examination was CF at 2 m on the RE and 0.3 on the LE, accompanied by the reduced color vision and bilateral central scotoma. On examination, bilateral hyperemic ONH and tortuous vessels were seen (Fig. 3). FA showed circumpapillary telangiectatic microangiopathy without leakage. Electrophysiological testing at the time of acute presentation showed reduced RE PERG N95 and delayed RLE VEP P100 (Fig. 3). This corresponded to better LE VA. In the following months, his visual function further deteriorated and within 5 months his VA was HM bilaterally with pale ONHs. Electrophysiology at that time revealed bilaterally reduced PERG N95 amplitude and prolonged and reduced VEP responses (Fig. 3). He had no systemic nor neurological diseases, except for a clinically insignificant aortic stenosis diagnosis diagnosed at the age 10 years. Both their mothers (II-2, II-3) were without visual symptoms at the ages 56 and 59 years and with normal clinical features (Fig. 1, photograph of II-2 is available). The maternal grandmother (I-2) suffered from bilateral VA loss at the age 58 years, followed by bilateral ONH atrophy (Fig. 1), which at that time was diagnosed in the local hospital as atypical optic neuritis, while the neurological examination was normal. No follow-up data are available since the patient in the meantime passed away. The possibility of LHON was attributed 14 years later when her grandson (III-3) presented with LHON-like optic neuropathy. During the follow-up period of affected patients (proband - 17 years and III-5 - 15 years), their VA and visual fields (VF) remained unchanged. Proband started generic idebenone in 2009, 5 years after the disease onset, and FIG. 1. Pedigree of the family with G13042T/ND5 A236S pathogenic variant. 342 Petrovi c Paji c et al: J Neuro-Ophthalmol 2023; 43: 341-347 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution FIG. 2. Phenotypic characteristics of the proband on the onset and last follow-up visit. switched to Raxone in 2016. Subjectively, his VA temporarily improved with the drug intake. He reported “flashes of light,” with a “blurry or sandy” picture. Objectively, his VA and VF remained unchanged. At the last visit, his VA was 0.015 bilaterally, and in patient III-5, 0.005 bilaterally, without significant VF improvement (Figs. 2, 3). Our patients had no neurological symptoms at the presentation nor during the follow-up, other than a headache in proband before VA loss. Head MRI was normal, without signs of demyelination or expansive processes. No cataracts were found in any of the patients. There were no signs of retinopathy on fundoscopy or OCT; however, some changes were observed in full-field ERG responses after 15 and 17 years of follow-up period at the age 26 and 37 years. Both patients had bilaterally reduced amplitudes of the cone and 30 Hz responses while patient III-5 also had lower rod responses on the RE as well. Electrophysiological findings of both affected male individuals at the initial assessment were previously described (7). Electrophysiology showed decreased PERG N95 wave and almost undetectable VEP P100 wave in both patients during the whole follow-up period (Figs. 2, 3). The slope of the N95/P50 ratio deterioration in the first couple of months was steep in the proband and a bit flatter in his younger cousin (III-5). During the follow-up period, this ratio FIG. 3. Phenotypic characteristics of patient III-5 at the onset and last follow-up visit. Petrovi c Paji c et al: J Neuro-Ophthalmol 2023; 43: 341-347 343 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution remained abnormal, under 1.1 (see Supplementary Digital Content, Figure 2, http://links.lww.com/WNO/A677). In patient III-5, a slight temporary improvement in VA was noticed 2.5 years after the disease onset which resulted in an increase in the N95/P50 ratio (see Supplementary Digital Content, Figure 1, http://links.lww.com/WNO/A677). Over the follow-up period, significant thinning of the overall retina, RNFL, ganglion cell layer, and inner plexiform layer was found in all ETDRS quadrants (8). Interestingly, the outer nuclear layer was significantly thicker in comparison with the normal population as already reported (8, 9). Ring analysis of the RNFL around the ONH showed decreased RNFL in all quadrants (Figs. 2, 3). Genetic Analysis Genetic analysis revealed a novel homoplasmic missense change m.13042G . T (A236S) in the MT-ND5 gene, haplogroup K1a1a1 in both patients. According to the recommendations for classifying mitochondrial variants, we classified this variant as a variant of unknown significance (PM2, PM5 supporting) (10). Exome sequencing did not reveal the presence of variants associated with optic atrophy in the nuclear genes (Optic neuropathy PanelApp version 2.75). The presence of the same homoplasmic variant was confirmed in both of their mothers, unaffected carriers (Fig. 1). We could not confirm the mtDNA change in the maternal grandmother (I-2) because the patient passed away. Still, considering the maternal inheritance and homoplasmy in both daughters, it is most likely that she had the same homoplasmic mtDNA variant. The same pathogenic variant was confirmed in subject III-1 who is currently asymptomatic. Subjects II-1 and III-2 were not available for clinical evaluation and genetic analyses but were reportedly not affected. A literature search identified that alternative missense change m.13042G . A (A236T) in the MT-ND5 gene has been reported as pathogenic in several clinical presentations (Table 1 and see Supplementary Digital Content, Table S1, http://links.lww.com/WNO/A678). DISCUSSION Detailed phenotypic analysis of a Slovenian family with 3 individuals who suffered from LHON-like optic atrophy and 2 unaffected relatives is presented. The 2 affected individuals had profound visual loss at an early age (11 and 20 years) while their grandmother had a late bilateral presentation at the age 58 years. The visual loss was characterized by centrocecal scotoma, abnormal PERG N95 and VEP, and retinal nerve fiber thinning on the OCT. During the followup period, there was no improvement in visual function (VA, VF, electrophysiology, or OCT). Genetic analysis identified a novel homoplasmic mtDNA missense variant m.13042G . T (A236S) in the MT-ND5 gene of unknown significance. TABLE 1. Overview of m.13042G . A (Ala236Thr) variant and associated phenotypes reported in the literature Reference https:// doi: 10.1097/ 01.gim.0000237782. 94878.05 (van Eijsden et al, Genet Med, 2006) https://doi: 10. 1136/jmg.2005. 037507 (Valentino et al, J Med Genet, 2006) https://doi: 10. 1001/archneur.62.3. 473 (Naini et al, Arch Neurol, 2005) https://doi: 10. 1136/jmg.2006. 045716 (Blok et al, J Med Genet. 2007) Variant Phenotype Method Comment MitoChip resequencing (Affymetrix) 1 patient (variant of unknown significance) m.13042G.A (Ala236Thr) 84% heteroplasmy in muscle OXPHOS disorder m.13042G.A (Ala236Thr) 4%–41% urine (5 patients), 91% muscle (1 patient) LHON-like phenotype, mtDNA sequencing retinopathy, and cataract m.13042G.A (Ala236Thr) 90% heteroplasmy MELAS, MERRF in muscle, 50% heteroplasmy in blood 77% heteroplasmy Leigh-like syndrome in blood, 84% muscle, 86% fibroblasts m.13042G.A (Ala236Thr) m.13042G.A https://doi: 10. (Ala236Thr) 1136/jclinpath2012-200778 (Slawek et al, J Clin Pathol 2012) 344 Heteroplasmy 69% heteroplasmy Mitochondrial encephalomyopathy in muscle, 30% (more MERRF-like) heteroplasmy in blood ND5 gene sequencing DHPLC analysis of mtDNA and mtDNA sequencing mtDNA sequencing 1 Italian family (3 retinopathy, 2 LHON-like OA, retinopathy, and cataract) 1 patient 1 patient 1 patient (de novo variant) Petrovi c Paji c et al: J Neuro-Ophthalmol 2023; 43: 341-347 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution There are several lines of evidence in favor of the pathogenicity of identified variant. First, it is a missense variant at a position where a different nucleotide change was reported as pathogenic. In addition, this amino acid residue is located in a highly conserved domain across vertebrates (Conservation Score phyloP100: 9.101) of the ND5 subunit gene of complex I (11). Second, this variant is ultra-rare and was not as yet reported in the available mitochondrial DNA databases (Mitomap database http://www.mitomap.org/) and is absent in the Slovenian control population (UMC Ljubljana, Clinical Institute of Genomic Medicine, database with more than 8,000 exomes with mtDNA sequencing). Third, the variant segregates with the disease in the family (proband and patient III-5 and their mothers). Mothers were not affected, most likely because of incomplete and sexdependent penetrance typical for LHON. However, proband’s grandmother showed the clinical characteristics of LHON-like optic neuropathy (patient I-2). The same pathogenic variant was confirmed in subject III-1 who is currently asymptomatic. Furthermore, the identified variant is in a homoplasmic state which is typical for LHON pathogenic variants. Our patients’ phenotype was predominantly associated with ocular features (patients had normal MRI and no systemic changes, other than clinically insignificant aortic valve stenosis in patient III-5). An alternative pathogenic heteroplasmic variant MTND5:13042G . A (A236T) with different amino acid change at the same position and a different haplogroup (H) was reported and proven by functional studies as pathogenic (by a skeletal muscle mitochondrial dysfunction, as evidenced by the consistent increase of serum lactic acid and mitochondrial proliferation at muscle biopsy) in an Italian family with neurological and visual symptoms (3). There was a clear difference in the clinical features between our patients and the previously reported Italian family (3): The onset of visual acuity loss in the Italian family was later and was associated with good visual acuity after the cataract surgery. The most striking difference between Italian and our patients was the presence of neurological features. The Italian family had inflammatory changes in the arachnoid specimen and changes in the brain MRI which were not found in our patients. The unusual symptoms in our proband were the presence of strong headaches and flashes, which lasted for a short period of time before the visual acuity loss. Similar headaches were described in 1 family member reported by Valentino et al (3). Rarely LHON patients reported seeing flashes, colors, dizziness, headaches, Uhthoff’s phenomenon etc. (1). Our patients had no other neurological symptoms or signs. Besides the optic neuropathy, the 2 affected individuals in the Italian family and some of the unaffected carriers had cataract and retinopathy characterized by yellowish dots distributed both in the peripheral and central retina (3). Our patients had no retinopathy based on the clinical findings nor in the OCT; however, mild changes were observed in full-field ERG responses. A similar Petrovi c Paji c et al: J Neuro-Ophthalmol 2023; 43: 341-347 reduction of the cone responses was also observed in a Brazilian family with LHON (12). Cataract was not present in either our patients or their mothers. Visual evoked potentials (VEPs) and electroretinograms were normal in the Italian family, while our patients had prolonged and decreased VEP, decreased amplitude of N95 wave, and a decreased P50/N95 ratio similar to previous electrophysiology findings in LHON (12,13). Patient III-5 had decreased P50/N95 ratio already in the first weeks of the disease onset, reflecting primary ganglion cell dysfunction. Similar findings were seen in the proband as well. Our patients experienced a profound visual acuity loss at an early age (11 and 20 years) which never recovered (proband:0.005, patient III-5:0.015). The childhood onset LHON as in the case of the patient III-5 is rare (less than 10% in children younger than 12 years) (14, 15). Although Majander et al (14) reported a better outcome at early ages with the spontaneous visual recovery to $0.5 Snellen decimal in at least 1 eye in 39%, our patients’ VA did not recover. Barboni et al reported worse visual acuity outcomes in children with m.3460G . A/MT-ND1(p.A52T) pathogenic variant (15). This pathogenic variant affects the electron transfer, directly impinging on the redox activity of CI, decreasing the quinol formation and downstream electron flow and proton pumping resulting in increased ROS production (16). The mutations in ND4 and ND6 (other 2 common mutations m.11778G . A/MT-ND4 and m.14484T . C/MT-ND6) affect the proton translocation across the inner mitochondrial membrane and energy conserving efficiency but do not affect electron transfer (12). Similar to the mutations in ND1, mutations in the ND5 subunit also affect electron transport by changing the putative quinone‐reactive site on ND5 subunit (5). There are several possible explanations for such different phenotypes in the Italian family with different pathogenic variant at the same position: First, the different amino acid change might have modified the protein structure and consequently function and metabolism of the mitochondria in a less favorable way. We analyzed this effect in silico, compared the impact of the Ala236Ser variant, and compared it with the previously published pathogenic variant affecting the same residue (Ala236Thr). The analysis using EBI’s VarSite tool has demonstrated that the Ala236Ser variant is predicted the result in an amino acid substitution of an aliphatic alanine to a neutrally charged serine, with a minor anticipated change in hydrophobicity and charge (17). This is strikingly similar to the previously reported pathogenic substitution’s anticipated effect on this amino acid (3). Figure 4 A and B illustrates the position of Ala236 in the interior of MT-ND5 and flanking sequence of Ala236 from Met201 to Leu280. Ala236 is located between 2 helical structures on positions 211–233 and 241–261, respectively. The highlighted hydroxyl group could compete for hydrogen bonds with the hydroxyl group of the neighboring Ser235 or interfere with its other neighbor, 345 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution FIG. 4. Possible consequence of the Ala236Ser mutation in MT-ND5. A. Ala236 location in the interior of MT-ND5 highlighted with a red circle on the 3D structure of MT-ND5 protein (PDB: 5XTC). B. The flanking sequence of Ala236 from Met201 to Leu280. The protein chain is colored by amino acid hydrophilia (red) or hydrophobia (blue). C. The highlighted hydroxyl group could compete for hydrogen bonds with the hydroxyl group of the neighboring Ser235 or interfere with its other neighbor, Met237. D. Analogous to Ala236Thr, the Ala236Ser mutation also introduces a hydroxyl group, likely leading to similar consequences. Images were sourced from UniProt (A), generated by SWISS-MODEL (B), or are available under GNU free documentation license (C, D). analogous to Ala236Thr, the Ala236Ser mutation also introduces a hydroxyl group, likely leading to similar consequences (Fig. 4 C, D). In addition, the theoretical predictions obtained using APOGEE indicate that both variants are predicted as pathogenic (18). The second possibility is homoplasmy because in the Italian family, more serious phenotypes were also associated with higher heteroplasmy percentages (3). There is also a possibility that polymorphisms within the haplogroup changed the penetrance of the presented G13042T mtDNA pathogenic variant. In European LHON patients, haplogroups J2, J1, and K increase the risk of blindness in patients with m.11778G . A, m.14484T . C, and m.3460G . A, and haplogroup H is suggested to be a protective factor for LHON patients with m.11778G . A (19,20). Whether the haplogroups have the same influence on the variants of unknown significance remains unanswered. In conclusion, the novel homoplasmic variant m.13042G . T(A236S) in MT-ND5 gene in our family was associated with the LHON-like phenotype. However, predicting the pathogenicity of a novel ultra-rare missense variant in the mitochondrial ND5 gene is challenging. Genetic counseling should consider genotypic and pheno346 typic heterogeneity, incomplete penetrance, haplogroup type, and tissue-specific thresholds. STATEMENT OF AUTHORSHIP Conception and design: S. Petrovi c Paji c, M. Jarc-Vidmar, M. Hawlina; Acquisition of data: S. Petrovi c Paji c, M. Jarc-Vidmar, M. Su star Habjan, A. Fakin, M. Volk; Analysis and interpretation of data: S. 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