Whole Genome Sequencing Identifies a Partial Deletion of RTN4IP1 in a Patient With Isolated Optic Atrophy

Clinical Correspondence Section Editors: Robert Avery, DO Karl C. Golnik, MD Caroline Froment, MD, PhD An-Guor Wang, MD Whole Genome Sequencing Identifies a Partial Deletion of RTN4IP1 in a Patient With Isolated Optic Atrophy Neringa Jurkute, MD, FEBO, Gavin Arno, PhD, Genomics England Research Consortium, Andrew R. Webster, FRCOphth, MD(Res), Patrick Yu-Wai-Man, BMedSci, MBBS, PhD, FRCPath, FRCOphth R TN4IP1 (reticulon 4 interacting protein 1)-associated disease is an autosomal recessive mitochondrial disor- Genetics Department, Moorfields Eye Hospital NHS Foundation Trust (NJ, GA, ARW, PY-W-M), London, United Kingdom; Institute of Ophthalmology (NJ, GA, ARW, PY-W-M), University College London, London, United Kingdom; North Thames Genomic Laboratory Hub, Great Ormond Street Hospital for Children (GA), London, United Kingdom; Cambridge Eye Unit, Addenbrooke’s Hospital (PYW-M), Cambridge University Hospitals, Cambridge, United Kingdom; and John van Geest Centre for Brain Repair and MRC Mitochondrial Biology Unit (PY-W-M), Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom. N. Jurkute is supported by Moorfields Eye Charity (GR001203), National Eye Research Centre (SAC051), and National Institute of Health Research Biomedical Research Centre (NIHR-BRC) at Moorfields Eye Hospital and UCL Institute of Ophthalmology. G. Arno is supported by Moorfields Eye Charity (GR001203), National Eye Research Centre (SAC051), Fight For Sight UK Early Career Investigator Award (5045/46), National Institute of Health Research Biomedical Research Centre (NIHR-BRC) at Moorfields Eye Hospital and UCL Institute of Ophthalmology, and NIHR-BRC at Great Ormond Street Hospital Institute for Child Health. P. Yu-Wai-Man is supported by an Advanced Fellowship Award (NIHR301696) from the UK National Institute of Health Research (NIHR) and a Clinician Scientist Fellowship Award (G1002570) from the UK Medical Research Council (MRC). P. Yu-Wai-Man also receives funding from Fight for Sight (UK), the Isaac Newton Trust (UK), Moorfields Eye Charity (GR001376), the Addenbrooke’s Charitable Trust, the National Eye Research Centre (UK), the International Foundation for Optic Nerve Disease (IFOND), the NIHR as part of the Rare Diseases Translational Research Collaboration, the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014), and the NIHR Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. 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 full text and PDF versions of this article on the journal’s Web site (www. jneuro-ophthalmology.com). Genomics England Research Consortium: For this study, data were available through the 100,000 Genomes Project. To reflect staff members’ contribution to the Project, Genomics England Research Consortium should be included as an author. These guidelines are available at: https://www.genomicsengland.co.uk/about-gecip/ publications/. Supplementary file with the names is provided. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health. Address correspondence to Patrick Yu-Wai-Man, BMedSci, MBBS, PhD, FRCPath, FRCOphth, John van Geest Centre for Brain Repair and MRC Mitochondrial Biology Unit, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom; E-mail: [e-mail] e142 der caused by pathogenic variants involving both alleles (biallelic) of the RTN4IP1 gene, which encodes for the mitochondrial RTN4IP1, previously known as Nogointeracting mitochondrial protein (1). It has previously been reported that pathogenic RTN4IP1 variants can cause isolated or syndromic optic atrophy (OA, OMIM 616732) (2,3). This study reports biallelic candidate pathogenic RTN4IP1 variants identified in an individual diagnosed with early-onset isolated OA, highlighting the utility of whole genome sequencing (WGS) to identify missing alleles in previously unsolved cases. This study adhered to the tenets of the Declaration of Helsinki, and it was granted the relevant ethical and institutional approvals. The male proband from a nonconsanguineous family (Fig. 1A) was born after an uneventful pregnancy. He was found to have reduced visual acuity bilaterally at the age of 2–3 years and was subsequently diagnosed with bilateral OA. A comprehensive neuro-ophthalmological workup excluded possible acquired causes for his OA. At the age of 4 years, the patient experienced a single episode of febrile convulsion, but he was otherwise healthy with no neurological deficits. He underwent visual electrophysiology testing (visual-evoked cortical potentials and pattern electroretinogram) at the age of 9 years, which was consistent with bilateral optic nerve dysfunction. At the last clinic visit when the patient was aged 19 years, his visual acuity was 1.08 logMAR with reduced Ishihara color vision of 1/17 in both eyes. Over this 15-year follow-up period, visual acuity did not deteriorate and any transient worsening was related to a change in refractive error. The patient was myopic with progression to a moderate myopic astigmatism. Fundus examination revealed temporal optic disc pallor (Fig. 1B) that was confirmed by SPECTRALIS spectral domain optical coherence tomography (SD-OCT). SD-OCT of the optic nerve showed marked peripapillary retinal nerve fiber layer (pRNFL) thinning within the temporal quadrant (Fig. 1C). Macular scans indicated pronounced RNFL and retinal ganglion cell (RGC) layer thinning bilaterally (Fig. 1C). Previous genetic screening for the 3 most common LHON mitochondrial DNA variants, OPA1, OPA3, TNEM126A, and ACO2, did not identify any pathogenic variants. Because there was no relevant family history, an autosomal recessive Jurkute et al: J Neuro-Ophthalmol 2023; 43: e142-e145 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Clinical Correspondence FIG. 1. Pedigree and multimodal imaging of affected individual. A. Pedigree of the family (GC22844). Arrow indicates proband. M1 and M2 correspond to candidate pathogenic RTN4IP1 variants. B. Color images of fundus and the optic nerve head show temporal optic disc pallor. C. SD-OCT B-scan of the disc (left panel) indicates marked peripapillary RNFL (pRNFL) thinning within the temporal quadrant (white arrow). B-scan of the macular region (middle panel) shows thinning of RNFL (white arrow) and RGC (red arrow) layers. GCL volume thickness maps (right panel) demonstrate pronounced macular ganglion cell layer thinning in all sectors in the affected individual. Healthy control single eye (right eye) SD-OCT B-scans of both, optic disc and macular, and GCL thickness map provided in the bottom for comparison. Abbreviations: GCL, ganglion cell layer; LE, left eye; M, mutation; pRNFL, peripapillary retinal nerve fiber layer; RE, right eye; RNFL, retinal nerve fiber layer; WT, wild type. inherited optic neuropathy (ION) was suspected. To reach a molecular diagnosis, the patient and his unaffected father were recruited into the United Kingdom’s 100,000 Genomes Project. No pathogenic biallelic protein-altering genotypes were identified after the clinical interpretation pipeline. Subsequent reanalysis performed by applying the ION gene panel (https:// panelapp.genomicsengland.co.uk/panels/186/, accessed April 2019) revealed 2 rare (minor allele frequency, MAF,0.01) variants in the proband: RTN4IP1 (NM_032730.5) c.307C.T, p.(Arg103Cys) and MT-ATP6 m.9088T.C, p.(Ser188Pro), with the latter previously reported as likely benign (4). The candidate RTN4IP1 variant, c.307C.T, p.(Arg103Cys), is found in 4 alleles in the gnomAD database (v3.1, allele frequency of 0.00002629), and it has not been previously reported in the literature. The affected Jurkute et al: J Neuro-Ophthalmol 2023; 43: e142-e145 amino acid residue showed strict evolutionary conservation across orthologues (Fig. 2A). An adjacent variant (c.308G.A, p.(Arg103His)), which disrupts the same residue, has been reported in multiple unrelated families, and it is predicted to be pathogenic (see Supplemental Digital Content, Table S1, http://links.lww.com/WNO/A583). This suggests that this particular amino acid residue is clinically significant, and variants disrupting it may be pathogenic. Based on in silico analysis and the ACMG guidelines, the p.(Arg103Cys) variant was predicted to be diseasecausing and likely pathogenic, respectively. Because autosomal recessive disease was suspected and the p.(Arg103Cys) variant was present in the proband, but absent from the father’s genome, it was assumed that a second RTN4IP1 variant may have remained undetected on the paternal allele. This led to further WGS data interrogation e143 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Clinical Correspondence FIG. 2. RTN4IP1 variants identified in the study family. A. The evolutionary conservation of the affected amino acid residue across orthologues indicating strict conservation (blue box) across diverse species. B. Schematic diagram of the RTN4IP1 gene (introns not to scale) and protein (Uniprot Q8WWV3). The candidate variants identified in proband are indicated in bold red at the corresponding positions of the gene and protein. Previously reported variants are shown on the top of the bar at the corresponding exons of the gene. C. An IGV visualization of a large deletion spanning the exon 4 (chr6:106610328_106620366del) of RTN4IP1 identified in the proband and father. A candidate missense variant indicated in proband’s reads and is absent from father’s DNA sample. D. Segregation analysis and sequence electropherogram of mother’s DNA sample shows RTN4IP1 c.307C.T, p.(Arg103Cys) variant (red arrow). E. RTN4IP1 three-dimensional protein model (molecule A of a WT RTN4IP1 2VN8 template). Residue and side chain at position 103 indicated in red. Region of interest (rectangle) is zoomed in the bottom panel. The bottom left panel shows WT RTN4IP1 at the position 103. Red arrows indicate hydrogen bonds formed between Arg103 and Lys109, Leu99, Asn100, and a water-mediated ionic interaction. The bottom middle panel shows the previously reported missense p.(Arg103His) mutant RTN4IP1 model. The bottom right panel shows mutant RTN4IP1 model of a candidate missense p.(Arg103Cys) variant identified in this study. Both mutant RTN4IP1 protein models indicate loss of hydrogen bond with Asn100 and water-mediated ionic interaction. Abbreviations: ADH_N, alcohol dehydrogenase GroES-like domain; ADH_zinc, alcohol dehydrogenase with zinc-binding motif domain; NDP, NADPH dihydro-nicotinamide-adenine-dinucleotide phosphate; WT, wild type. for rare noncoding and structural variants in RTN4IP1 shared by the proband and his unaffected father. This led to the identification of a 10,038bp deletion spanning exon 4 (chr6:106610328_106620366del) (Fig. 2B, C), which is predicted to result in premature termination after a frameshift (p.(Arg207Cysfs*35)). The deletion is absent from the gnomAD database (SVs v2.1). However, another 9,989bp deletion (chr6:106610359_106620348del) spanning the same exon is present on 1 allele. Segregation analysis confirmed the p.(Arg103Cys) variant to be on the maternal allele (Fig. 2D). Three-dimensional modeling of the mutant RTN4IP1 protein showed that WT Arg103 forms hydrogen bonds with Lys109, Leu99, Asn100, and an ionic interaction with a water e144 molecule, whereas both the His103 and Cys103 mutants lose hydrogen bonds with Asn100 and disrupting the interaction with water (Fig. 2E). We hypothesize that both missense variants act similarly, affecting the protein’s structural stability and plasticity, and leading ultimately to protein dysfunction. IONs are genetically heterogeneous with pathogenic variants identified in both the mitochondrial and nuclear genomes. The eye is particularly vulnerable to mitochondrial dysfunction, and the most common ophthalmological manifestations include OA, retinopathy, and ophthalmoplegia. RTN4IP1 localizes to the mitochondrial outer membrane (1,2). It interacts with reticulon 4 (RTN4), which regulates neurite fasciculation, branching, and extension in the Jurkute et al: J Neuro-Ophthalmol 2023; 43: e142-e145 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Clinical Correspondence developing nervous system. RTN4IP1 is, therefore, a key regulator of RTN4, and dysfunction of this interaction resulted in abnormal neurite outgrowth in mouse pups, and in significant alteration in RGC numbers in mouse and zebrafish animal models (2). To date, 28 affected individuals (21 families) have been reported in the literature. Of those, 15 of 28 (53.6%) had isolated ocular disease, with the remaining 13 of 28 (46.4%) exhibiting a broad spectrum of neurological manifestations (see Supplemental Digital Content, Table S1, http://links.lww.com/WNO/ A583). Interestingly, most of the affected individuals (9 of 13, 69.2%) with syndromic disease were compound heterozygous (with 2 different pathogenic variants in RTN4IP1) and with 1 allele carrying a loss-of-function (LOF) variant. The remaining 4 of 13 syndromic cases were homozygous for missense variants. By contrast, only 1 of 15 (6.7%) individuals with isolated OA had a LOF variant. Although the p.(Arg103His) variant is believed to cause a milder isolated ocular phenotype, neurological impairment with generalized chorea, intellectual disability, and midbrain MRI abnormalities have been observed in 1 homozygote (5). To date, only 1 case has been reported with a large deletion spanning the entire gene in RTN4IP1-associated disease. In summary, we have identified a likely pathogenic biallelic RTN4IP1 genotype in a patient with isolated OA. We highlight the necessity for complete interrogation of coding, noncoding, and structural variants by WGS to identify the missing heritability in unsolved cases. Owing to the relative rarity of RTN4IP1-associated disease, establishing clear genotype–phenotype correlations remains challenging. Nevertheless, LOF alleles are seemingly enriched in syndromic disease, although the current patient did not exhibit any extraocular features at the time of examination. STATEMENT OF AUTHORSHIP Conception and design: N. Jurkute, G. Arno, P. Yu-Wai-Man. Acquisition of data: N. Jurkute, G. Arno, Genomics England Research Consortium. Analysis and interpretation of data: N. Jurkute, G. Arno, Genomics England Research Consortium, A. R Webster, P. Yu-Wai-Man. Drafting the manuscript: N. Jurkute, G. Arno, P. Yu-Wai-Man. Revising the manuscript for intellectual content: N. Jurkute, G. Arno, Genomics England Research Consortium, A. R Webster, P. Yu-Wai-Man. Final approval of the completed manuscript: N. Jurkute, G. Arno, Genomics England Research Consortium, A. R Webster, P. Yu-Wai-Man. Jurkute et al: J Neuro-Ophthalmol 2023; 43: e142-e145 ACKNOWLEDGMENTS The authors thank the family for participation in this study. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly-owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research United Kingdom, and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. REFERENCES 1. 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