Exome sequencing identifies novel missense and deletion variants in RTN4IP1 associated with Optic Atrophy, Global Developmental Delay, Epilepsy, Ataxia, and Choreoathetosis

Abstract

Inherited optic neuropathies (IONs) are neurodegenerative disorders characterized by optic atrophy with or without extra-ocular manifestations. Optic atrophy-10 (OPA10) is an autosomal recessive ION recently reported to be caused by mutations in RTN4IP1, which encodes reticulon 4 interacting protein 1 (RTN4IP1), a mitochondrial ubiquinol oxydo-reductase. Here we report novel compound heterozygous mutations in RTN4IP1 in a male proband with developmental delay, epilepsy, optic atrophy, ataxia, and choreoathetosis. Workup was notable for transiently elevated lactate and lactate-to-pyruvate ratio, brain magnetic resonance imaging with optic atrophy and T2 signal abnormalities, and a non-diagnostic initial genetic workup, including chromosomal microarray and mitochondrial panel testing. Exome sequencing identified a paternally inherited missense variant (c.263T>G, p.Val88Gly) predicted to be deleterious and a maternally inherited deletion encompassing RTN4IP1. To our knowledge, this is the first report of a non-single nucleotide pathogenic variant associated with OPA10. This case highlights the expanding phenotypic spectrum of OPA10, the association between “syndromic” cases and severe RTN4IP1 mutations, and the importance of non-biased genetic testing, such as ES, to analyze multiple genes and variants types, in patients suspected of having genetic disease.

Introduction:

Inherited optic neuropathies (IONs) are neurodegenerative disorders characterized by optic atrophy with or without extra-ocular manifestations. Optic atrophy-10 (OPA10) with or without ataxia, developmental delay/intellectual disability, and seizures (OMIM 616732) is an autosomal recessive ION recently reported to be caused by mutations in RTN4IP1 [Angebault and others 2015], which encodes RTN4IP1, a mitochondrial ubiquinol oxydo-reductase. Here, we report a male child with optic atrophy, developmental delay, epilepsy, ataxia, and choreoathetosis. Metabolic testing from plasma, urine, and cerebrospinal fluid (CSF), chromosomal microarray (CMA), targeted sequencing, and brain magnetic resonance imaging (MRI) were non-diagnostic apart from transiently elevated lactate and lactate-to-pyruvate ratio. Exome sequencing (ES) identified a paternally inherited missense variant and a maternally inherited deletion in RTN4IP1, classified as likely pathogenic and pathogenic respectively based on the American College of Medical Genetics guidelines for variant interpretation [Richards and others 2015]. To our knowledge, this is the first case reporting a pathogenic copy number variant (CNV) in RTN4IP1, highlighting the importance of analyzing different variant classes.

Case Report:

The male proband was the product of an in vitro fertilization twin pregnancy and born at 36 weeks gestation. He was admitted to the neonatal intensive care unit for respiratory distress, required continuous positive airway pressure for one day, and was discharged home at one week on supplemental oxygen for an additional four weeks. He briefly required nasogastric tube feeds and was noted to choke on feeds. Newborn screening was normal. At 3 months he was found to have bilateral horizontal nystagmus. At 4 months he had a normal brain MRI and ophthalmology evaluation noted severe hyperopia and slow optic nerve transmission on visual evoked potential (VEP) testing. At 7 months neurology evaluation noted hypotonia, ataxia, and choreoathetosis. Plasma amino acid and urine organic acid testing were non-diagnostic. At 10 months brain MRI revealed optic atrophy, pituitary pars intermedia cyst, T2 hyperintensities in the central tegmental tracts, and mild T2 hyperintensity in the globi pallidi and parietooccipital white matter (Figure 1A). VEP testing confirmed slow optic nerve transmission; electroencephalogram (EEG), electroretinography, and auditory evoked potentials were normal.

Figure 1: Compound heterozygous RTN4IP1 variants identified in the proband.

(A) MRI Brain at 10 months: Axial T2-weighted image shows mild T2 hyperintensity in the globi pallidi (arrowheads) and parietooccipital white matter (thin arrows). (B) MRI Brain at 29 months: Axial T2-weighted image shows abnormal T2 hyperintensity and decreased size of the bilateral thalami (block arrows), T2 hyperintensity in the globi pallidi (arrowheads) and parietooccipital white matter (thin arrows), and mild sulcal prominence in the bilateral frontal and temporal lobes (curved arrows). Coronal T2-weighted image shows small prechiasmatic segments of the optic nerves (black arrows). (C) The RTN4IP1 (NM_032730) missense variant (c.263T>G, p.Val88Gly) is present in the proband (01) and his father (03) and not his mother (02). (D) The deletion (hg19 chr6:106987026-107077832) is present in the proband (blue line) and his mother (purple line) with copy number (CN) = 1 and not present in his father (green line) with CN = 2. (E) Val88 is fairly conserved across species. (F) Pedigree of the proband and his family with variants as described above. (G) 3D RTN4IP1 modeling (green with secondary structures) shows that (H) Val88 (red) is located near the NADPH binding site (blue) and forms hydrophobic interactions with Phe379 and Val382 (purple) while (I) Val88Gly (orange) removes the hydrophobic side chain and likely reduces hydrophobic interactions with the surrounding area.

He underwent a multidisciplinary evaluation in the United States. Family history was notable for a healthy fraternal twin brother, maternal hypothyroidism, and paternal gout. His exam was notable for short stature, macrocephaly, occasional horizontal nystagmus, distinctive facial features, high arched palate, widely spaced nipples, optic atrophy, ataxia, choreoathetotic movements, truncal hypotonia, intermittent appendicular hypertonia, and decreased deep tendon reflexes. He babbled but had no words. At 12 months he was able to sit unsupported; at 18 months he was starting to pull to stand and could briefly hold and pass an object between his hands. Ophthalmology evaluation noted poor vision and bilateral optic atrophy. Feeding evaluation noted oropharyngeal dysphagia and aspiration. Plasma, urine, and CSF amino acids, plasma very long chain fatty acids, urine organic acids, purine and pyrimidine panel, and 3-methylglutaconate, and CSF succinyladenosine, 5-hydroxyindoleacetic acid, homovanillic acid, 3-o-methyldopa, neopterin, tetrahydrobiopterin, and 5-methyltetrahydrofolate were non-diagnostic (Supplementary Table 1). Plasma lactate (4.5 mmol/L; reference range 0.5-2.2 mmol/L) and plasma lactate-to-pyruvate ratio (~42) were elevated, suggestive of a mitochondrial respiratory chain defect; however, these normalized on repeat. Genetic workup, including CMA and SLC2A1 and OPA3 sequencing and deletion/duplication analysis, were negative.

At 23 months, he was no longer able to sit unsupported or pull to stand and had stopped babbling, concerning for developmental regression. Ophthalmology exam noted bilateral optic nerve pallor/atrophy. Feeding evaluation noted continued aspiration risk and he had a gastrostomy tube placed. Plasma acylglycine and acylcarnitine profiles were normal. Brain MRI showed abnormal mild T2 hyperintensity in the globi pallidi and parietooccipital white matter, decreased size of both thalami (Figure 1B), and prominent adenoids, and brain magnetic resonance spectroscopy (MRS) was normal. He had his adenoids removed. Sequencing and deletion/duplication analysis of 139 genes in the “Combined Mito Genome Plus Mito Nuclear Gene Panel” (GeneDx) identified 3 heterozygous missense variants of uncertain significance (VUSs) in AARS2, AUH, and NDUFS8, all associated with AR disorders, and thus not thought to be disease causing. Mitochondrial genome sequencing and deletion testing was negative. Nerve conduction velocity studies and electromyography were normal. Global metabolomics assisted pathway screening (Baylor Genetics) was non-diagnostic.

At 4 years he developed generalized tonic-clonic seizures which evolved to focal seizures: eyes open with dilated pupils, eyelid and mouth twitching, residual left facial droop, unresponsive, lasting 30-40 seconds. EEG revealed frequent bilateral discharges initially in the right and later the left hemisphere, more prominent during sleep, and diffuse slow background (Supplementary Figure 1). He is currently having 2-3 seizures for 2-3 days 3-4 times per year, and is treated with valproic acid (43.7 mg/kg/day), levetiracetam (50 mg/kg/day), and clonazepam (0.045 mg/kg/day). Currently, he speaks 3 Spanish words, seems to understand some words, shows some appropriate social interactions, and is able to move independently by lying on his back and pushing backwards with his legs. He has not had further regression.

Methods:

A male proband and his family were enrolled in the IRB-approved protocol of the Gene Discovery Core of the Manton Center for Orphan Disease Research at Boston Children’s Hospital. ES for single nucleotide variant (SNV) and CNV analysis was performed as described previously [Edvardson and others 2017; Babadi and others 2017; https://github.com/broadinstitute/gatk-protected/blob/master/docs/CNVs/CNV-methods.pdf].

Results:

The proband was referred to the Manton Center for further evaluation. SNV analysis of ES data identified what appeared to be a homozygous missense variant in exon 1 of RTN4IP1 in the proband (hg19 chr6:107076634A>C, c.263T>G, p.Val88Gly; NM_032730) (Figure 1C), confirmed in the proband by Sanger sequencing, but seen as a heterozygous change in the father and absent in the mother. This led us to hypothesize that the proband either inherited a deletion carried by the mother or has uniparental disomy from the father. CNV analysis from exome data identified a 90.8kb maternal deletion (hg19 chr6:106987026-107077832) encompassing the entire gene (Figure 1D), consistent with deletion of RTN4IP1 inherited from the mother. The deletion includes part of AIM1, which may be associated with human melanoma, and part of QRLS1, which is associated with an AR disorder, thus not likely disease-causing although could act as modifiers. The missense variant is absent from gnomAD, EVS, and 1000 Genomes, conserved across species (variant PhyloP score 4.5 and amino acid identical or similar) (Figure 1E), and predicted to be deleterious by multiple in silico tools, including Polyphen-2, PROVEAN, SIFT, and Mutation Taster [Adzhubei and others 2010; Choi and other 2012; Genomes Project and others 2015; Karczewski and others 2020; Ng and Henikoff 2001; NHLBI Exome Variant Server; Schwarz and others 2014]. The variant was not detected in his sibling (Figure 1F). Val88 is located near the NADPH binding site and forms hydrophobic interactions with Phe379 and Val382 (Figure 1G-H). The mutated Val88Gly removes a hydrophobic side chain and likely reduces hydrophobic interactions in the surrounding area, which may lead to reduced NADPH binding (Figure 1I).

Discussion:

OPA10 is a rare AR ION caused by mutations in RTN4IP1. Fourteen mutations in 22 individuals from 17 families have been previously described [Angebault and others 2015; Charif and others 2018; Okamoto and others 2017; Zou and others 2019]. Here, we present two novel mutations in an affected male, including a heterozygous deletion encompassing the entire RTN4IP1 gene. To our knowledge, this is the first report of a pathogenic variant that is not a SNV. As summarized in Supplementary Table 2, the patients can be divided into “non-syndromic” with isolated optic atrophy and “syndromic” with other major manifestations.

Syndromic patients have been reported to present with seizures and/or developmental delay, and less commonly with ataxia, deafness, stridor, abnormal brain MRI and/or abnormal EEG. Our patient initially presented at 3 months with nystagmus, noted in 9/23 patients, and was found to have bilateral optic atrophy. He had severe developmental delay and concern for regression; developmental delay was reported in 10/23 patients, including one other patient with severe developmental delay who was also nonverbal [Charif and others 2018]. In addition, our patient had ataxia and choreoathetosis, which, to our knowledge, has not been previously reported. While the exact pathogenesis is unknown, choreoathetosis is likely due to disease in the striatum, with MRIs showing T2 hyperintensities in the globi pallidi, while ataxia may be due to cerebellar dysfunction as he exhibits jerky tremors during actionable movements. He also had hypotonia, noted in 3/23 patients. Epilepsy has been reported in 8/22 patients (unknown in one), and our patient had generalized tonic-clonic and focal seizures. His initial brain MRI in Ecuador was reportedly normal. In addition to optic atrophy, subsequent MRIs showed mild T2 hyperintensity in the globi pallidi and parietooccipital white matter and decreased thalami size. There were abnormal central tegmental tract T2 hyperintensities on the scan at 10 months, which have been reported as a physiological process that may be modified by additional factors [Aguilera-Albesa and others 2012]. Brain MRIs for previously reported patients have been varied: half had normal MRIs (8/16), one quarter had optic nerve or tract abnormalities (4/16), and the remainder had various signal abnormalities or atrophy. Many were suspected to have metabolic disorders, and the most common abnormal laboratory finding was lactate elevation, reported in 7/23 patients, including our patient.

We used ES data for SNV and CNV analysis, and identified biallelic variants in RTN4IP1 in our proband: a maternally inherited deletion and a paternally inherited missense variant. Our patient previously had fairly extensive genetic testing, including CMA, single-gene testing (SLC2A1 associated with GLUT1 deficiency and OPA3 associated with AD IONs), GeneDx “Combined Mito Genome Plus Mito Nuclear Gene Panel”, and mitochondrial genome testing. These genetic tests had been negative apart from three VUSs identified in genes associated with AR diseases, deeming him a carrier for these conditions. ES allowed us to perform a broad genetic analysis, including genes recently reported to be associated with IONs which may not have been included on predetermined panels, assessing SNVs and larger deletions/duplications.

RTN4IP1 is located on chromosome 6 and has nine exons. The protein has an N-terminal mitochondrial targeting sequence and two domains, an alcohol dehydrogenase GroES-like domain located near the N-terminus, which the paternally inherited missense variant is located in, and a zinc-binding motif located near the C-terminus [Charif and others 2018]. The majority of reported pathogenic variants lie within the two domains (11/15, not including the deletion). It has been suggested that non-syndromic cases tend to be caused by milder variants, such as those leading to conservative amino acid substitutions, while syndromic cases tend to be caused by more severe changes, such as loss of function variants [Charif and others 2018]. Indeed, all reported patients with splicing or nonsense variants have extra-ocular manifestations (9/9).

In conclusion, we report novel compound heterozygous mutations in RTN4IP1 in a male patient with optic atrophy, developmental delay, epilepsy, ataxia, choreoathetosis, and hypotonia. To our knowledge, the maternally inherited deletion encompassing the entire RTN4IP1 gene is the first structural variant associated with OPA10. This case highlights the expanding phenotypic spectrum of OPA10, the association between syndromic cases and severe RTN4IP1 variants, and the importance of considering unbiased genetic testing, such as ES, to analyze multiple genes and variant classes in patients suspected of having a genetic disease. Further, this report is an example of successful utilization of ES to determine pathogenic copy number variant/s.

Data Availability Statement:

The variants have been submitted to ClinVar (SUB7440994).