A Novel Ornithine Aminotransferase Splice Site Mutation Causes Vitamin B6-Responsive Gyrate Atrophy

Abstract Purpose Gyrate atrophy of the choroid and retina (GACR) is a rare congenital disorder and mutations in the ornithine aminotransferase (OAT) gene has been specified as the underlying cause. Patients show a high level of ornithine in body fluids which may be controlled by low protein diets. Pyridoxine (vitamin B6) supplementation may also be effective, however, most patients appear to be nonresponsive to this modality of treatment. Case Report Here, we report a characterized case of a vitamin B6-responsive GACR who had a splicing mutation in the OAT gene. The GACR diagnosis was confirmed through the clinical features, imaging, biochemical findings, and whole-exome sequencing (WES) results. WES data revealed the splicing mutation in intron 4 of the OAT gene (NM_001322967: c.425-1G > A). Conclusion Our knowledge about the diagnosis and treatment of GACR can be improved by identifying novel mutations in the OAT gene and accurate follow-up of the patients to determine how they respond to treatment.


INTRODUCTION
Gyrate atrophy of the choroid and retina (GACR-OMIM: 258870) is a rare autosomal recessive ophthalmic disorder with the highest incidence in Finland. [1]GACR is a phenotypically variable disorder with inconsistent age of onset (childhood-40s).Night blindness and constriction of the visual field which are caused by multiple round areas of chorioretinal atrophy in the periphery are the first complaints noticed in patients.Progression of the atrophic areas leads to central visual loss after 40 years of age. [2]Ocular symptoms are the major manifestation in most GACR patients; however, type II fibers atrophy, muscle weakness, intellectual disability, neonatal hyperammonemia, and peripheral nervous system abnormalities have also been reported in some cases. [3,4] CR is caused by homozygous or compound heterozygous mutations in the OAT gene. [5]he OAT gene, which consists of 11 exons, encodes the mitochondrial enzyme ornithine aminotransferase which is a key enzyme in the pathway that converts arginine and ornithine into the neurotransmitters glutamate and Gamma-aminobutyric acid (GABA); where vitamin B6 works as a co-factor for the enzyme. [6]AT enzyme deficiency caused by OAT gene mutations leads to significant elevation of ornithine concentration in plasma (about 10 fold) and other body fluids.Long-term exposure to high concentrations of ornithine in the blood causes retinal toxicity in patients along with gyrate atrophy of the choroid and retina.[2,6] Introduction of a low-protein diet and vitamin B6 therapy can slow the progression of chorioretinal degeneration and both have been recommended as treatment for all newly diagnosed patients with gyrate atrophy.[7][8][9][10] Molecular analysis for the detection of OAT mutations is recommended for accurate diagnosis of disease and determination of the vitamin B6responsive variations.
Here, we present an Iranian male patient diagnosed with GACR.Whole-exome sequencing (WES) detected a splicing mutation, c.425-1G>A in the OAT gene.In addition, we have summarized the entire list of reported OAT mutations that correlate with GACR patients as a literature review.

CASE REPORT
The patient was a 24-year-old Mazandarani male from a healthy and non-consanguineous family.He signed the consent form and allowed the publishing of the results of his examination and tests.The Ethical Committee of the Shahid Beheshti University of Medical Sciences approved all study procedures.The patient presented with gradual visual loss, which was first noted when he was five years old.He also had a history of decreased night vision since the age of 15.His best-corrected visual acuity in each eye was 4/10.The refractive error was -13.5-0.75 × 50 for his right eye and -13.5-1.00 ×150 for his left eye.Anterior segment examination showed mild posterior subcapsular cataracts in both eyes.Fundus examination of both eyes revealed sharply demarcated areas of the choroidal and retinal atrophy in gyrate shape involving the mid periphery, however, the macula was not affected [Figure 1].An electroretinogram demonstrated a reduction of both cone and rod responses.
Furthermore, biochemical tests showed elevation of plasma ornithine levels (1463.2micmol/L; Normal range: 27-83 micmol/L).Introduction of a low protein diet and vitamin B6 therapy significantly decreased the amount of ornithine to 818.8 micmol/L after three months.DNA sequencing revealed a mutation in the OAT gene and confirmed the ophthalmological examination, imaging, and biochemical test findings for gyrate atrophy diagnosis.

DNA Sequencing and Analyses
DNA was extracted from peripheral blood of the patient and his parents using the saltingout method.After quality and concentration assessment of the DNA samples, they were subjected to WES.WES enrichment was performed using an Agilent Sure Select V6 Target Enrichment Kit, and the library was sequenced on the Illumina Hiseq 4000 platform, performed by Macrogen, South Korea.All exons and flanking 10 bps were detected and analyzed.Then, rare variants (MAF < 0.01) were analyzed in silico for pathogenicity predicting using predictor tools databases.The final assessment from WES data revealed a homozygote splicing mutation in the last nucleotide of the intron 4 of the OAT gene (NM_001322967: c.425-1G>A).In addition, the Iranome database was accessed as a local reference for exome variants (http://www.iranome.ir/).The mutation was predicted to be pathogenic and damaging according to different prediction tools such as DANN score, mutation taster, FATHMM, SIFT, PROVEN, and REVEL database.
To confirm the WES result, primer was designed to amplify a short sequence containing the targeted mutation using PCR.Then Sanger sequencing was performed in both forward and reverse directions for the amplified sequence.Also, the patient's parents were investigated and both were heterozygous for the mentioned mutation [Figure 2].

Search Method of Literature Review
In this study, a review in genotype and phenotype of previously reported GACR cases was conducted up to June 2020, which is summarized in Table 1.We used PubMed (https://pubmed.ncbi.nlm.nih.gov) and Google Scholar (http://scholar.google.com)databases to search published papers and abstracts using the following keywords: OAT mutation, Gyrate Atrophy, and GACR, and also Google using keywords: OAT OMIM, Clinvar OAT, and LOVD OAT.Only studies published in English were included.The following information was extracted from each article: Nucleotide change, Amino acid change, Mutated exon/intron, Mutation type, Zygosity status, Age of onset (years), Clinical symptoms, Plasma ornithine levels (µmol/L), Response to vitamin B6 therapy and origin of reported cases.This table included missense, frameshift, nonsense, and splicing mutations, however, large structural indels were excluded.

DISCUSSION
GACR is a rare metabolic disorder due to ornithine aminotransferase enzyme deficiency, which in turn results in progressive vision loss, myopia, cataracts, and night blindness in patients. [2]As a result of the common symptoms that exist between GACR and retinitis pigmentosa (RP), misdiagnosis is probable.Therefore, molecular and biochemical analysis could help in the differential diagnosis. [11]OAT is a homohexameric enzyme which has a vital role in proline and GABA synthesis from ornithine and arginine. [12]The OAT enzyme deficiency caused by different mutations in the OAT gene leads to hyperornithinemia and chorioretinal degeneration.
To date, about 80 mutations have been reported in the OAT gene.Distribution of the mutations in the OAT protein is shown in Figure 3.We reviewed the literature to investigate and compare the reported mutations, the resulting phenotypes, and their response to vitamin B6 therapy [Table 1].Gradual vision loss, night blindness, and early-onset cataract were clinically diagnosed in our patient.Hyperornithinemia was the early laboratory finding in the patient and genetic study confirmed the GACR diagnosis.Hyperornithinemia was significantly decreased by treating with a low protein diet and vitamin B6 intake as a supplement.
According to the literature, only a few GACR patients have been recognized to be responsive to vitamin B6 therapy. [13,14] enotype-phenotype correlation for vitamin B6-responsive patients has not yet been determined.Here, we described a B6responsive case of GACR in the Iranian population.Low protein diet and vitamin B6 as a medical supplement led to a 44% reduction in plasma ornithine levels after three months and decreased adverse ocular changes in the long term.
The patient had a homozygote splice mutation in the OAT gene in the 3′ splice acceptor site (AG) of intron 4 (c.425-1G>A).[18][19][20][21] Splice site mutations can disrupt the binding of splicing enhancers, silencers, and spliceosome elements and lead to exon skipping.They primarily result in an aberrant transcript and a truncated protein. [22] 9-bp deletion (c.425-4_429del) spanning 3′acceptor of exon 5 of OAT has been reported in a Danish/Swedish GACR patient in a study by McClatchey et al. [23] This mutation resulted in the exon 5 skipping without any disruption in the    Table 1.reading frame.An A to G substitution at the 3′ splice acceptor site of intron 4 (c.425-2A>G) was reported in the OAT gene in a study by Mashima et  al.They also identified the exon 5 skipping in the mRNA in another study.[17] Therefore, the mutation in our patient (c.425-1G>A) probably caused the same result, which is exon 5 skipping, loss of 32 amino acid residues and generating a truncated OAT enzyme.The truncated enzyme is possibly an inactive one that is not functional in the ornithine metabolic pathway and therefore has led to the GACR phenotype in our patient.The mutation's location and its vicinity has been reported for other mutations in previous studies.3,34] Kim et al reported the c.425G>A mutation in a pair of 19-year-old Korean female identical twins whose clinical manifestations were consistent with GACR.These two patients were also responsive to B6 therapy.[19] Early diagnosis of patients with GACR, specifically those who respond positively to a low protein diet and vitamin B6 supplement intake, can significantly help in the successful treatment of the condition.Therefore, identifying OAT mutations that are responsive to treatment and determination of the respective genotype-phenotype correlation in GACR patients is essential.

Figure 3 .
Figure 3. Distribution of the OAT gene mutations on OAT protein.This figure shows OAT protein domains and distribution and numbers of mutations in OAT domains according to their type.Aminotran_3 domain is shown as the main domain of OAT enzyme.Mutation diagram circles are colored with respect to the corresponding mutation types.In the case of different mutation types at a single position, the circle's color is determined with respect to the most frequent mutation type.Mutation types and corresponding color codes are as follows: Missense mutations (Green), truncating mutations: Nonsense, nonstop, frameshift deletion, frameshift insertion, splice site (Black), inframe mutations: Inframe deletion, inframe insertion (Brown), and splice mutations (Orange).The location of the detected mutation in our study is a site for five previously reported mutations, as shown in this figure.