Genetic and Phenotypic Landscape of PRPH2-Associated Retinal Dystrophy in Japan

Peripherin-2 (PRPH2) is one of the causative genes of inherited retinal dystrophy. While the gene is relatively common in Caucasians, reports from Asian ethnicities are limited. In the present study, we report 40 Japanese patients from 30 families with PRPH2-associated retinal dystrophy. We identified 17 distinct pathogenic or likely pathogenic variants using next-generation sequencing. Variants p.R142W and p.V200E were relatively common in the cohort. The age of onset was generally in the 40’s; however, some patients had earlier onset (age: 5 years). Visual acuity of the patients ranged from hand motion to 1.5 (Snellen equivalent 20/13). The patients showed variable phenotypes such as retinitis pigmentosa, cone-rod dystrophy, and macular dystrophy. Additionally, intrafamilial phenotypic variability was observed. Choroidal neovascularization was observed in three eyes of two patients with retinitis pigmentosa. The results demonstrate the genotypic and phenotypic variations of the disease in the Asian cohort.


Introduction
Inherited retinal dystrophy (IRD) refers to a group of diseases characterized by progressive retinal cell death, particularly photoreceptor cell death, caused by genetic mutations. More than 300 causative genes have been reported to date, with a considerable overlap [1]. Recently, gene therapy has become available to patients with pathogenic variants of a specific gene, and other trials are ongoing [2]. Thus, identifying causative genes is becoming increasingly important.
Peripherin-2 (PRPH2; online Mendelian inheritance in man ID: 179605, https://www. omim.org/ accessed on 17 November 2021) is one of the causative genes of IRD. The gene is located on chromosome 6p21.2 and contains three exons. The gene was also called retinal degeneration slow (RDS) because the ortholog was found in a classic animal model, rds mice [3]. PRPH2 encodes PRPH2 protein, which consists of 346 amino acids and is essential for the proper outer segment formation and maintenance of outer segment disc alignment, both in rod and cone photoreceptors [4]. The gene is generally associated with an autosomal-dominant inheritance pattern, but autosomal recessive [5] and digenic patterns in conjunction with retinal outer segment membrane protein 1 (ROM1) has also been reported [6,7]. Pathogenic variants of PRPH2 may cause diverse phenotypes such as retinitis pigmentosa (RP), retinitis punctata albescens, cone/cone-rod dystrophy (CRD), and macular dystrophies (MD) [3,[8][9][10]. Variable phenotypes were observed in a single family sharing the same variant [11][12][13]. The presence of ROM1 variants may modify these phenotypic appearances [14] or increase the severity of the disease [15].
Pathogenic variants of PRPH2 are one of the major causes of IRD. It has been reported that 5.2% of patients with IRD are associated with PRPH2 in the United Kingdom [13] and 3.9% of patients with RP are associated with PRPH2 in Spain [16]. The prevalence is particularly high in patients with autosomal dominant CRD or MD; 12% in CRD/MD [17], 19.5% in autosomal dominant CRD/MD [18], and 10.3% in autosomal dominant RP were associated with PRPH2 [19], respectively.
Meanwhile, the prevalence of PRPH2 as a causative gene of IRD in Asian populations is relatively low. The prevalence of PRPH2 as a causative gene of RP is 0.06% in China [20], 1.6% in Korea [21], and 0-3.4% in Japan [22][23][24]. Moreover, the prevalence of PRPH2 as a causative gene of CRD or MD is 2.3-6.1% [25,26]. Thus, little is known about the genetic and phenotypic spectrum of PRPH2-associated IRD in Asia.
In this multicenter joint study, we recruited patients with PRPH2-associated IRD from all over Japan and reported their phenotypic and genotypic characteristics.

Materials and Methods
This study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committees of the participating institutions in Japan (National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center [Reference: R18-029] and Kyoto University Graduate School of Medicine [Reference: G0746]). All patients who participated in the study provided written informed consent.

Clinical Examinations
All patients underwent comprehensive ophthalmological examination, including best-corrected visual acuity (BCVA) measurement, slit-lamp ophthalmoscopy, fundus photography, fundus autofluorescence imaging, optical coherence tomography (OCT), visual field test, electroretinogram (ERG), and electrooculogram (EOG), if available. ERG and EOG were recorded in accordance with the standards of the International Society for Clinical Electrophysiology of Vision [27][28][29]. Clinical diagnosis was made at each institution and reviewed by the consortium. In the present study, phenotype subgroups were defined based on clinical manifestations reported in a previous study. RP was defined as a progressive retinal dystrophy initially often presenting peripheral atrophy with generalized rod dysfunction greater than cone dysfunction. CRD was defined as a progressive retinal dystrophy initially often presenting macular atrophy with generalized cone dysfunction greater than rod dysfunction. MD was defined as a progressive retinal dystrophy presenting macular atrophy with confined macular dysfunction despite no abnormalities of generalized cone and rod functions [30].
In addition to the phenotype subgroups, we investigated the presence of clinical factors such as macular atrophy, peripheral atrophy, Best disease-like deposits, and multiple flecks on retinal imaging because some patients showed overlapping phenotypes and clinical diagnosis may obscure the characteristics (Figure 1).
We obtained family history and assumed the mode of inheritance as autosomal dominant if two generations or more were affected; autosomal recessive if there was parental consanguinity or siblings from normal parents were affected; X-linked if the diseased occurred in multiple generations but without male-to-male transmission and only males were affected.

Genetic Screening
While this study focused on PRPH2, the screening was conducted as a part of comprehensive genetic screening of patients with IRD. Genomic DNA was extracted from peripheral blood samples and analyzed using next-generation sequencer as previously reported. [22,[30][31][32][33]. Briefly, we employed targeted exome sequencing for case series of 2014 [22], whole genome sequencing for Kyoto University cases after 2014 [32], and whole exome sequencing for the other participants. Subsequently, target analysis of retinal disease-associated genes was performed. We analyzed all the variants in exons and their boundaries (±2 bp) that were detected on the genes registered in Retinal Information Network (RetNet; Available online: https://sph.uth.edu/retnet/ (accessed on 1 September 2021)). The identified variants were filtered based on the allele frequency in the Human Genetic Variation database (HGVD, a database of allele frequency in the general Japanese cohort; Available online: http://www.hgvd.genome.med. kyoto-u.ac.jp/ (accessed on 1 September 2021), Genome Aggregation Database (gnomAD; Available online: https://gnomad.broadinstitute.org/), and 1000 Genomes (Available online: http://www.internationalgenome.org/ (accessed on 1 September 2021)). Missense variants were evaluated using seven in silico prediction programs: MutationTaster  [34]. Candidate variants were confirmed by Sanger sequencing in the index patient and their family members, if possible.

Statistical Analysis
Decimal visual acuity was converted to logarithm of the minimum angle of resolution (logMAR) for statistical analysis. Counting finger and hand motion were regarded as log-MAR 2.0 and 2.3, respectively [35]. Comparisons between the two groups were performed using the Mann-Whitney U test or chi-square test, as appropriate. Associations between the clinical factors were assessed using the Spearman's rank correlation test. All statistical analyses were performed using IBM SPSS Statistics 26 (IBM Japan, Tokyo, Japan).

Results
A total of 40 patients from 30 families with 17 distinct PRPH2 variants were identified. Details of the patients are summarized in Table 1. Some cases have been reported previously [36,37]. Twenty patients were men and 20 were women. Based on comprehensive examinations, patients were phenotypically classified into the RP (n = 16), CRD (n = 7), and MD (n = 17) subgroups. Among patients with MD, four had Best disease-like deposits, and seven had Stargardt disease or pattern dystrophy-like multiple flecks. Three of the four patients with Best disease-like deposits had subnormal EOG. Seven of 16 patients with RP had macular atrophy in addition to typical peripheral atrophy. Meanwhile, two patients with CRD and one patient with MD had peripheral atrophy. Common primary complaints were reduced visual acuity or central visual field loss (16 patients, 40%), night blindness (12 patients, 30%), photophobia (4 patients, 10%), and peripheral visual field loss (1 patient, 2.5%). Three (7.5%) patients had no symptoms at the time of diagnosis.
Most patients (from 16 families) had autosomal dominant inheritance of PRPH2associated retinal dystrophy, whereas 11 patients had sporadic disease. We could not determine the inheritance pattern in one patient. The pedigree charts of two patients were not available. Some discrepancies in the phenotype subgroups within families were noted. Illustrative cases are presented in Figure 2. An 88-year-old woman had patterndystrophy-like MD and her 61-year-old daughter had RP. The mother showed a lower limit of normal range but still recordable rod and cone responses in ERG, the daughter showed a non-recordable rod and barely recordable cone responses.   The age of the patients ranged from 28 to 88 years. The age of onset was generally in the 40 s; however, some patients had the onset as early as 5 years of age. No association was found between the age of onset and sex or visual acuity; however, patients with RP tended to develop symptoms earlier than patients with CRD or MD (31.2 vs. 40.9 years, p = 0.161).
Visual acuity of the patients ranged from hand motion (logMAR equivalent 2.3) to 1.5 (Snellen equivalent 20/13, logMAR equivalent −0.18). No significant difference in BCVA was noted among patients with RP, those with CRD, and those with MD. As expected from the irreversible and progressive nature of the disease, BCVA tended to be worse in elderly patients (Spearman's correlation = 0.36, p = 0.22).
The data of the detected variants are presented in Tables 2 and 3. Eleven variants were previously reported, whereas six were novel. Twelve variants were missense, two were splice site, one was a frameshift, one was a stop gain, and one was an in-frame deletion. The locations of the variants in the amino acid sequence are illustrated in Figure 4. All missense variants were located in the D2 loop of the protein. None of the cases had likely pathogenic or pathogenic variants in ROM1.  [3]. Sequence is based on NP_000313.2). Missense or in-frame deletions are shown as colored residues and premature termination and frameshift variants are shown as colored bars. When a locus is associated with various phenotype, they were indicated by mixed colors e.g., red + yellow = orange, yellow + blue = green, red + blue = purple, and red + yellow + blue = brown.

Discussion
Here, we present the clinical and genetic features of 40 patients with PRPH2-associated retinal dystrophy. To the best of our knowledge, this is the largest cohort among Asian ethnicities. This study confirmed phenotypic variability, even within the same family. The present data provide novel insights into genotype-phenotype associations in East Asian ethnicity.
Previous reports have suggested association between the location of the variant in PRPH2 and clinical phenotype. Specifically, variants are mostly found in the D2 loop [13,43,[46][47][48], which is critical for protein-protein interactions. Variants that cause autosomal dominant RP tend to accumulate between Lys193 and Glu226 [3]. Particularly, missense mutations in Pro210 to Pro216 cause autosomal dominant RP [3]. Patients diagnosed with CRD, RP, and STGD tend to have variants in exon 1, whereas patients with Best disease and pattern dystrophy tend to have variants in exon 2 [13,43]. Patients with p.Arg172Trp present earlier onset than those with other alleles [43]. Some of these findings are compatible with the findings of the present cohort; 14 of 17 (82.3%) identified variants were in the D2 loop. Six of 16 patients (from two of ten families) with RP had variants between Lys193 and Glu226. The Best disease-or pattern dystrophy-like deposits were found in 10 patients. Five of these 10 patients had variants in exon 2. However, the age of onset in four patients with p.Arg172Trp was 45-60 years. The results reveal the difficulty in establishing a clear genotype-phenotype correlation.
CNV was relatively common (2/40, 5%) in our cohort. CNV occasionally occurs in IRD, but is not a common complication, especially in RP [59]. One of the patients was considered to have myopic CNV. The other patient might have been complicated with neovascular age-related macular degeneration (AMD), but drusen, a hallmark of AMD, was not evident. We assumed that the patient developed dystrophy-associated CNV. The development of CNV is generally discussed in association with pattern dystrophy [3,60]; however, it can be seen in the RP phenotype as previously reported [61]. Considering that anti-vascular endothelial growth factor therapy is an effective treatment for CNV. Patients should be advised to visit the eye clinic when they notice acute vision loss and/or metamorphopsia.
This study has some limitations. First, the prevalence of PRPH2-associated dystrophy could not be determined. Each institution recruited patients independently and the criteria to proceed to genetic testing may have been different in each institution. Patients with a dominant inheritance pattern may be more willing to undergo genetic testing. Nevertheless, 40 cases were sufficient to determine the phenotypic variability within the cohort. Second, the pathogenicity of each variant is based on the standard criteria but not on solid biological evidence. Although we systematically applied ACMG guidelines and graded all identified variants as pathogenic or likely pathogenic, there is still a chance that some of these variants are bystanders and pathogenic variants are present in other loci or genes. Finally, we did not intensively investigate the disease modifying effect of ROM1 variants. While none of the patients had likely pathogenic or pathogenic variants in ROM1, variants filtered out or beyond the target lesion may modify the phenotype.

Conclusions
We analyzed 40 Japanese patients with PRPH2-associated retinal dystrophy and confirmed the genotypic and phenotypic variations of the disease in the Japanese population. Further studies involving multiple ethnicities would enhance our understanding of the disease.