December 2024
Volume 65, Issue 14
Open Access
Retina  |   December 2024
Clinical and Imaging Characteristics of PRPH2 Retinopathies in a Longitudinal Cohort and Diagnostic Implications
Author Affiliations & Notes
  • Johanna M. Seddon
    Department of Ophthalmology and Visual Sciences, University of Massachusetts Chan Medical School, Worcester, Massachusetts, United States
  • Dikha De
    Department of Ophthalmology and Visual Sciences, University of Massachusetts Chan Medical School, Worcester, Massachusetts, United States
  • Laura Grunenkovaite
    Department of Ophthalmology and Visual Sciences, University of Massachusetts Chan Medical School, Worcester, Massachusetts, United States
  • Daniela Ferrara
    Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States
    Roche Personalized Healthcare, Genentech, Inc., South San Francisco, California, United States
  • Correspondence: Johanna M. Seddon, Department of Ophthalmology and Visual Sciences, University of Massachusetts Chan Medical School, 281 Lincoln St., Worcester, MA 01605, USA; [email protected]
Investigative Ophthalmology & Visual Science December 2024, Vol.65, 31. doi:https://doi.org/10.1167/iovs.65.14.31
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      Johanna M. Seddon, Dikha De, Laura Grunenkovaite, Daniela Ferrara; Clinical and Imaging Characteristics of PRPH2 Retinopathies in a Longitudinal Cohort and Diagnostic Implications. Invest. Ophthalmol. Vis. Sci. 2024;65(14):31. https://doi.org/10.1167/iovs.65.14.31.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The purpose of this study was to define genotypic-phenotypic correlations related to PRPH2-associated retinopathies in an observational longitudinal cohort and to improve diagnostic accuracy.

Methods: Individuals with PRPH2 variants were identified by genetic sequencing of 263 individuals (including 59 families). Ocular examinations with multimodal imaging were evaluated.

Results: Two pathogenic/likely pathogenic PRPH2 variants were identified in 22 individuals with retinopathies, low genetic susceptibility to age-related macular degeneration (AMD) and younger age of onset. The mean follow-up was 14 years. One family and 4 independent cases (n = 7) were heterozygous for the variant rs121918563 L185P (p.Leu185Pro). The individuals developed retinopathy compatible with autosomal dominant pattern dystrophy (PD), including adult-onset vitelliform macular dystrophy and butterfly macular dystrophy in their fourth to fifth decades of life, evolving to retinal pigment epithelial (RPE) irregularities and central macular atrophy 20 years later. Two families and an independent case (n = 15) had the rs281865373 splice-site variant c.828+3A>T (IVS2+3A>T) presenting as retinal flecks consistent with adult-onset fundus flavimaculatus with macular dystrophy and diffuse RPE atrophy consistent with central areolar chorioretinal dystrophy (CACD) in the fifth decade of life progressing to extensive atrophy in the sixth to eighth decades. The L185P variant was associated with better visual acuity (VA) during follow-up versus c.828+3A>T variant. Some individuals were initially misdiagnosed with geographic atrophy secondary to AMD.

Conclusions: Individuals with the L185P variant had less severe disease with clinical manifestation typical of PD and better VA. More advanced disease with CACD and worse VA were associated with the c.828+3A>T variant. Results contribute to knowledge about genotypic-phenotypic associations of PRPH2 retinopathies and inform clinical and therapeutic end points.

Diagnosis of retinal and macular degenerations or dystrophies can be clinically challenging due to overlapping or atypical phenotypes, especially in the late stages of the disease. Late onset Stargardt disease with macular atrophy and geographic atrophy (GA) secondary to age-related macular degeneration (AMD) may have similar clinical presentations, but retinal flecks that may be present in Stargardt disease are not a typical feature in AMD. Pattern dystrophies (PDs) of the retinal pigment epithelium (RPE) with macular pigmentary abnormalities1,2 may resemble AMD, but various other forms of RPE abnormalities that occur in PD might deviate from AMD diagnostic criteria. Retinal atrophy that is not confined to the macula and extends beyond the vascular arcades may also suggest a diagnosis other than AMD. Nonetheless, some cases of these retinopathies are misdiagnosed as AMD and may be inadvertently included in clinical studies specific for AMD. Utilizing multimodal retinal imaging beyond color fundus photography (CFP) can be helpful to distinguish characteristic features that are relevant for the differential diagnosis of these conditions, including fundus autofluorescence (FAF), near infrared reflectance (IR), and optical coherence tomography (OCT) imaging. Family history, age of onset, and genetic sequencing may also aid in the differential diagnosis of these conditions. 
In this study, we selected individuals from our longitudinal database who presented with macular abnormalities in the clinical spectrum of differential diagnosis of AMD, but were likely to carry genetic variants other than those related to AMD due to their low genetic susceptibility to AMD and/or relatively younger age of onset.37 We investigated genetic variants associated with retinopathies using genetic sequencing methods and identified two peripherin-2 gene (PRPH2) or retina degeneration slow (RDS) OMIM #179605 variants in affected families and independent cases.8,9 We present herein longitudinal results for a missense and splice-site PRPH2 variant in three families and five independent cases using whole exome sequencing, multimodal imaging, and visual acuity scores, and describe the genotypic-phenotypic correlations which may further improve diagnostic accuracy. Preliminary results were presented at the Association for Research in Vision and Ophthalmology meeting in 201710 and the American Academy of Ophthalmology meeting in 2018. 
Methods
Study Population and Subject Selection
All participants were enrolled in ongoing genetic and epidemiologic studies of AMD and retinal dystrophies, and were followed prospectively to determine the course of disease and visual outcomes (the Seddon Longitudinal Cohort Study or SLCS).11 From the SLCS database, probands and family members were selected if at least one family member had low polygenic risk scores for AMD, as determined by a composite of genes known to predict AMD.7 This suggested that the clinical phenotypes could be unrelated to AMD and possibly due to other genetic variants.37,12,13 Other inclusion criteria included macular disease that did not conform to typical AMD presentation in at least one family member, such as relatively younger age of onset compared to AMD. Probands and family members were excluded if affected relatives carried known rare variants related to AMD, as previously reported, including CFH R1210C, CFH R53G, CFH D90G, CFH P503A, and C3 K155Q and C9 P167S.5,6,14 Based on these selection criteria, 263 individuals including 59 families and unrelated individuals were selected for whole exome sequencing (WES). 
The research protocol received approval from institutional review boards and all research adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants. 
Ocular Examinations and Phenotyping
Participants underwent comprehensive ocular examinations conducted by board-certified ophthalmologists. These included a funduscopic examination, CFP, FAF, IR, fluorescein angiography (FA), OCT, and OCT angiography (OCTA) with Heidelberg Spectralis (Heidelberg Engineering, Heidelberg, Germany), Zeiss Cirrus (Carl Zeiss Meditec AG, Jena, Germany), and/or Optovue RTVue XR Avanti (Optovue Inc., Fremont, CA, USA). Expert retina specialists and image graders (authors J.M.S. and D.F.) evaluated multimodal retinal images to determine the presence, location, and overall aspect of the following imaging biomarkers: drusen or lipofuscin deposition; RPE abnormalities; RPE, retinal and choroidal atrophy; and any signs of macular neovascularization (NV). Eyes were classified according to the Clinical Age-Related Maculopathy Staging (CARMS) system at the time of enrollment and subsequent follow-up in SLCS, based on size of drusen and pigmentary abnormalities: 1 = no AMD; 2 = early AMD, or 3 = intermediate AMD; 4 = central or non-central atrophy corresponding to complete RPE and outer retinal atrophy; and 5 = retinal and subretinal NV disease.15 Macular signs not typical of AMD, such as retinal degenerations and macular dystrophies, were classified as grade 6. 
Genetic Sample Preparation and Whole Exome Sequencing
Genomic DNA was extracted from the blood of all individuals using a standard protocol. WES was performed.5,6 The exomic sequence was targeted following the SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library 6.1 protocol from Agilent. Following the exome library preparation, the samples were sequenced using the Illumina HiSeq2000 Sequencing System. The sequenced samples had an average of 96.6% of the exome covered at ≥ 10 times. 
Read Mapping, Variant Detection, and Annotation
Following deconvolution of barcodes from each lane, individual reads were aligned to the human reference genome (hg19) using the Burrows-Wheeler Aligner, resulting in BAM files.16 Variant calling was performed using the best recommendations from the tools present in the Genome Analysis Toolkit (GATK) version 3.1 suite.1719 For each sample, distinct genomic variant call format files (gVCF) containing variant calls for all loci were created using the HaplotypeCaller tool. The gVCFs were combined, and joint genotyping was performed across individuals using the GenotypeGVCFs tool. The resulting raw genotype calls in the VCF file were filtered for low-quality genotypes using the Variant Quality Score Recalibration tool. Other than high-quality variants assigned “PASS” by variant quality score recalibration (VQSR), we also included only those variants in lower tranches with truth sensitivity between 99.0 and 100 that were also separately recorded in the exome sequencing project database of 6500 samples.20 After filtering, 598,065 high-quality variants were identified. Functional effects for the variants were annotated using the Ensembl Variant Effect Predictor.21 
Variant Classification
To screen for rare genetic variants that segregated with macular diseases within these families, we used the xBrowse (https://xbrowse.broadinstitute.org) tool to filter variants in a stepwise manner. We prioritized variants that fulfilled the following criteria: (1) were rare (minor allele frequency [MAF] < 0.1%) in the 1000 Genomes Project and the ExAC databases), (2) belonged to a potentially damaging functional annotation class (i.e. nonsense, missense, splice site, and frameshift), (3) were predicted to impact protein function (i.e. in silico prediction of damaging or deleterious by PolyPhen-2,22 SIFT,23 MutationTaster2,24 FATHMM,25 and Human Splicing Finder26 depending on the type of mutation), and (4) followed the inheritance pattern of disease within each family. In some cases, we performed Sanger sequencing or targeted sequencing after identifying potential causative variants in the WES, as previously described.5,6,14 
Targeted Sequencing
A custom SureSelectXT Kit was used to capture genomic sequences of coding exons, splice junctions, 5′ UTR, and 3′ UTR regions in the selected genes with indexing barcodes for each sample. Hybridized library fragments were isolated, quantitated by qPCR and sequenced as paired-end reads with the Illumina HiSeq2000 sequencing platform. We required sequencing data for each sample to have over 10× coverage at greater than 90% targeted regions and over 20× coverage at greater than 80% targeted regions. Sequences were aligned to the human reference genome (NCBI build 37.3, hg19) with BWA (version 0.59).16 We called the consensus genotypes in the target regions with GATK version 2.18 with the workflow and parameters recommended in the best practice variant detection with GATK version 4.17,27 We applied GATK duplicate removal, indel realignment, base quality score recalibration, and performed multi-sample SNP and indel discovery and genotyping across all samples simultaneously using VQSR. Other than high-quality variants assigned “PASS” by VQSR, we also included only those variants in lower tranches with truth sensitivity between 99.0 and 100 that were also separately recorded in the exome sequencing project database of 6500 samples.20 Variant annotation was performed with snpEff (version 2.05).28 We further excluded SNPs failing the Hardy-Weinberg test in controls (P < 10−6) and alleles that had high missing genotype data (>1%), likely due to systematic low coverage or difficulty mapping reads across many samples. We also excluded samples with high missing genotype data (>1%) for common alleles with >1% frequency in our data set. For burden testing, we only tested those genes on autosomes that obtained >10× coverage at an average of >90% of the targeted region and had rare coding variants. 
Assessment of Other Known Pathogenic Variants of Retinal Dystrophies
To confirm that the disease observed in these individuals was not the result of another known variant associated with retinal dystrophies, we checked for the presence of risk alleles for known pathogenic and rare novel variants in BEST1, ABCA4, CTNNA1, IMPG1, and IMPG2. This was done using the exome and targeted sequencing data. Although none of the individuals in this study were clinically diagnosed with retinitis pigmentosa (RP), we also checked for the presence of ROM1 Gly80 (1-base pair insertions) and ROM1 Leu114 (1-base pair insertions) known to be inherited with the PRPH2 L185P variant in patients affected with digenic RP.8 
Visual Acuity Analyses
Best corrected visual acuity (BCVA) scores were extracted from medical records during the clinical course of the disease and was converted to logarithm of the minimal angle of resolution (logMAR). Distributions of initial and final visual acuity (logMAR) for each genetic variant were plotted where the width of each violin plot is proportional to the density for the range of values. For the longitudinal analysis, we ran mixed effects linear regression, with a compound symmetry correlation structure to account for the correlation between fellow eyes to assess the rate of change of visual acuity (logMAR) over time (1 line of Snellen vision = 0.1 logMAR). The regression model included time (years), genetic variant, and the cross product of time × genetic variants. For this analysis, we censored an eye if it reached BCVA of counting fingers or worse at a particular clinical visit. We calculated the change in visual acuity (logMAR) per year for each genetic variant. 
Results
Overview
We identified two PRPH2 rare variants rs121918563 L185P (p.Leu185Pro, c.554T>C) and splice-site rs281865373 c.828+3A>T (IVS2+3A>T; Table 1) in 22 individuals (44 eyes), all with retinal diffuse and/or macular abnormalities, among which 17 were members of 3 families and 5 were independent cases. The genotypic and phenotypic characterizations of a total of 25 individuals (50 eyes), including 3 additional family members with no PRPH2 variants and no abnormalities on fundus exam, are described herein. 
Table 1.
 
Functional Interpretation of PRPH2 Variants Associated With AMD Status
Table 1.
 
Functional Interpretation of PRPH2 Variants Associated With AMD Status
Fourteen of the 25 individuals (56%) were women, and all were of European ancestry. The mean age at diagnoses or the earliest clinical record for individuals with the PRPH2 L185P variant was 56 years (n = 7, range = 39–66 years) and the mean age of individuals with the PRPH2 c.828+3A>T variant was 48 years (n = 15, range = 24–70 years). Among those with longitudinal data, the mean follow-up time was 14 years (range = 1–27 years). 
Familial Phenotype of PRPH2 rs121918563 L185P (p.Leu185Pro, c.554T≥C)
Pedigree A
Four individuals in this two-generation family were enrolled in the study. Genetic analysis of this family under the hypothesis of an autosomal dominant inherited coding variant revealed a list of 20 variants. Due to the disease prevalence in this family, we narrowed our search to coding variants that are predicted to have a deleterious effect on the protein, with a resulting list of only 4 variants: TAS1R2 L581P, ANGPT1 R493Q, THBD R106C, and PRPH2 L185P (Table 2). Of note, from this small list, we identified the missense variant in PRPH2, rs121918563, a transition from adenine to guanine that leads to a change from leucine to proline at amino acid 185 (PRPH2 L185P).8 
Table 2.
 
Filtering of Variants in Exome Sequencing Data
Table 2.
 
Filtering of Variants in Exome Sequencing Data
There were 3 affected individuals with the heterozygous PRPH2 L185P variant (Table 3Fig. 1), including the proband (II:1), his sister (II:3), and his father (I:1). All of these individuals exhibited macular abnormalities compatible with PD. Notably, both the proband and the father had manifestations of adult-onset vitelliform macular dystrophy (AVMD) displaying bilateral and symmetric well delineated, circular, yellowish pseudo-vitelliform lesions in the center of the macula measuring less than one disc diameter, surrounded by pigmentary abnormalities. The proband's sister had pigmentary clumping in the center of the macular area typical of PD. The proband's brother did not have this variant (II:2) and had a normal fundus examination. 
Table 3.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree A With PRPH2 Rare Variant rs121918563 (L185P)
Table 3.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree A With PRPH2 Rare Variant rs121918563 (L185P)
Figure 1.
 
(A) Pedigree A, heterozygous for the PRPH2 rare variant rs121918563 (L185P). (B) Phenotypes of Proband II:1 (arrow in A). (a, b) Small round yellowish lesion in the center of the macula in both eyes typical of PD presenting as AVMD on CFP. (c, d) Red-free retinography documenting the same central macular lesions in OU. (e, f) OCT with dense hyper-reflective material accumulation in the subretinal space in a vitelliform pattern, typical of AVMD. (g, h) Ultra-high resolution OCT shows pseudo-vitelliform lesions OU with heterogeneous material, and RPE migration at the dome of the pseudo-vitelliform lesions (h). (i, j) CFP 7 years after baseline. (k, l) FAF OD showing mottled hyper and hypo-autofluorescence, and near IR OS showing pigmentary accumulation and clumping, associated with the pseudo-vitelliform lesions typical of AVMD in the center of the macula OU. (m, n) OCT shows thinning and disruption of the retinal layers and disappearance of the foveal lesion OD after a retinal detachment repair, and typical pseudo-vitelliform lesion in the center of the macula OS. (o) OCT OS, 11 years after baseline showing RPE abnormalities and outer retinal atrophy, likely secondary to natural history of the pseudo-vitelliform lesion. (C) Phenotypes of other family members in Pedigree A. I:1 (a, b) CFP and (c, d) OCT of proband's father showing central macular pseudo-vitelliform lesions in different stages OU typical of AVMD. II:2 (a, b) Normal OCT OU from sibling of proband without the variant. II:3 (a–o) Multimodal retinal imaging of the sister of the proband. (a) Red free and (b) FA at baseline showing central foveal lesions typical of AVMD. Images 14 years later include (c, d) CFP, (e, f) FAF, and (g, h) OCT showing RPE and outer retina irregularities including pigmentary clumping in both eyes and incipient central macular atrophy in the OS, typical of AVMD. (i–o) OCT and IR images 17 to 18 years after baseline show RPE irregularities in a pattern configuration, with RPE and outer retina atrophy OU. AVMD, adult-onset vitelliform macular dystrophy; CFP, color fundus photography; FA, fluorescein angiography; FAF, fundus autofluorescence; IR, infrared reflectance; OCT, optical coherence tomography; OD, right eye; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 1.
 
(A) Pedigree A, heterozygous for the PRPH2 rare variant rs121918563 (L185P). (B) Phenotypes of Proband II:1 (arrow in A). (a, b) Small round yellowish lesion in the center of the macula in both eyes typical of PD presenting as AVMD on CFP. (c, d) Red-free retinography documenting the same central macular lesions in OU. (e, f) OCT with dense hyper-reflective material accumulation in the subretinal space in a vitelliform pattern, typical of AVMD. (g, h) Ultra-high resolution OCT shows pseudo-vitelliform lesions OU with heterogeneous material, and RPE migration at the dome of the pseudo-vitelliform lesions (h). (i, j) CFP 7 years after baseline. (k, l) FAF OD showing mottled hyper and hypo-autofluorescence, and near IR OS showing pigmentary accumulation and clumping, associated with the pseudo-vitelliform lesions typical of AVMD in the center of the macula OU. (m, n) OCT shows thinning and disruption of the retinal layers and disappearance of the foveal lesion OD after a retinal detachment repair, and typical pseudo-vitelliform lesion in the center of the macula OS. (o) OCT OS, 11 years after baseline showing RPE abnormalities and outer retinal atrophy, likely secondary to natural history of the pseudo-vitelliform lesion. (C) Phenotypes of other family members in Pedigree A. I:1 (a, b) CFP and (c, d) OCT of proband's father showing central macular pseudo-vitelliform lesions in different stages OU typical of AVMD. II:2 (a, b) Normal OCT OU from sibling of proband without the variant. II:3 (a–o) Multimodal retinal imaging of the sister of the proband. (a) Red free and (b) FA at baseline showing central foveal lesions typical of AVMD. Images 14 years later include (c, d) CFP, (e, f) FAF, and (g, h) OCT showing RPE and outer retina irregularities including pigmentary clumping in both eyes and incipient central macular atrophy in the OS, typical of AVMD. (i–o) OCT and IR images 17 to 18 years after baseline show RPE irregularities in a pattern configuration, with RPE and outer retina atrophy OU. AVMD, adult-onset vitelliform macular dystrophy; CFP, color fundus photography; FA, fluorescein angiography; FAF, fundus autofluorescence; IR, infrared reflectance; OCT, optical coherence tomography; OD, right eye; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
None of the members of this family had any known pathogenic or other rare variants in BEST1, IMPG1, or IMPG2, nor did they carry either of the ROM1 variants known to be carried by patients with digenic RP. The sister of the proband (II:3), heterozygous for L185P, also had the ABCA4 R943Q variant (rs1801581). This ABCA4 missense variant has conflicting reports of pathogenicity in ClinVar. It has been noted in patients diagnosed with AMD, Stargardt disease, and in individuals who have no clinical manifestations of retinal disease.29 
Familial Phenotype of PRPH2 Splice-Site rs281865373 c.828±3A≥T (IVS2±3A≥T)
Pedigree B
This 3-generation family had 13 individuals enrolled in the study. Analysis of this family began by conducting WES on five affected members (II:4, II:8, II:10, III:3, and III:6). When considering all members as affected with the same disease, the inheritance pattern was autosomal dominant. After filtering out variants not shared by all sequenced members, common variants, and variants from classes not expected to have an impact on the protein, eight variants remained (see Table 2). After this, only one variant was predicted to be damaging by multiple prediction software. This variant, rs281865373, is a thymine to adenine transversion in the PRPH2 gene within the splice donor site between exons 2 and 3 (PRPH2 c.828+3A>T). After identifying this variant, we used Sanger sequencing to genotype the remaining members of Pedigree B, including unaffected family members to confirm that the variant segregated with the disease. 
Twelve of the 13 family members had the PRPH2 c.828+3A>T heterozygous variant and all were clinically affected (Table 4Fig. 2). The proband (II:8), her identical twin (II:7), and 3 siblings (II:1, II:4, II:10) had this PRPH2 splice-site variant. They exhibited diffuse RPE abnormalities and macular atrophy, and, in some cases, RPE atrophy extended beyond the retinal vascular arcades (see Fig. 2), whereas the sibling (II:5) was the only wild-type individual and had a normal fundus examination. The son of the proband (III:6), five of her nieces and nephews (III:2–7), and a grandniece (IV:1) also had the variant and were clinically affected, four of whom (III:2, III:3, III:6, and IV:1) presented with yellow flecks in the macular area extending throughout the temporal retinal vascular arcades, consistent with adult-onset fundus flavimaculatus with macular dystrophy (AFMD). Over time, multimodal retinal imaging showed disease progression starting from diffuse atrophy of the retina and the RPE, advancing to the macular area and extending beyond the retinal vascular arcades throughout the posterior pole. This was consistent with central areolar chorioretinal dystrophy (CACD), among the cases with or without retinal flecks that had follow-up records available. Two nieces (III:3 and III:5) and one nephew (III:7) also developed macular NV. 
Table 4.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree B With PRPH2 Rare Variant rs281865373 (c.828+3A>T)
Table 4.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree B With PRPH2 Rare Variant rs281865373 (c.828+3A>T)
Figure 2.
 
(A) Pedigree B, heterozygous for the PRPH2 rs281865373 (c.828+3A>T) variant. (B) Phenotypes of proband II:8 (arrow in A) and identical twin sister (II:7). Proband II:8 (a, b) CFP shows bilateral extensive macular and peripapillary retinal atrophy extending beyond the retinal vascular arcades, consistent with CACD. II:7 Identical twin of the proband. (a–d) CFP shows a very similar clinical presentation in comparison to the proband, with further worsening of the diffuse retinal atrophy over a follow-up time of 6 years. II:1 Sibling of the proband. (a, b) Large areas of retinal and RPE atrophy extending throughout the macula (CACD). II:10 Sibling of the proband. (a–d) CFP OU show patches of retinal atrophy in the central macula evolving over a period of 16 years to retinal atrophy involving the entire macular area and extending to the retinal vascular arcades (CACD). (C) III:3 Niece of proband. (a–i) Multimodal retinal imaging from baseline to 19 years of follow-up shows yellow retinal flecks as seen in AFMD on (a, b) CFP and (c, d) FA. Central macular atrophy resembling GA secondary to AMD developed over a follow-up time of 19 years as seen in (e, f) CFP and (g, h) red-free retinography. (i, j) Last documentation with OCT shows outer retinal atrophy throughout the macula OU and residual intraretinal fluid OS after intravitreal anti-vascular endothelial growth factor for secondary macular NV OU. (D) Other nieces and nephews of the proband and 1 great-niece. III:2 Niece of proband. (a–d) CFP demonstrates RPE irregularities and retinal flecks resembling AFMD, which progressed over a period of 16 years to large areas of retinal atrophy affecting the macular area and extending beyond the retinal vascular arcades in OU. III:6 Nephew of the proband. (a–d) CFP OU show central area of macular atrophy that resembles GA secondary to AMD, which evolved over a follow-up time of 14 years to develop extensive retinal atrophy throughout the macular area and extending beyond the retinal vascular arcades, more typical of CACD. III:4 Nephew of the proband. (a, b) CFP OU show central macular atrophy that resemble GA secondary to AMD with foveal sparing, which evolved over 19 years to more widespread retinal atrophy extending nasally to the optic nerve and in the far retinal periphery, as shown on (c, d) CFP and (e, f) FAF. III:5 Niece of the proband. (a, b) CFP OU exhibits large areas of macular atrophy and subretinal fibrosis involving the central macular area with history of bilateral macular NV. III:7 Nephew of the proband. (a, b) CFP and (c, d) FAF show central macular atrophy and significant abnormalities of the retina and RPE extending throughout the macular area and beyond the retinal vascular arcades OU. (e, f) OCT shows outer retinal atrophy and intraretinal cystic spaces in both eyes. IV:1 Daughter of III:2. (a, b) CFP OU show retinal flecks as seen on AFMD at baseline. AFMD, adult-onset fundus flavimaculatus with macular dystrophy; AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; FAF, fundus autofluorescence; GA, geographic atrophy; NV, neovascularization; OCT, optical coherence tomography; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 2.
 
(A) Pedigree B, heterozygous for the PRPH2 rs281865373 (c.828+3A>T) variant. (B) Phenotypes of proband II:8 (arrow in A) and identical twin sister (II:7). Proband II:8 (a, b) CFP shows bilateral extensive macular and peripapillary retinal atrophy extending beyond the retinal vascular arcades, consistent with CACD. II:7 Identical twin of the proband. (a–d) CFP shows a very similar clinical presentation in comparison to the proband, with further worsening of the diffuse retinal atrophy over a follow-up time of 6 years. II:1 Sibling of the proband. (a, b) Large areas of retinal and RPE atrophy extending throughout the macula (CACD). II:10 Sibling of the proband. (a–d) CFP OU show patches of retinal atrophy in the central macula evolving over a period of 16 years to retinal atrophy involving the entire macular area and extending to the retinal vascular arcades (CACD). (C) III:3 Niece of proband. (a–i) Multimodal retinal imaging from baseline to 19 years of follow-up shows yellow retinal flecks as seen in AFMD on (a, b) CFP and (c, d) FA. Central macular atrophy resembling GA secondary to AMD developed over a follow-up time of 19 years as seen in (e, f) CFP and (g, h) red-free retinography. (i, j) Last documentation with OCT shows outer retinal atrophy throughout the macula OU and residual intraretinal fluid OS after intravitreal anti-vascular endothelial growth factor for secondary macular NV OU. (D) Other nieces and nephews of the proband and 1 great-niece. III:2 Niece of proband. (a–d) CFP demonstrates RPE irregularities and retinal flecks resembling AFMD, which progressed over a period of 16 years to large areas of retinal atrophy affecting the macular area and extending beyond the retinal vascular arcades in OU. III:6 Nephew of the proband. (a–d) CFP OU show central area of macular atrophy that resembles GA secondary to AMD, which evolved over a follow-up time of 14 years to develop extensive retinal atrophy throughout the macular area and extending beyond the retinal vascular arcades, more typical of CACD. III:4 Nephew of the proband. (a, b) CFP OU show central macular atrophy that resemble GA secondary to AMD with foveal sparing, which evolved over 19 years to more widespread retinal atrophy extending nasally to the optic nerve and in the far retinal periphery, as shown on (c, d) CFP and (e, f) FAF. III:5 Niece of the proband. (a, b) CFP OU exhibits large areas of macular atrophy and subretinal fibrosis involving the central macular area with history of bilateral macular NV. III:7 Nephew of the proband. (a, b) CFP and (c, d) FAF show central macular atrophy and significant abnormalities of the retina and RPE extending throughout the macular area and beyond the retinal vascular arcades OU. (e, f) OCT shows outer retinal atrophy and intraretinal cystic spaces in both eyes. IV:1 Daughter of III:2. (a, b) CFP OU show retinal flecks as seen on AFMD at baseline. AFMD, adult-onset fundus flavimaculatus with macular dystrophy; AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; FAF, fundus autofluorescence; GA, geographic atrophy; NV, neovascularization; OCT, optical coherence tomography; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Thus, we confirmed the variant was exclusively present in the affected family members. None of the family members had any known pathogenic or other rare variants in ABCA4, BEST1, CTNNA1, IMPG1, IMPG2, or ROM1. 
Pedigree C
Given the known phenotypic presentations of the PRPH2 variants identified in Pedigrees A and B, we searched all previously collected genetic data for individuals in our database to identify additional individuals with PRPH2 variants. A query of our targeted sequencing data identified one subject who was initially diagnosed with GA secondary to AMD based on clinical examination (II:2) and was heterozygous for PRPH2 c.828+3A>T. This subject was part of an expanded family unit within our database for which we had phenotypic and genetic data on two additional siblings. Investigating these 3 sisters, 2 were heterozygous for the PRPH2 c.828+3A>T variant and were also clinically affected (Table 5Fig. 3). The proband (II:1) showed macular atrophy in both eyes that could be misdiagnosed as GA secondary to AMD but showed RPE abnormalities in spoke-like configuration in the central macular area more prominent in one eye, which is more commonly seen in PD. The area of macular atrophy enlarged over 7 years in both eyes. Similarly, the proband's sister (II:2) had a comparable phenotype with macular atrophy and enlargement of the lesions in both eyes over 15 years. The other sister (II:3) did not have this variant and had a normal fundus examination. 
Table 5.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree C With PRPH2 Rare Variant rs281865373 (c.828+3A>T)
Table 5.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree C With PRPH2 Rare Variant rs281865373 (c.828+3A>T)
Figure 3.
 
(A) Pedigree C, heterozygous for the PRPH2 rs281865373 (c.828+3A>T) variant. (B) Phenotypes of Proband II:1. (a, b) FA OU shows central macular atrophy that may resemble GA secondary to AMD, but with RPE abnormalities in spoke-like configuration similar to pattern dystrophy more evident OS. (c, d) CFP OU demonstrates worsening of central macular atrophy that could be confounded with GA secondary to AMD but also fit in the differential diagnosis of early CACD. II:2 Sibling of the Proband. (a–d) CFP OU exhibits clinical presentation very similar to the Proband, with central macular atrophy in the differential diagnosis of GA secondary to AMD or CACD, with enlargement of the lesions 7 years after baseline. AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; GA, geographic atrophy; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 3.
 
(A) Pedigree C, heterozygous for the PRPH2 rs281865373 (c.828+3A>T) variant. (B) Phenotypes of Proband II:1. (a, b) FA OU shows central macular atrophy that may resemble GA secondary to AMD, but with RPE abnormalities in spoke-like configuration similar to pattern dystrophy more evident OS. (c, d) CFP OU demonstrates worsening of central macular atrophy that could be confounded with GA secondary to AMD but also fit in the differential diagnosis of early CACD. II:2 Sibling of the Proband. (a–d) CFP OU exhibits clinical presentation very similar to the Proband, with central macular atrophy in the differential diagnosis of GA secondary to AMD or CACD, with enlargement of the lesions 7 years after baseline. AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; GA, geographic atrophy; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Phenotypic Presentation of Independent Cases
Five independent cases were also determined to be heterozygous for the PRPH2 variants by genetic sequencing. Four were heterozygous for the PRPH2 L185P variant (cases A to D), one individual was heterozygous for the splice-site variant PRPH2 c.828+3A>T (case E), and all 5 cases were clinically affected (Table 6Fig. 4). Some cases were initially misdiagnosed as GA secondary to AMD before being referred to our study, and one of them had been previously enrolled in a clinical trial for AMD. 
Table 6.
 
Demographic, Clinical, and Genetic Characteristics of Independent Cases With PRPH2 Rare Variants
Table 6.
 
Demographic, Clinical, and Genetic Characteristics of Independent Cases With PRPH2 Rare Variants
Figure 4.
 
Phenotypes of independent cases, heterozygous for the PRPH2 rs121918563 (L185P) variant (cases A–-D) and the PRPH2 rs281865373 (c.828+3A>T) variant (case E). Case A. (a, b) CFP OU show central pseudo-vitelliform macular lesions which evolved to small circular area of central RPE and outer retinal atrophy, also shown on (c, d) fundus autofluorescence (FAF) and (e, f) near IR as well as on (g, h) OCT typical of AVMD. Of note, IR images show a pattern configuration of pigmentary clumps. Case B. (a, b) CFP exhibits RPE irregularities in a butterfly shape OD and central atrophy OS. Case C. (a, b) FA OU show small central macular atrophy surrounded by increased fluorescence due to pigmentary mobilization and marked fluorescein staining, and (c, d) FAF OU shows small areas of distinct hypo-autofluorescence correspondent to the central macular atrophy, and increased areas of FAF in a distinct butterfly pattern correspondent to the areas of pigmentary mobilization. (e, f) OCT OU show small focal areas of RPE and outer retinal atrophy, and shallow “low-lying” RPE detachments in both eyes, without evident signs of exudation. (g, h) OCTA OU show macular NV. Case D. (a, b) Red-free retinography in both eyes show RPE abnormalities in a butterfly pattern configuration. (c, d) FAF OU show progression to central macular atrophy 9 years after baseline, and in the same visit (e, f) OCT OU show central macular atrophy and subretinal fibrosis secondary to macular NV. Case E. (a, b) CFP OU show multifocal areas of RPE atrophy. (c, d) CFP OU show that 6 years after baseline the areas of macular atrophy enlarged, resembling GA secondary to AMD earlier in the disease presentation but that could fit in the differential diagnosis of CACD as the disease progressed. AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; FAF, fundus autofluorescence; GA, geographic atrophy; IR, infrared reflectance; NV, neovascularization; OCT, optical coherence tomography; OCTA, optical coherence tomography angiography; OD, right eye; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 4.
 
Phenotypes of independent cases, heterozygous for the PRPH2 rs121918563 (L185P) variant (cases A–-D) and the PRPH2 rs281865373 (c.828+3A>T) variant (case E). Case A. (a, b) CFP OU show central pseudo-vitelliform macular lesions which evolved to small circular area of central RPE and outer retinal atrophy, also shown on (c, d) fundus autofluorescence (FAF) and (e, f) near IR as well as on (g, h) OCT typical of AVMD. Of note, IR images show a pattern configuration of pigmentary clumps. Case B. (a, b) CFP exhibits RPE irregularities in a butterfly shape OD and central atrophy OS. Case C. (a, b) FA OU show small central macular atrophy surrounded by increased fluorescence due to pigmentary mobilization and marked fluorescein staining, and (c, d) FAF OU shows small areas of distinct hypo-autofluorescence correspondent to the central macular atrophy, and increased areas of FAF in a distinct butterfly pattern correspondent to the areas of pigmentary mobilization. (e, f) OCT OU show small focal areas of RPE and outer retinal atrophy, and shallow “low-lying” RPE detachments in both eyes, without evident signs of exudation. (g, h) OCTA OU show macular NV. Case D. (a, b) Red-free retinography in both eyes show RPE abnormalities in a butterfly pattern configuration. (c, d) FAF OU show progression to central macular atrophy 9 years after baseline, and in the same visit (e, f) OCT OU show central macular atrophy and subretinal fibrosis secondary to macular NV. Case E. (a, b) CFP OU show multifocal areas of RPE atrophy. (c, d) CFP OU show that 6 years after baseline the areas of macular atrophy enlarged, resembling GA secondary to AMD earlier in the disease presentation but that could fit in the differential diagnosis of CACD as the disease progressed. AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; FAF, fundus autofluorescence; GA, geographic atrophy; IR, infrared reflectance; NV, neovascularization; OCT, optical coherence tomography; OCTA, optical coherence tomography angiography; OD, right eye; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
All four affected individuals (cases A to D) with the PRPH2 L185P variant had fundus features typical of PD, similar to Pedigree A. Two cases (A and D) presented with macular abnormalities typical of PD in their 60s which evolved to the central area of macular atrophy in their 70s. Two other cases (B and C) demonstrated the so-called butterfly shaped presentation of PD with a central area of macular atrophy. In case C, the OCTA revealed type 1 macular NV adjacent to the area of RPE atrophy in both eyes, which was not suspected based on clinical history but was associated with shallow, low-lying RPE detachment on OCT. Case D also presented with central areas of macular atrophy and developed exudative macular NV in both eyes. 
One individual who was heterozygous for PRPH2 c.828+3A>T (case E) evolved to extensive atrophy of the retina and RPE in both eyes, similar to some phenotypes observed in Pedigrees B and C with the same variant. This subject presented at baseline with bilateral, multifocal areas of macular atrophy which enlarged to central and paracentral retinal atrophy in both eyes, and which could clinically resemble GA secondary to AMD at this stage. Over a follow-up period of 10 years, the atrophic lesions progressed beyond the topography of the retinal vascular arcades and posterior pole consistent with CACD, based on clinical records. 
None of these independent cases have any known pathogenic or other rare variants in BEST1, CTNNA1, IMPG1, or IMPG2, and ROM1 variants. One of these individuals (case B) was heterozygous for ABCA4 V2050L (rs41292677), a rare missense variant listed as having uncertain significance as per American College of Medical Genetics and Genomics (ACMG) criteria in ClinVar.30,31 This variant has been reported as a genetic modifier seen in conjunction with PRPH2 R172W and ROM1 variants, but not with the PRPH2 L185P variant present in this subject.32 
Best Corrected Visual Acuity Results
Figure 5 displays distribution of visual acuity in both variants at the initial and final visits and trends during the follow-up period. Visual acuity (logMAR) in individuals with the PRPH2 c.828+3A>T variant was worse at the final visit compared to the initial visit, whereas less vision loss from baseline was seen in individuals heterozygous for L185P (see Fig. 5A). For individuals heterozygous for PRPH2 c.828+3A>T, visual acuity change per year was +0.021 (±0.006), and for individuals heterozygous for PRPH2 L185P the slope was +0.006 (±0.005; see Fig. 5B). Therefore, a greater rate of vision loss for the PRPH2 splice site variant was observed, aligning with more severe phenotypes seen in affected individuals carrying this variant compared to the missense variant (P < 0.001 for difference in rates of decline over time between the two variants). Because the first BCVA measurement in this study was collected once the macular abnormalities were already clinically significant, and medical records are not available at the onset of their retinal disease, the overarching impact of the genetic variants on rate of visual decline may be underestimated. 
Figure 5.
 
(A) Violin plots showing initial and final visual acuity (logMAR) for individuals heterozygous for PRPH2 rs281865373 (c.828+3A>T) (orange) and PRPH2 rs121918563 (L185P) (blue) variants. The width of each violin plot is proportional to the density for the range of values and the medians are represented by horizontal bars within the boxplots. (B) Comparison of change in visual acuity (logMAR) per year between individuals heterozygous for PRPH2 rs281865373 (c.828+3A>T) (orange) and PRPH2 rs121918563 (L185P) (blue) estimated using a mixed effects linear regression with a compound symmetry correlation structure. Standard error is illustrated by vertical error bars (***P < 0.001). logMAR, logarithm of the minimal angle of resolution; PRPH2, peripherin-2 gene.
Figure 5.
 
(A) Violin plots showing initial and final visual acuity (logMAR) for individuals heterozygous for PRPH2 rs281865373 (c.828+3A>T) (orange) and PRPH2 rs121918563 (L185P) (blue) variants. The width of each violin plot is proportional to the density for the range of values and the medians are represented by horizontal bars within the boxplots. (B) Comparison of change in visual acuity (logMAR) per year between individuals heterozygous for PRPH2 rs281865373 (c.828+3A>T) (orange) and PRPH2 rs121918563 (L185P) (blue) estimated using a mixed effects linear regression with a compound symmetry correlation structure. Standard error is illustrated by vertical error bars (***P < 0.001). logMAR, logarithm of the minimal angle of resolution; PRPH2, peripherin-2 gene.
Discussion
In this study, we used whole exome sequencing to explore genetic underpinnings of maculopathies and we identified two PRPH2 genetic mutations (L185P and c.828+3A>T) in unrelated families and independent cases, which were not explained by other variants. Genotypic-phenotypic evaluation with longitudinal multimodal imaging revealed varied clinical manifestations described in PD or CACD. Some cases showed variable clinical manifestation throughout the follow-up period and some evolved to macular atrophy with or without macular NV over time, underscoring the importance of accurate diagnoses enabled by next generation sequencing, especially for maculopathies associated with earlier onset or other clinical features atypical for AMD. These results provide additional information about the natural history associated with these variants which have been classified as likely pathogenic/pathogenic using the 2015 ACMG criteria in ClinVar (www.ncbi.nih.gov/clinvar),33,34 and the Human Gene Mutation Database (www.hgmd.cf.ac.uk).35 
Clinical Manifestations of the PRPH2 Variants
The phenotypes were meaningfully different between the two PRPH2 variants, whereas they were more similar but not identical within each variant. Retinal abnormalities described in this study included clinical manifestations characteristic of PD, such as AVMD, AFMD, and CACD. In all individuals with the PRPH2 L185P variant (rs121918563), fundus findings typical of PD were observed. Some affected individuals with the L185P variant had AVMD which developed in their 40s and 50s which evolved to RPE irregularities and central areas of macular atrophy in their mid-60s. Others exhibited PD which presented as butterfly shaped RPE irregularities that evolved to small areas of central macular atrophy in their 70s. A few individuals later developed macular NV. Two cases with the L185P variant each had a different ABCA4 variant, which may have affected the severity of the phenotypes, however, neither case had retinal flecks which can occur with fundus flavimaculatus or Stargardt disease. In summary, all individuals with the L185P variant in our study manifested various clinical presentations of PD, evolving over time to macular areas of RPE and outer retinal atrophy. 
Affected individuals with the PRPH2 c.828+3A>T splice-site variant (rs281865373) exhibited a different phenotype with more diffuse RPE and outer retinal atrophy worsening over time, and some were consistent with CACD. Some individuals had flecks, such as fundus flavimaculatus and/or multifocal areas of retinal atrophy confined to the macula in their early 50’s. These lesions further evolved to diffuse retinal atrophy, extending beyond the macula and the topography of retinal vascular arcades in their late 60s to 80s. Phenotypes associated with this splice-site variant resemble those reported in a cross-sectional study.36 In our longitudinal study, one individual with the splice-site variant had a presentation of PD, as seen in some individuals with the L185P variant. 
Another correlation between genotype and phenotype was the degree of visual acuity decline over time. Individuals with the splice-site variant had more severe visual loss over the follow-up period. In contrast, individuals with the missense L185P variant had mild to moderate visual impairment over time. The trends for differences in retinopathies and visual outcomes suggest patterns that warrant further investigation to inform clinical trial end points. 
Previous Literature About PRPH2 Variants
There are numerous reported PRPH2 variants and many have been associated with retinal disorders3741 including PD,4244 RP,8,45 CACD,36,46,47 retinitis punctata albescens,48 cone-rod dystrophy,49,50 AMD-like late-onset maculopathy,51 and Leber congenital amaurosis.52 The splice site variant c.828+3A>T is one of the most common PRPH2 variants and has been shown to exhibit variable expressivity similar to what we have reported,36,37,53,54 whereas the missense L185P is less common.41,45,5456 Of note, a vast spectrum of PRPH2 variants and phenotypic presentations (including those reported in this paper) have been reported and/or reviewed by Sears et al. (2001),37 Boon et al. (2008),38 Shankar et al. (2015-16),36,53 Reeves et al. (2020),54 Peeters et al. (2021),41 and Heath Jeffrey et al. (2024).57 
Genomics for PRPH2 Gene and Genetic Mutations
The PRPH2 gene encodes the protein Peripherin-2 and consists of 3 exons located on Chromosome 6p21. The protein is membrane-associated glycoprotein crucial for photoreceptor outer segment function. 
The PRPH2 L185P variant is located in the second intradiscal loop of the protein, the portion of the protein where over 70% of disease-causing variants are located. This loop is vital to the formation of PRPH2 homotetramers and PRPH2/ROM1 heterotetramers, the impairment of which results in abnormal disc morphogenesis, photoreceptor degeneration, and subsequent visual loss.58,59 Another study showed that when the PRPH2 L185P was expressed by itself in COS-1 cells, it was unable to form homotetramers, but when expressed along with wild type ROM1 protein, the PRPH2 L185P mutant proteins were still able to form heterotetramers.60 These heterotetramers are involved in the formation of a higher-order complex that extends over the entire circumference of the disc.61 The individuals in this study have the PRPH2 L185P variant but are wild type at the positions in ROM1 known to cause digenic RP. We hypothesize that individuals who carry PRPH2 L185P are able to form heterotetramers with ROM1, but are unable to form the higher-order complexes consisting of both homo- and heterotetramers.61 This inability to form homotetramers, and in turn higher-order complexes, may result in the manifestations of PD seen in Pedigree A and four independent cases. Modifier genes could also explain variable phenotypes and different penetrance in patients with the same variant described in this study. 
The other variant described in this report, PRPH2 c.828+3A>T, is a splice-site variant located between the second and third exons and leads to the addition of 10 amino acids to exon 2 and then a stop codon is encountered. This premature stop results in the last exon being absent from the protein. This last exon encodes the final piece of the protein that is located within the discal membrane and the C terminal of the protein that is in the cytoplasmic space. So, this variant leads to the formation of a nonfunctional truncated protein. It is considered pathogenic as per reports in ClinVar and HGMD.3335 
Strengths and Limitations
Strengths of this study include the identification of protein-coding variants using whole exome genetic sequencing, although non-coding regions would not be detected. The SLCS database enabled us to determine families and independent individuals with PRPH2 retinopathies who had the variants and to assess differences in phenotypes. This comprehensive evaluation with longitudinal clinical examinations and multimodal retinal imaging provides further insights into the natural history of disease progression, benefited also by remarkably long follow-up periods in some cases. Our results also contribute to the body of knowledge on differential diagnosis of retinal dystrophies and AMD, which could be crucial for evaluation of new therapeutics. Limitations include acquisition of ocular data from a study database augmented by real-world clinical care at variable follow-up times. The cohort with PRPH2 variants was relatively small and electrophysiological findings were not available for most cases. 
Conclusions
In conclusion, this report underscores important clinical phenotypic features associated with variants of the PRPH2 gene and demonstrates the autosomal dominant pattern of inheritance with variable expressivity. There was a trend for individuals with the same variant to have similar phenotypes and visual outcomes, with more marked differences seen between the two variants. Results contribute to the characterization of clinically heterogeneous cases of PRPH2 retinopathies associated with two distinct variants of the gene, adds information about the natural untreated course of these diseases, and provides new information about their visual outcomes over time. Detailed clinical assessment paired with comprehensive genetic analysis is integral for accurate diagnosis, and for a better understanding of the natural history and underlying pathophysiology of inherited retinal disorders, with relevance for both clinical research and clinical practice. 
Acknowledgments
The authors thank Bernard Rosner, PhD, for his statistical advice, William Casazza, PhD, for review of the manuscript, and Luis Nakayama, MD, for assistance with literature review. 
Supported in part by National Institutes of Health, National Eye Institute R01-EY011309, R01-EY028602, Bethesda, MD; Carl Marshall Reeves and Mildred Almen Reeves Foundation, Inc., Fenton, MO; American Macular Degeneration Foundation, Northampton, MA; Macular Degeneration Center of Excellence, University of Massachusetts Chan Medical School, Department of Ophthalmology and Visual Sciences, Worcester, MA. The sponsor or funding organization had no role in the design or conduct of this research. 
Disclosure: J.M. Seddon, Gemini Therapeutics (I), Apellis Pharmaceuticals (I), Laboratoires Théa, Inc. (C); D. De, None; L. Grunenkovaite, None; D. Ferrara, Roche (I), Genentech, Inc. (E) 
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Figure 1.
 
(A) Pedigree A, heterozygous for the PRPH2 rare variant rs121918563 (L185P). (B) Phenotypes of Proband II:1 (arrow in A). (a, b) Small round yellowish lesion in the center of the macula in both eyes typical of PD presenting as AVMD on CFP. (c, d) Red-free retinography documenting the same central macular lesions in OU. (e, f) OCT with dense hyper-reflective material accumulation in the subretinal space in a vitelliform pattern, typical of AVMD. (g, h) Ultra-high resolution OCT shows pseudo-vitelliform lesions OU with heterogeneous material, and RPE migration at the dome of the pseudo-vitelliform lesions (h). (i, j) CFP 7 years after baseline. (k, l) FAF OD showing mottled hyper and hypo-autofluorescence, and near IR OS showing pigmentary accumulation and clumping, associated with the pseudo-vitelliform lesions typical of AVMD in the center of the macula OU. (m, n) OCT shows thinning and disruption of the retinal layers and disappearance of the foveal lesion OD after a retinal detachment repair, and typical pseudo-vitelliform lesion in the center of the macula OS. (o) OCT OS, 11 years after baseline showing RPE abnormalities and outer retinal atrophy, likely secondary to natural history of the pseudo-vitelliform lesion. (C) Phenotypes of other family members in Pedigree A. I:1 (a, b) CFP and (c, d) OCT of proband's father showing central macular pseudo-vitelliform lesions in different stages OU typical of AVMD. II:2 (a, b) Normal OCT OU from sibling of proband without the variant. II:3 (a–o) Multimodal retinal imaging of the sister of the proband. (a) Red free and (b) FA at baseline showing central foveal lesions typical of AVMD. Images 14 years later include (c, d) CFP, (e, f) FAF, and (g, h) OCT showing RPE and outer retina irregularities including pigmentary clumping in both eyes and incipient central macular atrophy in the OS, typical of AVMD. (i–o) OCT and IR images 17 to 18 years after baseline show RPE irregularities in a pattern configuration, with RPE and outer retina atrophy OU. AVMD, adult-onset vitelliform macular dystrophy; CFP, color fundus photography; FA, fluorescein angiography; FAF, fundus autofluorescence; IR, infrared reflectance; OCT, optical coherence tomography; OD, right eye; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 1.
 
(A) Pedigree A, heterozygous for the PRPH2 rare variant rs121918563 (L185P). (B) Phenotypes of Proband II:1 (arrow in A). (a, b) Small round yellowish lesion in the center of the macula in both eyes typical of PD presenting as AVMD on CFP. (c, d) Red-free retinography documenting the same central macular lesions in OU. (e, f) OCT with dense hyper-reflective material accumulation in the subretinal space in a vitelliform pattern, typical of AVMD. (g, h) Ultra-high resolution OCT shows pseudo-vitelliform lesions OU with heterogeneous material, and RPE migration at the dome of the pseudo-vitelliform lesions (h). (i, j) CFP 7 years after baseline. (k, l) FAF OD showing mottled hyper and hypo-autofluorescence, and near IR OS showing pigmentary accumulation and clumping, associated with the pseudo-vitelliform lesions typical of AVMD in the center of the macula OU. (m, n) OCT shows thinning and disruption of the retinal layers and disappearance of the foveal lesion OD after a retinal detachment repair, and typical pseudo-vitelliform lesion in the center of the macula OS. (o) OCT OS, 11 years after baseline showing RPE abnormalities and outer retinal atrophy, likely secondary to natural history of the pseudo-vitelliform lesion. (C) Phenotypes of other family members in Pedigree A. I:1 (a, b) CFP and (c, d) OCT of proband's father showing central macular pseudo-vitelliform lesions in different stages OU typical of AVMD. II:2 (a, b) Normal OCT OU from sibling of proband without the variant. II:3 (a–o) Multimodal retinal imaging of the sister of the proband. (a) Red free and (b) FA at baseline showing central foveal lesions typical of AVMD. Images 14 years later include (c, d) CFP, (e, f) FAF, and (g, h) OCT showing RPE and outer retina irregularities including pigmentary clumping in both eyes and incipient central macular atrophy in the OS, typical of AVMD. (i–o) OCT and IR images 17 to 18 years after baseline show RPE irregularities in a pattern configuration, with RPE and outer retina atrophy OU. AVMD, adult-onset vitelliform macular dystrophy; CFP, color fundus photography; FA, fluorescein angiography; FAF, fundus autofluorescence; IR, infrared reflectance; OCT, optical coherence tomography; OD, right eye; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 2.
 
(A) Pedigree B, heterozygous for the PRPH2 rs281865373 (c.828+3A>T) variant. (B) Phenotypes of proband II:8 (arrow in A) and identical twin sister (II:7). Proband II:8 (a, b) CFP shows bilateral extensive macular and peripapillary retinal atrophy extending beyond the retinal vascular arcades, consistent with CACD. II:7 Identical twin of the proband. (a–d) CFP shows a very similar clinical presentation in comparison to the proband, with further worsening of the diffuse retinal atrophy over a follow-up time of 6 years. II:1 Sibling of the proband. (a, b) Large areas of retinal and RPE atrophy extending throughout the macula (CACD). II:10 Sibling of the proband. (a–d) CFP OU show patches of retinal atrophy in the central macula evolving over a period of 16 years to retinal atrophy involving the entire macular area and extending to the retinal vascular arcades (CACD). (C) III:3 Niece of proband. (a–i) Multimodal retinal imaging from baseline to 19 years of follow-up shows yellow retinal flecks as seen in AFMD on (a, b) CFP and (c, d) FA. Central macular atrophy resembling GA secondary to AMD developed over a follow-up time of 19 years as seen in (e, f) CFP and (g, h) red-free retinography. (i, j) Last documentation with OCT shows outer retinal atrophy throughout the macula OU and residual intraretinal fluid OS after intravitreal anti-vascular endothelial growth factor for secondary macular NV OU. (D) Other nieces and nephews of the proband and 1 great-niece. III:2 Niece of proband. (a–d) CFP demonstrates RPE irregularities and retinal flecks resembling AFMD, which progressed over a period of 16 years to large areas of retinal atrophy affecting the macular area and extending beyond the retinal vascular arcades in OU. III:6 Nephew of the proband. (a–d) CFP OU show central area of macular atrophy that resembles GA secondary to AMD, which evolved over a follow-up time of 14 years to develop extensive retinal atrophy throughout the macular area and extending beyond the retinal vascular arcades, more typical of CACD. III:4 Nephew of the proband. (a, b) CFP OU show central macular atrophy that resemble GA secondary to AMD with foveal sparing, which evolved over 19 years to more widespread retinal atrophy extending nasally to the optic nerve and in the far retinal periphery, as shown on (c, d) CFP and (e, f) FAF. III:5 Niece of the proband. (a, b) CFP OU exhibits large areas of macular atrophy and subretinal fibrosis involving the central macular area with history of bilateral macular NV. III:7 Nephew of the proband. (a, b) CFP and (c, d) FAF show central macular atrophy and significant abnormalities of the retina and RPE extending throughout the macular area and beyond the retinal vascular arcades OU. (e, f) OCT shows outer retinal atrophy and intraretinal cystic spaces in both eyes. IV:1 Daughter of III:2. (a, b) CFP OU show retinal flecks as seen on AFMD at baseline. AFMD, adult-onset fundus flavimaculatus with macular dystrophy; AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; FAF, fundus autofluorescence; GA, geographic atrophy; NV, neovascularization; OCT, optical coherence tomography; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 2.
 
(A) Pedigree B, heterozygous for the PRPH2 rs281865373 (c.828+3A>T) variant. (B) Phenotypes of proband II:8 (arrow in A) and identical twin sister (II:7). Proband II:8 (a, b) CFP shows bilateral extensive macular and peripapillary retinal atrophy extending beyond the retinal vascular arcades, consistent with CACD. II:7 Identical twin of the proband. (a–d) CFP shows a very similar clinical presentation in comparison to the proband, with further worsening of the diffuse retinal atrophy over a follow-up time of 6 years. II:1 Sibling of the proband. (a, b) Large areas of retinal and RPE atrophy extending throughout the macula (CACD). II:10 Sibling of the proband. (a–d) CFP OU show patches of retinal atrophy in the central macula evolving over a period of 16 years to retinal atrophy involving the entire macular area and extending to the retinal vascular arcades (CACD). (C) III:3 Niece of proband. (a–i) Multimodal retinal imaging from baseline to 19 years of follow-up shows yellow retinal flecks as seen in AFMD on (a, b) CFP and (c, d) FA. Central macular atrophy resembling GA secondary to AMD developed over a follow-up time of 19 years as seen in (e, f) CFP and (g, h) red-free retinography. (i, j) Last documentation with OCT shows outer retinal atrophy throughout the macula OU and residual intraretinal fluid OS after intravitreal anti-vascular endothelial growth factor for secondary macular NV OU. (D) Other nieces and nephews of the proband and 1 great-niece. III:2 Niece of proband. (a–d) CFP demonstrates RPE irregularities and retinal flecks resembling AFMD, which progressed over a period of 16 years to large areas of retinal atrophy affecting the macular area and extending beyond the retinal vascular arcades in OU. III:6 Nephew of the proband. (a–d) CFP OU show central area of macular atrophy that resembles GA secondary to AMD, which evolved over a follow-up time of 14 years to develop extensive retinal atrophy throughout the macular area and extending beyond the retinal vascular arcades, more typical of CACD. III:4 Nephew of the proband. (a, b) CFP OU show central macular atrophy that resemble GA secondary to AMD with foveal sparing, which evolved over 19 years to more widespread retinal atrophy extending nasally to the optic nerve and in the far retinal periphery, as shown on (c, d) CFP and (e, f) FAF. III:5 Niece of the proband. (a, b) CFP OU exhibits large areas of macular atrophy and subretinal fibrosis involving the central macular area with history of bilateral macular NV. III:7 Nephew of the proband. (a, b) CFP and (c, d) FAF show central macular atrophy and significant abnormalities of the retina and RPE extending throughout the macular area and beyond the retinal vascular arcades OU. (e, f) OCT shows outer retinal atrophy and intraretinal cystic spaces in both eyes. IV:1 Daughter of III:2. (a, b) CFP OU show retinal flecks as seen on AFMD at baseline. AFMD, adult-onset fundus flavimaculatus with macular dystrophy; AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; FAF, fundus autofluorescence; GA, geographic atrophy; NV, neovascularization; OCT, optical coherence tomography; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 3.
 
(A) Pedigree C, heterozygous for the PRPH2 rs281865373 (c.828+3A>T) variant. (B) Phenotypes of Proband II:1. (a, b) FA OU shows central macular atrophy that may resemble GA secondary to AMD, but with RPE abnormalities in spoke-like configuration similar to pattern dystrophy more evident OS. (c, d) CFP OU demonstrates worsening of central macular atrophy that could be confounded with GA secondary to AMD but also fit in the differential diagnosis of early CACD. II:2 Sibling of the Proband. (a–d) CFP OU exhibits clinical presentation very similar to the Proband, with central macular atrophy in the differential diagnosis of GA secondary to AMD or CACD, with enlargement of the lesions 7 years after baseline. AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; GA, geographic atrophy; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 3.
 
(A) Pedigree C, heterozygous for the PRPH2 rs281865373 (c.828+3A>T) variant. (B) Phenotypes of Proband II:1. (a, b) FA OU shows central macular atrophy that may resemble GA secondary to AMD, but with RPE abnormalities in spoke-like configuration similar to pattern dystrophy more evident OS. (c, d) CFP OU demonstrates worsening of central macular atrophy that could be confounded with GA secondary to AMD but also fit in the differential diagnosis of early CACD. II:2 Sibling of the Proband. (a–d) CFP OU exhibits clinical presentation very similar to the Proband, with central macular atrophy in the differential diagnosis of GA secondary to AMD or CACD, with enlargement of the lesions 7 years after baseline. AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; GA, geographic atrophy; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 4.
 
Phenotypes of independent cases, heterozygous for the PRPH2 rs121918563 (L185P) variant (cases A–-D) and the PRPH2 rs281865373 (c.828+3A>T) variant (case E). Case A. (a, b) CFP OU show central pseudo-vitelliform macular lesions which evolved to small circular area of central RPE and outer retinal atrophy, also shown on (c, d) fundus autofluorescence (FAF) and (e, f) near IR as well as on (g, h) OCT typical of AVMD. Of note, IR images show a pattern configuration of pigmentary clumps. Case B. (a, b) CFP exhibits RPE irregularities in a butterfly shape OD and central atrophy OS. Case C. (a, b) FA OU show small central macular atrophy surrounded by increased fluorescence due to pigmentary mobilization and marked fluorescein staining, and (c, d) FAF OU shows small areas of distinct hypo-autofluorescence correspondent to the central macular atrophy, and increased areas of FAF in a distinct butterfly pattern correspondent to the areas of pigmentary mobilization. (e, f) OCT OU show small focal areas of RPE and outer retinal atrophy, and shallow “low-lying” RPE detachments in both eyes, without evident signs of exudation. (g, h) OCTA OU show macular NV. Case D. (a, b) Red-free retinography in both eyes show RPE abnormalities in a butterfly pattern configuration. (c, d) FAF OU show progression to central macular atrophy 9 years after baseline, and in the same visit (e, f) OCT OU show central macular atrophy and subretinal fibrosis secondary to macular NV. Case E. (a, b) CFP OU show multifocal areas of RPE atrophy. (c, d) CFP OU show that 6 years after baseline the areas of macular atrophy enlarged, resembling GA secondary to AMD earlier in the disease presentation but that could fit in the differential diagnosis of CACD as the disease progressed. AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; FAF, fundus autofluorescence; GA, geographic atrophy; IR, infrared reflectance; NV, neovascularization; OCT, optical coherence tomography; OCTA, optical coherence tomography angiography; OD, right eye; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 4.
 
Phenotypes of independent cases, heterozygous for the PRPH2 rs121918563 (L185P) variant (cases A–-D) and the PRPH2 rs281865373 (c.828+3A>T) variant (case E). Case A. (a, b) CFP OU show central pseudo-vitelliform macular lesions which evolved to small circular area of central RPE and outer retinal atrophy, also shown on (c, d) fundus autofluorescence (FAF) and (e, f) near IR as well as on (g, h) OCT typical of AVMD. Of note, IR images show a pattern configuration of pigmentary clumps. Case B. (a, b) CFP exhibits RPE irregularities in a butterfly shape OD and central atrophy OS. Case C. (a, b) FA OU show small central macular atrophy surrounded by increased fluorescence due to pigmentary mobilization and marked fluorescein staining, and (c, d) FAF OU shows small areas of distinct hypo-autofluorescence correspondent to the central macular atrophy, and increased areas of FAF in a distinct butterfly pattern correspondent to the areas of pigmentary mobilization. (e, f) OCT OU show small focal areas of RPE and outer retinal atrophy, and shallow “low-lying” RPE detachments in both eyes, without evident signs of exudation. (g, h) OCTA OU show macular NV. Case D. (a, b) Red-free retinography in both eyes show RPE abnormalities in a butterfly pattern configuration. (c, d) FAF OU show progression to central macular atrophy 9 years after baseline, and in the same visit (e, f) OCT OU show central macular atrophy and subretinal fibrosis secondary to macular NV. Case E. (a, b) CFP OU show multifocal areas of RPE atrophy. (c, d) CFP OU show that 6 years after baseline the areas of macular atrophy enlarged, resembling GA secondary to AMD earlier in the disease presentation but that could fit in the differential diagnosis of CACD as the disease progressed. AMD, age-related macular degeneration; CACD, central areolar chorioretinal dystrophy; CFP, color fundus photographs; FA, fluorescein angiography; FAF, fundus autofluorescence; GA, geographic atrophy; IR, infrared reflectance; NV, neovascularization; OCT, optical coherence tomography; OCTA, optical coherence tomography angiography; OD, right eye; OS, left eye; OU, both eyes; PRPH2, peripherin-2 gene; RPE, retinal pigment epithelium.
Figure 5.
 
(A) Violin plots showing initial and final visual acuity (logMAR) for individuals heterozygous for PRPH2 rs281865373 (c.828+3A>T) (orange) and PRPH2 rs121918563 (L185P) (blue) variants. The width of each violin plot is proportional to the density for the range of values and the medians are represented by horizontal bars within the boxplots. (B) Comparison of change in visual acuity (logMAR) per year between individuals heterozygous for PRPH2 rs281865373 (c.828+3A>T) (orange) and PRPH2 rs121918563 (L185P) (blue) estimated using a mixed effects linear regression with a compound symmetry correlation structure. Standard error is illustrated by vertical error bars (***P < 0.001). logMAR, logarithm of the minimal angle of resolution; PRPH2, peripherin-2 gene.
Figure 5.
 
(A) Violin plots showing initial and final visual acuity (logMAR) for individuals heterozygous for PRPH2 rs281865373 (c.828+3A>T) (orange) and PRPH2 rs121918563 (L185P) (blue) variants. The width of each violin plot is proportional to the density for the range of values and the medians are represented by horizontal bars within the boxplots. (B) Comparison of change in visual acuity (logMAR) per year between individuals heterozygous for PRPH2 rs281865373 (c.828+3A>T) (orange) and PRPH2 rs121918563 (L185P) (blue) estimated using a mixed effects linear regression with a compound symmetry correlation structure. Standard error is illustrated by vertical error bars (***P < 0.001). logMAR, logarithm of the minimal angle of resolution; PRPH2, peripherin-2 gene.
Table 1.
 
Functional Interpretation of PRPH2 Variants Associated With AMD Status
Table 1.
 
Functional Interpretation of PRPH2 Variants Associated With AMD Status
Table 2.
 
Filtering of Variants in Exome Sequencing Data
Table 2.
 
Filtering of Variants in Exome Sequencing Data
Table 3.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree A With PRPH2 Rare Variant rs121918563 (L185P)
Table 3.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree A With PRPH2 Rare Variant rs121918563 (L185P)
Table 4.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree B With PRPH2 Rare Variant rs281865373 (c.828+3A>T)
Table 4.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree B With PRPH2 Rare Variant rs281865373 (c.828+3A>T)
Table 5.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree C With PRPH2 Rare Variant rs281865373 (c.828+3A>T)
Table 5.
 
Demographic, Clinical, and Genetic Characteristics of Pedigree C With PRPH2 Rare Variant rs281865373 (c.828+3A>T)
Table 6.
 
Demographic, Clinical, and Genetic Characteristics of Independent Cases With PRPH2 Rare Variants
Table 6.
 
Demographic, Clinical, and Genetic Characteristics of Independent Cases With PRPH2 Rare Variants
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