From left to right, images are color photographs, fundus autofluorescence photographs, and optical coherence tomography images. A, Images of patient AR6; visual acuity, 1.3 OD and 1.3 OS; c.199C>T variant. B, Images of patient AR5; visual acuity, 1.6 OD and 1.4 OS; c.1354dup variant. C, Images of patient AR9; visual acuity, 1.7 OD and 1.6 OS; c.1142-1G>A variant. D, Images of patient AR10; visual acuity, light perception in both eyes; c.1853T>G variant. E, Images of patient AD5; visual acuity, 0.1 OD and 0.0 OR; c.1117C>T variant.
The mean logMAR score between the right and left eyes is plotted against age. CF indicates counting fingers; HM, hand movements; NPL, no perception of light; and PL, perception of light.
The PROM1 protein is shown as a white bar with the respective protein domains depicted in different colors. Recessive variants are shown above, and dominant variants are shown below. Variants from the current study appear in bold, and novel variants appear in red. For splice site and frameshift variants, the arrow indicates the location of the first affected amino acid. Variants without a reliable prediction on the protein (eg, splice site) are marked as p.? with their c. nomenclatures shown underneath.
OS indicates outer segment.
eTable 1.PROM1 Variants Reported to Date Including Their Respective References
eTable 2. Detailed Description of Sequence Variations in PROM1 in the Series With Predicted Effect on Protein Function
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Cehajic-Kapetanovic J, Birtel J, McClements ME, et al. Clinical and Molecular Characterization of PROM1-Related Retinal Degeneration. JAMA Netw Open. Published online June 14, 20192(6):e195752. doi:10.1001/jamanetworkopen.2019.5752
What are the clinical and molecular characteristics of PROM1-related retinal degeneration?
In this case series of 19 patients with PROM1-related retinal degeneration, recessive variants were associated with early-onset, severe panretinal degeneration, whereas the dominant disease was associated with the c.1117C>T variant and a late-onset, milder phenotype that predominantly involves the macula. In addition, the dominant variant was preferentially associated with cone photoreceptors.
A better understanding of the clinical and molecular characteristics of PROM1-related retinal degeneration may aid development of future treatments, including gene therapy and optogenetics.
The PROM1 gene, commonly associated with cone-rod dystrophies, may have dominant or recessive phenotypes that influence disease onset and severity.
To characterize the clinical phenotype and molecular genetic variations in patients with PROM1 variants.
Design, Setting, and Participants
This case-series study was conducted at 2 specialist retinal genetics clinics and examined 19 consecutively enrolled patients with PROM1-related retinal degeneration. Data were collected and analyzed from May 2018 to December 2018.
Main Outcomes and Measures
Results of ophthalmic examination, retinal imaging, and molecular genetic analysis by next-generation sequencing.
Of 19 patients, 13 (68%) were women, and age ranged from 11 to 70 years. All patients presented with central visual loss, with or without photophobia. Individuals with recessive variants commonly had severe loss of visual acuity by their 20s, whereas the dominant variant was associated with a milder phenotype, with most patients retaining good vision into late adulthood. The recessive cases were associated with a panretinal dystrophy of cone-rod phenotype with early macular involvement, whereas the dominant variants were associated with a cone-rod phenotype that was restricted to the macula with predominantly cone dysfunction. Next-generation sequencing identified 3 novel and 9 previously reported variants in PROM1. Recessive mutations included 6 truncating variants (3 nonsense and 3 frameshift), 4 splice site variants, and 1 missense variant. All 6 dominant variants were associated with a c.1117C>T missense variant. The variants were distributed throughout the PROM1 genomic sequence with no specific clustering on protein domains.
Conclusions and Relevance
In this case-series study, PROM1 recessive variants were associated with early-onset, severe panretinal degeneration. The similar phenotypes observed in patients with homozygous missense variants and splice site variants compared with similarly aged patients with truncating variants suggests that all recessive variants have a null (or loss of function close to null) outcome on PROM1 function. In contrast, the dominant missense cases were associated with a milder, cone-driven phenotype, suggesting that the dominant disease is preferentially associated with cones. This has implications for the development of treatments for this severely blinding disease, and adeno-associated viral vector–based gene therapy and optogenetics could become successful treatment options.
Inherited retinal degenerations are a heterogeneous group of disorders that lead to the progressive degeneration of photoreceptors and loss of vision. On the more severe end of the spectrum lie cone-rod dystrophies, in which there is preferential involvement of the central retina with early manifestations of cone-driven symptoms, including photophobia, dyschromatopsia, reduced visual acuity, and central scotomas. As the disease progresses, there is variable involvement of rods, affecting peripheral and night vision.
Prominin 1 (PROM1; OMIM 604365) is commonly associated with cone-rod dystrophies.1 However, throughout the literature, variations in PROM1 have been implicated in extremely varied and overlapping phenotypes that have been described as retinitis pigmentosa2-8; retinitis pigmentosa with macular involvement9; retinal dystrophy8; cone-rod dystrophy1,8,10-17; bull’s-eye maculopathy18; bull’s-eye maculopathy associated with rod, rod-cone, and macular dystrophy19; Stargardt-like disease20-23; macular dystrophy24; and maculopathy with rod-cone dystrophy.25 The age at onset, presenting symptoms, and severity of disease vary with sequence variations. Moreover, variants can be inherited in recessive and dominant fashions.15
Despite many genetic reports that focus on identifying PROM1 variants, the genotype-phenotype correlation remains poorly understood. In addition, the molecular roles of these sequence variations and their effects on protein function are largely unknown. Moreover, it remains unclear if PROM1 has a more important role in cone or rod photoreceptors and how variants in the same gene can have a recessive and/or dominant effect. The aim of this study is to analyze the sequence variations in the PROM1 gene in a series of patients to understand better their potential for pathophysiological consequences on retinal degeneration. This has important implications in the development of potential gene-based therapies and optogenetics for this complex genetic condition, especially as the successful treatment of macular photoreceptors could be sufficient to preserve good visual acuity in affected individuals.
The study design adhered to the tenets of the Declaration of Helsinki.26 This study follows the reporting guideline for case series. Institutional review boards at Oxford University Hospitals and the University of Bonn approved the studies, and patients provided written informed consent.
Patients were identified from genetic databases between July 1, 2014, and May 1, 2018, at 2 clinical genetic centers in Oxford, United Kingdom, and Bonn, Germany. The data analysis was conducted from May 1, 2018, to December 1, 2018. Medical records of patients with PROM1 variants were reviewed for information on family, general history, and ophthalmic history. Details of clinical assessments, including visual acuity and dilated fundal examinations, were collected. Retinal imaging studies, including color photography, fundus autofluorescence (55° and 30°), and optical coherence tomography, were taken with the Heidelberg Spectralis system (Heidelberg Engineering). In addition, widefield fundus imaging was performed with the Optos 200Tx confocal scanning laser ophthalmoscopy camera (Optos).
Sequence variations in PROM1 were identified by targeted next-generation sequencing techniques. For patients at the Oxford site, enrichment for PROM1 was achieved as part of a customized HaloPlex enrichment system kit (Agilent Technologies) designed to capture the coding exons and 10 base pairs of the flanking introns of 117 retinal genes. HaloPlex reactions were prepared per manufacturer’s instructions. Libraries were pooled into batches of 14 and sequenced on an Illumina MiSeq instrument (Illumina) using a MiSeq version 3 kit per manufacturer’s instructions. Reads were aligned using Burrows-Wheeler Aligner 2,27 and variants were called using Platypus.28 All variants identified by next-generation sequencing were confirmed by Sanger sequencing. For patients at the Bonn site, the next-generation sequencing methodology was performed as described previously.1
Sequence variations were assessed for pathogenic association with protein function based on in silico analysis using Alamut Visual (Alamut Interactive Biosoftware). Nonsense variants and frameshift variants were considered pathogenic unless at the 3′ end of the gene. The likely pathogenicity of missense variants was crosschecked against the PolyPhen-2, SIFT, and Grantham matrix (for those variants that led to a stop codon) algorithm scores. The allelic frequency of variants was evaluated in gnomAD, which includes the Exome Aggregation Consortium data set. Geneious bioinformatics software version 11.1.5 (Geneious) was used for the PROM1 nucleotide sequence analysis and mapping of specific variants to protein domains. For patients with autosomal recessive inheritance patterns and compound heterozygous variants (6 of 13 patients [46%]), no further genotyping analysis was carried out in individuals’ parents to exclude the rare possibility that the 2 variants were present on the same chromosome.
Overall, 19 patients (13 [68%] women) aged 11 to 70 years were included in this study. Inheritance was autosomal recessive in 13 patients (68%) and autosomal dominant in 6 patients (32%). Ethnic origins included 11 British patients (58%), 5 German patients (26%), 2 Turkish patients (11%), and 1 Ukrainian patient (5%). Details of participant demographic characteristics, history, clinical findings, and genotype analysis are summarized in the Table. Most participants (17 [89%]) were unrelated, except AD4 and AD5, who are related.
The clinical phenotypes of recessive and dominant variant classes are shown in Figure 1. Independent of variant class (ie, nonsense, frameshift, splice site, and missense), recessive genotypes showed severe panretinal dystrophy with macular involvement and peripheral pigment spicules. Fundus autofluorescence showed areas of macular hypoautofluorescence with variable degrees of patchier hypoautofluorescence patterns in the periphery. Optical coherence tomography revealed widespread loss or thinning of the outer retina in all recessive genotypes. In contrast, at a comparable age, the dominant genotype showed much milder changes, largely restricted to the macula on fundus autofluorescence and optical coherence tomography imaging, and no peripheral bone spicule pigmentations.
The age at onset of retinal degeneration ranged from early childhood (recessive genotype) to 40 years (dominant genotype), with all patients presenting with central visual loss, indicating early cone involvement. Photophobia was present in 6 patients (32%) with recessive and dominant genotypes. The association of visual acuity with patient age is depicted in Figure 2 and eTable 1 in the Supplement, both of which include, to our knowledge, all PROM1 cases reported with sufficient data to date. There is a decline in visual acuity in recessive variants from an early age, with visual acuity of 1.0 logMAR (Snellen equivalent, 20/200) or less in patients older than 20 years, and most patients having visual acuity of counting fingers or less after the age of 30 years. In contrast, dominant PROM1 mutations in this series were associated with a later onset and a slower decline in visual function, with most patients having visual acuity of at least 1.0 logMAR at the time of presentation. (Three of 6 patients with the dominant variant were in their 50s.) Previously reported19,24 dominant PROM1-related dystrophies (2 variants, c.1117C>T [31 patients] and c.2485G>A [1 patient]) show a similar trend.
Other ophthalmic features appeared in patients with recessive variations including myopia (4 patients [21%]), cataract or history of cataract extraction (8 patients [42%]), nystagmus (4 patients [21%]), oscillopsia (1 patient [5%]), and strabismus (1 patient [5%]). There were no consistently associated systemic diseases, although 1 patient had polymyalgia rheumatica and another had psoriasis.
We identified 12 mutations in PROM1 that may be associated with the disease phenotype, of which 3 were novel (Table) (eTable 2 in the Supplement). All patients with dominant inheritance had the known c.1117C>T missense variant in PROM1. In patients with autosomal recessive disease, 6 variants were truncating variants (3 nonsense and 3 frameshift), 4 were splice site variants, and 1 was a missense variant (Table). Overall, 7 of 13 patients with recessive disease were homozygous for the identified PROM1 variant.
Detailed in silico analysis confirmed a pathogenic association with protein function, with all sequence variations having a highly likely pathogenic effect, including the supporting evidence for missense variants from the PolyPhen2, SIFT, and Grantham matrix algorithm scores (eTable 2 in the Supplement). The novel variants were not previously reported in the literature, and they were crosschecked against normal variants based on the gnomAD dataset (eTable 2 in the Supplement).
A schematic diagram of all PROM1 mutations reported to date depicts their distribution throughout the protein, with no specific clustering relative to protein domains (Figure 3) (eTable 1 in the Supplement). Most PROM1 variants (34 of 41 reported with known inheritance) were found to be recessive (31 of 34: 11 nonsense variants, 9 frameshift variants, 8 splice site variants, and 3 missense variants), whereas only 3 of 34 were dominant (all missense variants). The dominant cases in the current study had the same variant (p.Arg373Cys). The inheritance pattern of reported variant p.Leu3fs*15 remains unclear (excluded from Figure 3) (shaded in green in eTable 1 in the Supplement). The inheritance pattern of 6 variants is not available in the reporting studies8,23 (excluded from Figure 3) (shaded in yellow in eTable 1 in the Supplement).
In this case-series study, we analyzed the sequence variations in the PROM1 gene, and we identified 3 novel sequence variations. The types of variant as well as the mode of inheritance were associated with disease phenotype, age at onset of symptoms, and severity of retinal degeneration.
The PROM1 gene encodes a pentaspan transmembrane domain glycoprotein,29 which is expressed ubiquitously in plasma membrane protrusions. It is best known as a surface marker of endothelial progenitor, hematopoietic stem cells (AC133 and CD133) and cancer cells in the central nervous system. In retinal photoreceptors, it has a critical structural role. The protein specifically localizes to membrane protrusions at the base of rod and cone outer segments, where it plays a key role in disc morphogenesis30 and subsequent photopigment sorting.31 More recently, PROM1 has been found to be associated with the regulation of photoreceptor autophagy in retinal pigment epithelium cells.32
Given the ubiquitous expression of PROM1, a remarkable feature of PROM1-associated retinal dystrophy in the current series was the absence of an extraocular phenotype. We found no syndromic cases and 2 systemic conditions that were unlikely to be associated with PROM1 expression. This is in keeping with the current literature, with only 1 study reporting a family with the p.Arg373Cys variant having some subclinical systemic features.33 Despite the rarity of an observed extraocular PROM1 phenotype, we nonetheless need be aware of possible systemic manifestations of the PROM1 variant when faced with such patients in a genetic clinic. In our study, we noted that, among other ocular features, one-third of patients had myopia. Although it is plausible that PROM1 is implicated in the pathogenicity of myopia, a more likely explanation is that the myopia is induced by sensory deprivation owing to blurring of vision in patients with PROM1 sequence variations.
Although previous genetic reports have described numerous PROM1-associated retinal dystrophy phenotypes,1-25 in the current study, we found that the morphological phenotype was associated with cone-rod dystrophy in all cases. The main distinction in phenotypes lies between recessive and dominant forms of the disease. The recessive disease was associated with early-onset, severe panretinal degeneration with early central loss of vision, whereas the dominant disease was associated with late-onset dystrophy predominantly involving the macula. In contrast to some previously reported cases,2,3 we did not find any patients who presented with night vision problems that would suggest early rod-driven functional deficiencies. It is possible, owing to the relatively fast progression of PROM1-associated recessive cone-rod dystrophy, that cone and rod dysfunction occur in close succession or simultaneously, making it difficult for patients to distinguish which symptoms (ie, central blurring of vision or nyctalopia) came first. In addition, as the disease progresses, pigmentation indistinguishable from rod-cone dystrophy appears in the retinal periphery.
Our analysis of PROM1 variations showed that the entire protein is associated with sequence variations, with no major clustering in a domain-dependent manner, suggesting that the entire protein is important to its function. The severe homozygous recessive phenotype is likely associated with null variants that abolish the function of alleles, leading to the absence of PROM1. Most frameshift and nonsense variants result in a premature stop codon, leading to a truncated modified RNA that is quickly degraded by nonsense-mediated decay before undergoing translation.2 In addition, the missense and splice site variants involved in the recessive disease are likely associated with a null (or loss of function close to null) effect, as evident from the similar phenotypes in our patients with homozygous missense and splice site variants compared with similarly aged patients with truncating mutations. These loss-of-function variants are associated with disorganized optic disc membranes and photoreceptor degeneration, as shown in PROM1 knockout mice.31
In contrast, a milder, dominant phenotype, as observed in our study as well as in most other studies, is associated with a dominant negative effect of a missense variant. This appears to result in a stable variant protein that is associated with the mislocalization of the variant protein. It also appears to interfere with the function of the wild-type protein as well as CDHR1 and actin,30 exerting a dominant negative effect. As cysteine residues are evolutionarily conserved in PROM1, it is possible that the addition of another cysteine residue in p.Arg373Cys disrupts disulfide bridges, impairing homophilic protein interactions. The cone-associated phenotype observed in the dominant disease suggests that the dominant variant is preferentially associated with cones. A 2008 study30 that implicates PROM1 in disc morphogenesis used a transgenic mouse model carrying the Arg373Cys variant expressed in rods only under the control of rhodopsin promoter, thereby making it difficult to study the direct effect of this variant in cones. However, in a PROM1 knockout mouse model,31 the null protein impairs disc morphogenesis as well as causes ectopic accumulation of visual pigment in rods and cones. Since cone opsins are more prone to mislocalization than rod opsins,34 this could explain why cones become affected first and with greater severity in PROM1 degeneration. In addition, there is some evidence of a differential distribution of wild-type PROM1 protein between rods and cones.29,35 In contrast to rods, the cones’ outer segment lamellar membrane is suggested to have a wide distribution of PROM1 (Figure 4). The open lamellar structure of cones’ outer segments makes cone proteins more exposed to extracellular space and to interference from other proteins, predisposing cones to premature degeneration.
Clarifying the mechanisms underlying PROM1 degeneration has important implications in the development of potential treatments. Subretinal delivery of adeno-associated viral vector–carrying PROM1 (2.5 kilobase pairs) to photoreceptors at an early stage of recessive dystrophy could replace the null protein and potentially rescue the phenotype. However, the dominant variant would need to be silenced first, eg, RNA silencing via a mirtron,36 followed by a gene replacement therapy of the wild-type protein, ie, block-and-replace therapy. In more advanced instances of disease, where irreversible photoreceptor damage has occurred, restoring sight by optogenetic treatment37 may be an alternative therapy. Moreover, the ubiquitous expression of PROM1 will facilitate the development of in vitro models of PROM1 gene therapy and related functional assays.
This noninterventional case series of 19 patients with PROM1 variants is limited by the retrospective, uncontrolled nature of the study. In our series and in other reports (with the exception of 2 cases), the dominant disease was caused by a single c.1117C>T variant. Nonetheless, our study, which, to our knowledge, is the largest reported PROM1 case series to date, adds substantially to the knowledge and understanding of PROM1-related retinal disease and possible future therapies. Further longitudinal studies will shed more light on the natural progression of the disease and the timing of therapeutic interventions.
To our knowledge, this is the largest reported case series on PROM1-related retinal degeneration to date. This study shows that PROM1 recessive variants were associated with early-onset, severe retinal degeneration, whereas the c.1117C>T variant, which was associated with autosomal dominant inheritance, showed a milder, cone-driven phenotype. This clinical and molecular characterization has deepened understanding of the disease and will aid in the design of future randomized clinical trials and therapeutic approaches.
Accepted for Publication: April 30, 2019.
Published: June 14, 2019. doi:10.1001/jamanetworkopen.2019.5752
Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2019 Cehajic-Kapetanovic J et al. JAMA Network Open.
Corresponding Author: Jasmina Cehajic-Kapetanovic, FRCOphth, PhD, Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, Oxford University, Level 6 West Wing, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, United Kingdom (email@example.com).
Author Contributions: Dr MacLaren had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Concept and design: Cehajic-Kapetanovic, MacLaren.
Acquisition, analysis, or interpretation of data: Cehajic-Kapetanovic, Birtel, McClements, Shanks, Clouston, Downes, Charbel Issa.
Drafting of the manuscript: Cehajic-Kapetanovic, MacLaren.
Critical revision of the manuscript for important intellectual content: Cehajic-Kapetanovic, Birtel, McClements, Shanks, Clouston, Downes, Charbel Issa, MacLaren.
Statistical analysis: MacLaren.
Obtained funding: MacLaren.
Administrative, technical, or material support: Cehajic-Kapetanovic, Birtel, McClements, Shanks, Clouston.
Conflict of Interest Disclosures: Dr McClements reported receiving personal fees from and consulting for Nightstar Therapeutics outside the submitted work. Dr Downes reported receiving grants from Retina UK and Fight for Sight outside the submitted work. No other disclosures were reported.
Funding/Support: This work was supported by the National Institute for Health Research and Oxford Biomedical Research Centre. Dr Cehajic-Kapetanovic is a recipient of the Global Ophthalmology Awards Programme Fellowship.
Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Disclaimer: The authors alone are responsible for the content and writing of this article. The views expressed are those of the authors and not necessarily those of the National Health Service, the National Institute for Health Research, or the Department of Health.
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