Introduction
Leber Congenital Amaurosis/Early-Onset Severe Retinal Dystrophy (LCA/EOSRD) are genetically and phenotypically heterogeneous groups of inherited retinal diseases, with widely overlapping features. Clinical presentation includes severe congenital/early infancy visual loss, nystagmus, amaurotic pupils and a markedly abnormal or undetectable full-field electroretinogram (ERG) [
1]. The most common causative genes are
CEP290 [
2],
GUCY2D [
3],
CRB1 [
4], and
RPE65 [
5‐
8]
. In total, the reported LCA/EOSRD-associated genes (n = 25) account for approximately 70–80% of cases [
6,
7], with more genes yet to be identified.
Advances in ocular genetics, retinal imaging and molecular biology, have been pivotal in developing treatments for inherited retinal diseases. The first FDA- and EMA-approved gene therapy is available for LCA/EOSRD-associated with
RPE65 [
9,
10]
, and there are multiple other trials underway for LCA/EOSRD and other inherited retinal diseases. LCA/EOSRD is a disease with arguably the poorest visual prognosis. It is of paramount importance to molecularly characterize LCA/EOSRD patients, in order to facilitate access to, and potential benefit from, the on-going advances in the field.
Herein we describe the detailed retinal phenotype of five cases, from four unrelated families, with LCA/EOSRD harboring disease-causing sequence variants in genes not usually associated with the disease. We thereby extend the phenotypic spectrum associated with PRPF8, PRPH2, RP1, and RPGR, and the genotypic spectrum of LCA/EOSRD.
Discussion
Herein we presented in detail five cases, with the clinical diagnosis of LCA/EOSRD and the molecular confirmation for disease-causing variants in genes either only very rarely or not previously associated with the disease.
PRPF8 (OMIM: 607300) encodes for precursor mRNA-processing factor 8, a spliceosome component [
16], a gene known to cause autosomal dominant retinitis pigmentosa (ADRP, RP13 MIM: 600059). The
PRPF8 variant (c.5804G > A, p.Arg1935His) identified in our patient, has been previously reported in the heterozygous state in a patient with childhood-onset ADRP [
17], as well as in 3 patients with ADRP screened by the Manchester Centre for Genomic Medicine. Recently, in a large EOSRD/LCA genotyping study, a patient with EOSRD was genotyped as
PRPF8 (p.X2336Serext*41, age of onset: 3 years old) [
7]. However the presented clinical information is limited to age of onset and the clinical diagnosis of EOSRD/LCA. The patient described in the current report is the youngest patient affected with
PRPF8-associated disease (including the identified families in Moorfields Eye Hospital, n = 15) [
18], and the first detailed clinical report in the literature.
PRPH2 (OMIM: 179605) is a five-exon gene encoding peripherin-2, a cell surface glycoprotein in the outer segments, with an essential role in disc morphogenesis [
19‐
21].
PRPH2 is associated with a wide range of clinical phenotypes, including: AD central areolar choroidal dystrophy (CACD, MIM: 613105), AD macular pattern dystrophy (MPD, MIM: 169150), AD vitelliform macular dystrophy (MIM: 608161), AD/AR retinitis punctata albescens (MIM: 136880), AD/AR RP (MIM: 608133) and AR LCA/EOSRD (LCA18, MIM: 608133). Homozygous variants in
PRPH2 have been reported in two individuals with LCA (c.637 T > C, p.C213R and c.554 T > C, p.L185P) [
22]. Family members heterozygous for the variant were asymptomatic but showed butterfly-shaped MPD on clinical examination [
22]. In a pedigree harboring the variant p.Gln238Ter, intrafamilial variability was also observed; with the heterozygous members exhibiting CACD and MPD, and the patient in the homozygous state exhibiting LCA/EOSRD [
23]. The reported homozygous variant herein is novel to the best of our knowledge (c.620_627delinsTA, p.Asp207_Gly208del). In our pedigree, clinical examination and detailed retinal imaging was performed in heterozygous parents, with only a small drusenoid deposit identified in the one eye of the father of patient 2 at age of 28 years. We provide the detailed phenotype of the fourth patient described with LCA18, and consolidate
PRPH2 as a cause of LCA; with previous descriptions often lacking clinical detail.
RP1 (OMIM 603937) encodes for oxygen-regulated photoreceptor protein 1 (ORP1), and has been associated with ADRP and ARRP (RP1, MIM: 180100) [
24,
25]. The majority of cases in the literature follow an AD mode of inheritance [
26]. Previous studies reported that the development of ADRP and ARRP depended on the location of the
RP1 variants [
25,
26]. Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that leads to a degradation of the transcripts with introns in the 3′ untranslated region, preventing the synthesis of truncated proteins that may have toxic effects, such as dominant negative interactions [
27,
28]. Chen et al. suggested four classes of truncation variants in the
RP1 gene with different effects on the inheritance mode of RP: (i) Class I are NMD-sensitive truncations located in exons 2 and 3, (ii) Class II are NMD-insensitive truncations located in a region spanning approximately p.500 to p.1053 in exon 4 (deleterious—dominant negative effect), (iii) Class III are NMD-insensitive truncations located in the regions p.264 to p.499 and p.1054 to p.1751 (loss-of-function, ARRP), and iv) Class IV includes NMD-insensitive truncations located after p.1816 in exon 4, with the resulting truncated proteins expected to have normal function [
25]. The identified novel variant in the current study (c.4147_4151delGGATT) is a class III variant according to the aforementioned classification, and is expected to cause AR disease in the homozygous state and carriers to be asymptomatic, in agreement with the presented pedigree. Initially class IV variants were perceived as non-pathogenic and only recently have been associated with AR macular dystrophy and cone-rod dystrophy [
29]. Kabir et al. reviewed all published
RP1 variants (2016) and observed that the heterozygous variants responsible for ADRP resided between amino acid residues 617–1551, and the homozygous variants responsible for ARRP cluster in two regions: amino acids 193–736 and amino acids 1243–1890 [
26]. The overlap of ADRP and ARRP phenotypes for variants in the amino acid residues 617–736 and 1243–1551, lead to the speculation that the nature of the variant and not only its location dictates the inheritance pattern. Recently one case of EOSRD (age of disease onset: 4 years old) was reported with compound heterozygous class III variants [
7]. Our truncating variant (within the 1243–1551 amino acids group), presented with the earliest disease onset of all the reported cases in the literature and the clinical diagnosis of LCA/EOSRD, in both siblings; further extending the genetic and clinical spectrum and validating
RP1 as a rare cause of LCA/EOSRD.
RPGR (OMIM: 312610) encodes for retinitis pigmentosa GTPase regulator and is a nineteen exon gene that gives rise to two alternatively spliced retinal isoforms, encoded by exons 1–19 and 1–15 (+ part of intron 15) respectively [
30]. The latter isoform, also known as exon open reading frame 15 (ORF15), is the most highly expressed retinal variant and a mutational hotspot [
31‐
33]. Most disease-causing variants in
RPGR result in RP (RP3, MIM: 300029) [
34], but those leading to cone and cone-rod dystrophy (MIM: 304020) are preferentially located at the 3′ end of the ORF15 region [
35,
36].
RPGR variants lead also to atrophic macular degeneration (MIM: 300834) and ciliopathy (MIM: 300455) [
36]. Identical intrafamilial sequence variants in
RPGR may lead to distinctly different phenotype [
37,
38]. Our
RPGR patient is a female, and while female
RPGR carriers can be affected to a variable extent, depending on X-chromosome inactivation ratios, carriers are usually mildly affected or unaffected [
39], whereas some can progress to moderate or severe vision loss after the third decade of life [
40]. A high proportion of adult female carriers of XLRP manifest significant ERG abnormalities [
41], although typically much milder than in male patients and rarely as severe as in case 5, even in older individuals. In most female heterozygotes, retinal dysfunction is manifest as asymmetrical LA 30 Hz flicker ERG delay and reduction in rod-mediated scotopic strong flash ERG a-waves, the asymmetry being highly unusual for a genetically-determined retinal disorder
. Phenotypic diversity can be attributed to a certain extend to genetic factors (
i.e. allelic heterogeneity and genetic modifiers) [
38]. Single nucleotide polymorphism (SNP) screen of
RPGR patients displaying varying disease severity showed that SNPs in
IQCB1 (I393N) and
RPGRIP1L (R744Q) may be associated with disease severity [
38]. Our patient was negative for both those SNPs, however, no study to date has performed whole genome sequencing to identify genome wide modifiers in a cohort of
RPGR patients. Our patient represents the first report of an
RPGR variant causing LCA/EOSRD, with the family being another example of marked intrafamilial phenotypic variability.
At present, whilst there are no specific proven treatments for the genotypes discussed herein, several avenues of intervention show promise for molecularly characterized patients, with several challenges and limitations including mode of inheritance and gene size. The AD inheritance of
PRPF8 limits the utility of 'simple' gene augmentation therapy and requires more complex approaches, including gene editing technology, which are not as advanced. In contrast, adeno-associated viruses (AAV) and compacted DNA nanoparticles carrying
PRPH2 have been successfully used to mediate gene transfer and improvement in the
rds−/− and
rds+/− mouse models [
42]. Successful integration and material transfer of donor- or stem cell-derived cone photoreceptors in Prph2
rd2/rd2 murine models of the disease are also promising [
43]. A pharmacological approach, with inhibition of the overactive poly-ADP-ribose polymerase (PARP), with the PARP inhibitor PJ34, has demonstrated a decrease in the levels of poly-ADP-ribosylation and photoreceptor cell death, in this same model [
44]. For recessive
RP1 disease, gene supplementation may be a potential therapeutic intervention; however,
RP1 consists of 6468 base pairs, making it too large for current AAV vectors [
45]. Human treatment trials of gene replacement therapy are already underway for
RPGR-associated RP (NCT03252847, NCT03116113, and NCT03316560).
The current study provides clinical and genetic evidence, which extends the phenotypic spectrum of PRPF8-, PRPH2-, RP1-, and RPGR-associated disease, and the genotypic spectrum of LCA/EOSRD. All cases had disease onset within the first year of life, with lifelong morbidity for those patients. Molecular genetics, segregation results, and retinal and functional phenotypes are presented in detail, the latter revealing unexpectedly severe disease in some cases. The study emphasizes the critical need for molecular confirmation of disease but also highlights phenotypic variability and the need for detailed electrophysiological and retinal characterization. Comprehensive phenotyping of patients with inherited retinal disease is of vital importance to identify suitable candidates and to enable benefit from future therapeutic advancements.
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