Retinal dystrophies are a complex group of disorders characterized by the degeneration of retinal tissue and visual dysfunction. These diseases can be classified into three broad categories based upon the primary photoreceptor cell type that is affected. In rod and rod-cone dystrophies, the rods are primarily affected, and patients present with nyctalopia and progressive peripheral vision loss that may manifest at varying ages.1 Clinical findings include pigmentary changes, vascular attenuation, and optic nerve palor.2 In cone and cone-rod dystrophies, photopic and color vision are affected primarily, often early in life. On examination, the macula, which is involved more prominently than the periphery, may appear atrophic or may present with retinal pigment deposits.1 Generalized retinal dystrophies involve degeneration of both rod and cone function typically resulting in severe, progressive loss of vision, often from an early age.
Determining the genetic etiologies of retinal dystrophies is complex due to phenotypic heterogeneity and the number of genetic variants/mutations associated with retinal pathology. In the era of gene therapy, identifying causative mutations has become clinically relevant, particularly since the U.S. Food and Drug Administration approval of voretigene neparvovec-rzyl (Luxturna; Spark Therapeutics, Philadelphia, PA), an adeno-associated viral vector based gene therapy for the treatment of Leber's congenital amaurosis (LCA) due to biallelic RPE65 mutations.3 Although most cases cannot currently be treated with gene therapy, genetic testing helps foster understanding of phenotypic and genotypic characteristics of retinal dystrophies. With the advent of next generation sequencing, genetic screens can now be offered to patients in a rapid, cost-effective manner.4–7
In this study, the frequency of mutation in 31 genes commonly associated with inherited retinal disorders was investigated in a cohort of 37 patients with a clinical diagnosis of retinal dystrophy. Patients' phenotypic characteristics were also collected and analyzed.
Patients and Methods
A cohort of 37 patients, aged 15 to 79, who carry a clinical diagnosis of retinal dystrophy underwent genetic testing. All patients were evaluated by and received their genetic testing from practitioners at a retina-only group practice. The initiative was free of charge to patients and insurers and was sponsored by Spark Therapeutics and executed by Prevention Genetics. A comprehensive genetic counseling program was also provided free of charge. Clinical data were extracted from electronic medical records and included disease onset and progression, associated systemic diseases, visual acuity (VA), color vision testing, ophthalmoscopic findings, and the results of imaging tests, and electroretinogram (ERG) findings when available.
Results of genetic testing were analyzed, and the specific mutations and their zygosities were recorded for each individual who tested positive for a variant in any of the target genes. The frequency of involvement of individual genes regardless of specific mutation was also calculated. Gene variants were excluded from data analysis if they did not result in a change in the affiliated protein (eg, the mutation was synonymous and did not result in an amino acid substitution, or the change occurred in an intronic region of the affected gene).
The Snellen VA of each patient at the time of genetic testing was recorded. Patients' color vision, if available, was reported as the percent of Ishihara plates that were correctly identified for each eye out of either 10 or 13 total plates.
Clinical diagnoses were determined by patient symptoms and exam findings. ERG findings were reported when they were available. The patient-reported age of symptom onset was recorded. For those patients who described the onset of symptoms as starting within a certain decade of life (eg, 20s, 30s, etc.), the midpoint of the decade (eg, 20s as 25) was recorded. Duration of disease was calculated by subtracting the reported age of onset from the age of the patient on the visit closest to when genetic testing was conducted.
The patients' symptoms were extracted from the office notes that corresponded to the most recent office visit relative to the date of genetic testing. The frequency of each reported symptom, as well as the percentage of patients who experienced each symptom, was then calculated and recorded. Of the patients who reported each symptom, the number of those patients who tested positive for a mutation in one or more of the genes examined was also recorded. The same procedure was used to analyze the frequency of fundus exam and optical coherence tomography (OCT) findings.
Of the 37 patients in the cohort, 18 (48.6%) tested positive for a variant(s) in one or more of the 31 genes tested. Variants were found in 14 genes. The frequency of those variations is depicted in Figure 1. USH2A was the most frequently mutated gene (22.9%). The age of symptom onset was comparable for both gene-positive and gene-negative patients (median age: 25 and 24, respectively). Figure 2 represents the age distribution of symptom onset as a function of gene mutation. The data demonstrated heterogeneity between genotypic characteristics and onset of clinical symptoms. Mean VA was 0.61/0.67 and 0.55/0.65 (right eye [OD]/left eye [OS]) in gene-positive and gene-negative patients, respectively. Mean VA was similar between groups. Results of color vision testing, although variable among patients (range: 0% to 100%), was similar between gene-positive and gene-negative patients (average percentage correct OD/OS: 75%/75% in gene-positive patients, 61%/69% in gene-negative patients) and tended to be impaired overall for most patients. Of the 33 patients for whom Ishihara testing had been attempted, 13 (39%) were unable to perform the test, due to severely compromised VA, and 79% of all patients had at least some degree of color vision impairment. Tables 1 and 2 summarize the VA, color vision, and length of disease (where available) for each individual patient in the gene-positive and gene-negative groups, respectively. Given the small sample size, variation in age of symptom onset, length of disease course, and heterogeneity of genetic results, it is not possible to correlate these outcomes precisely with gene status.
Frequency of genetic variations.
Age distribution of symptom onset by gene. Dark blue: age < 20; Medium blue: age 20–40; Light blue: age > 40.
Clinical Characteristic of Patients and Genetic Variant
Acuity and Color Vision in Patients With No Known Genetic Variant
Table 1 summarizes the specific gene variants identified in each patient who tested positive, as well as the zygosity of each mutation. Two of the patients (Patient 4 and Patient 26) are siblings and tested positive for the same specific mutations in two genes (CRB1 c.2977G>A and USH2A c.478G>A). Patient 4 had two additional variants not found in Patient 26, including a deletion in CEP290 (ie, CEP290 c.1864_1865del), which is a known pathological variant in the homozygous state (this patient was heterozygous). The two siblings experienced virtually the same symptoms (ie, nyctalopia, dyschromatopsia, and decreased VA), which started at around the same age: 16 and 17, respectively. The older sibling (Patient 4) had slightly worse VA. On funduscopic exam, Patient 4 had focal arteriolar attenuation, trace epiretinal membrane, and midperipheral pigment mottling in both eyes. In addition, ERG study showed diffuse retinal dysfunction, which was greater in rods than cones. Funduscopic exam for Patient 26 revealed pigment mottling and mild fundus flecks. OCT for both patients was relatively normal, with the exception of retinal thinning in both eyes (foveal thickness OD/OS: 165/160 μm and 174/174 μm in Patients 4 and 26, respectively). These findings in both patients are consistent with their diagnoses of retinitis pigmentosa (RP). Both patients also experience seizures. Although several patients in this study had variations in the same gene, there were no other incidences of patients with identical mutations.
The majority of gene-positive patients in our cohort (12 of 18) had more than one mutation discovered, and most of the gene variants were of unknown clinical significance. Seven patients (ie, Patients 4, 11, 14, 21, 27, 29, and 30) harbored mutations that were either reported or predicted to be pathogenic in the homozygous or hemizygous state. Of these, one patient (Patient 29) was homozygous for a pathogenic RPE65 mutation (RPE65 c.271C>T), developed early symptoms (by age 7), and had severe vision loss (LP). Funduscopic examination revealed peripapillary atrophy, mild disc pallor, retinal pigment epithelial (RPE) scarring, and severe bone spicule changes in both eyes. Patient 11 was found to be hemizygous for a previously unreported frameshift mutation in the X-linked gene CHM, leading to early protein termination (CHM c.46_49dup). The patient was 24 years old at the time of testing, had peripheral visual field loss for 3 years, and carried a clinical diagnosis of RP. His VA was 20/20-2 with normal color vision in both eyes. Funduscopic examination showed temporal peripapillary atrophy, a normal macula, and peripheral RPE changes in both eyes. On OCT, both eyes had a normal retinal contour with no evidence of macular edema or subretinal fluid. No additional patients were homo- or hemizygous for any given mutation, nor did any individual carry a gene mutation that conferred disease in an autosomal dominant fashion.
In order to determine trends in disease characteristics, the patients' symptoms and clinical findings were analyzed. Table 3 summarizes the most frequent symptoms experienced. Eighty-four percent of patients reported decreased VA, with 43% of patients having developed legal blindness in one or both eyes. Dyschromatopsia, nyctalopia, photopsia, and reduced peripheral vision were also common. Common clinical findings include RPE changes (70%), vascular attenuation (35%), and the presence of bone spicules (27%). Additional findings include pigment clumping, epiretinal membrane, and disc pallor (Table 4). The most common findings on OCT include loss of normal retinal contour, disrupted inner segment/outer segment layer, epiretinal membrane, RPE atrophy/changes, and retinal thinning (Table 5). In each table, the number of patients with each finding who are also gene-positive is indicated in the right-hand column.
Symptoms at Time of Genetic Testing
Retinal Examination Findings
In this study, we report on the disease characteristics and the frequency of gene mutations in a cohort of 37 patients with known or suspected inherited retinal dystrophy (IRD). We were able to detect mutations in genes that are commonly disrupted in inherited retinal disorders in roughly half of our patients (18 patients and 48.6% of the total sample). In addition, among these patients, 30 variants were discovered in 14 of the 31 genes represented on the testing panel. Mutations in USH2A were particularly common, representing 27% of all the reported variations. Limitations of the present study include its retrospective nature, small sample size, and lack of uniform ERG testing and standardized fundus imaging. These issues might be addressed with a larger prospective study. Although our sample size was too small and the heterogeneity in genotype too large to derive any meaningful statistical correlations between gene status and disease presentation/progression, we believe that our data demonstrate several important findings regarding the genetic basis of IRD. Given that about half of our patients tested positive for one or more mutations, despite the fact that the number of genes tested was relatively low and that our sample size was very small speaks to the frequency with which these mutations are associated with these clinical presentations. However, the fact that half of the sample was gene-negative and that most of the gene-positive patients harbored multiple heterozygous mutations of unknown clinical significance indicates that there may be a host of additional mutations in genes that were not included on the testing panel in this study that are also contributing significantly to the development of IRD, and that many complex genotypic pathways can converge upon a similar phenotype.
Although the genotypic heterogeneity of our sample, both within and between individual patients, makes it difficult in most cases to conclude with certainty which of these genetic variants will produce disease and under which circumstances, there were a few instances in which the case was more clear cut. Our pair of siblings did not test positive for a singular causative mutation to explain their condition; however, the fact that they shared identical gene variants and presented in an almost identical fashion and at around the same age gives us reasonable confidence that their condition is of a heritable nature, which is important information to have in terms of genetic counseling and family planning. One patient with biallelic RPE65 mutations and disease onset at 7 years of age displayed a fairly classic presentation for early onset severe retinal dystrophy, which is considered to be a similar, milder form of LCA8. Although his gene status qualifies him for Luxturna gene therapy, his duration of disease and minimal residual visual function likely preclude him from benefitting from treatment. As genetic testing becomes more and more readily available, utilizing this powerful technology to screen for patients such as these earlier in their disease course will become the mainstay of effective, vision-sparing intervention.
Interestingly, another of our patients was hemizygous for a duplication causing a frameshift in CHM leading to early termination of the REP1 protein (p.Gly17Aspfs*5). Due to the severe truncation of the protein, this mutation, although previously unreported, is predicted to be pathogenic, as other truncating mutations in this region have resulted in disease. Because of its location on the X chromosome, a hemizygous male such as our patient would be predicted to develop disease. Mutations in CHM are known to cause choroideremia, which is a progressive disease caused by diffuse loss of RPE, photoreceptors, and choriocapillaris. It is characterized by early nyctalopia, followed by progressive tunnel vision and eventual loss of VA.9 Although the rate of progression among patients varies, the disease is ultimately blinding. Due to the extensive similarity in symptoms, patients with choroideremia are often misdiagnosed with RP, among other conditions.1,10 Our patient was 24 years old when he presented and had been experiencing worsening nyctalopia since the age of 21. On presentation, he also reported decreasing peripheral vision, photopsia, and difficulty with transitioning from darkness to light. His VA and color vision were not yet adversely affected, but his funduscopic exam showed peripheral RPE changes. Appropriately, he was given a diagnosis of RP. It was only his genetic testing that pointed to an alternative diagnosis, particularly this early in his disease course. This is vitally important in this particular patient's case, because just as Luxturna offers the hope of retaining vision to those with biallelic RPE65 mutations, phase 1 and 2 trials testing the safety and efficacy of AAV.REP1 in choroideremia patients have shown improved visual function following treatment (12).11 Phase 3 trials are currently underway, and candidacy for treatment will be contingent upon genetic diagnosis.
This small study demonstrates the utility of utilizing next generation sequencing based genetic screening in a patient population suspected of inherited retinal dystrophies, with a frequency of mutations identified approaching 50%. This screening panel, provided free of charge to patients, was comprised of 31 genes commonly implicated in IRD. There are now hundreds of genes known to be associated with heritable retinal dystrophies and current testing at our center involves a panel of roughly 250 genes, a broad rather than targeted approach. A robust genetic counseling program is also essential to accompany broad genetic screening tests to help patients and their families interpret the implications of the test results. At present, gene therapy for inherited retinal dystrophies is in its infancy, with a single approved clinically available agent. This therapeutic arena is expected to expand significantly in the upcoming years. Knowledge of genetic mutations associated with IRD may help to identify patients that may be amenable to therapy, to provide diagnostic and prognostic information to patients and their family members, and to further our understanding of genotype-phenotype correlations in inherited retinal dystrophies.
- Nash BM, Wright DC, Grigg JR, Bennetts B, Jamieson RV. Retinal dystrophies, genomic applications in diagnosis and prospects for therapy. Transl Pediatr. 2015;4(2):139–163. PMID:26835369
- Verbakel SK, van Huet RAC, Boon CJF, et al. Non-syndromic retinitis pigmentosa. Prog Retin Eye Res. 2018;66:157–186. doi:10.1016/j.preteyeres.2018.03.005 [CrossRef] PMID:29597005
- Russell S, Bennett J, Wellman JA, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390(10097):849–860. doi:10.1016/S0140-6736(17)31868-8 [CrossRef] PMID:28712537
- Lacey S, Chung JY, Lin H. A comparison of whole genome sequencing with exome sequencing for family-based association studies. BMC Proc. 2014;8(S1)(Suppl 1 Genetic Analysis Workshop 18Vanessa Olmo):S38–S38. doi:10.1186/1753-6561-8-S1-S38 [CrossRef] PMID:25519383
- Audo I, Bujakowska KM, Léveillard T, et al. Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases. Orphanet J Rare Dis. 2012;7(1):8. doi:10.1186/1750-1172-7-8 [CrossRef] PMID:22277662
- Bernardis I, Chiesi L, Tenedini E, et al. Unravelling the Complexity of Inherited Retinal Dystrophies Molecular Testing: Added Value of Targeted Next-Generation Sequencing. BioMed Res Int. 2016;2016:6341870. doi:10.1155/2016/6341870 [CrossRef] PMID:28127548
- 2010 ASN Abstracts - 2010 - Journal of Neuroimaging. Wiley Online Library. https://onlinelibrary-wiley-com.proxy.libraries.rutgers.edu/doi/abs/10.1111/j.1552-6569.2009.00451.x. Accessed June 11, 2019.
- Kumaran N, Moore AT, Weleber RG, Michaelides M. Leber congenital amaurosis/early-onset severe retinal dystrophy: clinical features, molecular genetics and therapeutic interventions. Br J Ophthalmol. 2017;101(9):1147–1154. doi:10.1136/bjophthalmol-2016-309975 [CrossRef] PMID:28689169
- Coussa RG, Traboulsi EI. Choroideremia: a review of general findings and pathogenesis. Ophthalmic Genet. 2012;33(2):57–65. doi:10.3109/13816810.2011.620056 [CrossRef] PMID:22017263
- Lee TKM, McTaggart KE, Sieving PA, et al. Clinical diagnoses that overlap with choroideremia. Can J Ophthalmol. 2003;38(5):364–372. doi:10.1016/S0008-4182(03)80047-9 [CrossRef] PMID:12956277
- Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial- ClinicalKey. https://www-clinicalkey-com.proxy.libraries.rutgers.edu/#!/content/playContent/1-s2.0-S0140673613621170?returnurl=null&referrer=null. Accessed June 11, 2019.
Clinical Characteristic of Patients and Genetic Variant
|Patient||Gene||Variant||Zygosity||VA OD||VA OS||Color OD||Color OS||Dx||Onset||Disease Duration (Years)|
Acuity and Color Vision in Patients With No Known Genetic Variant
|Patient||VA OD||VA OS||Color OD||Color OS||Diagnosis||Onset (Age)||Disease Duration (Years)||Electrophysiology|
|18||0.00||0.00||92%||92%||BD||22||9||EOG: reduced light peak OS|
|22||0.30||0.66||60%||0%||RP||57||1||Retinal dysfunction rods > cones|
|36||0.70||0.30||80%||100%||RP||?||?||Retinal dysfunction rods > cones|
|37||0.88||0.88||8%||8%||CD||childhood||?||Retinal dysfunction affecting cones only|
Symptoms at Time of Genetic Testing
|Symptoms||Number (%)||Number With Known Gene Variant|
|Decreased visual acuity||31 (84)||14|
|Acuity ≤ 20/200 (one or both eyes)||16 (43)||8|
|Reduced visual fields / peripheral vision||6 (16)||3|
Retinal Examination Findings
|Retina Findings||Number (%)||Number With Known Gene Variant|
|RPE changes||26 (70)||11|
|Attenuated vessels||13 (35)||6|
|Bone spicules||10 (27)||3|
|Pigment clumping||8 (22)||1|
|Epiretinal membrane||8 (22)||3|
|Disc pallor||7 (19)||2|
|OCT Findings||Number (%)||Number With Known Gene Variant|
|Loss of normal retinal contour||17 (46)||8|
|IS/OS layer disrupted||16 (43)||6|
|Epiretinal membrane||15 (41)||7|
|RPE atrophy||12 (32)||3|
|RPE changes||9 (24)||5|
|Retinal thinning||9 (24)||3|