Corresponding Author: Christopher Seungkyu Lee, MD, PhD. Institute of Vision Research, Department of Ophthalmology, Severance Hospital, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea. Choroideremia is X-linked recessively inherited retinal disorder, caused by CHM gene mutation, which encodes Rab escort protein-1 (REP-1) [1]. REP-1 is an essential component for Rab geranyl geranyl transferase (GGTase) complex, which controls intracellular traffic [2]. REP-2, encoded by autosomal CHM-like (CHML) gene, is the other isoform of REP-1 and can compensate for REP-1 deficiency in most tissues except retinal pigment epithelium (RPE), photoreceptors, and choroid [3,4]. As such, loss-of-function mutations in this REP-1 encoding gene leads to progressive degeneration of RPE, photoreceptors, and choroid. Choroideremia can be clinically misdiagnosed as other retinal dystrophies with similar clinical features such as X-linked retinitis pigmentosa (RP) and gyrate atrophy.
Male patients characteristically experience night blindness in the first decade of life followed by progressive peripheral visual field (VF) restriction. Visual acuity of the central VF is relatively preserved until the advanced degeneration affects the macula, usually after fourth to fifth decade of life [5,6]. Although female carriers might often demonstrate fundus changes such as yellow crystalline spots in the macula or irregular mottled pigmentation in the peripheral retina, most carriers have no symptoms or mild night blindness. Rarely, severe cases of female carriers presenting with significant retinal and choroidal atrophy leads to visual impairment and are attributable to the phenomenon of skewed X-inactivation [7,8].
Choroideremia is a rare disease with estimated prevalence of 1 in 50,000 to 1 in 100,000 people of European descent [9]. The prevalence of Korean population is largely unknown. We share and analyze the genotype and phenotype of Korean male patients and female carriers who visited our clinic.
Materials and Methods
Ethics statements
This retrospective study protocol was reviewed and approved by the Institutional Review Board of Severance Hospital (No. 4-2022-0830) in 2022 and adhered to the tenets of the Declaration of Helsinki. The requirement for written informed consent was waived due to the retrospective study design and minimal risk of the subjects.
Subjects
This study included six male choroideremia patients and three female carriers who initially visited the Department of Ophthalmology of Severance Hospital (Seoul, Korea) and Gangnam Severance Hospital (Seoul, Korea) between September 2018 and November 2020.
Clinical evaluation
Demographic and medical information, including age, sex, other medical history, previous ocular surgery history, and family history of the retrospectively enrolled subjects, was obtained from patients’ electronic medical records. All subjects were examined with necessary ophthalmologic assessments: best-corrected visual acuity, intraocular pressure, slit-lamp examination, color fundus photography, fundus autofluorescence (FAF), optical coherence tomography (OCT), and VF test.
Genetic analysis
Genetic diagnosis was confirmed by panel-based next-generation sequencing or whole exome sequencing. Genomic DNA extracted from the individual’s sample was used for library preparation and target capture using a custom panel targeting candidate genes. Target enrichment was performed using a customized target enrichment kit (Celemics Inc). Next-generation sequencing was done on the NextSeq 550Dx (Illumina) or NovaSeq 6000 System (Illumina). Databases used for analysis and variant annotation include Online Mendelian Inheritance in Man (OMIM), Human Gene Mutation Database (HGMD), Clin-Var, dbSNP, 1000 Genome, Exome Aggregation Consortium (ExAC), Exome Sequencing Project (ESP), and Korean Reference Genome Database (KRGDB). All pathogenic and likely pathogenic variants were further confirmed by Sanger sequencing.
Results
Clinical features
Six male patients were diagnosed with choroideremia at the median age of 25 years old. Only two patients had chief complaints of choroideremia-associated symptoms including nyctalopia and VF narrowing. The other four patients visited eye clinic for irrelevant reasons such as hordeolum, ocular trauma, or routine exam before military service. Regardless of chief complaints, four patients (66.7%) reported nyctalopia that started around late teenage years. Before genetic analysis, choroideremia was clinically suspected in three patients (50.0%), while RP was suspected in the other three (50.0%).
One female carrier (case 7) was referred for abnormal fundus findings during evaluation for cataract surgery by a local ophthalmologist. She brought her son (case 1) together, who had been clinically diagnosed with RP before. The other two female carriers were mother and sister (cases 8 and 9, respectively) of a patient (case 2), who wanted evaluation as family members.
Genetic analysis
Six male patients showed hemizygous CHM variants, including two nonsense variants, c.715C>T:p.(Arg239*) and c.799C>T:p.(Arg267*); two frameshift variants, c.1584_1587del:p.(Val529Hisfs*7) and c.403_404del:p.(Asp-135Phefs*9); one splicing variant c.1511-28_1511-2del; and one exon 2-8 duplication. The latter three variants were novel. Two female carriers had heterozygous exon 2-8 duplication and the other one female carrier had heterozygous nonsense variant c.715C>T:p.(Arg239*) (Table 1).
Multimodal imaging
Widefield color fundus photography of the choroideremia patients showed diffuse yellow-whitish scleral reflex due to the atrophy of choroid and RPE, and granular pigmented lesions (Fig. 1A, 1B). FAF showed multiple patchy hypofluorescence with sparing of the macula that showed relative hyperfluorescence. The hypofluorescence sites were more prominent at the nasal retina, compared to the temporal retina (Fig. 2A, 2B). OCT showed decreased thickness of outer nuclear layer, ellipsoid zone, RPE layer, and choroid, which was more prominent at the periphery. OCT showed other associated structural variations including interlaminar bridges (ILBs), outer retinal tubulations (ORTs), microcysts in the inner nuclear layer (Fig. 3A, 3B). The VF test showed characteristic ring scotoma pattern with small amount of remaining central field.
All female carriers were asymptomatic with normal VF, but variable fundus findings including moth-eaten appearance, pigmentary mottling, drusen-like deposits were observed (Supplementary Fig. 1). Mild changes in ellipsoid zone were detectable in OCT images. Widefield fundus photography, fundus, autofluorescence, Humphrey visual field test results of the subjects are described in Supplementary Fig. 1-3.
Discussion
Choroideremia is a rare cause of inherited retinal disease and less than 10 cases have been described previously in Korea as case report, small case series, or part of genetic profiling studies [10-14]. Our study demonstrated genotype and phenotype spectrum of six Korean patients with choroideremia and identified three novel variants, c.403_404del:p.(Asp135Phefs*9), c.1511-28_1511-2del, and exon 2-8 duplication. Clinical manifestations were largely similar to previous studies.
FAF is mainly produced by retinoid byproducts of the visual cycle that accumulate in the RPE and choroideremia patients often show characteristic FAF pattern: patchy areas of RPE atrophy with hypofluorescence, in contrast to the remaining islands with preserved RPE with normal or hyperfluorescence due to lipofuscin accumulation [15-17]. Jolly et al. [18] found that temporal side of the fovea showed preferential preservation of AF than nasal side and hypothesized that temporal preservation is due to higher rod photoreceptor concentration and relatively more dense choroidal blood supply in the temporal retina than the nasal retina. This pattern of preferential temporal preservation in FAF was also observed in the present study.
The borders of AF islands corresponded to the beginning of acute decline in outer nuclear layer thickness in OCT. Attenuation of the ellipsoid zone and external limiting membrane was also identified in the areas of decreased outer nuclear layer thickness. ORT, first described by Zweifel et al. [19] in 2009 refers to the structure located in the outer nuclear layer of retina that typically appears as round or ovoid hyporeflective spaces with hyperreflective borders in OCT. ORT has been speculated to represent remodeling of degenerating photoreceptors and was commonly reported in choroideremia patients [20-23]. ILBs are wedge-shaped hyperreflective structures that have been described in choroideremia [21-25]. ILBs are identified at the junction of normal and atrophic retina and thought to represent hyperplastic Müller cells in response to the degenerative process. Retinal microcysts have been reported in choroideremia patients, as well [21,22]. Typical cystic macular edema or cystic spaces in retina have been described in 38.0% to 62.5% of choroideremia patients [26,27].
Patients with choroideremia generally show patchy loss of midperipheral vision in earlier stages, which progresses to absolute scotoma in the midperipheral region. The remaining central island gradually shrinks irregularly, leading to central visual loss towards blindness at the latest age of life [28]. Male patients in this study were relatively young with the median age of 25 years old and showed relatively good central vision. However, VF outside 10° from fixation showed variable VF defects. The youngest patient (case 6) in our series showed considerable VF defect at the age of 7 years (Fig. 4A, 4B).
Female carriers of our study were asymptomatic with normal VFs but showed mild fundus changes mostly at periphery. Conventionally, the skew X-chromosome inactivation was considered the main cause of disease manifestations in female carriers. Recently, however, the potential influence of the presence of biallelic expression of CHM has been proposed [29]. CHM variants that could not produce a final protein product (i.e., deletion, frameshift, nonsense mutations) would have manifestations depending only on the skew X-chromosome inactivation. However, if a particular in-frame variant produces mutant REP-1 that bind to Rab geranylgeranyl transferase, making them unavailable for prenylation of Rab proteins, it would act with a dominant-negative effect. This may potentially hypothesize the genotype-phenotype relationships of female carriers and explain the reason why only some female carriers show severe clinical manifestations, but further studies are needed [29].
To summarize, three novel variants in CHM gene were identified through this study, expanding the spectrum of choroideremia-related mutations. Multimodal imaging and VF tests were useful for clinical diagnosis and the results accorded with the previous knowledge about choroideremia. Considering the developing gene therapy for choroideremia, this work will not only increase our understanding of the disease but may potentially lead to hope for the realization of curing this blinding disease [30,31].
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