OBJECTIVE: Bietti crystalline dystrophy (BCD) is an inherited retinal degeneration disease caused by mutations in the CYP4V2 gene. Currently, there is no clinical therapy approach available for BCD patients. Previous research has suggested that polyunsaturated fatty acids (PUFAs) may play a significant role in the development of BCD, implicating the involvement of ferroptosis in disease pathogenesis. In this work, we aimed to investigate the interplay between ferroptosis and BCD and to detect potential therapeutic strategies for the disease.
METHODS: Genetic-edited RPE cell line was first established in this study by CRISPR-Cas9 technology. Cyp4v3 (the homologous gene of human CYP4V2) knock out (KO) mice have also been used. Lipid profiling and transcriptome analysis of retinal pigment epithelium (RPE) cells from Cyp4v3 KO mice have been conducted. Ferroptosis phenotypes have been first investigated in BCD models in vitro and in vivo, including lipid peroxidation, mitochondrial changes, elevated levels of reactive oxygen species (ROS), and altered gene expression. Additionally, an iron chelator, deferiprone (DFP), has been tested in vitro and in vivo to determine its efficacy in suppressing ferroptosis and restoring the BCD phenotype.
RESULTS: Cyp4v3 KO mice exhibited progressive retinal degeneration and lipid accumulation, similar to the BCD phenotype, which was exacerbated by a high-fat diet (HFD). Increased levels of PUFAs, such as EPA (C22:5) and AA (C20:4), were observed in the RPE of Cyp4v3 KO mice. Transcriptome analysis of RPE in Cyp4v3 KO mice revealed changes in genes involved in iron homeostasis, particularly an upregulation of NCOA4, which was confirmed by immunofluorescence. Ferroptosis-related characteristics, including mitochondrial defects, lipid peroxidation, ROS accumulation, and upregulation of related genes, were detected in the RPE both in vitro and in vivo. Abnormal accumulation of ferrous iron was also detected. DFP, an iron chelator administration suppressed ferroptosis phenotype in CYP4V2 mutated RPE. Oral administration of DFP also restored the retinal function and morphology in Cyp4v3 KO mice.
CONCLUSIONS: This study represented the first evidence of the substantial role of ferroptosis in the development of BCD. PUFAs resulting from CYP4V2 mutation may serve as substrates for ferroptosis, potentially working in conjunction with NCOA4-regulated iron accumulation, ultimately leading to RPE degeneration. DFP administration, which chelates iron, has demonstrated its ability to reverse BCD phenotype both in vitro and in vivo, suggesting a promising therapeutic approach in the future.

300 million people live with at least one of 6,000 rare diseases worldwide. However, rare disease research is not always reviewed with scrutiny, making it susceptible to what the author refers to as nontransparent science. Nontransparent science can obscure animal model flaws, misguide medicine regulators and drug developers, delay or frustrate orphan drug development, or waste limited resources for rare disease research. Flawed animal models not only lack pharmacologic relevance, but also give rise to issue of clinical translatability. Sadly, these consequences and risks are grossly overlooked. Nontransparency in science can take many forms, such as premature publication of animal models without clinically significant data, not providing corrections when flaws to the model are discovered, lack of warning of critical study limitations, missing critical control data, questionable data quality, surprising results without a sound explanation, failure to rule out potential factors which may affect study conclusions, lack of sufficient detail for others to replicate the study, dubious authorship and study accountability. Science has no boarders, neither does nontransparent science. Nontransparent science can happen irrespective of the researcher\'s senority, institutional affiliation or country. As a patient-turned researcher suffering from Bietti crystalline dystrophy (BCD), I use BCD as an example to analyze various forms of nontransparent science in rare disease research. This article analyzes three papers published by different research groups on Cyp4v3-/-, high-fat diet (HFD)-Cyp4v3-/-, and Exon1-Cyp4v3-/- mouse models of BCD. As the discussion probes various forms of nontransparent science, the flaws of these knockout mouse models are uncovered. These mouse models do not mimic BCD in humans nor do they address the lack of Cyp4v3 (murine ortholog of human CYP4V2) expression in wild type (WT) mouse retina which is markedly different from CYP4V2 expression in human retina. Further, this article discusses the impact of nontransparent science on drug development which can lead to significant delays ultimately affecting the patients. Lessons from BCD research can be helpful to all those suffering from rare diseases. As a patient, I call for transparent science in rare disease research.

Bietti crystalline corneo-retinal dystrophy (BCD) is an autosomal recessive inherited retinal dystrophy characterized by multiple shimmering yellow-white deposits in the posterior pole of the retina in association with atrophy of the retinal pigment epithelium (RPE), pigment clumps, and choroidal atrophy and sclerosis. Blindness and severe visual damage are common in late-stage BCD patients. We generated a Cyp4v3 knockout mouse model to investigate the pathogenesis of BCD. This model exhibits decreased RPE numbers and signs of inflammation response in the retina. Rod photoreceptors were vulnerable to light-induced injury, showing increased deposits through fundoscopy, a decrease in thickness and a loss of cells in the ONL, and the degeneration of rod photoreceptors. These results suggest that an inflammatory response might be an integral part of the pathophysiology of BCD, suggesting that it might be reasonable for BCD patients to avoid strong light, and the results provide a useful model for evaluating the effects of therapeutic approaches.

OBJECTIVE: To investigate the clinical characteristics and genetic features of a Bietti crystalline dystrophy (BCD) proband in a Chinese family.
METHODS: A Chinese female diagnosed with BCD complicated by bilateral choroidal neovascularization (CNV) and her parents underwent complete ophthalmic examinations, including fundus autofluorescence (AF), fundus photography (FP), fundus fluorescein angiography (FFA), visual field testing, full-field electroretinography (ERG), optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA). The sequencing of the CYP4V2 gene was performed to the whole family.
RESULTS: Bilateral tiny glittering crystal-like deposits and differing extent of atrophy of the retinal pigment epithelium (RPE) were found in the posterior pole of her fundus. The diffuse hypo-fluorescence shown on AF images and window defects shown on FFA both indicated the atrophy of the RPE and choriocapillaris. OCT showed the thinning of the RPE and choriocapillaris layer, ellipsoid zone (EZ) band defect and CNV in both eyes. OCTA images proofed bilateral type 2 CNV. The visual field test showed central and paracentral scotoma. ERG showed a slightly decreased b-wave in scotopic ERG. Gene sequencing identified three mutations of the CYP4V2 gene, c.802_807del, c.810delT, and c.1388G>A. The mutation c.1388G>A was a novel substitution mutation.
CONCLUSIONS: The novel mutation c.1388G>A may be a possible cause that could induce the clinical phenotype of BCD.

OBJECTIVE: We wanted to investigate the radial peripapillary capillary (RPC) network in patients with Bietti crystalline dystrophy (BCD).
METHODS: We compared RPC densities in the disk and different peripapillary regions, obtained using optical coherence tomography angiography in 22 patients with BCD (37 eyes) and 22 healthy subjects (37 eyes). The BCD group was then divided into Stage 2 and Stage 3 subgroups based on Yuzawa staging, comparing the RPC densities of the two.
RESULTS: The disk area RPC density was 38.8% ± 6.3% in the BCD group and 49.2% ± 6.1% in the control group ( P < 0.001), and peripapillary region RPC density was significantly lower in the BCD group than in the control group (49.1% ± 4.7% and 54.1% ± 3.0%, respectively, P < 0.001). There were no significant RPC density differences between the tempo quadrant and inside disk of Stages 2 and 3 subgroups; the other areas showed a significantly lower RPC density in Stage 3 than in Stage 2 BCD.
CONCLUSIONS: The BCD group RPC density was significantly lower than the control group. The reduction of RPC density in the tempo quadrant occurred mainly in the Stage 1 BCD. In contrast, the reduction of RPC density in superior, inferior, and nasal quadrants occurred mainly in Stage 2.

OBJECTIVE: Bietti crystalline dystrophy (BCD) is a rare degenerative eye disease caused by mutations in the CYP4V2 gene, and Cyp4v3 is the murine ortholog to CYP4V2. To better understand the molecular pathogenesis of this disease and to explore the potential treatment we have established a Cyp4v3 knock-out mouse model.
METHODS: Cyp4v3-/- mice were generated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in embryonic stem cells of C57BL/6J mice. Ocular morphologic characteristics were evaluated via fundus imaging, histologic analysis of rods and cones via immunofluorescence, and phalloidin stain to observe retinal pigment epithelium (RPE) in whole-mounts, electroretinogram (ERG) was also conducted to examine the retinal function.
RESULTS: The characteristic features of BCD recurred in the Cyp4v3-/- mice, including retinal crystalline deposits, atrophy and degeneration of RPE cells, and ERG amplitude decline of dark and light adapted a- and b- wave; however, the immunofluorescence stain of rod and cone cells did not show obvious differences when compared with the wild type (WT) mice. In the early stage of the disease, no crystal-like deposits were found in the fundus, ERG detection of the retinal function did not find a significant decline, and the morphological structure and quantity of the neural retina and RPE did not change significantly. Crystalline deposits occurred and converged when the Cyp4v3-/- mice at the end of 6 months, and the deposits disappeared when the Cyp4v3-/- mice at the end of 12 months. The ERG amplitude started to decline when the Cyp4v3-/- mice at the end of 6 months and deteriorated at the end of 12 months. The RPE cells of the 12-month old Cyp4v3-/- mice showed irregular shape by phalloidin staining of F-actin. The Cyp4v3-/- mice behaved normally and were viable and fertile when maintained under specific pathogen-free (SPF) housing conditions.
CONCLUSIONS: Just like BCD patients, the disease progress of Cyp4v3-/- mouse is correlated with the age, which provides a good model for pathogenesis and gene therapy study in the future. The atrophy and degeneration of RPE take the lead in progressing of the disease, but the mechanism is not clear yet.

BACKGROUND: Bietti crystalline dystrophy (BCD) is an autosomal recessive genetic disorder that causes progressive vision loss. Here, 12 patients were followed up for 1-5 years with fundus fluorescein angiography (FFA) to observe BCD disease progression.
METHODS: FFA images were collected for 12 patients with BCD who visited our clinic twice or more over a 5-year period. Peripheral venous blood was collected to identify the pathogenic gene related to the clinical phenotype.
RESULTS: We observed two types in FFA images of patients with BCD. Type 1 showed retinal pigment epithelium (RPE) atrophy in the macular area, followed by choriocapillaris atrophy and the subsequent appearance of RPE atrophy appeared at the peripheral retina. Type 2 showed RPE atrophy at the posterior pole and peripheral retina, followed by choriocapillaris atrophy around the macula and along the superior and inferior vascular arcades and the nasal side of the optic disc. The posterior and peripheral lesions of both type 1 and type 2 BCD subsequently extended to the mid-periphery; finally, all the RPEs and choriocapillaris atrophied, exposing the choroid great vessels, but type 2 macular RPE atrophy could last longer.
CONCLUSIONS: The characterization of two different types of BCD development provides a better understanding of the phenotype and the progression of the disease for a precise prognosis and prediction of pathogenesis.

BACKGROUND: Bietti crystalline dystrophy (BCD) is a distinct entity of retinitis pigmentosa with a wide range of genotypic and phenotypic variabilities. The goal of the present study was to investigate the morphological, functional and genetic features of BCD.
METHODS: A full series of multimodal imaging was performed in four Chinese patients with BCD, including fundus photography, fundus autofluorescence, fundus fluorescein angiography (FFA), indocyanine green (ICG) angiography, optical coherence tomography (OCT) and microperimetry. Electrophysiological tests including full-field electroretinography (ERG) and multifocal ERG were employed. CYP4V2 gene sequencing was performed.
RESULTS: Intraretinal crystalline deposits were observed in fundus photographs in all patients. The crystals were better appreciated in infrared images. Autofluorescence imaging demonstrated multifocal patchy hypofluorescence, suggesting massive RPE atrophy. FFA and ICG angiography further confirmed atrophy of the RPE and the underlying choroidal vessels. OCT revealed disruption of the photoreceptors, RPE and the choroid. Outer retinal tubulations (ORTs) confining to the outer nuclear layer were detected in three out of four patients. Full-field ERG showed markedly diminished responses. Multifocal ERG displayed reduced central and peripheral responses in a patient with normal vision. Gene sequencing identified two deletion mutations in CYP4V2, c.802_807del and c.810delT. BCD complicated by choroidal neovascularization (CNV) was diagnosed in one patient, and intravitreal anti-vascular endothelial growth factor (VEGF) injection was given with favorable response.
CONCLUSIONS: Multimodal imaging features and electrophysiological findings of BCD patients were comprehensively discussed. A novel deletion mutation, c.802_807del, in the CYP4V2 gene was reported. ORTs are important changes in the outer retina of BCD patients, further investigation of this structure may provide insights into pathology of BCD. Intravitreal anti-VEGF therapy was effective for treatment of BCD complicated by CNV.