Detailed functional and structural phenotype of Bietti crystalline dystrophy associated with mutations in CYP4V2 complicated by choroidal neovascularization

Purpose: To describe in detail the phenotype of a patient with Bietti crystalline dystrophy (BCD) complicated by choroidal neovascularization (CNV) and the response to intravitreal Bevacizumab (Avastin^®; Genentech/Roche).

Methods: A 34-year-old woman with BCD and mutations in CYP4V2 (c.802-8_806del13/p.H331P:c992A>C) underwent a complete ophthalmic examination, full-field flash electroretinography (ERG), kinetic and two-color dark-adapted perimetry, and dark-adaptometry. Imaging was performed with spectral domain optical coherence tomography (SD-OCT), near infrared (NIR) and short wavelength (SW) fundus autofluorescence (FAF), and fluorescein angiography (FA).

Results: Best-corrected visual acuity (BCVA) was 20/20 and 20/60 for the right and left eye, respectively. There were corneal paralimbal crystal-like deposits. Kinetic fields were normal in the peripheral extent. Retinal crystals were most obvious on NIR-reflectance and corresponded with hyperreflectivities within the RPE on SD-OCT. There was parafoveal/perifoveal hypofluorescence on SW-FAF and NIR-FAF. Rod > cone sensitivity loss surrounded fixation and extended to ~10° of eccentricity corresponding to regions of photoreceptor outer segment-retinal pigmented epithelium (RPE) interdigitation abnormalities. The outer nuclear layer was normal in thickness. Recovery of sensitivity following a ~76% rhodopsin bleach was normal. ERGs were normal. A subretinal hemorrhage in the left eye co-localized with elevation of the RPE on SD-OCT and leakage on FA, suggestive of CNV. Three monthly intravitreal injections of Bevacizumab led to restoration of BCVA to baseline (20/25).

Conclusion: crystals in BCD were predominantly located within the RPE. Photoreceptor outer segment and apical RPE abnormalities underlie the relatively extensive retinal dysfunction observed in relatively early-stage BCD. Intravitreal Bevacizumab was effective in treating CNV in this setting.     

携带CYP4V2基因突变的结晶样视网膜色素变性患者的表型特征

目的 分析国人结晶样视网膜色素变性（BCD）患者CYP4V2基因型及表型特征。设计 回顾性病例系列。研究对象 北京同仁医院结晶样视网膜色素变性患者138例。方法 回顾患者病历记录及眼科检查结果。先证者、直系亲属及家系患病成员采集外周静脉血,Sanger测序及实时定量荧光PCR检测先证者CYP4V2基因突变,并对致病性突变进行家系共分离验证。主要指标 最佳矫正视力、眼前节及眼底情况、CYP4V2基因突变。结果 138例BCD患者均携带CYP4V2基因双突变,突变c.802-810del17insCG、c.1091-2A>G和p.H331P是本组患者的常见突变。患者平均最佳矫正视力（LogMAR）（1.17±0.95）,平均发病年龄（29.19±9.64）岁,视力损伤程度与病程相关,不受发病年龄影响。所有患者眼底均可见细小颗粒状黄白色结晶沉积,结晶沉积范围及密度随病程而加重。结论 本研究拓展了BCD致病基因CYP4V2突变谱,BCD患者临床特征明显,结合多种影像学检查方法可有效进行诊断,致病基因突变的确定可为患者提供相应的遗传咨询帮助。（眼科, 2020, 29： 93-97）     

Crystal deposits on the lens capsules in Bietti crystalline corneoretinal dystrophy associated with a mutation in the CYP4V2 gene

Purpose: We report a patient (Case 1) with Bietti crystalline corneoretinal dystrophy (BCD) associated with previously unknown findings of crystal‐like deposits on the anterior and posterior lens capsules. This patient is one of four (Cases 1–4) in whom we have found BCD associated with the same mutation in the CYP4V2 gene. Methods: We present a case report with molecular diagnosis. A 45‐year‐old man (Case 1) was referred to our clinic with complaints of gradual progression of visual disturbances and night blindness. His visual acuity was limited to hand movement bilaterally. Slit‐lamp biomicroscopy disclosed glistening, crystal‐like deposits on the anterior and posterior lens capsules, as well as on the corneal stroma near the corneoscleral limbus. No such deposit was found in the lens stroma. Fundus examination disclosed profound chorioretinal atrophy with scarce crystal deposits. Full‐field electroretinography showed extinguished responses of isolated rods, isolated cones, and mixed rods and cones. Results: Molecular genetic analysis revealed that the subject had a homozygous mutation in the CYP4V2 gene (IVS6–8delTCATACAGGTCATCGCG/insGC), which is most commonly found in Japanese patients with BCD. Three other cases (Cases 2–4) of BCD associated with the same mutation did not show such crystal‐like deposits on the lens surface. Conclusions: Although their exact origin remains unknown, crystal‐like deposits may appear on the lens capsule of patients with BCD associated with a mutation in the CYP4V2 gene.     

结晶样视网膜色素变性临床与基础研究的机遇与挑战

结晶样视网膜色素变性（Bietti crystalline dystrophy,BCD）是视网膜色素变性中的一种特殊类型,其在东亚人群特别是中国人和日本人中较为常见。BCD的遗传方式为常染色体隐性遗传,唯一的致病基因为CYP4V2。对BCD患者自然史和CYP4V2突变导致BCD机制研究,将对确定BCD患者基因治疗的时间窗、临床研究中治疗效果评定以及探索治疗BCD其他方法均至关重要。（眼科,2020,29： 84-86）     

结晶样视网膜色素变性中与CYP4V2基因相关的致病突变

目的：使用Sanger测序法鉴定2个中国结晶样视网膜色素变性(BCD)家系中CYP4V2基因的突变位点。

方法：收集2019-01/09临床诊断为BCD患者的临床相关资料。采集患者、患者家系成员的外周血,提取DNA,利用Sanger测序法鉴定突变位点。

结果：共收集2个BCD先证者,先证者均表现为渐进性视力下降,眼底均可见典型的结晶样物质沉积。测序发现先证者1及其患病的哥哥,妹妹均在CYP4V2基因上存在c.802-8_810del17insGC的纯合突变。而先证者2则在CYP4V2基因上存在c.219T>A(p.F73L)、c.802-8_810del17insGC杂合突变。

结论： 中国BCD患者中最常见的c.802-8_810del17insGC突变在先证者1家系中为纯合突变,是其家系的致病突变。而先证者2则携带了中国BCD患者最常见的c.802-8_810del17insGC杂合突变,同时先证者2还携带c.219T>A(p.F73L)错义突变,突变均影响了CYP4V2基因的正常编码,进而导致疾病。     

Novel CYP4V2 mutations associated with Bietti crystalline corneoretinal dystrophy in Chinese patients

AIM: To analyze the CYP4V2 mutations in five unrelated Chinese patients with Bietti crystalline corneoretinal dystrophy (BCD) and to provide clinical features of these patients. BCD is a rare monogenic autosomal recessively inherited disorder characterized by the presence of crystals in the retina and retinal pigment epithelium atrophy. Mutations in the CYP4V2 gene have been found to be causative for BCD.METHODS:Ophthalmic examinations were carried out in the affected individuals. Peripheral blood samples were collected and genomic DNA was extracted. All exons and flanking intronic regions of the CYP4V2 gene were amplified with polymerase chain reaction and screened for mutations by direct DNA sequencing. One hundred control chromosomes were also screened to exclude nonpathogenic polymorphisms.RESULTS: Fundus examination revealed the presence of tiny yellowish-sparkling crystals at the posterior pole of the fundus and atrophy of the retinal pigment epithelium in all patients. Choroid neovascularization was noted in one patient. Five different CYP4V2 mutations were identified, including two missense mutations (p.F73L, p.R400H), two splice site mutations (c.802-8_810del17insGC, c.1091-2A>G), and one single base-pair deletion (p.T479TfsX7 or c.1437delC). The two splice site mutations were identified in three of the patients with BCD. Mutation p.T479TfsX7 was a novel mutation not observed in any of 100 ethnically matched control chromosomes.CONCLUSION: Mutation c.802-8_810del17insGC and c.1091-2A>G are common mutations in Chinese patients with BCD. Our results expand the allelic heterogeneity of BCD.     

结晶样视网膜变性患者五种血清离子的异常变化

目的　分析结晶样视网膜变性（Ｂｉｅｔｔｉｃｒｙｓｔａｌｌｉｎｅｄｙｓｔｒｏｐｈｙ,ＢＣＤ）患者血清中镁、钙、钾、钠及氯离子浓度的变化情况。方法　采用回顾性序列病例研究方法,选取临床诊断为ＢＣＤ患者５０例纳入ＢＣＤ组,另选取９９位正常人作为正常对照组,按盲法由专业技术人员完成血清离子浓度检测,并分别行ＢＣＤ组和正常对照组比较。结果　ＢＣＤ组患者血清中,钙离子和钾离子浓度分别为（２．３２３±０．０９２）ｍｍｏｌ·Ｌ－１和（４．１２３±０．３５５）ｍｍｏｌ·Ｌ－１,与正常对照组相比均显著升高（ｔ＝－４．２６６,Ｐ＝０．０００;ｔ＝－４．０８１,Ｐ＝０．０００）;镁离子浓度为（０．８４５±０．０５２）ｍｍｏｌ·Ｌ－１,与正常对照组血清中镁离子浓度（０．８７４±０．０７６）ｍｍｏｌ·Ｌ－１相比显著下降（ｔ＝２．７６７,Ｐ＝０,００６）;钠和氯两种离子血清浓度与正常对照组相比差异均无统计学意义（均为Ｐ＞０．０５）。结论　ＢＣＤ患者血清中钙离子和钾离子浓度升高,镁离子浓度降低,其可能与ＢＣＤ的发病有关。     

Indocyanine green angiographic interpretation of reticular dystrophy of the retinal pigment epithelium complicated by choroidal neovascularization

A 37-year-old woman presented with flashes in her left eye and bilateral visual distortion. Fundal examination revealed a reticular 'fishnet' pattern of retinal pigmentation in both eyes consistent with reticular dystrophy of the retinal pigment epithelium. In the left eye there was a small haemorrhage and a shallow serous macular detachment. Fluorescein angiography demonstrated subfoveal choroidal neovascularization. Indocyanine green angiography (ICG) revealed more extensive involvement than fluorescein angiography, with small areas of intense hyperfluorescence amongst reticular areas of hypofluorescence. These changes, as interpreted in light of the known histopathological localization of ICG, are consistent with varying stages of dysfunction of the retinal pigment epithelium in this disease.     

结晶样视网膜色素变性患者血脂浓度的变化

背景 结晶样视网膜色素变性(BCD)为一种先天性遗传性眼病,已有研究认为其与血清中脂肪酸代谢异常有关,但目前鲜见关于BCD与血清中脂质水平变化关系的报道. 目的 分析BCD患者血清脂质浓度的变化情况.方法 采用前瞻性研究设计,本研究经过北京同仁医院伦理委员会批准,受检者受检前均签署知情同意书.纳入2011年11月至2013年3月于北京同仁医院眼科就诊的双眼BCD患者50例及同期进行健康体检、年龄和性别匹配的健康体检者50人,采集受检者外周静脉血3 ml,由检验科专业人员检测受检者血清三酰甘油(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)浓度,结果的判定参照《中国成人血脂异常防治指南》(2007版)中成年人的正常值标准,并将BCD患者组检测结果与正常对照组进行比较.结果 50例BCD患者中血脂水平异常者占58.00％(29/50),其中高TG血症者占34.48％ (10/29);高TC血症者占34.48％ (10/29);混合型高脂血症者占27.59％(8/29).BCD患者血清TG、TC、LDL-C浓度分别为(1.63±1.19)、(5.10±1.05)、(3.27±0.97) mmol/L,明显高于正常对照组的(0.93±0.33)、(4.33±0.56)、(2.63±0.51) mmol/L,差异均有统计学意义(t=4.036、4.496、4.095,均P=0.000). 结论 血脂异常可能与BCD发病有关.     

Characterization of Bietti crystalline dystrophy patients with CYP4V2 mutations

PURPOSE: Mutations of the CYP4V2 gene, a novel family member of the cytochrome P450 genes on chromosome 4q35, have recently been identified in patients with Bietti crystalline dystrophy (BCD). The aim of this study was to investigate the spectrum of mutations in this gene in BCD patients from Singapore, and to characterize their phenotype. METHODS: Nine patients with BCD from six families were recruited into the study. The 11 exons of the CYP4V2 gene were amplified from genomic DNA of patients by polymerase chain reaction and then sequenced. Detailed characterization of the patients' phenotype was performed with fundal photography, visual field testing, fundal fluorescein angiography, and electroretinography (ERG). RESULTS: Three pathogenic mutations were identified; two mutations, S482X and K386T, were novel and found in three patients. The third mutation, a previously identified 15-bp deletion that included the 3' splice site for exon 7, was found in all nine patients, with six patients carrying the deletion in the homozygous state. Haplotype analysis in patients and controls indicated a founder effect for this deletion mutation in exon 7. Clinical heterogeneity was present in the patients. Compound heterozygotes for the deletion in exon 7 seemed to have more severe disease compared to patients homozygous for the deletion. There was good correlation between clinical stage of disease and ERG changes, but age did not correlate with disease severity. CONCLUSIONS: This study identified novel mutations in the CYP4V2 gene as a cause of BCD. A high carrier frequency for the 15-bp deletion in exon 7 may exist in the Singapore population. Phenotype characterization showed clinical heterogeneity, and age did not correlate with disease severity.     

Suppression of choroidal neovascularization by intramuscular polymer-based gene delivery of vasostatin

The purpose of this study was to evaluate the efficacy of gene delivery of angiogenesis inhibitor, vasostatin (VS), in suppressing experimental model of choroidal neovascularization (CNV). A mammalian expression vector carrying VS, pCMV3-VS, was constructed and evaluated for its ability to produce VS in transfected cells using western blot analysis and a cell viability assay. CNV was induced in Brown Norway rats by fundus argon laser photocoagulation and evaluated by fundus fluorescein angiography (FAG). Ten days post-laser treatment, gene delivery was achieved by intramuscular (IM) injection of poly-(N-vinyl pyrrolidone) (PVP) polymer conjugated with pCMV3-VS (PVP-VS) or a control vector (PVP-vector). Systemic VS expression was analysed by western blot analysis and enzyme linked immunosorbent assay (ELISA), and the extent of CNV was monitored by FAG analysis at different time intervals post-PVP treatment. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Besides, IM injection of PVP-VS, but not PVP-vector, led to elevated VS level in plasma for 30 days. After laser photocoagulation, rats injected with PVP-VS exhibited significantly lower incidence of CNV comparing with animals of control groups (P < 0.01) for at least 42 days. Moreover, rats treated with PVP-VS also showed a significant reduction in the CNV lesions compared with control groups (P < 0.001) for at least 42 days. Above all, no overt adverse effects were observed in rats received PVP-VS. These results demonstrate the potential of IM VS gene delivery for CNV treatment.     

氪激光诱导大鼠脉络膜新生血管基因芯片研究

目的运用基因芯片技术研究氪激光诱导大鼠脉络膜新生血管的差异表达基因．为脉络膜新生血管的治疗提供实验依据。方法氪激光光凝视网膜后7d，分剐提取实验组和对照组脉络膜组织的总RNA，将纯化后的mRNA进行反转录，制备杂交探针，应用含有5705条Oligo DNA的表达谱芯片对实验组和对照组脉络膜组织进行差异表达基因检测。结果氪激光诱导脉络膜新生血管组织的差异表达基因共有427条，其中上调基因为189条，下调基因为238条。上调最高的基因是MMP12基因。在差异表达基因中有代谢、细胞粘附、细胞运动、发育、运输、信号传导、分化等基因。结论氪激光诱导脉络膜新生血管的发生发展涉及多基因改变。[眼科新进展2005；25（5）：408—410]     

可溶性Tie2融合蛋白抑制小鼠实验性脉络膜血管新生

目的:观察可溶性Tie2融合蛋白(soluble Tie2fusion pro-tein,sTie-Fc)对小鼠脉络膜血管新生(choroidal neovascu-larization,CNV)的抑制作用。方法:采用激光击射的方法诱导成年C57BL/6小鼠CNV模型,术前及术后3,6,9d选取1眼玻璃体腔注入人sTie-Fc0.67μg,对侧眼在相同时点注入人IgG作为对照。激光术后10d处死小鼠,行脉络膜灌流铺片及组织病理学检查观察CNV的发展,定量评价sTie-Fc对于实验性CNV的抑制作用。结果:术后10d所有激光光斑均发展为实验性CNV,脉络膜灌流铺片(16.7±4.0)×10-3mm2vs(30.2±5.1)×10-3mm2,P<0.01)及组织病理学检查(1.70±0.56vs3.07±0.91,P<0.01)结果一致表明sTie-Fc明显抑制了小鼠CNV的发展。结论:sTie-Fc可以抑制实验性CNV的发展。     

Antiangiogenic effect of oral 2-methoxyestradiol on choroidal neovascularization in mice

We evaluated the efficacy of systemic 2-methoxyestradiol (2ME2) in a laser-induced murine model of choroidal neovascularization (CNV). C57BL/6J mice (8-week-old males) were used in this study and divided into four groups. After laser treatment, daily oral treatment with vehicle control, and 30, 50, and 75 mg/kg of 2ME2 was started. Two weeks after laser treatment, digital images of CNV were obtained from fluorescein isothiocyanate-dextran (FITC-dextran) angiography and choroidal flat mount after FITC-dextran perfusion. These images were quantified by NIH image software. Analysis of images from both FITC-dextran angiography and choroidal flat mount with FITC-dextran perfusion demonstrated that the 2ME2 treated groups showed a statistically significant, dose-dependent decrease in CNV. No toxicity or weight loss was observed during the treatment. Significant antiangiogenic effects of oral 2ME2 on laser induced CNV were observed. Since 2ME2 (Panzem) has demonstrated good safety in phase I/II trials for cancer, it has the potential to be used as a novel oral treatment for age-related macular degeneration.     

Fixation pattern and macular sensitivity in eyes with subfoveal choroidal neovascularization secondary to age-related macular degeneration. A microperimetry study

Purpose. To investigate the effects of subfoveal choroidal neovascularizzation (CNV) secondary to age-related macular degeneration (AMD) on macular functional parameters quantified with an automatic fundus perimeter. Methods. 118 eyes of 98 consecutive patients with subfoveal CNV secondary to AMD were evaluated. Best corrected visual acuity (ETDRS charts), fundus photography, and fluorescein angiography were performed. Microperimetry (fundus-related perimetry) was used to quantify macular sensitivity and fixation pattern (location and stability). Results. Of 118 eyes: 26 (21.9%) had central, 18 (15.1%) poor central and 74 (63.0%) eccentric fixation; 31 (26.0%) had stable, 42 (35.6%) relatively unstable and 45 (38.4%) unstable fixation. In 75 eyes (63.4%) a dense central scotoma was found. Angiographic classification of subfoveal CNV (occult versus classic) was not significantly related to fixation pattern (location: P = 0.274; stability: P = 0.385), and presence of dense scotoma (P = 0.41). Conclusion. Microperimetric quantification of macular sensitivity and fixation pattern in eyes with subfoveal CNV secondary to AMD offers new data about the impact of visual impairment in these eyes. Moreover, microperimetry improves the functional evaluation of subfoveal CNV in AMD.     

考布他汀A4隐形/靶向脂质体抑制脉络膜新生血管的研究

目的观察考布他汀A4（CA4）隐形/靶向脂质体对激光诱导的棕色挪威（BN）大鼠脉络膜新生血管（CNV）的抑制作用。方法成年BN大鼠20只,647 nm氪红激光光凝视网膜至Bruch膜破裂诱导CNV,脂质体药物的制备采用薄膜分散-超声法。动物模型随机分为考布他汀A4磷酸盐（CA4P）组、连接精氨酸-甘氨酸-天冬氨酸三肽（RGD）靶头的CA4靶向脂质体（RGD-SSL-CA4）组、连接RGD靶头的空白靶向脂质体（RGD-SSL）组、不连接RGD靶头的CA4隐形脂质体（SSL-CA4）组和生理盐水（NS）组,于激光光凝后3 d和10 d经大鼠尾静脉给药。光凝后1周和2周进行荧光素眼底血管造影（FFA）检查,观察激光斑处荧光素渗漏情况。激光光凝后2周行FITC-Dextran灌注后处死大鼠制作脉络膜铺片,计算CNV面积。结果 RGD-SSL-CA4组视网膜光凝斑处的荧光素渗漏明显较其他组轻。RGD-SSL-CA4组CNV面积为（25 360±14 050）μm2,NS组为（46 500±17 230）μm2,RGD-SSL-CA4组较NS组明显缩小,差异有统计学意义（P〈0.05）;CA4P组为（38 290±19 290）μm2,RGD-SSL组为（44 150±16 410）μm2,SSL-CA4组为（39 690±17 850）μm2,与NS组比较差异均无统计学意义（P〉0.05）。结论 RGD-SSL-CA4具有长循环、靶向的双重优势,可显著抑制激光诱导的CNV。