ELOVL4基因过表达对n3和n6超长链多不饱和脂肪酸生物合成效率的影响

目的：ELOVL4是常染色体显性Stargardt氏黄斑变性的致病基因,ELOVL4蛋白酶参与n3和n6超长链多不饱和脂肪酸（very long chain polyunsaturated fatty acid,VLC-PUFA）的合成,比较两者的生物合成效率,对治疗Stargardt氏黄斑变性有指导意义。方法：构建携带ELOVL4基因和绿色荧光蛋白的重组腺病毒,转入培养的PC12细胞,将细胞分成三组：PC12、PC12＋Ad-GFP和PC12＋Ad-ELOVL4,前两组为对照组,通过qRT-PCR定量分析ELOVL4基因的表达量,Western Blot检测ELOVL4蛋白的表达;等浓度（1∶1）加入EPA（n3PUFA）和AA（n6 PUFA）,孵育48h之后进行脂肪酸提取,通过气相色谱-质谱法（gas chromatography-mass spectrometry,GC-MS）分析超长链脂肪酸的成分。结果：GC-MS检测到分别用EPA和AA处理后的PC12＋Ad-ELOVL4的细胞中有n3 VLC-PUFA的表达,34∶5n3和36∶5n3是20∶5n3/EPA的主要产物,分别为0.71%和1.6%;34∶4n6和36∶4n6是20∶4n6/AA的主要产物,分别为0.46%和0.61%;EPA所产生的n3 VLC-PUFAs总和是AA所产生的n6 VLC-PUFAs总和的2倍。结论：在ELOVL4蛋白作用下,EPA合成VLC-PUFAs的效率高于AA,饮食中给予适当比例的n3/n6 PUFAs,可能是治疗STGD3疾病的方式之一。     

先天性无虹膜合并先天性白内障家系致病基因突变分析

目的：对先天性无虹膜合并先天性白内障家系进行PAX6基因突变位点筛查,丰富该致病基因的突变谱。

方法：选取就诊于北京同仁医院眼科门诊的1个先天性无虹膜合并先天性白内障家系和100名健康志愿者,采集外周静脉血,提取基因组DNA,采用直接测序法进行PAX6基因突变位点的筛查。

结果：该家系中先证者和其他患者均表现为无虹膜合并白内障,PAX6基因测序结果显示,该致病基因第11外显子无义突变c.991 C>T,造成PAX6基因编码的蛋白截短(R331X),从而使该蛋白失去功能,且该突变在家系内与疾病表型共分离,不存在于家系内及家系外健康样本的基因中。

结论：PAX6 R331X突变与先天性无虹膜合并先天性白内障的发生有关。     

特发性黄斑裂孔患者玻璃体术后黄斑结构和中央凹视网膜厚度变化

目的：分析特发性黄斑裂孔患者接受23G玻璃体手术治疗后黄斑结构的修复情况,以及视力和黄斑中央凹视网膜厚度的变化。

方法：将2016-06/2017-12在我院进行择期手术的单眼特发性黄斑裂孔患者85例85眼纳入研究,其中男37例,女48例,平均年龄64.7±10.1岁。所有患者均接受23G玻璃体切割术,应用OCT观察术后黄斑裂孔闭合情况; 应用OCT观察术前和术后1、3、6mo黄斑中央凹视网膜厚度的变化; 观察术前和术后1、3、6mo患者最佳矫正视力的变化。

结果：术后所有患者获得良好的黄斑裂孔闭合。术后3、6mo时所有患者平均最佳矫正视力显著高于术前和术后1mo(P<0.05); 术后6mo平均最佳矫正视力显著高于术后3mo,差异有统计学意义(t=7.983,P=0.037)。术后1mo黄斑中央凹视网膜厚度显著高于术前和术后3、6mo(P<0.05); 术后3、6mo的黄斑中央凹视网膜厚度显著低于术前(P<0.05)。

结论：应用23G玻璃体切除术治疗特发性黄斑裂孔具有较高的裂孔成功闭合率,患者的视力明显提高。     

后巩膜加固术在超长眼轴黄斑裂孔性视网膜脱离手术中的应用

目的：评价后巩膜加固术(PSR)辅助超长眼轴黄斑裂孔性视网膜脱离患者玻璃体切除手术(PPV)的临床效果。

方法：采用临床随机对照研究。纳入眼轴≥29mm的超长眼轴黄斑裂孔性视网膜脱离患者46例46眼,随机分为两组：A组采用PSR+PPV+硅油填充,B组采用单纯PPV +硅油填充。随访1a,分析各组术后BCVA、黄斑裂孔愈合情况、视网膜脱离复位率、再手术率等指标。

结果：两组治疗后平均BCVA(LogMAR视力)均较术前改善,A组由1.61±0.02提升为0.85±0.22(t=10.36,P<0.01),B组由1.59±0.04提升为1.08±0.16(t=7.92,P<0.01),其中A组改善幅度大于B组(t=-2.38,P=0.03)。两组术前术后眼轴均无明显改变(P>0.05)。A组一次手术黄斑裂孔愈合率为91%(21/23),取油前再手术率4%(1/23),取油后无再脱离患者,1a随访期内所有术眼完成了硅油取出。B组一次手术黄斑裂孔愈合率为65%(15/23),取油前再手术率35%(8/23),取油后再脱离比例26%(6/23),1a随访期取出硅油比例74%(17/23)。

结论：后巩膜加固术有助于提高病理性近视黄斑裂孔性视网膜脱离的手术成功率,减少复发及再手术的几率。     

激光联合复方血栓通胶囊治疗视网膜静脉阻塞的临床观察

目的：观察激光联合复方血栓通胶囊治疗视网膜静脉阻塞的临床疗效。

方法：将64例视网膜静脉阻塞患者随机分为激光治疗组(对照组,32例)和激光联合复方血栓通胶囊治疗组(观察组,32例),观察对比两组治疗后的视力、视网膜黄斑厚度和有效率。

结果：观察组临床总有效率和视力进步比例均显著高于对照组,两组间差异具有统计学意义(P<0.05或P<0.01); 两组治疗后1、3、6mo的黄斑中心凹厚度均较治疗前显著降低(P<0.05),但观察组治疗后1、3、6mo的黄斑中心凹厚度均明显小于对照组同期(P<0.05)。

结论：激光联合复方血栓通胶囊治疗视网膜静脉阻塞伴黄斑水肿有较好的疗效,能有效提高视力,降低视网膜黄斑厚度,值得进一步研究。     

硅油填充眼黄斑下积液手术疗效观察

目的：分析硅油填充眼黄斑下积液行硅油取出联合视网膜内界膜剥除、C₃F₈填充术后的临床疗效。

方法：分析2007-01/2013-12于我院行玻璃体切除联合硅油填充术后出现黄斑下积液6mo的患者31例31眼,根据手术方式分为A、B组,A组行硅油取出术,B组行硅油取出联合内界膜剥除、C₃F₈填充术,手术后随访时间为6～12(平均为8.33±1.45)mo,对比分析术后3、6mo两组的LogMAR最佳矫正视力提高率、黄斑平均神经上皮层厚度。

结果：术后3mo比较各组平均LogMAR 最佳矫正视力(best-corrected visual acuity,BCVA)均较术前有提高,差异有统计学意义(t=2.326、2.577,P<0.05),A组和B组LogMAR BCVA提高率比较,差异有统计学意义(χ²=5.473,P<0.05); 术后6mo比较各组平均LogMAR BCVA均较术前有提高,差异有统计学意义(t=4.216、3.801,P<0.05),A组和B组LogMAR BCVA提高率比较,差异有统计学意义(χ²=4.210,P<0.05)。术后各组黄斑平均神经上皮层厚度变薄。

结论：硅油取出联合视网膜内界膜剥除、C₃F₈填充术是治疗硅油填充眼持续性黄斑下积液的有效方法。     

特发性黄斑前膜手术前后脉络膜厚度分析

目的：研究特发性黄斑前膜手术前后膜脉络膜厚度的变化及视功能与脉络膜厚度的关系。

方法：选取 2016-01/2017-12于我院接受手术治疗的特发性黄斑前膜患者30例30眼进行临床研究,排除手术禁忌,采用25G玻璃体切割系统行标准三切口经睫状体平坦部玻璃体切除术联合剥除黄斑前膜及内界膜,观察术前、术后1wk,1、3mo患者的BCVA、黄斑中心凹脉络膜厚度,分析BCVA与脉络膜厚度的相关性。

结果：术前的BCVA显著低于术后3mo(P=0.011),术后3mo BCVA与术前BCVA显著相关(r=0.610,P<0.01),术前术后不同时间的脉络膜厚度无差异(P=0.999)。手术前后BCVA与手术前后脉络膜厚度无线性相关关系。

结论：特发性黄斑前膜手术前后脉络膜厚度无明显变化,患者视功能与脉络膜厚度也无明显相关性。     

激光光凝术联合VEGF抑制剂治疗糖尿病性黄斑水肿的临床研究

目的：探讨激光光凝术、血管内皮生长因子抑制剂单用或联用对糖尿病性黄斑水肿(diabetic macular edema,DME)患者疗效和安全性的影响。

方法：研究对象选取我院2014-10/2016-10收治的DME患者150例156眼,以随机数字表法分为A组50例52眼,B组50例51眼和C组50例53眼,分别采用激光光凝术单用、血管内皮生长因子抑制剂单用和激光光凝术+血管内皮生长因子抑制剂联合方案治疗。比较三组患者治疗前后最佳矫正视力、黄斑中心凹厚度、视网膜新生血管渗漏面积和并发症发生率。

结果：B、C组患者治疗后3、6、12mo最佳矫正视力水平较A组均显著提高(P<0.05); B、C组患者治疗后3、6、12mo黄斑中心凹厚度较A组均显著降低(P<0.05); B、C组患者治疗后3mo视网膜新生血管渗漏面积较A组均显著缩小(P<0.05); C组患者治疗后6mo和12mo视网膜新生血管渗漏面积较A、B组均显著缩小(P<0.05); 同时三组患者术后并发症发生率比较,差异无统计学意义(P>0.05)。

结论：激光光凝术联合血管内皮生长因子抑制剂治疗DME可有效改善视力水平,降低黄斑中心凹厚度,控制视网膜新生血管渗漏,且未增加不良反应,效果优于激光光凝术和血管内皮生长因子抑制剂单用。     

两种内界膜剥离方式治疗MHCI<0.7特发性黄斑裂孔的疗效

目的：观察玻璃体切割术中不同内界膜剥离方式治疗黄斑裂孔闭合指数(MHCI)<0.7特发性黄斑裂孔(IMH)的临床效果。

方法：将2014-05/2017-05收治的MHCI<0.7 IMH患者88例88眼随机分为A组(44眼,行扩大内界膜剥离术)和B组(44眼,行标准内界膜剥离术),观察两组患者黄斑裂孔闭合情况、最佳矫正视力(BCVA)、中心暗点及并发症发生情况。

结果：术后6mo,A组患者黄斑裂孔闭合率明显高于B组(91% vs 75%,P<0.05),BCVA优于B组(0.47±0.05 vs 0.74±0.14,P<0.05),中心暗点眼数占比低于B组(4% vs 23%,P<0.05),且两组患者并发症发生率无明显差异(11% vs 9%,P>0.05)。

结论：临床治疗MHCI<0.7的IMH采用扩大内界膜剥离术较标准内界膜剥离术疗效更突出,前者视网膜功能恢复效果更佳。     

特发性黄斑前膜患者内界膜剥除术后视功能的影响因素分析

目的：探讨影响特发性黄斑前膜患者内界膜剥除术后视功能恢复的危险因素。

方法：回顾我院2016-01/2020-04收治的特发性黄斑前膜行视网膜内界膜剥除术的患者118例118眼。术后随访6mo评价手术疗效,观察术前、术后1、3、6mo的视力变化、视物变形程度、黄斑中心凹平均厚度、黄斑区容积。分析术前及术后黄斑中心凹平均厚度、黄斑区容积与术后视力及视物变形评分的相关性,评估术后视功能恢复不良的危险因素。

结果：本组患者96眼视功能恢复良好,手术治疗改善率为81.4%。与术前相比,术后1、3、6mo术眼视力明显提升(P<0.05),水平方向视物变形评分明显缩小(P<0.05),术眼黄斑中心凹平均厚度、黄斑区容积明显减小(P<0.05)。术前与术后6mo黄斑中心凹平均厚度、黄斑区容积与术后6mo视力均呈负相关(P<0.05),与术后6mo水平方向视物变形评分均呈正相关(P<0.05)。IMEM病程、术前矫正视力、术前水平或垂直方向视物变形、术前黄斑水肿是患者术后视功能恢复情况的影响因素(均P<0.05),其中术前矫正视力差(OR=3.062)、术前存在水平方向视物变形(OR=2.438)、术前存在黄斑水肿(OR=2.000)是导致患者术后视功能恢复不良的危险因素。

结论：内界膜剥除术治疗特发性黄斑前膜效果良好,可有效改善术眼视力,减轻视物变形。病程越长、术前矫正视力越差、术前视物变形越严重、术前有黄斑水肿的患者内界膜剥除术后视功能恢复越差。     

Long-term follow-up of autosomal dominant Stargardt macular dystrophy (STGD3) subjects enrolled in a fish oil supplement interventional trial

Background: Earlier studies have raised the notion that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) supplementation could be a useful intervention in autosomal dominant Stargardt macular dystrophy (STGD3). We sought to assess whether fish oil supplementation has a beneficial effect on the clinical course of STGD3 secondary to a mutation in the ELOVL4 gene.

Materials and Methods: Eleven patients with STGD3 were enrolled in an 8-year open-label, clinical interventional study of over-the-counter fish oil supplements at a recommended daily dose of 650 mg EPA and 350 mg DHA (NCT00420602). Subjects had annual eye examinations with complete imaging, visual function testing, and blood lipid analyses. Compliance with therapy was measured by periodic patient self-report and with serum and red blood cell biomarkers of lipid consumption. Paired sample t-tests were used to measure differences in mean values of visual acuity, lipid biomarkers, and contrast sensitivity obtained at baseline and the last follow-up.

Results: All subjects showed progression of their maculopathy, and we could not discern a beneficial effect of the intervention. Compliance with the recommended fish oil supplement intervention was poor as assessed by patient self-report and biomarkers of lipid consumption.

Conclusions: Our inability to detect a benefit of fish oil could be the result of small subject numbers, poor compliance, or intervention too late in the course of the disease. We still advise STGD3 patients to consume fish or fish oil regularly, and we recommend that pre-symptomatic children with ELOVL4 mutations should be especially targeted for these interventions.     

Genetics and molecular pathology of Stargardt-like macular degeneration

Stargardt-like macular degeneration (STGD3) is an early onset, autosomal dominant macular degeneration. STGD3 is characterized by a progressive pathology, the loss of central vision, atrophy of the retinal pigment epithelium, and accumulation of lipofuscin, clinical features that are also characteristic of age-related macular degeneration. The onset of clinical symptoms in STGD3, however, is typically observed within the second or third decade of life (i.e., starting in the teenage years). The clinical profile at any given age among STGD3 patients can be variable suggesting that, although STGD3 is a single gene defect, other genetic or environmental factors may play a role in moderating the final disease phenotype. Genetic studies localized the STGD3 disease locus to a small region on the short arm of human chromosome 6, and application of a positional candidate gene approach identified protein truncating mutations in the elongation of very long chain fatty acids-4 gene (ELOVL4) in patients with this disease. The ELOVL4 gene encodes a protein homologous to the ELO group of proteins that participate in fatty acid elongation in yeast. Pathogenic mutations found in the ELOVL4 gene result in altered trafficking of the protein and behave with a dominant negative effect. Mice carrying an Elovl4 mutation developed photoreceptor degeneration and depletion of very long chain fatty acids (VLCFA). ELOVL4 protein participates in the synthesis of fatty acids with chain length longer than 26 carbons. Studies on ELOVL4 indicate that VLCFA may be necessary for normal function of the retina, and the defective protein trafficking and/or altered VLCFA elongation underlies the pathology associated with STGD3. Determining the role of VLCFA in the retina and discerning the implications of abnormal trafficking of mutant ELOVL4 and depleted VLCFA content in the pathology of STGD3 will provide valuable insight in understanding the retinal structure, function, and pathology underlying STGD3 and may lead to a better understanding of the process of macular disease in general.     

Elovl4 5-bp deletion knock-in mouse model for Stargardt-like macular degeneration demonstrates accumulation of ELOVL4 and lipofuscin

The mechanism underlying photoreceptor degeneration in autosomal dominant Stargardt-like macular degeneration (STGD3) due to mutations in the elongation of very long chain fatty acids-4 (ELOVL4) gene is not fully understood. To evaluate the pathological events associated with STGD3, we used a mouse model that mimics the human STGD3 phenotype and studied the progression of retinal degeneration. Morphological changes in the retina of Elovl4 5-bp deletion knock-in mice (E_mut^+/−) were evaluated at 22 months of age. The localization of ELOVL4, and the expression pattern of inner retinal tissue marker proteins, and ubiquitin were determined by immunofluorescence labeling of retinal sections. Levels of the retinal pigment epithelium (RPE) lipofuscin fluorophores were measured by quantitative HPLC. Morphological evaluation of the retina revealed an accumulation of RPE debris in the subretinal space. A significant increase in the amount of ELOVL4 was observed in the outer plexiform layer in E_mut^+/− mice compared to controls. Apart from the accumulation of ELOVL4, E_mut^+/− mice also exhibited high expression of ubiquitin in the retina. Analysis of lipofuscin fluorophores in the RPE showed a significant elevation of A2E and compounds of the all-trans-retinal dimer series in retinas from four and ten month old E_mut^+/− mice compared to wild-type littermates. These observations suggest that abnormal accumulation of ELOVL4 protein and lipofuscin may lead to photoreceptor degeneration in E_mut^+/− mice.     

Association of n3 and n6 polyunsaturated fatty acids in red blood cell membrane and plasma with severity of normal tension glaucoma

AIM:To determine whether red blood cell (RBC) membrane and plasma lipids, particularly long-chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA) are significantly correlated with severity of normal tension glaucoma (NTG).METHODS:This study included 35 patients with NTG and 12 healthy normal control subjects, matched for age and sex with the study group. The stage of glaucoma was determined according to the Hodapp-Parrish-Anderson classification. Lipids were extracted from RBC membranes and plasma, and fatty acid methyl esters prepared and analyzed by gas chromatography-mass spectrometry (GC-MS).RESULTS:When RBC lipids were analyzed, the levels of EPA, the levels of DHA and the ratio of n3 to n6 were positively associated with the Humphrey Perimetry mean deviation (MD) score (r=0.617, P<0.001; r=0.727, P<0.001 and r=0.720, P<0.001, respectively), while the level of AA was negatively associated with the MD score (r=-0.427, P=0.001). When plasma lipids were analyzed, there was a significant positive relationship between the levels of EPA and the MD score (r=0.648, P<0.001), and the levels of AA were inversely correlated with the MD score (r=-0.638, P<0.001).CONCLUSION:The levels of n3 and n6 polyunsaturated fatty acids in RBC membrane and plasma lipids were associated with severity of NTG.     

Elovl4 5-bp deletion knock-in mouse model for Stargardt-like macular degeneration demonstrates accumulation of ELOVL4 and lipofuscin

The mechanism underlying photoreceptor degeneration in autosomal dominant Stargardt-like macular degeneration (STGD3) due to mutations in the elongation of very long chain fatty acids-4 (ELOVL4) gene is not fully understood. To evaluate the pathological events associated with STGD3, we used a mouse model that mimics the human STGD3 phenotype and studied the progression of retinal degeneration. Morphological changes in the retina of Elovl4 5-bp deletion knock-in mice (E_mut^+/−) were evaluated at 22 months of age. The localization of ELOVL4, and the expression pattern of inner retinal tissue marker proteins, and ubiquitin were determined by immunofluorescence labeling of retinal sections. Levels of the retinal pigment epithelium (RPE) lipofuscin fluorophores were measured by quantitative HPLC. Morphological evaluation of the retina revealed an accumulation of RPE debris in the subretinal space. A significant increase in the amount of ELOVL4 was observed in the outer plexiform layer in E_mut^+/− mice compared to controls. Apart from the accumulation of ELOVL4, E_mut^+/− mice also exhibited high expression of ubiquitin in the retina. Analysis of lipofuscin fluorophores in the RPE showed a significant elevation of A2E and compounds of the all-trans-retinal dimer series in retinas from four and ten month old E_mut^+/− mice compared to wild-type littermates. These observations suggest that abnormal accumulation of ELOVL4 protein and lipofuscin may lead to photoreceptor degeneration in E_mut^+/− mice.     

Elovl4 haploinsufficiency does not induce early onset retinal degeneration in mice

ELOVL4 was first identified as a disease-causing gene in Stargardt macular dystrophy (STGD3, MIM 600110.) To date, three ELOVL4 mutations have been identified, all of which result in truncated proteins which induce autosomal dominant juvenile macular degenerations. Based on sequence homology, ELOVL4 is thought to be another member within a family of proteins functioning in the elongation of long chain fatty acids. However, the normal function of ELOVL4 is unclear. We generated Elovl4 knockout mice to determine if Elovl4 loss affects retinal development or function. Here we show that Elovl4 knockout mice, while perinatal lethal, exhibit normal retinal development prior to death at day of birth. Further, postnatal retinal development in Elovl4 heterozygous mice appears normal. Therefore haploinsufficiency for wildtype ELOVL4 in autosomal dominant macular degeneration likely does not contribute to juvenile macular degeneration in STGD3 patients. We found, however, that Elovl4+/- mice exhibit enhanced ERG scotopic and photopic a and b waves relative to wildtype Elovl4+/+ mice suggesting that reduced Elovl4 levels may impact retinal electrophysiological responses.     

Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.

PURPOSE: A 5-bp deletion in ELOVL4, a photoreceptor-specific gene, has been associated with autosomal dominant (ad) macular dystrophy phenotypes in five related families, in which phenotypes range from Stargardt-like macular dystrophy (STGD3; Mendelian Inheritance in Man 600110) to pattern dystrophy. This has been the only mutation identified in ELOVL4 to date, which is associated with macular dystrophy phenotypes. In the current study, the potential involvement was investigated of an ELOVL4 gene variation in adSTGD-like and other macular dystrophy phenotypes segregating in a large unrelated pedigree from Utah (K4175). METHODS: The entire open reading frame of the ELOVL4 gene was analyzed by direct sequencing in a proband from the K4175 family. The combination of denaturing high-performance liquid chromatography (DHPLC) analysis and direct sequencing of all available family members was used to further assess segregation of identified ELOVL4 variants in the pedigree. RESULTS: A complex mutation, two 1-bp deletions separated by four nucleotides, was detected in all affected members of the family. The mutation results in a frameshift and the truncation of the ELOVL4 protein, similar to the effect of the previously described 5-bp deletion. CONCLUSIONS: The discovery of a second mutation in the ELOVL4 gene segregating with macular dystrophy phenotypes confirms the role of this gene in a subset of dominant macular dystrophies with a wide range of clinical expressions and suggests a role for modifying genes and/or environmental factors in the disease process.     

Pax6基因在视网膜母细胞瘤中的表达及临床意义

目的:探讨Pax6基因在视网膜母细胞瘤(retinoblastoma,Rb)中的表达及临床意义。方法:选择我院2001-01/2012-12收存在眼科病理室的15例Rb组织切片设为观察组,再选取15例正常视网膜组织切片设为对照组。应用Western-Blot及RT-PCR(逆转录酶链反应)法分别对正常视网膜组织及Rb组织中的Pax6蛋白和Pax6 mRNA的表达进行检测,同时应用Western-Blot法对Pax6基因下游的BRN3b及MATH5促分化基因在蛋白水平的表达进行检测,最后进行组间比较,进而对Pax6基因在Rb中的表达及临床意义进行探讨。结果:观察组Pax6基因mRNA表达平均值为0.99±0.03,Pax6基因蛋白表达平均值为2.07±0.15,BRN3b蛋白表达平均值为0.195±0.016,MATH5蛋白表达平均值为0.190±0.031,均明显高于对照组,差异有统计学意义(P<0.05)。结论:异常表达的Pax6基因可能对Rb的出现起到促进作用。