Leber先天性黑矇致病基因研究及基因治疗进展

Leber先天性黑矇(LCA)是一种严重的遗传性视网膜病变,具有遗传异质性与表型多样性的特点。近年来的遗传学研究相继发现多个致病基因与LCA发病相关,并对这些致病基因的发病机制作了进一步研究。LCA基因治疗已经从临床前期动物研究阶段进入Ⅰ期临床试验阶段,尤其是在LCAⅡ型患者中进行的RPE65基因治疗方面取得了突破性进展,为其他遗传性视网膜疾病的基因治疗打下良好的基础。     

利用全外显子组测序法筛选Leber先天性黑矇一家系候选致病基因的实验研究

目的利用全外显子组测序法筛选Leber先天性黑矇(LCA)一个家系的候选致病基因,为补充或验证LCA致病基因的研究奠定基础。方法采用横断面研究方法。收集一个在深圳市眼科医院就诊的中国汉族LCA家系的临床资料。详细询问并记录全部家系成员的疾病史、家族史及婚育史,并对全部家系成员进行全面的身体检查。其中,眼部检查项目包括视力、眼位、眼压、验光、眼球运动情况、眼前段、眼底照相、光学相干断层扫描及视网膜电图检查等。提取先证者、另外1例患者及其父母的静脉血基因组DNA,采用磁珠提取法对DNA进行提纯。使用高通量测序平台对质量检测通过的DNA文库进行测序。在进行生物信息学分析时,对采集的数据行标准信息分析流程处理,同时对数据进行质控检测。采用GATK基因组分析网络工具库检索单核苷酸多态性位点和缺失标记位点的数量。测序结果经生物信息学分析工具Seattle Seq Annotation138注释后,与人类HAPMAP、db SNP138、Exome Sequencing Project及Exome Aggregation Consortium数据库进行比对。过滤掉已报道的常见变异后,再过滤掉位于非编码区的变异和同义突变,并进一步筛选出该LCA家系的候选致病基因。结果该家系成员共有3代16人。其中,3名LCA患者均为第Ⅱ代成员,第Ⅰ代及第Ⅲ代成员均无发病者,符合常染色体隐性遗传规律。经测序及分析后,得到17个LCA的候选致病基因,分别为ACTN1、C1QTNF3、FAN1、MMP28、MYO9B、NAV1、NUP62、PHLDB3、PRDM12、SLC24A4、ST3GAL3、TCIRG1、TP53、USP54、YIF1B、ZDHHC17及ZNF107。这些基因均与目前已报道的24个LCA相关的致病基因不同。结论对该家系的DNA进行分析后,发现了17个新的LCA候选致病基因,可为进一步明确该家系LCA致病基因的研究奠定了基础。     

Leber先天性黑矇的基因治疗研究进展

Leber先天性黑矇(LCA)是一种严重的先天性致肓遗传性视网膜疾病.近1O年来,随着分子遗传学的发展及基因治疗技术的进步,以腺相关病毒载体介导的LCA基因治疗研究取得了令人鼓舞的进展,尤其是对LCAⅡ患者进行的RPE65基因治疗的Ⅰ期临床试验的成功使其成为眼科遗传性疾病基因治疗领域中的先行者,为今后进行其他遗传性视网膜疾病的基因治疗开辟了光明的前景.本文就目前LCA基因治疗的临床前研究及Ⅰ期临床试验的进展等方面作一综述.

Abstract：

Leber congenital amaurosis (LCA)is an early onset retinal dystrophy that causes severe visual impairment. With the development of molecular genetics and the therapeutic gene replacement technology, the adeno-associated viral (AAV) vector-mediated gene therapy for LCA achieved encouraging progress in the past decade. The success of the Phase Ⅰ clinical trials of human RPE65 gene therapy for LCA Ⅱ patients makes it a pioneer in the field of retinal gene therapy and brings light to the cure of other hereditary retinopathy. This article briefly reviews the recent developments in the preclinical animal experiments and Phase Ⅰ clinical trials for LCA.     

Leber先天性黑朦致病基因及相关临床表型研究进展

Leber先天性黑嚎（LCA）是导致先天性盲的主要遗传性视网膜疾病,具有遗传异质性与临床表型多样性的特点。近年来其分子遗传学研究成为国内外热点,相继明确了20个与LCA相关的致病基因。多项研究表明LCA的基因型和临床表型之间存在关联,了解不同致病基因对应的临床表型特点有助于致病基因的筛查。就当前发现的LCA致病基因、可能的发病机制以及特定基因型与临床表型的关系进行综述,以期有助于临床诊断和咨询。     

CRB1突变型Leber先天性黑矇和早发视网膜萎缩患儿基因型与表型特征研究

目的:观察 CRB1突变型Leber先天性黑矇（LCA）和早发视网膜萎缩（EOSRD）患儿基因型与表型关系特征。 方法:回顾性临床研究。2013年1月至2019年12月于上海交通大学医学院附属新华医院眼科临床及基因诊断的 CRB1突变LCA和EOSRD患儿10例纳入研究。二代测序及致病...     

RPGRIP1基因新突变致Leber先天性黑矇一家系

目的确定1个汉族Leber先天性黑矇(LCA)家系的致病基因突变。方法回顾性研究。2018年10月在河南省立眼科医院就诊的LCA一家系1例患者和3名家系成员纳入研究。详细询问患者病史并行物体注视性质、追随试验、裂隙灯显微镜、散瞳验光、眼底照相及全视野ERG检查;家系成员行BCVA、裂隙灯显微镜联合前置镜、验光、眼底照相及全视野ERG检查。采集先证者及其兄长、父母的外周静脉血5 ml,提取全基因组DNA。应用包含441个致病基因的遗传眼病捕获芯片进行靶向捕获富集高通量测序以获得致病基因及突变。对可疑致病突变位点通过Sanger进行验证,并行生物信息学分析确定基因突变位点的致病性。结果患者表现为自幼不追物但有明显畏光和眼球震颤;双眼眼前节及眼底无异常;全视野ERG检查可见双眼视锥、视杆系统功能严重下降。基因检测结果显示,患者RPGRIP1基因存在c.1635dupA和c.3565C>T两个突变。其中,RPGRIP1 c.1635dupA为新发突变。RPGRIP1基因c.1635dupA和c.3565C>T构成复合杂合突变。生物信息学分析结果显示,c.3565C>T为致病突变,c.1635dupA为可能致病突变。结论RPGRIP1基因新发突变c.1635dupA与c.3565C>T构成复合杂合突变可能是本家系的致病原因。     

临床特征不典型的Leber遗传性视神经病变线粒体DNA基因检测结果分析

Leber遗传性视神经病变(LHON)是由线粒体DNA(mtDNA)突变引起的母系遗传性视神经疾病,mtDNA基因检测是LHON确诊的必要手段,目前已有35个与LHON相关的mtDNA突变位点被发现[1-3].     

Leber先天性黑矇患者血清脂质浓度的变化

目的：分析Leber先天性黑矇(Leber congenital amaurosis,LCA)患者血清脂质浓度的变化。

方法：采用回顾性序列病例研究方法,选取临床诊断为LCA的患者50例作为LCA组,选取52例正常人作为正常对照组,按盲法由专业技术人员测量两组受检者血清中低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、甘油三酯(triglycerides,TG)、总胆固醇(total cholesterol,TC)含量,并对两组受检者的测量结果进行比较。

结果：LCA患者50例中,血脂水平异常者占46%,其中低HDL-C血症者占26%,高TG血症者占48%,高TC血症者占17%,混合型高脂血症者占9%。LCA患者血清HDL-C浓度为1.221±0.317mmol/L,较正常对照组明显下降,差异有统计学意义(P<0.05); 血清TG浓度(1.377±1.171mmol/L)和TC浓度(4.506±0.694mmol/L)与正常对照组相比均明显升高,差异有统计学意义(均P<0.01); LDL-C浓度与正常对照组相比,差异无统计学意义(P>0.05)。

结论：LCA患者血清HDL-C、TG和TC浓度异常变化可能与LCA的发病相关。     

应用新一代目标区域捕获测序揭示视网膜色素变性患者的EYS基因突变

目的应用新一代测序技术研究一例散发视网膜色素变性（RP）患者的致病基因突变位点及其临床表型。方法实验研究。 收集一散发的RP家系,共3名家庭成员参与研究。其中1名患者,2名正常家属。采集3 ml外周静脉血,提取基因组DNA,运用目标区域捕获测序技术来筛查目前已报道的201个遗传性视网膜疾病的致病基因,测序结果运用生物信息学分析得到候选基因,最后用Sanger测序验证。结果临床检查结果显示患者呈现典型的RP临床症状。遗传学筛查结果证实患者在EYS基因上存在2个复合杂合突变：1个杂合的错义突变（c.6416G>A,p.C2139Y）,1个杂合的无义突变（c.8012T>A,p.L2671X）。Sanger测序结果证实为阳性并且分别遗传自父亲母亲,为常染色体隐性遗传。EYS基因被报道为RP的主要致病基因之一。结论本例患者的EYS基因存在2个杂合突变,是导致RP的致病原因。     

应用目标基因捕获结合二代测序技术检测无遗传性眼病家族史的遗传性视网膜疾病患者的致病基因

背景 遗传性视网膜疾病(HRDs)是以原发性视网膜病变为主要病理改变、视力和色觉等视敏度严重损害为主要临床表现的致盲眼病,其与遗传因素密切相关,是临床上难治性盲的首要原因. 目的 应用目标基因捕获结合二代测序技术检测HRDs的致病基因. 方法 选择2014年1月至2015年1月在宁夏眼科医院就诊的无遗传性眼病家族史的20例HRDs患者作为研究对象.收集所有患者及其家庭成员临床资料,完善眼科检查,抽取患者和家庭正常成员外周静脉血,提取DNA.针对232个HRDs已知致病基因的目标序列设计并定制目标序列捕获芯片,借助高通量二代测序技术检测患者的遗传变异,数据经分析滤过,候选突变进行Sanger测序验证和致病性评估,明确致病基因和突变,并对表型和基因型间的关系进行分析.结果 20例HRDs患者中有11例患者检测到致病性突变位点,共涉及9个基因,阳性率为55％.其中有8例患者检测到复合杂合突变,3例患者为纯合子突变,均为新发现的突变位点.通过对家系成员的基因筛查分析,6例HRDs患者为常染色体隐性遗传,5例遗传类型不确定.结合临床表型以及基因检测结果分析,11例HRDs患者的诊断分别为锥杆细胞营养不良5例,致病基因分别为ABCA4、RPE65、USH2A、RIMS1和RHO;Leber先天性黑曚3例,致病基因分别为CRB1(2例)和LCA5;先天性静止性夜盲l例,致病基因为PRPF3;Best卵黄样黄斑营养不良1例,致病基因为BEST1基因;Stargardt病1例,致病基因为ABCA4基因. 结论 目标基因捕获结合二代测序技术可以对视网膜疾病患者进行快速、有效的基因诊断,结合临床特征分析有助于提高隐性遗传及遗传方式不明的HRDs的临床诊断水平.     

Comprehensive analysis of genetic variations in strictly-defined Leber congenital amaurosis with whole-exome sequencing in Chinese

AIM: To make a comprehensive analysis of the potential pathogenic genes related with Leber congenital amaurosis (LCA) in Chinese. METHODS: LCA subjects and their families were retrospectively collected from 2013 to 2015. Firstly, whole-exome sequencing was performed in patients who had underwent gene mutation screening with nothing found, and then homozygous sites was selected, candidate sites were annotated, and pathogenic analysis was conducted using softwares including Sorting Tolerant from Intolerant (SIFT), Polyphen-2, Mutation assessor, Condel, and Functional Analysis through Hidden Markov Models (FATHMM). Furthermore, Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of pathogenic genes were performed followed by co-segregation analysis using Fisher exact Test. Sanger sequencing was used to validate single-nucleotide variations (SNVs). Expanded verification was performed in the rest patients. RESULTS: Totally 51 LCA families with 53 patients and 24 family members were recruited. A total of 104 SNVs (66 LCA-related genes and 15 co-segregated genes) were submitted for expand verification. The frequencies of homozygous mutation of KRT12 and CYP1A1 were simultaneously observed in 3 families. Enrichment analysis showed that the potential pathogenic genes were mainly enriched in functions related to cell adhesion, biological adhesion, retinoid metabolic process, and eye development biological adhesion. Additionally, WFS1 and STAU2 had the highest homozygous frequencies. CONCLUSION: LCA is a highly heterogeneous disease. Mutations in KRT12, CYP1A1, WFS1, and STAU2 may be involved in the development of LCA.     

Leber先天性黑蒙临床与GUCY2D基因突变分析

目的 分析Leber先天性黑蒙的临床特点及其候选基因变异情况。方法 连续收集分析27例年龄4mo-18a的Leber先天性黑蒙先证者临床资料,应用PCR-异源双链-SSCP法分析GUCY2D基因外显子2和8,寻找可能的变异结果 27例患者均在2a以内出现视力差或对光、物体无反应,最最好视力小于0.1。症状年龄21例在3mo内,27例均有眼球震颤（其中15例为眼球扫视运动）,22例有眼底异常,5例眼底未见异常,3例有家族史并呈常染色体隐性遗传,ERG锥杆反应重度下降或记录不到波,未发现GUCY2D基因突变。结论 Leber先天 性黑蒙临床表现多样,诊断有赖于ERG。本组病例可能与GUCY2D外显子2、8突变无关。     

Advantage of Whole Exome Sequencing over Allele-Specific and Targeted Segment Sequencing in Detection of Novel TULP1 Mutation in Leber Congenital Amaurosis

Background: Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. A broader next-generation sequencing (NGS) strategy, such as whole exome sequencing, provides an improved molecular genetic diagnostic capacity for patients with these conditions.

Materials and Methods: In a child with LCA, an allele-specific assay analyzing 135 known LCA-causing variations, followed by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and SnpEff to annotate the variants.

Results: No disease-causing variants were found using the allele-specific or targeted segment Sanger sequencing assays. Analysis of variants in the exome sequence data revealed a novel homozygous nonsense mutation (c.1081C?>?T, p.Arg361*) in TULP1, a gene with roles in photoreceptor function where mutations were previously shown to cause LCA and retinitis pigmentosa. The identified homozygous variant was the top candidate using both bioinformatic pipelines.

Conclusions: This study highlights the value of the broad sequencing strategy of exome sequencing for disease gene identification in LCA, over other existing methods. NGS is particularly beneficial in LCA where there are a large number of causative disease genes, few distinguishing clinical features for precise candidate disease gene selection, and few mutation hotspots in any of the known disease genes.     

遗传性Leber视神经病变家系致病基因突变分析

目的 探讨遗传性Leber视神经病变(LHON)家系的致病基因突变位点.方法 家系调查研究.对LHON家系成员进行临床检查,抽取部分家系成员外周血提取基因组DNA,应用聚合酶链反应(PCR)和DNA序列测定法,检测家系成员线粒体DNA上的突变位点.结果 该家系3代14例成员中共有7例患者,其中男性2例,女性5例,家系图分析符合母系遗传特征.对其中4例患者及3例未发病家系成员进行线粒体DNA突变检测,显示4例患者及2例未发病家系成员携带G11778A位点突变.结论 线粒体DNA的G11778A突变位点是导致该家系患者发病的致病基因.对尚未发病的基因突变携带者进行早期干预治疗,有可能延缓或阻止疾病的发生与发展.     

Publication trends of Leber congenital amaurosis researches: a bibliometric study during 2002-2022

AIM: To analyze the changes in scientific output relating to Leber congenital amaurosis (LCA) and forecast the study trends in this field. METHODS: All of the publications in the field of LCA from 2002 to 2022 were collected from Web of Science (WOS) database. We analyzed the quantity (number of publications), quality (citation and H-index) and development trends (relative research interest, RRI) of published LCA research over the last two decades. Moreover, VOSviewer software was applied to define the co-occurrence network of keywords in this field. RESULTS: A total of 2158 publications were ultimately examined. We found that the focus on LCA kept rising and peaked in 2015 and 2018, which is consistent with the development trend of gene therapy. The USA has contributed most to this field with 1162 publications, 56 674 citations and the highest H-index value (116). The keywords analysis was divided into five clusters to show the hotspots in the field of LCA, namely mechanism-related, genotype-related, local phenotype-related, system phenotype-related, and therapy-related. We also identified gene therapy and anti-retinal degeneration therapy as a major focus in recent years. CONCLUSION: Our study illustrates historical research process and future development trends in LCA field. This may help to guide the orientation for further clinical diagnosis, treatment and scientific research.     

Leber先天性黑矇患者五种血清离子的异常变化

目的：分析Leber先天性黑矇(Leber congenital amaurosis,LCA)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,纳入临床诊断的LCA患者50例,正常对照组99例,按盲法由专业技术人员完成血清离子浓度检查,并分别行LCA组和正常对照组比较。

结果：在LCA患者检测的血清离子浓度中,钙离子浓度和钾离子浓度分别为2.338±0.090mmol/L和4.164±0.356 mmol/L,分别与正常组相比均显著升高(均P<0.05); 镁离子浓度为0.835±0.059mmol/L,与正常组相比显著下降(P<0.05); 其他两种离子血清浓度与正常组相比无统计学差异(均P>0.05)。

结论：LCA患者血清镁、钙和钾离子浓度异常变化应该引起眼科医师的关注,其可能与LCA的发病有关。     

A new novel nonsense mutation in AIPL1 in a LCA4 family

ABSTRACT

Purpose: To investigate the disease-causing gene in a Chinese family with Leber congenital amaurosis 4 (LCA4).

Materials and methods: Four members of an LCA family underwent ophthalmological examination and systemic assessment. DNA samples were obtained from their peripheral blood. Whole exome sequencing (WES) was performed in the two patients. After data filtering, Sanger sequencing was performed to verify the mutation within this family.

Results: The two patients were diagnosed as having LCA4 and with keratoconus (KCN). The older brother also has intellectual disability, epilepsy, Tourette syndrome and an abnormal gait, while the younger one has an abnormal bulge at the end of his sternum. A novel p.Gln81* mutation in the AIPL1 gene was determined as causing LCA4 in this family. Protein structure change prediction showed AIPL1 p.Gln81* mutation coded a very short AIPL1 peptide and could not form a normal structure as an normal AIPL1 protein.

Conclusion: Although KCN has been associated with LCA4, this type of LCA is typically moderate in severity and variable between patients. The present cases also have some systemic abnormalities.