一个X连锁显性遗传Nance-Horan综合征家系致病突变的遗传学研究

目的对中国东北辽宁地区一个三代后极性先天性白内障合并小眼球小角膜家系进行致病变异筛查,为临床明确诊断及遗传咨询提供必要的遗传学依据。 方法2017年4月采集该家系中2例患者和1例正常对照者的外周静脉血,提取基因组脱氧核糖核酸,对先证者进行眼部检查。通过MiSeq测序平台对4813个临床疾病相关基因进行高通量测序,寻找致病变异位点。 结果该家系表现为后极性先天性白内障伴有小眼球小角膜,呈X连锁显性遗传。对家系中3个成员基因组脱氧核糖核酸进行测序,发现家系内患者携带X染色体NHS基因无义突变(NHS c．742C>T,p.R248^*),该突变在家系中与疾病表型共分离。通过查询人类基因突变数据库,该突变为Nance-Horan综合征致病突变,患者临床表现与该综合征相符。 结论该家系诊断为Nance-Horan综合征,NHS基因c.742C>T(p.R248^*)突变是该家系的致病变异。     

目标区域捕获测序检测到一视网膜色素变性家系ABCA4基因新突变

目的研究我国一个常染色体隐性遗传的视网膜色素变性（RP）家系患者的临床表型及致病基因突变,并分析表型与基因型间的关系。方法实验研究。收集了宁夏人民医院眼科诊治的一个RP家系的临床资料,共有7名家庭成员参与研究,其中包括2名患者和5名正常成员。完善家系内所有参与成员的眼科检查,包括最佳矫正视力（BCVA）、视野、眼底照相、光学相干断层扫描（OCT）及全视野视网膜电流图（ERG）等。针对180个已知的遗传性视网膜疾病致病基因及9个高度可疑的候选基因设计目标区域捕获芯片,利用该捕获芯片对先证者（Ⅳ-4）进行目标区域内的高通量二代测序,借助优化的生物信息学分析对捕获的遗传变异进行筛查过滤,最终通过家系内共分离验证确认致病突变,进一步分析该突变与患者表型间的关系。结果临床检查结果表明该家系内的2名患者的临床表现均符合典型的RP改变,遗传学分析结果证实了ABCA4基因c.419G>A突变是该家系的致病突变。该突变导致了ABCA4基因所编码的蛋白第140号氨基酸由精氨酸变为谷氨酰胺（p.Arg140Gln）,保守性分析显示该突变位点在各物种中高度保守,PloyPhen-2软件预测结果表明该突变具有较高的致病性。结论本研究借助基于高通量二代测序平台的目标区域捕获测序,首次发现了ABCA4基因新突变p.Arg140Gln是一个常染色体隐性遗传RP家系的致病突变,进一步扩充了ABCA4基因的遗传突变谱及表型谱。     

Novel mutations in CRYBB1/CRYBB2 identified by targeted exome sequencing in Chinese families with congenital cataract

AIM: To summarize the phenotypes and identify the underlying genetic cause of the CRYBB1 and CRYBB2 gene responsible for congenital cataract in two Chinese families. METHODS: Detailed family histories and clinical data were collected from patients during an ophthalmologic examination. Of 523 inheritable genetic vision system-related genes were captured and sequenced by targeted next-generation sequencing, and the results were confirmed by Sanger sequencing. The possible functional impacts of an amino acid substitution were performed with PolyPhen-2 and SIFT predictions. RESULTS: The patients in the two families were affected with congenital cataract. Sixty-five (FAMILY-1) and sixty-two (FAMILY-2) single-nucleotide polymorphisms and indels were selected by recommended filtering criteria. Segregation was then analyzed by applying Sanger sequencing with the family members. A heterozygous CRYBB1 mutation in exon 4 (c.347T>C, p.L116P) was identified in sixteen patients in FAMILY-1. A heterozygous CRYBB2 mutation in exon 5 (c.355G>A, p.G119R) was identified in three patients in FAMILY-2. Each mutation co-segregated with the affected individuals and did not exist in unaffected family members and 200 unrelated normal controls. The mutation was predicted to be highly conservative and to be deleterious by both PolyPhen-2 and SIFT. CONCLUSION: The CRYBB1 mutation (c.347T>C) and CRYBB2 mutation (c.355G>A) are novel in patients with congenital cataract. We summarize the variable phenotypes among the patients, which expanded the phenotypic spectrum of congenital cataract in a different ethnic background.     

A novel MIP mutation in a Chinese family with congenital cataract

Purpose: To identify the disease-causing gene of a four-generation Chinese family with congenital cataract.

Methods: To screen the disease-causing gene of the family, six disease genes of congenital cataract are screened by direct DNA sequencing, the cDNA of wild-type (WT) MIP gene, and P191R mutant MIP gene (MT) were constructed into pEGFP-C1 vector and pGH19 vector. The recombinant plasmids of pEGFP-C1, WT, and mutant MIP were transfected into Hela cell to check the localization and HEK293T cells to detect expression level of protein. The cRNA of WT and MT MIP gene were injected into Xenopus oocytes to measure the swelling rate.

Results: A novel missense mutation c.572C>G（p.P191R）at exon 3 of the MIP gene was identified and co-segregated with disease in the Chinese family. The same amount of pEGFP-WT MIP and pEGFP- P191R MIP plasmids were transfected in Hela cells. Confocal microscopy imaging showed that WT MIP protein predominantly localized on the plasma membrane, the mutant protein was rich in the cytoplasm in Hela cells. Western blot results show that the expression level of P191R mutant MIP was significantly lower than WT MIPincell membrane enriched lysates in HEK293T cells. Xenopus oocytes swelling assay showed that the P191R mutation reduces the swelling rate of Xenopus oocytes.

Conclusions: The novel missense mutation c.572C>G（p.P191R）at exon 3 of the MIP gene was identified in a Chinese family of congenital cataract. The mutation affects the traffic of MIP protein in the cells and reduces the expression level of MIP protein in the cell membrane. The mutation of MIP gene reduces the swelling ratio of Xenopus oocytes.     

全外显子组测序检测到一视网膜色素变性家系ABCA4基因致病剪切新突变

目的探讨一个中国视网膜色素变性（RP）家系的致病基因及其位点。方法实验研究。对一个RP家系的成员进行病史采集、视力检查、眼底检查及多焦视网膜电图（mfERG）检查。绘制家系图,对家系成员采血,进行DNA提取、全外显子组测序、数据分析和Sanger测序验证,并在500例健康对照者中进行测序验证。结果共纳入该家系成员5例,含2例患者。患者表现为青少年期发病,夜盲,进行性周边视力受损,逐渐累及中央区。眼底检查显示视网膜色素沉着。mfERG显示a波、b波振幅明显下降甚至记录不到。家系特点为2例患者为姐妹,另一位姐妹正常,父母均正常,符合常染色体隐性遗传模式特征。经外显子组测序、数据分析和Sanger测序验证后,发现ABCA4变异性剪切位点c.1761-2A>G发生纯合突变。另外,在500例健康对照者中,Sanger测序没有发现该位点纯合突变。结论本研究发现了一个新的视网膜色素致病基因突变位点c.1761-2A>G,扩大了ABCA4致病基因位点谱,为临床的诊断和治疗提供了新靶点。     

先天性晶状体半脱位一家系FBN1基因突变研究及手术治疗

目的 对一个先天性晶状体半脱位家系进行FBN1基因突变筛查,总结该家系晶状体半脱位的治疗方法。设计 回顾性病例系列。研究对象 2014年5月就诊于北京同仁医院白内障中心一个先天性晶状体半脱位家系人员25例。方法 提取该家系成员外周血全基因组DNA,利用目标基因捕获技术,收集目标基因组DNA,再利用HiSeq2000进行高通量测序,确定突变位点,用Sanger法验证测序结果。晶状体半脱位90°~180°者4例（8眼）行一期囊袋张力环IOL植入术（Phaco+CTR+IOL）,二期晶状体囊袋复合体（IOL-CTR)单点巩膜缝合固定术。晶状体半脱位大于180°者1例（2眼）行晶状体玻璃体切除IOL植入,巩膜缝合固定术（PPL+PPV+IOL）。2例（4眼）行晶状体囊内摘除术（ICCE）,1例（1眼）行玻璃体切除术（PPV）。主要指标 FBN1基因测序结果、术式、最佳矫正视力（BCVA）。结果 该家系所有患者FBN1基因38号外显子mRNA第4588位碱基均出现C→T杂合性错义改变,导致患者均携带c.4588C>T（p.R1530C）突变体,而家系正常个体15人无此突变。行Phaco+CTR+IOL联合二期IOL-CTR术后BCVA 0.5~0.8。行PPL+PPV+IOL术后BCVA 0.3~0.5。结论 FBN1基因c.4588C>T（p.R1530C）突变体是导致该家系的致病原因。采用一期囊袋内张力环IOL植入及二期IOL-CTR单点巩膜缝合固定术矫正IOL偏位效果较好。（眼科, 2018,  27： 85-90）     

A novel p.R890C mutation in EPHA2 gene associated with progressive childhood posterior cataract in a Chinese family

AIM:To identify the genetic defect in a Chinese family with bilateral progressive childhood posterior cataract.METHODS: A two-generation family was recruited in this study. Family history and clinical data were recorded. All reported candidate genes associated with congenital posterior cataract were screened by direct DNA sequencing.RESULTS: All affected individuals presented posterior opacities in the lens. Direct sequencing of the candidate genes showed a heterozygous c. 2668C>T variation in EPHA2 gene, which resulted in the replacement of arginine by cysteine at codon 890 (p. R890C). This mutation was found in two affected individuals, but was not observed in 200 normal controls.CONCLUSION: We report a novel mutation (p. R890C) in the EPHA2 receptor tyrosine kinase gene. The finding expands the mutation spectrum of EPHA2 in association with posterior cataract.     

珊瑚状先天性白内障一家系致病基因筛查与鉴定

目的：对一个珊瑚状先天性白内障家系进行致病基因的筛查。方法：采集家系中2例患者和1例正常对照者的外周静脉血,提取基因组DNA。选择与珊瑚状白内障相关的候选基因GJA3、GJA8、CRYGC及CRYGD设计引物,进行聚合酶链反应（ PCR）扩增候选基因,并对扩增片段进行Sanger测序。结果：该家系疾病表型为珊瑚状白内障,呈常染色体显性遗传。通过对扩增产物测序,发现家系内患者CRYGD第2个外显子第70位有1个C＞A碱基的杂合突变（ c．70C＞A）,正常对照未见该点突变。结论：CRYGD基因的错义突变c．70C＞A是该珊瑚状白内障家系的致病原因。     

A novel CRX mutation by whole-exome sequencing in an autosomal dominant cone-rod dystrophy pedigree

AIM: To identify the disease-causing gene mutation in a Chinese pedigree with autosomal dominant cone-rod dystrophy (adCORD). METHODS: A southern Chinese adCORD pedigree including 9 affected individuals was studied. Whole-exome sequencing (WES), coupling the Agilent whole-exome capture system to the Illumina HiSeq 2000 DNA sequencing platform was used to search the specific gene mutation in 3 affected family members and 1 unaffected member. After a suggested variant was found through the data analysis, the putative mutation was validated by Sanger DNA sequencing of samples from all available family members. RESULTS: The results of both WES and Sanger sequencing revealed a novel nonsense mutation c.C766T (p.Q256X) within exon 5 of CRX gene which was pathogenic for adCORD in this family. The mutation could affect photoreceptor-specific gene expression with a dominant-negative effect and resulted in loss of the OTX tail, thus the mutant protein occupies the CRX-binding site in target promoters without establishing an interaction and, consequently, may block transactivation. CONCLUSION: All modes of Mendelian inheritance in CORD have been observed, and genetic heterogeneity is a hallmark of CORD. Therefore, conventional genetic diagnosis of CORD would be time-consuming and labor-intensive. Our study indicated the robustness and cost-effectiveness of WES in the genetic diagnosis of CORD.     

A novel splice site mutation of CRYBA3/A1 gene associated with congenital cataract in a Chinese family

AIM: To identify the disease-causing mutation responsible for the presence of congenital cataract in a Chinese family. METHODS: The study recruited a four-generation Chinese pedigree affected by autosomal dominant congenital cataract (ADCC). Family history and the history of cataract extraction were recorded. Blood samples were collected from individuals for DNA extraction. Direct sequencing of congenital cataract-associated genes was performed. Single-strand conformational polymorphism and bioinformatic analysis were conducted to further study the mutation. RESULTS: Direct sequencing revealed a novel splice site mutation of c.30-2 A>G in the CRYBA3/A1 gene. The mutation co-segregated within all affected individuals in the family and was not found in unaffected members or 100 unrelated normal controls. These results were further confirmed by single-strand conformational polymorphism and bioinformatic analysis using the Human Splicing Finder and MaxEnt online software and Annovar computer software. CONCLUSION: c.30-2 A>G mutation of CRYBA3/A1 gene is a novel mutation and broadens the genetic spectrum of ADCC.     

Exome sequencing analysis identifies novel homozygous mutation in ABCA4 in a Chinese family with Stargardt disease

AIM: To identify the disease-associated mutations in a Chinese Stargardt disease(STGD) family, extend the existing spectrum of disease-causing mutations and further define the genotype-phenotype correlations.METHODS: A Chinese STGD family and 200 normal controls were collected. Whole exome sequencing(WES) and bioinformatics analysis were performed to find the pathogenic gene mutation. Physico-chemical parameters of mutant and wildtype proteins were computed by Prot Param tool. Domains analysis was performed by SMART online software. HOPE online software was used to analyze the structural effects of mutation. Immunofluorescence, quantitative real-time polymerase chain reaction and Western blotting were used for expression analysis.RESULTS: Using WES, a novel homozygous mutation(NM_000350: c.G3190 C, p.G1064 R) in ABCA4 gene was identified. This mutation showed co-segregation with phenotype in this family. It was not found in the 200 unrelated health controls and absent from any databases. It was considered "Deleterious" as predicted by five function prediction softwares, and was highly conserved during evolution. ABCA4 was expressed highly in the human eye and mouse retina. The p.G1064 R was located in AAA domain, may force the local backbone into an incorrect conformation, disturb the local structure, and reduce the activity of ATPase resulting in the disease pathology. CONCLUSION: We define a novel pathogenic mutation(c.G3190 C of ABCA4) of STGD. This extends the existing spectrum of disease-causing mutations and further defines the genotype-phenotype correlations.     

一个后极性白内障家系致病基因的筛查与鉴定

目的　对中国一个具有常染色体显性遗传特点的后极性白内障家系进行致病基因的筛查。方法　分别采集家系成员外周静脉血,提取基因组ＤＮＡ,根据临床表型选取６个候选基因（ＣＲＹＡＡ、ＣＲＹＡＢ、ＰＩＴＸ３、ＣＨＭＰ４Ｂ、ＭＩＰ、ＣＲＹＧＤ）设计引物,通过ＰＣＲ扩增ＤＮＡ片段,琼脂糖凝胶电泳分离ＤＮＡ片段,直接测序法寻找致病基因及突变位点。结果　该家系符合常染色体显性遗传家系特征,先证者表型为后极性白内障,通过候选基因外显子直接测序,发现家系内患者ＣＲＹＧＤ基因第２个外显子第１２７位碱基有１个Ｔ→Ｃ的点突变,此突变导致蛋白第４３位的色氨酸被精氨酸取代（Ｗ４３Ｒ）。结论　ＣＲＹＧＤ基因ｃ．Ｔ１２７Ｃ（ｐ．Ｗ４３Ｒ）突变是该后极性白内障家系的致病原因。     

第二代测序技术在一中国先天性白内障家系致病基因检测中的应用

背景 某些遗传性疾病具有高度遗传异质性,因此传统的Sanger测序技术已经不能满足医学研究及临床工作的需求.第二代测序(NGS)技术由于具有费用低及检测速度快的优点而得到广泛应用,但在先天性眼病突变基因的检测中的应用效果有待验证.目的 探讨NGS技术对先天性白内障患者进行致病基因诊断和产前诊断的可行性.方法 于2013年1月收集来自河南省洛阳市的一汉族先天性白内障家系,抽取家系中3例患者(Ⅱ2、Ⅲ3、Ⅲ4)和3名表型正常者(Ⅱ3、Ⅲ1、Ⅲ2)的外周血各2 ml,EDTA抗凝,在河南省医学遗传研究所应用NGS技术对先证者进行基因突变位点检测,并采用Sanger测序技术对NGS结果进行验证,然后用Sanger测序技术对该家系其他成员的外周血样本进行突变位点的序列分析,根据确定的致病突变位点对先证者的胎儿进行产前诊断.本研究遵循赫尔辛基宣言,检测方案经河南省人民医院医学伦理委员会批准,所有研究对象均签署知情同意书.结果 该家系4代14位成员中共5例患者,其中男2例,女3例,分布于Ⅰ、Ⅱ、Ⅲ代中,其他家系成员表型正常,符合常染色体显性遗传方式.NGS检测发现先证者Ⅲ3CRYBB1基因第6外显子上存在c.682T＞C(p.S228P)杂合突变,Sanger法验证了该点突变.Sanger法检测发现家系中患者均存在CRYBB1基因c.682 T＞C突变,而家系中表型正常者CRYBB1基因的c.682位点基因型为T/T野生型.产前诊断结果显示胎儿(Ⅳ1)CRYBB1基因c.682位点基因型为T/T野生型.结论 NGS可用于先天性白内障基因突变的快速检测,该家系致病性基因为CRYBB1基因c.682T＞C突变,应用NGS技术结合一代测序技术成功对先证者进行了产前诊断.     

Novel compound heterozygous mutation in the POC1B gene underlie peripheral cone dystrophy in a Chinese family

Purpose: To describe the clinical characteristics of a Chinese family with peripheral cone dystrophy (PCD) and identify the gene mutations causing PCD.

Methods: The Chinese PCD pedigree underwent comprehensive ophthalmic examinations, including visual acuity, slit lamp examination, fundoscopy, visual field examination, autofluorescence, fluorescence fundus angiography and indocyanine green angiography, full-field electroretinograms, and spectral-domain optical coherence tomography. The targeted next-generation sequencing of COD or cone-rod dystrophy (CORD) genes was used to identify the causative mutation.

Result: The fundus characteristics of the Chinese patient were consistent with PCD. The novel compound heterozygous mutation, c.1354C>T and c.710A>G, in POC1B was identified in the patient, the mutations were segregated with the PCD phenotype in the family and were absent from ethnically matched control chromosomes. Prediction analysis demonstrated the novel missense mutation, POC1B c.710A>G, might be damaging.

Conclusions: PCD was a type of COD or CORD and the novel compound heterozygous mutation in POC1B was responsible for PCD phenotype in the family.     

A novel mutation of LIM2 causes autosomal dominant membranous cataract in a Chinese family

AIM: To determine the prevalence and associations of non-retinopathy ocular conditions among older Australian adults with diabetes. METHODS: Multistage random-cluster sampling was used to select 3098 non-indigenous Australians aged 50y or older (46.4% male) and 1738 indigenous Australians aged 40y or older (41.1% male) from all levels of geographic remoteness in Australia. Participants underwent a standardised questionnaire to ascertain diabetes history, and a clinical examination to identify eye disease. We determined the prevalence of uncorrected refractive error, visually significant cataract, cataract surgery, age-related macular degeneration, glaucoma, ocular hypertension, retinal vein occlusion and epiretinal membrane among those with and without self-reported diabetes. RESULTS: Participants with self-reported diabetes had a higher prevalence of cataract surgery than those without diabetes (28.8% vs 16.9%, OR 1.78, 95%CI: 1.35-2.34 among non-indigenous Australians, and 11.3% vs 5.2%, OR 1.62, 95%CI: 1.22-2.14 among indigenous Australians). Diabetic retinopathy (DR) increased the odds of cataract surgery among self-reported diabetic indigenous and non-indigenous Australians (OR 1.89, P=0.004 and OR 2.33, P<0.001 respectively). Having diabetes for ≥20y and having vision-threatening DR increased the odds of cataract surgery among indigenous Australians with diabetes (OR 3.73, P=0.001 and 7.58, P<0.001, respectively). CONCLUSION: Most non-retinopathy ocular conditions are not associated with self-reported diabetes. However, to account for Australia’s worsening diabetes epidemic, interventions to reduce the impact of diabetes-related blindness should include increased cataract surgery services.     

一个中国全色盲家系 CNGB3基因新突变

目的:探讨全色盲一家系的致病基因突变。方法:采用家系调查研究方法,于2018年11月对河南省立眼科医院收集的来自河南省洛阳市的汉族全色盲一家系进行基因测序。详细采集患者的病史资料,对患者及其家系成员进行最佳矫正视力(BCVA)测定,采用裂隙灯显微镜和前置镜检查眼前节和眼底,采用客观和主觉验光法对受检者进行屈光度检查,采...     

Use of high-throughput targeted exome sequencing in genetic diagnosis of Chinese family with congenital cataract

AIM: To identify disease-causing mutation in a congenital cataract family using enrichment of targeted genes combined with next-generation sequencing. METHODS: A total of 371 known genes related to inherited eye diseases of the proband was selected and captured, followed by high-throughput sequencing. The sequencing data were analyzed by established bioinformatics pipeline. Validation was performed by Sanger sequencing. RESULTS: A recurrent heterozygous non-synonymous mutation c.130G>A (p.V44M) in the GJA3 gene was identified in the proband. The result was confirmed by Sanger sequencing. The mutation showed co-segregation with the disease phenotype in the family but was not detected in unaffected controls. CONCLUSION: Targeted exome sequencing is a rapid, high-throughput and cost-efficient method for screening known genes and could be applied to the routine gene diagnosis of congenital cataract.     

眼白化病Ⅰ型家系致病基因GPR143新突变

目的：对收集的一例眼白化病1型家系的致病基因GPR143进行突变检测。方法抽取先证者两兄弟及其母亲的5ml外周血,酚-氯仿法抽提基因组DNA,通过聚合酶链反应（polymerase chain reaction,PCR）扩增眼白化病1型致病基因GPR143外显子及其相邻的内含子,并进行直接测序。结果 PCR结果显示两眼白化病1型的兄弟第五外显子均无产物,而其他8个外显子均有产物,相同反应条件下其母亲及正常对照组所有外显子都可扩增出产物,证明该眼白化病患者的致病基因GPR143的第五外显子缺如。结论本研究在眼白化病1型的患者的致病基因中发现了整个外显子的缺失突变,扩展了OA1致病基因的突变频谱。     

先天性无虹膜家系中发现PAX6基因新的致病突变

目的对中国一先天性无虹膜家系进行PAX6基因突变检测,以确定其突变位点。方法实验研究。收集一先天性无虹膜家系,采集该家系患者及健康成员的外周静脉血,收集100名健康人外周血作为正常对照,采用Sanger测序的方法对PAX6基因的11个外显子（外显子4-14）以及外显子-内含子连接区域进行测序,随后进行家系共分离分析以及正常样本的对照分析。结果该家系8名成员经全面眼科检查,3名确诊为先天性无虹膜,且合并有复杂的眼部表型,包括不同程度的角膜病变、不同类型的白内障、黄斑发育不良、轻度上睑下垂和轻度眼球水平震颤等。在该家系患者中发现一个新杂合突变[c.569_570delinsACGG（p.Ile190Asnfs*18）],该突变可致PAX6基因编码的蛋白截短,该突变符合共分离且在100名正常对照者中未检测到。结论PAX6基因第8外显子上一个新的杂合突变[c.569_570delinsACGG（p.Ile190Asnfs*18）]为本研究中先天性无虹膜家系的致病突变,该突变与先天性无虹膜有关,本研究扩大了PAX6基因的突变频谱。