视网膜母细胞瘤病人体细胞中RB1基因突变的检测

目的检测视网膜母细胞瘤(RB)病人体细胞中RBI基因突变，探讨RBI基因突变的分子生物学机制。方法应用PCR—SSCP技术筛查RB病人白细胞基因组DNA，测序分析确定突变。结果在12例RB病人中，确定3例RBI基因生殖细胞性突变。他们分别是第10外显子中T至A杂合性突变，将苯丙氨酸编码序列变为酪氨酸编码序列；第10外显子1bp碱基缺失(GAT—AT)，发生阅读框架改变；第22外显子中A至T杂合性突变，将精氨酸序列变为终止密码。结论这3例RBI基因生殖细胞性突变的方式为点突变和小缺失，这些突变改变了RBI基因的遗传信息，致使异常的RBI基因蛋白产生，导致视网障母细胞瘤的发生。     

变性高效液相色谱技术在视网膜母细胞瘤基因诊断中的应用

视网膜母细胞瘤（RB）的发生与13q14的Rb1基因失活有关,其中约90％的Rb1基因失活是由于Rb1基因的缺失、点突变或甲基化所致。新近的变性高效液相色谱技术（DHPLC）可简便、快速、准确检测基因的点突变、甲基化状态及其含量。目前DHPLC已经在RB患者及其家庭成员的基因诊断和遗传咨询中起重要作用。（中华眼科杂志,2007,43：564-566）     

视网膜母细胞瘤患者RB1基因突变筛选

目的：了解视网膜母细胞瘤患RBl基因的突变特点。方法：应用PCR-SSCP技术筛奄RB患白细胞基因组DNA，测序分析确定突变。结果：在RB患10例中，确定双眼RB患1例第8外显子37位核苷酸A-T突变，引起12位谷氨酸变为天门冬酰氨酸(G12A)。其母亲同样为第8外显子36位核苷酸A-T突变，引起12位谷氨酸变为天门冬酰氨酸(G12A)。结论：本组双眼RB患1例为遗传性，其RB1基因突变为微小突变，其母亲为突变基因携带，本研究为遗传咨询提供一些有价值的资料。     

Thiel-Behnke角膜营养不良家系的TGFBI基因突变研究

目的探讨我国Thiel—Behnke角膜营养不良中TGFBI基因突变的类型,了解5代常染色体显性遗传性Thiel—Behnke角膜营养不良家系的基因突变位点。方法对1个常染色体显性遗传性Thiel-Behnke角膜营养不良家系成员中10例患者和2名正常人进行眼科常规检查,抽取5ml外周血,盐析法提取基因组DNA,利用TGFBI基因第4、7、8、11、12外显子特异性引物,进行PCR扩增,对扩增产物测序进行突变检测。结果对TGFBI第4、7、8、11、12外显子扩增产物进行直接双向测序,在TGFBI基因第12外显子发现G→A的改变,此改变位于基因第12外显子,导致编码蛋白质第555位精氨酸被谷氨酰胺取代（R555Q）。这一序列的改变见于家系所有受累成员,而家系其他正常个体均无此改变。结论本研究Thiel—Behnke角膜营养不良家系患者的角膜病变由TGFBI基因R555Q突变引起,两者密切相关。     

视网膜母细胞瘤细胞系SO—Rb50瘤细胞Rb基因突变动态变化研究

目的：研究SO－Rb50细胞系瘤细胞Rb基因突变的动态变化。方法：1．用Southern blot杂交法分析SO－Rb50细胞系第327代瘤细胞DNA。2．用PCR－SSCP法对SO－Rb50细胞系第415代及第713代瘤细胞的Rb基因27个外显子和1个启动子逐个筛查。3．将SO－Rb50细胞系的第775代克隆出3个细胞株：MC2、MC3及MC4。用PCR－SSCP－HA法对MC2、MC3及MC4第11代细胞和MC3第138代细胞的Rb基因的27个外显子逐个筛查。结果：SO－Rb50细胞系第327代细胞DNA缺失3．5Kb、2.9Kb及1.0Kb的三条带,证明SO－Rb50细胞系瘤细胞Rb基因缺失。第415代瘤细胞Rb基因第23外显子少一条单链;第713代第25外显子发生新的突变。SO－Rb50细胞系第775代的3个克隆株MC2、MC3及MC4的第11代细胞,只有MC4第24外显子突变;而MC3第138代第24外显子突变,提示MC3经多次传代后第24外显子发生新突变。结论：SO－Rb50细胞系在长期培养传代过程中,瘤细胞的Rb基因突变有动态变化。     

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。     

超声微泡介导Rb94联合野生型p53基因对鼠视网膜母细胞瘤的抑制

背景研究表明,野生型p53（wtp53）和Rb94基因对人视网膜母细胞瘤（RB）的生长均有抑制作用,这2个基因是诱导和维持细胞衰老信号通路中重要的参与基因,因此2个基因联合应用是否对RB的生长抑制效果更好是近来关注的问题。目的观察超声微泡介导Rb94联合wtp53基因转染裸鼠RB后对其凋亡的影响。方法将HXO—Rb44细胞悬液种植至40只雌性SPF级BALB／e裸鼠视网膜下腔建立RB动物模型,造模成功的裸鼠按随机数字表法平均分为模型对照组、wtp53质粒组（含wtp53质粒的微泡悬液）、Rb94质粒组（含Rb94质粒）和wtp53＋Rb94质粒组（联合组）（含wtp53质粒及Rb94质粒）,其中模型对照组不做任何处理,其他3个组造模后第7天均由鼠尾静脉注入含相应基因的微泡悬液后,每天以0．5W／cm^2。超声波辐照眼球4S,间隔24S,循环2次。转染基因超声辐照7d摘除肿瘤组织,利用半定量逆转录PCR（RT—PCR）法检测肿瘤组织中wtp53mRNA及Rb94mRNA的表达;采用Western blot法检测基因转染的肿瘤组织中wtp53及Rb94蛋白的表达;用免疫组织化学法测定肿瘤组织中血管内皮生长因子（VEGF）的表达;采用TUNEL法检测基因转染后肿瘤组织的凋亡情况,计算凋亡指数（AI）。结果HXO-Rb44细胞悬液视网膜下腔注射后移植瘤构建的成功率为80％（32／40）,常规组织病理学检查提示检测样本的细胞异型性明显。基因转染后7d,模型对照组无wtp53mRNA及Rb94mRNA的表达条带;wtp53组wtp53mRNA相对值为0．65±0．07,wtp53＋Rb94组为0．32±0．02,差异有统计学意义（t=11．743,P=0．000）;Rb94组Rb94mRNA相对值为0．42±0．03,wtp53＋Rb94组为0．23±0．03,差异有统计学意义（t=5．041,P=0．001）。wtp53、Rb94或wtp53＋Rb94转染后,各组可见与转染相应的蛋白表达条带,wtp53＋Rb94组可同时检测到wtp53及Rb94蛋白的表达条带,但模型对照组无任何基因反应条带。免疫组织化学染色表明,wtp53＋Rb94组肿瘤细胞中VEGF阳性反应强度明显弱于wtp53组、Rb94组和模型对照组。wtp53＋Rb94组AI为37．35±2．14,明显高于模型对照组的0．46±0．05、wtp53组的5．05±O．80和Rb94组的6．43±1．02,差异均有统计学意义（t=-34．395、-28．206、-26．006,P〈0．01）。结论超声微泡造影剂可介导双基因联合转染RB移植瘤,且Rb94联合wtp53基因对RB细胞的促凋亡作用较单基因转染增强。     

PCR-SSCP联合序列分析法在RB1基因突变检测中的应用

用聚合酶链反应-单链构象多态(PCR-SSCP)联合序列分析，对视网膜母细胞瘤肿瘤组织RB1基因存在状态进行检测。对RB1基因全部27个外显子分别进行PCR扩增后，选用适当的限制性核酸内切酶将扩增产物切割成200bp左右的片段，加热至双链解离后，6%聚丙烯酰胺凝胶电泳。电泳后对有异常电泳迁移率条带所属标本的同一外显子行PCR扩增、序列分析，进一步证实突变的位置和类型。本研究发现包括点突变、缺失、插入等RBl基因改变，显示PCR-SSCP联合序列分析在已知序列基因突变检测中的敏感性和可靠性。 （中华眼底病杂志，1994，10：17-20）     

108例视网膜母细胞瘤Rb基因突变的特征

目的；研究RB患者肿瘤及体细胞内Rb基因的存在状态、细微结构、Rb基因突变的特征、起源与传递。 方法；DNA分子杂交、SSCP分析、DNA序列测定。结果：108例RB肿瘤标本中80例(?4％)存在Rb基因点突变，其中44例为纯合型，20例有二个独立发生的杂合型点突变，16例只检出一个杂合型点突变。通过对比分析RB患者肿瘤与白细胞DNA、RB患者家庭成员白细胞DNARlo基因的结构，揭示了Rb基因点突变的起源与遗传特征。 结论：Rb基因是与RB肿瘤形成关系最为密切的基因。RB肿瘤发生需二次突变事件导致Rb基因的二个等位基因失活，第一次突变事件突出表现为点突变；第二次突变事件主要是LOH，其次是点突变。 （中华眼底病杂志，1997，13：12-16）     

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。     

湖北地区原发性先天性青光眼患儿CYP1B1基因的新突变研究

目的探讨湖北地区汉族原发性先天性青光眼（PCG）患儿的基因突变情况并为基因诊断奠定基础。方法对47例无关个体PCG患儿及100例健康正常儿童进行基因分析。采取外周静脉血4ml,制备外周白细胞基因组DNA,参照文献所报道的引物序列,聚合酶链反应（PCR）分别扩增CYP1B1基因的第2、3外显子,琼脂糖凝胶电泳鉴定产物后,应用单链构象多态性（SSCP）、变性高效液相色谱分析（DHPLC）及DNA序列分析技术对患儿和对照组进行基因分析。结果7例PCG患儿呈现CYP1B1基因的第3外显子第385密码子的第1个碱基C→T碱基点突变,致对应的亮氨酸转变为苯丙氨酸（L385F）。这一变异在正常对照组成员中未检出,且检索国内外文献尚未见报道。结论湖北地区汉族PCG患者存在CYP1B1基因第3外显子突变,这一新突变位点位于P450蛋白的重要功能区,可能为病理性突变。在汉族PCG患者中进行CYP1B1基因突变的深入研究并寻找其他致病基因,对探讨PCG发病机制具有重要意义。     

分子遗传学实验研究在角膜营养不良临床诊断中的应用

目的 :通过对相关基因突变的检测 ,探讨以分子遗传学为基础的新的临床分类方法在角膜营养不良临床诊断中的应用。方法 :取我院门诊及病房收治的角膜营养不良患者 2 0例及 10例正常人外周静脉血 2ml,采取快速法提取白细胞DNA ,合成特异引物 ,分别行BIGH3基因第 4及第 12外显子PCR扩增 ,将PCR扩增产物进行纯化和测序。结果 :所有角膜营养不良患者均有BIGH3基因突变 ,其中 ,13例为R12 4H杂合子 ,确诊为Avellino角膜营养不良 ;3例为R5 5 5W杂合子 ,确诊为颗粒状角膜营养不良 ;4例为R12 4C杂合子 ,确诊为格子状角膜营养不良Ⅰ型。所有正常对照者均无BIGH3基因突变。结论 :通过本研究证实 ,以分子遗传学为基础的新的临床分类方法使诊断更为准确 ,同时使更为准确地预测疾病的发生、发展及预后成为可能 ,并为今后进行更为根本、有效的治疗包括基因治疗打下了良好的基础。     

Meesmann corneal dystrophy (MECD): report of 2 families and a novel mutation in the cornea specific keratin 12 (KRT12) gene

PURPOSE: Meesmann corneal dystrophy (MECD) is an autosomal dominant disorder affecting the corneal epithelium. It is caused by heterozygous mutations in KRT3 or KRT12 gene. Actually, 14 mutations have been reported, 1 in KRT3 and 13 in KRT12. These genes were screened in several patients suffering from MECD. METHODS: Patients from 2 families were screened for mutation in KRT3 and KRT12. Exons were PCR-amplified and directly sequenced. The new mutation was checked by DHPLC in 51 control individuals of Swiss origin. RESULTS/CONCLUSIONS: In one family, the M129T heterozygous mutation was observed in KRT12. In the second family, we identified a novel I426S heterozygous mutation in exon 6 of KRT12.     

Rapid mutation detection by the transgenomic wave analyser DHPLC identifies MYOC mutations in patients with ocular hypertension and/or open angle glaucoma

AIMS: To rapidly screen Scottish patients with a family history of open angle glaucoma (OAG) or ocular hypertension (OHT) for mutations in the myocilin gene (MYOC) and develop a new rapid screening method for MYOC mutation detection. METHODS: All three exons of the MYOC gene were amplified by PCR from genomic DNA and subjected to direct DNA sequencing. Mutation detection methodology was also developed based on denaturing high performance liquid chromatography (DHPLC). A recurrent mutation was investigated by analysis of microsatellite haplotypes at the MYOC gene locus. RESULTS: Mutations were identified by DNA sequencing in four families. MYOC mutation Q368X was found in three kindreds and the fourth family carried the mutation G367R. The Q368X mutation was found to be associated with the same haplotype for markers closely flanking the MYOC gene. The mutations were identified by direct sequencing and were also readily detected by DHPLC analysis of PCR fragments, demonstrating that this is a robust method for MYOC analysis in future. CONCLUSIONS: Mutations in the MYOC gene were identified in patients presenting with highly variable phenotypes from normal through OHT to severe OAG. Haplotype analysis showed that mutation Q368X is likely to be an ancestral mutation in this population. DHPLC analysis is an accurate, rapid and cost effective method for MYOC mutation analysis in large population samples.     

A clinical and molecular genetic study of autosomal-dominant stromal corneal dystrophy in British population

AIMS: To identify the underlying mutations in our British families and sporadic patients with different types of corneal dystrophies (CDs) and to establish a phenotype-genotype correlation. METHODS: Twenty-nine patients, 9 sporadic and 20 patients from 7 families were subjected to both clinical and genetic examination. Slit lamp examination was performed for all patients who participated in the study to assess their corneal phenotype. Genomic DNA was extracted from 10 ml venous blood, and the BIGH3 gene was amplified exon by exon to perform heteroduplex analysis. Exons that displayed double bands were then analysed by direct bi-directional sequencing and restriction digest analyses. RESULTS: Clinically our patients showed three distinct phenotypes of CD: 16 with Thiel-Behnke corneal dystrophy or corneal dystrophy of Bowman layer type 2 (CDB2), 8 with granular CD (GCD), and 5 with lattice CD type I (LCDI). Three different missense mutations have been detected in the coding region of BIGH3 gene, R555Q, in 16 CDB2 patients, R555W in 8 GCD patients, and R124C in 5 LCDI patients. These mutations were the same as to those previously reported in patients from other ethnic origins. Also,we identified seven nucleotide substitutions that did not change the amino acid sequence of the encoded protein of which four were novel. CONCLUSIONS: In our patients of British origin, each phenotype of CD has been linked to a particular point mutation of the BIGH3 gene. Our study also highlights the importance of codons 124 and 555 as mutation hot spots in the BIGH3 gene in the British population.     

Molecular genetic analysis of ABCR gene in Japanese dry form age-related macular degeneration

PURPOSE: To explore whether the mutation in the retina-specific ATP-binding cassette transporter (ABCR) gene, the Stargardt's disease gene, contributes to the prevalence of the dry form of age-related macular degeneration (dry AMD) in Japanese unrelated patients. METHODS: Twenty-five Japanese unrelated patients with dry AMD who were diagnosed by fluorescein angiography and indocyanine green angiography were chosen as the dry AMD group. None of these cases had apparent choroidal neovascularization. To detect the mutations in the ABCR gene, genomic DNA was extracted from leukocytes of peripheral blood, and 26 exons of the ABCR gene were amplified by polymerase chain reaction (PCR). All the PCR products were then directly sequenced. When a mutation was detected, the occurrence of a mutation was compared between these AMD patients and the control group. RESULTS: After direct sequencing, a point mutation in exon 29 was found in one of the 25 dry AMD patients. In addition, a polymorphism in exon 45 was found in two other patients, and three sequence variations in exon 23 were detected in all patients. The incidence in AMD patients in whom a mutation in exon 29 (4%) was detected was less than that in controls (5%). Screening of the intron-exon boundaries also led to the identification of intronic mutation in intron 33. CONCLUSION: In this study we found no relationship between allelic variation in the ABCR gene and the prevalence of dry AMD in Japanese unrelated patients.     

Single 5'green-3'red Hybrid Gene in Protanopia

Purpose: To disclose the structure of visual pigment gene for a protanopia with specific variation.Methods: Exon 5 fragments of the red andgreen visual pigment genes from the protanopia with specific varnation as well as controls were amplified by poly-merase chain reaction (PCR). The PCR products were put through heteroduplex-SSCP analysis and PCR-RFLP (restriction fragement length polymorphism) analysis to clarify the specific variation. The specific variation of the exon 5 DNA fragment from the protanopia was identified by sequencing.Results: A novel 5'green-3'red hybrid gene fragment without the normal red and green visual pigment gene was discovered in the protanopia. He should only have a single visual pigment gene, 5'green-3'red hybrid gene, on his X chromosome. The fusion point is between codon 285 and codon 296 in exon 5. Conclusion : Unequal intragenic recombination may occur in exon 5 as well as its upstream. A 5'green-3'red hybrid gene may present independently on the X chromosome without ac