常染色体显性视网膜色素变性一家系基因排除定位研究

目的对1个四代常染色体显性视网膜色素变性(autosomal dominant retinitis pigmentosa，ADRP)家系在包含已知ADRP致病基因的全部9条染色体上进行基因连锁定位。方法对家系中的所有患者进行眼科及电生理检查。在家系中对第1、3、6、7、8、11、14、17、19号染色体进行基因扫描、基因型分析及连锁分析。结果在全部9条染色体范围内未发现微卫星位点与该家系疾病表型共分离，最大lod score小于-2。结论此家系中排除了目前已知的所有ADRP位点，此家系中应存在新的ADRP连锁位点位于上述9条染色体之外。     

先天性白内障一家系致病基因的排除性定位

目的 明确一个国人常染色体显性先天性白内障（ADCC）家系致病基因的染色体位点是否位于已知的22个非综合征型ADCC致病位点内，从而初步定位该ADCC家系致病基因的染色体位点。设计 家系遗传研究。研究对象 一个先天性白内障家系。方法 对26例家系成员中的16例进行临床检查、采集静脉血样、提取基因组DNA；在已知的22个非综合征型ADCC致病位点内，分别选取3-6个多态性微卫星标记，对该ADCC家系进行遗传连锁分析。主要指标 先天性白内障临床表型、Lod值。结果 该家系患者为晶状体前囊膜及前囊膜下混浊；所有多态性微卫星标记与致病基因两点间的Lod值均≤-2，证实微卫星标记所在的染色体区域与该ADCC家系的致病基因不连锁。结论 该ADCC家系致病基因的染色体位点不在已知的22个非综合征型ADCC致病位点内，可能是一个新的致病基因导致了该家系的临床表型。     

常染色体显性视网膜色素变性家系基因定位的研究

目的 对一个常染色体显性视网膜色素变性大家系进行基因定位。方法 收集视网膜色素变性家系,并对该家系成员进行详细眼部检查;抽取外周血3－5ml并提取DNA;采用多个已知遗传标记与该家系致病基因位点进行连锁分析。结果 两点连锁分析结果显示该家系致病基因位点与遗传位标D3S1292连锁,在θ＝0．1时间到最大LOD值2．73。结论 D3S1292位于3号染色体长臂2区1带（3q21),从而将该家系致病基因位点大致定位于3q21附近。同时有文献报道视紫红质基因位点也位于3q染色体上,且与D3S1292邻近,因此该家系的致病基因很可能是视紫红质基因。     

晶状体蛋白基因在一先天性白内障家系中的突变筛查

目的研究晶状体蛋白基因与先天性白内障的关系。方法收集1个先天性白内障家系,制备外周血白细胞基因组DNA。除对CRYGD基因直接测序外,在距离已知晶状体蛋白基因5个厘摩范围内选取微卫星标记进行连锁分析,计算微卫星位点与致病基因之间的最大优势对数值(LOD SCORE),来确定晶状体蛋白基因与此家系致病基因的关系。结果当重组分数分别为0.1、0.2、0.3、0.4时,所选取的位于晶状体蛋白基因附近的14个微卫星位点与该家系致病基因之间连锁的LOD值均为负值,故排除连锁。CRYGD基因测序后在基因编码区及启动子、内含子与外显子连接处的剪切位点均未见任何碱基改变。结论晶状体蛋白基因非此家系的致病基因,为进一步定位与克隆该家系的致病基因,需进行全基因组扫描,以探求先天性白内障的分子发病机制。     

汉族常染色体显性视网膜色素变性一家系的连锁分析及突变筛查

背景 原发性视网膜色素变性(RP)有明显的遗传异质性和表型异质性,目前已确定的致病基因较多,确定患病家系的致病基因是进行基因治疗的基础. 目的 对患常染色体显性遗传性RP(ADRP)的一个汉族家系进行致病基因的定位和基因突变分析.方法 此家系的5代21名成员纳入研究,包括12例ADRP患者和9名表型正常者.12例患者进一步接受中心视野、间接检眼镜、眼电图(EOG)、视网膜电图(ERG)检查.对22个已知的ADRP致病基因所在染色体位点进行连锁分析,以确定该家系与疾病连锁的染色体区域,随后对该区域附近的候选基因视紫红质(RHO)进行直接测序评估其突变情况. 结果 间接检眼镜检查该家系先证者眼底表现符合原发性RP表现,EOG和ERG表现为波形记录不到,视野呈向心性缩小.两点连锁分析结果显示,该家系致病基因位点与遗传标记D3S1292连锁,在θ=0.0时得到最大优势对数(LOD)值为3.6671.候选的RHO基因直接测序结果发现,该家系所有患者第53位密码子的第2个核苷酸均出现了C→G的突变,致其氨基酸由脯氨酸变为精氨酸(Pro53Arg),而该家系正常成员中未发现此突变. 结论 RHO基因的错义突变Pro53Arg与RP疾病出现共分离现象,可确定为该ADRP家系的致病基因.     

中国东北一个先天性核性白内障家系致病基因的初步研究

目的定位一个先天性白内障家系的致病基因。方法根据以往研究得到证实的与先天性白内障有关的三类晶状体蛋白基因在染色体上的位置,分别选取3～4个用Fam或Hex荧光标记的微卫星标记物,多重PCR产物经美国ABI公司3700测序仪毛细管电泳,由GeneMapperV3.0软件处理,结合外显率和发病率对该家系进行连锁分析。结果三类晶状体蛋白5个侯选突变基因周围的18对微卫星标记位点的LOD值均<0.50,致病基因同已知基因之间不存在连锁关系。结论该家系致病基因不是三类已知晶状体蛋白基因,其致病基因的定位有待进一步研究。     

中国人一个显性视网膜色素变性家系8号染色体连锁分析

目的用连锁分析法对中国人一个显性视网膜色素变性家系8号染色体进行分析，确定致病基因。方法随机选取8号染色体RP1基因上下约10厘摩(cm)范围内的10对微卫星标记(marker)，确立单倍体型，用两点法计算最大优势对数(LOD SCORE)值。结果所选微卫星标记与该家系表型间最大LOD值小于1。结论RP1基因可能为该家系的非致病性基因，用连锁分析法进行致病基因排除对最终确立致病基因所在染色体的范围具有重要的价值。     

一常染色体显性遗传性白内障家系致病基因的排除性定位

目的对常染色体显性遗传先天性白内障家系进行致病基因的定位研究。方法对4代11例家系成员（6例患者）进行眼部和全身检查,采集静脉血,提取基因组DNA,选取已报道的与遗传性白内障相关位点附近的微卫星标记,PCR扩增后进行基因型分析,用连锁分析进行排除;没有排除的位点,基因外显子测序。结果 35例家系成员中,追溯调查共有10例患者,其中第1代1例,第2代2例,第3代5例,第4代2例。该家系患者表型为完全性白内障;绝大多数位点,患者没有共享基因型;微卫星标记与致病基因间的2点连锁Lod值〈-2,证实这些位点与该家系的致病基因不连锁;有3个多态性标记（D10S1239、D22S286、D22S926）0〈Lod值≤0.6,Lod值虽然不是〈-2,但在家系患者中没有共享等位基因;测序未发现外显子有突变。结论此家系的致病基因不是已报道位点的致病基因,其致病基因有待进一步研究。     

先天性眼组织缺损一家系基因排除定位研究

目的对1个3代常染色体显性遗传性先天性眼组织缺损家系进行致病基因的定位。方法对家系所有成员进行详细的临床检查,排除其他系统疾患。提取家系成员外周血DNA,选取20个位于4、7、10、11号染色体上已知与先天性眼组织缺损相关的4个致病基因及已知基因位点20q13.1附近的微卫星标记物进行多重PCR扩增,经ABI3130型遗传分析仪,Genscan2.1收集数据,Genotyper2.1进行基因分型,Linkage软件计算两点LOD值。研究过程遵循赫尔辛基宣言。结果未发现所选微卫星位点与该家系疾病表型共分离,LOD值均为负值。致病基因与已知的先天性眼组织缺损候选基因不存在连锁关系。结论该家系的遗传与目前已知的致病基因无关,是否存在新的致病基因有待进一步研究。     

一个常染色体显性遗传先天性白内障家系致病基因筛查

目的: 对中国一个常染色体显性遗传先天性白内障家系(ADCC)的已知候选基因进行筛查以寻找致病位点。方法: 收集一个ADCC家系的临床资料并采集静脉血。在24个已知与ADCC相关基因附近选择微卫星标记,利用Linkage软件Mlink软件包进行连锁分析计算Lod值。结果: 此家系白内障类型为核性白内障,24个候选基因附近50个微卫星Lod值均小于0,微卫星所在区域与此家系致病基因无连锁关系。结论: 此ADCC家系致病基因不是已知的与ADCC相关基因,可能是一个新的致病基因突变导致此家系疾病发生。     

Analysis of rhodopsin gene in patients with retinitis pigmentosa using allele-specific polymerase chain reaction.

Point mutations within the rhodopsin gene have been found recently in some patients with autosomal dominant retinitis pigmentosa (ADRP). Currently, four types of point mutations at codons 23, 58 and 347 have been identified. The purposes of this study were to establish simple methods for screening patients with retinitis pigmentosa (RP) to detect these point mutations, and to apply these methods to determine if these mutations are found in Japanese patients with RP. Utilizing the polymerase chain reaction (PCR), a one-step method was developed to detect point mutations at codon 23. This method was then applied to screen genomic DNAs from 30 patients with various types of RP, including ADRP, autosomal recessive RP, simplex RP, Leber's congenital amaurosis or Usher's syndrome. Subsequently, point mutations at codons 58 and 347 were detected by restriction enzyme digestion (Dde I or Msp I) of exons 1 and 5 amplified by PCR. To date, no mutations have been found in codons 23 and 58 in Japanese patients. By using the allele-specific PCR, however, two patients from one pedigree of ADRP were confirmed to have a C-to-T transition at the second nucleotide of codon 347, which results in the substitution of leucine for proline. Our findings indicated the availability of this simple method for detecting these point mutations.     

Expression of the TIGR gene in the iris,ciliary body,and trabecular meshwork of the human eye

Purpose: To identify mutations in the rhodopsin ( RHO ) gene in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) and to measure the prevalence rate of RHO mutations in Chinese ADRP cases. Methods: Thirteen Chinese families with ADRP were clinically characterized. The complete coding region and intron splice sites of RHO were analyzed for mutations with single-strand conformation polymorphism (SSCP) analysis and direct genomic sequencing. Results: One of the 13 Chinese families with ADRP was found to have a new, previously unidentified RHO mutation, a change from GAG to TAG at codon 341. The mutation (E341X) results in an in-frame stop codon, leading to the truncation of the rhodopsin protein. Mutation E341X was not detected in 100 normal control individuals. Patients carrying mutation E341X reported night blindness and showed optic atrophy, vessel attenuation, and a few bone spicule-like pigments in peripheral retina at the age of 23–25 years. At the age of 30 years, visual acuity was severely impaired, peripheral visual field was greatly constricted, rod and cone ERG was not detectable, and only a slight left cone response remained. Conclusion: We have identified a novel rhodopsin mutation (E341X) in a Chinese family with ADRP. The location and character of the mutation expand the spectrum of RHO mutations causing RP. Identification of a RHO mutation in one of the 13 ADRP families studied suggests that only 7.7% of the ADRP cases in a Chinese population were caused by RHO mutations, a ratio significantly lower than that from North America or Europe.     

Progress in pathogenesis and therapeutic research in retinitis pigmentosa and age-related macular degeneration

Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are designated special targeted eye diseases by the Welfare and Labor Ministry of Japan. We have been studying the pathogenesis, diagnosis, treatment, and evaluation of these diseases. The development of molecular genetic analyses of RP revealed that the type and frequency of mutations varied with the ethnic population. In our present study, we focused on the genetic analysis and clinical examinations for autosomal dominant retinitis pigmentosa (ADRP). We screened 96 unrelated ADRP families with 9 genes, which included rhodopsin, peripherin/RDS, RP 1, NRL, FSCN 2, PRPF 31, PRPC 8, HPRP 3, IMPDH 1. We also showed the correlations we have found between the phenotype and genotype of hereditary retinal diseases in Japanese patients. Our mutation screenings suggested that Japanese patients with ADRP might have a unique mutation, because the mutation in the FSCN 2 gene has been found only in Japanese patients. On the other hand, the Pro347Leu and Pro23His mutations in the rhodopsin, the Arg677X mutation in the RP 1, and the Asp226Asn mutation in the IMPDH 1 genes are representative mutations for ADRP, and are not found or are very rare in Japanese patients with ADRP. The results of randomized controlled trials of low-dose radiation for wet-type age-related macular degeneration located at the fovea centralis indicate the effectiveness of this treatment for maintaining visual acuity and regression of choroidal neovascular membrane (CNV) for at least one-year. Simple surgical removal of CNV or transplantation of autologous cultured iris pigment epithelium (IPE) with vitreous surgery showed some improvement of vision. In either RP or AMD, photoreceptors die, in most cases by apoptosis. Neurotrophic factors (NT) are effective for reducing these processes and preventing photoreceptor cell death in animal models. To apply these methods to humans, the procedures are as follows: 1) obtaining IPE by peripheral iridectomy, 2) culturing it with autologous serum and transfecting the cDNA of NT, and then 3) transplantation of these cells under the retina. We used cDNA of brain-derived neurotrophic factor (BDNF) with adeno-associated virus (AAV) as a vector. These ex vivo procedures were safe and very effective for preventing photoreceptor cell death in animal models, such as RCS rats and light-damaged rats. In the future, these procedures could be applied for RP or AMD and might show some clinical effects for maintaining or improving the vision of patients.     

Exclusion of the autosomal dominant retinitis pigmentosa gene from a substantial region of chromosome 1: Study of a large Australian family

In an attempt to map the gene(s) responsible for autosomal dominant retinitis pigmentosa (ADRP), the technique of reverse genetics was used on a large multigenerational Australian pedigree. The family demonstrated a form of the disease which appears to be less severe than that observed in the Irish pedigree. It was typed for 10 restriction fragment length polymorphism (RFLP) markers on chromosome 1. The data from the linkage study was analysed using the programs LIPED 3; six markers gave informative results. The ADRP gene was excluded from this family from 102 cM using previously prepared chromosome 1 maps. This accounts for 36% of chromosome 1 which is estimated to be the longest human chromosome.     

Exclusion of the autosomal dominant retinitis pigmentosa gene from a substantial region of chromosome 1: study of a large Australian family

In an attempt to map the gene(s) responsible for autosomal dominant retinitis pigmentosa (ADRP), the technique of reverse genetics was used on a large multigenerational Australian pedigree. The family demonstrated a form of the disease which appears to be less severe than that observed in the Irish pedigree. It was typed for 10 restriction fragment length polymorphism (RFLP) markers on chromosome 1. The data from the linkage study was analysed using the programs LIPED 3; six markers gave informative results. The ADRP gene was excluded from this family from 102 cM using previously prepared chromosome 1 maps. This accounts for 36% of chromosome 1 which is estimated to be the longest human chromosome.     

视紫红质基因突变相关的视网膜色素变性

视紫红质基因突变是导致视网膜色素变性最常见的原因,占常染色体显性遗传视网膜色素变性的25％～30％.在视网膜色素变性中已发现的视紫红质基因突变多达150余种.视紫红质基因突变可引起内质网应激、蛋白聚集、膜受体异常激活,从而导致视网膜色素变性.P23H和T17M等突变小鼠转基因模型的建立,为深入探讨视紫红质在视网膜色素变性中的作用,以及为干扰突变基因表达,替换突变基因等潜在治疗策略提供理论依据.     

Mutation screening of Pakistani families with congenital eye disorders

To map the disease loci several Pakistani families suffering from autosomal recessive retinitis pigmentosa with preserved para-arteriolar retinal pigment epithelium and Leber congenital amaurosis (LCA) were analyzed. Analysis revealed close genetic linkage between the disease phenotype of some of the families (3330RP, 111RP and 010LCA) and the microsatellite markers on chromosome 1q31. Mutation screening of the candidate gene CRB1 revealed a G to A transversion in exon 7 in arRP family 330RP and a T to C substitution in another arRP family, 111RP. In exon 9 of the CRB1 gene a T to C transversion was found in the family suffering from LCA (010LCA).The LCA phenotype of another family (011LCA) in which the CRB1 locus was excluded, showed linkage with microsatellite markers D17S1294 and D17S796 on chromosome 17p13.1. The association of the candidate gene GUCY2D (17p13.1) with the disease phenotype was excluded as no disease-associated mutation was found in any of its exons. Mutation screening of another candidate gene, AIPL1 located in the same region, showed a novel homozygous C to A substitution in exon 2. These sequence changes are unique for the Pakistani families and some of these have not been reported previously.     

视网膜色素变性患者视紫红质E341ter基因突变及临床表型分析

目的 探讨常染色体显性遗传视网膜色素变性患者视紫红质基因突变及其与临床表型的关系。方法 应用聚合酶链反应（polymerase chain reaction,PCR)和单链构象多态性（single strand conformation polymorphism,SSCP)技术,对13个常染色体显性遗传视网膜色素变性家系的27例成员,进行视紫红质基因整个编码区的突变筛选,对SSCP检测有变异带的外显子PCR产物进行测序;同时应用裂隙灯、眼底镜、动静态视地和视网膜电流图（ERG）对患者进行临床检测。随机收集30例正常人进行对照检测。结果 发现1个家系患者有视紫红质E341ter突变,呈杂合子,密码子341第一个碱基由G变成T。该家系临床表现为青年期出现夜盲,视力和视野损害较重,ERG检查杆体和锥体无反应或仅有较小的锥体反应。结论 视紫红质基因突变家系的视网膜色素变性病史开始于杆体功能的丢失,进而累及锥体系统,并最终导致视功能严重丧失。视紫红质E341ter突变被认为是该家系的病因。