常染色体隐性遗传视网膜色素变性家系视紫红质基因突变的检测分析

目的 观察一个近亲婚配常染色体隐性遗传视网膜色素变性（ARRP）家系中视紫红质基因（RHO）的突变特征，并探讨其视网膜色素变性(RP)发病机制。 方法 抽取8例该ARRP家系成员及10例正常对照者的外周静脉血5~8 ml；提取基因组DNA；采用聚合酶链反应（PCR）方法扩增RHO基因第1~5外显子和第1内含子基因 片段 ，用直接DNA测序法筛查RHO基因突变。 结果 来自同一家系3例患者RHO 基因的第5外显子第344密码子发生了A→G碱基的错义突变，导致了谷氨酰胺变成了精氨酸(G ln344Arg)，3例患者为该突变的纯合子。患者近亲婚配父母及1例未患病家庭成员为该突变 的杂合子。2例未患病家庭成员及10例正常对照者均未发现RHO基因突变。 结论 Gln344Arg突变可能是该ARRP家系的致病原因；在近亲婚配ARRP家系中RHO基因突变频率可能增加。 （中华眼底病杂志，2004，20：145-148）     

Reis-Bucklers角膜营养不良家系的TGFBI基因序列分析

目的：应用基因测序的方法,对一个常染色体显性遗传的角膜营养不良家系进行基因筛查,确定该家系的基因缺陷。方法收集一个中国人Reis-Bucklers地图状角膜营养不良家系,共4代30人,其中患者12人。对所有家系患病成员及正常同胞进行详细的临床检查及TGFBI基因编码序列分析,包括TGFBI基因的全部17个外显子,5＇及3＇端与外显子拼接的内含子序列。结果对TGFBI基因直接测序显示家系中所有患者在TGFBI基因的第4外显子存在杂合性突变,mRNA第371位碱基出现G→T的杂合性碱基改变,导致了野生型基因编码的Arg被Leu替代,使第124编码子发生了R124L的突变,并且该突变与疾病表型共分离。结论经序列分析确定该家系角膜营养不良患者均存在TGFBI基因第4外显子的R124L突变。     

常染色体显性遗传视网膜色素变性家系视紫红质基因突变分析

目的:观察常染色体显性遗传视网膜色素变性(autosomaldominantRP,ADRP)家系视紫红质基因(rhodopsin,RHO)的突变特征。方法:抽取11个ADRP家系成员的外周血3～5mL,提取DNA;应用聚合酶链反应(polymerasechainreaction,PCR)扩增RHO基因的第1至5外显子基因片断,对PCR产物进行直接测序。结果:在1个家系中有3例ADRP患者297密码子存在杂合的2种类型的密码子(AGC和AGT)。另外,该家系在第3外显子3'端下游第4个碱基处发生C-T转换,呈T纯合子的1例,8例呈杂合子状态。结论:Ser-297-Ser系基因多态现象。另外,RHO基因第3外显子3'端下游内含子处发生的C/T多态性是否与RP的发生存在相关性,需进一步研究。     

常染色体显性遗传RP患者视紫红质基因突变的检测分析

目的研究常染色体显性遗传视网膜色素变性（ADRP）患者视紫红质（RHO）基因的突变特征及其在视网膜色素变性（RP）发病机制中的作用。方法应用变性高效液相色谱分析（DHPLC）技术和直接及克隆测序方法对RHO基因进行突变检测。结果一家系4例ADRP患者RHO基因的第297密码子存在杂合的两种类型密码子（AGC和AGT）。该家系的另3名患者未检测到该突变，对照组发现1例此类型沉默型突变。该家系在第3外显子3’端下游第4个碱基处发生C—T转换，其11个成员中该位点呈T纯合子1例，呈杂合子状态8例。对照组发现2例该位点的杂合子状态。结论视紫红质基因Ser-297-Ser突变与RP疾病未出现“共分离”现象，因此该沉默型突变不是该ADRP家系的致病原因，系RHO基因的多态现象。     

我国常染色体显性遗传视网膜色素变性家系中PRPF31基因新的剪切位点突变

目的　研究我国一个4代常染色体显性遗传视网膜色素变性(RP)家系患者的致病基因突变位点及临床表型特征。方法　对RP家系中的所有患者进行眼部及视觉电生理检查;对全部家系成员进行全基因组扫描及连锁分析, 对候选基因直接测序并通过限制性内切酶反应证实突变位点。结果　RP家系患者致病基因定位于染色体带19q13 4,微卫星标记物D19S589和D19S254之间不到4Mb区域。在所有患者的PRPF31基因内含子8的第一个碱基处发现一新的杂合突变(G>C),使内含子8的剪切供体由GT变为CT。RP家系患者的临床表型符合早期发病且弥漫型的RP患者类型。结论　我国该4代RP家系中的患者由PRPF31基因中一新的剪切位点的杂合突变致病(IVS8+1G>C)。     

土家族中一个先天性无虹膜家系的临床特点和PAX6基因突变位点分析

目的：确认土家族中一个先天性无虹膜家系的PAX6基因致病突变并分析其临床特点。方法：实验研究。详细询问家族病史并对该家系中所有7 例成员（4 例患者,3 例正常人）进行详细的眼部检查,采集家系成员及100例（50例土家族人和50例汉族人）正常对照者的外周静脉血,提取DNA;对先证者PAX6基因的全部外显子进行PCR扩增及测序;对家系中所有成员和正常对照者进行PAX6基因突变位点的验证检测。结果：该家系中患者主要以虹膜缺损、白内障、眼球震颤、黄斑中心凹发育不良和角膜病变为主要临床表现,虹膜缺损轻重不一,角膜病变和白内障情况随年龄增加而加重。该家系的4 例患者均在第3 外显子与内含子3 交界处出现一个杂合突变（c.357+1G > A）,正常家系成员及正常对照者均无此突变。结论：该先天性无虹膜家系患者虹膜缺损程度不一。PAX6是该家系的致病基因,该家系患者PAX6基因的突变位点是杂合突变(c.357+1G > A）。     

视网膜色素变性患者特异性光感受器细胞核受体基因突变检测分析

目的 观察特异性光感受器细胞核受体(NR2E3)基因在宁夏地区视网膜色素变性(RP)患者中的突变频率及特征,探讨其在RP发病机制中的作用。方法 经检查确诊的120例RP患者纳入研究。其中,常染色体显性遗传RP (ADRP)患者33例,来自18个家系;常染色体隐性遗传RP (ARRP)患者20例,来自15个家系;散发型RP(SRP)患者67例。选取100名健康成年人作为对照组。采用聚合酶链反应(PCR)和直接测序方法,检测NR2E3基因全编码区和邻近剪切位点的内含子区域序列突变。多因素分析研究NR2E3基因突变位点对RP的作用。结果 120例RP患者NR2E3基因检测出变异位点12个。其中,非编码区5个;第4、6、7外显子上7个。12个变异位点中,新发现变异位点6个。外显子上7个变异位点中,同义突变3个;错义突变4个。统计学分析结果显示,所有变异位点均为NR2E3基因多态性。多因素Logistic回归分析显示,变异位点均与RP发生无相关性。18例ADRP先证者、67例SRP患者和正常对照组中,分别有1、3、2例NR2E3基因第4外显子上发现p.Glu121Lys变异。发生该位点变异的ADRP患者家系（NXRP-1）另外8例患者中,出现p.Glu121Lys位点变异5例,未出现变异3例。出现变异的6例患者发病年龄较未出现p.Glu121Lys位点变异的3例患者早,且较早出现明显的中心视力损害。结论 宁夏地区RP患者NR2E3基因致病突变率小于1％,NR2E3基因的p.Glu121Lys变异发生率较低。     

中国北方先天性白内障家系中缝隙连接蛋白基因突变的筛查

目的：在一中国人先天性白内障家系中进行缝隙连接蛋白基因（GJA3、GJA8）突变筛查.方法：通过聚合酶链反应（polymerase chain reaction,PCR）对一先天性白内障家系中的全部患者进行GJA3基因及GJA8基因外显子的扩增,扩增产物进行直接测序.结果：该家系GJA8基因的外显子及其邻近的内含子未发现任何突变.先证者GJA3基因外显子非编码区发现碱基CA的缺失.结论：缝隙连接蛋白基因为该先天性白内障家系的非致病性基因.     

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。     

视网膜色素变性1基因在常染色体显性遗传视网膜色素变性家系中的突变分析

目的:研究常染色体显性遗传视网膜色素变性(autosomal dominant retinitis pigmentosa,ADRP)家系中视网膜色素变性1(retinitis pigmentosa-1,RP1)基因的突变特征及其在RP发病机制中的作用。方法:运用聚合酶链反应和直接测序方法,对6个ADRP家系的47例成员和50例对照者进行了RP1基因全编码区和邻近剪切位点的内含子区域序列突变的筛选与检测。运用单因素分析、多因素Logistic回归分析研究RP1基因点突变在RP发病中的作用。结果:ADRP家系成员和对照组RP1基因第4外显子上检测出2个变异位点。在1691和1725密码子存在杂合的两种类型的密码子(S1691P,Ser-Pro,TCT→CCT;Q1725Q,Gln-Gln,CAA→CAG)。ADRP家系成员中Ser-1691-Pro及Gln-1725-Gln位点突变率显著高于正常对照组(χ2=11.202,P<0.05)。结论:RP1基因Ser-1691-Pro及Gln-1725-Gln位点多态性可增高RP的危险性,具有潜在的致病性,考虑为ADRP家系的易感基因。     

Novel mutations in PDE6B causing human retinitis pigmentosa

AIM: To identify the genetic defects of a Chinese patient with sporadic retinitis pigmentosa (RP). METHODS: Ophthalmologic examinations were performed on the sporadic RP patient, 144 genes associated with retinal diseases were scanned with capture next generation sequencing (CNGS) approach. Two heterozygous mutations in PDE6B were confirmed in the pedigree by Sanger sequencing subsequently. The carrier frequency of PDE6B mutations of reported PDE6B mutations based on the available two public exome databases (1000 Genomes Project and ESP6500 Genomes Project) and one in-house exome database was investigated. RESULTS: We identified compound heterozygosity of two novel nonsense mutations c.1133G>A (p.W378X) and c.2395C>T (p.R799X) in PDE6B, one reported causative gene for RP. Neither of the two mutations in our study was presented in three exome databases. Two mutations (p.R74C and p.T604I) in PDE6B have relatively high frequencies in the ESP6500 and in-house databases, respectively, while no common dominant mutation in each of the database or across all databases. CONCLUSION: We demonstrates that compound heterozygosity of two novel nonsense mutations in PDE6B could lead to RP. These results collectively point to enormous potential of next-generation sequencing in determining the genetic etiology of RP and how various mutations in PDE6B contribute to the genetic heterogeneity of RP.     

Frequency of mutations in the gene encoding the alpha subunit of rod cGMP-phosphodiesterase in autosomal recessive retinitis pigmentosa.

PURPOSE: To determine the mutation spectrum of the PDE6A gene encoding the alpha subunit of rod cyclic guanosine monophosphate (cGMP)phosphodiesterase and the proportion of patients with recessive retinitis pigmentosa (RP) due to mutations in this gene. METHODS: The single-strand conformation polymorphism (SSCP) technique and a direct genomic sequencing technique were used to screen all 22 exons of this gene for mutations in 164 unrelated patients with recessive or isolate RP. Variant DNA fragments revealed by SSCP analysis were subsequently sequenced. Selected alleles that altered the coding region or intron splice sites were evaluated further through segregation analysis in the families of the index cases. RESULTS: Four new families were identified with five novel mutations in this gene that cosegregated with disease. Combining the data presented here with those published earlier by the authors, eight different mutations in six families have been discovered to be pathogenic. Two of the mutations are nonsense, five are missense, and one affects a canonical splice-donor site. CONCLUSIONS: The PDE6A gene appears to account for roughly 3% to 4% of families with recessive RP in North America. A compilation of the pathogenic mutations in PDE6A and those reported in the homologous gene PDE6B encoding the beta subunit of rod cGMP-phosphodiesterase shows that the cGMP-binding and catalytic domains are frequently affected.     

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.     

马凡综合征一家系的致病基因筛查

目的：对中国辽宁省一个具有常染色体显性遗传特点的马凡综合征( Marfan syndrole, MFS )家系进行突变基因的筛查。
　　方法：分别采集家系成员的外周静脉血,提取基因组DNA,通过对与马凡综合征相关的致病基因进行遗传学研究和分析,选取候选基因并设计引物,应用聚合酶链式反应( PCR)扩增DNA片段后进行琼脂糖凝胶电泳分离,利用直接测序法确定致病基因及其突变位点。
　　结果：该家系遗传方式符合常染色体显性遗传,先证者表型为双眼晶状体向鼻上方脱位,通过对候选基因外显子直接测序,发现该家系内患者原纤维蛋白基因-1(fibrillin-1 gene,FBN1)第7个外显子第640位碱基有1个A>G的点突变,此突变导致蛋白第214位的甘氨酸被丝氨酸取代(G214S)。
　　结论：FBN1基因 c. A640G(p. G214S)突变为该马凡综合征家系的致病因素。     

视网膜色素变性遗传致病基因peripherin/RDS的突变筛选

目的 了解中国视网膜色素变性患者（RP）中peripherin/RDS基因的突变谱及突变率。方法 应用聚合酶链-异源双链-单链构象多态性（PCR-SSCP）及DNA序列分析技术对收集的15个常染色体显性遗传视网膜色谱变性家系和55例散发视网膜色素变性患者peripherin/RDS基因的第一，第二外显子进行检测。结果 15个家系及55例散发患者未检测到peripherin/RDS基因突变。结论 本研究所检测的视网膜色素变性患者与RDS基因无关，显示视网膜色素变性的遗传异质性。     

Evaluation of cGMP-phosphodiesterase (PDE) subunits for causal association with rod-cone dysplasia 2 (rcd2), a canine model of abnormal retinal cGMP metabolism.

Rod-cone dysplasia types 1 (rcd1; Irish setter) and 2 (rcd2; collie) in dogs are early onset forms of progressive retinal atrophy (PRA) which serve as models of retinitis pigmentosa (RP) in humans. As both rcd1 and rcd2 result from abnormal retinal cGMP metabolism associated with a deficiency in cGMP-phosphodiesterase (PDE) activity, and a nonsense mutation in the PDE6B subunit gene has been shown to cause rcd1, the genes encoding the four subunits of the PDE complex (PDE6A, PDE6B, PDE6G and PDE6D) make compelling candidates for the rcd2 locus. We adopted diverse strategies to evaluate causal association of the four PDE subunit genes with the rcd2 phenotype. Identification in an informative pedigree of obligate recombinations between intragenic polymorphisms within PDE6A and PDE6D and the rcd2 locus unequivocally excludes these two genes. PDE6B was excluded by a breeding strategy demonstrating nonallelism of rcd1 and rcd2. Direct sequencing of PDE6G from an rcd2 -homozygous collie dog revealed no abnormality in the entire genomic sequence. To evaluate cosegregation between PDE6G and rcd2, advantage was taken of prior knowledge that PDE6G and Galactokinase 1 (GALK1) localize to the same canine-rodent somatic hybrid cell line. Linkage analysis using a single nucleotide polymorphism (SNP) in the PDE6G gene, and a (CA)n repeat polymorphism in the GALK1 gene, which were both segregating in an unrelated pedigree, established close linkage of these two genes (theta = 0; Z = 4.21). Identification of obligate recombinations between GALK1 and the rcd2 locus in an informative rcd2 pedigree thus excluded PDE6G as a candidate gene for rcd2; the exclusion distance between GALK1 and rcd2 is at least 0.35 cM. These results therefore exclude the entire set of genes coding for the rod PDE complex as candidates for rcd2.     

cGMP phosphodiesterase-alpha mutation causes progressive retinal atrophy in the Cardigan Welsh corgi dog.

PURPOSE: To screen the alpha-subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6A) as a potential candidate gene for progressive retinal atrophy (PRA) in the Cardigan Welsh corgi dog. METHODS: Single-strand conformation polymorphism (SSCP) analysis was used to screen short introns of the canine PDE6A gene for informative polymorphisms in members of an extended pedigree of PRA-affected Cardigan Welsh corgis. After initial demonstration of linkage of a polymorphism in the PDE6A gene with the disease locus, the complete coding region of the PDE6A gene of a PRA-affected Cardigan Welsh corgi was cloned in overlapping fragments and sequenced. SSCP-based and direct DNA sequencing tests were developed to detect the presence of a PDE6A gene mutation that segregated with disease status in the extended pedigree of PRA-affected Cardigan Welsh corgis. Genomic DNA sequencing was developed as a diagnostic test to establish the genotype of Cardigan Welsh corgis in the pet population. RESULTS: A polymorphism within intron 18 of the canine PDE6A gene was invariably present in the homozygous state in PRA-affected Cardigan Welsh corgis. The entire PDE6A gene was cloned from one PRA-affected dog and the gene structure and intron sizes established and compared with those of an unaffected animal. Intron sizes were identical in affected and normal dogs. Sequencing of exons and splice junctions in the affected animal revealed a 1-bp deletion in codon 616. Analysis of PRA-affected anti obligate carrier Cardigan Welsh corgis showed that this mutation cosegregated with disease status. CONCLUSIONS: A single base deletion at codon 616 in the PDE6A gene cosegregated with PRA status with zero discordance in Cardigan Welsh corgis with PRA. A lod score of 4.816 with a recombination fraction (theta) of zero strongly suggests that this mutation is responsible for PRA in the breed. The mutation is predicted to lead to a frame shift resulting in a string of 28 altered codons followed by a premature stop codon. The authors suggest that this type of PRA be given the name rod-cone dysplasia 3 (rcd3).     

Mutations in the ABCA4 gene in a family with Stargardt's disease and retinitis pigmentosa (STGD1/RP19)

BACKGROUND: Demonstrating the types of ABCA4 mutations in the STGD1 gene in a family manifesting both Stargardt's disease and retinitis pigmentosa (RP19). METHODS: Clinical ophthalmological examination included funduscopy, ERG, Arden Colour contrast test, fluorescein angiography in one patient, perimetry and SLO perimetry. The 50 exons of the ABCA4 gene were screened using a combination of denaturating gradient gel electrophoresis (DGGE), high performance electrophoresis (dHPLC) and SSCP analysis. RESULTS: Patient I/1 showed typical signs of Stargardt's disease, while her son, II-1 demonstrated functional signs and morphological features of retinitis pigmentosa. Mutational analysis of the ABCA4 gene revealed a missense mutation in exon 42 (G5882G > A) and a frameshift mutation in exon 43 (5917delG) of patient I-1. Patient II/1 demonstrated a homozygous 5917delG mutation in exon 43, resulting in a functional null-mutation. CONCLUSIONS: The combination of ABCA4 alleles with various functional consequences to protein activity can lead to different clinical phenotypes in one and the same family, resulting either in typical Stargardt's disease or in autosomal recessive retinitis pigmentosa (RP19).     

A new Leu253Arg mutation in the RP2 gene in a Japanese family with X-linked retinitis pigmentosa

PURPOSE: To identify the clinical findings in a Japanese family with X-linked retinitis pigmentosa associated with mutation in codon 253 (Leu253Arg) in the RP2 gene. METHODS: Case reports included clinical features and results of fluorescein angiography, electroretinogram, kinetic visual field testing, and DNA analysis. Two affected hemizygotes with retinitis pigmentosa associated with transversion mutations in codon 253 (Leu253Arg) of the RP2 gene and the obligate carriers were examined. RESULTS: A novel Leu253Arg mutation of the RP2 gene was found to cosegregate with retinal degeneration in two affected males and two carriers in female heterozygote in a Japanese family. The ophthalmic findings in hemizygote showed severe retinal degeneration. In the obligate carrier, mild chorioretinal degeneration was observed in both eyes but a tapetal-like reflex of the fundus was not apparent. CONCLUSIONS: The mutation at codon 253 of the RP2 gene is the first mutation reported in a Japanese family. It is concluded that the mutation of the RP2 gene also causes the X-linked retinitis pigmentosa in Japanese patients.