Truncations in the TIGR gene in individuals with and without primary open-angle glaucoma

PURPOSE: To investigate the coding exons in the trabecular meshwork-induced glucocorticoid response protein (TIGR) gene for mutations in primary open-angle glaucoma (POAG) in Chinese subjects. METHODS: Ninety-one Chinese patients with POAG and 113 of their family members without glaucoma were screened for sequence alterations in the TIGR gene by polymerase chain reaction, conformation-sensitive gel electrophoresis, and DNA sequencing. One hundred thirty-two unrelated individuals without glaucoma, aged 50 years or more, were studied as control subjects. RESULTS: Five sequence variants that lead to amino acid changes were identified. One was novel: Arg91Stop in one patient with POAG. Four had been reported: Arg46Stop in subjects with and without POAG, including an unaffected 77-year-old woman homozygous for Arg46Stop; Gly12Arg in subjects without glaucoma; and Asp208Glu and Thr353Ile in subjects with and without POAG. The previously reported 1-83(G-->A) and Arg76Lys polymorphisms were detected in both patients and controls and always occurred together. CONCLUSIONS: A different pattern of TIGR sequence variants exists in the Chinese than in non-Chinese populations. No common TIGR mutation that causes POAG was found. The occurrence of subjects without glaucoma who are heterozygous or homozygous for Arg46Stop suggests that reduction in the amount of TIGR protein does not cause glaucoma. Thus, the TIGR missense mutations known to cause POAG probably do not cause glaucoma by inactivating a normal TIGR function, but rather through the gain of a pathologic function.     

TIGR/MYOC gene sequence alterations in individuals with and without primary open-angle glaucoma

PURPOSE: To discover sequence alterations in the TIGR/MYOC gene associated with primary open-angle glaucoma (POAG) in Chinese subjects. METHODS: Two hundred one unrelated Chinese patients with POAG and 291 unrelated individuals without glaucoma, aged 50 years or more, were screened for sequence alterations in the TIGR/MYOC gene by polymerase chain reaction, conformation sensitive gel electrophoresis, and DNA sequencing. Up to 111 more control subjects were screened for some of the alterations. RESULTS: Fourteen sequence variants that lead to amino acid changes were identified. Seven were novel: Pro16Leu, Ala17Ser, Leu95Pro, Leu215Pro, Glu300Lys, Glu414Lys, and Tyr471Cys. Of these, Glu300Lys and Tyr471Cys were found only in POAG. Arg46Stop was found in 4 patients with POAG (2.0%) and 9 of 402 control subjects (2.2%); one control subject was homozygous. IOP showed a trend (P = 0.11) toward a decrease of 1.5 mm Hg among the control subjects, with Arg46Stop compared with matched control subjects without Arg46Stop. Gly12Arg occurred four times as frequently in control subjects as in patients, but the difference was not statistically significant. CONCLUSIONS: Gly12Arg might be negatively associated with POAG, suggesting a protective effect. Three patients with POAG had a sequence change not found in control subjects, for a frequency of possible disease-causing TIGR/MYOC mutations of 1.5% (95% confidence interval [CI] = 0.3%-4.3%). Arg46Stop occurred with similar frequency in patients with POAG and control subjects, suggesting that the reduced amount of TIGR/MYOC predicted to result from this truncation does not dramatically increase or decrease risk of glaucoma.     

重庆地区开角型青光眼患者及其亲属和正常人群中小梁网糖皮质激素诱导反应蛋白基因突变的研究

目的 研究开角型青光眼患者及其亲属和正常人群中小梁网糖皮质激素诱导反应蛋白(TIGR)基因突变的情况。方法 (1)应用聚合酶链反应(PCR)方法,对15例开角型青光眼患者及其10例一级亲属和20例正常对照者的TIGR基因的3个外显子、部分内含子及部分启动子(7对引物)的各个片段进行扩增,应用单链空间构象多态(SSCP)分析法,筛选可能存在突变的PCR产物。(2)将筛选出的PCR产物交上海基康公司测序。(3)对突变片段行生物信息学分析。结果 (1)在重庆地区15例原发性开角型青光眼患者中,共发现TGR基因突变者5例,其中青少年型青光眼4例;在10例青光眼患者亲属中发现TIGR基因突变者2例;正常对照者20例中未发现TIGR基因突变。(2)PCR产物测序共发现4个序列改变,其中编码区2个,非编码区2个。编码区的2个突变位点(Ser55Thl、Asp247Stop)及第二内含子区bp35c→t的突变,均未见文献报道;而在启动子区域bp-83c→t的突变有文献报道为1个多态位点。(3)生物信息学分析结果显示编码区的突变可导致氨基酸序列、蛋白质的二级结构及等电点、抗原结合位点等发生改变。结论 TIGR基因突变与青少年开角型青光眼的发生密切相关,由此可推测青光眼患者的亲属发病率较正常人高。TIGR基因突变可引起TIGR蛋白结构及理化特性的变化,这些改变可能是引发开角型青光眼的危险因素之一。     

Different optineurin mutation pattern in primary open-angle glaucoma

PURPOSE: The optineurin gene (OPTN) is the second gene besides MYOC in which mutations have been identified to be associated with primary open-angle glaucoma (POAG). In this study, sequence alterations in the OPTN gene associated with POAG in Chinese subjects were investigated. METHODS: All the coding exons of OPTN were screened, including the intron-exon boundaries, for sequence alterations in a Chinese sample of 119 sporadic patients with POAG and 126 unrelated control subjects by polymerase chain reaction-conformation-sensitive gel electrophoresis and DNA sequencing. RESULTS: Sixteen sequence changes were identified: 3 had been reported (T34T, M98K, and R545Q) and 13 were novel (T49T, E103D, V148V, P199P, T202T, H486R, IVS6-5T-->C, IVS6-10G-->A, IVS7+24G-->A, IVS8+20G-->A, IVS13+21C-->G, IVS15+10G-->A, and IVS15-48C-->A). Among them, only E103D, H486R, V148V, and IVS13+21C-->G were found exclusively in patients with POAG, whereas P199P, T202T, and IVS8+20G-->A were present only in control subjects. The genotype of IVS7+24G-->A showed a significant association with POAG (P = 0.02, Fisher two-tailed exact test) and with and increased cup-to-disc ratio in these patients (P = 0.005, Mann-Whitney test). CONCLUSIONS: The findings in the current study enrich the evidence on the OPTN gene as a causative gene for POAG and suggest a different mutation pattern of OPTN in Chinese than in whites. The wide spectrum of putative mutations detected in this study suggests that both structural and functional disruptions in OPTN may contribute to the pathogenesis of glaucoma.     

原发性开角型青光眼MYOC- TIGR基因突变研究

目的 筛选并研究原发性开角型青光眼(POAG)小梁网糖皮质激素诱导反应蛋白MYOC- TIGR基因突变情况。方法 病例对照研究。抽取2002年1至12月就诊并诊断为POAG患者118例和150例非POAG对照者的外周静脉血4～8ml,采用酚-氯仿抽提全基因组DNA,应用聚合酶链反应(PCR)技术扩增全基因组DNA,以单核苷酸构象多态性(SSCP)分析MYOC基因的3个外显子（7对引物）编码区域序列改变。对SSCP分析异常者,采用双向测序法进一步证实。应用Cfr13I、Hinfl及BsmA1等限制性内切酶检测对照者MYOC基因编码区序列改变。POAG与非POAG人群MYOC基因各位点突变率比较采用x2检验。结果 基因序列分析发现G12R、I288M及Y353I共3个基因序列改变。118例POAG患者中仅有5例(4.23％)发生G12R位点突变,150例对照者中未见G12R位点改变;两组G12R位点突变率比较差异有统计学意义(x2=4.37,P=0.037)。POAG组和对照组均有I288M和Y353I位点改变,但两组突变率比较差异无统计学意义(x2=0.07,P=0.791和x2=0.56,P=0.453)。POAG患者基因突变率4.23％。结论 MYOC基因突变可能与POAG发病有关,MYOC基因突变可能是POAG发病的分子机制之一。     

我国原发性开角型青光眼患者TIGR基因突变筛选、克隆及序列分析

目的　研究我国原发性开角型青光眼 (primaryopenangleglaucoma,POAG)患者小梁网糖皮质激素诱导反应蛋白 (trabecularmeshworkinducedglucocorticoidresponseprotein ,TIGR)基因的突变情况。方法　 (1)应用聚合酶链式反应 (polymerasechainreaction ,PCR)方法扩增 70例POAG患者、2 0例正常对照组的TIGR基因 3个外显子 (7对引物 )的各个片段 ,应用单链构像多态 (single strandedconformationpolymorphism ,SSCP)筛选可能突变的PCR产物。 (2 )将筛选出的样本 (PCR产物 ) ,克隆到PT Adv载体 ,以EcorI酶切鉴定重组质粒 ,然后用ABI 373自动测序仪行双向测序。结果　 (1)用SSCP方法筛选的 2例POAG患者在TIGR基因第 3外显子中部 (第 6对引物 )片段出现单链带型异常 ;正常对照组未见异常。 (2 )克隆测序的 2例样品 ,1例在 388密码子位置出现由GAT突变为AAT ,氨基酸由天冬氨酸替换为天冬酰胺 ,即Asp388Asn ;另 1例碱基序列无变化。提示我国POAG患者TIGR基因突变仅为 1 4% (1/ 70 ) ,远较国外为低。结论　我国POAG患者的发病机制可能与TIGR基因突变有关 ,但突变率较国外低 ,提示POAG发病存在地区或种族之间的差异。     

Clinical phenotype of a Japanese family with primary open angle glaucoma caused by a Pro370Leu mutation in the MYOC/TIGR gene.

PURPOSE: To present the phenotype of two patients with primary open angle glaucoma (POAG) caused by a mutation of the myocilin/trabecular meshwork-inducible glucocorticoid response (MYOC/TIGR) gene. METHODS: Complete ocular examinations were performed on the 13-year-old proband, her father, mother, and sister. DNA analysis was performed to detect the mutant gene. RESULTS: The proband and her father were found to have a mutation of the MYOC/TIGR gene. Both patients carried a heterozygous mutation in the 1,109th nucleotide, which corresponds to the 370th amino acid residue of the MYOC/TIGR gene. The clinical characteristics of both patients were: (1) development of POAG at an early age, (2) high peaks of intraocular pressure. and (3) poor response to medical treatment. CONCLUSIONS: The phenotype of these patients with a mutation of the MYOC/TIGR gene agreed with reports of other patients with mutations at other loci in this gene. The discovery of the MYOC/TIGR gene not only makes early detection of glaucoma possible, but also presents a new direction for investigating the pathogenesis of glaucoma.     

Myocilin and glaucoma: A TIGR by the tail?

In 1997, Stone and 14 colleagues from 7 laboratories reported the identification of a gene (TIGR) associated with juvenile open-angle glaucoma (JOAG). Screening of adults with primary open-angle glaucoma (POAG) revealed that about 4% also carried a mutation of the coding region of this gene. The mutations were found through genetic linkage analysis of families with JOAG. Juvenile open-angle glaucoma was a logical starting point in the search for genetic causes of open-angle glaucoma: it shows a strong autosomal-dominant inheritance pattern, occurs at an early age, demonstrates obvious phenotypic signs (dramatic elevation of intraocular pressure and subsequent optic nerve damage), and is likely to be found in multiple generations as parents of affected children are still living. These factors, however, also serve to distinguish it from adult-onset POAG, which generally has a lower intraocular pressure and a less severe course. The discovery of the actual gene represented a true advance over previous studies that had mapped the gene to a segment of a chromosome but did not identify the specific gene. How the mutant gene causes glaucoma is unknown and is the subject of intense research. To date, 26 mutations in the TIGR gene sequence (the term TIGR has been replaced by the term myocilin, abbreviated MYOC) have been described, all associated with either JOAG or adult-onset POAG. A correlation between specific mutations in MYOC and the clinical course of glaucoma has been found. Not all cases of JOAG or POAG have mutations in the MYOC gene, however, indicating that more discoveries of other genes are yet to come. Arch Ophthalmol. 2000;118:974-978     

The attempt to identify mutations in TIGR gene in Polish patients with primary open angle glaucoma

PURPOSE: The case work presents studies on the identification of the most frequent TIGR mutations in Polish population with primary open angle glaucoma. The TIGR gene was identified in a GLC1A locus on chromosome 1 (1q) in a family with juvenile primary open angle glaucoma. The gene encodes TIGR protein (trabecular meshwork inducible gluco-corticoid response protein)--trabecular meshwork glucoprotein. MATERIAL AND METHODS: Ophthalmologic examination was performed in twenty subjects with juvenile primary open angle glaucoma. The blood samples were taken for DNA analyses. RESULTS: Neither any mutations nor polymorphic changes in TIGR gene were found. CONCLUSION: Our studies have not identified any mutations in exon 3 of TIGR gene. We cannot exclude, however, that mutations are localised in other exons or regulatory region of examined gene. The questions how many genes, how many mutations of these genes and how often they contribute to glaucoma in general population are still open? These are important questions to answer in order to get closer to understanding extremely complicated aetiology of glaucoma.     

Variants in optineurin gene and their association with tumor necrosis factor-alpha polymorphisms in Japanese patients with glaucoma

PURPOSE: To investigate sequence variations in the optineurin (OPTN) gene and their association with TNF-alpha polymorphisms in Japanese patients with glaucoma. METHODS: The OPTN gene was analyzed in blood samples from 629 Japanese subjects. There were 194 patients with primary open-angle glaucoma (POAG), 217 with normal-tension glaucoma (NTG), and 218 with no eye disease (control subjects). The gene was screened for mutations by denaturing high-performance liquid chromatography. Genotyping of three polymorphisms of -308G-->A, -857C-->T, and -863C-->A in the TNF-alpha promoter region was performed. The associations between the genotypes and age, intraocular pressure (IOP), and visual field defects at the time of diagnosis were examined. RESULTS: A possible glaucoma-causing mutation, His26Asp, was identified in 1 of the 411 Japanese patients with glaucoma. A c.412G-->A (Thr34Thr) polymorphism in the OPTN gene was significantly associated with POAG (genotype frequency, P = 0.011; allele frequency, P = 0.003). The frequency of TNF-alpha/-857T and optineurin/412A carriers was significantly higher (P = 0.006) in patients with POAG than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-857T, the optineurin/412A carriers had significantly worse (P = 0.020) visual field scores than the non-optineurin/412A ones. The frequency of TNF-alpha/-863A and optineurin/603A (or Lys98) carriers was significantly higher in patients with POAG (P = 0.008) or NTG (P = 0.027) than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-863A, the ones with optineurin/603A (or Lys98) had significantly worse (P = 0.026) visual field scores than did those with non-optineurin/603A (or Lys98). CONCLUSIONS: These findings demonstrated that the OPTN gene is associated with POAG rather than NTG in the Japanese. Statistical analysis showed a possible interaction between polymorphisms in the OPTN and the TNF-alpha genes that would increase the risk for glaucoma.     

高度近视与原发性开角型青光眼

目的:从流行病学、临床特征及诊断、遗传学说及机制等方面对高度近视与原发性开角型青光眼的关系及研究进展进行综述。方法:仔细分析了28篇原文,总结出高度近视与原发性开角型青光眼的可能机理。结果:高度近视与原发性开角型青光眼密切相关,其可能的机制有:①升压基因学说。②胶原基因学说。结论:高度近视与原发性开角型青光眼密切相关,根据TIGR基因理论和胶原基因理论,高度近视与原发性开角型青光眼都与TIGR基因突变和胶原疾病有关。     

Molecular analysis of the myocilin gene in Chinese subjects with chronic primary-angle closure glaucoma

PURPOSE: Mutations in the myocilin (MYOC) gene have been implicated in juvenile as well as late-onset primary open-angle glaucoma (POAG). Overall, MYOC mutations account for 3% to 5% of cases of POAG worldwide, making it the most significant gene identified so far in glaucoma. Although there are some similarities in the phenotype of POAG and in particular chronic primary angle-closure glaucoma (PACG), little is known about the role of MYOC in the causation of PACG. To address this, the MYOC gene was screened in a cohort of 106 patients with chronic PACG. METHODS: Genomic DNA was extracted from leukocytes of the peripheral blood and exons 1 to 3 of the MYOC gene were PCR amplified and subjected to bidirectional sequencing and analysis. RESULTS: One hundred six patients with chronic PACG of Chinese ethnicity were studied. Sequencing of the MYOC gene in these patients revealed eight sequence variants. Of these, one was a nonsense change, three were missense changes, two were synonymous codon changes, and two were changes in noncoding sequences. These included the Arg46Stop and Thr353Ile mutations, which have been reported in individuals with POAG. However, all the sequence alterations identified have been found in normal Chinese subjects. CONCLUSIONS: The results of this study do not support a role for MYOC mutations in the pathogenesis of chronic PACG in the Chinese.     

陕西省两个原发性开角型青光眼家系MYOC基因突变的研究

目的:研究陕西省两个原发性开角型青光眼家系MYOC基因突变情况。方法:分析陕西省两个原发性开角型青光眼家系。从先证者、家族成员及正常对照者外周静脉血中提取基因组DNA;根据MYOC基因编码序列合成7对特异性引物;应用PCR扩增MYOC基因3个外显子序列,DNA测序法双向测序筛选突变位点。结果:家系1中并未发现MYOC基因编码序列的突变位点;家系2中三个患者MYOC基因均存在异常(c.1021T>C杂合突变),导致myocilin蛋白第341位氨基酸由丝氨酸(S)转变为脯氨酸(P)即Ser341Pro错义突变,该家系正常成员及100例对照者中均未发现此突变。结论:MYOC基因Ser341Pro突变可能为家系2原发性开角型青光眼的致病原因。     

MYOC/TIGR基因与原发性开角型青光眼

近年来,原发性开角型青光眼的分子遗传学研究取得了飞速发展,尤其是1997年首次发现位于GLCIA基因位点上的开角型青光眼的致病基因小梁网糖皮质激素诱导反应蛋白基因(TIGR)以来,该领域得到了迅速发展.人们对TIGR基因及其编码产物的研究正在逐步深入,就该基因的发现、结构、功能、突变以及在原发性开角型青光眼病变过程中可能的作用作一综述.     

Polymorphisms in the myocilin promoter unrelated to the risk and severity of primary open-angle glaucoma

PURPOSE: To investigate the proximal 2.5 kb promoter in the myocilin (MYOC) gene for mutations in Chinese patients with primary open-angle glaucoma (POAG). PATIENTS AND METHODS: We screened for sequence alterations in the MYOC promoter in 88 unrelated Chinese patients with POAG and 94 unrelated individuals without glaucoma, aged 50 years or above, as control subjects. In addition, the specific MYOC.mt1 polymorphism was determined in a total of 212 POAG patients and 221 control subjects. The relationships between POAG phenotype and the identified polymorphisms were studied by univariate analysis, multivariable logistic regression analysis, and haplotype analysis. RESULTS: All polymorphisms identified in this study followed Hardy-Weinberg equilibrium (P > 0.12) both in POAG patients and controls. Both univariate and multivariable logistic regression analyses showed no polymorphism that was significantly associated with the risk of POAG, P > 0.08 and P > 0.044 respectively. Haplotype analysis further indicated no association of MYOC promoter polymorphisms with the susceptibility for POAG (P > 0.1). On the other hand, there was no difference of POAG phenotypes among different genotypes of MYOC.mt1 (P > 0.31). CONCLUSIONS: In this study on the Chinese population, polymorphisms in the MYOC promoter are not related to the risk of POAG. There is no association between the MYOC.mt1 promoter polymorphism with the severity of POAG.     

Genetic screening in a large family with juvenile onset primary open angle glaucoma

AIMS: A number of genetic loci have been implicated in the pathogenesis of primary open angle glaucoma (POAG). The aim of this study was to identify the genetic cause of POAG in a large Scottish family and, if possible, offer genetic screening and advice to family members. METHODS: Family members were examined to determine their disease status. Base excision sequence scanning was carried out in order to test for the presence of a POAG causing mutation at known genetic loci. Direct DNA sequencing was performed in order to determine the mutation sequence. RESULTS: All family members of known affected disease status and two family members of unknown disease status were found to have a mutation in the TIGR gene. The mutation resulted in the substitution of a glycine residue with an arginine residue at codon 252 (Gly252Arg). No other sequence variations were present in any members of the family. CONCLUSION: The Gly252Arg mutation in the TIGR gene results in the development of POAG in this family. It was possible to identify younger, currently unaffected, members of the family who carry the mutation and who are therefore at a very high risk of developing POAG themselves. This is the first demonstration that Gly252Arg can be a disease causing mutation rather than a benign polymorphism. The possible pathogenic mechanisms and wider implications of the mutation are considered.     

ABCA4 gene mutations in Japanese patients with Stargardt disease and retinitis pigmentosa

PURPOSE: To evaluate photoreceptor cell-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) gene mutations in Japanese patients with Stargardt disease (STGD) and the correlation of these mutations to clinical phenotypes. METHODS: Serum was obtained from 10 unrelated Japanese patients with STGD and 96 unrelated Japanese patients with autosomal recessive retinitis pigmentosa (arRP). All 50 ABCA4 gene exons of the patients with STGD were screened for mutations by a combination of single-strand conformation polymorphism analysis and polymerase chain reaction (PCR) direct-sequencing techniques. By restriction enzyme digestion, primer extension analysis, and PCR direct sequencing techniques, the patients with arRP were screened for three segregated, presumably null ABCA4 gene mutations observed in Japanese patients with STGD. RESULTS: Three novel, presumably null mutations of the ABCA4 gene, IVS7-45_952delinsTCTGACC, IVS12+2T-->G, and 1894delA, were identified. The Arg2149stop mutation that had been found in a white patient with STGD in a prior study was also found in a Japanese patient. Two arRP-affected siblings and two unrelated patients with STGD were found to be homozygous for the same IVS12+2T-->G mutation, and three other arRP-affected siblings were carriers of the IVS12+2T-->G mutation and/or the IVS7-45_952delinsTCTGACC mutation. These three siblings with arRP showed only atrophic degeneration in the macula early after the onset of the disease, and STGD had been diagnosed. CONCLUSIONS: Three novel ABCA4 gene mutations were identified in Japanese patients with STGD and arRP. Mutations in the ABCA4 gene can cause panretinal degeneration that changes its clinical appearance from STGD to arRP over time.     

MYOC Mutations in Black South African Patients with Primary Open-angle Glaucoma: Genetic Testing and Cascade Screening

Background: Primary Open Angle Glaucoma (POAG) is an important cause of irreversible blindness in South Africa. Mutations in the MYOC gene are important in monogenic POAG. This study aimed to characterize potentially pathogenic MYOC mutations in this population.

Materials and Methods: Self-identified black South African POAG patients (215) and unaffected control participants (214) had ophthalmological examinations and DNA extraction. Potentially pathogenic MYOC variants were genotyped in the study population. Family members of participants with the mutations were screened for glaucoma clinically and for the mutations using Sanger sequencing.

Results: The following mutations were genotyped: Gly374Val (2 POAG patients), Lys500Arg (3 POAG patients) and Tyr453del (5 POAG patients). None of the relatives screened for Gly374Val had the mutation or POAG. The Lys500Arg mutation did not co-segregate with the disease in an affected family. The Tyr453del mutation co-segregated with the disease, but demonstrated incomplete penetrance. POAG patients with the Tyr453del mutation had adult-onset POAG with high intraocular pressures and advanced cupping.

Conclusions: Overall, 3.3% of black South Africans with POAG have a Gly374Val or Tyr453del MYOC mutation. The Tyr453del mutation is incompletely penetrant. That the mutation is necessary but insufficient introduces a counseling dilemma. Mutation screening can, however, identify high-risk individuals who can be monitored to detect early signs of the disease. The Gly374Val mutation is predicted to be damaging to MYOC. The Lys500Arg mutation is predicted to be benign and tolerated. This study has important implications for the management and counseling of black South African patients with POAG and their families.     

高度近视儿童中未检测到TIGR基因突变

目的:在具有家族史的高度近视儿童中进行TIGR基因突变筛查。方法:对高度近视儿童中TIGR基因第三外显子进行毛细血管电泳测序检测,并对结果进行序列分析。结果:在近视组和对照组中均未发现TIGR基因突变。结论:未能发现TIGR基因突变与高度近视相关证据。