Polymorphism Analysis of VSX1 and SOD1 Genes in Greek Patients with Keratoconus

Background: A number of mutations in the VSX1 and SOD1 genes have been reported to be associated with keratoconus (KC), however the results from different studies are controversial. In this study, we conducted the genotyping of common polymorphisms [VSX1: D144E, H244R, R166W, G160D; SOD1: intronic 7-base deletion (c.169?+?50delTAAACAG)], in a case–control sample panel of the Greek population.

Materials and methods: A case–control panel, with 33 KC patients and 78 healthy controls, were surveyed. DNA from each individual was tested for the VSX1: D144E, H244R, R166W, G160D and SOD1: intronic 7-base deletion (c.169?+?50delTAAACAG) polymorphisms by direct sequencing.

Results: We observed no polymorphisms of the VSX1 gene in the case–control panel. Concerning the SOD1 intronic 7-base deletion (c.169?+?50delTAAACAG), our findings suggest that heterozygous carriers are over-represented among KC cases compared to healthy controls (p?=?0.002).

Conclusions: We cannot confirm the previously reported association of the polymorphism in the VSX1 gene with KC. Our results suggest a possible causative role of SOD1 in the pathogenesis of KC. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of KC.     

No VSX1 gene mutations associated with keratoconus

PURPOSE: To determine whether mutations of the VSX1 gene play a pathogenetic role in the development of keratoconus (KTCN). METHODS: DNA extraction, PCR amplification, and direct sequencing of the VSX1 gene were performed in 100 unrelated patients with diagnoses of clinical and topographic features of KTCN. RESULTS: Of the four previously identified presumed pathogenic mutations in the VSX1 gene (Leu17Pro, Asp144Glu, Leu159Met, and Arg166Trp), only Asp144Glu was identified in a single affected patient. Two novel single nucleotide polymorphisms (SNPs), both resulting in synonymous substitutions, were identified: c.53G>T (Ser6Ser) in four affected patients and c.209G>T (Pro58Pro) in two affected patients. Two previously reported SNPs were also identified: c.426C>A (Arg131Ser) in one affected patient and c.581A>G (Ala182Ala) in 51 of the 100 affected patients. CONCLUSIONS: Only one of the presumed pathogenic mutations in the VSX1 gene, Asp144Glu, was identified in a single member of the cohort of affected patients. However, as previously demonstrated, Asp144Glu is a non-disease-causing polymorphism. The absence of pathogenic mutations in the VSX1 gene in a large number of unrelated KTCN patients indicates that other genetic factors are involved in the development of this disorder.     

VSX1 mutational analysis in a series of Italian patients affected by keratoconus: detection of a novel mutation

PURPOSE: Keratoconus is a noninflammatory corneal disorder that is clinically and genetically heterogeneous. Mutations in the VSX1 (visual system homeobox 1) gene have been identified for two distinct, inherited corneal dystrophies: posterior polymorphous corneal dystrophy and keratoconus. To evaluate the possible role of the VSX1 gene in a series of Italian patients, 80 keratoconus-affected subjects were screened for mutations. METHODS: The diagnosis of keratoconus was made on the basis of clinical examination and corneal topography. The whole coding region and the exon-intron junctions of the VSX1 gene were analyzed by direct sequencing. RESULTS: Three already-described changes, D144E, G160D, and P247R, and a novel L17P mutation were found in 7 of 80 unrelated patients (8.7%). Two undescribed intronic polymorphisms are also reported. CONCLUSIONS: Mutational analysis of the VSX1 gene in a series of Italian patients revealed one novel mutation and confirmed an important role played by this gene in a significant proportion of patients affected by keratoconus, when it is inherited as an autosomal dominant trait with variable expressivity and incomplete penetrance.     

视觉同源基因多态性与散发性圆锥角膜易感性的关联研究

背景圆锥角膜是双侧、非炎性、角膜中央进行性变薄的疾病。研究圆锥角膜的相关致病基因对于明确该病的发病机制和建立基因早期诊断标准及治疗措施意义重大。目的探讨视觉同源基因（VSXl）多态性与散发性圆锥角膜患病风险的关系。方法采用病例一对照研究方法。收集散发性圆锥角膜患者101例和健康志愿者80人,受试者均为汉族,所有患者经裂隙灯（Vogt条纹、Fleischer环、Munson症等）和角膜地形图检查确诊。运用MassARRAY单核苷酸多态性（SNP）分型技术对VSXl基因SNP进行检测,采用统一的调查问卷收集每个研究对象详细的人口学资料和相关危险因素暴露资料,用x。检验和二元Logistic回归模型分析比较圆锥角膜组和正常对照组问各位点等位基因频率及基因型频率分布的差异,并分析其与圆锥角膜患病风险的关系。结果中国汉族人群中仅发现rs743018（C．843＋140C〉T）和rs6138482（R217HC〉T）两个位点具有多态性,其余位点未见多态性。与正常对照组比较,圆锥角膜组中2个SNP位点基因型频率与等位基因频率差异均无统计学意义（P〉0．05）。校正年龄、性别因素后,各SNP位点与圆锥角膜患病风险之间无显著相关性[隐性模型下：rs743018（C〉T）校正P=0．35,OR=0．72,95％CI：0．37～1．43;rs6138482（C〉T）校正P=0．48,OR=0．76,95％C1：0．35～1．64]。结论本研究发现中国汉族人群VSX1基因有2个SNP位点,但其多态性可能与圆锥角膜的患病无关。由于基因型和等位基因频率存在着种族差异性,VSX1基因在圆锥角膜发病机制中的作用仍存在争议,有待进一步研究证实。     

Six different mutations of TGFBI (betaig-h3, keratoepithelin) gene found in Japanese corneal dystrophies

PURPOSE: To investigate mutations of the human transforming growth factor beta-induced gene (TGFBI), transforming growth factor-beta-induced gene product (betaig-h3, keratoepithelin), in Japanese patients with Avellino corneal dystrophy (ACD), lattice corneal dystrophy (LCD), granular corneal dystrophy (GCD), and Reis-Bücklers corneal dystrophy (RBCD). METHODS: Genomic DNA was extracted from the peripheral blood of 75 patients and 7 unaffected relatives from 60 families with ACD, 34 patients and 8 unaffected relatives from 21 families with LCD, 4 patients and 4 unaffected relatives from 4 families with GCD, and 4 patients and an unaffected relative from 3 families with RBCD. Fifty normal volunteers served as controls. Exons 4, 11, and 12 of the TGFBI gene were amplified by polymerase chain reaction and were directly sequenced. RESULTS: Six different heterozygous missense mutations were detected in codons R124, L518, L527, and R555 of the TGFBI gene in the 117 patients from 88 families. A R124H mutation was detected in the patients with ACD. A R124C mutation was detected in the patients with LCD type 1 (LCD1), L518P was in atypical LCDI, and L527R in LCD with opacities deep in stroma. A R555W mutation was detected in the patients with GCD. A R555Q mutation was detected in the patients with RBCD. CONCLUSIONS: We conclude that codons R124 and R555 of the TGFBI gene are also hot spots in Japanese patients with ACD, LCD, GCD, and RBCD. Many Japanese patients with CD had ACD with R124H mutation. GCD with R555W mutation was rare.     

Candidate gene screening for posterior polymorphous dystrophy

PURPOSE: To perform candidate gene screening for posterior polymorphous corneal dystrophy (PPCD). The initial 3 genes chosen, ID1, BCL2L1, and VSX1, lie within the region on chromosome 20 to which the PPCD gene has been linked, and mutations in VSX1 have previously been identified in patients with PPCD. METHODS: DNA extraction, PCR amplification, and direct sequencing of the VSX1, BCL2L1, and ID1 genes were performed in 14 affected patients (12 families) as well as in unaffected family members and healthy control subjects. RESULTS: No coding region mutations in the BCL2L1 or ID1 genes were identified in affected patients. In the VSX1 gene, the previously identified Gly160Asp missense change was not present in any of our 12 probands, and the Asp144Glu mutation was identified in 1 affected patient as well as 1 unaffected control individual. Additionally, 2 synonymous substitutions were identified, Ala182Ala (8 affected patients from 8 families) and Gly239Gly (1 affected patient and 1 unaffected patient from the same family). In the ID1 gene, the synonymous substitution Gly216Gly was observed in 2 affected patients (2 families) who also demonstrated a single nucleotide change in both the 5'UTR (2129T>C) and 3'UTR (3267A>G). Another 5'UTR change, 2177T>C, was identified in 1 affected patient and his unaffected parent, both of whom also demonstrated the 2129T>C and 3267A>G changes. CONCLUSIONS: None of the 12 probands with PPCD demonstrated the previously described Gly160Asp mutation within the VSX1 gene. The Asp144Glu missense change, present in an affected patient as well as an unaffected control individual, appears to be a rare polymorphism, not a disease-causing mutation. No coding region changes were identified in the ID1 or BCL2L1 genes. Therefore, although we report a number of novel polymorphisms in the VSX1 and ID1 genes, the failure to identify any sequence variants that sort with the disease phenotype suggests that other genetic factors are involved in PPCD.     

评价VSX1突变与圆锥角膜合并角膜颗粒状营养不良在伊朗家族中的关联

目的:为了评估视觉系统同源框1(VSX1)基因的突变和圆锥角膜(KCN)以及颗粒状角膜营养不良(GCD)之间的关联.方法:对一个同时患有KCN和GCD的四代伊朗人家系进行了直接测序,鉴别出一个包含四代人同时患有GCD的伊朗KCN家系.从全血样品中提取基因组DNA.然后,为了研究KCN和GCD之间可能的连锁关系,通过PCR在每个样品中扩增VSX1基因的整个编码区和内含子-外显子边界.随后,对PCR产物进行直接测序,并在患者和对照组中进行突变分析.结果:VSX1基因突变分析未发现KCN和GCD疾病与VSX1基因相关的证据.我们的数据排除了VSX1作为该特定家系中KCN / GCD致病基因的可能性.结论:尽管患有GCD的KCN患者与VSX1基因变异无关联,但是仍需要对其它可能与KCN合并GCD发病机制相关的基因进行研究.     

Mutations in the CHST6 gene in patients with macular corneal dystrophy: immunohistochemical evidence of heterogeneity

PURPOSE: Macular corneal dystrophy (MCD) is an autosomal recessive disorder leading to severe visual impairment. The carbohydrate sulfotransferase 6 (CHST6) gene, which encodes the corneal N-acetylglucosamine 6-O-sulfotransferase on 16q22 has been identified as a causative gene for MCD. The purpose of this study was to identify mutations in CHST6 in Japanese patients with MCD and evaluate them by means of immunohistochemistry. METHODS: CHST6 was screened in 7 patients and 45 healthy control subjects. Genomic DNA was isolated, and the open reading frame (ORF) of CHST6 was amplified by polymerase chain reaction (PCR). PCR products were analyzed by direct sequencing and restriction enzyme digestion. Immunohistochemistry with a monoclonal anti-keratan sulfate (KS) antibody was performed on corneas of four patients from three families. RESULTS: Three novel mutations (P204Q, R205L, and R177H) and two previously reported mutations (R211W and A217T) were identified in the ORF of CHST6. P204Q, R205L, and R211W were found to be homozygous and R177H and A217T compound heterozygous with R211W on another allele. Immunohistochemistry revealed that R205L homozygous cornea had negative reactivity against the anti-KS antibody, representing type I MCD, and that R211W homozygous and R211W/A217T compound heterozygous corneas had negative or very weak reactivity in the stroma with antibody positive deposits, which were distinct from any previously reported types. CONCLUSIONS: Two mutations (homozygoous R211W and compound heterozygous R211W/A217T) should be subclassified immunohistochemically into new phenotypes of MCD. This heterogeneity could provide further insights into the pathogenesis of MCD.     

Keratoconus: epidemiology, risk factors and diagnosis

Keratoconus is a bilateral, non-inflammatory and progredient corneal ectasia with an incidence of approximately 1 per 2,000 in the general population. Within the second decade of life the cornea develops a conical shape, due to thinning of the corneal stroma with subsequent irregular astigmatism and myopia leading to marked impairment of vision. The most common presentation of the keratoconus is as a sporadic disorder, but it has long been recognized that a significant minority of patients exhibit a family history as an autosomal dominant mode of inheritance. Most investigators suggest complete penetrance of predisposing factors with variable phenotypic expression. In some patients heterozygous mutations in the VSX1 gene are described as the underlying gene defect. An association with Down syndrome, monosomia X (Turner syndrome), Leber's congenital amaurosis, mitral valve prolaps, collagenosis, retinitis pigmentosa and Marfan syndrome is described. The role of corneal cells in the pathogenesis of keratoconus is supported by the published reports of recurrence of keratoconus in eyes after penetrating keratoplasty due to graft repopulation by the recipient cells. Placido-based computeed videokeratographic corneal curvature mapping systems, linked with pachymetry, are useful for identifying overt and subclinical cases of keratoconus. Different indices may quantify the clinical features of keratoconus and may improve the classification. We compared videokeratometric data (Fourier series harmonic analysis and wavefront analysis) in eyes with keratoconus to answer the question of which parameters are useful for early diagnosis of keratoconus.     

FZD4基因与家族性渗出性玻璃体视网膜病变关系的研究

目的 研究家族性渗出性玻璃体视网膜病变（familial exudative vitreoretinopathy,FEVR）患者卷曲蛋白4 基因（Frizzled 4,FZD4） 突变类型和频率,探讨其基因型-表型之间的关系。设计 实验研究。 研究对象 2004年11月至2012年3月北京大学人民医院及北京同仁医院FEVR患者51例,其中家系病例39例,散发病例12例;同期北京大学人民医院体检100例正常对照个体。方法 应用聚合酶链反应（PCR）扩增全基因组DNA,用直接测序法检测FZD4所有外显子及邻近的非编码区序列。FZD4基因突变用生物信息法分析突变可能引起的蛋白结构和功能性改变。主要指标 基因序列。结果 在FZD4基因所有2个外显子及邻近的非编码区共检出12个致病性突变,其中9个为新发现的突变：1个缺失突变（P14fsX57）、1个无义突变（S491X）、7个错义突变（G22E、E180K、T237R、R253C、F328S、A339T、D470N）。3个已报道的致病突变：H69Y、M105V和W496X。在2个家系中同时发现携带2个不同的FZD4基因突变（分别为：H69Y和E180K;H69Y和W496X）,且这些患者临床表型严重于携带1个突变的患者。FZD4基因在FEVR患者中的突变率为29.4 % （15/51）;且均为常染色体显性遗传。结论 FZD4基因突变与中国人FEVR发病有关,其突变率约30%。突变率和其他人种相似,但突变谱具有明显的特异性。（眼科,2013,22：224-229）     

TGFBI gene mutation analysis in families with hereditary corneal dystrophies from Ukraine

In our study, 5 previously reported mutations of the TGFBI gene - R124C, R124H, R124L (exon 4), R555W, R555Q (exon 12) - were analyzed using polymerase chain reaction followed by restriction digestion in 48 individuals from 19 unrelated families with different forms of corneal dystrophy from different regions of Ukraine. The R555W mutation was detected in 6 patients from 4 families with granular corneal dystrophy. The R124C mutation was detected in 1 unaffected 10-year-old individual and in 24 patients from 8 families with lattice corneal dystrophy. As far as the R124C mutation detected in 1 patient with clinically diagnosed Reis-Bucklers corneal dystrophy is concerned, we concluded that this patient was misdiagnosed. The obtained results show that TGFBI gene mutation analysis is important as well for the early differential diagnosis of corneal dystrophies and genetic consulting in high-risk families.     

角膜营养不良与BIGH3基因突变研究

对角膜营养不良患者进行分子遗传学分析,探讨我国人角膜营养不良中BIGH3基因突变的类型。方法2000年7～12月收集15例颗粒状、Avellino、Reis-Bācklers角膜营养不良患者和5例无任何角膜病变的正常对照者外周血10ml,提取白细胞DNA,利用合成的BIGH3基因第4和第12外显子特异性引物,进行PCR扩增,并对扩增产物进行直接测序,分析相应基因序列。结果15例患者均检出BIGH3基因突变,其中R124H突变10例,包括纯合子2例,杂合子8例;R124L突变2例,均为杂合子;R555W突变3例,均为杂合子;正常对照者均未检出BIGH3基因突变。结论15例角膜营养不良患者的角膜病变均由BIGH3基因突变引起,与国外文献报道相同。突变基因对角膜病变严重程度的影响具有剂量效应,纯合子病情严重。R124L突变患者的临床表现重于R124H突变患者。     

Mutation analysis of the carbohydrate sulfotransferase gene in Vietnamese with macular corneal dystrophy

PURPOSE: Mutations in a new carbohydrate sulfotransferase gene (CHST6) encoding corneal N-acetylglucosamine-6-sulfotransferase (C-GlcNac-6-ST) have been identified as the cause of macular corneal dystrophy (MCD) in various ethnicities. This study was conducted to examine the CHST6 gene in Vietnamese with MCD. METHODS: Nineteen unrelated families, including 35 patients and 38 unaffected relatives were examined clinically. Blood samples were collected. Fifty normal Vietnamese individuals served as control subjects. Genomic DNA was extracted from leukocytes. Analysis of the CHST6 gene was performed with polymerase chain reaction and direct sequencing. Corneal buttons were studied histopathologically. RESULTS: A slit lamp examination revealed clinical features of MCD with gray-white opacities and stromal haze between. On histopathology, corneal sections showed positive staining with colloidal iron. Sequencing of the CHST6 gene revealed six homozygous and three compound heterozygous mutations. The homozygous mutations, including L59P, V66L, R211Q, W232X, Y268C, and 1067-1068ins(GGCCGTG) were detected, respectively, in two, one, eight, one, one, and two families. Compound heterozygous mutations R211Q/Q82X, S51L/Y268C, and Y268C/1067-1068ins(GGCCGTG) were identified, each in one family. A single heterozygous change at codon 76 (GTG-->ATG) was detected in family L, resulting in a valine-to-methionine substitution (V76M). None of these mutations was detected in the control group. CONCLUSIONS: Mutations identified in the CHST6 gene cosegregated with the disease phenotype in all but one family studied and thus caused MCD. Among these, the R211Q detected in 9 of 19 families may be the most common mutation in Vietnamese. These data also indicate that significant allelic heterogeneity exists for MCD.     

Tyrosinase gene (TYR) mutations in Chinese patients with oculocutaneous albinism type 1

Background: To identify the sequence variants of the tyrosinase (TYR) gene in Chinese families with oculocutaneous albinism. Methods: Three families with oculocutaneous albinism type 1 and 95 unrelated healthy Chinese individuals with normal pigmentation were screened for mutations in the TYR gene by direct sequencing. Computational algorithms were used to characterize the biological significance of the mutants. Results: Four previously reported mutations (R299C, R299H, W400L and frame‐shift c.930insC) and one novel mutation (F214del) were identified, and probands had homozygous or compound heterozygous TYR mutant alleles. None of the mutants were identified among the 95 normal control subjects. Computational analysis predicted that the R299C mutant inactivates the tyrosinase enzyme by misfolding of protein tertiary structure and/or retention of the misfolded tyrosinase within the endoplasmic reticulum, and F214del causes dysfunction of tyrosine enzyme by affecting the copper binding sites and altering substrate orientation and electronic transfers during catalytic reactions for melanosynthesis. Conclusion: We have identified five different TYR mutations, including one novel mutation, which caused oculocutaneous albinism type 1 in Chinese. Further analysis of the patients will be useful to determine the effects of these mutations on the tyrosinase activities.     

REP-1 gene mutations in Japanese patients with choroideremia

· Background: Choroideremia (CHM) is an X-linked progressive dystrophy of the choroid, retinal pigment epithelium, and retina. Recently, the REP-1 gene was isolated and the causative mutations in the gene were detected in patients with CHM. In a previous study, we described a Japanese family with CHM who had a mutation in the REP-1 gene. In the present study, we performed extensive analysis of the REP-1 gene in patients with CHM from several institutions in Japan. · Methods: Twenty-six patients with CHM and 5 unaffected females from 22 independently ascertained families were examined. Exons 1–15 of the REP-1 gene were screened by single-strand conformation polymorphism. The DNA fragments suspected of any variations were directly sequenced. · Results: Fifteen different mutations, including one previously reported mutation, were detected in 18 families. In addition, carrier status was proven in four unaffected females found to be heterozygous for the mutant allele. · Conclusions: Fifteen different mutations of the REP-1 gene were detected in 18 Japanese families. There were no hot spots for the mutations and no missense mutations. The results show that REP-1 gene defects cause CHM in Japanese patients, and the mutations in these Japanese patients differed from the mutations reported for CHM patients in Europe, Canada, and America except for R267X and 1313delTC. These findings suggest that the mutations occurred independently in the Japanese patients. Received: 13 August 1998 Revised version received: 16 November 1998 Accepted: 9 December 1998     

H244R VSX1 is associated with selective cone ON bipolar cell dysfunction and macular degeneration in a PPCD family

PURPOSE: To elucidate the retinal dysfunction and the molecular basis of posterior polymorphous corneal dystrophy (PPCD) associated with macular dystrophy, both inherited in a dominant manner through a three-generation family. METHODS: Ophthalmologic examinations including slit lamp examination, visual acuity tests, fundus visualization by scanning laser ophthalmoscopy, fluorescein angiography, color vision tests, electro-oculography, photopic and scotopic electroretinography (ERG) according to the International Society for Clinical Electrophysiology of Vision (ISCEV) protocols, and oscillatory potential (OP) recordings were conducted on affected family members. Corneal button from one affected patient was examined by transmission electron microscopy. All exons and intron-exon boundaries of the VSX1 and the COL8A2 genes were amplified by polymerase chain reaction and sequenced. RESULTS: The presence of endothelial cells that have epithelial-like features with multiple layers, desmosomal junctions, and microvillous projections supports the diagnosis of PPCD. Sequence analysis indicated that the H244R variant in the VSX1 segregated with corneal and macular disease phenotypes in this family. Electrophysiologic studies indicated normal scotopic ERG findings, decreased amplitude of the photopic b-wave, photopic OP2 and OP3 barely recordable with a preserved OP4 amplitude, and variably decreased 30-Hz flicker amplitude. CONCLUSIONS: The human VSX1 is required for cone ON bipolar cell function but not for rod and cone OFF bipolar cells, giving a unique example of such a selective heritable retinal defect in humans. Furthermore, the authors provide the first clinical support for a new alternative role of VSX1 in cone biology, probably similar to that proposed for its goldfish ortholog during retinal differentiation.     

Inheritance of a novel COL8A2 mutation defines a distinct early-onset subtype of fuchs corneal dystrophy

PURPOSE: To characterize the genetic basis and phenotype of inherited Fuchs corneal dystrophy (FCD). METHODS: DNA from blood was used for genome-wide linkage scans with tandem repeat polymorphisms. Mutation detection involved sequencing PCR-amplified exons. Families with FCD were clinically evaluated and graded on the Krachmer severity scale. Confocal specular microscopy visualized the morphology of endothelial guttae, small protrusions of Descemet's membrane that are characteristic of FCD. RESULTS: Linkage was obtained to 1p34.3-p32 for the autosomal dominant kindred originally reported by Magovern in 1979. All 21 cases with FCD and one with posterior polymorphous dystrophy were heterozygous for L450W, a novel point mutation in the COL8A2 gene. Of 62 independent cases of familial FCD, none had the previously reported mutations in COL8A2. Corneal guttae in COL8A2 patients were small, rounded, and associated with the endothelial cell center. This contrasts with common FCD, in which guttae were larger, sharply peaked, and initially positioned at edges of endothelial cells. The profile of age and disease severity for the L450W FCD kindred suggested that disease onset occurred in infancy, compared with an average age of onset of 50 years estimated for 201 familial FCD patients in 62 other families. CONCLUSIONS: A novel pathogenic L450W COL8A2 mutation was identified and its highly distinctive pathology characterized. This indicates that COL8A2 mutations give rise to a rare subtype of FCD. This study also provides the first direct evidence that COL8A2-FCD progresses from early to late stages in 25 years, a rate similar to that estimated for late-onset FCD.     

Compound heterozygous CNGA3 mutations (R436W, L633P) in a Japanese patient with congenital achromatopsia

Congenital achromatopsia is a stationary retinal disorder with autosomal recessive inheritance that is characterized by loss of color discrimination, low visual acuity, photophobia, and nystagmus. This disorder has been shown to be associated with CNGA3, CNGB3, and GNAT2 mutations, and the frequency of mutations in the CNGA3 gene (encoding alpha subunit of the cone-specific cGMP-gated cation channel) was 23-33% in European populations. The aim of this study was to test the hypothesis that CNGA3 mutations are also responsible for congenital achromatopsia in Japanese patients. DNA from venous blood samples from a total of 14 patients from 13 Japanese pedigrees was prepared. Mutation screening of the CNGA3 gene was performed using direct sequencing and PCR-single-strand conformation polymorphism analysis. Compound heterozygous missense mutations (p.R436W and p.L633P, the latter of which was novel) were identified in one patient only, a 22-year-old female. Neither of these two mutations was found in 150 Japanese control individuals. The patient's parents and sister carried one of these mutations each but were not affected. No mutations in the CNGB3 or GNAT2 genes were identified in the patient. Clinically, best-corrected visual acuity was 0.1 in both eyes. No specific findings were obtained in funduscopy. Optical coherence topography revealed a normal foveal thickness but a 20% decrease in parafoveal thickness. Ganzfeld full-field electroretinograms (ERGs) showed normal responses in rod and mixed rod-plus-cone ERGs but no response in cone or 30-Hz flicker ERGs. Spectral sensitivity on a white background revealed a curve with only one peak at around 500 nm, which fits the absorption spectrum of human rhodopsin. L633, conserved among vertebrate orthologs of human CNGA3, is a hydrophobic residue forming part of the carboxy-terminal leucine zipper (CLZ) domain, which is functionally important in the mediation of intracellular interactions. To our knowledge, this is the first report of a Japanese complete achromat with CNGA3 mutations, and of any patient with a missense mutation within the CLZ domain. The outcome suggests low frequency (7%, 1/14) of CNGA3 mutations in Japanese patients.     

Frizzled 4 gene (FZD4) mutations in patients with familial exudative vitreoretinopathy with variable expressivity

AIMS: To search for mutations in the frizzled 4 (FZD4) gene in patients with familial exudative vitreoretinopathy (FEVR) and to delineate the defective gene associated clinical features. METHODS: Direct sequencing following polymerase chain reaction of exons of FZD4 was performed for 24 probands with FEVR (18 familial and six sporadic), and some of their families. Clinical symptoms among individuals with mutations were assessed. RESULTS: Four novel mutations were identified in four patients with familial and one with sporadic FEVR. Three of these mutations were missense (M105V, R417Q, and G488D) and one was a nonsense change (W319X). M105V, R417Q, and G488D co-segregated with the disease. None of these sequence changes was found among 300 chromosomes from 150 healthy volunteers. The severity of vitreoretinopathy in the individuals involved in this study varied, but no patient with mutations in FZD4 exhibited rhegmatogenous retinal detachment although this pathology is thought to be the most common type of retinal detachment in FEVR. CONCLUSION: FZD4 gene mutations were found in some cases of autosomal dominant and sporadic FEVR. FZD4 mutations were responsible for FEVR with variable clinical manifestations.     

H626R and R124C mutations of the TGFBI (BIGH3) gene caused lattice corneal dystrophy in Vietnamese people

BACKGROUND/AIMS: Mutations of the human transforming growth factor beta induced gene (TGFBI) were reported to cause lattice corneal dystrophy (LCD) in various nationalities. This study analysed the TGFBI gene in Vietnamese people with LCD. METHODS: 13 unrelated families, including 34 patients and 21 unaffected members were examined. 50 normal Vietnamese people served as controls. Blood samples were collected. Genomic DNA was extracted from leucocytes. Analysis of TGFBI gene was performed using the polymerase chain reaction and direct sequencing. Corneal buttons were studied histopathologically. RESULTS: Two clinically distinguishable forms of LCD were revealed: one was typical of LCDI; the other was characterised by the late onset, thick lattice lines, and asymmetry between two eyes. Sequencing of the TGFBI gene revealed R124C mutation in three families and H626R mutation in 10 families. Congo red staining of the H626R-LCD cornea showed amyloid deposits in the subepithelial and stromal layers. CONCLUSIONS: R124C and H626R mutations of TGFBI gene caused LCD in Vietnamese people. R124C, a common cause of LCDI in many nationalities, was relatively rare, whereas H626R reported in several white people but not yet in Asians was most common (>75%) in Vietnamese people. Since the phenotype caused by H626R represents a new variant intermediate between LCDI and LCDIIIA, we proposed to consider it as LCD type IIIB.