TGFBI Research Articles

Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR

BackgroundHypermethylation of the TGFBI promoter has been shown to correlate with decreased expression of this gene in human tumor cell lines. In this study, we optimized a methylation-specific polymerase chain reaction (MSP) method and investigated the methylation status of the TGFBI promoter in human lung and prostate cancer specimens.MethodsMethylation-specific primers were designed based on the methylation profiles of the TGFBI promoter in human tumor cell lines, and MSP conditions were optimized for accurate and efficient amplification. Genomic DNA was isolated from lung tumors and prostatectomy tissues of prostate cancer patients, bisulfite-converted, and analyzed by MSP.ResultsAmong 50 lung cancer samples, 44.0% (22/50) harbored methylated CpG sites in the TGFBI promoter. An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the TGFBI promoter was associated with a metastatic phenotype, with 42.9% (6/14) of metastatic lung cancer samples demonstrating dense methylation vs. only 5.6% (2/36) of primary lung cancer samples (p < 0.05). Similar to these lung cancer results, 82.0% (41/50) of prostate cancer samples harbored methylated CpG sites in the TGFBI promoter, and dense methylation of the promoter was present in 38.9% (7/18) of prostate cancer samples with the feature of locoregional invasiveness vs. only 19.4% (6/31) of prostate cancer samples without locoregional invasiveness (p < 0.05). Furthermore, promoter hypermethylation correlated with highly reduced expression of the TGFBI gene in human lung and prostate tumor cell lines.ConclusionWe successfully optimized a MSP method for the precise and efficient screening of TGFBI promoter methylation status. Dense methylation of the TGFBI promoter correlated with the extent of TGFBI gene silencing in tumor cell lines and was related to invasiveness of prostate tumors and metastatic status of lung cancer tumors. Thus, TGFBI promoter methylation can be used as a potential prognostic marker for invasiveness and metastasis in prostate and lung cancer patients, respectively.

TGFBI gene mutations in the Ukrainian patients with inherited corneal stromal dystrophies

Mutations Arg124Cys, Thr538Arg, Arg555Thr, Arg555Gln, Leu558Pro, and His626Arg in TGFBI gene were analyzed by polymerase chain reaction and restriction in 84 patients with various forms of corneal stromal dystrophies from 49 unrelated families and 29 clinically healthy relatives of these patients. A new mutation in TGFBI gene, Leu558Pro, was identified in the patients with atypical lattice dystrophy. The haplotypes of four microsatellite markers surrounding TGFBI gene region were analyzed in 22 families. The data on association of genotype and phenotype suggest that the analysis of TGFBI gene mutations is important for differential diagnostics of corneal dystrophies.

Spectrum of TGFBI gene mutations among Polish patients with corneal dystrophies

Abstract Purpose To report the clinical and molecular findings in Polish patients with stromal corneal dystrophies caused by TGFBI gene mutations. Methods Patients with clinically diagnosed granular (n=14; 9 unrelated families) and lattice (n=2; 2 unrelated families) corneal dystrophies participated in the study. Corneal phenotypes were assessed by slit lamp and confocal microscopy in vivo. Genomic DNA was obtained from blood samples and exons 4, 12‐14, known to contain mutation hot spots, were PCR amplified and sequenced on both strands. Results Molecular genetic testing revealed a heterozygous R555W (exon 12) mutation in eight (5 families) patients diagnosed with granular Groenouw type I corneal dystrophy. In one patient a heterozygous R124H mutation (Avellino corneal dystrophy, GCD type II) was found. In three families with “atypical granular” dystrophy affecting mainly the anterior stroma no mutation was detected in the analyzed regions. Heterozygous T538R (exon 12) and H626R (exon 14) mutations were identified, respectively, in two patients diagnosed with lattice corneal dystrophy. Conclusion Our results show that TGFBI gene mutations located in exons 4, 12 and 14 are frequently (8/11, 72%) found in Polish patients with corneal dystrophies of granular and lattice type. This indicates that a relatively straightforward molecular analysis can be a practical use in diagnosis of these conditions and associated genetic counseling.

Granular Corneal Dystrophy in 830-nm Spectral Optical Coherence Tomography

Spectral optical coherence tomography (SOCT) is a new imaging technique that can provide high-resolution tomograms much faster and with higher sensitivity than conventional Time domain (TdOCT) systems. Its usefulness in producing cross-sectional imaging of different corneal pathologies in vivo has already been presented. The aim of this case report is to show 830-nm SOCT findings in granular corneal dystrophy. A 48-year-old woman with granular corneal dystrophy was examined with a slit-lamp, confocal microscope (Confoscan 4) and a prototype SOCT instrument constructed at the Institute of Physics, Nicolaus Copernicus University, Torun, Poland. A genetic examination showed a mutation of arginine 555-to-tryptophan (Arg555Trp) in the TGFBI gene that confirmed the clinical diagnosis. SOCT tomograms showed multiple hyperreflective changes throughout the corneal stroma that corresponded to hyaline deposits. Precise and objective assessment of the localization, size, shape, and light scattering properties of the pathologic changes was possible. Three-dimensional rendering of the acquired data allowed a comprehensive evaluation of the deposits in the central cornea. SOCT (830 nm) provides clinically valuable 2- and 3-dimensional assessments of pathomorphologic changes in granular corneal dystrophy in vivo.

Phenotypic non-penetrance in granular corneal dystrophy type II

To report a possible case of phenotypic non-penetrance in granular corneal dystrophy type II (GCD-II). DNA analysis was performed on 11 patients with white granular corneal opacities and 50 normal controls after informed consent was obtained. The TGFBI gene was analyzed by sequencing DNA from epidermal keratinocytes obtained using adhesive tape. The heterozygous R124H mutation of TGFBI gene was found in all 11 patients. Although 49 normal controls had no mutation in the TGFBI gene, one normal control, a 26-year-old man, had the heterozygous R124H mutation of TGFBI gene. His 55-year-old father had the same mutation, but no corneal opacities. As not all mutations are expressed in the phenotype, GCD-II gene mutation may have non-penetrance. This report documents a possible case of phenotypic non-penetrance in GCD-II.

A Novel Variant of Combined Granular-Lattice Corneal Dystrophy Associated With the Met619Lys Mutation in the TGFBI Gene

To report a novel mutation in TGFBI (GenBank NM_000358), p.Met619Lys, associated with a variant of combined granular-lattice corneal dystrophy. Slitlamp examination and DNA collection from the proband and affected and unaffected relatives. All 17 exons of TGFBI were amplified and sequenced in the proband. Exon 14 was amplified and sequenced in the proband's family members and in 100 controls. Histopathologic examination of the excised corneal buttons from the proband and 3 family members was also performed. Affected individuals demonstrated an age-dependent phenotype, with the progression from central subepithelial needlelike deposits in younger individuals to polymorphic anterior stromal opacities in older family members. Screening of TGFBI in the proband demonstrated a novel mutation, p.Met619Lys, which was also present in all affected family members. Histopathologic examination revealed stromal deposits that stained with the Congo red and Masson trichrome stains as well as an antibody to the protein product of TGFBI. We present a unique corneal dystrophy phenotype associated with the novel p.Met619Lys mutation in TGFBI. Clinical Relevance The atypical and variable phenotype and the demonstration of both hyaline and amyloid stromal deposits indicate that neither clinical nor histopathologic features may be relied on to accurately diagnose and classify the corneal dystrophies.

Association study in the 5q31-32 linkage region for schizophrenia using pooled DNA genotyping

BackgroundSeveral linkage studies suggest that chromosome 5q31-32 might contain risk loci for schizophrenia (SZ). We wanted to identify susceptibility genes for schizophrenia within this region.MethodsWe saturated the interval between markers D5S666 and D5S436 with 90 polymorphic microsatellite markers and genotyped two sets of DNA pools consisting of 300 SZ patients of Bulgarian origin and their 600 parents. Positive associations were followed-up with SNP genotyping.ResultsNominally significant evidence for association (p < 0.05) was found for seven markers (D5S0023i, IL9, RH60252, 5Q3133_33, D5S2017, D5S1481, D5S0711i) which were then individually genotyped in the trios. The predicted associations were confirmed for two of the markers: D5S2017, localised in the SPRY4-FGF1 locus (p = 0.004) and IL9, localized within the IL9 gene (p = 0.014). Fine mapping was performed using single nucleotide polymorphisms (SNPs) around D5S2017 and IL9. In each region four SNPs were chosen and individually genotyped in our full sample of 615 SZ trios. Two SNPs showed significant evidence for association: rs7715300 (p = 0.001) and rs6897690 (p = 0.032). Rs7715300 is localised between the TGFBI and SMAD5 genes and rs6897690 is within the SPRY4 gene.ConclusionOur screening of 5q31-32 implicates three potential candidate genes for SZ: SMAD5, TGFBI and SPRY4.

Дослідження мутацій гена TGFBI у хворих на дистрофію строми рогівки, які проживають в Україні

Дослідження мутацій гена TGFBI у хворих на дистрофію строми рогівки, які проживають в Україні

Phenotype Associated with the H626P Mutation and Other Changes in the TGFBI Gene in Czech Families

Aims: To evaluate mutations in the transforming-growth-factor-β-induced (TGFBI) gene in patients of Czech origin with autosomal dominant corneal dystrophies. Methods: The coding sequence of the TGFBI gene was analysed in 22 affected Czech individuals from 7 apparently unrelated families. Comparison of phenotype to genotype was performed. Results: A H626P mutation, previously only described in a family with a variant of lattice corneal dystrophy (LCD), was detected in one family with superficial geographic corneal opacities. Light microscopy of 2 samples obtained following either a prior superficial keratectomy or keratoplasty showed amyloid but no fuchsinophilic deposits. In a family with LCD type I, an R124C mutation was identified. The R124L mutation was shown to be causative of Reis-Bücklers corneal dystrophy in 2 families. A family with Thiel-Behnke corneal dystrophy exhibited an R555Q mutation. In 2 families with granular corneal dystrophy type I, the typical R555W change was identified. Conclusion: The phenotype of the family with the H626P mutation differed from the phenotype previously reported for this change.

First molecular characterization and immunolocalization of keratoepithelin in adult human skeletal muscle

Keratoepithelin (KE) is an extracellular matrix protein that binds collagens, fibronectin, decorin, biglycan and integrins, interconnecting extracellular matrix components with resident cells in several tissues. KE has a molecular mass of 68 kDa and harbours four FAS1 domains named after those identified in the insect cell adhesion molecule fasciclin I. In humans, KE is preferentially expressed by the corneal epithelial layer and liberated towards the corneal stroma but it was also detected in the lung and in the bladder smooth muscle. No detailed information is available on the distribution of this protein in other human tissues. In this work, we have raised a polyclonal antibody against the recombinantly expressed human fourth FAS1 domain which is able to specifically detect KE in human skeletal muscle tissue extracts. Immunofluorescence experiments indicate that KE is localized around the perimysium and endomysium of each skeletal muscle fiber. The same kind of analysis shows that in muscle sections from patients affected by different forms of muscular dystrophy KE is upregulated and widely distributed in fibrotic tissues. The muscle specific expression of KE was also demonstrated by RT-PCR. In human skeletal muscle, KE may help to build up a bridge between collagen VI and yet unidentified muscle receptor(s), adding to the complexity of the adhesive molecular network established between muscle fibers and the surrounding basement membrane.

Progress in the study of molecular genetics of BIGH3 related corneal dystrophy

BIGH3相关性角膜营养不良是临床上一种较常见的致盲性遗传眼病,大多为单基因常染色体显性遗传.其防治的关键在于通过揭示更多BIGH3基因的突变位点及其相关发病机制,以提供准确的临床诊断和基因治疗.近年来的研究表明,位于BIGH3基因4、11、12、14号突变位点外显于上的突变类型可能通过改变其表达蛋白(KE蛋白)的结构、诱导凋亡、影响蛋白的黏附爬行功能等机制引起角膜异常蛋白沉积而致病.就目前国内外该病分子遗传学方面的研究做一简要综述。

Analysis of the H626R, A546T, and T538R mutations in the TGFBI gene in patients with corneal stroma lattice dystrophy from Ukraine

An analysis of the mutations H626R (exon 14) and A546T and T538R (exon 12) of the TGFBI gene using the polymerization chain reaction method with subsequent restriction, performed on 52 individuals from 22 unrelated families, together with a clinical diagnosis of different types of lattice corneal dystrophy is carried out. The H626R mutation was discovered in patients in 12 of the 17 families examined with a clinical diagnosis of lattice dystrophy with late manifestation and in six individuals in whom no clinical manifestations had yet appeared. Interestingly, the T538R and H626R mutations, which are associated with lattice dystrophy with late manifestation of the disease, were discovered in two patients with preliminary clinical diagnosis of lattice dystrophy (type I), a condition which is characterized by early manifestation of the disease. The A546T mutation was not found in any of our patients. Possible features of the mutant protein tgfbi and its involvement in the pathogenesis of the disease are also discussed. The results obtained indicate that the analysis of mutations in the TGFBI gene is of considerable importance for differential diagnosis of corneal dystrophy with predictive and therapeutic use as well as for genetic counseling in high-risk families.

A Novel Variant Lattice Corneal Dystrophy Caused by Association of Mutation (V625D) in TGFBI Gene

To characterize the molecular defect in the TGFBI gene in a Chinese family affected with an atypical lattice corneal dystrophy. Case report and experimental study. Molecular genetic analysis was performed on the DNA extracted from peripheral leucocytes from a Chinese family with atypical lattice corneal dystrophy. Fifty normal unrelated subjects of Chinese origin were used as controls. All exons of the TGFBI gene were amplified by polymerase chain reaction and directly sequenced. Bilateral, symmetrical, ridgy round pattern of opacities with uneven surfaces and thin lattice lines were noted in the proband. Analysis of exon 14 revealed a heterozygous T to A transition on codon 625. The mutation was not detected in the unaffected family member and 50 unaffected individuals. The novel TGFBI gene mutation (V625D) is associated with an early-onset variant of lattice corneal dystrophy. This case highlights the utility of molecular genetic analysis in differentiating corneal dystrophies associated with an atypical phenotype from nondystrophic conditions.

TGFBI gene mutation analysis in a Chinese family with Thiel-Behnke corneal dystrophy

To identify the gene mutation in autosomal dominant Thiel-Behnke corneal dystrophy affecting a five-generation Chinese family. To study the TGFBI gene mutation in Chinese patients with Thiel-Behnke corneal dystrophy by molecular genetic analysis. Ophthalmologic examinations were performed in 10 patients and two normal family members in an autosomal dominant Thiel-Behnke corneal dystrophy family. Five ml peripheral blood was collected and Genomic DNA was extracted using salt fractionation. The exons 4, 7, 8, 11 and 12 of the TGFBI gene were amplified by PCR and mutation analysis was performed by direct sequencing. Mutation analysis of the exons 4, 7, 8, 11 and 12 of the TGFBI gene identified a G-->A missense mutation in the exon 12 by bidirectional sequencing. This mutation resulted in a substitution of glutamine for arginine at amino acid 555 (R555Q) of the protein. This mutation existed in all of the patients, but not in unaffected individuals. Thiel-Behnke corneal dystrophy in this family is caused by R555Q mutation of the TGFBI gene, the phenotypes of this corneal dystrophy are closely correlated with TGFBI mutation.

In Vivo Laser Confocal Microscopy Findings for Bowman’s Layer Dystrophies (Thiel–Behnke and Reis–Bücklers Corneal Dystrophies)

To investigate microstructures in patients with genetically confirmed Bowman's layer dystrophies (Thiel-Behnke or Reis-Bücklers corneal dystrophy) using an in vivo laser scanning confocal microscope. Single-center, prospective, comparative small case series. Two patients from one pedigree (a 29-year-old woman and 58-year-old man) with Thiel-Behnke corneal dystrophy (Arg555Gln [R555Q] heterozygous missense mutation of human transforming growth factor beta-induced [TGFBI] gene) and 3 patients from one pedigree (a 70-year-old woman, 58-year-old man, and 14-year old man) with Reis-Bücklers corneal dystrophy (Arg124Leu [R124L] heterozygous missense mutation of the TGFBI gene) were examined. Two patients with Reis-Bücklers corneal dystrophy exhibited recurrence after corneal transplantation. All patients were examined by slit-lamp biomicroscopy. The center and the peripheral cornea of both eyes also were examined by in vivo laser scanning confocal microscopey. Image analysis was used to identify the corneal epithelial and stromal deposits correlated with each disorder. Selected images of the corneal layers were evaluated qualitatively for the shape and degree of light reflection of the deposits. In each dystrophy, distinct characteristic deposits were observed in the epithelium and Bowman's layer, respectively, by in vivo laser scanning confocal microscopy. In Thiel-Behnke corneal dystrophy, the deposits in the epithelial basal cell layer showed homogeneous reflectivity with round edges accompanying dark shadows. In contrast, deposits in Reis-Bücklers corneal dystrophy in the same cell layer showed extremely high reflectivity from small granular materials without any shadows in all cases. In each dystrophy, Bowman's layer was replaced totally with pathological materials; the reflectivity of those materials is much higher in Reis-Bücklers corneal dystrophy than in Thiel-Behnke corneal dystrophy. In vivo laser scanning confocal microscopy is capable of identifying in vivo corneal microstructural changes related to Thiel-Behnke and Reis-Bückler corneal dystrophy with a higher resolution than is available with slit-lamp biomicroscopy or in vivo white-light confocal microscopy. As a result, this device may enable differentiation of Thiel-Behnke and Reis-Bücklers corneal dystrophy in vivo. In vivo laser scanning confocal microscopy also may be a valuable tool for further research into the corneal dystrophies, especially to follow the natural course.

Identification of an amyloidogenic region on keratoepithelin via synthetic peptides

Mutations of keratoepithelin (KE) gene in human chromosome 5q31 have been linked with corneal epithelial or stromal dystrophies characterized by the abnormal deposits of amyloid fibrils and/or non-amyloid aggregations in corneal tissue. We report herein that synthetic peptide containing amino acid (a.a.) residues of 515–532 of native KE protein can readily form β-sheet-containing amyloid fibrils in vitro. Amyloid fibrils formed in various conditions from short synthetic peptides (containing a.a. 515–532 and 515–525, respectively) were characterized by thioflavin T (ThT) fluorescence assay, Congo red staining, electron microscopy (EM) and circular dichroism (CD). Triple-N-methylation of the synthetic peptides prevented the β-sheet polymerization and related amyloid fibril formation. Comparison study with ThT fluorescence further demonstrated that synthetic peptides containing corneal dystrophy-related mutations within this region formed amyloid fibrils to various extents. Our results suggest that each individual dystrophy-related mutation by itself does not necessarily potentiate amyloid fibril formation of KE. Roles of these intrinsically amyloidogenic foci in abnormal KE aggregations and amyloid deposits of stromal corneal dystrophies await further investigation.

Molecular genetic study on corneal dystrophy related to BIGH3 in China

目的 探讨中国BIGH3基因相关性角膜营养不良患者的基因突变类型.方法 从40例颗粒状、格子状、Reis-Bucklers角膜营养不良患者的静脉血中提取全血白细胞DNA,应用聚合酶链反应(PCR)技术,分别合成BIGH3基因第4、12、14外显子PCR产物,并对各产物进行DNA直接测序,同时行裂隙灯显微镜检查并照裂隙灯显微镜外眼像.45例正常人采集静脉血样进行对照检测.结果 40例患者均检出BIGH3基因突变,其中R124H突变为22例、R555W突变6例、R124C突变5例、H626R突变5例、R124L突变2例.结论 中国人与亚洲的日本和韩国人相似,在BIGH3基因相关的角膜营养不良患者中,Avellino角膜营养不良(ACD,R124H突变)最为常见。

Molekulargenetische und histopathologische Untersuchungen zur Genotyp-Phänotyp-Analyse bei Patienten mit TGFBI-gekoppelten Hornhautdystrophien

Different missense mutations in the TGFBI gene cause granular (Groenouw CDGG1, Avellino CDA, Reis-Bücklers CDB1) and lattice (Type I; Biber-Haab-Dimmer; CDL1) corneal dystrophies and, in some reports, corneal dystrophy Thiel-Behnke (CDB2). We report on the mutation spectrum and the genotype-phenotype correlations on the basis of clinical and histopathological examinations of 13 German families with TGFBI-linked corneal dystrophies. In 31 patients with different corneal dystrophies, DNA was extracted from leukocytes of the peripheral blood and mutation analysis was performed by direct sequencing of the TGFBI gene. Clinical and histopathological findings were compared with the molecular genetic findings for genotype-phenotype correlations. In 6 patients (2 families/one single person) with clinical and histopathological CDL1 we found a Missense mutation Arg124Cys and in 7 patients (3 families/one single person) with clinical and histopathological CDA we found a Missense mutation Arg124His in the exon 4 of the TGFBI gene. In 12 patients (4 families/2 single persons) with clinical and histopathological CDGG1 we found a Missense mutation Arg555Trypt in the codon 12 of the TGFBI gene. In all five patients (1 family/4 single persons) with clinical and histopathological CDB2 we could not find any mutation in the TGFBI gene. In one patient with exceptional clinical and histopathological findings we found a Missense mutation Ala546Asp, which was reported before only twice in connection with polymorphous corneal amyloidosis. In comparison of our clinical and histopathological findings and the molecular genetic results we found a strong genotype-phenotype correlation in patients with TGFBI-linked corneal dystrophies. Rare mutations can lead to exceptional clinical and histopathological findings which cannot be classified into the different groups of corneal dystrophies. In our patients with CDB2 we could not find any molecular genetic correlation to the TGFBI gene.

Distrofia corneal granular autosómica dominante causada por mutación del gen TGFBI en una familia mexicana

To describe the clinical data and the results of molecular analyses of the TGFBI gene in a patient with classic granular stromal corneal dystrophy (type I). A female patient aged 60-years complaining of a long-standing decrease of visual acuity bilaterally associated with photophobia and foreign body sensation, underwent a complete ophthalmologic examination. Molecular analyses of DNA from the patient and from an affected brother included PCR amplification of exons 4, 11, 12, and 14 of the TGFBI gene and direct automated sequencing of the PCR products. The affected patient showed a pattern of corneal stromal lesions that was compatible with a diagnosis of classic granular dystrophy. No involvement of other corneal layers was evident. Molecular analysis disclosed a point mutation in exon 14 of the TGFBI gene which consisted of an adenine to guanine change at nucleotide position 1924, predicting a substitution of arginine instead of histidine at residue 626 of the TGFBI protein (H626R). An identical mutation was detected in DNA from her affected brother. This is the first time that a case of stromal granular dystrophy has been demonstrated to be caused by the H626R mutation, a molecular defect classically detected in the phenotypically distinct lattice corneal dystrophy. Our data indicate that the same molecular defects in the TGFBI gene lead to different phenotypes in stromal dystrophies, thus expanding the genotypic-phenotypic spectrum in this group of corneal diseases.

No Pathogenic Mutations Identified in the TGFBI Gene in Polymorphic Corneal Amyloid Deposition

To determine whether primary, polymorphic, corneal amyloid deposition is associated with a mutation of the TGFBI gene. Interventional case series of 8 patients. Slit lamp examination of all patients and photodocumentation of 5 patients were performed. Genomic DNA was isolated from buccal mucosal swabs obtained from all patients and all 17 exons of the TGFBI gene were amplified and sequenced. Multiple polymorphic, refractile deposits were noted throughout the central corneal stroma in all patients. The deposits appeared gray-white on direct illumination and translucent on retroillumination, characteristic of amyloid. In 2 patients, linear, branching opacities, reminiscent of lattice corneal dystrophy, were identified. Histopathologic examination confirmed the presence of stromal amyloid in the cornea of 1 patient who required corneal transplantation for pseudophakic corneal edema. Screening of the entire coding region of the TGFBI gene revealed 4 previously described synonymous substitutions, Leu217Leu, Val327Val, Leu472Leu, and Phe540Phe. A previously unreported missense change, Asp299Asn, was identified in one affected patient but not in her affected sister. No pathogenic mutations, including the Ala546Asp missense mutation previously associated with polymorphic corneal amyloidosis, were identified in any of the patients. TGFBI gene mutations were not identified in a series of patients with polymorphic corneal amyloid deposition. As bilateral, discrete stromal amyloid deposits may be dystrophic or degenerative, differentiation between these phenotypically similar conditions is facilitated with the use of molecular genetic analysis.