Nguyen Thanh Ha

Leu518Pro mutation of the βig-h3 gene causes lattice corneal dystrophy type I

Abstract PURPOSE: To describe a Japanese family with lattice corneal dystrophy type I, which segregates with a novel mutation, Leu518Pro of the βig-h3 gene. METHODS: DNA was extracted from leukocytes in four members (three affected and one unaffected) of a Japanese family with lattice corneal dystrophy type I. Exon 12 of the βig-h3 gene was amplified and analyzed with a molecular biologic method. Clinical data were also collected. RESULTS: Three generations of this family have been positively diagnosed with lattice corneal dystrophy, indicating autosomal dominant inheritance. We found a heterozygous point mutation that segregates with the disease phenotype. It was a single base-pair transition (CTG to CCG, Leu to Pro). CONCLUSIONS: Although it is extremely rare compared with the Arg124Cys mutation of the βig-h3 gene, Leu518Pro mutation of the βig-h3 also causes lattice corneal dystrophy type I.

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 (&bgr;ig-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 LCD1, 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.

Microarray analysis identified differentially expressed genes in keratocytes from keratoconus patients

Purpose. To identify differentially expressed genes in human keratoconus keratocyte by cDNA microarray. Methods. Normal and keratoconic cornea were cultured for keratocytes. RNA was extracted. cDNA probe labeled with fluorescence dye was made from Poly A+ RNA, hybridized with microarray slide, containing 164 human apoptosis genes. Signal intensity was measured. Expression ratio between keratoconus and normal was determined using ImaGene Ver.3.0. Identified genes were further evaluated by RT-PCR and real-time PCR. Results. Five over-expressed and four under-expressed genes were identified. Of these, differential expression of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), human insulin-like growth factor binding protein 5 (IGFBP5), and IGFBP3 were verified and confirmed by RT-PCR. Real-time PCR showed TNFAIP6 increased by 3.3 folds, while IGFBP5, IGFBP3 decreased by 14 and 11 folds respectively. Conclusions. The identified genes could be important and deserve further investigation. Significant differential expression of TNFAIP6, IGFBP5, and IGFBP3 may indicate an important role of these genes in the mechanism underlying stromal thinning.

Q118X mutation of M1S1 gene caused gelatinous drop-like corneal dystrophy: the p501t of BIGH3 gene found in a family with gelatinous drop-like corneal dystrophy☆

PURPOSE To analyze BIGH3 and M1S1 genes in two Japanese brothers with gelatinous drop-like corneal dystrophy and five unaffected family members. METHODS DNA was extracted, and each part of the two genes was amplified and directly sequenced. RESULTS On the BIGH3 gene, a heterozygous P501T mutation was found in the elder brother and three unaffected family members. On the M1S1 gene, both brothers with gelatinous drop-like corneal dystrophy showed a homozygous Q118X mutation, whereas all unaffected members were heterozygous. CONCLUSIONS The Q118X mutation of M1S1 gene caused gelatinous drop-like corneal dystrophy. Although the P501T of the BIGH3 gene found in this pedigree was precisely the one reported for lattice corneal dystrophy IIIA, no clinical feature was shown, even in the 85-year-old father. This fact shows that the P501T mutation for LCDIIIA has low penetrance.

A novel mutation of the TGFBI gene found in a Vietnamese family with atypical granular corneal dystrophy

BACKGROUND Mutation of the human transforming growth factor beta-induced (TGFBI) gene causes granular corneal dystrophy (GCD) in various ethnic groups. In this report, we identify the genetic defect on the TGFBI gene in a Vietnamese family with atypical GCD . CASES The patient and her relatives were examined clinically. Genomic DNA was extracted from blood leukocytes. Fifty normal Vietnamese were used as controls. Analysis of the TGFBI gene was performed using polymerase chain reaction and direct sequencing. OBSERVATIONS The 42-year-old proband clinically showed multiple white dot-like opacities scattered in the anterior and mid-stroma of the central cornea. Unlike GCD, these deposits were smaller, localized deeper and less severe. DNA analysis revealed a nucleotide transversion at codon 123 (GAC --> CAC), causing Asp --> His substitution (D123H). This mutation was also detected in 3 out of 5 unaffected family members, but was absent in the 50 normal controls. CONCLUSIONS The novel D123H mutation of the TGFBI gene was not co-segregated with GCD in the family studied, and did not exist in the control population. It probably was a disease-causing mutation, thus expected to cause a novel variant of GCD in the proband. The detection of the D123H mutation in three unaffected family members indicates that it has low penetrance for GCD.

Identification of novel mutations of the CHST6 gene in Vietnamese families affected with macular corneal dystrophy in two generations.

Purpose. To report the clinical and genetic findings of Vietnamese families affected with macular corneal dystrophy (MCD) in 2 generations. Methods. Two families, including 7 patients and 3 unaffected members, were examined clinically. Blood samples were collected. Fifty normal Vietnamese individuals were used as controls. Genomic DNA was extracted from leukocytes. Analysis of the carbohydrate sulfotransferase (CHST6) gene was performed using polymerase chain reaction and direct sequencing. Results. The typical form of MCD was recognized in family B, in which sequencing of CHST6 gene revealed an nt 1067-1068ins(GGCCGTG) mutation (frameshift after 125V) homozygously in MCD patients and heterozygously in the unaffected members. Family N also showed clinical features of MCD, moderate in the mother but severe in the affected son. Sequencing revealed a single heterozygous Arg211Gln in the mother, compound heterozygous Arg211Gln+ Gln82Stop in the affected son, and heterozygous Arg211Gln mutation in the unaffected members. The identified mutations in these pedigrees were excluded from normal controls. Conclusions. The novel frameshift and compound heterozygous mutations might be responsible for MCD in the families studied. The phenotypic variation between affected parents and offspring was unclear. In family N, severe MCD phenotype seen in the affected son may be due the fact that he had an early stop codon mutation (Gln82Stop).

Mutation Analysis of the TGFBI Gene in Vietnamese with Granular and Avellino Corneal Dystrophy

PurposeMutations of the human transforming growth factor Β-induced gene (TGFBI) were reported to cause granular (GCD) and Avellino (ACD) corneal dystrophy in various nationalities. In this study we examined the TGFBI gene in a Vietnamese population with GCD and ACD.MethodsEight unrelated Vietnamese families, including 20 affected and 24 unaffected individuals, were examined; 50 normal Vietnamese individuals were used as controls. Genomic DNA was extracted from peripheral blood leukocytes. The TGFBI gene was analyzed using the polymerase chain reaction and direct sequencing. The corneal button was studied.ResultsSlit-lamp examination revealed typical features of GCD in most cases. A few features of ACD and a patient with an atypical form of GCD were also seen. Histopathological analysis of a GCD cornea showed deposits that stained bright red with Masson trichrome. Sequencing revealed three distinct mutations: R555W in six families, R124H in one family, and D123H in another.ConclusionsR555W and R124H mutations were co-segregated with the disease phenotype and thus caused GCD and ACD, respectively, in the families studied. The R555W detected in six of the eight families indicates that the GCD phenotype may be the most common in Vietnamese individuals, unlike in other Asians (Japanese and Korean), where ACD is most common (≫90%). The D123H mutation may cause an atypical variant of GCD. Jpn J Ophthalmol 2004;48:12–16

Expression of Estrogen Receptors α and β, Androgen Receptors and Progesterone Receptors in Human Cornea

PURPOSE Using immunohistochemical techniques and reverse transcription polymerase chain reaction (RT-PCR), we examined the localization of estrogen receptors alpha and beta (ER alpha, beta), androgen receptor (AR), and progesterone receptor (PR) in human corneas. MATERIALS AND METHODS Using formalin-fixed donor human cornea, we did immunohistochemical staining after making transverse sections, and examined the localization of receptors. Also, we extracted mRNA from primary culture cells of the corneal epithelium and stroma as well as the endothelial cell layer and epithelial layer of the cornea, and we performed RT-PCR and examined the expression of each receptor. RESULTS Immunohistochemical staining revealed that ER alpha was localized in corneal epithelial cells as well as in corneal stromal cells, and ER beta, AR and PR were localized in corneal epithelial, stromal, and endothelial cells. ER alpha, ER beta, and AR mRNA expression was observed in cultured and in vivo epithelium and cultured stroma cells. PR mRNA was expressed not only in cultured and in vivo epithelium and in cultured stroma cells but also in endothelium. CONCLUSIONS We detected the localization of estrogen receptors alpha and beta, androgen receptors, and progesterone receptors in the human cornea.