In vivo histology and p.L132V mutation in KRT12 gene in Japanese patients with Meesmann corneal dystrophy.

PURPOSE: To report genetic mutational analysis and in vivo histology of Meesmann corneal dystrophy. STUDY DESIGN: Prospective, case control study. METHODS: Six patients from three independent families with clinically diagnosed Meesmann corneal dystrophy were enrolled in this study. Slit-lamp biomicroscopy with fluorescein vital staining, anterior segment optical coherence tomography (AS-OCT), and in vivo laser confocal microscopy (IVCM) were performed on selected patients. Mutational screening for the keratin genes KRT3 and KRT12 was performed in all six patients and selected unaffected family members. RESULTS: Slit-lamp biomicroscopy revealed numerous intraepithelial microcysts in all affected individuals. AS-OCT revealed hyperreflectivity and high corneal epithelial layer thickness (mean, 64.8µm) in all individuals tested (3/3). By using IVCM, multiple epithelial microcysts and hyperreflective materials (6/6), subepithelial nerve abnormalities (6/6), tiny punctate hyperreflective material (6/6), and needle-like hyperreflective materials (4/6) were observed in the corneal stromal layer. A heterozygous genetic mutation in the KRT12 gene (c.394 C>G, p.L132V) was identified in all six patients. No pathological mutation was observed in the KRT3 gene. CONCLUSION: We identified a heterozygous genetic mutation (c.394 C>G, p.L132V) in the KRT12 gene in six Japanese patients with inherited Meesmann corneal dystrophy. This is the first study to confirm this genetic mutation in Japanese Meesmann corneal dystrophy patients. This mutation has been independently reported in an American Meesmann corneal dystrophy patient, confirming its pathogenicity. AS-OCT and IVCM proved to be useful tools for observing corneal epithelial layer pathology in this dystrophy. Furthermore, IVCM reveals corneal stromal layer pathological changes not previously reported in this dystrophy.

A novel exon 17 deletion mutation of RPGRIP1 gene in two siblings with Leber congenital amaurosis.

PURPOSE: To investigate mutations of causal genes in two affected male siblings of a Japanese family with suspected Leber congenital amaurosis (LCA) and to characterize the related clinical features. METHODS: After obtaining informed consent, genomic DNA was extracted from peripheral blood of the proband and his family members. Mutation screening was initially performed with microarrays. The PCR and direct sequencing were successively done for confirmation of mutation detected by microarray, and the two patients who are the subjects of this study were also clinically examined. RESULTS: Results of the microarray suggested deletion of exon 17 of RPGRIP1. Confirmation by PCR and direct sequencing following microarray analysis revealed that both siblings had homozygous deletion of exon 17 of the RPGRIP1 gene, while their unaffected parents were heterozygous carriers. Length of the deletion was 1339 bp including exon 17 at the position of c.2710+372_2895+76del1339. Clinical features of the two siblings showed nystagmus, poor visual acuity, hyperopia, and photophobia since early childhood; but there was no oculo-digital sign, vessel attenuation or RPE mottling from the mid-retina to the periphery. Full-field single flash ERG was recordable but 30 Hz flicker ERG was not detectable. CONCLUSIONS: Although the present patients did not show sufficient clinical findings as LCA, PCR findings and direct sequencing following microarray analysis confirmed that they were LCA. Genetic analyses are helpful for confirmation of clinical diagnosis.

Familial cases of Norrie disease detected by copy number analysis.

PURPOSE: Norrie disease (ND, MIM#310600) is an X-linked disorder characterized by severe vitreoretinal dysplasia at birth. We report the results of causative NDP gene analysis in three male siblings with Norrie disease and describe the associated phenotypes. METHODS: Three brothers with suspected Norrie disease and their mother presented for clinical examination. After obtaining informed consent, DNA was extracted from the peripheral blood of the proband, one of his brothers and his unaffected mother. Exons 1-3 of the NDP gene were amplified by polymerase chain reaction (PCR), and direct sequencing was performed. Multiplex ligation-dependent probe amplification (MLPA) was also performed to search for copy number variants in the NDP gene. RESULTS: The clinical findings of the three brothers included no light perception, corneal opacity, shallow anterior chamber, leukocoria, total retinal detachment and mental retardation. Exon 2 of the NDP gene was not amplified in the proband and one brother, even when the PCR primers for exon 2 were changed, whereas the other two exons showed no mutations by direct sequencing. MLPA analysis showed deletion of exon 2 of the NDP gene in the proband and one brother, while there was only one copy of exon 2 in the mother. CONCLUSION: Norrie disease was diagnosed in three patients from a Japanese family by clinical examination and was confirmed by genetic analysis. To localize the defect, confirmation of copy number variation by the MLPA method was useful in the present study.

OPA1 mutations in Japanese patients suspected to have autosomal dominant optic atrophy.

PURPOSE: To report three types of heterozygous mutations in the OPA1 gene in five patients from three families with autosomal dominant optic atrophy (ADOA, MIM#165500). METHODS: DNA was extracted from the leukocytes of the peripheral blood. For mtDNA, mutations were examined at positions 11778, 3460 and 14484. For the OPA1 gene, the exons were amplified by PCR and mutations were detected by restriction enzymes or the dye terminator method. RESULTS: We detected three types of OPA1 mutation but no mtDNA mutations. In the OPA1 gene, heterozygous frameshift mutations from codon 903 due to a four-base pair deletion in exon 27 were detected in three patients from one family (c.2708_2711delTTAG, p.V903GfsX905). A heterozygous mutation due to a three-base pair deletion in exon 17, leading to a one-amino acid deletion (c.1618_1620delACT, p.T540del), and a heterozygous mutation due to a one-base substitution in exon 11, leading to a stop codon (c.1084G>T, p.E362X), were detected in sporadic cases. CONCLUSION: OPA1 mutations existed in three Japanese families with ADOA. After a detailed clinical assessment of the proband, the screening of the OPA1 gene may be helpful for precise diagnosis of ADOA, provided the relevant information of the family members is limited.

A novel nonsense mutation in rhodopsin gene in two Indonesian families with autosomal recessive retinitis pigmentosa.

PURPOSE: To report a novel, identical nonsense mutation in the rhodopsin (RHO) gene in two Indonesian families with autosomal recessive retinitis pigmentosa (arRP). METHODS: Mutation screening for the RHO gene was performed in 38 unrelated patients with retinitis pigmentosa (RP) by direct sequencing. Clinical features were also characterized, through complete ophthalmologic examination. Family members of RP patients testing positive for the RHO gene were subjected to genetic and clinical examination. To assess the founder effect in the two families, haplotype analysis also was performed. RESULTS: A novel homozygous nonsense mutation was detected in two patients by a G to A transition at nucleotide position 482 in exon 2 of the RHO gene, resulting in substitution of a tryptophan-to-stop at codon 161 (c.482G>A, p.W161X). Examination of family members of these 2 patients showed that the affected members were homozygous and unaffected carriers were heterozygous for the p.W161X mutation. Haplotype analysis revealed that members of the two families carried the same disease-associated variants in markers (IVS1 RHO and D3S2322). No p.W161X mutations were detected in 45 normal Indonesian subjects, nor were any mutations detected in exons 1-5 of the RHO gene in the remaining 36 RP patients. CONCLUSION: We detected a novel, recessive nonsense mutation (p.W161X) in the RHO gene of two families through mutation screening of RHO in 38 Indonesian RP patients. Haplotype analysis suggested that p.W161X was the founder mutation.

Distribution of goblet cells and MUC5AC mRNA in the canine nictitating membrane.

This study evaluated the distribution of goblet cells and the expression of MUC5AC mRNA in the canine nictitating membrane. The distribution of goblet cells in the nictitating membrane and temporal bulbar conjunctiva of beagle dogs was examined by histochemical analysis of impression cytology specimens and frozen sections. MUC5AC mRNA was detected by the reverse transcription polymerase chain reaction (RT-PCR). The distribution of MUC5AC mRNA was also examined by in situ hybridization using digoxigenin-labeled antisense and sense RNA probes. Histochemical analysis showed that the canine nictitating membrane epithelium contained many more periodic acid-Schiff positive goblet cells, particularly on the palpebral side, compared with the temporal bulbar conjunctiva. RT-PCR revealed that MUC5AC was expressed in both the nictitating membrane and in conjunctival tissue. When the distribution of MUC5AC mRNA was assessed by in situ hybridization, its expression was high on the palpebral side of the nictitating membrane and low in the temporal bulbar conjunctiva. MUC5AC mRNA expression corresponded with the distribution of goblet cells by histochemical examination. In conclusion, there were numerous goblet cells in the canine nictitating membrane epithelium, particularly on the palpebral side, and MUC5AC mRNA was expressed in the nictitating membrane epithelium at locations corresponding to the goblet cells.

Mutation analysis of CHST6 gene in Chinese patients with macular corneal dystrophy.

PURPOSE: To examine the carbohydrate sulfotransferase 6 (CHST6) gene in Chinese patients with macular corneal dystrophy (MCD). METHODS: Nineteen unrelated Chinese families with MCD, including 24 patients and 3 unaffected relatives, were examined. Genomic DNA was extracted from peripheral blood leukocytes. The coding region of the CHST6 gene was amplified by the polymerase chain reaction, and the DNA fragments were directly sequenced. Fifty unrelated normal Chinese volunteers served as the controls. RESULTS: Eighteen different mutations in the CHST6 gene (including 15 novel mutations) were identified, of which 12 were missense mutations, 5 were nonsense mutations, and 1 was a frameshift mutation. Six families had homozygous mutation, and 13 families had compound heterozygous mutation. None of these mutations were detected in the normal controls. CONCLUSIONS: CHST6 mutations may be responsible for the pathogenesis of MCD in Chinese patients. The Q298X mutation detected in 5 of 19 families (6 of 38 alleles, 15.8%) may be the founder mutation in Chinese patients. However, our findings also indicate a high level of allelic heterogeneity of the CHST6 gene in Chinese patients and in other ethnic groups.

A case of oculodentodigital dysplasia syndrome with novel GJA1 gene mutation.

PURPOSE: To report a case of oculodentodigital dysplasia syndrome (ODDD) with a heterozygous mutation in GJA1 (connexin 43) gene. METHODS: A 9-year-old girl visited our hospital complaining of visual disturbances. The patient had microphthalmia, a small nose with hypoplastic alae nasi, and syndactyly. Visual acuity with prescribed glasses improved to 0.5 (1.2) OU 2 months after the first visit. She was satisfied with the new glasses and the improvement in visual acuity. Genomic DNA was extracted from leukocytes of the patient's peripheral blood in accordance with standard procedures, after obtaining parental informed consent. We amplified GJA1 exon 2 from her genomic DNA by the PCR method, and sequenced the product using the dye terminator method. RESULTS: S5C (c. 13A > T), a novel mutation in exon 2 of GJA1, was found in the patient. The parents had no mutation of GJAI, nor was there any sign of abnormality in other family members. No similar mutation could be found in the 50 genotyped normal subjects in the control group. CONCLUSIONS: A novel GJA1 mutation was detected in a Japanese ODDD patient. Glaucoma complications associated with ODDD have already been reported. Careful long-term monitoring and treatment are also necessary.

In vivo laser confocal microscopy findings and mutational analysis for Schnyder's crystalline corneal dystrophy.

OBJECTIVE: To identify any mutation of the UbiA prenyltransferase domain-containing protein 1 (UBIAD1) gene in Japanese patients with Schnyder's crystalline corneal dystrophy (SCCD) and to investigate in vivo microstructural phenotype and genotype correlations using laser scanning confocal microscopy (Heidelberg Retina Tomograph 2 Rostock Cornea Module; Heidelberg Engineering GmbH, Dossenheim, Germany). DESIGN: Small, comparative case series. PARTICIPANTS: Three patients from 3 pedigrees (3 males) with clinically diagnosed SCCD and their relatives (2 males, 1 female) participated in this study. TESTING: All participants were examined genetically and by slit-lamp biomicroscopy and in vivo laser confocal microscopy. MAIN OUTCOME MEASURES: Genomic DNA from the patients and 100 unrelated healthy volunteers (200 chromosomes) was isolated from blood samples and used for mutation screening of the UBIAD1 gene. Selected confocal images of corneal layers were evaluated qualitatively for shape and degree of light reflection of deposits. RESULTS: Novel mutations in the UBIAD1 gene (Y174C, K181R, and N233H) were identified. Additionally, cosegregation of the mutation (Y174C) and SCCD was confirmed in 1 pedigree, indicating that the mutation of the UBIAD1 gene is causative for SCCD. The 3 mutations were absent in all 200 control chromosomes. In vivo laser confocal microscopy demonstrated subepithelial highly reflective crystals in 4 cases; the shapes of the crystals were needle-shaped (3 cases) or rectangular (1 case). A phenotype and genotype correlation was demonstrated in 1 pedigree, and phenotypic heterogeneity (SCCD with or without crystals caused by a same mutation of Y174C in the UBIAD1 gene) also was demonstrated in 1 pedigree. CONCLUSIONS: Nonsynonymous novel mutations in the UBIAD1 gene were detected in 3 unrelated Japanese pedigrees with SCCD, confirming the genetic heterogeneity of this disorder. In vivo laser confocal microscopy is capable of identifying characteristic corneal microstructural changes related to genetically mapped SCCD with high resolution, and phenotypic heterogeneity was presented. Further confocal and mutational analysis using a larger number of patients with SCCD is required to elucidate in vivo microstructural phenotype and genotype correlations. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

A novel mutation (967-970+2)delAAAGGT in the choroideremia gene found in a Japanese family and related clinical findings.

PURPOSE: To investigate the choroideremia (CHM) gene of one affected male and one obligate carrier in a Japanese family with choroideremia, and to characterize the related clinical features. METHODS: We examined one affected man and one carrier woman from a Japanese family. Genomic DNA was extracted from leukocytes of peripheral blood collected from the affected man and his daughter, who is an obligate carrier of choroideremia. Exons 1-15 of the CHM gene were amplified by polymerase chain reaction (PCR) and directly sequenced. We performed ophthalmic examinations including best-corrected visual acuity, slit-lamp examination, fundus examination, electroretinography, and Goldmann perimetry. RESULTS: A novel (967-970+2)delAAAGGT mutation was detected in the CHM gene. The affected man was hemizygous and had night-blindness, chorioretinal atrophy spreading from the posterior pole to the mid-periphery, and bareness of the sclera. His daughter was a heterozygous carrier who had chorioretinal atrophy and mottled appearance of the retinal pigment epithelium. CONCLUSION: A novel (967-970+2)delAAAGGT mutation existed in the CHM gene of a Japanese family with choroideremia.

A novel mutation in the cornea-specific keratin 12 gene in Meesmann corneal dystrophy.

PURPOSE: To report a novel mutation in the keratin 12 gene (KRT12) found in a Japanese family in association with Meesmann corneal dystrophy (MECD). METHODS: After informed consent was obtained, genomic DNA was extracted from the leukocytes of the peripheral blood of the proband, her affected father, normal mother, and 50 normal unrelated volunteers. Exons 1-8 of the KRT12 gene were amplified by polymerase chain reaction and directly sequenced. RESULTS: A novel heterozygous T to G transversion at the second nucleotide position of codon 433 (CTG>CGG), resulting in the replacement of leucine by arginine at codon 433 of the KRT12 gene (L433R), was detected in the proband and her affected father but not in her normal mother or the 50 controls. CONCLUSIONS: The novel L433R mutation of the KRT12 gene found in two members of this Japanese family caused MECD.

Expression and distribution of junctional adhesion molecule-1 in the human cornea.

PURPOSE: To investigate the expression and distribution of junctional adhesion molecule 1 (JAM-1) in human corneal tissue and cells. METHODS: Reverse transcriptase-polymerase chain reaction was used to detect the expression of JAM-1, ZO-1, and occludin mRNAs in corneal cells, while the presence of JAM-1 protein was analyzed by flow cytometry (FACS). Double immunofluorescence staining was used to determine the tissue distribution of JAM-1 and occludin in human corneas. RESULTS: Strong expression of JAM-1, ZO-1, and occludin mRNAs was observed in primary cultured corneal epithelial and endothelial cells, but not in primary cultured keratocytes. The expression of JAM-1 protein in cultured epithelial and endothelial cells was confirmed by FACS. When keratocytes were cultured in medium with 10% fetal calf serum for several passages, differentiation into corneal myofibroblasts occurred. The expression of JAM-1 was detected in these corneal myofibroblasts at both RNA and protein levels. JAM-1 immunoreactivity was seen at cell borders throughout the entire epithelium, but not in keratocytes from normal corneal tissue. On the other hand, JAM-1 immunoreactivity was detected in the cytoplasm of corneal endothelial cells. CONCLUSIONS: JAM-1 is expressed by human corneal epithelial and endothelial cells, but not by keratocytes, although its expression is induced in corneal myofibroblasts.

A novel variant lattice corneal dystrophy caused by association of mutation (V625D) in TGFBI gene.

PURPOSE: To characterize the molecular defect in the TGFBI gene in a Chinese family affected with an atypical lattice corneal dystrophy. DESIGN: Case report and experimental study. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

In vivo laser confocal microscopic findings of corneal stromal dystrophies.

OBJECTIVE: To investigate in vivo laser confocal microscopic findings of genetically mapped corneal stromal dystrophies and their relationship to histopathologic findings. METHODS: Seven patients with Avellino corneal dystrophy, 2 patients with lattice corneal dystrophy, and 2 patients with macular corneal dystrophy were examined genetically and using slitlamp biomicroscopy and in vivo laser confocal microscopy. Corneal specimens obtained after surgery in selected patients were histopathologically studied. RESULTS: In Avellino corneal dystrophy (Arg124His mutation of human transforming growth factor beta-induced gene [TGFBI]), highly reflective granular materials with irregular edges were observed in the superficial stroma. In lattice corneal dystrophy (Arg124Cys and Leu527Arg mutations of TGFBI), highly reflective branching filaments of variable width were observed in the stroma. In macular corneal dystrophy (Ala217Thr mutation of the carbohydrate sulfotransferase gene [CHST6]), homogeneous reflective materials with dark striaelike images were observed throughout the stroma. All confocal findings correlated well with histopathologic findings. CONCLUSIONS: In vivo laser confocal microscopy is capable of high-resolution visualization of characteristic corneal microstructural changes related to 3 types of genetically mapped corneal stromal dystrophies. The use of laser confocal microscopy may be valuable in the differential diagnosis of corneal stromal dystrophies, especially when diagnosis is otherwise uncertain.

[Ocular pulse amplitude in patients with open-angle glaucoma, normal-tension glaucoma, and ocular hypertensionby dynamic observing tonometry].

PURPOSE: The aim of this study was to compare the ocular pulse amplitude (OPA) in patients with different types of glaucoma, and also to evaluate the usefulness of OPA for the elucidation of normal-tension glaucoma (NTG). OPA is thought to reflect choroidal circulation. SUBJECTS: Sixty-six patients with normal-tension glaucoma (NTG), 52 patients with primary open angle glaucoma (POAG), 42 with ocular hypertension (OH) and 68 normal controls (NC) were enrolled in this study. METHODS: OPA was measured in all participants by dynamic observing tonometry(DOT). The correlation between OPA and the following parameters [IOP, refraction error (Ref), blood pressure, pulse pressure (PP), MD of Humphrey field analyzer 30-2, type of groups] was analyzed by linear and multiple regression analysis (MRA). Multiple logistic regression analysis (MLR) was used to estimate the adjusted odds ratio (OR) for evaluation of the association between OPA (including other factors) and the proportion of NTG. RESULTS: In MRA, IOP, Ref (< -3 D), PP and type of groups were significantly associated with OPA. The OPA in NTG was significantly lower than NC (p < 0.05). MLR demonstrated that OPA [OR 0.26 (95% CI, 0.12-0.57), p = 0.001] was associated with increased risk of having NTG. CONCLUSIONS: Lower OPA in patients with NTG suggests that there is insufficiency of ocular circulation in NTG. Evaluation of OPA may be useful for the elucidation of the pathogenesis of glaucoma.

[Clinical phenotype of a Japanese family with primary open-angle glaucoma caused by a Ala 363 Thr mutation in the MYOC gene].

BACKGROUND: Myocilin is a gene that causes primary open-angle glaucoma (POAG). We report a family whose members had an Ala 363 Thr mutation in the myocilin gene. We present the clinical phenotype of this family. CASE: The proband was a 57-year-old man diagnosed with POAG. His younger sister (50 years old) was also diagnosed with POAG. Visual field impairment did not worsen and ocular pressure decreased with eyedrop treatment. Although two of their children in their 30s had ocular hypertension, they did not have any sign of glaucomatous optic neuropathy. Genetic analysis revealed that all four family members had an Ala 363 Thr mutation in myocilin gene. CONCLUSION: Ala 363 Thr mutation was considered to be the cause of open-angle glaucoma. In this family, age at onset was comparatively high The two patients in their 30s had high intraocular pressure but no loss in visual acuity. The family members who had POAG and those who did not have POAG were not different from each other in the results of standard ocular examinations, only in age. Patients with this mutation will develop high intraocular pressure after 30 years of age and glaucomatous neuropathy after 50 years of age. When this gene mutation is detected in juvenile patients, careful follow-up and early therapy are necessary.

[Analysis of gene mutation in Chinese patients with Reis-Bücklers corneal dystrophy].

OBJECTIVE: To identify the mutation of the TGFBI gene in Chinese patients with Reis-Bücklers corneal dystrophy, and to study the relationship between the gene mutation and the clinical appearance. METHODS: Ten patients and 2 unaffected family members from 2 unrelated families with corneal dystrophy were studied. Molecular genetic analysis was performed on DNA extracted from peripheral leucocytes, and exons 4 and 12 of the TGFBI gene were amplified by polymerase chain reaction for direct sequencing. RESULTS: Both pedigrees showed an autosomal dominant inheritance. The clinical appearance of the cornea consisted of fine granular, subepithelial opacities which spread and become confluent with time, and resembled geographic type of Reis-Bücklers corneal dystrophy. Direct sequencing of all affected members revealed a G-to-T transition at codon 124 (CGC to CTC), producing R124L mutation of TGFBI gene. CONCLUSIONS: R124L mutation of the TGFBI gene is found in two Chinese families with Reis-Bücklers corneal dystrophy. The phenotype of Reis-Bücklers corneal dystrophy in both families belongs to the geographic type. Molecular genetic approach may be useful for the proper diagnosis of this type of corneal dystrophy.

Novel mutation (V505D) of the TGFBI gene found in a Chinese family with lattice corneal dystrophy, type I.

PURPOSE: To report a novel V505D mutation of the human transforming growth factor beta-induced (TGFBI) gene found in a Chinese family with lattice corneal dystrophy, type I (LCDI). METHODS: Genomic DNA was extracted from peripheral leukocytes from eight affected and four unaffected members of a Chinese family with LCDI. Exons of the TGFBI gene were amplified by polymerase chain reaction and directly sequenced. Fifty normal Chinese individuals were also analysed as controls. Histopathological examination of a corneal button was performed after keratoplasty of the proband. RESULTS: A heterozygous single-base-pair transversion (GTC to GAC, valine to aspartic acid) at codon 505 in exon 11 of the TGFBI gene (V505D) was detected in all of the affected members. No mutation was found in the unaffected members or in the 50 normal controls. The mutation cosegregated with the disease phenotype throughout three generations. Although a slit-lamp examination showed features of LCDI in most cases, the age at onset of the symptoms was several years later than that in cases of LCDI with an R124C mutation. By histopathological examination, numerous amyloid deposits were observed in the stroma, including beneath Bowman's membrane. CONCLUSION: A novel V505D mutation in the TGFBI gene causes LCDI in this Chinese family. It is the fourth reported mutation of the TGFBI gene associated with LCDI.