Noriyuki Azuma

Mutations of the PAX6 Gene Detected in Patients with a Variety of Optic-Nerve Malformations

The PAX6 gene is involved in ocular morphogenesis and is expressed in the developing central nervous system and numerous ocular tissues during development. PAX6 mutations have been detected in various ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, congenital cataracts, and foveal hypoplasia. However, it has not been identified in patients with optic-nerve malformations. Here, we identified novel mutations in eight pedigrees with optic-nerve malformations, including coloboma, morning glory disc anomaly, optic-nerve hypoplasia/aplasia, and persistent hyperplastic primary vitreous. A functional assay demonstrated that each mutation decreased the transcriptional activation potential of PAX6 through the paired DNA-binding domain. PAX6 and PAX2 are each thought to downregulate the expression of the other. Four of the detected mutations affected PAX6-mediated transcriptional repression of the PAX2 promoter in a reporter assay. Because PAX2 gene mutations were detected in papillorenal syndrome, alternation of PAX2 function by PAX6 mutations may affect phenotypic manifestations of optic-nerve malformations.

Missense mutation in the alternative splice region of the PAX6 gene in eye anomalies

The PAX6 gene is involved in ocular morphogenesis, and PAX6 mutations have been detected in various types of ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, congenital cataract, and foveal hypoplasia. The gene encodes a transcriptional regulator that recognizes target genes through its paired-type DNA-binding domain. The paired domain is composed of two distinct DNA-binding subdomains, the N-terminal subdomain (NTS) and the C-terminal subdomain (CTS), which bind respective consensus DNA sequences. The human PAX6 gene produces two alternative splice isoforms that have the distinct structure of the paired domain. The insertion, into the NTS, of 14 additional amino acids encoded by exon 5a abolishes the DNA-binding activity of the NTS and unmasks the DNA-binding ability of the CTS. Thus, exon 5a appears to function as a molecular switch that specifies target genes. We ascertained a novel missense mutation in four pedigrees with Peters anomaly, congenital cataract, Axenfeldt anomaly, and/or foveal hypoplasia, which, to our knowledge, is the first mutation identified in the splice-variant region. A T-->A transition at the 20th nucleotide position of exon 5a results in a Val-->Asp (GTC-->GAC) substitution at the 7th codon of the alternative splice region. Functional analyses demonstrated that the V54D mutation slightly increased NTS binding and decreased CTS transactivation activity to almost half.

PAX6 expression in the developing human eye

AIMS To investigate the changes in PAX6 expression in the developing human eye. METHODS Six developing human eyes from 6 to 22 weeks’ gestation were evaluated. Frozen sections were immunohistochemically stained with monoclonal antibody to chick Pax6 (amino acids 1–223). To verify antibody specificity, western blot analysis was carried out using cell lysates from P19 cells transfected with the human PAX6 gene. RESULTS Western blot analysis demonstrated that the antibody reacted to human PAX6 protein. Positive immunostainings for PAX6 were seen in the surface ectoderm, lens vesicle, inner and outer layers of the optic cup, and optic stalk at 6 weeks, and in the corneal epithelia and conjunctiva, lens, and non-pigmented ciliary epithelia from 8 to 22 weeks. In the retina, positive cells were seen in the entire retina from 8 to 10 weeks, and were restricted to the ganglion cell layer and the inner and outer portions of the inner nuclear layer after 21 weeks. CONCLUSIONS PAX6 is expressed on the surface and neuroectoderms at an early stage, then in the differentiating cells in the cornea, lens, ciliary body, and retina through development. PAX6 may play a role in determining cell fate in the morphogenesis of various human ocular tissue.

Generation of retinal ganglion cells with functional axons from human induced pluripotent stem cells.

We generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells. After development of the optic vesicle from the induced stem cell embryoid body in three-dimensional culture, conversion to two-dimensional culture, achieved by supplementation with BDNF, resulted in differentiation of RGCs at a rate of nearly 90% as indicated by a marginal subregion of an extruded clump of cells, suggesting the formation of an optic vesicle. Axons extended radially from the margin of the clump. Induced RGCs expressed specific markers, such as Brn3b and Math5, as assessed using by quantitative PCR and immunohistochemistry. The long, prominent axons contained neurofilaments and tau and exhibited anterograde axonal transport and sodium-dependent action potentials. The ability to generate RGCs with functional axons uniformly and at a high rate may contribute to both basic and clinical science, including embryology, neurology, pathognomy, and treatment of various optic nerve diseases that threaten vision.

Two novel mutations in the EYS gene are possible major causes of autosomal recessive retinitis pigmentosa in the Japanese population.

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease including autosomal recessive (ar), autosomal dominant (ad), and X-linked inheritance. Recently, arRP has been associated with mutations in EYS (Eyes shut homolog), which is a major causative gene for this disease. This study was conducted to determine the spectrum and frequency of EYS mutations in 100 Japanese arRP patients. To determine the prevalence of EYS mutations, all EYS exons were screened for mutations by polymerase chain reaction amplification, and sequence analysis was performed. We detected 67 sequence alterations in EYS, of which 21 were novel. Of these, 7 were very likely pathogenic mutations, 6 were possible pathogenic mutations, and 54 were predicted non-pathogenic sequence alterations. The minimum observed prevalence of distinct EYS mutations in our study was 18% (18/100, comprising 9 patients with 2 very likely pathogenic mutations and the remaining 9 with only one such mutation). Among these mutations, 2 novel truncating mutations, c.4957_4958insA (p.S1653KfsX2) and c.8868C>A (p.Y2956X), were identified in 16 patients and accounted for 57.1% (20/35 alleles) of the mutated alleles. Although these 2 truncating mutations were not detected in Japanese patients with adRP or Lebers congenital amaurosis, we detected them in Korean arRP patients. Similar to Japanese arRP results, the c.4957_4958insA mutation was more frequently detected than the c.8868C>A mutation. The 18% estimated prevalence of very likely pathogenic mutations in our study suggests a major involvement of EYS in the pathogenesis of arRP in the Japanese population. Mutation spectrum of EYS in 100 Japanese patients, including 13 distinct very likely and possible pathogenic mutations, was largely different from the previously reported spectrum in patients from non-Asian populations. Screening for c.4957_4958insA and c.8868C>A mutations in the EYS gene may therefore be very effective for the genetic testing and counseling of RP patients in Japan.

Detection of cytotoxic anti-LEDGF autoantibodies in atopic dermatitis

In the last two decades, atopic dermatitis (AD) has been of increasing clinical significance in Japan. Eight-20% of patients with AD developed progressive cataracts (cataract-AD) and lens epithelial cells (LECs) were severely damaged. Lens epithelium-derived growth factor (LEDGF) is a newly isolated survival factor. In the presence of LEDGF, LECs survive well and in the absence of LEDGF, they become highly susceptible to stress. We investigated (1) whether auto-antibody (auto-Ab) to LEDGF is present in sera of AD patients and (2) whether depletion of LEDGF by the auto-Ab kills LECs. In sera from 26 patients with AD using ELISA, we found significantly higher levels of auto-Ab to LEDGF than that in a normal control group. Affinity purified auto-Ab to LEDGF from these sera killed LECs without complement activation. Levels of histamine in the AD group were significantly higher and levels of prostaglandin E2 were significantly lower than in the normal group. However, statistically there are no differences between sera from AD and cataract-AD in levels of Ab to LEDGF, histamine, prostaglandin E2 (PGE2), immunoglobulin E (IgE) and eosinophiles. We speculate that cataract-AD may be induced, in part, by a combination of high levels of serum histamine and eye rubbing which could break the blood-aqueous barrier to allow the entry of Ab to LEDGF into the privileged compartment, thus, reducing LEDGF levels, resulting in damage to LECs, and cataract formation.

Ultrastructural and immunohistochemical findings in five patients with vitreomacular traction syndrome.

Objective: To evaluate pathologic features of vitreomacular traction syndrome (VMT). Methods: Preretinal membranes removed from five patients during vitreous surgery for VMT syndrome were evaluated by electron microscopy (n = 4) and immunohistochemistry (n = 1). Results: Electron microscopic examination revealed large segments of internal limiting lamina (ILL) in three of the four cases. Other extracellular features included two types of abnormal collagen fibrils, determined to be type I and fibrous long‐spacing collagen. The myofibrocyte was the predominant cell type in all cases. Collagen types I, II. and III, as well as glial fibrillary acidic protein and vimentin, were identified by immunohistochemistry. Conclusions: Epiretinal membranes in eyes with VMT syndrome adhered tightly to the ILL with abnormal collagen and contractile elements that included myofibrocytes.

Phenotype of cytochrome P4501B1 gene (CYP1B1) mutations in Japanese patients with primary congenital glaucoma

Aim: To investigate the phenotypes associated with cytochrome P4501B1 gene (CYP1B1) mutations in Japanese patients with primary congenital glaucoma (PCG). Methods: 66 Japanese patients with PCG were screened for sequence mutations in the CYP1B1 gene using single strand conformation polymorphism analysis followed by automated DNA sequencing. 11 cases had a CYP1B1 mutation in both alleles (the mutation group) and 21 cases did not have a CYP1B1 mutation (the “no mutation” group). The clinical features, such as age of onset, sex, intraocular pressure, and Descemet‘s membrane rupture, of the two groups were compared. Results: The clinical symptoms and signs did not differ for the two groups. The mean age at onset was 1.7 months in the mutation group and 3.1 months in the no mutation group, and the male:female ratio was 6:5 in the mutation group and 19:2 in the no mutation group. Both of these differences were statistically significant. Conclusions: In clinically diagnosed cases of PCG, a subgroup shows a CYP1B1 gene mutation. Age at onset was earlier in PCG patients with CYP1B1 mutations than in patients without mutations. Women were more prevalent among patients with mutations than those without mutations.

Vascular abnormalities in aggressive posterior retinopathy of prematurity detected by fluorescein angiography.

PURPOSE To evaluate fluorescein angiography (FA) in eyes with aggressive posterior retinopathy of prematurity (AP-ROP). DESIGN Retrospective, nonrandomized case series. PARTICIPANTS Three patients (6 eyes) with AP-ROP. METHODS Three patients (6 eyes) diagnosed with AP-ROP during ROP screening between July 2007 and July 2008 were included in this study. Fundus photographs and FA were obtained before and after laser and surgical treatment using a wide-field digital pediatric imaging system. MAIN OUTCOME MEASURES Fluorescein angiography and fundus photographs. RESULTS At the initial stage of AP-ROP, FA showed vascular abnormalities, including capillary nonperfusion throughout the vascularized retina, shunting in the vascularized retina, a circumferential demarcation line, and limited vessel development, which was difficult to identify only by ophthalmoscopy. After treatment, FA showed poorly developed retinal vessels, including 4 small major vessels without an arcade pattern, small macular vessels, an inhomogeneous capillary bed, and absence of a capillary-free zone in the fovea. CONCLUSIONS Capillary bed loss throughout the vascularized posterior retina is characteristic of AP-ROP and may exacerbate retinopathy.

p38 Mitogen-Activated Protein Kinase Controls a Switch Between Cardiomyocyte and Neuronal Commitment of Murine Embryonic Stem Cells by Activating Myocyte Enhancer Factor 2C-Dependent Bone Morphogenetic Protein 2 Transcription

Many studies have shown that it is possible to use culture conditions to direct the differentiation of murine embryonic stem (ES) cells into a variety of cell types, including cardiomyocytes and neurons. However, the molecular mechanisms that control lineage commitment decisions by ES cells remain poorly understood. In this study, we investigated the role of the 3 major mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) in ES cell lineage commitment and showed that the p38 MAPK-specific inhibitor SB203580 blocks the spontaneous differentiation of ES cells into cardiomyocytes and instead induces the differentiation of these ES cells into neurons. Robust p38 MAPK activity between embryoid body culture days 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of p38 MAPK activity at this time results in ES cell differentiation into neurons rather than cardiomyocytes. At the molecular level, inhibition of p38 MAPK activity suppresses the expression of bmp-2 mRNA, whereas treatment of ES cells with bone morphogenetic protein 2 (BMP-2) inhibits the neurogenesis induced by SB203580. Further, luciferase reporter assays and chromatin immunoprecipitation experiments showed that BMP-2 expression in ES cells is regulated directly by the transcription factor myocyte enhancer factor 2C, a well-known substrate of p38 MAPK. Our findings reveal the molecular mechanism by which p38 MAPK activity in ES cells drives their commitment to differentiate preferentially into cardiomyocytes, and the conditions under which these same cells might develop into neurons.