Gergely Losonczy

Differentially Expressed Genes Associated with Human Limbal Epithelial Phenotypes: New Molecules That Potentially Facilitate Selection of Stem Cell-Enriched Populations

PURPOSE The aim of this study was to identify differentially expressed genes in the human limbal epithelium by microarray analysis. METHODS Total RNA isolates of human limbal and central corneal epithelia were used after transcription for hybridization on whole human genome expression microarrays. A set of differentially expressed genes detected by both microarrays was established. In the case of eight selected molecules, microarray results were confirmed by qRT-PCR, and protein expression in the cornea was examined by confocal immunofluorescence microscopy. Colocalization with the putative stem cell marker C/EBPδ was also examined. RESULTS The authors established a database of 126 limbal overexpressed genes. qRT-PCR confirmed microarray results in all examined cases (SPON1, IFITM1, ITM2A, PHLDA1, CXCR4, FZD7, DCT, DKK4). Limbal localization of the protein product of SPON1, IFITM1, ITM2A, CXCR4, and DKK4 was shown with confocal immunofluorescence microscopy. SPON1, IFITM1, and ITM2A signals mostly colocalized with C/EBPδ-positive putative resting limbal stem cells. CONCLUSIONS By detecting several new differentially expressed genes in the human corneal limbus, this study further expands current knowledge on the molecular signature of limbal epithelial stem cells. Plasma membrane localization of IFITM1 and ITM2A suggests their potential usefulness as targets to select stem cell-enriched populations from the limbal epithelium.

Analysis of complement factor H Y402H, LOC387715, HTRA1 polymorphisms and ApoE alleles with susceptibility to age‐related macular degeneration in Hungarian patients

Purpose:  Recent studies strongly support the role of genetic factors in the aetiology of age‐related macular degeneration (AMD). We investigated the frequency of Tyr402His polymorphism of the complement factor H (CFH) gene, Ser69Ala polymorphism at LOC387715, rs11200638 polymorphism of the HTRA1 gene and different apolipoprotein E (ApoE) alleles in Hungarian patients with AMD in order to determine the disease risk conferred by these factors.

Segregation analysis suggests that keratoconus is a complex non-mendelian disease.

Complex segregation analysis of 60 unrelated sporadic keratoconus (KC) families was performed to reveal the presumed mode of inheritance in our dataset.

ADAMTS9 locus associates with increased risk of wet AMD

Institute of Clinical MedicineNeurology, University of Eastern Finland,Kuopio, Finland; Department of Neurology, Kuopio University Hospital, Kuopio, Finland; Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland; Department of Ophthalmology, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary; Department of Ophthalmology, Semmelweis University, Budapest, Hungary; Department of Clinical Nutrition, Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, Finland; Department of Ophthalmology, Kuopio University Hospital, University of Eastern Finland, Kuopio, Finland

Effect of the Gas6 c.834+7G>A Polymorphism and the Interaction of Known Risk Factors on AMD Pathogenesis in Hungarian Patients

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Numerous genetic factors contribute to the development of the multifactorial disease. We performed a case-control study to assess the risk conferred by known and candidate genetic polymorphisms on the development of AMD. We searched for genetic interactions and for differences in dry and wet AMD etiology. We enrolled 213 patients with exudative, 67 patients with dry AMD and 106 age and ethnically matched controls. Altogether 12 polymorphisms in Apolipoprotein E, complement factor H, complement factor I, complement component 3, blood coagulation factor XIII, HTRA1, LOC387715, Gas6 and MerTK genes were tested. No association was found between either the exudative or the dry form and the polymorphisms in the Apolipoprotein E, complement factor I, FXIII and MerTK genes. Gas6 c.834+7G>A polymorphism was found to be significantly protective irrespective of other genotypes, reducing the odds of wet type AMD by a half (OR = 0.50, 95%CI: 0.26–0.97, p = 0.04). Multiple regression models revealed an interesting genetic interaction in the dry AMD subgroup. In the absence of C3 risk allele, mutant genotypes of both CFH and HTRA1 behaved as strongly significant risk factors (OR = 7.96, 95%CI: 2.39 = 26.50, p = 0.0007, and OR = 36.02, 95%CI: 3.30–393.02, p = 0.0033, respectively), but reduced to neutrality otherwise. The risk allele of C3 was observed to carry a significant risk in the simultaneous absence of homozygous CFH and HTRA1 polymorphisms only, in which case it was associated with a near-five-fold relative increase in the odds of dry type AMD (OR = 4.93, 95%CI: 1.98–12.25, p = 0.0006). Our results suggest a protective role of Gas6 c.834+7G>A polymorphism in exudative AMD development. In addition, novel genetic interactions were revealed between CFH, HTRA1 and C3 polymorphisms that might contribute to the pathogenesis of dry AMD.

A novel homozygous mutation (1619delC) in GPIIb gene associated with Glanzmann thrombasthenia, the decay of GPIIb-mRNA and the synthesis of a truncated GPIIb unable to form complex with GPIIIa

The absence of agonist-induced platelet aggregation and the lack of fibrinogen receptor (GPIIb/IIIa) on the platelet surface demonstrated that the severe hemorrhagic complications of a child of Romany descent were caused by Glanzmann thrombasthenia. DNA sequencing revealed a novel homozygous deletion of a cytosine (1619delC) in the GPIIb gene causing a frameshift and predicting a novel stop codon at position 533 following 24 altered amino acids. Both parents possessed the same deletion in heterozygous form. The amount of GPIIb mRNA in the patients platelets was 0.06% of the amount measured in control platelets. Neither GPIIb nor its truncated form could be detected in the platelets of the patient by Western blotting, while a small amount of GPIIIa was demonstrated. Quantitative flowcytometric analysis showed an elevated number of vitronectin receptors, a component of which is GPIIIa, on the patients platelets. The surface expression of vitronectin receptor on thrombasthenic, but not on normal platelets was further increased by activation with thrombin receptor agonist peptide. BHK cells transfected with wild type GPIIIa andmutated GPIIb failed to express any mature GPIIb or pro-GPIIb. Immunoprecipitation with a polyclonal antibody recognizing both GPIIb and GPIIIa recovered a 60 kDa truncated form of GPIIb. This band was absent when immunoprecipitation was carried out with an antibody recognizing GPIIIa, suggesting that the truncated protein, lacking calf-1, calf-2 domains and major part of the thigh domain, is unable to form complex with GPIIIa.

Myopia and Late-Onset Progressive Cone Dystrophy Associate to LVAVA/MVAVA Exon 3 Interchange Haplotypes of Opsin Genes on Chromosome X

Purpose Rare interchange haplotypes in exon 3 of the OPN1LW and OPN1MW opsin genes cause X-linked myopia, color vision defect, and cone dysfunction. The severity of the disease varies on a broad scale from nonsyndromic high myopia to blue cone monochromatism. Here, we describe a new genotype-phenotype correlation attributed to rare exon 3 interchange haplotypes simultaneously present in the long- and middle-wavelength sensitive opsin genes (L- and M-opsin genes). Methods A multigenerational family with X-linked high myopia and cone dystrophy was investigated. Results Affected male patients had infantile onset myopia with normal visual acuity and color vision until their forties. Visual acuity decreased thereafter, along with the development of severe protan and deutan color vision defects. A mild decrease in electroretinography response of cone photoreceptors was detected in childhood, which further deteriorated in middle-aged patients. Rods were also affected, however, to a lesser extent than cones. Clinical exome sequencing identified the LVAVA and MVAVA toxic haplotypes in the OPN1LW and OPN1MW opsin genes, respectively. Conclusion Here, we show that LVAVA haplotype of the OPN1LW gene and MVAVA haplotype of the OPN1MW gene cause apparently nonsyndromic high myopia in young patients but lead to progressive cone-rod dystrophy with deuteranopia and protanopia in middle-aged patients corresponding to a previously unknown disease course. To the best of our knowledge, this is the first report on the joint effect of these toxic haplotypes in the two opsin genes on chromosome X.

Cultivation of Human Oral Mucosal Explants on Contact Lenses

ABSTRACT Purpose/Aim: Autologous cultivated oral mucosal (OM) epithelial transplantation has been successfully used as corneal epithelial replacement in bilateral limbal stem cell deficiency. Recently, lotrafilcon A contact lens (CL) surface was described as a suitable carrier for cultured stem cells in corneal epithelial transplantation. Our aim was to establish explant cultures from human OM on CL carriers that are free of animal-derived materials and feeder cells. Materials and methods: Human cadaveric 2 mm OM explants were sutured onto CL surfaces and cultivated with fetal calf serum (FCS) or human serum (HS) supplemented culture medium without feeder cells. Confluent cultures were harvested and evaluated morphologically with hematoxylin and eosin stain and with immunofluorescence microscopy for the presence of p63, vimentin and cytokeratins (CK) 3, 4, 13 and 14. Results: Confluent cell sheets covering the whole CL surface were produced from OM explants after 2 weeks of culture with HS and after 3 weeks with FCS. A basal layer consisted of small, vimentin, p63 and CK14 positive putative stem/progenitor cells, which were present in the whole cell sheet. Large, CK3, CK4 and CK13 positive, differentiated cells appeared to spread above this confluent layer. Conclusions: We have established an animal-free culture system from human OM explants on CL surface. The cultured OM sheets contain large numbers of putative stem cells including limbal-like CK3 and CK14 positive cells. This method can be adapted to good manufacturing practice (GMP) conditions and has, therefore, great potential for clinical use.

Presence of Fleischer ring and prominent corneal nerves in keratoconus relatives and normal controls.

AIM To examine the occurrence of commonly known clinical signs of keratoconus (KC), i.e. Fleischer ring, prominent corneal nerves and thinning, among unaffected family members of KC patients and healthy control individuals. METHODS Data of both eyes of 117 relatives of KC patients having no manifest disease based on videokeratography indices (KC relatives), and 142 controls were used for Pearson correlation and t-test statistics. Correlation of Fleischer ring, prominent corneal nerves and central pachymetry data were tested with each other and with videokeratography indices (KSI, KISA, 3 and 6 mm Fourier asymmetry, and I-S). RESULTS A moderate correlation was found between Fleischer ring and all examined topographical indices. Most important correlation was present with 6 mm Fourier asymmetry, and corneal pachymetry (r=0.272, P<0.001; r=-0.234, P=0.027, respectively). Similar correlations were found with prominent corneal nerves (r=0.234, P<0.001 for 6 mm Fourier asymmetry and r=-0.235, P=0.0265 for pachymetry). KC family members who exhibited Fleischer ring or prominent nerves had thinner and more asymmetric corneas than those without Fleischer ring or prominent corneal nerves (P<0.05 for pachymetry and topographic indices with t-test and Mann-Whitney rank sum test). Though rarely, Fleischer ring and prominent corneal nerves occurred among normal controls, indicating the existence of forme fruste cases in the normal population. Control subjects, who had corneal Fleischer ring or prominent nerves had corneas more similar to KC than other controls (t-test: increased KSI and KISA, P=0.048 and 0.012, respectively). CONCLUSION In KC family members and healthy individuals, Fleischer ring and prominent corneal nerves are associated with features of KC and may suggest a possibility of forme fruste KC. Searching for the possible presence of Fleischer ring or prominent nerves on the cornea may help in the decision whether or not to diagnose subclinical KC in a borderline case.

Relative anterior microphthalmos in oculodentodigital dysplasia

Here, we report a patient with oculodentodigital dysplasia (ODDD) caused by the c. 413G>A, p.Gly138Asp mutation in the gap junction protein alpha-1 gene. The patient suffered from characteristic dysmorphic features of ODDD. Ophthalmological investigation disclosed microcornea and a shallow anterior chamber, as expected. Surprisingly, the patient had a normal axial length and moderate myopia on both eyes. To the best of our knowledge, this is the first report on ODDD associated with relative anterior microphthalmos and myopia.