Publications by authors named "Pavlina Skalicka"

22 Publications

  • Page 1 of 1

Comprehensive phenotypic and functional analysis of dominant and recessive FOXE3 alleles in ocular developmental disorders.

Hum Mol Genet 2021 May 27. Epub 2021 May 27.

Department of Pediatrics and Children's Research Institute at the Medical College of Wisconsin and Children's Hospital of Wisconsin, Milwaukee, WI 53226, USA.

The forkhead transcription factor FOXE3 is critical for vertebrate eye development. Recessive and dominant variants cause human ocular disease but the full range of phenotypes and mechanisms of action for the two classes of variants are unknown. We identified FOXE3 variants in individuals with congenital eye malformations and carried out in vitro functional analysis on selected alleles. Sixteen new recessive and dominant families, including six novel variants, were identified. Analysis of new and previously reported genetic and clinical data demonstrated a broad phenotypic range with an overlap between recessive and dominant disease. Most families with recessive alleles, composed of truncating and forkhead-domain missense variants, had severe corneal opacity (90%; sclerocornea in 47%), aphakia (83%), and microphthalmia (80%), but some had milder features including isolated cataract. The phenotype was most variable for recessive missense variants, suggesting that the functional consequences may be highly dependent on the type of amino acid substitution and its position. When assessed, aniridia or iris hypoplasia were noted in 89% and optic nerve anomalies in 60% of recessive cases, indicating that these defects are also common and may be underrecognized. In dominant pedigrees, caused by extension variants, normal eye size (96%), cataracts (99%), and variable anterior segment anomalies were seen in most, but some individuals had microphthalmia, aphakia, or sclerocornea, more typical of recessive disease. Functional studies identified variable effects on the protein stability, DNA binding, nuclear localization and transcriptional activity for recessive FOXE3 variants, whereas dominant alleles showed severe impairment in all areas and dominant-negative characteristics.
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http://dx.doi.org/10.1093/hmg/ddab142DOI Listing
May 2021

Non-Penetrance for Ocular Phenotype in Two Individuals Carrying Heterozygous Loss-of-Function Alleles.

Genes (Basel) 2021 Apr 30;12(5). Epub 2021 Apr 30.

Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, 128 08 Prague, Czech Republic.

loss-of-function (LoF) alleles are known to cause a rare autosomal dominant disorder-posterior polymorphous corneal dystrophy type 3 (PPCD3). To date, 50 pathogenic LoF variants have been identified as disease-causing and familial studies have indicated that the PPCD3 phenotype is penetrant in approximately 95% of carriers. In this study, we interrogated in-house exomes ( = 3616) and genomes ( = 88) for the presence of putative heterozygous LoF variants in . Next, we performed detailed phenotyping in a father and his son who carried a novel LoF c.1279C>T; p.(Glu427*) variant in (NM_030751.6) absent from the gnomAD v.2.1.1 dataset. Ocular examination of the two subjects did not show any abnormalities characteristic of PPCD3. GnomAD ( = 141,456 subjects) was also interrogated for LoF variants, notably 8 distinct heterozygous changes presumed to lead to haploinsufficiency, not reported to be associated with PPCD3, have been identified. The NM_030751.6 transcript has a pLI score ≥ 0.99, indicating extreme intolerance to haploinsufficiency. In conclusion, LoF variants are present in a general population at an extremely low frequency. As PPCD3 can be asymptomatic, the true penetrance of LoF variants remains currently unknown but is likely to be lower than estimated by the familial led approaches adopted to date.
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http://dx.doi.org/10.3390/genes12050677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146820PMC
April 2021

The micronucleus cytome assay - A fast tool for DNA damage screening in human conjunctival epithelial cells.

Ocul Surf 2021 04 4;20:195-198. Epub 2021 Mar 4.

Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.

Purpose: To assess whether the micronucleus cytome assay (MCyt) reliably detects DNA damage occurring in control and pathological superficial epithelial cells from human conjunctiva.

Methods: Impression cytology samples from the bulbar conjunctiva of 33 healthy controls, eight patients with conjunctival intraepithelial neoplasia (CIN) and eight with mucous membrane pemphigoid (MMP) were examined using the MCyt modified for the ocular surface.

Results: The mean number of micronuclei (MNi) in control samples was 0.94 MNi/1000 epithelial cells, with no significant difference between conjunctival quadrants and independent of sex and age. The MCyt assay applied to CIN-affected eyes showed a significantly higher frequency of MNi (18.63/1000 cells), apoptotic cells, nuclear enlargement, multinucleated cells, and keratolysis compared with the corresponding unaffected paired eyes and with the control value. Although the mean MNi frequency in MMP eyes was also higher (1.73 MNi/1000 cells), it did not prove to be statistically different from the control samples. On the other hand, the MMP-affected eyes revealed significantly elevated percentages of cells with snake-like chromatin, multinucleated cells, apoptotic cells, and nuclear buds compared with controls.

Conclusions: Micronucleus cytome assay was adapted as a rapid screening test for genomic instability on the ocular surface. We have determined reference levels for MNi and other nuclear alterations on healthy conjunctiva and demonstrated that particularly frequencies of MNi are significantly elevated in conjunctiva affected by CIN. We demonstrate that MNi are more specific than other nuclear abnormalities and thus can be used for screening of ocular surface neoplasia.
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http://dx.doi.org/10.1016/j.jtos.2021.02.011DOI Listing
April 2021

A multi-ethnic genome-wide association study implicates collagen matrix integrity and cell differentiation pathways in keratoconus.

Commun Biol 2021 03 1;4(1):266. Epub 2021 Mar 1.

Institute for Translational Genomics and Population Sciences, The Lundquist Institute for Biomedical Innovation (formerly Los Angeles Biomedical Research Institute) at Harbor-UCLA Medical Center; Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA, USA.

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease.
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http://dx.doi.org/10.1038/s42003-021-01784-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921564PMC
March 2021

Pseudodominant Nanophthalmos in a Roma Family Caused by a Novel Variant.

J Ophthalmol 2020 10;2020:6807809. Epub 2020 May 10.

Research Unit for Rare Diseases, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, 128 08 Prague, Czech Republic.

Background: The aim of the study was to identify the molecular genetic cause of two different Mendelian traits with ocular involvement present in the members of a single consanguineous Czech Roma family.

Methods: We have performed ocular examination and review of medical records in two individuals diagnosed with nanophthalmos (proband and her father) and one individual followed for bilateral congenital cataract and microcornea (uncle of the proband). DNA of subjects with nanophthalmos was analysed by exome sequencing. Sanger sequencing was applied for targeted screening of potentially pathogenic variants and to follow segregation of identified variants within the family.

Results: A homozygous variant c.1509G>C; p.(Met503Ile), in was found in the two individuals affected with nanophthalmos. The change was absent from the gnomAD dataset, but two out of 118 control Roma individuals were also shown to be heterozygous carriers. Analysis of single nucleotide polymorphisms in linkage disequilibrium with the c.1509G>C in suggested a shared chromosomal segment. The nanophthalmos phenotype, characterized in detail in the younger individual, encompassed bilateral corneal steepening, retinal folds, buried optic head drusen, and restricted visual fields, but no signs of retinal dystrophy. A known pathogenic founder variant c.863+389C>T in a homozygous state was identified in the other family member confirming the suspected diagnosis of congenital cataracts, facial dysmorphism, and demyelinating neuropathy syndrome.

Conclusions: Herein, we report the first occurrence of nanophthalmos in the Roma population. We have identified pseudodominant inheritance for this phenotype caused by a novel variant in , representing a possible founder effect. Despite advances in genetic technologies such as exome sequencing, careful phenotype evaluation in patients from an isolated population, along with an awareness of population-specific founder effects, is necessary to ensure that accurate molecular diagnoses are made.
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http://dx.doi.org/10.1155/2020/6807809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212339PMC
May 2020

IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing.

Invest Ophthalmol Vis Sci 2019 07;60(8):3084-3090

Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.

Purpose: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing.

Methods: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts.

Results: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele.

Conclusions: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.
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http://dx.doi.org/10.1167/iovs.19-26930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6645617PMC
July 2019

Paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS): clinical findings in twelve patients including recurrence after keratoplasty.

Acta Ophthalmol 2019 Nov 2;97(7):e987-e992. Epub 2019 May 2.

Research Unit for Rare Diseases, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic.

Purpose: To describe the ocular findings of 12 subjects with paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS).

Methods: Ocular examination included corneal spectral domain optical coherence tomography. In three individuals with an initial diagnosis of a lattice or Thiel-Behnke corneal dystrophy, the TGFBI gene was screened by conventional Sanger sequencing.

Results: We confirmed a diagnosis of MGUS by systemic examination and serum protein electrophoresis in 12 individuals (9 males and 3 females), with a mean age at presentation of 52.2 years (range 24-63 years) and mean follow-up 6.4 years (range 0-17 years). The best-corrected visual acuity (BCVA) at presentation ranged from 1.25 to 0.32. In all individuals, the corneal opacities were bilateral. The appearances were diverse and included superficial reticular opacities and nummular lesions, diffuse posterior stromal opacity, stromal lattice lines, superficial and stromal crystalline deposits, superficial haze and a superficial ring of hypertrophic tissue. In one individual, with opacities first recorded at 24 years of age, we documented the progression of corneal disease over the subsequent 17 years. In another individual, despite systemic treatment for MGUS, recurrence of deposits was noted following bilateral penetrating keratoplasties. The three individuals initially diagnosed with inherited corneal dystrophy were negative for TGFBI mutations by direct sequencing.

Conclusion: A diagnosis of MGUS should be considered in patients with bilateral corneal opacities. The appearance can mimic corneal dystrophies or cystinosis. In our experience, systemic treatment of MGUS did not prevent recurrence of paraproteinemic keratopathy following keratoplasty.
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http://dx.doi.org/10.1111/aos.14123DOI Listing
November 2019

Brittle cornea syndrome: Disease-causing mutations in ZNF469 and two novel variants identified in a patient followed for 26 years.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2020 Jun 17;164(2):183-188. Epub 2019 Apr 17.

Research Unit for Rare Diseases, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.

Aims: Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder. The aim of this study was to review ZNF469 mutations associated with BCS type 1 to date and to describe an additional case of Czech/Polish background.

Methods: Whole genome sequencing was undertaken to identify the molecular genetic cause of disease in the proband. Sequence variants in ZNF469 previously reported as BCS type 1-causing were searched in the literature, manually curated and aligned to the reference sequence NM_001127464.2.

Results: The proband has been reviewed since childhood with progressive myopia and hearing loss. Aged 13 years had been diagnosed with Stickler syndrome. Aged 16.5 years, he developed acute hydrops in the left eye managed by corneal transplantation. At the age of 26, he experienced right corneal rupture after blunt trauma, also managed by grafting. He had a number of secondary complications and despite regular follow-up and timely management, the right eye became totally blind and the left eye had light perception at the last follow-up visit, aged 42. He was found to be a compound heterozygote for two novel mutations c.1705C>T; p.(Gln569*) and c.1402_1411del; p.(Pro468Alafs*31) in ZNF469. In total 22 disease-causing variants in ZNF469 have been identified, mainly in consanguineous families or endogamous populations. Only four probands, including the case described in the current study, harboured compound heterozygous mutations.

Conclusion: BCS occurs very rarely in outbred populations which may cause diagnostic errors due to poor awareness of the disease. Investigation into the underlying molecular genetic cause in patients with connective tissue disorders may lead to a re-evaluation of their clinical diagnosis.
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http://dx.doi.org/10.5507/bp.2019.017DOI Listing
June 2020

Coincidental Occurrence of Schnyder Corneal Dystrophy and Posterior Polymorphous Corneal Dystrophy Type 3.

Cornea 2019 Jun;38(6):758-760

Research Unit for Rare Diseases First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic First Faculty of Medicine, Charles University, Prague, Czech Republic.

Purpose: To report a simultaneous occurrence of 2 rare corneal dystrophies.

Methods: A 30-year-old man with a family history of posterior polymorphous corneal dystrophy type 3 (PPCD3) was invited for ophthalmic examination. Sanger sequencing of the coding regions and intron/exon boundaries of disease-associated genes, ZEB1 and UBIAD1, was performed.

Results: The clinical findings suggested co-occurrence of PPCD3 and Schnyder corneal dystrophy in the proband. This dual diagnosis was supported by genetic findings. He was identified to carry a previously reported heterozygous nonsense mutation in ZEB1: c.2157C>G; p.(Tyr719*), and a novel heterozygous missense mutation in UBIAD1: c.569T>C; p.(Ile190Thr). The mother of the proband only carried c.2157C>G in ZEB1, and slit-lamp examination of her corneas showed endothelial lesions characteristic of PPCD3. The sister of the proband carried c.569T>C in UBIAD1 and had corneal crystal deposition in her anterior stroma consistent with the diagnosis of Schnyder corneal dystrophy.

Conclusions: This case illustrates the coincidental occurrence of 2 rare and genetically distinct corneal dystrophies in a single patient. Furthermore, it highlights the need to perform comprehensive phenotyping in combination with appropriate genetic diagnostic testing to achieve an accurate diagnosis.
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http://dx.doi.org/10.1097/ICO.0000000000001930DOI Listing
June 2019

The utility of massively parallel sequencing for posterior polymorphous corneal dystrophy type 3 molecular diagnosis.

Exp Eye Res 2019 05 7;182:160-166. Epub 2019 Mar 7.

Research Unit for Rare Diseases, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, 128 08, Prague 2, Czech Republic; UCL Institute of Ophthalmology, 11-43 Bath Street, EC1V 9EL, London, United Kingdom; Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, U Nemocnice 2, 128 08, Prague, Czech Republic. Electronic address:

The aim of this study was to identify the molecular genetic cause of disease in posterior polymorphous corneal dystrophy (PPCD) probands of diverse origin and to assess the utility of massively parallel sequencing in the detection of ZEB1 mutations. We investigated a total of 12 families (five British, four Czech, one Slovak and two Swiss). Ten novel and two recurrent disease-causing mutations in ZEB1, were identified in probands by Sanger (n = 5), exome (n = 4) and genome (n = 3) sequencing. Sanger sequencing was used to confirm the mutations detected by massively parallel sequencing, and to perform segregation analysis. Genome sequencing revealed that one proband harboured a novel ∼0.34 Mb heterozygous de novo deletion spanning exons 1-7 and part of exon 8. Transcript analysis confirmed that the ZEB1 transcript is detectable in blood-derived RNA samples and that the disease-associated variant c.482-2A>G leads to aberrant pre-mRNA splicing. De novo mutations, which are a feature of PPCD3, were found in the current study with an incidence rate of at least 16.6%. In general, massively parallel sequencing is a time-efficient way to detect PPCD3-associated mutations and, importantly, genome sequencing enables the identification of full or partial heterozygous ZEB1 deletions that can evade detection by both Sanger and exome sequencing. These findings contribute to our understanding of PPCD3, for which currently, 49 pathogenic variants have been identified, all of which are predicted to be null alleles.
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http://dx.doi.org/10.1016/j.exer.2019.03.002DOI Listing
May 2019

Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants.

PLoS One 2019 11;14(2):e0211861. Epub 2019 Feb 11.

Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.

To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]-P2). Cells were characterized by alcian blue/periodic acid-Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0-P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211861PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370187PMC
November 2019

Schnyder corneal dystrophy and associated phenotypes caused by novel and recurrent mutations in the UBIAD1 gene.

BMC Ophthalmol 2018 Sep 17;18(1):250. Epub 2018 Sep 17.

Research Unit for Rare Diseases, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, 128 08, Prague 2, Czech Republic.

Background: The purpose of this study was to identify the genetic cause and describe the clinical phenotype of Schnyder corneal dystrophy (SCD) in six unrelated probands.

Methods: We identified two white Czech, two white British and two South Asian families with a clinical diagnosis of SCD. Ophthalmic assessment included spectral domain optical coherence tomography (SD-OCT) of one individual with advanced disease, and SD-OCT and confocal microscopy of a child with early stages of disease. UBIAD1 coding exons were amplified and Sanger sequenced in each proband. A fasting serum lipid profile was measured in three probands. Paternity testing was performed in one family.

Results: A novel heterozygous c.527G>A; p.(Gly176Glu) mutation in UBIAD1 was identified in one Czech proband. In the second Czech proband, aged 6 years when first examined, a previously described de novo heterozygous c.289G>A; p.(Ala97Thr) mutation was found. Two probands of South Asian descent carried a known c.305G>A; p.(Asn102Ser) mutation in the heterozygous state. Previously reported heterozygous c.361C>T; p.(Leu121Phe) and c.308C>T; p.(Thr103Ile) mutations were found in two white British families. Although crystalline deposits were present in all probands the affected area was small in some individuals. Corneal arcus and stromal haze were the most prominent phenotypical feature in two probands. In the Czech probands, SD-OCT confirmed accumulation of reflective material in the anterior stroma. Crystalline deposits were visualized by confocal microscopy. Mild dyslipidemia was found in all three individuals tested.

Conclusion: Although de novo occurrence of mutations in UBIAD1 is extremely rare, SCD should be considered in the differential diagnosis of bilateral corneal haze and/or crystal deposition, especially in children.
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http://dx.doi.org/10.1186/s12886-018-0918-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142341PMC
September 2018

Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions.

Exp Eye Res 2018 11 19;176:20-28. Epub 2018 Jun 19.

Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Albertov 4, 128 00 Prague 2, Czech Republic; Ophthalmology Department of 3rd Medical Faculty and University Hospital Kralovske Vinohrady, Šrobárova 1150/50, 100 34 Prague 10, Czech Republic.

Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.
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http://dx.doi.org/10.1016/j.exer.2018.06.019DOI Listing
November 2018

Antisense Therapy for a Common Corneal Dystrophy Ameliorates TCF4 Repeat Expansion-Mediated Toxicity.

Am J Hum Genet 2018 04 8;102(4):528-539. Epub 2018 Mar 8.

UCL Institute of Ophthalmology, London ECIV 9EL, UK. Electronic address:

Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.
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http://dx.doi.org/10.1016/j.ajhg.2018.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985359PMC
April 2018

Ectopic GRHL2 Expression Due to Non-coding Mutations Promotes Cell State Transition and Causes Posterior Polymorphous Corneal Dystrophy 4.

Am J Hum Genet 2018 03;102(3):447-459

UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK; Moorfields Eye Hospital, London EC1V 2PD, UK. Electronic address:

In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.
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http://dx.doi.org/10.1016/j.ajhg.2018.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985340PMC
March 2018

Familial Limbal Stem Cell Deficiency: Clinical, Cytological and Genetic Characterization.

Stem Cell Rev Rep 2018 Feb;14(1):148-151

Research Unit for Rare Diseases; Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, Praha 2, 128 08, Prague, Czech Republic.

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http://dx.doi.org/10.1007/s12015-017-9780-yDOI Listing
February 2018

Analysis of KERA in four families with cornea plana identifies two novel mutations.

Acta Ophthalmol 2018 Feb 5;96(1):e87-e91. Epub 2017 Jul 5.

Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Praha, Czech Republic.

Purpose: To identify the molecular genetic cause in four families of various ethnic backgrounds with cornea plana.

Methods: Detailed ophthalmological examination and direct sequencing of the KERA coding region in five patients of Czech and Turkish origin and their available family members.

Results: Compound heterozygosity for a novel missense mutation c.209C>T; p.(Pro70Leu) and a novel splice site mutation c.887-1G>A in KERA were detected in two affected siblings of Czech origin. In silico analysis supported the pathogenicity of both variants. The second proband of Czech origin harboured c.835C>T; p.(Arg279*) in a homozygous state. Homozygous mutations c.740A>G; p.(Asn247Ser) and c.674C>T; p.(Ile225Thr) were identified in the Turkish probands, both born out of consanguineous marriages. Observed ocular phenotypes were typical of cornea plana with the exception of one Czech patient who also had marked thinning and protrusion in the superior part of the left cornea (mean keratometry 47.2 D). No corneal endothelial cell pathology was found by specular microscopy in seven eyes, in three eyes visualization of the posterior corneal surface was unsuccessful.

Conclusion: KERA mutation c.740A>G has been identified to date in three different populations, which makes it the most frequently occurring mutation in patients with cornea plana. Marked corneal thinning and ectasia are a very rare finding in this disorder and longitudinal follow-up needs to be performed to determine its potential progressive nature.
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http://dx.doi.org/10.1111/aos.13484DOI Listing
February 2018

Active transforming growth factor-β2 in the aqueous humor of posterior polymorphous corneal dystrophy patients.

PLoS One 2017 17;12(4):e0175509. Epub 2017 Apr 17.

Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.

Purpose: Posterior polymorphous corneal dystrophy (PPCD) is characterized by abnormal proliferation of corneal endothelial cells. It was shown that TGF-β2 present in aqueous humor (AH) could help maintaining the corneal endothelium in a G1-phase-arrest state. We wanted to determine whether the levels of this protein are changed in AH of PPCD patients.

Methods: We determined the concentrations of active TGF-β2 in the AH of 29 PPCD patients (42 samples) and 40 cadaver controls (44 samples) by ELISA. For data analysis the PPCD patients were divided based on either the molecular genetic cause of their disease as PPCD1 (37 samples), PPCD3 (1 sample) and PPCDx (not linked to a known PPCD loci, 4 samples) or on the presence (17 samples) or absence (25 samples) of secondary glaucoma or on whether they had undergone penetrating keratoplasty (PK, 32 samples) or repeated PK (rePK, 7 samples).

Results: The level of active TGF-β2 in the AH of all PPCD patients (mean ± SD; 386.98 ± 114.88 pg/ml) in comparison to the control group (260.95 ± 112.43 pg/ml) was significantly higher (P = 0.0001). Compared to the control group, a significantly higher level of active TGF-β2 was found in the PPCD1 (P = 0.0005) and PPCDx (P = 0.0022) groups. Among patients the levels of active TGF-β2 were not significantly affected by gender, age, secondary glaucoma or by the progression of dystrophy when one or repeated PK were performed.

Conclusion: The levels of active TGF-β2 in the AH of PPCD patients are significantly higher than control values, and thus the increased levels of TGF-β2 could be a consequence of the PPCD phenotype and can be considered as another feature characterizing this disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175509PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393593PMC
April 2017

Novel TGFBI mutation p.(Leu558Arg) in a lattice corneal dystrophy patient.

Ophthalmic Genet 2016 12 30;37(4):473-474. Epub 2016 Mar 30.

a Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty of Medicine , Charles University in Prague and General University Hospital in Prague , Prague , Czech Republic.

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http://dx.doi.org/10.3109/13816810.2015.1126615DOI Listing
December 2016

Apoptosis of conjunctival epithelial cells before and after the application of autologous serum eye drops in severe dry eye disease.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2016 Jun 29;160(2):271-5. Epub 2016 Feb 29.

Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, General University Hospital and First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.

Aims: To assess the impact of autologous serum eye drops on the level of ocular surface apoptosis in patients with bilateral severe dry eye disease.

Methods: This prospective study was conducted on 10 patients with severe dry eye due to graft versus host disease (group 1) and 6 patients with severe dry eye due to primary Sjögren's syndrome (group 2). Impression cytology specimens from the bulbar conjunctiva were obtained before and after a three-month treatment with 20% autologous serum eye drops applied a maximum of 12 times a day together with regular therapy with artificial tears. The percentage of apoptotic epithelial cells was evaluated immunochemically using anti-active caspase 3 antibody.

Results: In group 1, the mean percentage of apoptotic cells was 3.6% before the treatment. The three-month treatment led to a significant decrease to a mean percentage of 1.8% (P = 0.028). The mean percentage of apoptotic conjunctival cells decreased from 5.4% before the treatment to 3.8% in group 2; however, these results did not reach the level of significance.

Conclusion: Three-month autologous serum treatment led to the improvement of ocular surface apoptosis, especially in the group of patients with severe dry eye due to graft versus host disease. This result supports the very positive effect of autologous serum on the ocular surface in patients suffering from severe dry eye.
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http://dx.doi.org/10.5507/bp.2016.001DOI Listing
June 2016

Severe retinal degeneration in women with a c.2543del mutation in ORF15 of the RPGR gene.

Mol Vis 2014 20;20:1307-17. Epub 2014 Sep 20.

Department of Ophthalmology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic ; Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders; First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic ; UCL Institute of Ophthalmology, London, United Kingdom.

Purpose: To describe the genotype-phenotype correlation and serial observations in a five-generation Czech family with X-linked retinitis pigmentosa (XLRP) associated with severe visual impairment in women.

Methods: Comprehensive ophthalmological examination including spectral domain optical coherence tomography (SD-OCT) was performed. Based on the pedigree structure and women being severely affected, autosomal dominant inheritance was suspected, and screening for known mutations by genotyping microarray was performed. Subsequently, direct sequencing of ORF15 RPGR was undertaken.

Results: Eighteen family members (nine women and nine men) were examined. A pathogenic variant, c.2543del in ORF15 of RPGR, was found to segregate with disease. The oldest woman and her two sisters had no perception of light in their sixth decade. Four women and five men had signs and symptoms of typical XLRP, including moderate to high myopia. Three other women also had moderate to high myopia and myopic astigmatism but without the presence of bone spicule-like formation. Severe disruption of macular architecture on SD-OCT was equally common in both sexes. Only one 32-year-old female carrier had clinically normal findings. Subfoveal choroidal thickness was decreased in all affected men and in all female carriers, except the only carrier with a normal fundus examination.

Conclusions: The c.2543del mutation in ORF15 of RPGR is associated with a severe phenotype in the women in this family. The presence of a significant myopic refractive error, in the absence of male-to-male transmission, may be indicative of X-linked inheritance. Measurements of choroidal thickness may help in clinically identifying carrier status.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169777PMC
June 2015
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