Fuchs Endothelial Corneal Dystrophy: Strong Association with

Research ArticleFuchs Endothelial Corneal DystrophyStrong Association with rs613872 Not Paralleled byChanges in Corneal Endothelial TCF4 mRNA Level

Monika OBdak12 Ewelina Ruszkowska123 Monika Udziela4 Dominika OziwbBo1

Ewelina BiNczyk4 Aneta UciehyNska2 RafaB PBoski5 and Jacek P Szaflik4

1Department of Genetics World Hearing Center Institute of Physiology and Pathology of Hearing Warsaw Poland2Department of Histology and Embryology Medical University of Warsaw Warsaw Poland3Postgraduate School of Molecular Medicine Medical University of Warsaw Warsaw Poland4Department of Ophthalmology Medical University of Warsaw Warsaw Poland5Department of Medical Genetics Medical University of Warsaw Warsaw Poland

Correspondence should be addressed to Monika Ołdak moldakifpsorgpl

Received 27 March 2015 Revised 5 June 2015 Accepted 8 June 2015

Academic Editor Alessandro Lambiase

Copyright copy 2015 Monika Ołdak et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fuchs endothelial corneal dystrophy (FECD) is a common corneal endotheliopathy with a complex and heterogeneous geneticbackground Different variants in the TCF4 gene have been strongly associated with the development of FECD TCF4 encodes theE2-2 transcription factor but the link between the strong susceptibility locus and disease mechanism remains elusive Here weconfirm a strong positive association between TCF4 single nucleotide polymorphism rs613872 and FECD in Polish patients (OR= 1295 95 CI 863ndash1942 1205942 = 1895 119901 lt 00001) We show that TCF4 expression at the mRNA level in corneal endothelium(119899 = 63) does not differ significantly between individuals with a particular TCF4 genotype It is also not altered in FECD patientsas compared to control samples The data suggest that changes in the transcript level containing constitutive TCF4 exon encodingthe amino-terminal part of the protein seem not to contribute to disease pathogenesis However considering the strong associationof TCF4 allelic variants with FECD genotyping of TCF4 risk alleles may be important in the clinical practice

1 Introduction

Fuchs endothelial corneal dystrophy (FECD) affects approx-imately 4 of the population over the age of 40 and is themost common genetic disorder of the corneal endotheliumThe disease usually has a late onset and presents clinicallyduring the fifth or sixth decade of life [1] It is characterizedby thickening of Descemetrsquos membrane and deposition ofextracellular matrix in the form of guttae [2] Patients withFECD have a reduced density of corneal endothelial cells Asthe cells regulate corneal hydration and maintain its trans-parency their lossmay eventually progress to corneal oedemaand vision loss [1] For the end-stage disease transplantsurgery represents the only definitive treatment FECD is aleading indication for corneal transplantation andDescemetrsquos

Stripping Automated Endothelial Keratoplasty (DSAEK) isconsidered a standard procedure for these patients [3]

Genetic basis for FECD is complex and heterogeneousEarly-onset form of endothelial dystrophy with some of thephenotypic features of FECD occurs rarely and displays anautosomal dominant mode of inheritance with mutations intheCOL8A2 gene (1p343MIMlowast120252)Themore commonlate-onset FECD begins usually after the age of 40 years andboth familial and sporadic FECD cases have been describedFamilial late-onset FECD is inherited in autosomal dominantfashion However the majority of late-onset FECD casesare sporadic with a negative family history It is muchmore common and severe in females than in males (3-4 1) [4] which contradicts a strictly autosomal dominanttransmission but follows multifactorial inheritance Higher

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 640234 6 pageshttpdxdoiorg1011552015640234

2 BioMed Research International

Table 1 Loci genes and genetic variants related to late-onset FECD

Chromosome Locusgenevariant Late-onset FECD1p351 rs760594 F5q123 rs1301475 F5q331ndashq352 FCD3 F7q223 rs257376 F8p213 rs2466216 F8p211 rs9797 F8q2113 rs1380229 F9p221ndashp241 FCD4 F10p1122 ZEB1 FS10q242 rs1889974 F13pter-q1213 FCD1 F15q222 rs235512 F15q2231 rs352476 F15q253 AGBL1 FS17q253 rs938350 F18q211 LOXHD1 FS18q212 TCF4 FS18q212ndashq2132 FCD2 F20p13 SLC4A11 FS20p122 rs674630 FXq28 rs1990383 FF familial S sporadic

risk of cornea guttata was independently associated witholder age female sex and a thinner central corneal thickness[5] as well as genetic variants [6]

To date four different genes ZEB1 (10p1122MIMlowast189909) AGBL1 (15q253 MIMlowast615496) LOXHD1(18q211 MIMlowast613072) and SLC4A11 (20p13 MIMlowast610206)as well as four causal loci on chromosomes 5q331ndashq352(FCD3) 9p221ndashp241 (FCD4) 13pter-q1213 (FCD1) and18q212ndashq2132 (FCD2) together with a number of suscep-tibility loci have been implicated in the pathogenesis ofFECD (summarized in Table 1) [7 8] A strong associ-ation has been established between TCF4 gene (18q212MIMlowast602272) variants and FECD A genome-wide associ-ation study has identified a common biallelic deep intronicTCF4 single nucleotide polymorphism (SNP rs613872NG 0117161g50559CgtA) as a highly significant risk factorfor FECD [6] In addition to that a separate study reporteda trinucleotide expansion (CTG181) in the intron regionof TCF4 which is highly prevalent in FECD individuals[9] The role of these associations in FECD developmentremains poorly understood In the study we set out to analyzewhether the presence of TCF4 rs613872 risk allele affects theexpression of TCF4 in corneal endothelial cells which wouldprovide a functional link between a risk factor and diseasemechanism in FECD

2 Patients and Methods

The study conformed to the tenets of the Declaration ofHelsinki and was approved by the local Ethics Committee

Patients were recruited from the Department of Ophthal-mology Medical University of Warsaw and gave informedconsent prior to participation The diagnosis of FECD wasbased on the visualization of ldquoguttaerdquo and stromal oedema byslit-lamp examination confocal microscopy in vivo (IVCMConfoscan 4 Nidek Technologies) and anterior segmentoptical coherence tomography (CASIACorneaAnterior Seg-ment OCT SS-1000 Tomey) (Figure 1)

21 Genotyping Genomic DNAwas isolated from peripheralblood samples of sporadic unrelated FECD patients (119899 =252 187 females and 65 males) with a standard salting-outprocedure Control DNA samples (119899 = 323) representative ofthe background population of central Poland came from therepository of the Department of Medical Genetics [10] TheTCF4 rs613872 was genotyped using ABI Custom TaqManSNP Genotyping Assay (Applied Biosystems) and the real-time PCR system (Viia7 Thermo Fisher Scientific) Theaccuracy of genotyping was confirmed by Sanger sequencingin selected subjects

22 RNA Isolation and Quantitative Real-Time PCR Cornealendothelial cell monolayers attached to Descemetrsquos mem-brane were obtained from FECD patients (119899 = 40) duringendothelial keratoplasty (DSAEK) or excised from donorcorneoscleral buttons (119899 = 23) that were not used fortransplantation The FECD patients and controls includedin the RNA studies derived from the group genotyped atrs613872 (FECD group TT 119899 = 3 TG 119899 = 31 GG 119899 = 6control group TT 119899 = 14 TG 119899 = 6 GG 119899 = 3) Thespecimens were submerged in the RNAlater storage solution(Ambion Austin TX USA) and frozen immediately TotalRNA was extracted using Trizol (Thermo Fisher ScientificWaltham MA USA) and cDNA was generated from 500 ngof RNA with the Maxima First Strand cDNA Synthesis Kit(Thermo Fisher Scientific) Gene expression was measuredby real-time PCR (Viia7 Thermo Fisher Scientific) usingPCR primers (Oligo Warsaw Poland) and probes (RocheUniversal Probe Library) designed with the ProbeFindersoftware version 250 (Roche) The analyzed coding regionof TCF4 is present in both TCF4-A and TCF4-B isoformsIt is located in the amino-terminal part of the protein closeto the activation domain 2 (AD2) For TCF4 quantitativePCR (qPCR) the primer pair 51015840-GCACTTTCCCTAGCT-CCTTCT-31015840 and 51015840-GCATAGCCAGGCTGATTCAT-31015840 andprobe 25 for RPL13A (60S ribosomal protein L13a) qPCRthe primer pair 51015840-CTGGTGCTTGATGGTCGAG-31015840 and51015840-GTTGATGCCTTCACAGCGTA-31015840 and probe 77 wereused Expression values for TCF4 were calculated usingthe modified double delta Ct (ΔΔCt) method and absolutequantification and normalized to RPL13A [11]

23 Statistical Analysis Hardy-Weinberg equilibrium(HWE) was analyzed with the 1205942 test in the patient andcontrol groups The odds ratios (ORs) with 95 confidenceintervals (95 CI) and 119901 values were calculated with theWeb-Assotest program (httpwwwekstroemcomassotestassotesthtml) Differences in gene expression between

BioMed Research International 3

(a)

(b) (c)

Figure 1 Characteristic features of FECD (a) Slit-lamp photography shows the presence of pathological guttae focal excrescences ofDescemetrsquos membrane at the level of corneal endothelium in a patient with FECD (b) Confocal microscopy image of the corneal endotheliumin a control subject demonstrates a regular mosaic of the endothelial monolayer with bright cell bodies and dark hexagonal cell boundaries(c) Confocal microscopy in a patient with FECD reveals pleomorphism and polymegathism of the endothelium and typical guttae as darkbodies with a central bright reflex

Table 2 Genotype distribution and allele frequency of TCF4 rs613872 in patients with FECD and control subjects

TCF4 rs613872genotype

Genotype counts 119899 () OR dominant model(95 CI)

Allele counts 119899 () 1205942 statistic119901TT TG GG Total T G

FECD patients 46 (1825) 170 (6746) 36 (1429) 252 1295(863ndash1942)

262 (5198) 242 (4802) 1567

lt00001Controls 240 (7430) 74 (2291) 9 (279) 323 554 (8576) 92 (1424)

the groups were analyzed with a two-sided unpaired 119905-test(Statistica StatSoft Poland) Pearsonrsquos correlationwas used toassess the degree of the relationship between gene expressionand age or sex 119901 lt 005 was considered to indicate statisticalsignificance

3 Results

Both the patient and control groups were genotyped forthe TCF4 SNP rs613872 In FECD patients the distributionof the TT TG and GG genotypes showed a significant

deviation from Hardy-Weinberg equilibrium (1205942 = 311 119901 lt00001) while in control subjects the genotype distributionremained in the Hardy-Weinberg equilibrium (1205942 = 13119901 = 0265) Allele G was significantly more prevalent inpatients with FECD than among control subjects (Table 2)The odds ratio (OR) for two copies of the risk allele (GGhomozygotes versus TT homozygotes) was 2087 (95 CI942ndash4624) whereas the OR for one copy of the risk allele G(TG heterozygotes versus TT homozygotes) was 1199 (95CI 790ndash1819) Testing the association between FECD andthe TCF4 genotype under dominant additive or recessivemodels we found that the most plausible model was the


00

05

10

15

20

25

TT TG GG

ns

ns

FECD patients

Relat

ive e

xpre

ssio

n of

TCF4

mRN

A

(a)

00

05

10

15

20

25

Controls FECD patients

ns

Relat

ive e

xpre

ssio

n of

TCF4

mRN

A

(b)

Figure 2 Expression ofTCF4 in corneal endothelial cellsThe amount ofTCF4mRNAwas quantified by real-time PCR in relation to RPL13A(a) Expression of TCF4 in FECD patients with respect to a different TCF4 genotype at rs613872 (TT 119899 = 3 TG 119899 = 31 and GG 119899 = 6) isshown ns nonsignificant (b) Average values of TCF4mRNA expression in control samples (white bars) and FECD patients (black bars) areshown ns nonsignificant

dominant one whereas the additive and recessive modelscould be formally rejected as indicated by the 119901 values formodel fit (119901 lt 00001) The dominant model (combiningthe GG and TG into one category) conferred the highest ORof all the tested models (OR = 1295 95 CI 863ndash19421205942= 1895 119901 lt 00001)Expression of TCF4 was not age or sex dependent There

were no differences in TCF4 mRNA levels in FECD patientswith a particular TCF4 SNP rs613872 genotype (Figure 2(a))but also when TCF4 genotypes of patients and controlswere analyzed together (data not shown) No increase in theamount of TCF4 mRNA transcripts was observed in FECDpatients as compared to the control group (Figure 2(b))

4 Discussion

Our data are in accord with previous studies showing thatthe presence of a TCF4 risk allele at rs613872 is much morecommon in Caucasian patients with FECD and stronglypredisposes to the development of the corneal dystrophy[6 12ndash16] Indian FECD patients do not show a distinctassociation with this TCF4 variant which may be explainedby a small sample size studied [17] In Chinese FECD patientsno association could be observed as the population is notpolymorphic at this genomic position [18ndash20] In theseFECD patients TCF4 genetic variants adjacent to rs613872(eg rs17089887 in both Indian and Chinese subjects) werestrongly associated with FECD which suggested the presenceof significant disease-causing changes in the nearby regions ofthese alleles [17 19 20] All the relevant SNPs in the respectivepopulations are in strong linkage disequilibrium with anewly identified CTG181 trinucleotide repeat expansion inthe TCF4 gene [9 17 20 21] which confers a transethnicassociation with FECD

TCF4 is now widely recognized as a major contributor toFECD One may assume that patients carrying TCF4 risks

alleles and thus having an increased susceptibility to FECDshould more often have follow-up ophthalmic examinationsDiagnosis of cataract in these patients might be an indi-cation for an earlier cataract surgery before a full-blownclinically significant FECD develops and the patients requirea combined procedure of corneal transplantation and cataractsurgery

TCF4 genetic variants are strongly associated with FECDbut so far little is known about a possible involvement ofTCF4gene products in the development of FECD Hypothesizingthat the strong genetic association may indicate a causal rela-tionship and the TCF4 genetic variant may represent a tissue-specific regulatory element TCF4 mRNA studies in cornealendothelial cells were performed Analyzing more than 60different corneal endothelium and Descemetrsquos membranecomplexes we have not found any significant differences inthe expression of TCF4 at mRNA level in FECD patientsas compared to control samples There were no differencesin TCF4 expression between the carriers and noncarriers ofthe TCF4 risk allele neither in the group of FECD patientsalone nor when FECD patients and controls were pooledtogether The data suggest that changes in the transcriptlevel containing constitutiveTCF4 exon encoding the amino-terminal part of the protein seem not to contribute to diseasepathogenesis

Recently unaltered TCF4 expression was also reportedin another group of FECD patients [22] In the absence ofdistinguished differences in the amount of mRNA transcriptactivity still may be altered or posttranslational mechanismsmay affect protein structure level or cellular distributionA novel and so far the only identified link between TCF4susceptibility and FECD disease mechanism is the formationof toxic poly(CUG)n RNA and missplicing events in patientswith TCF4 repeat expansion In corneal endothelial cells ofthese patients the TCF4 repeats are actively transcribed andseem to preferentially accumulate into poly(CUG) RNA foci


A protein found to be immobilized in RNA foci is MBNL1 asplicing regulator Consequently differential splicing eventswere found in corneal endothelium of FECD patients [23]

Our study confirms and extends the association of TCF4with FECD by testing a novel previously not analyzedpopulation It advances our knowledge on the role of TCF4in the development of FECD Genotyping of TCF4 risk allelesmight be considered as a diagnostic procedure as the geneticresults have an important predictive value and are of clinicalutility

Conflict of Interests

The authors declare no conflict of interests regarding thispaper

Acknowledgments

This work was supported by the Medical University ofWarsaw Grants 1M15NM42011 1M15N2015 and 2WFN2015 and the project entitled ldquoIntegrated System of ToolsDesigned for Diagnostics and Telerehabilitation of the SenseOrgans Disorders (Hearing Vision Speech Balance TasteSmell)rdquo INNOSENSE cofinanced by the National Centrefor Research and Development within the STARTEGMEDProgram

References

[1] J Zhang and D V Patel ldquoThe pathophysiology of Fuchsrsquoendothelial dystrophymdasha review of molecular and cellularinsightsrdquo Experimental Eye Research vol 130 pp 97ndash105 2015

[2] S G Levy J Moss H Sawada P J C Dopping-Hepenstal andA C E McCartney ldquoThe composition of wide-spaced collagenin normal and diseased Descemetrsquos membranerdquo Current EyeResearch vol 15 no 1 pp 45ndash52 1996

[3] M A Nanavaty X Wang and A J Shortt ldquoEndothelial ker-atoplasty versus penetrating keratoplasty for Fuchs endothelialdystrophyrdquo Cochrane Database of Systematic Reviews no 7Article ID CD008420 2011

[4] G K Klintworth ldquoCorneal dystrophiesrdquo Orphanet Journal ofRare Diseases vol 4 no 1 article 7 2009

[5] A Higa H Sakai S Sawaguchi et al ldquoPrevalence of and riskfactors for cornea guttata in a population-based study in asouthwestern island of Japan the Kumejima studyrdquo Archives ofOphthalmology vol 129 no 3 pp 332ndash336 2011

[6] K H Baratz N Tosakulwong E Ryu et al ldquoE2-2 proteinand Fuchsrsquos corneal dystrophyrdquo The New England Journal ofMedicine vol 363 no 11 pp 1016ndash1024 2010

[7] B W Iliff S A Riazuddin and J D Gottsch ldquoThe genetics ofFuchsrsquo corneal dystrophyrdquo Expert Review of Ophthalmology vol7 no 4 pp 363ndash375 2012

[8] S A Riazuddin S Vasanth N Katsanis and J D GottschldquoMutations in AGBL1 cause dominant late-onset Fuchs cornealdystrophy and alter protein-protein interactionwith TCF4rdquoTheAmerican Journal of HumanGenetics vol 93 no 4 pp 758ndash7642013

[9] E D Wieben R A Aleff N Tosakulwong et al ldquoA commontrinucleotide repeat expansion within the transcription factor4 (TCF4 E2-2) gene predicts Fuchs corneal dystrophyrdquo PLoSONE vol 7 no 11 Article ID e49083 2012

[10] J P Szaflik M Ołdak R B Maksym et al ldquoGenetics ofMeesmann corneal dystrophy a novel mutation in the keratin 3gene in an asymptomatic family suggests genotype-phenotypecorrelationrdquoMolecular Vision vol 14 pp 1713ndash1718 2008

[11] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquoNucleic Acids Research vol 29 no9 article e45 2001

[12] S A Riazuddin E J McGlumphy W S Yeo J Wang NKatsanis and J D Gottsch ldquoReplication of the TCF4 intronicvariant in late-onset Fuchs corneal dystrophy and evidence ofindependence from the FCD2 locusrdquo Investigative Ophthalmol-ogy and Visual Science vol 52 no 5 pp 2825ndash2829 2011

[13] J F Stamler B R Roos M D Wagoner et al ldquoConfirmationof the association between the TCF4 risk allele and Fuchsendothelial corneal dystrophy in patients from the MidwesternUnited Statesrdquo Ophthalmic Genetics vol 34 no 1-2 pp 32ndash342013

[14] Y-J Li M A Minear J Rimmler et al ldquoReplication of tcf4through association and linkage studies in late-onset fuchsendothelial corneal dystrophyrdquo PLoS ONE vol 6 no 4 ArticleID e18044 2011

[15] R P Igo Jr L J Kopplin P Joseph et al ldquoDiffering rolesfor TCF4 and COL8A2 in central corneal thickness and fuchsendothelial corneal dystrophyrdquo PLoS ONE vol 7 no 10 ArticleID e46742 2012

[16] A Kuot AWHewitt K Griggs et al ldquoAssociation of TCF4 andCLU polymorphisms with Fuchsrsquo endothelial dystrophy andimplication of CLU and TGFBI proteins in the disease processrdquoEuropean Journal of HumanGenetics vol 20 no 6 pp 632ndash6382012

[17] G G Nanda B Padhy S Samal S Das and D P AloneldquoGenetic association ofTCF4 intronic polymorphismsCTG181and rs17089887 with Fuchsrsquo endothelial corneal dystrophy inan Indian populationrdquo Investigative Ophthalmology amp VisualScience vol 55 no 11 pp 7674ndash7680 2014

[18] K J Wang V Jhanji J Chen et al ldquoAssociation of transcriptionfactor 4 (TCF4) and protein tyrosine phosphatase receptor typeG (PTPRG) with corneal dystrophies in southern ChineserdquoOphthalmic Genetics vol 35 no 3 pp 138ndash141 2014

[19] AThalamuthu CCKhorDVenkataraman et al ldquoAssociationof TCF4 gene polymorphisms with Fuchsrsquo corneal dystrophy inthe Chineserdquo Investigative Ophthalmology amp Visual Science vol52 no 8 pp 5573ndash5578 2011

[20] C Xing X Gong I Hussain et al ldquoTransethnic replicationof association of CTG181 repeat expansion of TCF4 gene withFuchsrsquo corneal dystrophy in Chinese implies common causalvariantrdquo Investigative Ophthalmology amp Visual Science vol 55no 11 pp 7073ndash7078 2014

[21] V VMootha X Gong H-C Ku and C Xing ldquoAssociation andfamilial segregation of CTG181 trinucleotide repeat expansionof TCF4 gene in Fuchsrsquo endothelial corneal dystrophyrdquo Inves-tigative Ophthalmology and Visual Science vol 55 no 1 pp 33ndash42 2014

[22] V V Mootha I Hussain K Cunnusamy et al ldquoTCF4 tripletrepeat expansion and nuclear RNA foci in Fuchsrsquo endothelial


corneal dystrophyrdquo InvestigativeOpthalmologyampVisual Sciencevol 56 no 3 pp 2003ndash2011 2015

[23] J Du RAAleff E Soragni et al ldquoRNA toxicity andmissplicingin the common eye disease fuchs endothelial corneal dystro-phyrdquo The Journal of Biological Chemistry vol 290 no 10 pp5979ndash5990 2015

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Behavioural Neurology

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Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Loci genes and genetic variants related to late-onset FECD

Chromosome Locusgenevariant Late-onset FECD1p351 rs760594 F5q123 rs1301475 F5q331ndashq352 FCD3 F7q223 rs257376 F8p213 rs2466216 F8p211 rs9797 F8q2113 rs1380229 F9p221ndashp241 FCD4 F10p1122 ZEB1 FS10q242 rs1889974 F13pter-q1213 FCD1 F15q222 rs235512 F15q2231 rs352476 F15q253 AGBL1 FS17q253 rs938350 F18q211 LOXHD1 FS18q212 TCF4 FS18q212ndashq2132 FCD2 F20p13 SLC4A11 FS20p122 rs674630 FXq28 rs1990383 FF familial S sporadic

risk of cornea guttata was independently associated witholder age female sex and a thinner central corneal thickness[5] as well as genetic variants [6]

To date four different genes ZEB1 (10p1122MIMlowast189909) AGBL1 (15q253 MIMlowast615496) LOXHD1(18q211 MIMlowast613072) and SLC4A11 (20p13 MIMlowast610206)as well as four causal loci on chromosomes 5q331ndashq352(FCD3) 9p221ndashp241 (FCD4) 13pter-q1213 (FCD1) and18q212ndashq2132 (FCD2) together with a number of suscep-tibility loci have been implicated in the pathogenesis ofFECD (summarized in Table 1) [7 8] A strong associ-ation has been established between TCF4 gene (18q212MIMlowast602272) variants and FECD A genome-wide associ-ation study has identified a common biallelic deep intronicTCF4 single nucleotide polymorphism (SNP rs613872NG 0117161g50559CgtA) as a highly significant risk factorfor FECD [6] In addition to that a separate study reporteda trinucleotide expansion (CTG181) in the intron regionof TCF4 which is highly prevalent in FECD individuals[9] The role of these associations in FECD developmentremains poorly understood In the study we set out to analyzewhether the presence of TCF4 rs613872 risk allele affects theexpression of TCF4 in corneal endothelial cells which wouldprovide a functional link between a risk factor and diseasemechanism in FECD

2 Patients and Methods

The study conformed to the tenets of the Declaration ofHelsinki and was approved by the local Ethics Committee

Patients were recruited from the Department of Ophthal-mology Medical University of Warsaw and gave informedconsent prior to participation The diagnosis of FECD wasbased on the visualization of ldquoguttaerdquo and stromal oedema byslit-lamp examination confocal microscopy in vivo (IVCMConfoscan 4 Nidek Technologies) and anterior segmentoptical coherence tomography (CASIACorneaAnterior Seg-ment OCT SS-1000 Tomey) (Figure 1)

21 Genotyping Genomic DNAwas isolated from peripheralblood samples of sporadic unrelated FECD patients (119899 =252 187 females and 65 males) with a standard salting-outprocedure Control DNA samples (119899 = 323) representative ofthe background population of central Poland came from therepository of the Department of Medical Genetics [10] TheTCF4 rs613872 was genotyped using ABI Custom TaqManSNP Genotyping Assay (Applied Biosystems) and the real-time PCR system (Viia7 Thermo Fisher Scientific) Theaccuracy of genotyping was confirmed by Sanger sequencingin selected subjects

22 RNA Isolation and Quantitative Real-Time PCR Cornealendothelial cell monolayers attached to Descemetrsquos mem-brane were obtained from FECD patients (119899 = 40) duringendothelial keratoplasty (DSAEK) or excised from donorcorneoscleral buttons (119899 = 23) that were not used fortransplantation The FECD patients and controls includedin the RNA studies derived from the group genotyped atrs613872 (FECD group TT 119899 = 3 TG 119899 = 31 GG 119899 = 6control group TT 119899 = 14 TG 119899 = 6 GG 119899 = 3) Thespecimens were submerged in the RNAlater storage solution(Ambion Austin TX USA) and frozen immediately TotalRNA was extracted using Trizol (Thermo Fisher ScientificWaltham MA USA) and cDNA was generated from 500 ngof RNA with the Maxima First Strand cDNA Synthesis Kit(Thermo Fisher Scientific) Gene expression was measuredby real-time PCR (Viia7 Thermo Fisher Scientific) usingPCR primers (Oligo Warsaw Poland) and probes (RocheUniversal Probe Library) designed with the ProbeFindersoftware version 250 (Roche) The analyzed coding regionof TCF4 is present in both TCF4-A and TCF4-B isoformsIt is located in the amino-terminal part of the protein closeto the activation domain 2 (AD2) For TCF4 quantitativePCR (qPCR) the primer pair 51015840-GCACTTTCCCTAGCT-CCTTCT-31015840 and 51015840-GCATAGCCAGGCTGATTCAT-31015840 andprobe 25 for RPL13A (60S ribosomal protein L13a) qPCRthe primer pair 51015840-CTGGTGCTTGATGGTCGAG-31015840 and51015840-GTTGATGCCTTCACAGCGTA-31015840 and probe 77 wereused Expression values for TCF4 were calculated usingthe modified double delta Ct (ΔΔCt) method and absolutequantification and normalized to RPL13A [11]

23 Statistical Analysis Hardy-Weinberg equilibrium(HWE) was analyzed with the 1205942 test in the patient andcontrol groups The odds ratios (ORs) with 95 confidenceintervals (95 CI) and 119901 values were calculated with theWeb-Assotest program (httpwwwekstroemcomassotestassotesthtml) Differences in gene expression between


(a)

(b) (c)







262 (5198) 242 (4802) 1567

lt00001Controls 240 (7430) 74 (2291) 9 (279) 323 554 (8576) 92 (1424)


3 Results




00

05

10

15

20

25

TT TG GG

ns

ns

FECD patients

Relat

ive e

xpre

ssio

n of

TCF4

mRN

A

(a)

00

05

10

15

20

25


ns

Relat

ive e

xpre

ssio

n of

TCF4

mRN

A

(b)




4 Discussion











Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b) (c)







262 (5198) 242 (4802) 1567

lt00001Controls 240 (7430) 74 (2291) 9 (279) 323 554 (8576) 92 (1424)


3 Results




00

05

10

15

20

25

TT TG GG

ns

ns

FECD patients

Relat

ive e

xpre

ssio

n of

TCF4

mRN

A

(a)

00

05

10

15

20

25


ns

Relat

ive e

xpre

ssio

n of

TCF4

mRN

A

(b)




4 Discussion











Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

20

25

TT TG GG

ns

ns

FECD patients

Relat

ive e

xpre

ssio

n of

TCF4

mRN

A

(a)

00

05

10

15

20

25


ns

Relat

ive e

xpre

ssio

n of

TCF4

mRN

A

(b)




4 Discussion











Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Fuchs Endothelial Corneal Dystrophy: Strong Association with