inherited limbal stem cell deficiency
TRANSCRIPT
1
Familial limbal stem cell deficiency: clinical, cytological and genetic
characterization
Lubica Dudakova1, Sek-Shir Cheong2, Stanislava Reinstein Merjava1, Pavlina Skalicka1,3,
Marcela Michalickova3, Michalis Palos3, Gabriela Mahelkova4, Deli Krizova5, Martin
Hlozanek4,MarieTrkova6,JenaL.Chojnowski7,EnkelaHrdlickova4,NikolasPontikos8,Vincent
Plagnol8, Viera Veselá1, Katerina Jirsova1, Alison J. Hardcastle2, Martin Filipec9, James D.
Lauderdale7,PetraLiskova1,3
1ResearchUnitforRareDiseases;DepartmentofPediatricsandAdolescentMedicine,FirstFaculty of Medicine, Charles University and General University Hospital in Prague, CzechRepublic2UCLInstituteofOphthalmology,London,UK3DepartmentofOphthalmology, First FacultyofMedicine,CharlesUniversity andGeneralUniversityHospitalinPrague,CzechRepublic4DepartmentofOphthalmology,SecondFacultyofMedicine,CharlesUniversityandMotolUniversityHospital,Prague,CzechRepublic5 Ophthalmology Department, Third Faculty ofMedicine, Charles University and TeachingHospitalKralovskeVinohrady,Prague,CzechRepublic6GENNET,Prague,CzechRepublic7DepartmentofCellularBiology,UniversityofGeorgia,Athens,GA,30602,USA8UCLGeneticsInstitute,London,UK9EuropeanEyeClinicLexum,Prague,CzechRepublic
Correspondingauthor:
PetraLiskova,Assoc.Prof.
DepartmentofPediatricsandAdolescentMedicine,FirstFacultyofMedicine
CharlesUniversityandGeneralUniversityHospitalinPrague
KeKarlovu2,Praha2,12808,Prague,CzechRepublic
Tel:+420224967139
ConflictofInterest:Theauthorsdeclarenopotentialconflictsofinterest.
2
Sir
Limbalstemcelldeficiency(LSCD)ischaracterizedbyadecreasedabilitytoreplenish
the population of corneal epithelial cells, resulting in corneal neovascularization, surface
defects,scarringandreplacementofthecornealepitheliumwithconjunctivalepithelialcells.
FamilialLSCDwithminimalornootherocularpathology isanextremelyrarecondition. In
somebutnotinallfamiliescodingorsplicingpathogenicmutationsinthepairedboxgene6
(PAX6,MIM*607108)havebeendetected[1,2].
Theproband firstdevelopedocular symptomsat theageof3years,whenshehad
beenexaminedforrednessandtearingoftherighteye(RE).Peripheralsuperficialcorneal
vascularization, roughuneven corneal surface and scarring in theREweredocumented in
historicalnotes.Attheageof12yearsherbestcorrectedvisualacuity(BCVA)intheREwas
recorded to be handmotion. Apart fromdecreasedBCVA, hermain subjective symptoms
havebeenrecurrentperiodsof lightsensitivityandforeignbodysensationassociatedwith
profound redness occurring often in theRE andoccasionally in the left eye (LE). Since 25
yearsofage,thesesymptomshavebeenmanagedbydailycontact lenswear intheRE. In
addition to the recommendation to administer regularly artificial tears, she has been
prescribed intermittentlymild steroiddrops, topical cyclosporineAandanti-VEGF therapy
(bevacizumab); however, these compounds provided little or no relief. Examination at 30
years of age revealed a marked asymmetry in her clinical features. In the RE, extensive
vascularization of the entire cornea and stromal scarring with yellowish deposits was
observed(Figure1A).BCVAwashandmotion.IntheLE,acircularperipheralvascularization
wasobservedthatwasmostpronouncedinthesuperiorcorneawhereitextendedabout3
mmfromthe limbus;thecornealstromawasotherwiseclear.Uncorrectedvisualacuity in
the LEwas 1.0. Re-examination of the proband at the age of 36 years did not reveal any
3
progression(Figure1D,E).MildcorectopialossofthelimbalpalisadesofVogtwasobserved
bilaterally (Figure 1A, B, D, E). Spectral domain optical coherence tomography (SD-OCT;
Spectralis; Heidelberg EngineeringGmbH, Heidelberg, Germany) scans of the right cornea
showedirregularcornealthicknessandthinningupto251μm(Figure1C).SD-OCTimaging
ofthemaculadidnotrevealanyabnormalitiesinbotheyes(SupplementaryFigure1).
Thesonoftheprobandwasbornfromanuneventfulpregnancy.Hepresentedsince
theageof1monthwithbilateraltearing,lightsensitivity,conjunctivalinjectionandepisodes
ofmucousdischarge, forwhichheunderwent lacrimalprobing.Bilateral cornealepithelial
defectswere notedwhen hewas 4months old and topical treatmentwith acyclovir and
artificialtearswasstartedashewassuspectedtosufferfromherpetickeratitis.Attheageof
5 months, ophthalmic examination was performed under general anaesthesia using an
operatingmicroscope.Bilateral corneal vascularizationextending1-2mm from the limbus
nasallyandinferiorly,cornealepitheliopathywithpunctuatefluorescentstaining,andahaze
of the entire corneal surfacewere found. No gross abnormalities of the iris or lenswere
present, intraocular pressure and fundus examination were also bilaterally normal.
Subsequent re-examination under general anaesthesia at the age of 1 year and 1month
showed bilateral progression of the vascularization extending diffusely from limbus in all
fourquadrantsoverthecornealcentre.At2yearsand8monthsofage,bothofhiscorneas
wereentirelyvascularizedwithirregularsurfaceandcentralopacity(Figure1F,G).Thechild
hasbeenphotophobicsinceanearlyage,andforthisreasoncouldnotundergoophthalmic
examinationat the last follow-upat theageof7years. Itwasnoted thathis lefteyewas
esotropic.Hehasbeentreatedwithartificial tearsandtopicalcyclosporineA,howeverno
majorimprovementofhissymptomswasobserved.
4
Nosystemicabnormalitiesweredetectedintheaffectedchildwhoalsounderwent,
before the age of 5months, blood tests for general inflammatory response and serologic
testsforinfectiousdisordersknowntocausecongenitalcornealclouding.
Impressioncytologyperformed inboth individuals confirmed thediagnosisof LSCD
by histological detection of goblet and inflammatory cells in corneal imprints and by
immunocytochemical visualization of conjunctivalization using KRT7 as a marker [3]
(SupplementaryMaterialandSupplementaryFigure2).
PAX6 exons 1-13 and 5a (reference sequence ENST00000638914.1) were Sanger
sequenced[4].Next,wholeexomesequencing(WES)oftheprobandwasperformed.Reads
were aligned to the human reference sequence (Ensembl Genome browser hg19) and
annotated (Supplementary Material). Minor allele frequency cut-off value ≤ 0.005
(population frequency databases used are listed in Supplementary Material) was chosen
taking intoconsideration the rarityof theconditionstudied,and thepresumedautosomal
dominantinheritancewithunknownpenetrance.Copynumbervariation(CNV)analysiswas
alsoperformedintheproband(SupplementaryMaterial).
Investigationoftheproband’sWESdataidentified81rareheterozygousvariants,of
which39wereunique.Cross referencingof thisdatasetwith genespossibly implicated in
anteriorsegmentdysgenesis,cornealdystrophyandcornealvascularization(Supplementary
Tables1-3) identifiedauniqueheterozygousvariant inKRT12 (keratin12;MIM*601687),
c.1111G>C; p.(Asp371His) (reference sequence ENST00000251643.4). Mutations in KRT12
have been reported in Meesman corneal dystrophy patients presenting with corneal
neovascularizationasanadditionalfeaturetotheclassicalphenotype[5].Sangersequencing
howevershowedthattheaffectedsonoftheprobandwaswildtype(SupplementaryFigure
5
3), indicating that the KRT12 variant in the proband is a benign polymorphism. No CNVs
weredetected.
Clinical findings inthetwoaffected individualswereconsistentwithdescriptionsof
familialLSCDwithoutsystemicabnormalities[2,6].Tothebestofourknowledge,thisisthe
first report on the earliest onset of this condition at 1 month of age in the son of the
proband. We also extended our understanding of the effects of LSCD on the corneal
architecturethroughuseofacombinationof imagingandmolecularapproaches.Although
weappliedstate-of-the-arttechniques,includingWESandCNVanalysiswewerenotableto
findthemoleculargeneticcauseofLSCD inthefamilystudied.This finding, inconjunction
withtheobservationthatthemoleculardefectisunknownforsomefamilialcasesofLSCD
publishedtodate[2],raisesthepossibilitythat, inadditiontospecificgenessuchasPAX6,
theunderlyingpathogenicmechanismsofinheritedand/orcongenitalLSCDwithminimalor
no irisabnormalitiesmaybeassociatedwithnon-codingregionsof thegenomeand/orbe
multifactorial.
ACKNOWLEDGEMENTS
InstitutionalsupportwasprovidedbyUNCE204011andPROGRES-Q26/LF1programsofthe
CharlesUniversity.PSwassupportedbyGAUK227015/2017andSVV260367/2017.SRM,VV
and KJwere supported byNorwegian FinancialMechanism28477/2014, project 7F14156.
GM was supported by MH CZ – DRO, Motol University Hospital, Prague, Czech Republic
00064203. Support also provided by the Sharon Stewart Aniridia Research Trust and the
Children’s Glaucoma Foundation to JDL. We thank The National Center for Medical
6
Genomics (LM2015091) for providing ethnically matched population genotype frequency
data(projectCZ.02.1.01/0.0/0.0/16_013/0001634).
REFERENCES
1. MirzayansF,PearceWG,MacDonaldIM,WalterMA.MutationofthePAX6genein
patientswithautosomaldominantkeratitis.AmJHumGenet1995;57:539-48.
2. SkeensHM,BrooksBP,Holland EJ. Congenital aniridia variant:minimally abnormal
irideswithseverelimbalstemcelldeficiency.Ophthalmology2011;118:1260-4.
3. JirsovaK,DudakovaL,KalasovaS,VeselaV,MerjavaS.TheOV-TL12/30cloneofanti-
cytokeratin 7 antibody as a new marker of corneal conjunctivalization in patients
withlimbalstemcelldeficiency.InvestOphthalmolVisSci2011;52:5892-8.
4. Love J, Axton R, Churchill A, vanHeyningen V, Hanson I. A new set of primers for
mutationanalysisofthehumanPAX6gene.HumMutat1998;12:128-34.
5. Hassan H, Thaung C, Ebenezer ND, Larkin G, Hardcastle AJ, Tuft SJ. Severe
Meesmann'sepithelialcornealdystrophyphenotypeduetoamissensemutation in
thehelix-initiationmotifofkeratin12.Eye(Lond)2013;27:367-73.
6. EspanaEM,GrueterichM,RomanoAC,TouhamiA,TsengSC.Idiopathiclimbalstem
celldeficiency.Ophthalmology2002;109:2004-10.
7
FIGURELEGEND
Figure 1. Clinical findings in familial limbal stem cell deficiency. Anterior segment
photograph of the proband’s right eye (age 30 years) showing completely vascularized
cornea,withthebloodvesselslocatedprimarilyintheanteriorstroma,andstromalscarring
withyellowishdeposits[A];temporallimbalareaindetail(age36years)demonstratingloss
of the limbal palisades ofVogt [B]. SD-OCT imagingdocumenting irregular thinning in the
rightcornea(age36years)[C].Anteriorsegmentphotographoftheproband’slefteye,note
onlymildperilimbalvascularization[D]andsuperiorlimbalareaindetail[E];(bothattheage
of 36 years). Anterior segment photographs of the son (age 2 years and 8months) show
advancedcornealvascularizationandcentralopacityinhisright[F]andleft[G]cornea.