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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.

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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

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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.

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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

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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

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Genomics (LM2015091) for providing ethnically matched population genotype frequency

data(projectCZ.02.1.01/0.0/0.0/16_013/0001634).

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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.

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