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RESEARCHOpenAccessFabrydiseaseintheSpanishpopulation:observationalstudywithdetectionof77patientsIreneVieitez1,OlgaSouto-Rodriguez1,LorenaFernandez-Mosquera2,BeatrizSanMillan1,3,SusanaTeijeira1,JulianFernandez-Martin1,4,FelisaMartinez-Sanchez5,LuisJoseAldamiz-Echevarria6,MonicaLopez-Rodriguez7,CarmenNavarro8andSaidaOrtolano1,9*AbstractBackground:Fabrydiseaseisamultisystemiclysosomalstoragedisordercausedbytheimpairmentofα-galactosidaseA.
Theincidenceofthisrarediseaseisunderestimatedduetodelayeddiagnosis.
Moreover,themanagementoftheidentifiedsubjectsisoftencomplicatedbythedetectionofvariantsofuncleardiagnosticinterpretation,usuallyidentifiedinscreeningstudies.
Weperformedanobservationalstudybasedonbiochemicalandgeneticanalysisof805driedbloodspotsamplesfrompatientswithclinicalsymptomsorfamilyhistoryofthispathology,whichwerecollectedfrom109Spanishhospitals,alloverthecountry.
Results:Weidentified77newdiagnosedpatientswithmutationsrelatedtoclassicalFabrydisease,aswellas2subjectswithc.
374A>T;p.
His125Leu,apossiblenewmutationthatneedtobeconfirmed.
Additionally,wedetected8subjectscarryinggeneticvariantspossiblylinkedtolateonsetFabrydisease(p.
Arg118Cysandp.
Ala143Thr),4caseswithpolymorphismp.
Asp313Tyrand36individualswithsinglenucleotidepolymorphismsinintronicregionsofGLA.
Fiveoftheidentifiedmutations(c.
431delG;c.
1182delA;c.
374A>T;c.
932T>C;c.
125T>A;c.
778G>A),whichwereassociatedwithaclassicalphenotypehavenotbeenpreviouslydescribed.
Moreover3subjectspresentingcomplexhaplotypesmadeupbytheassociationofintronicvariantspresentedimpairedlevelsofGLAtranscriptsandGb3depositsinskinbiopsy.
Conclusions:EnzymaticscreeningforFabryDiseaseinriskpopulation(2ormoreclinicalmanifestationsorfamilyhistoryofthedisease)helpedtoidentifyundiagnosedpatientsandunraveltheimpairmentofGLAexpressioninsomesubjectswithcomplexhaplotypes.
Keywords:Fabrydisease,Lysosomalstoragedisorders,Enzymaticscreening,Intronicvariants,GLAcomplexhaplotypeBackgroundFabrydisease(FD,OMIM#301500)isanX-linkedlyso-somalstoragedisorder(LSD),causedbytheimpairmentofα-galactosidaseA(α-GalA,EC3.
2.
1.
22),whichleadstotheprogressiveaccumulationofglobotriaosylcera-mide(Gb3),anditsderivatives(eg.
lyso-Gb3).
Todate,over700geneticdefectshavebeenidentifiedinGLA,howevernofrequentmutationsweredescribedanditisdifficulttoestablishagenotype-phenotypecorrelation[1].
TheclassicalformofFDisgenerallyassociatedwithaverylowresidualα-GalAactivityinmen(1%)andexclusivelycardiacorrenalmanifesta-tions[4,5].
Duetoaselectionbiasinthescreeningstudies,thesevariantsareofuncleardiagnosticinter-pretationwhichcomplicatesthemanagementoftheidentifiedsubjects[6]andprecludeduptodatethein-clusionofFDintheuniformscreeningpanelsrecom-mendedbytheAdvisoryCommitteeonHereditableDisordersinNewbornsandChildren[7].
Moreover,thepoorspecificityofFDphenotypeoftenresultsindelayeddiagnosis,whichisusuallyestablishedinadulthood,evenifthesymptomsstartedduringchild-hood[8].
ItwasoriginallyreportedthattheincidenceofFDisof1:117,000[9]forthegeneralpopulation;how-ever,pilotstudiesperformedinasymptomaticnewbornsestimatedanincidencerangingbetween1:3000and1:7000livebirths[10,11].
Thesedataandtheresultsofseveralscreeningstudiesperformedinpatientswithmedicalconditionsofdiverseetiology(i.
e.
stroke,renalfailure,ventricularhypertrophy)[4,12,13]highlightthelargenumberofundetectedFDpatients,whoareprob-ablystillunidentifiedormisdiagnosed.
Avoidingdelayeddiagnosisiscrucialtoincreasethequalityandexpectancyoflifeofthepatientswhoneedaspecifictreatment.
InEuropethereare,currently,threeavailabletreatmentswithspecificindicationforFD(agalsidasealfa,agalsidasebetaandmigalastathydro-chloride).
Thesedrugsaremoreefficientinimprovingpatients'conditionwhenthetherapystartsbeforeexten-sivetissuedegenerationisachieved[14,15].
Theaimofthisworkwastoperformanobservationalstudybasedonbiochemicalandgeneticanalysisofsub-jectswhomanifestacombinationofsymptomsandsignssuggestiveofFD(i.
e.
acroparesthesia,cardiachypertrophy,strokeintheyoung,renalfailure)orhaveafamilyhistoryofthedisease.
Thiswillallowtheidentifi-cationofFDpatientswithnon-specificdiagnosis,whocanthereforeaccessmoreappropriatetherapeuticproto-cols.
Theresultsofthestudywillalsoallowtodefineap-propriatecriteriaandparameterstodesignafuturevalidateddiagnosticstudy.
MethodsDesignofthestudyWecarriedoutanobservationalstudybasedonbiochem-icalscreeningofα-GalAandGLAsequenceinsubjectswithagecomprisedbetween1and85years(averageage47.
05±18.
53,standarddeviation(SD)),whopresentedmorethanoneclinicalsymptomorsignassociatedtoFD(i.
e.
ventricularhypertrophy,renalinsufficiency,strokeintheyoung,acroparesthesia,angiokeratome,intolerancetoheathorcold,neuropathicpain,hypertrophicmyocardio-pathy),orwhoweregenetically-linkedrelativesofprevi-ouslydiagnosedpatients.
Enzymeactivitywasquantifiedindriedbloodspotssamples(DBS)byfluorimetricassay.
Aconfirmatorygenetictestwasperformedinmalesub-jectswithactivityT;c.
932T>C;c.
125T>A;c.
778G>A).
Wealsoidentifiedafamilyof2subjects(1maleand1female),whopre-sentedwithclinicalfeaturescompatiblewiththeclassicformofFD,andcarryanewlydescribedvariantinGLA(c.
374A>T;p.
His125Leu),whichneedtobefurtherstudiedtoconfirmitspathogenicstatus.
MoredetailsaboutthesesubjectsaredescribedinTable1.
Thec.
431delGframeshiftmutationwasidentifiedinasubjectbelongingtoafamilyofFDpatients,whohadbeendiagnosedbyenzymeassay.
Theclinicalfeaturesofthisfamily(eg.
angiokeratomas,acroparestesias,cardiachypertrophyandrenaldysfunction)weredescribedbyBarba-Romeroetal.
[22].
Ultrastructuralanalysisofskinbiopsiesfromtwopreviouslyidentifiedmembersofthefamilyrevealedthepresenceofabundantzebrabodiesindifferentstructures(fibroblast,bloodvessel,smoothmuscleandnerveendings)[3].
Thec.
1182delAwasadenovomutationfoundina47-year-oldman(Patient#69),presentingmultiplepara-pelviccystsinbothkidneys,serumcreatinineof1.
6mg/dlandproteinuriaof1g/24h,whoeventuallyunder-wentrenalfailureandtransplant.
Thepatientalsopre-sentedwithcardiomyopathy,smallfiberneuropathy,corneaverticillataandhypohidrosis.
Lowlevelsofα-GalAactivitywereconfirmed,followingmeasurementinplasma(0.
5nmol/h/ml)andleukocytes(4.
2nmol/h/mg),soenzymereplacementtherapywasstarted[23].
ThisVieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page3of13Table1PatientswithdefinitivediagnosisofFabryDisease.
Ageatdiagnosisisindicated.
M:hemizygousmenF:heterozygousfemales.
Activityofɑ-GalAisexpressedinμmol/Lh.
FS:familystudy;X:notindicated,NM.
notmeasured;Symptoms1:RenalFailure2:VentricularHypertrophy;3:Acroparesthesia,4:Angiokeratome;5:Cardiomegaly;6:Fabryfacies;7:Hernia;8:Hypohidrosis;9:Intolerancetoheatorcold;10:Neuropathicpain;11:Cornealclouding;12:Strokeintheyoung,13:Hypertrophic,myocardiopathyCodeAgeSexMedicalDepartmentSymptomsɑ-GalAActivityMutationReference152MNephrology1,2,130.
59±0.
14c.
613C>T;p.
Pro205Ser[35]230MFS0.
76±0.
43346FFS2.
97±0.
16425MFS1.
27±0.
06556FFS3.
33±0.
33647FFS4.
36±1.
58776FFS1.
83±0.
24855FFS2.
24±0.
02924FFS1.
82±0.
381022FFS0.
58±0.
371129MFS1.
04±0.
071262FFS4.
40±0.
031344FFS3.
67±1.
451460MFS0.
28±0.
111582FFS6.
41±0.
011621MFS1.
04±0.
611734MFS0.
48±0.
28180.
5FFS3.
7±1.
73198MGeneticsFS0.
92±0.
1c.
713G>A;p.
Ser238Asn[4]2012MFS1.
2±1.
812157MFS1.
12±0.
1222XFFS3.
76±0.
362352FFS2.
47±0.
222431FFS2.
54±0.
052539FPediatricsFS4.
98262FFS1.
30274MFS0.
692874MCardiology2,FS0.
58±0.
382949F2,31.
01±0.
343052M2,FS0.
56±0.
253115FFS4.
68±0.
3032XFFS4.
61±0.
423310FNephrologyFS4.
75±0.
113439MFS1.
35±0.
013570MFS1.
48±0.
563664MFS1.
89±0.
653721MFS1.
45±0.
73817FFSNM39XMFS1.
67±0.
124038MFS1.
20±0.
104156FFS2.
4±0.
07Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page4of13Table1PatientswithdefinitivediagnosisofFabryDisease.
Ageatdiagnosisisindicated.
M:hemizygousmenF:heterozygousfemales.
Activityofɑ-GalAisexpressedinμmol/Lh.
FS:familystudy;X:notindicated,NM.
notmeasured;Symptoms1:RenalFailure2:VentricularHypertrophy;3:Acroparesthesia,4:Angiokeratome;5:Cardiomegaly;6:Fabryfacies;7:Hernia;8:Hypohidrosis;9:Intolerancetoheatorcold;10:Neuropathicpain;11:Cornealclouding;12:Strokeintheyoung,13:Hypertrophic,myocardiopathy(Continued)CodeAgeSexMedicalDepartmentSymptomsɑ-GalAActivityMutationReference4262FFS4.
9±1.
164330MInternalMedicine2,4,6,101.
27±0.
10c.
836A>G;p.
Gln279Arg[36]4428M2,100.
92±0.
24551F2,4,6,104.
02±0.
014626MFS1.
5±0.
744753M21.
98±0.
444848FFS3.
13±0.
324948F27.
45±0.
365023FFS7.
13±0.
565122MInternalMedicine3,4,5,6,7,8,9,100.
75±0.
05c.
422C>T;p.
Thr141Ile[37]5255F2,4,51.
01±0.
015353M3,4,5,9,102.
25±0.
065448MCardiology21.
05±0.
54c.
679C>T;p.
Arg227*[38]5548M1,20.
86±0.
195646MCardiology21.
19±0.
05c.
242G>C;p.
Trp81Ser[36]5713FInternalMedicine31.
48±0.
3c.
242G>C;p.
Trp81Ser[36]5812FNephrologyFS3.
16±0.
2c.
1232G>A;p.
Gly411Asp[39]5965FFS5.
95±1.
256027MNephrology1,6,100.
94±0.
3c.
778G>A;p.
Gly260ArgNotdescribed6159F123.
58±0.
596253MNeurology1,50.
13±0.
01c.
374A>T;p.
His125LeuNotdescribed(possiblenewmutationthatneedtobeconfirmed)6377FFS1.
04±0.
4264XMGeneticsFS1.
9±0.
84c.
463G>C;p.
Asp155His[40]65XFFSNM6642MCardiologyFS1.
87±0.
53p.
Arg227Gln[41]67XMFS1.
96±0.
476835FInternalMedicine1,3,4,95.
88±0.
37c.
431delG;p.
Gly144Alafs*21Notdescribed6948MGenetics1,10,130.
84±0.
18c.
1182delA;p.
Phe396Serfs*8Notdescribed7037MNephrology10.
63±0.
03c.
509A>T;p.
Asp170Val[42]7123FInternalMedicine3,8,111.
02±0.
13c.
1277_1278delAA;p.
Lys426Argfs*11[43]7219FInternalMedicineFS2.
43±0.
58c.
132G>T;p.
Trp44Cys[44]73XMInternalMedicine2,3,4,8,9,101.
21±0.
54c.
932T>C;p.
Leu311ProNotdescribed7435FNephrology3,6,10,111.
35±0.
01c.
155G>C;p.
Cys52Ser[45]7546MInternalMedicine2,4,80.
85±0.
34c.
572T>C;p.
Leu191Pro[46]7671FGenetics1,10NMc.
337T>C;p.
Phe113Leu[42]7743FNephrologyFS8.
37±0.
84c.
298A>T;p.
Arg100*[47]Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page5of13frameshiftmutationwillcausesthesubstitutionof6aminoacidsintheC-terminalpartoftheproteinandtheformationofaprematurestopcodon(26aminoacidsaremissingcomparedtothewild-typesequence)(Additionalfile1:FigureS3).
Asforthemajorityoftheframeshiftmutations,thetranscriptcarryingthisvariantismostlikelydegradedattheendoplasmicreticulumlevelduetoinstability.
Patient#73withthec.
932T>C;p.
Leu311Propre-sentedseveralclinicalmanifestationsrelatedtoclassicalFD,suchasventricularhypertrophy,acroparesthesias,angiokeratoma,hypohidrosis,neuropathicpainwithintolerancetoheatorcoldandrenalimpairment(albu-minuria160μg/min).
α-GalAactivitywasalsomea-suredinplasma(1.
1nmol/ml/h)andleukocytes(0.
13nmol/mg/h),whileGb3/Ga2ratiowas1.
6.
Since2011thispatientisunderenzymereplacementtherapy.
Patient#79withthec.
125T>A;p.
Met42Lysmuta-tionspresentedmainlyneurologicalsymptoms(acuteischemicstrokewithlacunarhemibulbarlesion)associ-atedtochronicrenaldysfunctionwithincreasedprotein-uria/creatinineratio(1.
50mg/mg).
ThepatientalsopresentwithclassichallmarksofFD,suchasangiokera-tomasandcorneaverticillata.
Thediagnosiswascon-firmedbymeasuringLyso-Gb3levelsinplasma,whichwerequantifiedin16ng/ml(Cut-offvalues0–3.
5).
Patient#60withthec.
778G>A;p.
Gly260Argpre-sentedsevereperipheralsensorialneuropathy,hypohi-drosis,corneaverticillata,microalbumin,abdominalpainandmultipleinflammatorymesentericandin-guinaladenopathiesconfirmedbybiopsy,whichalsoshowedevidencesofcellulardeposits.
Thispatientisunderenzymereplacementtherapysince2011[24].
Patient#61,wasidentifiedbyfamilialstudy,followingdefinitivediagnosisoftheindexcase[#60]inhiscen-teroforigin.
Thesubject#62,whobearsthec.
374A>T;p.
His125Leumutation,wasderivedfromaneurologyde-partmentandpresentedwithstrokeatyoungageassoci-atedtorenalinsufficiencyandcardiomegaly,thispatientunderwentEnzymereplacementtherapy.
Hismother(subject#63)isheterozygousforthesamemutation.
Additional51cases(6malesand45females)pre-sentedgeneticvariantsinGLAofuncertainsignificance.
Polymorphismp.
Asp313Tyr(c.
937G>T)wasfoundin7samples(4malesand3females).
Wealsodetectedthecontrovertedlate-onsetvariantsp.
Ala143Thr(c.
427G>A,foundin3femalesand1male)andp.
Arg118Cys(c.
352C>T,foundin4females)(Table2).
Moreover,singlenucleotidepolymorphisms(SNPs)inintronicsequencesweredetectedin36individuals(1maleand35females).
Complexhaplotypesmadeupof2ormoreintronicsubstitutionswerepresentin19ofthesubjectswithintronicSNPs(Additionalfile1:TableS2).
Intheremaining675cases(435malesand240females),mutationswerenotidentifiedinthecodingregionofthegene,norincloseintronicfragments(around50bpineachdirection).
Thepositivepredictivevaluefortheenzymatictestinmaleswas80.
43%(CI95%:67.
88–92.
99%);themethodaccuracywas98.
12%(CI95%:96.
80–99.
44%)andthefalsepositiveratewas1.
88%.
Thesevalueswerecalculatedtakingintoaccountthe37malepatientswithconfirmedFDdiagnosis,amongthe479malesubjectsthatwerescreened.
Meanactivityinthewholemalecohortwasof7.
6μmol/Lh±4.
02andmedianwas3.
08±1.
57.
Thegeneticstudywasnotgenerallyperformedinmaleswithactivityabovethecut-offvalue,however22samplesfrommenwithα-GalAactivitybetween2.
6and5μmol/Lhweresequencedbyspecificrequestoftheresponsiblephysicians,duetotheirclinicalmanifesta-tions.
Amongthesepatientsweidentifiedthe4subjectswiththep.
Asp313Tyrpolymorphism,theindividualwiththecontrovertedlateonsetvariantp.
Ala143Thrandthesubjectwithacomplexintronichaplotypeinintronicre-gion(rs2071225,rs5903184,rs2071397andrs2071228).
ComplexhaplotypesanalysisDuetotheclinicalfeaturespresentedby3ofthesubjects(#114;#115and#116),whowerefoundtobearacomplexassociationofintronicvariantsinGLA,weanalyzedtran-scriptlevelsandGb3depositspresenceinleukocytessam-plesandskinbiopsy,respectively,inordertodetectpossibledefectsingeneexpression.
Table1PatientswithdefinitivediagnosisofFabryDisease.
Ageatdiagnosisisindicated.
M:hemizygousmenF:heterozygousfemales.
Activityofɑ-GalAisexpressedinμmol/Lh.
FS:familystudy;X:notindicated,NM.
notmeasured;Symptoms1:RenalFailure2:VentricularHypertrophy;3:Acroparesthesia,4:Angiokeratome;5:Cardiomegaly;6:Fabryfacies;7:Hernia;8:Hypohidrosis;9:Intolerancetoheatorcold;10:Neuropathicpain;11:Cornealclouding;12:Strokeintheyoung,13:Hypertrophic,myocardiopathy(Continued)CodeAgeSexMedicalDepartmentSymptomsɑ-GalAActivityMutationReference7855FCardiology9,47.
61±0.
63c.
53T>G;p.
Phe18Cys[48]7927MClinicalPathology1,121.
98±0.
25c.
125T>A;p.
Met42LysNotdescribedVieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page6of13Individuals#115and#116werea7-year-oldgirlandher42-year-oldfather,whopresentedhaplotypers2071225,rs5903184,rs2071397andrs2071228.
Thefatherreferredhearinglossandacroparesthesiaassociatedtomicroalbuminuriaandrenaldysfunction.
Thegirlreferrederraticpainattheextremities,withoutpaincrisis,recurrentabdominalpainrelatedtofoodin-gestionandpoorsweating.
Angiokeratomas,skinlesionsororganomegalywerenotdetected.
Cardiorespiratoryandneurologicalexaminationswerenormal,aswellasrenalfunction.
GLAtranscriptionwassignificantlyimpairedinthesesubjectsatmRNAlevel(mRNA13%ofcontrolin#115and7%ofcontrolin#116),(Fig.
1a),aswellasatproteinlevel(α-GalAexpressionisdecreasedcomparedtoactinendogenouscontrolintheWesternblot),(Fig.
1b).
AlowpercentageofGb3deposits(7affectedfibro-blastsperexaminedsection)wasdetectedbyCD77immunolabelingindermisfibroblastsof#115skinbiopsy(Fig.
2a,B).
PositiveGb3cellswerealsofoundinoneeccrineepithelialgland(Fig.
2c).
WewerenotabletodetectthepresenceofGb3depositsinendothelialcellsofbloodvesselsornervefibers.
Biopsyofpatient#116wasnotavailable.
AlthoughtheseresultsshowthatGLAtranscriptionisimpairedinpatients#115and#116,wecannotconfirmthediagnosisofFDinthesesubjects,sincewedidnotmeasureGb3orLyso-Gb3levelsinurineorplasma,nei-therweshowedthepresenceoflamellarbodies.
Patient#114wasa47-year-oldwoman,presentingtheassociationofintronicvariantsrs2071225andrs2071228andα-GalAactivityof4.
6μmol/Lh.
ShereportedmultipleclinicalsymptomsrelatedtoclassicalFDin-cludingacroparesthesia,neuropathicpain,hypohidrosis,skinlesionsandmuscleweakness.
Similarlytothepreviouslyanalyzedsubjects,thiswomanpresentedimpairedGLAtranscription(30%ofcontrol)andGb3depositsinskinfibroblasts(10affectedcellsforeachexaminedsection)(Fig.
3)wereidentifiedthroughimmunohistochemistry.
AlsointhiscaseGb3orLyso-Gb3levelsinurineorplasmaarenotavailabletoconfirmFDdiagnosis.
DiscussionInthispaper,wehaveshownhowalargenumberofpatientswithFDcanbeidentifiedthroughscreeningofriskpopulations(2ormoresymptomsrelatedtoFDorfamilyhistory),whichstressesthattherateofdelayeddiagnosisinFDremainsexcessivelyhigh.
LatediagnosisisdetrimentalinFD,sinceitpreventspa-tientsfrombeingtreatedpromptlywiththeavailablespecifictherapies.
VanderToletal.
[25]conductedasystematicre-viewofFDdiagnosisstudiesincludingscreeningofnewbornsandriskpopulation,torecalculatetheprevalenceofthedisease.
AccordingtothisarticletheestimatedprevalenceofclassicalFDinhighriskpopulations(45studiescomputed)is0.
12%,althoughTable2SubjectswithlateonsetvariantsinGLAorp.
Asp313Tyrpolymorphism,Activityofɑ-GalAisexpressedinμmol/Lh±Standarddeviation.
Ageatdiagnosisisindicated.
M:hemizygousmenF:heterozygousfemales.
FS:familystudy;X:notindicated,NM.
Notmeasured;Symptoms1:RenalFailure2:VentricularHypertrophy;3:Acroparesthesia,4:Angiokeratome;5:Cardiomegaly;6:FabryFacies;7:Hernia;8:Hypohidrosis;9:Intolerancetoheatorcold;10:Neuropathicpain;11:Cornealclouding;12:Strokeintheyoung,13:Hypertrophic,myocardiopathyCode#AgeSexMedicalDepartmentSymptomsɑ-GalAActivityVariant8042FNeurologyFS3.
6±1.
76c.
352C>T;p.
Arg118Cys8151FFS2.
33±0.
438228FFS5.
83±0.
138331FFS6.
64±0.
1384XMGenetics6,134.
21±2.
02c.
427G>A;p.
Ala143Thr8545FFSNM86XF2,13NM8717FNeurologyFS3.
05±0.
178875MCardiologyFS3.
77±0.
22c.
937G>T;p.
Asp313Tyr8957FNephrologyFS4.
28±0.
7990XMGeneticsFS5.
2±0.
879135MNeurologyFS3.
8±1.
57921FNephrology18.
69±1.
939348MNeurology3,55.
5±0.
419445FClinicalPathology2,56.
63±0.
56Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page7of13thisvalueincreasesto0.
62%whenincludingallthedetectedvariantsinGLA(variantsofuncertainsig-nificanceorneutralvariants),whileitreportedaprevalenceofGLAvariantsof0.
04%innewbornscreenings(6studiescomputed).
ThemostrecentnewbornscreeningsperformedinWashington[10]andMissouri[11]reportedanFDprevalenceof0.
012%inlivebirthsor0.
034%respectively.
ThepercentageofpatientswithclassicFDinthepresentstudywasof7.
72%inthemalecohortand9.
56%inthewholegroupofsubjects;however,thesevaluesarenotrepresentativeofthegeneralpopulation,abFig.
1GLAmRNAlevels(a)andWesternblotofα-GalAandactin(b)measuredinleukocyteextractsofsamplesfromsubjects#115and#116.
InpanelA,barsrepresentrelativelevelsofmRNAexpression(ΔΔCts)normalizedtothecontrol.
Twodifferentcontrols(healthyvolunteers'samples)wereusedinindependentexperiments(controlbar).
InpanelB,Control()representsthesamplefromahealthyvolunteerandControl(+)representsthesamplefromapatientdiagnosedwithFDcarryingamissensepathogenicmutationacbdFig.
2ImmunofluorescenceofGb3depositinskinbiopsyfromsubject#115.
Depositsarestainedingreen(CD77_FITC)andaffectedfibroblastsareindicatedwitharrows(a,b),bluestainingrepresentsnuclei(DAPI)andredstainingpolymerizedactin(Rhodamine-Phalloidin).
Panelcshowsaneccrineglandwithgreendepositsandpaneldshowsanon-affectedfieldofthesamebiopsy.
Collagenfibersareeasilydetectedinallpanelsduetoauto-fluorescenceofthespecimenVieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page8of13sincethesamplesizeisrelativelysmallandsubjectsselectioncriteriaincludedindividualswithhighclinicalsuspicion(2ormoresymptoms),aswellasgenetically-linkedrelativesofFDpatients,whocompriseobligatecarriersofmutantalleles.
Moreover,malesubjects'sampleswerepreferentiallysentforanalysistoourcenter,bytheirresponsiblephy-sicians,sincetheFDphenotypeisusuallymoreeasilyrecognized.
Thelackofhomogeneousselectioncriteriainourcohort,justifiestheextremelyhighpercentageofpositivecases,whichthereforecannotbedirectlycom-paredwiththeprevalencevalueinriskhomogeneouspopulationsestimatedbyVanderTolletal.
intheirmeta-analysisoftheliterature.
Ourgrouprecentlypub-lishedtheresultsofascreeningperformedintheSpan-ishnewborns[26],whichdeterminethattheprevalenceofthediseaseis0.
013%inmalesfromtheNorthwestofSpain.
Therefore,weestimatethattherealprevalenceofFDinourcountrycouldbearound0.
013%orslightlyhigherconsideringthatthemajorityofthedefinitivediagnosisofSpanishadultpatientsdescribedinthepresentstudywasdetectedintheMediterraneanarea,whilethenewbornscreeningonlyincludessubjectsfromtheNorthwest-Atlanticregion.
Althoughthisstudypursuedreachingadefinitivediagnosisintheanalyzedsamples,duetothelackofhomogeneousselectioncriteria,itwasdesignedasanobservationalstudy,whichcanhelptodefinepreciseparametersforavalidateddiagnosticstudy.
Thecut-offpointthatwedefinedsettingupthepresentscreeningwasof2.
6μmol/Lh,estimatedmeasuringactivityinDBSfrom5FDpatientswithdefinitivediagnosisand15healthyvolunteers.
Thelownumberofpositivepatientsavailablewhensettinguptheassaymayhavecausedanoverestimationofthecut-offpoint,whichconsequentlyledtoahighfalseposi-tiverateandalowpositivepredictivevalue.
Ifwerecalculatethethresholdforα-GalAactivitybasedonthedataobtainedinthemalecohortanalyzedinthepresentwork,wewillobtainavalueof2.
14μmol/Lh,(percentileofpositivepatients+0.
5%).
Ifwewouldhaveappliedthiscut-offtoourcohortthepositivepredictedvalueoftheassaywouldhavebeen90.
24%(CI79.
94–100%)thefalsepositiverate0.
84%andmethodaccuracy99.
16%(CI98.
25–100%),(orMethodaccuracy99.
3%,positivepredictivevalue92.
68%andfalsepositiverate0.
63%,assumingthatp.
His125Leuisconfirmedtobepathogenic.
)Thelargestfamilyidentifiedinthisstudy,countedwith19members(11femalesand6males)withthep.
Pro205Sermutation(Fig.
4).
Theindexcasewasamalepatientwithseverehypertrophicmyocardiopathywhowasdiagnosedafter25yearsofdialysisandtworenaltransplants.
Thisisaperfectexampleofthedramaticconsequencesofdelayeddiagnosisforpatientsandfam-ilies,whichalsodeterminesimportanteconomicreper-cussionstotheHealthCareSystem.
Nevertheless,withoutimplementingasystematicscreeningforFDincertainriskconditions(i.
e.
renalfail-ure,strokeintheyoung,etc),thenon-specificityofthesymptomsmakesdifficulttoestablishadefinitivediag-nosis.
Indeed,inthelargefamilymentioned,theclinicalfeaturesweredifferentineachmember.
Althoughmostofthemareaffectedbyrenalandcardiacsymptoms,twomalespresentedneurologicalsymptomsratherthanrenaldysfunctionandoneofthemprematurelydiedduetostroke.
Noneofthesepatientspresentedcorneaverticil-lata,whichistraditionallyconsideredasahallmarkofFD.
Thepresentstudyalsocontributedtotheidentifica-tionoffivenewmutations,whichhadnotbeenabcFig.
3ImmunofluorescenceofGb3deposits(CD77_FITC_green,affectedfibroblastsindicatedwithyellowarrows)inskinbiopsyfrompatient#114(a:affectedfield;b:non-affectedfield)andGLAmRNAlevelsinleukocyteextractformpatient#114(c)comparedtosamplefrom2healthyvolunteers(controlbar)Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page9of13previouslydescribed(c.
431delG;c.
1182delA;c.
932T>C;c.
125T>A,c.
778G>A).
PathogeniccharacterofthesemutationswaspredictedbyinsilicoanalysisanddefinitiveFDdiagnosiswasconfirmedforthesepatients,asdetailedintheresultssection,byα-GalAactivitymeasuredinleukocytes(formalesonly),LysoGb3/Gb3levelsinplasmaorurineorbiopsyexamination.
Moreover,wedetectedafamilybringingthepossiblepathogenicmutationc.
374A>T,p.
His125Leu,whichneedtobefurtherstudied.
Theindexcaseofthisfamilyisamalesubjectwithaα-GalAactivitymeasuredinDBSof±0.
130.
01μmol/hL,whopresentswithclinicalsymptomscompatiblewithFD,howeverwewerenotabletocollectdatatoconfirmadefinitivediagnosisofthesesubjects(ej.
Gb3levelsorbiopsy).
Ontheotherhand,wealsodetected8individualswithpossiblelate-onsetFD,aswellas36subjectswithSNPsinintronicregions(oftenformingcomplexhaplotypes).
Thesekindsofvariantsaregenerallynon-pathogenicpolymorphismsandtheirpresenceisnotsufficienttodetermineFDphenotype[27],forthisreasontheywerenotincludedinthemethodaccuracycalculation.
None-theless,frequentvariantssuchasp.
Arg118Cys(0.
04%)andp.
Ala143Thr(0.
09%)havebeenassociatedtolateonsetFD[4,13,25]andthedeficientexpressionofα-GalAhasbeenreportedinsubjectswithcomplexintronichaplotypes[28,29].
AsstatedbySchiffmannetal.
[6],theclinicalhetero-geneityofFD,eveninpatientscarryingthesamemuta-tion,isanevidencethattheindividualriskofdevelopingcomplicationsmaydependonα-GalAdeficiencyinter-playwithotherfactors(i.
e.
genetic,epigeneticorenvir-onmental).
Anaccuratefollow-upofsubjectspresentingvariantsofunknownsignificanceandabiopsyexamin-ationarenecessarytoolstodetermineifFDwillbeeventuallydeveloped.
Inthepresentstudy,weanalyzedGLAtranscriptionandGb3expressioninthreesubjectspresentingintronicSNPs,sincetheirclinicalfeatureswerehighlysuggestiveofFD.
Subjects#115and#116presentedacomplexhaplotype,previouslyassociatedwithFD[30–32].
Gervasetal.
[32]describedGLAtranscriptionim-pairmentinaSpanishfamilywiththiscomplexhaplo-type.
Theaffectedmembersofthefamilypresentedgalactosphingolipidaccumulationinfibroblastsand,insomesubjects,glycolipidstoragewasalsopresentinrenaltubulesandglomeruli.
Asaddressedinthisarticle,thetran-scriptiondefectcouldbetheconsequenceofthealterationofanuclearproteinbindingsite,causedbySNPrs2071225.
Schelleckesetal.
[30]alsoreportedFD-relatedcryptogenicstrokeandsmallfibreneuropathyintwofamilieswithin-tronicSNPsintheGLApromoterregionandshowedthatgenetranscriptionisimpairedduetoinefficientbindingofthetranscriptionfactorEBtothepromoter.
Subjects#115and#116camefromthesamegeographicareaofthefamilydescribedbyGervasetal.
andwecannotruleoutthattheybelongtothesamefamily.
Ourresultsarecomparabletothosedescribedinthisarticle[32]intermsofGLAmRNAlevels,althoughthenumberofaffectedfibroblastsislowinthebiopsyweexamined.
Sincethisbiopsywasobtainedfroma7-year-oldgirl,thelowamountofGb3inthisspecimenmayreflectanearlystageofanFDprogressivecondition.
Duetotheidentifiedfunctionalandquantitativedefi-ciencyofα-GalAassociatedtoincipientformationofGb3depositinskinbiopsy,weconcludethatitispos-siblethatsubjects#115-and#116are,infact,FDpa-tients;however,foradefinitivediagnosis,abiopsyexaminationisrequiredforsubject#116andasecondbiopsyexaminationisnecessarytoconfirmstoragepro-gressioninsubject#115.
Wealsoanalyzedthecaseofsubject#114whoisheterozygousforthers2071225andrs2071228SNPs,butshowsmanyclinicalsignsusuallypresentinclassicFDpatients.
Similarlyto#115and#116,thissubjectpresentsapartialimpairmentofGLAlevelsandfunc-tionality,aswellasalowdetectableamountofGb3depositsdetectedintheskinbiopsy.
Ascommentedpreviously,thisdataarenotsufficienttoestablishdefini-tivediagnosis,althoughanaccuratefollow-upofthesubjectisadvisable.
Indeedthepresenceoftypicalclinicalmanifestationssuggeststhatα-GalAactivityinFig.
4GenetictreeofafamilywithmembersaffectedbyFD,whocarrythep.
Pro205Sermutation.
Circlesindicatefemales,squaresindicatemales.
Blacksquaresarethehomozygousmalemembersofthefamilyandblack/whiteconcentriccirclesindicatethefemaleheterozygouspatientsVieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page10of13functionalorganssuchaskidneyandheartcouldbesignificantlyimpaired,inspiteoftheresidualactivityof4.
66±0.
67μmol/LhmeasuredinDBS.
Asmentionedinreference[6],aheart-kidney-cerebrovascularsystemgra-dientseemstohavebeenestablishedforFDandatruethresholdforpathogenicenzymeactivityintissuesisdifficulttoestimate.
Moreoverexternalfactors,suchasrandomX-chromosomeinactivation,hormoneregulatedtranscriptionmechanismsandotherepigeneticfactors,cancontributetodevelopthephenotype.
Forallthesereasons,abiopsyexaminationisstronglyrecommendedforspecificdiagnosisofsubject#114,ideallyperformedinkidneyorheart.
SkinbiopsyisavaluabletoolfordiagnosisofFDpatients,sinceitisabarelyinvasivemethodwhichallowsmonitoringofglycosphingolipidstoragelocalizationandamountindifferentstructures(nerves,glands,bloodvessels)[2];howeveraminorpresenceofdepositsintheskindoesnotexcludeamoreseverefunctionalimpairmentinothertissues.
Whileseveralrecentworkspointtoanonpathogenicroleofvariantssuchasp.
Ala143Thrandp.
Arg118Cys[33,34],ourresultsaswellasotherreportsofthelitera-turesuggestthatcomplexhaplotypesofintronicvariantsmaypredisposetoGb3accumulationintissues,andthereforetothedevelopmentofFDphenotypeincertainsubjects,presumablywhenadditionalgeneticorepigen-eticgenemodifiersarepresent.
Indeed,alsothevariantp.
Ala143Thrhasbeenoccasionallyassociatedtothede-velopmentofclassicFD[34].
Furtherinvestigationisre-quiredtovalidatethishypothesisandelucidatethemechanismsinvolvedinGLAexpressionimpairmentinpresenceofspecificfrequentpolymorphisms.
ConclusionsThisobservationalstudyperformedinsubjectswithhighrisktopresentFD(twoormoreclinicalmanifestationsorfamilyhistoryofthedisease)helpedtoidentify77newconfirmedpatientsand2individualswithapossiblediagnosisofFD,carryingthenewlydescribedvariantp.
His125Leu(amongatotalof805analyzedsamples).
Additionally,wedetected8subjectswithvariantsrelatedtopossiblelateonsetFDand3subjectswithSNPsinintronicregionassociatedtofunctionalandquantitativedeficiencyofα-GalA.
TheseresultsconfirmthatFDisstillunder-diagnosedinthepopulationandthattherealprevalenceofthediseaseismorelikelyreflectedbythevaluesestimatedinrecentlyperformednewbornscreen-ingstudies.
Thewidevarietyofclinicalsymptomsandsignsshowedbyeachpatient,aswellastheidentifica-tionofimpairedenzymefunctionalityandpathologicalsignsinsubjectswithSNPsinGLA,alsosuggestthatadditionalfactors(i.
e.
geneticorepigenetic)maycon-tributetodetermineFDclinicalphenotype.
AdditionalfilesAdditionalfile1:TableS1.
ListoftheprimersusedforgeneticsequencingofGLA.
Primersaligntointronicsequencingflankingexonsregions.
TableS2.
SubjectswithSNPsinintronicregionsofGLAdetectedthroughgeneticstudy.
Enzymaticactivityisexpressedinμmol/Lh±SD.
NM=notmeasured;X=notindicated.
BoldlettersindicatethesubjectswhoseGLAtranscriptionlevelsandbiopsywereanalyzed.
FigureS1.
Distributionofcollectedsamples.
InpanelAarerepresentedtherelativepercentagesofmedicaldepartmentsthatcollectedthesamples.
InpanelBitisrepresentedthenumberofsamplescollectedineachSpanishregion.
FigureS2.
Anexampleofbloodcardusedtocollectsamples,showingcollectinginstructionsandsymptomslistwithcheckboxes.
FigureS3.
Denovomutationc.
1182delAinGLA,Sequencinganalysisofthesamplefrompatient#69:thechromatogramiscomparedwithawildtypecontrolsequence,mutationsiteisindicatedbyanarrow(A).
PanelBshowspredictedchangesintheaminoacidsequencedeterminedbythenucleotidedeletion.
Dotsattheendofthewildtypesequenceindicatesthatthenucleotidesequencecontinueswithadditional24tripletsbeforestopcodons.
(DOCX4204kb)AbbreviationsDBS:DriedBloodSpots;FD:FabryDisease;Gb3:Globotriaosylceramide;LSDs:LysosomalStorageDisorders;lyso-Gb3:Globotriaosylsphingosine;SD:Standarddeviation;SNPs:SingleNucleotidePolymorphisms;α-GalA:α-GalactosidaseAAcknowledgementsWearegratefultothepatientsforparticipatinginthestudyandtoallthephysicianswhosenttheirsamplesforscreeninganalysis.
WeacknowledgeTaniaVazquez-Santos(FundacionBiomedicaGaliciaSur,Spain)foreditorialsupport.
FundingThisstudywassponsoredbygrantsfromtheFondodeInvestigaciónSanitaria,SpanishNationalInstituteofHealthCarlosIIIISCIII(PI11/00842)toSO,whichisco-foundedbytheEuropeanUnionERDFandfromShireIbéricaHumanGeneticTherapies.
AvailabilityofdataandmaterialsThedatasetsusedandanalyzedduringthecurrentstudyareavailablefromthecorrespondingauthoronareasonablerequest,attheGaliciaSurHealthResearchInstitute,Vigo.
DriedBloodSpotssamplecollectionwasregisteredintheSpanishNationalBiobanksNetwork(Ref.
C.
0003361).
Authors'contributionsSO,correspondingauthor,designedthestudy,setuptheprotocol,performedtheanalysis,draftedthepaperandrevisedit.
Thisauthorisresponsibleforalltheinformationcontributedtothismanuscriptandapprovedthefinalversionofthearticleassubmitted.
IV:PerformedqPCRexperimentsandcontributedtothegeneticdataanalysis,aswellastomanuscriptpreparation.
OSR:Processedthesamples,performedenzymaticactivitymeasurementsandPCRprocessforgeneticstudy.
Shealsoprocessedthebiopsiesforpathologicalanalysis.
LFMCollectedandorganizedsampledata,performedenzymaticactivitymeasurementsandPCRprocessforgeneticstudyinthefirstpartofthestudydevelopment.
BSM:Sheperformedpathologicalanalysisofthebiopsies.
ST:ContributedtobiochemicalmeasurementsandWesternblotexperiments.
JFM:Collectedsamples,revisedclinicaldataandcontributedtomanuscriptpreparation.
LJAE:Contributedclinicaldataandcollaboratedtotherevisedversionofthemanuscript.
MLR:Contributedclinicaldataandcollaboratedtotherevisedversionofthemanuscript.
FMS:Collectedsamples,revisedclinicaldataandcontributedtomanuscriptpreparation.
CN:Designedthestudy,performedbiopsyanalysis,criticallyrevisedthepaper.
Allauthorsapprovedthecontentofthisarticleandconsentedtoitspublication.
EthicsapprovalandconsenttoparticipateThestudywasapprovedbytheGalicianClinicalResearchEthicsCommittee(Ref2009/182,up-dateonMarch29th2011).
Allpatientsinvolvedinthestudysignedtheinformedconsent.
Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page11of13ConsentforpublicationNotapplicable.
CompetinginterestsSO,CNwereprincipalinvestigatorsinprojectssponsoredbythepharmaceuticalcompanyShireIbéricaHumanGeneticTherapies.
SO,CN,BS,JFM,LJAE,MLRandFMS,havealsobeeninvitedtoattendorgiveconferencesbyShireIbéricaHumanGeneticTherapiesandorSanofi-Genzyme.
Publisher'sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
Authordetails1RareDiseasesandPediatricMedicineResearchGroup,GaliciaSurHealthResearchInstitute(IISGaliciaSur).
SERGAS-UVIGO,Vigo,Spain.
2InstituteofCellularBiology,UniversityMedicalCenterGoettingen,Goettingen,Germany.
3DepartmentofPathology,XerenciadeXestionIntegradadeVigo,Sergas,Vigo,Spain.
4DepartmentofInternalMedicine,XerenciadeXestionIntegradadeVigo,Sergas,Vigo,Spain.
5DepartmentofNephrology,HospitalofTorrecardenas,Almeria,Spain.
6MetabolicUnit,HospitaldeCruces,Barakaldo,Spain.
7CoordinadoraGrupoTrabajodeEnfermedadesMinoritariasdelaSEMI,Madrid,Spain.
8DepartmentofPathology,InstituteofBiomedicalResearchofVigo,ClinicalEmeritus,UniversityHospitalofVigo,Vigo,Spain.
9GaliciaSurHealthResearchInstitute(IISGaliciaSur)HospitalAlvaroCunqueiro,Pl.
2Bloquetecnico,zonaA,EstradaClaraCampoamor341,36312Vigo,Pontevedra,Spain.
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Weacceptpre-submissioninquiriesOurselectortoolhelpsyoutondthemostrelevantjournalWeprovideroundtheclockcustomersupportConvenientonlinesubmissionThoroughpeerreviewInclusioninPubMedandallmajorindexingservicesMaximumvisibilityforyourresearchSubmityourmanuscriptatwww.
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Theincidenceofthisrarediseaseisunderestimatedduetodelayeddiagnosis.
Moreover,themanagementoftheidentifiedsubjectsisoftencomplicatedbythedetectionofvariantsofuncleardiagnosticinterpretation,usuallyidentifiedinscreeningstudies.
Weperformedanobservationalstudybasedonbiochemicalandgeneticanalysisof805driedbloodspotsamplesfrompatientswithclinicalsymptomsorfamilyhistoryofthispathology,whichwerecollectedfrom109Spanishhospitals,alloverthecountry.
Results:Weidentified77newdiagnosedpatientswithmutationsrelatedtoclassicalFabrydisease,aswellas2subjectswithc.
374A>T;p.
His125Leu,apossiblenewmutationthatneedtobeconfirmed.
Additionally,wedetected8subjectscarryinggeneticvariantspossiblylinkedtolateonsetFabrydisease(p.
Arg118Cysandp.
Ala143Thr),4caseswithpolymorphismp.
Asp313Tyrand36individualswithsinglenucleotidepolymorphismsinintronicregionsofGLA.
Fiveoftheidentifiedmutations(c.
431delG;c.
1182delA;c.
374A>T;c.
932T>C;c.
125T>A;c.
778G>A),whichwereassociatedwithaclassicalphenotypehavenotbeenpreviouslydescribed.
Moreover3subjectspresentingcomplexhaplotypesmadeupbytheassociationofintronicvariantspresentedimpairedlevelsofGLAtranscriptsandGb3depositsinskinbiopsy.
Conclusions:EnzymaticscreeningforFabryDiseaseinriskpopulation(2ormoreclinicalmanifestationsorfamilyhistoryofthedisease)helpedtoidentifyundiagnosedpatientsandunraveltheimpairmentofGLAexpressioninsomesubjectswithcomplexhaplotypes.
Keywords:Fabrydisease,Lysosomalstoragedisorders,Enzymaticscreening,Intronicvariants,GLAcomplexhaplotypeBackgroundFabrydisease(FD,OMIM#301500)isanX-linkedlyso-somalstoragedisorder(LSD),causedbytheimpairmentofα-galactosidaseA(α-GalA,EC3.
2.
1.
22),whichleadstotheprogressiveaccumulationofglobotriaosylcera-mide(Gb3),anditsderivatives(eg.
lyso-Gb3).
Todate,over700geneticdefectshavebeenidentifiedinGLA,howevernofrequentmutationsweredescribedanditisdifficulttoestablishagenotype-phenotypecorrelation[1].
TheclassicalformofFDisgenerallyassociatedwithaverylowresidualα-GalAactivityinmen(1%)andexclusivelycardiacorrenalmanifesta-tions[4,5].
Duetoaselectionbiasinthescreeningstudies,thesevariantsareofuncleardiagnosticinter-pretationwhichcomplicatesthemanagementoftheidentifiedsubjects[6]andprecludeduptodatethein-clusionofFDintheuniformscreeningpanelsrecom-mendedbytheAdvisoryCommitteeonHereditableDisordersinNewbornsandChildren[7].
Moreover,thepoorspecificityofFDphenotypeoftenresultsindelayeddiagnosis,whichisusuallyestablishedinadulthood,evenifthesymptomsstartedduringchild-hood[8].
ItwasoriginallyreportedthattheincidenceofFDisof1:117,000[9]forthegeneralpopulation;how-ever,pilotstudiesperformedinasymptomaticnewbornsestimatedanincidencerangingbetween1:3000and1:7000livebirths[10,11].
Thesedataandtheresultsofseveralscreeningstudiesperformedinpatientswithmedicalconditionsofdiverseetiology(i.
e.
stroke,renalfailure,ventricularhypertrophy)[4,12,13]highlightthelargenumberofundetectedFDpatients,whoareprob-ablystillunidentifiedormisdiagnosed.
Avoidingdelayeddiagnosisiscrucialtoincreasethequalityandexpectancyoflifeofthepatientswhoneedaspecifictreatment.
InEuropethereare,currently,threeavailabletreatmentswithspecificindicationforFD(agalsidasealfa,agalsidasebetaandmigalastathydro-chloride).
Thesedrugsaremoreefficientinimprovingpatients'conditionwhenthetherapystartsbeforeexten-sivetissuedegenerationisachieved[14,15].
Theaimofthisworkwastoperformanobservationalstudybasedonbiochemicalandgeneticanalysisofsub-jectswhomanifestacombinationofsymptomsandsignssuggestiveofFD(i.
e.
acroparesthesia,cardiachypertrophy,strokeintheyoung,renalfailure)orhaveafamilyhistoryofthedisease.
Thiswillallowtheidentifi-cationofFDpatientswithnon-specificdiagnosis,whocanthereforeaccessmoreappropriatetherapeuticproto-cols.
Theresultsofthestudywillalsoallowtodefineap-propriatecriteriaandparameterstodesignafuturevalidateddiagnosticstudy.
MethodsDesignofthestudyWecarriedoutanobservationalstudybasedonbiochem-icalscreeningofα-GalAandGLAsequenceinsubjectswithagecomprisedbetween1and85years(averageage47.
05±18.
53,standarddeviation(SD)),whopresentedmorethanoneclinicalsymptomorsignassociatedtoFD(i.
e.
ventricularhypertrophy,renalinsufficiency,strokeintheyoung,acroparesthesia,angiokeratome,intolerancetoheathorcold,neuropathicpain,hypertrophicmyocardio-pathy),orwhoweregenetically-linkedrelativesofprevi-ouslydiagnosedpatients.
Enzymeactivitywasquantifiedindriedbloodspotssamples(DBS)byfluorimetricassay.
Aconfirmatorygenetictestwasperformedinmalesub-jectswithactivityT;c.
932T>C;c.
125T>A;c.
778G>A).
Wealsoidentifiedafamilyof2subjects(1maleand1female),whopre-sentedwithclinicalfeaturescompatiblewiththeclassicformofFD,andcarryanewlydescribedvariantinGLA(c.
374A>T;p.
His125Leu),whichneedtobefurtherstudiedtoconfirmitspathogenicstatus.
MoredetailsaboutthesesubjectsaredescribedinTable1.
Thec.
431delGframeshiftmutationwasidentifiedinasubjectbelongingtoafamilyofFDpatients,whohadbeendiagnosedbyenzymeassay.
Theclinicalfeaturesofthisfamily(eg.
angiokeratomas,acroparestesias,cardiachypertrophyandrenaldysfunction)weredescribedbyBarba-Romeroetal.
[22].
Ultrastructuralanalysisofskinbiopsiesfromtwopreviouslyidentifiedmembersofthefamilyrevealedthepresenceofabundantzebrabodiesindifferentstructures(fibroblast,bloodvessel,smoothmuscleandnerveendings)[3].
Thec.
1182delAwasadenovomutationfoundina47-year-oldman(Patient#69),presentingmultiplepara-pelviccystsinbothkidneys,serumcreatinineof1.
6mg/dlandproteinuriaof1g/24h,whoeventuallyunder-wentrenalfailureandtransplant.
Thepatientalsopre-sentedwithcardiomyopathy,smallfiberneuropathy,corneaverticillataandhypohidrosis.
Lowlevelsofα-GalAactivitywereconfirmed,followingmeasurementinplasma(0.
5nmol/h/ml)andleukocytes(4.
2nmol/h/mg),soenzymereplacementtherapywasstarted[23].
ThisVieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page3of13Table1PatientswithdefinitivediagnosisofFabryDisease.
Ageatdiagnosisisindicated.
M:hemizygousmenF:heterozygousfemales.
Activityofɑ-GalAisexpressedinμmol/Lh.
FS:familystudy;X:notindicated,NM.
notmeasured;Symptoms1:RenalFailure2:VentricularHypertrophy;3:Acroparesthesia,4:Angiokeratome;5:Cardiomegaly;6:Fabryfacies;7:Hernia;8:Hypohidrosis;9:Intolerancetoheatorcold;10:Neuropathicpain;11:Cornealclouding;12:Strokeintheyoung,13:Hypertrophic,myocardiopathyCodeAgeSexMedicalDepartmentSymptomsɑ-GalAActivityMutationReference152MNephrology1,2,130.
59±0.
14c.
613C>T;p.
Pro205Ser[35]230MFS0.
76±0.
43346FFS2.
97±0.
16425MFS1.
27±0.
06556FFS3.
33±0.
33647FFS4.
36±1.
58776FFS1.
83±0.
24855FFS2.
24±0.
02924FFS1.
82±0.
381022FFS0.
58±0.
371129MFS1.
04±0.
071262FFS4.
40±0.
031344FFS3.
67±1.
451460MFS0.
28±0.
111582FFS6.
41±0.
011621MFS1.
04±0.
611734MFS0.
48±0.
28180.
5FFS3.
7±1.
73198MGeneticsFS0.
92±0.
1c.
713G>A;p.
Ser238Asn[4]2012MFS1.
2±1.
812157MFS1.
12±0.
1222XFFS3.
76±0.
362352FFS2.
47±0.
222431FFS2.
54±0.
052539FPediatricsFS4.
98262FFS1.
30274MFS0.
692874MCardiology2,FS0.
58±0.
382949F2,31.
01±0.
343052M2,FS0.
56±0.
253115FFS4.
68±0.
3032XFFS4.
61±0.
423310FNephrologyFS4.
75±0.
113439MFS1.
35±0.
013570MFS1.
48±0.
563664MFS1.
89±0.
653721MFS1.
45±0.
73817FFSNM39XMFS1.
67±0.
124038MFS1.
20±0.
104156FFS2.
4±0.
07Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page4of13Table1PatientswithdefinitivediagnosisofFabryDisease.
Ageatdiagnosisisindicated.
M:hemizygousmenF:heterozygousfemales.
Activityofɑ-GalAisexpressedinμmol/Lh.
FS:familystudy;X:notindicated,NM.
notmeasured;Symptoms1:RenalFailure2:VentricularHypertrophy;3:Acroparesthesia,4:Angiokeratome;5:Cardiomegaly;6:Fabryfacies;7:Hernia;8:Hypohidrosis;9:Intolerancetoheatorcold;10:Neuropathicpain;11:Cornealclouding;12:Strokeintheyoung,13:Hypertrophic,myocardiopathy(Continued)CodeAgeSexMedicalDepartmentSymptomsɑ-GalAActivityMutationReference4262FFS4.
9±1.
164330MInternalMedicine2,4,6,101.
27±0.
10c.
836A>G;p.
Gln279Arg[36]4428M2,100.
92±0.
24551F2,4,6,104.
02±0.
014626MFS1.
5±0.
744753M21.
98±0.
444848FFS3.
13±0.
324948F27.
45±0.
365023FFS7.
13±0.
565122MInternalMedicine3,4,5,6,7,8,9,100.
75±0.
05c.
422C>T;p.
Thr141Ile[37]5255F2,4,51.
01±0.
015353M3,4,5,9,102.
25±0.
065448MCardiology21.
05±0.
54c.
679C>T;p.
Arg227*[38]5548M1,20.
86±0.
195646MCardiology21.
19±0.
05c.
242G>C;p.
Trp81Ser[36]5713FInternalMedicine31.
48±0.
3c.
242G>C;p.
Trp81Ser[36]5812FNephrologyFS3.
16±0.
2c.
1232G>A;p.
Gly411Asp[39]5965FFS5.
95±1.
256027MNephrology1,6,100.
94±0.
3c.
778G>A;p.
Gly260ArgNotdescribed6159F123.
58±0.
596253MNeurology1,50.
13±0.
01c.
374A>T;p.
His125LeuNotdescribed(possiblenewmutationthatneedtobeconfirmed)6377FFS1.
04±0.
4264XMGeneticsFS1.
9±0.
84c.
463G>C;p.
Asp155His[40]65XFFSNM6642MCardiologyFS1.
87±0.
53p.
Arg227Gln[41]67XMFS1.
96±0.
476835FInternalMedicine1,3,4,95.
88±0.
37c.
431delG;p.
Gly144Alafs*21Notdescribed6948MGenetics1,10,130.
84±0.
18c.
1182delA;p.
Phe396Serfs*8Notdescribed7037MNephrology10.
63±0.
03c.
509A>T;p.
Asp170Val[42]7123FInternalMedicine3,8,111.
02±0.
13c.
1277_1278delAA;p.
Lys426Argfs*11[43]7219FInternalMedicineFS2.
43±0.
58c.
132G>T;p.
Trp44Cys[44]73XMInternalMedicine2,3,4,8,9,101.
21±0.
54c.
932T>C;p.
Leu311ProNotdescribed7435FNephrology3,6,10,111.
35±0.
01c.
155G>C;p.
Cys52Ser[45]7546MInternalMedicine2,4,80.
85±0.
34c.
572T>C;p.
Leu191Pro[46]7671FGenetics1,10NMc.
337T>C;p.
Phe113Leu[42]7743FNephrologyFS8.
37±0.
84c.
298A>T;p.
Arg100*[47]Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page5of13frameshiftmutationwillcausesthesubstitutionof6aminoacidsintheC-terminalpartoftheproteinandtheformationofaprematurestopcodon(26aminoacidsaremissingcomparedtothewild-typesequence)(Additionalfile1:FigureS3).
Asforthemajorityoftheframeshiftmutations,thetranscriptcarryingthisvariantismostlikelydegradedattheendoplasmicreticulumlevelduetoinstability.
Patient#73withthec.
932T>C;p.
Leu311Propre-sentedseveralclinicalmanifestationsrelatedtoclassicalFD,suchasventricularhypertrophy,acroparesthesias,angiokeratoma,hypohidrosis,neuropathicpainwithintolerancetoheatorcoldandrenalimpairment(albu-minuria160μg/min).
α-GalAactivitywasalsomea-suredinplasma(1.
1nmol/ml/h)andleukocytes(0.
13nmol/mg/h),whileGb3/Ga2ratiowas1.
6.
Since2011thispatientisunderenzymereplacementtherapy.
Patient#79withthec.
125T>A;p.
Met42Lysmuta-tionspresentedmainlyneurologicalsymptoms(acuteischemicstrokewithlacunarhemibulbarlesion)associ-atedtochronicrenaldysfunctionwithincreasedprotein-uria/creatinineratio(1.
50mg/mg).
ThepatientalsopresentwithclassichallmarksofFD,suchasangiokera-tomasandcorneaverticillata.
Thediagnosiswascon-firmedbymeasuringLyso-Gb3levelsinplasma,whichwerequantifiedin16ng/ml(Cut-offvalues0–3.
5).
Patient#60withthec.
778G>A;p.
Gly260Argpre-sentedsevereperipheralsensorialneuropathy,hypohi-drosis,corneaverticillata,microalbumin,abdominalpainandmultipleinflammatorymesentericandin-guinaladenopathiesconfirmedbybiopsy,whichalsoshowedevidencesofcellulardeposits.
Thispatientisunderenzymereplacementtherapysince2011[24].
Patient#61,wasidentifiedbyfamilialstudy,followingdefinitivediagnosisoftheindexcase[#60]inhiscen-teroforigin.
Thesubject#62,whobearsthec.
374A>T;p.
His125Leumutation,wasderivedfromaneurologyde-partmentandpresentedwithstrokeatyoungageassoci-atedtorenalinsufficiencyandcardiomegaly,thispatientunderwentEnzymereplacementtherapy.
Hismother(subject#63)isheterozygousforthesamemutation.
Additional51cases(6malesand45females)pre-sentedgeneticvariantsinGLAofuncertainsignificance.
Polymorphismp.
Asp313Tyr(c.
937G>T)wasfoundin7samples(4malesand3females).
Wealsodetectedthecontrovertedlate-onsetvariantsp.
Ala143Thr(c.
427G>A,foundin3femalesand1male)andp.
Arg118Cys(c.
352C>T,foundin4females)(Table2).
Moreover,singlenucleotidepolymorphisms(SNPs)inintronicsequencesweredetectedin36individuals(1maleand35females).
Complexhaplotypesmadeupof2ormoreintronicsubstitutionswerepresentin19ofthesubjectswithintronicSNPs(Additionalfile1:TableS2).
Intheremaining675cases(435malesand240females),mutationswerenotidentifiedinthecodingregionofthegene,norincloseintronicfragments(around50bpineachdirection).
Thepositivepredictivevaluefortheenzymatictestinmaleswas80.
43%(CI95%:67.
88–92.
99%);themethodaccuracywas98.
12%(CI95%:96.
80–99.
44%)andthefalsepositiveratewas1.
88%.
Thesevalueswerecalculatedtakingintoaccountthe37malepatientswithconfirmedFDdiagnosis,amongthe479malesubjectsthatwerescreened.
Meanactivityinthewholemalecohortwasof7.
6μmol/Lh±4.
02andmedianwas3.
08±1.
57.
Thegeneticstudywasnotgenerallyperformedinmaleswithactivityabovethecut-offvalue,however22samplesfrommenwithα-GalAactivitybetween2.
6and5μmol/Lhweresequencedbyspecificrequestoftheresponsiblephysicians,duetotheirclinicalmanifesta-tions.
Amongthesepatientsweidentifiedthe4subjectswiththep.
Asp313Tyrpolymorphism,theindividualwiththecontrovertedlateonsetvariantp.
Ala143Thrandthesubjectwithacomplexintronichaplotypeinintronicre-gion(rs2071225,rs5903184,rs2071397andrs2071228).
ComplexhaplotypesanalysisDuetotheclinicalfeaturespresentedby3ofthesubjects(#114;#115and#116),whowerefoundtobearacomplexassociationofintronicvariantsinGLA,weanalyzedtran-scriptlevelsandGb3depositspresenceinleukocytessam-plesandskinbiopsy,respectively,inordertodetectpossibledefectsingeneexpression.
Table1PatientswithdefinitivediagnosisofFabryDisease.
Ageatdiagnosisisindicated.
M:hemizygousmenF:heterozygousfemales.
Activityofɑ-GalAisexpressedinμmol/Lh.
FS:familystudy;X:notindicated,NM.
notmeasured;Symptoms1:RenalFailure2:VentricularHypertrophy;3:Acroparesthesia,4:Angiokeratome;5:Cardiomegaly;6:Fabryfacies;7:Hernia;8:Hypohidrosis;9:Intolerancetoheatorcold;10:Neuropathicpain;11:Cornealclouding;12:Strokeintheyoung,13:Hypertrophic,myocardiopathy(Continued)CodeAgeSexMedicalDepartmentSymptomsɑ-GalAActivityMutationReference7855FCardiology9,47.
61±0.
63c.
53T>G;p.
Phe18Cys[48]7927MClinicalPathology1,121.
98±0.
25c.
125T>A;p.
Met42LysNotdescribedVieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page6of13Individuals#115and#116werea7-year-oldgirlandher42-year-oldfather,whopresentedhaplotypers2071225,rs5903184,rs2071397andrs2071228.
Thefatherreferredhearinglossandacroparesthesiaassociatedtomicroalbuminuriaandrenaldysfunction.
Thegirlreferrederraticpainattheextremities,withoutpaincrisis,recurrentabdominalpainrelatedtofoodin-gestionandpoorsweating.
Angiokeratomas,skinlesionsororganomegalywerenotdetected.
Cardiorespiratoryandneurologicalexaminationswerenormal,aswellasrenalfunction.
GLAtranscriptionwassignificantlyimpairedinthesesubjectsatmRNAlevel(mRNA13%ofcontrolin#115and7%ofcontrolin#116),(Fig.
1a),aswellasatproteinlevel(α-GalAexpressionisdecreasedcomparedtoactinendogenouscontrolintheWesternblot),(Fig.
1b).
AlowpercentageofGb3deposits(7affectedfibro-blastsperexaminedsection)wasdetectedbyCD77immunolabelingindermisfibroblastsof#115skinbiopsy(Fig.
2a,B).
PositiveGb3cellswerealsofoundinoneeccrineepithelialgland(Fig.
2c).
WewerenotabletodetectthepresenceofGb3depositsinendothelialcellsofbloodvesselsornervefibers.
Biopsyofpatient#116wasnotavailable.
AlthoughtheseresultsshowthatGLAtranscriptionisimpairedinpatients#115and#116,wecannotconfirmthediagnosisofFDinthesesubjects,sincewedidnotmeasureGb3orLyso-Gb3levelsinurineorplasma,nei-therweshowedthepresenceoflamellarbodies.
Patient#114wasa47-year-oldwoman,presentingtheassociationofintronicvariantsrs2071225andrs2071228andα-GalAactivityof4.
6μmol/Lh.
ShereportedmultipleclinicalsymptomsrelatedtoclassicalFDin-cludingacroparesthesia,neuropathicpain,hypohidrosis,skinlesionsandmuscleweakness.
Similarlytothepreviouslyanalyzedsubjects,thiswomanpresentedimpairedGLAtranscription(30%ofcontrol)andGb3depositsinskinfibroblasts(10affectedcellsforeachexaminedsection)(Fig.
3)wereidentifiedthroughimmunohistochemistry.
AlsointhiscaseGb3orLyso-Gb3levelsinurineorplasmaarenotavailabletoconfirmFDdiagnosis.
DiscussionInthispaper,wehaveshownhowalargenumberofpatientswithFDcanbeidentifiedthroughscreeningofriskpopulations(2ormoresymptomsrelatedtoFDorfamilyhistory),whichstressesthattherateofdelayeddiagnosisinFDremainsexcessivelyhigh.
LatediagnosisisdetrimentalinFD,sinceitpreventspa-tientsfrombeingtreatedpromptlywiththeavailablespecifictherapies.
VanderToletal.
[25]conductedasystematicre-viewofFDdiagnosisstudiesincludingscreeningofnewbornsandriskpopulation,torecalculatetheprevalenceofthedisease.
AccordingtothisarticletheestimatedprevalenceofclassicalFDinhighriskpopulations(45studiescomputed)is0.
12%,althoughTable2SubjectswithlateonsetvariantsinGLAorp.
Asp313Tyrpolymorphism,Activityofɑ-GalAisexpressedinμmol/Lh±Standarddeviation.
Ageatdiagnosisisindicated.
M:hemizygousmenF:heterozygousfemales.
FS:familystudy;X:notindicated,NM.
Notmeasured;Symptoms1:RenalFailure2:VentricularHypertrophy;3:Acroparesthesia,4:Angiokeratome;5:Cardiomegaly;6:FabryFacies;7:Hernia;8:Hypohidrosis;9:Intolerancetoheatorcold;10:Neuropathicpain;11:Cornealclouding;12:Strokeintheyoung,13:Hypertrophic,myocardiopathyCode#AgeSexMedicalDepartmentSymptomsɑ-GalAActivityVariant8042FNeurologyFS3.
6±1.
76c.
352C>T;p.
Arg118Cys8151FFS2.
33±0.
438228FFS5.
83±0.
138331FFS6.
64±0.
1384XMGenetics6,134.
21±2.
02c.
427G>A;p.
Ala143Thr8545FFSNM86XF2,13NM8717FNeurologyFS3.
05±0.
178875MCardiologyFS3.
77±0.
22c.
937G>T;p.
Asp313Tyr8957FNephrologyFS4.
28±0.
7990XMGeneticsFS5.
2±0.
879135MNeurologyFS3.
8±1.
57921FNephrology18.
69±1.
939348MNeurology3,55.
5±0.
419445FClinicalPathology2,56.
63±0.
56Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page7of13thisvalueincreasesto0.
62%whenincludingallthedetectedvariantsinGLA(variantsofuncertainsig-nificanceorneutralvariants),whileitreportedaprevalenceofGLAvariantsof0.
04%innewbornscreenings(6studiescomputed).
ThemostrecentnewbornscreeningsperformedinWashington[10]andMissouri[11]reportedanFDprevalenceof0.
012%inlivebirthsor0.
034%respectively.
ThepercentageofpatientswithclassicFDinthepresentstudywasof7.
72%inthemalecohortand9.
56%inthewholegroupofsubjects;however,thesevaluesarenotrepresentativeofthegeneralpopulation,abFig.
1GLAmRNAlevels(a)andWesternblotofα-GalAandactin(b)measuredinleukocyteextractsofsamplesfromsubjects#115and#116.
InpanelA,barsrepresentrelativelevelsofmRNAexpression(ΔΔCts)normalizedtothecontrol.
Twodifferentcontrols(healthyvolunteers'samples)wereusedinindependentexperiments(controlbar).
InpanelB,Control()representsthesamplefromahealthyvolunteerandControl(+)representsthesamplefromapatientdiagnosedwithFDcarryingamissensepathogenicmutationacbdFig.
2ImmunofluorescenceofGb3depositinskinbiopsyfromsubject#115.
Depositsarestainedingreen(CD77_FITC)andaffectedfibroblastsareindicatedwitharrows(a,b),bluestainingrepresentsnuclei(DAPI)andredstainingpolymerizedactin(Rhodamine-Phalloidin).
Panelcshowsaneccrineglandwithgreendepositsandpaneldshowsanon-affectedfieldofthesamebiopsy.
Collagenfibersareeasilydetectedinallpanelsduetoauto-fluorescenceofthespecimenVieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page8of13sincethesamplesizeisrelativelysmallandsubjectsselectioncriteriaincludedindividualswithhighclinicalsuspicion(2ormoresymptoms),aswellasgenetically-linkedrelativesofFDpatients,whocompriseobligatecarriersofmutantalleles.
Moreover,malesubjects'sampleswerepreferentiallysentforanalysistoourcenter,bytheirresponsiblephy-sicians,sincetheFDphenotypeisusuallymoreeasilyrecognized.
Thelackofhomogeneousselectioncriteriainourcohort,justifiestheextremelyhighpercentageofpositivecases,whichthereforecannotbedirectlycom-paredwiththeprevalencevalueinriskhomogeneouspopulationsestimatedbyVanderTolletal.
intheirmeta-analysisoftheliterature.
Ourgrouprecentlypub-lishedtheresultsofascreeningperformedintheSpan-ishnewborns[26],whichdeterminethattheprevalenceofthediseaseis0.
013%inmalesfromtheNorthwestofSpain.
Therefore,weestimatethattherealprevalenceofFDinourcountrycouldbearound0.
013%orslightlyhigherconsideringthatthemajorityofthedefinitivediagnosisofSpanishadultpatientsdescribedinthepresentstudywasdetectedintheMediterraneanarea,whilethenewbornscreeningonlyincludessubjectsfromtheNorthwest-Atlanticregion.
Althoughthisstudypursuedreachingadefinitivediagnosisintheanalyzedsamples,duetothelackofhomogeneousselectioncriteria,itwasdesignedasanobservationalstudy,whichcanhelptodefinepreciseparametersforavalidateddiagnosticstudy.
Thecut-offpointthatwedefinedsettingupthepresentscreeningwasof2.
6μmol/Lh,estimatedmeasuringactivityinDBSfrom5FDpatientswithdefinitivediagnosisand15healthyvolunteers.
Thelownumberofpositivepatientsavailablewhensettinguptheassaymayhavecausedanoverestimationofthecut-offpoint,whichconsequentlyledtoahighfalseposi-tiverateandalowpositivepredictivevalue.
Ifwerecalculatethethresholdforα-GalAactivitybasedonthedataobtainedinthemalecohortanalyzedinthepresentwork,wewillobtainavalueof2.
14μmol/Lh,(percentileofpositivepatients+0.
5%).
Ifwewouldhaveappliedthiscut-offtoourcohortthepositivepredictedvalueoftheassaywouldhavebeen90.
24%(CI79.
94–100%)thefalsepositiverate0.
84%andmethodaccuracy99.
16%(CI98.
25–100%),(orMethodaccuracy99.
3%,positivepredictivevalue92.
68%andfalsepositiverate0.
63%,assumingthatp.
His125Leuisconfirmedtobepathogenic.
)Thelargestfamilyidentifiedinthisstudy,countedwith19members(11femalesand6males)withthep.
Pro205Sermutation(Fig.
4).
Theindexcasewasamalepatientwithseverehypertrophicmyocardiopathywhowasdiagnosedafter25yearsofdialysisandtworenaltransplants.
Thisisaperfectexampleofthedramaticconsequencesofdelayeddiagnosisforpatientsandfam-ilies,whichalsodeterminesimportanteconomicreper-cussionstotheHealthCareSystem.
Nevertheless,withoutimplementingasystematicscreeningforFDincertainriskconditions(i.
e.
renalfail-ure,strokeintheyoung,etc),thenon-specificityofthesymptomsmakesdifficulttoestablishadefinitivediag-nosis.
Indeed,inthelargefamilymentioned,theclinicalfeaturesweredifferentineachmember.
Althoughmostofthemareaffectedbyrenalandcardiacsymptoms,twomalespresentedneurologicalsymptomsratherthanrenaldysfunctionandoneofthemprematurelydiedduetostroke.
Noneofthesepatientspresentedcorneaverticil-lata,whichistraditionallyconsideredasahallmarkofFD.
Thepresentstudyalsocontributedtotheidentifica-tionoffivenewmutations,whichhadnotbeenabcFig.
3ImmunofluorescenceofGb3deposits(CD77_FITC_green,affectedfibroblastsindicatedwithyellowarrows)inskinbiopsyfrompatient#114(a:affectedfield;b:non-affectedfield)andGLAmRNAlevelsinleukocyteextractformpatient#114(c)comparedtosamplefrom2healthyvolunteers(controlbar)Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page9of13previouslydescribed(c.
431delG;c.
1182delA;c.
932T>C;c.
125T>A,c.
778G>A).
PathogeniccharacterofthesemutationswaspredictedbyinsilicoanalysisanddefinitiveFDdiagnosiswasconfirmedforthesepatients,asdetailedintheresultssection,byα-GalAactivitymeasuredinleukocytes(formalesonly),LysoGb3/Gb3levelsinplasmaorurineorbiopsyexamination.
Moreover,wedetectedafamilybringingthepossiblepathogenicmutationc.
374A>T,p.
His125Leu,whichneedtobefurtherstudied.
Theindexcaseofthisfamilyisamalesubjectwithaα-GalAactivitymeasuredinDBSof±0.
130.
01μmol/hL,whopresentswithclinicalsymptomscompatiblewithFD,howeverwewerenotabletocollectdatatoconfirmadefinitivediagnosisofthesesubjects(ej.
Gb3levelsorbiopsy).
Ontheotherhand,wealsodetected8individualswithpossiblelate-onsetFD,aswellas36subjectswithSNPsinintronicregions(oftenformingcomplexhaplotypes).
Thesekindsofvariantsaregenerallynon-pathogenicpolymorphismsandtheirpresenceisnotsufficienttodetermineFDphenotype[27],forthisreasontheywerenotincludedinthemethodaccuracycalculation.
None-theless,frequentvariantssuchasp.
Arg118Cys(0.
04%)andp.
Ala143Thr(0.
09%)havebeenassociatedtolateonsetFD[4,13,25]andthedeficientexpressionofα-GalAhasbeenreportedinsubjectswithcomplexintronichaplotypes[28,29].
AsstatedbySchiffmannetal.
[6],theclinicalhetero-geneityofFD,eveninpatientscarryingthesamemuta-tion,isanevidencethattheindividualriskofdevelopingcomplicationsmaydependonα-GalAdeficiencyinter-playwithotherfactors(i.
e.
genetic,epigeneticorenvir-onmental).
Anaccuratefollow-upofsubjectspresentingvariantsofunknownsignificanceandabiopsyexamin-ationarenecessarytoolstodetermineifFDwillbeeventuallydeveloped.
Inthepresentstudy,weanalyzedGLAtranscriptionandGb3expressioninthreesubjectspresentingintronicSNPs,sincetheirclinicalfeatureswerehighlysuggestiveofFD.
Subjects#115and#116presentedacomplexhaplotype,previouslyassociatedwithFD[30–32].
Gervasetal.
[32]describedGLAtranscriptionim-pairmentinaSpanishfamilywiththiscomplexhaplo-type.
Theaffectedmembersofthefamilypresentedgalactosphingolipidaccumulationinfibroblastsand,insomesubjects,glycolipidstoragewasalsopresentinrenaltubulesandglomeruli.
Asaddressedinthisarticle,thetran-scriptiondefectcouldbetheconsequenceofthealterationofanuclearproteinbindingsite,causedbySNPrs2071225.
Schelleckesetal.
[30]alsoreportedFD-relatedcryptogenicstrokeandsmallfibreneuropathyintwofamilieswithin-tronicSNPsintheGLApromoterregionandshowedthatgenetranscriptionisimpairedduetoinefficientbindingofthetranscriptionfactorEBtothepromoter.
Subjects#115and#116camefromthesamegeographicareaofthefamilydescribedbyGervasetal.
andwecannotruleoutthattheybelongtothesamefamily.
Ourresultsarecomparabletothosedescribedinthisarticle[32]intermsofGLAmRNAlevels,althoughthenumberofaffectedfibroblastsislowinthebiopsyweexamined.
Sincethisbiopsywasobtainedfroma7-year-oldgirl,thelowamountofGb3inthisspecimenmayreflectanearlystageofanFDprogressivecondition.
Duetotheidentifiedfunctionalandquantitativedefi-ciencyofα-GalAassociatedtoincipientformationofGb3depositinskinbiopsy,weconcludethatitispos-siblethatsubjects#115-and#116are,infact,FDpa-tients;however,foradefinitivediagnosis,abiopsyexaminationisrequiredforsubject#116andasecondbiopsyexaminationisnecessarytoconfirmstoragepro-gressioninsubject#115.
Wealsoanalyzedthecaseofsubject#114whoisheterozygousforthers2071225andrs2071228SNPs,butshowsmanyclinicalsignsusuallypresentinclassicFDpatients.
Similarlyto#115and#116,thissubjectpresentsapartialimpairmentofGLAlevelsandfunc-tionality,aswellasalowdetectableamountofGb3depositsdetectedintheskinbiopsy.
Ascommentedpreviously,thisdataarenotsufficienttoestablishdefini-tivediagnosis,althoughanaccuratefollow-upofthesubjectisadvisable.
Indeedthepresenceoftypicalclinicalmanifestationssuggeststhatα-GalAactivityinFig.
4GenetictreeofafamilywithmembersaffectedbyFD,whocarrythep.
Pro205Sermutation.
Circlesindicatefemales,squaresindicatemales.
Blacksquaresarethehomozygousmalemembersofthefamilyandblack/whiteconcentriccirclesindicatethefemaleheterozygouspatientsVieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page10of13functionalorganssuchaskidneyandheartcouldbesignificantlyimpaired,inspiteoftheresidualactivityof4.
66±0.
67μmol/LhmeasuredinDBS.
Asmentionedinreference[6],aheart-kidney-cerebrovascularsystemgra-dientseemstohavebeenestablishedforFDandatruethresholdforpathogenicenzymeactivityintissuesisdifficulttoestimate.
Moreoverexternalfactors,suchasrandomX-chromosomeinactivation,hormoneregulatedtranscriptionmechanismsandotherepigeneticfactors,cancontributetodevelopthephenotype.
Forallthesereasons,abiopsyexaminationisstronglyrecommendedforspecificdiagnosisofsubject#114,ideallyperformedinkidneyorheart.
SkinbiopsyisavaluabletoolfordiagnosisofFDpatients,sinceitisabarelyinvasivemethodwhichallowsmonitoringofglycosphingolipidstoragelocalizationandamountindifferentstructures(nerves,glands,bloodvessels)[2];howeveraminorpresenceofdepositsintheskindoesnotexcludeamoreseverefunctionalimpairmentinothertissues.
Whileseveralrecentworkspointtoanonpathogenicroleofvariantssuchasp.
Ala143Thrandp.
Arg118Cys[33,34],ourresultsaswellasotherreportsofthelitera-turesuggestthatcomplexhaplotypesofintronicvariantsmaypredisposetoGb3accumulationintissues,andthereforetothedevelopmentofFDphenotypeincertainsubjects,presumablywhenadditionalgeneticorepigen-eticgenemodifiersarepresent.
Indeed,alsothevariantp.
Ala143Thrhasbeenoccasionallyassociatedtothede-velopmentofclassicFD[34].
Furtherinvestigationisre-quiredtovalidatethishypothesisandelucidatethemechanismsinvolvedinGLAexpressionimpairmentinpresenceofspecificfrequentpolymorphisms.
ConclusionsThisobservationalstudyperformedinsubjectswithhighrisktopresentFD(twoormoreclinicalmanifestationsorfamilyhistoryofthedisease)helpedtoidentify77newconfirmedpatientsand2individualswithapossiblediagnosisofFD,carryingthenewlydescribedvariantp.
His125Leu(amongatotalof805analyzedsamples).
Additionally,wedetected8subjectswithvariantsrelatedtopossiblelateonsetFDand3subjectswithSNPsinintronicregionassociatedtofunctionalandquantitativedeficiencyofα-GalA.
TheseresultsconfirmthatFDisstillunder-diagnosedinthepopulationandthattherealprevalenceofthediseaseismorelikelyreflectedbythevaluesestimatedinrecentlyperformednewbornscreen-ingstudies.
Thewidevarietyofclinicalsymptomsandsignsshowedbyeachpatient,aswellastheidentifica-tionofimpairedenzymefunctionalityandpathologicalsignsinsubjectswithSNPsinGLA,alsosuggestthatadditionalfactors(i.
e.
geneticorepigenetic)maycon-tributetodetermineFDclinicalphenotype.
AdditionalfilesAdditionalfile1:TableS1.
ListoftheprimersusedforgeneticsequencingofGLA.
Primersaligntointronicsequencingflankingexonsregions.
TableS2.
SubjectswithSNPsinintronicregionsofGLAdetectedthroughgeneticstudy.
Enzymaticactivityisexpressedinμmol/Lh±SD.
NM=notmeasured;X=notindicated.
BoldlettersindicatethesubjectswhoseGLAtranscriptionlevelsandbiopsywereanalyzed.
FigureS1.
Distributionofcollectedsamples.
InpanelAarerepresentedtherelativepercentagesofmedicaldepartmentsthatcollectedthesamples.
InpanelBitisrepresentedthenumberofsamplescollectedineachSpanishregion.
FigureS2.
Anexampleofbloodcardusedtocollectsamples,showingcollectinginstructionsandsymptomslistwithcheckboxes.
FigureS3.
Denovomutationc.
1182delAinGLA,Sequencinganalysisofthesamplefrompatient#69:thechromatogramiscomparedwithawildtypecontrolsequence,mutationsiteisindicatedbyanarrow(A).
PanelBshowspredictedchangesintheaminoacidsequencedeterminedbythenucleotidedeletion.
Dotsattheendofthewildtypesequenceindicatesthatthenucleotidesequencecontinueswithadditional24tripletsbeforestopcodons.
(DOCX4204kb)AbbreviationsDBS:DriedBloodSpots;FD:FabryDisease;Gb3:Globotriaosylceramide;LSDs:LysosomalStorageDisorders;lyso-Gb3:Globotriaosylsphingosine;SD:Standarddeviation;SNPs:SingleNucleotidePolymorphisms;α-GalA:α-GalactosidaseAAcknowledgementsWearegratefultothepatientsforparticipatinginthestudyandtoallthephysicianswhosenttheirsamplesforscreeninganalysis.
WeacknowledgeTaniaVazquez-Santos(FundacionBiomedicaGaliciaSur,Spain)foreditorialsupport.
FundingThisstudywassponsoredbygrantsfromtheFondodeInvestigaciónSanitaria,SpanishNationalInstituteofHealthCarlosIIIISCIII(PI11/00842)toSO,whichisco-foundedbytheEuropeanUnionERDFandfromShireIbéricaHumanGeneticTherapies.
AvailabilityofdataandmaterialsThedatasetsusedandanalyzedduringthecurrentstudyareavailablefromthecorrespondingauthoronareasonablerequest,attheGaliciaSurHealthResearchInstitute,Vigo.
DriedBloodSpotssamplecollectionwasregisteredintheSpanishNationalBiobanksNetwork(Ref.
C.
0003361).
Authors'contributionsSO,correspondingauthor,designedthestudy,setuptheprotocol,performedtheanalysis,draftedthepaperandrevisedit.
Thisauthorisresponsibleforalltheinformationcontributedtothismanuscriptandapprovedthefinalversionofthearticleassubmitted.
IV:PerformedqPCRexperimentsandcontributedtothegeneticdataanalysis,aswellastomanuscriptpreparation.
OSR:Processedthesamples,performedenzymaticactivitymeasurementsandPCRprocessforgeneticstudy.
Shealsoprocessedthebiopsiesforpathologicalanalysis.
LFMCollectedandorganizedsampledata,performedenzymaticactivitymeasurementsandPCRprocessforgeneticstudyinthefirstpartofthestudydevelopment.
BSM:Sheperformedpathologicalanalysisofthebiopsies.
ST:ContributedtobiochemicalmeasurementsandWesternblotexperiments.
JFM:Collectedsamples,revisedclinicaldataandcontributedtomanuscriptpreparation.
LJAE:Contributedclinicaldataandcollaboratedtotherevisedversionofthemanuscript.
MLR:Contributedclinicaldataandcollaboratedtotherevisedversionofthemanuscript.
FMS:Collectedsamples,revisedclinicaldataandcontributedtomanuscriptpreparation.
CN:Designedthestudy,performedbiopsyanalysis,criticallyrevisedthepaper.
Allauthorsapprovedthecontentofthisarticleandconsentedtoitspublication.
EthicsapprovalandconsenttoparticipateThestudywasapprovedbytheGalicianClinicalResearchEthicsCommittee(Ref2009/182,up-dateonMarch29th2011).
Allpatientsinvolvedinthestudysignedtheinformedconsent.
Vieitezetal.
OrphanetJournalofRareDiseases(2018)13:52Page11of13ConsentforpublicationNotapplicable.
CompetinginterestsSO,CNwereprincipalinvestigatorsinprojectssponsoredbythepharmaceuticalcompanyShireIbéricaHumanGeneticTherapies.
SO,CN,BS,JFM,LJAE,MLRandFMS,havealsobeeninvitedtoattendorgiveconferencesbyShireIbéricaHumanGeneticTherapiesandorSanofi-Genzyme.
Publisher'sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
Authordetails1RareDiseasesandPediatricMedicineResearchGroup,GaliciaSurHealthResearchInstitute(IISGaliciaSur).
SERGAS-UVIGO,Vigo,Spain.
2InstituteofCellularBiology,UniversityMedicalCenterGoettingen,Goettingen,Germany.
3DepartmentofPathology,XerenciadeXestionIntegradadeVigo,Sergas,Vigo,Spain.
4DepartmentofInternalMedicine,XerenciadeXestionIntegradadeVigo,Sergas,Vigo,Spain.
5DepartmentofNephrology,HospitalofTorrecardenas,Almeria,Spain.
6MetabolicUnit,HospitaldeCruces,Barakaldo,Spain.
7CoordinadoraGrupoTrabajodeEnfermedadesMinoritariasdelaSEMI,Madrid,Spain.
8DepartmentofPathology,InstituteofBiomedicalResearchofVigo,ClinicalEmeritus,UniversityHospitalofVigo,Vigo,Spain.
9GaliciaSurHealthResearchInstitute(IISGaliciaSur)HospitalAlvaroCunqueiro,Pl.
2Bloquetecnico,zonaA,EstradaClaraCampoamor341,36312Vigo,Pontevedra,Spain.
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Weacceptpre-submissioninquiriesOurselectortoolhelpsyoutondthemostrelevantjournalWeprovideroundtheclockcustomersupportConvenientonlinesubmissionThoroughpeerreviewInclusioninPubMedandallmajorindexingservicesMaximumvisibilityforyourresearchSubmityourmanuscriptatwww.
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