Jorge A. Bevilacqua1,6, Yves Mathieu2, MarOn Krahn2, Marc

PS2Group1-026/#478
CALPAINOPATHIESINCHILE
JorgeA.Bevilacqua1,6,YvesMathieu2,MarFnKrahn2,MarcBartoli2,ClaudiaCasFglioni3,KarinKleinsteuber4,JorgeDíaz5,FrancescaPuppo2,
MathieuCerino2,SebasFenCourrier2,SvetlanaGorokhova2,AlejandraTrangulao6,NataliaMiranda1,PatricioGonzález-Hormazábal6,
MaríadelosÁngelesAvaria4,J.A.UrFzberea7,PabloCaviedes6,LilianJara6,NicolasLevy2
1.UnidadNeuromuscular,DepartamentodeNeurologíayNeurocirugía,HospitalClínicoUniversidaddeChile,SanFago,Chile;
2.GénéFqueMédicaleetGénomiqueFoncFonnelle,INSERMUMR_S910,Universitéd'Aix-Marseille,FacultédeMédecinedelaTimone,Marseille,France;
3.UnidaddeNeuropediatría,ClínicaLasCondes,SanFago,Chile;4.Neuropediatría,HospitalRobertodelRío,UniversidaddeChile,SanFago,Chile;
5.CentrodeImagenología,HospitalClínicoUniversidaddeChile,SanFago,Chile;6.InsFtutodeCienciasBiomédicas,FacultaddeMedicina,UniversidaddeChile,SanFago,Chile;
7.UnitéNeuromusculaire,HôpitalMarindeHendaye,AP-HP,Hendaye,France.
BackgroundandGeneralPurpose
CalpainopathiesinChile
1.  Limb girdle muscular dystrophy 2A (LGMD2A; MIM#253600) is an autosomal recessive disorder
causedbymutaFonsofthecalpaingene(CAPN3)whichencodesforcalpain-3(CAPN3)
2.  Calpain-3 is a muscle specific calcium-acFvated neutral protease involved in remodelling of the
sarcomere
3.  No paFents with calpainopathy have been reported hitherto from Chile, a country of 17 million
inhabitantsandastrongBasqueascendance
4.  We describe five paFents belonging to four unrelated Chilean families, harbouring CAPN3
mutaFons
5.  TwoofthemutaFonsfoundinChileanpaFentsarenovel
SubjectsandMethods
1.  ThelocalEthicsCommiVeeoftheHospitalClínicoUniversidaddeChileandtheChileanNaFonalCommissionof
ScienFficResearchandTechnology(CONICYT)approvedthestudyprotocol.
2.  PaFentswerereferredfromallacrossthecountry
3.  Diagnosiswasbasedonclinicalfeatures,musclebiopsybyimmunohistochemistrytoexcludeotherlimbgirdle
dystrophyormyopathyandclinicalfindingsconsistentwithcalpainopathy(Figure1)
4.  Work-up consisted of FuncFonal Motor Measure (MFM), echocardiogram, baseline spirometry, standardized
electrophysiologicalprotocolsandsequenFalenFrebodymuscleMRI
5.  ThehighestserumCKacFvityvaluewasusedforanalysis.
Geneanalysis.
GeneFc screening for LGMD mutaFons was performed in Marseille, France in four paFents 1-4 (GénéFque
MédicaleetGénomiqueFoncFonnelle,INSERMUMR_S910,Universitéd'Aix-Marseille,FacultédeMédecinedela
Timone), on a NGS panel of 306 genes involved in neuromuscular diseases, using HaloPlex (Agilent
TechnologiesTM) enrichment method and sequenced on the NextSeq500 (IlluminaTM) by HelixioTM (Biopôle
Clermont-Limagne,France).
Families of paFents 1 to 4 were analysed in SanFago, Chile (Programa de GenéFca Humana, ICBM, Facultad de
Medicina, Universidad de Chile) or in Marseille, France (PaFent 5) by Sanger sequencing using primers flanking
eachmutaFon.PrimersequencesandtheamplificaFoncondiFonsareavailableonrequest.
Magne<cResonanceImaging.
SequenFalenFrebodyaxialT1WandSTIRMRIimageswereanalysedandscoredformusclefaVyreplacementand
oedema.
1.  Standardized protocols were applied using a 1.5T system (Magnetom Symphony Maestro-Class, Siemens,
Erlangen,Germany).ExploraFonwasdoneinsupineposiFon,coveringfromthetemporalregionstotheankle
level.AxialT1-weightedspin-echo(T1W)sequence(TR:610-640ms,TE:11-12ms)andaxialT2Short–Time
InversionRecovery(STIR)sequence(TR:3700-4500ms,TE:51-64ms,TI:150).Slicethicknesswas8mm,with
agapof4mm.Thefieldofviewwasbetween350-490mm,withamatrixofapproximately384x240.DICOM
OsiriXviewerwasusedforanalysis.STIRsequenceswereusedforedema
2.  ScorewasadaptedfromKornblumetal.(2006).Stage0,normal(score0);stage1,discretemoth-eaten,with
sporadicscaVeredT1hyperintenseareas(score1);stage2a,moderatemoth-eaten,withnumerousscaVered
T1 hyperintense areas (score 2); stage 2b, late moth-eaten, with numerous confluence areas of T1
hyperintensity(score3);stage3,completefaVydegeneraFonandfibrousreplacementofmusclebyconnecFve
Fssueandfat(score4)
Figure3.MuscleMRIfeatures.AttheboVomofeachpanelisindicateddiseaseduraFonatthemomentofMRI.Notetheearlier
involvementoftheposteriorcompartmentofthethighs,glutealandparaspinalmuscles.Atlaterstagesofdisease,theposterior
2, ater 34 years of
compartment of the legs, proximal paraspinal muscles and shoulder girdle become also involved. PaFent
diseaseshowsinvolvementofallmusclesegments,includinghearmsand.NoteinPaFent4,therelaFvesparingoftheforearms
attrunklevel.
Figure 2. Pedigree of the four Chilean families harbouring CPN3
muta<ons.NotethatthenovelmutaFonc.107delG/p.Gly36Valfs*21is
presentinthreeoutoffourfamilies,thussuggesFngapossiblefounder
effect.SeealsoTable1.
Table1.Summaryofmainclinicalfeaturesofthe5Chileanpatientswithcalpainopatthy.
ClinicalFeatures
DDyrs
Family Patient sex/age Onset(yrs) (progress.)
1
Figure1.MainClinicalFeaturesoffourpa<ents.A.PaFent2,44yr
old.,notehyperl.ordosis,Fp-toestandingispossible.B.PaFent3,37
yr old, scapular winging is severe, note the marked hypotrophy of
thelowerlimbs.PaFent1,at18yrold,Fp-toewalkingwasobliged
andhyperlordosisisalsopresent.D.PaFent5,at13yrold,thereisa
generalizedamyotrophy,symmetricalscapularwinging,butisableto
standinFp-toe
1
W/26
<8
Genetics
Exon/cDNAchange
ProteinChange
Ex1.HTZc.107delG
p.Gly36Valfs*21
Ex21.HTZc.2243G>A
p.Arg748Gln
18(C)
Tip-toewalking,
hyperlordosis,
asymmetricscapular
winging.
x45
N/N
x1.5
N/N
Ex22.HMZ
c.2362_2363delAGinsTCATCT
p.Arg788Serfs*14
2
W/44
7
37(C)
Earlyonsetwithtiptoewaking,Achilles
tenotomy,
hyperlordosis
3
W/40
25
15(A)
Lateonset,severe
scapularwinging
x5
N/N
Ex22.HMZ
c.2362_2363delAGinsTCATCT
p.Arg788Serfs*14
4
M/20
<12
9(A)
Earlyonset,mild
retractions
x32
N/N
Ex1.HMZc.107delG
p.Gly36Valfs*21
2
3
Otherfeatures
CKlevel
(UI/L)
EcoC/
Spir
Ex1.HTZc.107delG
p.Gly36Valfs*21
4
5
M/25
10
11(A)
x32
N/N
Ex22.HTZ
p.Arg788Serfs*14
c.2362_2363delAGinsTCATCT
Abbreviations:Mmale,Wwoman,DD:diseaseduration;progress.:progression(A),ambulant;(C)supportwithacane.EcoC:echocardiography;Spir.:Basal
spirometry.N:normal.CK[N]=30-135UI/L.
Earlyonset,mild
retractions
SummaryofResultsandConclusions
1. 
2. 
3. 
4. 
5. 
6. 
7. 
FivepaFents,fouraffectedChileanfamilies,(anaddi+onalpa+entwasrecentlyiden+fyied)
Variableonset,onaverageinthefirstdecade
Typicalclinicalphenotype,nonspecificbiopsyfindings,needofWBanalysisofCAPN3inmuscle
Nocardiacinvolvement
Giventhelackofspecificclinicalandlaboratoryfeatures,diagnosisisfrequentlydelayed
TwonovelMutaFons
PossiblefoundereffectgiventherecurrenceofnovelmutaFons
SupportedbyGrantsFONDECYT#1151383toJABR