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A new de novo mutation in the GCK gene causing MODY2

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A new de novo mutation in the GCK gene causing MODY2
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  Briefreport  A   newde   novomutation   intheGCK   gene   causingMODY2  AlessiaCappelli a,1 ,SerenaSilvest ri b,1 ,   StefanoT umini c  ,   SilviaCarinci c  ,   PaolaCipriano c  ,LucianoMassi d ,   PaoloStaffolani b ,   LuigiPianese b, *  a Scuola   di   Bioscienze   eBiotecnologie,   Universita` degli   studi   di   Camerino,   Italy b U.O.C.   Laboratorio    Analisi   Cliniche   eMicrobiologiche,   Settore   di   Medicina   Molecolare,    ASUR   ZT13,   Via   degli   iris,63100    Ascoli   Piceno   (AP),Italy c Dipartimento   di   Pediatria,   Universita` di   Chieti,   Italy d U.O.C.   Immunologia   eMedicina   Trasfusionale   SIT,    ASUR   ZT    13,Italy 1.Introduction Glucokinase   (GK),   a   key   enzyme   inglycolysis,   is   the   glucosesensor   that   maintains   glucose   homeostasis   in   thebeta-cell   [1].The   GCK   gene   (7p15.3–p15.1)   consists   of12   exons   and   encodesa   465-aminoacid   protein   [2].Heterozygous   inactivating    GCKmutations   cause   asubtype   of    Maturity-Onset   Diabetes   oftheYoung    (MODY),   known   as   MODY2   [3].   MODY2   isanautosomaldominantly   inherited   form   ofdiabetes   characterized   by   anearly   age   at   onset,   mild   non-progressive   hyperglycaemia   thatis   often   asymptomatic   which   is   present   at   birth   [4].   More   than600   GCK   mutations   have   been   reported   in   many   populations[5,6].   To   date,   however,   only   sixmutations   arising    de   novohave   been   described   [7–9].Herein,   we   describe   anew   de   novoGCK   mutation   in   a15-year-old   male   with   clinical   features   of MODY. 2.Methods 2.1.   Patient We   performed   MODY2   genetic   analysis   on   a   15-year-oldItalian   boy.   At   theageof5   years   he   came   to   our   attention   forhyperglycaemia.   The   glycated   haemoglobin   (HbA1c)   was   5.8%.Anti-glutamic   acid   decarboxylase   (anti-GAD)   antibodies   werenegative.   At   the   time,   his   BMI   was   15.7   (  0.11   DS).   Every   yearan   oralglucose   tolerance   test   (OGTT)   was   performed   andshowed   impaired   glucose   tolerance   (IGT)   without   hyperinsu-linemia.   At   the   ageof    10.5   (BMI   =20.5   kg/m 2  1.84   DS),the2hOGTT   result   revealed   levels   above   11.1   mmol/l,   and   diabeteswas   diagnosed.   Anti-GAD   antibodies   were   negative   and   HbA1cwas   6.6%.   Because   of    postprandial   hyperglycaemia,   thepatient   started   insulin   therapy   with   rapid-acting    insulin d   iab   e   t   es   res   e   ar   c   h   an   d   cli   n   i   c   a   lp   r   ac   t   i   ce9   3   (   2   0   1   1   )   e 4   1–   e 4   3 a   r   t   ic   l   ein   f   o  Articlehistory: Received22December2010Accepted4April2011Publishedonline22   April2011 Keywords: GCKMODY2Denovomutation a   bst   r   a   ct Analysisofglucokinase(GCK)geneina   15-year-oldmaleidentifieda   newframeshiftmutationinexon4   causedby   a   heterozygousguaninedeletionatposition382(c.382delG,p.E128Xfs).No   mutationwas   detectedintheparents.Polymorphicmarkers’studyexcludedfalsepaternityindicatingthatc.382delGis   a   novelde   novomutation. # 2011   ElsevierIrelandLtd.Allrightsreserved.* Correspondingauthor .   Tel.:+39   0736358309;fax:+39   0736   358205.E-mailaddresses:luigi.pianese@sanita.marche.it,   lpianese@yahoo.it(L.Pianese). 1 Theseauthorscontributedequallytothis   work. Co   n   ten   ts   li   s   tsava   ilabl   eatS   ci   e   n   c   eDi   r   ec   t DiabetesResearchandClinicalPractice journal   homepage:   www.elsevier.com/locate/diabres 0168-8227/$–see   front   matter #   2011ElsevierIrelandLtd.Allrights   reserved.doi:10.1016/j.diabres.2011.04.006  before   meals.   At   the   age   of14,   during    agrowth   spurt(BMI   =   24.2,    2.06   DS),   he   needed   the   introduction   oflong-acting    insulin   to   improve   his   fasting    glucose   levels.   There   wasno   family   history   of    diabetes.   The   patient   and   relativesprovided   written,   informed   consent   for   this   study   that   wascarried   out   in   accordance   with   the   principles   ofthe   Declara-tion   of    Helsinki. 2.2.   Genetic   analysis DNA   was   obtained   from   leucocytes   ofblood   using    the   MagNAPure   LC   extractor   (Roche   Diagnostics,   Mannheim,   Germany).PCR   and   direct   sequencing    of    GCK   gene   were   done   aspreviously   described   [10].   Mutation   was   confirmed   using    asecond,   independent   amplification   ofthe   affected   genomicregion   and   re-sequencing    onanother   day.As   this   new   mutation   is   located   at   187   bp   upstream   of    theheterozygous   rs2268573   A/C   polymorphism   in   intron   4(IVS4+87A > C),   it   was   further   characterized   by   AS-PCR,   result-ing    in   the   separate   amplification   of    mutant   and   wild   typealleles.   Notably   we   designed   two   reverse   primers   with   amismatch   in   their   last3 0 nucleotide   in   such   a   way   that   eachwas   specific   for   oneof    the   two   variants   A/C   ofthers2268573polymorphism   (4AR:   5 0 -CCAGATCTCCCTTCTGAGCACAT-3 0 ;and   4CR:   5 0 -CAGATCTCCCTTCTGAGCACAG-3 0 ).   The   allele-specific   reverse   primers   (4AR   and   4CR)   and   the   commonforward   primer   (4F:   5 0 -GCAGCGGAAGAGGAGAGGGA-3 0 )   locat-ed   upstream   in   the   new   mutation   were   combined   in   twoparallel   PCR.   PCR   products   were   sequenced   in   both   the   senseand   antisense   orientations.Paternity   was   analyzed   byusing    HLA   polymorphic   markers(HLA   Typing    A,   B,   C,DRB1   and   DQA1–DQB1   by   SSP   –LowResolution,   Invitrogen,   Carlsbad,   CA,   USA)   to   define   the   allelesin   the   patient   and   both   parents,   according    to   the   manufac-turers   instructions. 3.Results Direct   sequencing    of    GCK   gene   revealed   the   presence   of    anovel   heterozygous   frameshift   mutation   located   in   exon   4   andcaused   by   a   Gdeletion   at   position   382   (c.382delG;   p.E128Xfs)(Fig.   1a).   In   order   to   confirm   the   putative   new   mutation   weused   the   presence   ofthe   heterozygous   rs2268573   A/Cpolymorphism   in   intron   4   (IVS4nt+87A > C)   to   perform   aselective   PCR   amplification   by   AS-PCR.   Direct   sequencing    of the   specific   PCR   products   corresponding    tothe   two   rs2268573A/C   alleles   showed   the   presence   of    the   G   deletion   in   cis   withthe   A   allele   at   rs2268573   (Fig.1b   and   c).The   mutation   was   not   detected   in   DNA   from   the   parents.The   HLA   loci   analysis   excluded   false   paternity   indicating    thatp.E128Xfs   is   anovel   de   novo   mutation   (Fig.2). 4.   Discussion Wefound   anew   mutation   in   exon   4   of    the   GCK   gene   caused   bya   heterozygous   1-bp   deletion   ofguanine   at   position   382(c.382delG)   in   aproband   with   clinical   features   of    MODY.   Thismutation   causes   aframeshift   that   introduces   astop   codon   and Fig.   1   –Identification   ofthe   GCK   gene   mutation.   (a)   Direct sequencing   oftheexon   4   PCRproduct    revealed   that    theproband   washeterozygous   for   a   novel   1-bp   deletion(c.382delG),   resulting   in   a   frameshift    deletion   mutation(p.E128Xfs).   (b)Direct    sequencing   of    the   specific   AS-PCR product    corresponding   tothe   C   allele   at    rs2268573   showedthe   wild   type   sequence   ofexon   4.   (c)   Direct    sequencing   of the   specific   AS-PCR    product    corresponding   to   the   A   alleleat    rs2268573   showed   thepresence   oftheGdeletion   inexon   4.   The   localization   ofthe   deletion   is   indicated    by   thearrow.  p.E128Xf A/C A/A A/A A/C Fig.   2   –Pedigree   of    family   with   GCK   mutation   identified.The   arrow   indicates   theproband   carrying   the   p.E128Xfsmutation.   Alleles   for   rs2268573   A/C   polymorphism   areshown    below   each   symbol. d   i   ab   e   t   es   r   es   e   a   r   chan   dcli   n   i   calpr   a   ct   i   c   e   93(   2011   )   e 4   1   –   e   43 e42  generates   atruncated   protein   of    only   138   amino   acids   which   isthus   unable   to   function.   Notably,   the   truncated   proteincontains   the   amino   acids   1–127   ofthe   wild   type   (465residues)and   an   additional   11   amino   acids   from   the   shifted   reading,with   apremature   termination   (p.E128Xfs).Both   parents   were   normoglycaemic   and   did   not   have   themutation.   Based   on   the   genotyping    results   with   HLA   polymor-phic   markers   there   was   no   evidence   of    non   paternitysuggesting    that   the   p.E128Xfs   mutation   arose   de   novo.   Untilnow,   only   six   de   novo   mutations   in   MODY2   patients   have   beenreported   [7–9].In   addition,   it   has   been   shown   byAS-PCR   that   the   newmutation   was   in   cis   phase   with   theA   allele   atrs2268573polymorphism   in   the   heterozygous   A/C   proband.   As   thers2268573   genotype   of    the   patient’s   father   and   mother   wasrespectively   A/A   and   A/C   itsuggest   that   de   novo   mutation   hasarisen   on   afather’s   A   allele   (Fig.   2).This   case   illustrates   the   value   of    analyzing    the   GCK   gene   incases   with   clinical   features   of    MODY,   even   in   the   absence   of    afamily   history   of    the   disease. Conflict    of    interest  There   are   no   conflicts   ofinterest. r   e   f   e   r   en   c   e   s [1]   MatschinskyF,LiangY,   KesavanP,WangL,FroguelP,VelhoG,etal.Glucokinaseaspancreaticbetacellglucosesensoranddiabetesgene.JClinInvest1993;92:2092–8.[2]   IynedjianPB.Mammalianglucokinaseits   gene.BiochemJ1993;293:1–13.[3]Gidh-JainM,TakedatJ,   XuLZ,LangeAJ,VionnettN,   StoffeltM,etal.Glucokinasemutationsassociatedwithnon-insulin-dependent(type2)   diabetesmellitushavedecreasedenzymaticactivity:implicationsforstructure/functionrelationships.ProcNatl   AcadSciU   S   A1993;90:1932–6.[4]FajansSS,BellGI,PolonskyKS.Molecularmechanismsandclinicalpathophysiologyofmaturity-onsetdiabetesof    theyoung.NEnglJMed   2001;27:971–80.[5]StensonPD,MortM,   BallEV,HowellsK,   PhillipsAD,ThomasNS,etal.The   HumanGeneMutationDatabase:2008update.GenomeMed2009;1:13.[6]   OsbakKK,ColcloughK,   Saint-MartinC,BeerNL,Bellanne´-ChantelotC,EllardS,   etal.Updateon   mutationsinglucokinase(GCK),which   causematurity-onsetdiabetesof theyoung,permanentneonataldiabetes,andhyperinsulinemichypoglycemia.HumMutat2009;30:1512–26.[7]MassaO,MeschiF,Cuesta-MunozA,Caumo   A,CeruttiF,ToniS,etal.HighprevalenceofglucokinasemutationsinItalianchildrenwithMODY.   Influenceon   glucosetolerance,first-phaseinsulinresponse,insulinsensitivityand   BMI.Diabetologia2001;44:898–905.[8]HagerJ,   Blanche´ H,SunF,VaxillaireNV,Poller   W,Cohen   D,etal.   Sixmutationsin   theglucokinasegeneidentifiedinMODYby   usinga   nonradioactivesensitivescreening technique.Diabetes1994;43:730–3.[9]VelhoG,BlancheH,Vaxillaire   M,Bellanne-ChantelotC,PardiniVC,TimsitJ,etal.Identificationof    14newglucokinasemutationsanddescriptionoftheclinicalprofileof42MODY-2families.Diabetologia1997;40:217–24.[10]CappelliA,TuminiS,ConsoliA,CarinciS,Piersanti   C,RuggieroG,etal.NovelmutationsinGCKandHNF1AgenesinItalianfamilieswithMODYphenotype.   DiabetesResClinPract2009;83:72–4. d   iab   etes   r   es   ear   cha   n   d   cli   n   i   c   a   lpr   acti   c   e   9   3   (2011)   e   41– e 4   3   e43
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