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Biomolecular identification of (CCTG)n mutation in myotonic dystrophy type 2 (DM2) by FISH on muscle biopsy

Biomolecular identification of (CCTG)n mutation in myotonic dystrophy type 2 (DM2) by FISH on muscle biopsy
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  437 ©2004,European Journal of HistochemistryMyotonic dystrophy type 2 (DM2) is a dominantly inheriteddisorder with multisystemic clinical features,caused by aCCTG repeat expansion in intron 1 of the zinc finger protein9(ZNF9) gene.The mutant transcripts are retained in thenucleus forming multiple discrete foci also called ribonu-clear inclusions.The size and the somatic instability of DM2expansion complicate the molecular diagnosis of DM2.Inour study fluorescence-labeled CAGG-repeat oligonu-cleotides were hybridized to muscle biopsies to investigate if fluorescence in situ hybridization (FISH),a relatively quickand simple procedure,could be used as a method to diag-nose DM2.When FISH was performed with (CAGG) 5 probe,nuclear foci of mutant RNA were present in all geneticallyconfirmed DM2 patients (n = 17) and absent in all patientswith myotonic dystrophy type 1 (DM1; n = 5) or with other muscular disease (n = 17) used as controls.In contrast,fociwere observed both in DM1 and DM2 myonuclei when mus-cle tissue were hybridized with (CAG) 6 CA probe indicating that this probe is not specific for DM2 identification.The con-sistent detection of ribonuclear inclusions in DM2 musclesand their absence in DM1,in agreement with the clinicaldiagnosis and with leukocyte (CCTG)n expansion,suggeststhat fluorescence in situ hybridization using (CAGG) 5 probes,maybe a specific method to distinguish between DM1 andDM2.Moreover,the procedure is simple,and readily appli-cable in any pathology laboratory.Keywords: myotonic dystrophy type 2; diagnostic method;fluorescence in situ hybridization; FISH; CAGG probe; ribonu-clear inclusions.Correspondence: Prof.Giovanni Meola,Professor and Chair of Neurology,Dept.Neurology,University of Milan,Istituto Policlinico San Donato,Via Morandi,30,20097 San Donato Milanese,Milan – ItalyPhone: ++02.52774480Fax: ++02.5274717e-mail:  Paper accepted on September 9,2004.European Journal of Histochemistry 2004; vol.48 issue 4 (Oct-Dec):437-442 Biomolecular identification of (CCTG)n mutation in myotonic dystrophy  type 2 (DM2) by FISH on muscle biopsy  R. Cardani,* E. Mancinelli,* V. Sansone,** G. Rotondo,** G. Meola** *Dipartimento di Fisiologia e Biochimica Generali,Università di Milano,Italy; ** Istituto Policlinico SanDonato,Dipartimento di Neurologia,Cattedra di Clinica Neurologica,Università di Milano,Italy. M yotonic dystrophy type 2 (DM2) is anautosomal dominant,multisystemic dis-ease.It is characterized by muscle weak-ness,myotonia and by non-muscle manifestationsincluding cataracts,cardiac arrhytmias and hypog-onadism (Meola et al. ,1999;Moxley et al. ,1998,2002;Meola,2000;Day et al. ,2003;Udd et al. ,2003).Muscle biopsies are characterized by fiberswith increased number of central nuclei,angulatedatrophic fibers,occasional ring fibers,type I fiberpredominance and a preferential atrophy of type IIfibres.(Day et al. ,2003;Schoser et al. ,2004;Vihola et al. ,2003).Nuclear clump type II fiberatrophy is considered typical of DM2,although,inour experience,20% of patients with clinical andgenetic confirmation of DM2 mutation have normalhistograms and no nuclear clump type 2 fiber atro-phy (Meola et al. ,2004).The DM2 locus is mapped to a 10 c-M region ofchromosome 3q (Ranum et al. ,1998) and recentlyLiquori et al. (2001) have reported that DM2 iscaused by a large expansion of a CCTG repeat locat-ed in intron 1 of the zinc finger protein 9 (ZNF9)gene.ZNF9 contains seven zinc finger domains andis thought to be an RNA binding protein that isexpressed in many different tissues.In DM2,expand-ed allele sizes range from 75 to 11000 CCTGrepeats,with a mean of approximately 5000 repeats(Liquori et al. ,2001).The mutant ZFN9 gene istranscribed into RNA that contains expanded CCUGrepeats and these transcripts are retained in themuscle nucleus,producing foci of mutant RNA alsocalled ribonuclear inclusions (Liquori et al. ,2001;Mankodi et al. ,2001,2003).A large body of evi- dence indicates that these ribonuclear inclusions areresponsible for the clinical manifestations of DM2.Ithas been suggested that CCUG expansion expressedat RNA level could themselves be pathogenic andcause the multisystemic features of DM2 (Liquori et al. ,2001).Moreover recent studies have demon-strated that intranuclear RNA foci bind specific ORIGINAL PAPER  RNA binding proteins and muscleblind that aresequestered in DM2 nuclei,leading to depletion oftranscripts that require these proteins for normalgene expression (Mankodi et al. ,2001,2003; Fardaei et al. ,2002;Kanadia et al. ,2003).The discovery of DM2 mutation raised the possi-bility to use molecular testing for a more accuratediagnosis of patients with suspected DM2 since theclinical and histological features of DM2 are simi-lar even if not identical to those of myotonic dys-trophy type 1 (DM1) and it is not always easy todistinguish by routine histological and histochemi-cal technique on muscle biopsy these two diseases(Meola,2000;Moxley et al. ,2002).This is espe-cially true when family trees are uninformative andcore clinical findings,like clinical EMG areextremely fluctuating and maybe absent when thepatient is first seen.Similarly,cataracts,one of thecore clinical diagnostic features for DM2,may beabsent because evaluation occurs early in the dis-ease.However,molecular testing is not applicableas yet on a routine basis as is CTG expansion PCRor Southern Blot for DM1.The size and somaticinstability of the DM2 repeat in fact,complicatethe interpretation of data obtained by PCR andSouthern analysis that sometimes fail to detect theDM2 expansions (Liquori et al. ,2001;Bachinski et al. ,2003;Day et al. ,2003).To determine the pres-ence of DM2 mutation,Bachinski et al. (2003)developed a repeat-primed PCR (RP-PCR),whichused in combination with a Field InversionElectrophoresis (FIGE) Southern blot also deter-mines the size of the expansion.Furthermore,Day et al. (2003) describe a repeat assay (RA) basedon the amplification of the genomic region with therepeat expansion followed by the Southern analysisof the PCR products.Although these tests increasethe detection rate of the DM2 expansion,still theyare expensive,not available as routine diagnostictest and much time consuming methods for DM2diagnosis.Only recently nuclear clump type 2 fiberatrophy (Vihola et al. ,2003) and type 2 fiber atro- 438 R.Cardani et al. Table 1. F = female; M = male; LLW = lower limb weakness; MRC = Medical Research Council Scale (Medical Research Council. Aidsto the examination of the peripheral nervous system. Memorandum 45. 1976, Pendragon House, London.); CK = creatine kinase; AV= atrioventricular; OGTT = oral glucose tolerance test; γ -GT = γ -glutamyl transferase; SVEB = supraventricular ectopic beats. PatientAge/DiseaseAge SymptomsMegaMRCMRC bicepsmultisystem involvement sexdurationat onsetat onset  BC59/F1445proximal LLW1184,5hypoacusia,hyperthyroidism,CK x4CI47/F1037distal LLW1344,5diabetes,hyperthyroidism,cataracts,memory lossCS49/F1039proximal LLW1184cataractsFE60/M654lower limb stiffness1505visual-spatial deficitsGL55/F1045proximal LLW1184cataracts,visual spatial deficitsGE36/M2115myotonia1505neurosensory hypoacusia,2 nd degree AV block,CK x4GM43/F2518myalgia1385neurosensory hypoacusia,sacral cysts,uterine fibroma,high glucose levels after OGTT,CK x2,triglycerides x2,visual-spatial deficits,WMHL on brain MRING62/M1240proximal LLW1244neurosensory hypoacusia,paresthesias of the hands,coronary heart disease,cataracts,cerebellar vermis and corpus callosum atrophy,cholesterol x2,triglycerids x3,CX x2PV47/M1730myotonia1324,5testicular motility,baldness,CK x5, γ -GT x2,cholesterol x2,PL65/M1550proximal LLW1054,5cataracts,baldness,diabetes,high blood pressure,triglycerides x4,mild mitral prolapse,choreic movements,oro-mandibular dyskinesiaRG63/M2736proximal LLW1274,5cataracts,feet paresthesias,truncal lipomas,erectile dysfunction,visual-spatial deficits,bradycardia,neurosensory hypoacusia,lower limb venous incontinence,restless leg syndrome,CK x3, γ -globulin < 10%,low back pain RL32/F626myotonia1505noneRO40/F1535proximal LLW1464,5cataracts,visual spatial deficits,low back pain,hand paresthesias,neurosensory hypoacusia,infertility,tachycardia,CK x2, γ -globulin <10%RR44/F2816proximal LLW772,5visual spatial-deficits,low-back pain,cervical herniated discs,diffuse trunk and limb lipomas,initial baldness,fine distal tremor,SVEB,CK x2RS43/M2122proximal LLW1464,5visual spatial-deficits,low-back pain,lumbar herniated discs,initial baldness,SVEB,CK x2RA20/F218myotonia1505noneRAm71/F4625proximal LLW1123extrapyramidal signs,cataracts,1st AV block,chronic obstructive broncopulmonary disease  439 Original Paper  phy (Schoser et al. ,2004) have been found typicalof DM2 and could be considered an useful tool totarget subsequent biomolecular analysis. Fluorescence in situ hybridization (FISH) is apowerful technique used to localize nucleic acidsequences within the cell.In the present study,fluo-rescent labelled antisense oligonucleotide probes tothe CCUG repeat were hybridized to muscle biopsytissue from genetically confirmed DM2 patients forthe direct visualization of mutant RNA in myonuclei.Our aim was to asses if FISH could be used as amethod to diagnose DM2,since the procedures forthe application of this technique on tissues are quick,not very complex and share many similarities to thosefor immunohistochemistry,which is widely used inboth basic research and diagnostic laboratories. Materials and Methods Patients and tissue samples The diagnosis of DM2 was based upon the clini-cal diagnostic criteria set by the InternationalConsortium for Myotonic Dystrophies (Moxley et al. ,2002).A description of the clinical features ofthe DM2 patients used in this study are reported inTable I.Biceps brachii muscle biopsies from genet-ically confirmed DM2 patients (n=17) were pre-pared by snap-freezing in cooled isopentane.The histological diagnosis was performed on seri-al sections (10 µ m) of muscle biopsies from all ofthese patients processed for routine histological orhistochemical stainings.A standard myofibrillarATPase staining protocol was used after preincuba-tion at pH 4.3,pH 4.6,and pH 9.4 or 10.4(Dubowitz,1985).Antibodies against differentmyosin heavy chain (MHC) isotypes were used forimmunohistochemical identification of fast andslow MHC fibers according to Vihola et al. (2003).The genetic confirmation of DM2 was performed byrepeat assay described by Bachinski et al. (2003).Muscle biopsies from patients with myotonic dys-trophy type 1 (DM1;n=5) or with other musculardiseases (n=17) were used as controls.The biopsieswere used for this study after informed consentfrom patients. Fluorescence In Situ Hybridization (FISH) Transverse muscle cryostat sections (6 µ m) were Figure 1. Fluorescence in situ hybridiza-tion (FISH) with (CAGG) 5 probe on mus-cle tissue. A-D. Ribonuclear inclusions(red) are present in myonuclei (blue) of muscle tissue from DM2 patients (A:srcinal magnification 400 × ; D srcinalmagnification 1000 × ). B. Mutant RNAfoci (red) are also present in centralnuclei (blue) of muscle tissue: note therod shape of foci suggesting the conflu-ence of 2 or more spot signals (srcinalmagnification 630 × ). C. Multiple ribonu-clear inclusion in muscle nucleus in aDM2 patient (srcinal magnification1000 × ). E-F. Ribonuclear inclusions areabsent in myonuclei in DM1 patient (E:srcinal magnification 1000 × ) and inpatient with other muscular disease (F:srcinal magnification 1000 × )  440 R.Cardani et al. dried for 30 min at room temperature and fixed in2% paraformaldehyde for 30 min at 4°C.After fix-ation,sections were washed five times in phosphate-buffered saline (PBS) for 3 min at room tempera-ture,and permeabilized in 2% acetone PBS (pre-chilled at –20°C) for 5 min.After washing in PBS,sections were incubated in 30% formamide and2xSSC for 10 min at room temperature and thenhybridized with probe (1 ng/  µ L for 2 h at 37°C in30% formamide,2xSSC,0.02% BSA,67 ng/  µ Lyeast tRNA,2 mM vanadyl ribonucleoside com-plex.Sections were washed first in 30% formamideand 2xSSC at 45°C for 30 min,then in 1xSSC and165 nM 4,6 diamidino-2-phenylindole (DAPI) atroom temperature for 30 min.Sections were thenmounted with Gel Mount mounting medium(Biomedia Corp).Probes were HPLC-purified 2-O-methhyl RNA CAGG or CAG repeat 20-mers and5’Texas red labelled (IDT,Coralville,IA).Musclesections were examined using a fluorescence micro-scope or sometimes using a confocal microscope.Inorder to get the same treatment,one section fromboth DM2 and control patient muscle tissue wereplaced on the same slide. Results Fluorescently-labelled antisense oligonucleotide(CAGG) 5 probe was used for in situ hybridization todetect the ribonuclear inclusions containing theCCUG expansion in muscles from genetically con-firmed DM2 patients.Intense nuclear foci ofhybridization were observed in muscle tissue fromeach of 17 DM2 individuals considered (Figures1A-D).The number of foci was 1 to 3 per nucleuswith no foci detected in the cytoplasm (Figures 1A,B).In contrast,no nuclear foci were detected when(CAGG) 5 probe was hybridized to muscle tissuefrom DM1 patients or from subjects with othermuscle diseases used as controls (Figures 1E,F).(CAGG) 5 probe generated a high background in thenucleoplasm both in DM2,DM1 and control mus-cles,but foci of hybridization were easily distin-guished since they resulted more intensely fluores-cent than the background hybridization.When(CAG) 6 CA probe was hybridized to DM2 muscletissue,mutant RNA was also identified and RNAfoci appeared to be smaller and less fluorescentthan those identified by (CAGG) 5 (Figure 2C).Asexpected,(CAG) 6 CA probe also evidenced ribonu-clear inclusions myonuclei of DM1 muscle sections(Figure 2A,B).Indeed DM1 patients,unlike DM2patients,have an abnormally high number of CTGrepeats in the gene for myotonic dystrophy (DMprotein kinase gene) (Brook et al. ,1992;Mahadevan et al. ,1992).As for DM2,mutant tran-scripts containing the expanded repeats areretained in myonuclei producing inclusions similarto those of DM2 (Taneja et al. ,1995).Nuclear fociwere not observed in control muscle sectionshybridized with (CAG) 6 CA probe (Figure 2D).Nonuclear background was observed in any musclesections when hybridized with (CAG) 6 CA probe.Insummary our results show that (CAGG) 5 probe Figure 2. Fluorescence in situ hybridiza-tion (FISH) with (CAG) 6 CA probe onmuscle tissue. A,B. Ribonuclear inclu-sions (red) are present in nuclei (blue)of muscle tissue from DM1 patients (A:srcinal magnification 1000 × ; B srcinalmagnification 1000 × ). C. ribonuclearinclusion (red) are also present in nuclei(blue) of muscle tissue from DM2patient (srcinal magnification 400 × ).D.No foci are present in myonuclei inpatient with other muscular diseases (F:srcinal magnification 1000 × )  identifies ribonuclear inclusions in DM2 patientsonly,whereas (CAG) 6 CA probe can detect foci ofmutant RNA both in DM1 and DM2 patients. Discussion Conventional genetic tests used for myotonic dys-trophy type 1 diagnosis,such as Southern blotanalysis or PCR,are not reliable for DM2 identifi-cation (Liquori et al. ,2001;Bachinski et al. ,2003;Day et al. ,2003).Molecular diagnostic protocols,recently described by Bachinski et al. (2003) and byDay et al. (2003),can be used for detecting the DM2mutation,but are still too long and complicated to bepart of routine laboratory diagnostics.In agreementwith previous data (Liquori et al. ,2001;Mankodi et al. ,2003;Sallinen et al. ,2004),the present studyclearly demonstrates that the DM2 mutation can beidentified by FISH using CAGG probe to visualizenuclear foci in muscle biopsies.Moreover,our resultsshow a consistent detection of ribonuclear inclusionsby (CAGG) 5 in DM2 muscles and their absence inDM1 and in other control muscles,suggesting thatthe use of this probe allows a specific method forDM2 diagnosis.Performing FISH with CAG oligonu-cleotides instead,reveals ribonuclear inclusions bothin DM1 and DM2 muscles as previously reported byother Authors (Mankodi et al. ,2001,2003).This aspecific finding indicates that (CAG) 6 CA probe can-not be used for a differential diagnosis between DM2and DM1.It is worth emphasizing that ribonuclear inclu-sions by FISH,were present irrespective of degreeof muscle involvement or of severity of clinicalinvolvement.Three of 17 patients had normal his-tograms,with no preferential nuclear clump type IIfiber atrophy as is usually found in DM2.Yet FISHdemonstrated nuclear foci in these patients.The nuclear accumulation of mutant RNA inDM1 and DM2 muscle tissue,is a molecular fea-ture shared by these two disorders.This may indi-cate that some characteristic they share,likemyotonia,cardiac arrhythmias and insulin resist-ance,as well as other aspects of the multisystemicinvolvement of these disorders,result from a com-mon mechanism such as altered RNA splicing ofseveral genes.Alteration of the chloride channelsplicing leads to the loss of chloride channel proteinthat results in myotonia (Charlet et al. ,2002;Mankodi et al. ,2002);changes in cardiac troponinT splicing (Philips et al. ,1998) may be implicatedin the cardiac disfunction;insulin receptor splicingalterations in DM2 may be implicated in insulinresistance which characterizes the disease (Savkur et al. ,2001,2004).The role of ribonuclear inclu- sions in disease pathogenesis remains unclear.Nevertheless ribonuclear inclusions and splicingchanges are present before any histological abnor-mality manifestations (Mankodi et al. ,2001;Savkur et al. ,2004).This could be important for anearly diagnosis before the spectrum of clinical signsof muscle disease appear.In conclusion,FISH with (CAGG) 5 is a methodthat allows the direct visualization of the mutantRNA on muscle biopsy.Therefore it may be a sim-ple approach for DM2 diagnosis,which can be per-formed in a rapid and sensitive manner in anypathology laboratory.Moreover,it permits a differ-ential diagnosis between DM2 and DM1.We sug-gest that FISH with (CAGG) 5 should be consideredas a routine laboratory procedure to confirm orrefute the clinical suspicion of DM2.It should alsobe applied routinely to screen patients with myoton-ic disorders.This procedure may contribute forexample to differentiate chloride channelopathiesfrom DM2 and better target subsequent geneticscreening.It may also allow to recognize preclinicalor sporadic presentations of DM2 in patients withunknown neuromuscular disorders or asympto-matic high CK levels,in whom a biopsy is per-formed for diagnostic purposes.  Acknowledgements This study was supported by MURST 60% to G.Meola. References Bachinski LL,Udd B,Meola G,Sansone V,Bassez G,Eymard B,et al.Confirmation of the type 2 myotonic dystrophy (CCTG)n expansionmutation in patients with proximal myotonic myopathy/proximalmyotonic dystrophy of different European origins:a single sharedhaplotype indicates an ancestral founder effect.Am J Hum Genet2003;73:835-48.Brook JD,McCurrach ME,Harley HG,Buckler AJ,Church D,Aburatani H,et al.Molecular basis of myotonic dystrophy:expan-sion of a trinucleotide (CTG) repeat at the 3’end of a transcriptencoding a protein kinase family member.Cell 1992;68:799-808.Charlet BN,Savkur RS,Singh G,Philips AV,Grice EA,Cooper TA.Loss of the muscle-specific chloride channel in type 1 myotonic dys-trophy due to misregulated alternative splicing.Mol Cell 2002;10:45-53.Day JW,Ricker K,Jacobsen JF,Rasmussen LJ,Dick BA,Kress W,etal.Myotonic dystrophy type 2:molecular,diagnostic and clinicalspectrum.Neurology 2003;60:657-64.Dubowitz V.Muscle biopsy:a practical approach.Bailliére Tindall,London 1985;19-40.Fardaei M,Rogers MT,Thorpe HM,Larkin K,Hamshere MG,HarperPS,et al.Three proteins,MBNL,MBLL and MBXL,co-localize in 441 Original Paper 
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