Dutraetal.BMCResearchNotes2012,5:13 http://www.biomedcentral.com/1756-0500/5/13 RESEARCH ARTICLE Open Access Copy number variation in Williams-Beuren syndrome: suitable diagnostic strategy for developing countries Roberta L Dutra1,2,3*, Rachel S Honjo1, Leslie D Kulikowski2, Fernanda M Fonseca3, Patrícia C Pieri3, Fernanda S Jehee1, Debora R Bertola1 and Chong A Kim1 Abstract Background: Williams-Beuren syndrome (WBS; OMIM 194050) is caused by a hemizygous contiguous gene microdeletion at 7q11.23. Supravalvular aortic stenosis (SVAS), mental retardation, and overfriendliness comprise typical symptoms of WBS. Although fluorescence in situ hybridization (FISH) is considered the gold standard technique, the microsatellite DNA markers and multiplex ligation-dependent probe amplification (MLPA) could be used for to confirm the diagnosis of WBS. Results: We have evaluated a total cohort of 88 patients with a suspicion clinical diagnosis of WBS using a collection of five markers (D7S1870, D7S489, D7S613, D7S2476, and D7S489_A) and a commercial MLPA kit (P029). The microdeletion was present in 64 (72.7%) patients and absent in 24 (27.3%) patients. The parental origin of deletion was maternal in 36 of 64 patients (56.3%) paternal in 28 of 64 patients (43.7%). The deletion size was 1.55 Mb in 57 of 64 patients (89.1%) and 1.84 Mb in 7 of 64 patients (10.9%). The results were concordant using both techniques, except for four patients whose microsatellite markers were uninformative. There were no clinical differences in relation to either the size or parental origin of the deletion. Conclusion: MLPA was considered a faster and more economical method in a single assay, whereas the microsatellite markers could determine both the size and parental origin of the deletion in WBS. The microsatellite marker and MLPA techniques are effective in deletion detection in WBS, and both methods provide a useful diagnostic strategy mainly for developing countries. Background WBS is generally sporadic with frequency of approxi- Williams-Beuren syndrome (WBS; OMIM 194050) is a mately 1 in 7,500 live births with no ethnic or sex pre- neurodevelopmental disorder described independently ference, although familial cases have been reported with [1,2] as a syndrome involving facial appearance charac- apparent autosomal dominant inheritance [4,5]. Despite teristics, supravalvular aortic stenosis (SVAS) and men- the consistency of the overall clinical features, the broad tal retardation. In fact, WBS presents a wide collection spectrum of anomalies and phenotypic variability fre- of symptoms affecting blood vessels, growth, intelli- quently lead to a significant difference in the number of gence, and behavior. Children with this condition have patients diagnosed [6]. distinctive facial features, a hoarse voice associated with WBS is caused by a hemizygous contiguous gene growth, mental retardation and an overfriendly personal- microdeletion of the WBS critical region on chromo- ity; hyperacusis, infantile hypercalcemia, prematurely some 7 at position 7q11.23. The most common deletion wrinkled skin are also common symptoms [3]. is found in 90% to 95% of WBS patients and spans a genomic region of approximately 1.55 Mb. It is the result of mispairing between the centromeric and medial *Correspondence:[email protected] LCR (Low copy repeats) blocks B (Bcen and Bmid) [7]. 1DepartmentofGenetics,InstitutodaCriança,UniversidadedeSãoPaulo, In 5% to 10% of cases, the breakpoints are within the SãoPaulo,Brazil centromeric and medial LCR blocks A (Acen and Amid) Fulllistofauthorinformationisavailableattheendofthearticle ©2011Dutraetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited. Dutraetal.BMCResearchNotes2012,5:13 Page2of5 http://www.biomedcentral.com/1756-0500/5/13 and lead to an ~1.84-Mb deletion [8]. Atypical (approxi- mately 0.2 Mb to ~2.5 Mb) deletions may be the leading cause of the substantial phenotypic variability among WBS patients [9]. Duplication of the WBS region occurs at half the fre- quency of deletions with less distinctive and somehow opposite clinical features, such as deficits of social inter- action and an autistic-like phenotype [10,11]. Confirmation of clinical suspicion is essential for clini- cal monitoring of the patient and genetic counseling of the family. Although fluorescence in situ hybridization (FISH) is widely used and considered the gold standard for WBS molecular diagnosis, the use of microsatellite Figure 1 Genotyping by MLPA technique (SALSA kit P029) usingthesoftwareGeneMarker®foranalysis.Hemizygous DNA markers has also been widely used and is consid- contiguousgenemicrodeletion,canbevisualizedbyprobes21to ered highly informative and easily performed [12,13]. 32.WiththepresenceonecopythesegenesintheWBScritical Multiplex ligation-dependent probe amplification region,7q11.23. (MLPA) has been introduced into DNA diagnostic laboratories for the detection of deletions and/or dupli- kit that are in the same position (Figure 3). Considering cations in several disease genes [14]. MLPA kit for both techniques, there was no clinical difference in rela- WBS, makes possible a more precise mapping of the tion to either the size of deletion or the parental origin deletion in the critical region, compared with the FISH of deletion. [15]. In this study, the results obtained with microsatel- lite markers were compared with those obtained with Discussion MLPA. Microsatellite DNA markers and MLPA have been con- It can be argued that both techniques, together, are sidered highly informative and easily manageable for extremely valuable tools for the diagnosis of the WBS diagnostic confirmation of WBS. patients and that the implementation of both methods In our study, five microsatellite markers (D7S1870, should be considered. D7S489, D7S613, D7S2476, and D7S489A) were infor- mative, except in four cases. Results A total of 88 patients with the suspicion of a clinical diagnosis of WBS were tested. The five markers (D7S1870, D7S489, D7S613, D7S2476 and D7S489A) were informative in 84 patients and not informative in 4 patients. The most informative marker was D7S1870 (78.4% of patients), followed by D7S613 (68.2% of patients), D7S489 (65.9% of patients) and D7S2476 (57.9% of patients). The microdeletion was present in 64 (72.7%) patients and absent in 24 (27.3%) patients. The observed deletion size was 1.55 Mb in 57 of 64 patients (89.1%) and 1.84 Mb in 7 of 64 patients (10.9%). For the parental origin, the deletion was mater- nal in 36 of 64 patients (56.3%) and paternal in 28 of 64 patients (43.7%). Using the MLPA kit (P029), the results were concor- dant with the microsatellite marker analysis in 84 patients and on 4 cases the deletion was only detected by MLPA (Figure 1). FISH was performed in all patients and the results were concordant with those found by Figure 2 Representation of the 7q11.23 region and the microsatellites and MLPA. locationoftheprobesfromSALSAkitP029andthemarkers The microsatellite markers used in the present study, tested.Thepatients54and55wererepresentedinthefigureto are located in different regions in comparison with the illustratethelocalizationofthegenesintheregion7q11.23andthe probes in the P029 kit for WBS (Figure 2). Except the sizeofthedeletion.Neg.Negative;Pos.PositiveandUn. Uninformative. D7S489 marker and the FZD9 probe from MLPA P029 Dutraetal.BMCResearchNotes2012,5:13 Page3of5 http://www.biomedcentral.com/1756-0500/5/13 Figure3ComparisonbetweentheresultsobtainedbymicrosatellitemarkersandMLPA.Atotalof88patientsparticipatedofthestudy andnumbered1to107.ThecorrespondentprobetogeneFZD9islocalizedinthesameregionthatD7S489microsatellitemarker. However, in these particular cases, short stature, MLPA technique. These patients present a phenotypic microcephaly, and cardiovascular anomalies were absent, variability that often leads to diagnostic difficulties and but not in one patient that presented mitral and tricus- the confirmation of results only was possible using pid regurgitation and hiperacusis. MLPA technique. The D7S1870 microsatellite marker showed the high- The microsatellite markers were efficient in deletion est power of detection, able to identify 78.4% of the detection for WBS when compared to the MLPA. They cases by itself, which confirmed the results from pre- allowed for the detection of deletions larger than 1.55 vious studies [8,16-19]. MB and for detection of the parental origin of the Two best markers (D7S1870 and D7S613) in our deletion. study were able to detect the deletion in 93.2% of cases FISH is widely used and considered the gold standard when used together. When the D7S613 and D7S489 for WBS molecular diagnosis, however, FISH is labor- markers were included, informative detection increased intensive, time-consuming, and it does not allow the to almost 95%. detection of the exact size of the deletion [20]. The microsatellite marker D7S489A was effective in The cost of the microsatellite marker technique has the analysis of deletion size. The 1.55-Mb deletion was greatly decreased, and it can be deployed in molecular found in 57 of 64 (89.1%) patients, and the 1.84-Mb biology laboratories that have basic equipment for con- deletion was found in 7 of 64 patients (10.9%); these ventional PCR reactions and a vertical electrophoresis observed percentages are similar to those found in other system. studies in the literature [8]. The most important advantages of the MLPA are its Using markers to identify the parental origin, we relative simplicity, low cost, rapid turnaround (2 days), found no significant difference between the frequencies ease of multiplexing to permit high confidence in the of maternal and paternal deletions (56.3% and 43.7%, results, high accuracy of copy number estimation, and respectively), and the literature is concordant with our the potential for combination of copy number analysis findings [12,13,17,19]. with other applications, such as methylation detection There was also no relationship between clinical fea- or SNP genotyping [21]. tures with the size of the deletion and with the parental The accuracy of both diagnostic tests is well recog- origin. nized to be susceptible to technical problems and clini- Since the MLPA technique was developed [14], it has cal heterogeneity. In our study, FISH, markers and been tested as a diagnostic method in several diseases MLPA presented higher sensitivity (99.8%), similar to involving chromosomal disorders. In this study, we used others studies [22] and microsatellites markers presents the MLPA kit (P029) to observe the microdeletion in 64 lower specificity compared to FISH and MLPA (93%). (72.7%) patients and find it was absent in 24 (27.3%) Real-time quantitative polymerase chain reaction tech- patients. nique (QPCR) and array-based comparative genomic We find four discrepant results comparing the micro- hybridisation (array-CGH) are also being used for the satellite markers and the MLPA method in the detection molecular diagnosis of WBS. of deletions in the WBS critical region. In these patients QPCR is considered a robust methodology, with easy where the microsatellite markers are uninformative, interpretation, and simple to set up [23,24]. Conversely, detection of the deletion can be confirmed using the to perform this technique we need sophisticated Dutraetal.BMCResearchNotes2012,5:13 Page4of5 http://www.biomedcentral.com/1756-0500/5/13 equipments and specific primers for each target region, Microsatellite markers differently from MLPA, where the simultaneous hybridi- The five microsatellites markers used included D7S1870, zation of more than 40 different probes can be used in D7S489, D7S613 and D7S2476 inside the common 1.55- one single reaction. Mb deletion and D7S489A to distinguish deletions of Recently, array-CGH has also been proved also to be a 1.84 Mb. PCR reactions were carried out according to powerful and promising method to detect microdele- Dutra et al. (2011) [13]. tions and to identify novel cytogenetic abnormalities Patient genotypes were compared with those of their [25]. However, the resolution of array-CGH can vary parents. Deletions were diagnosed as maternal when the depending on the format and design of the array [26]. proband presented with gel bands representing the alle- Additionally, this method is relatively difficult and lic marker inherited only from the father. When by costly, and it requires a different setup as far as instru- chance both parents have the same alleles, the monoal- mentation is concerned [25]. lelic inheritance of the corresponding microsatellite Economic models are important to help health profes- marker by the proband indicated an uninformative sionals to take decisions based on available strategies. result. The molecular tests available together with socio eco- We first used a two-step algorithm to identify the nomic characteristics of the country is fundamental most common 1.55-Mb deletion. We then tested the when a new strategy is considered to be taken, especially D7S489A marker either to identify the larger 1.84-Mb in developing countries where resources are limited [27]. deletion (in those patients in which a deletion of at least one marker was detected in the first step) or to confirm Conclusions the lack of a deletion. The diagnosis of WBS based on clinical assessments may be difficult because of the great variability of its MLPA manifestations. Laboratory tests to detect the microdele- The MLPA (SALSA kit P029 - MRC-Holland, Amster- tions in 7q11.23 are essential to confirm the clinical dam, The Netherlands) containing probes for eight diagnosis of WBS. genes from the WBS critical region (FKBP6, FZD9, In summary, the microsatellite marker and MLPA TBL2, STX1A, ELN, LIMK1, RFC2 and CYLN2) were techniques are effective in deletion detection in WBS, used. The ELN and CYLN2 probes for various exons are and both methods improve complete molecular cover- present in the kit. Denaturation, overnight hybridisation, age in screening of the critical region mainly for devel- ligation and PCR were performed according to the man- oping countries. ufacturer’s instructions. MLPA products were separated on a MegaBACE™ Methods 1000 (GE Life Sciences, Waltham, USA) using Mega- Subjects BACE ET SIZE Standards ET550-R (GE Life Sciences, A total cohort of 88 patients with a clinical diagnosis of Waltham, USA). The analysis was performed using the WBS (56 boys and 32 girls) were followed through clini- GeneMarker, version 1.6, software (Softgenetics, State cal evaluation by geneticists of the Unit of Clinical College, PA, USA). The ratio of the probes’ peak heights Genetics - Instituto da Criança, Hospital das Clínicas - was determined by comparing the probes’ peak heights Universidade de São Paulo (ICr-HCFMUSP), Brazil. The obtained from patient samples to those obtained from inclusion criteria were dysmorphic facial features sug- three normal control samples. gestive of WBS and the presence of cardiovascular dis- orders, mainly SVAS. Statistical analysis The study was approved by the Institutional Review Pairwise comparisons between clinical features of WBS Board - Ethics Committee for Analysis of Research Pro- and the presence of deletion, clinical features and dele- jects HCFMUSP/Cappesq - and written consent was tion size and clinical features and parental origin of obtained from all participants. deletion were tested for significance using two-tailed Among the 88 patients, DNA from both parents was Fisher’s exact test. A 2 × 2 contingency table was used obtained in 80 cases; in 8 cases, the molecular analysis to compare clinical features. P analysis was performed was performed only with maternal DNA. Most of in SPSS 13.0 software and considered statistically signifi- patients had normal GTG band karyotype and FISH was cant when p ≤ 0.05. previously had been done in 24 patients. The molecular study was performed in Laboratory of Acknowledgements Genomic Pediatrics - LIM 36 - (Icr -HCFMUSP). DNA TheauthorsaregratefultoDr.UlyssesDória-Filho(NucleodeApoio was isolated from peripheral blood lymphocytes using a MetodológicodoInstitutodaCriança-FMUSP)forhishelpwiththe salt precipitation technique [28]. statisticalanalysisandtoDra.JulianaForteMazzeudeAraújowithtechnical Dutraetal.BMCResearchNotes2012,5:13 Page5of5 http://www.biomedcentral.com/1756-0500/5/13 assistanceintheMLPAtechnique.ThisworkwassupportedbyFAPESP 13. DutraRL,PieriPC,TeixeiraAC,HonjoRS,BertolaDR,KimCA:Detectionof grantsn°2008/55391-6,grantsn°2009/53105andCNPqgrantsn°401910/ deletionat7q11.23inWilliams-Beurensyndromebypolymorphic 2010-5. markers.Clinics(SaoPaulo)2011,66(6):959-964. 14. SchoutenJP,McElgunnCJ,WaaijerR,ZwijnenburgD,DiepvensF,PalsG: Authordetails Relativequantificationof40nucleicacidsequencesbymultiplex 1DepartmentofGenetics,InstitutodaCriança,UniversidadedeSãoPaulo, ligation-dependentprobeamplification.NucleicAcidsRes2002,30:e57. SãoPaulo,Brazil.2DepartmentofPathology,LIM03,UniversidadedeSão 15. vanHagenJM,EussenHJ,vanSchootenR,vanDerGeestJN,Lagers-van Paulo,SãoPaulo,Brazil.3LaboratoryofGenomicPediatrics-LIM36,Instituto HaselenGC,WoutersCH,DeZeeuwCI,GilleJJ:Comparingtwodiagnostic daCriança,UniversidadedeSãoPaulo,SãoPaulo,Brazil. laboratorytestsforWilliamssyndrome:fluorescentinsituhybridization versusmultiplexligation-dependentprobeamplification.GenetTest2007, Authors’contributions 11:321-327. RLDgraduatestudent(PhD),involvedindraftingthemanuscript, 16. Karmiloff-SmithA,GrantJ,EwingS,CaretteMJ,MetcalfeK,DonnaiD, participatedinthedesignofthestudyandcollaboratedwithanalysisand ReadAP,TassabehjiM:Usingcasestudycomparisonstoexplore interpretationofdata.RSHMedicalandgraduatestudent(PhD)participated genotype-phenotypecorrelationsinWilliams-Beurensyndrome.JMed inthedesignofthestudyandcollaboratedwithupdatingmedicalrecords Genet2003,40:136-140. andambulatorycareofWilliamssyndromepatients.LDKBiologistPhD, 17. PérezJuradoLA,PeoplesR,KaplanP,HamelBC,FranckeU:Molecular sponsorofCytogenomicsgroup,participatedinthedesignofthestudyand definitionofthechromosome7deletioninWilliamssyndromeand collaboratedwithfinalreviewofthemanuscript.FMFTechnicallaboratory parent-of-origineffectsongrowth.AmJHumGenet1996,59:781-792. LIM36,collaboratedwiththemicrosatellitesmarkersexperiments.PCP 18. Gilbert-DussardierB,BonneauD,GigarelN,LeMerrerM,BonnetD,PhilipN, BiologistinLIM36,collaboratedwiththestandardizationofmethods ServilleF,VerloesA,RossiA,AyméS,etal:AnovelmicrosatelliteDNA (microsatellitemarkers)andwithinterpretationofdata.FSJBiologistPhD, markeratlocusD7S1870detectshemizygosityin75%ofpatientswith collaboratedwiththestandardizationofmethods(MLPA)andwithanalysis Williamssyndrome.AmJHumGenet1995,56:542-544. andinterpretationofdata.DRBAssistantDoctorinUnitofGenetics,Instituto 19. Brøndum-NielsenK,BeckB,GyftodimouJ,HørlykH,LiljenbergU, daCriança,FMUSP,collaboratedwiththeambulatorycareofWilliams PetersenMB,PedersenW,PetersenMB,SandA,SkovbyF,etal: syndromepatients.CAKSponsoroftheresearchofthisworkand Investigationofdeletionsat7q11.23in44patientsreferredforWilliams- responsibleforUnitofGenetics,InstitutodaCriança,FMUSPand Beurensyndrome,usingFISHandfourDNApolymorphisms.HumGenet participatedwithrevisingitcriticallyforimportantintellectualcontent.All 1997,99:56-61. authorsreadandapprovedthefinalmanuscript. 20. MerlaG,Brunetti-PierriN,MicaleL,FuscoC:Copynumbervariantsat Williams-Beurensyndrome7q11.23region.HumGenet2010,128(1):3-26. Competinginterests 21. KozlowskiP,JasinskaAJ,KwiatkowskiDJ:Newapplicationsand Non-financialcompetinginterests. developmentsintheuseofmultiplexligation-dependentprobe amplification.Electrophoresis2008,29:4627-4636. Received:12October2011 Accepted:9January2012 22. BishopB:Applicationsoffluorescenceinsituhybridization(FISH)in Published:9January2012 detectinggeneticaberrationsofmedicalsignificance.Biohorizons2010, 3:85-95. References 23. HowaldC,MerlaG,DigilioMC,AmentaS,LyleR,DeutschS,ChoudhuryU, 1. WilliamsJC,Barratt-BoyesBG,LoweJB:Supravalvularaorticstenosis. BottaniA,AntonarakisSE,FryssiraH,DallapiccolaB,ReymondA:Twohigh Circulation1961,24:1311-1318. throughputtechnologiestodetectsegmentalaneuploidiesidentifynew 2. BeurenAJ,ApitzJ,HarmjanzD:Supravalvularaorticstenosisin Williams-Beurensyndromepatientswithatypicaldeletions.JMedGenet associationwithmentalretardationandacertainfacialappearance. 2006,43:266-273. Circulation1962,26:1235-1240. 24. SchubertC,LacconeF:Williams-Beurensyndrome:determinationof 3. MorrisCA,DemseySA,LeonardCO,DiltsC,BlackburnBL:Naturalhistory deletionsizeusingquantitativereal-timePCR.IntJMolMed2006, ofWilliamssyndrome:physicalcharacteristics.JPediatr1988,113:318-326. 18:799-806. 4. StrømmeP,BjørnstadPG,RamstadK:PrevalenceestimationofWilliams 25. SnijdersAM,NowakN,SegravesR,BlackwoodS,BrownN,ConroyJ, syndrome.JChildNeurol2002,17:269-271. HamiltonG,HindleAK,HueyB,KimuraK,etal:Assemblyofmicroarrays 5. MorrisCA,ThomasIT,GreenbergF:Williamssyndrome:autosomal forgenome-widemeasurementofDNAcopynumber.NatGenet2011, dominantinheritance.AmJMedGenet1993,47:478-481. 29:263-264. 6. AshkenasJ:Williamssyndromestartsmakingsense.AmJHumGenet 26. ShafferLG,BejjaniBA:Acytogeneticist’sperspectiveongenomic 1996,59:756-761. microarrays.HumReprodUpdate2004,10:221-226. 7. PeoplesR,FrankeY,WangYK,Pérez-JuradoL,PapernaT,CiscoM, 27. JeheeFS,TakamoriJT,MedeirosPF,PordeusAC,LatiniFR,BertolaDR, FranckeU:Aphysicalmap,includingaBAC/PACclonecontig,ofthe KimCA,Passos-BuenoMR:UsingacombinationofMLPAkitstodetect Williams-Beurensyndrome–deletionregionat7q11.23.AmJHumGenet chromosomalimbalancesinpatientswithmultiplecongenitalanomalies 2000,66:47-68. andmentalretardationisavaluablechoicefordevelopingcountries.Eur 8. BayésM,MaganoLF,RiveraN,FloresR,PérezJuradoLA:Mutational JMedGenet2011,54(4):e425-e432. mechanismsofWilliams-Beurensyndromedeletions.AmJHumGenet 28. MillerSA,DykesDD,PoleskyHF:Asimplesaltingoutprocedurefor 2003,73:131-151. extractingDNAfromhumannucleatedcells.NucleicAcidsRes1988, 9. GagliardiC,BonagliaMC,SelicorniA,BorgattiR,GiordaR:Unusual 16:1215. cognitiveandbehaviouralprofileinaWilliamssyndromepatientwith atypical7q11.23deletion.JMedGenet2003,40:526-530. dCoitie:1t0h.1is18a6rt/i1c7le56a-s0:5D00u-t5ra-1e3tal.:CopynumbervariationinWilliams- 10. BergJS,Brunetti-PierriN,PetersSU,KangSH,FongCT,SalamoneJ, Beurensyndrome:suitablediagnosticstrategyfordevelopingcountries. FreedenbergD,HannigVL,ProckLA,MillerDT,etal:Speechdelayand BMCResearchNotes20125:13. autismspectrumbehaviorsarefrequentlyassociatedwithduplicationof the7q11.23Williams-Beurensyndromeregion.GenetMed2007, 9:427-441. 11. VanderAaN,RoomsL,VandeweyerG,vandenEndeJ,ReyniersE, FicheraM,RomanoC,DelleChiaieB,MortierG,MentenB,etal:Fourteen newcasescontributetothecharacterizationofthe7q11.23 microduplicationsyndrome.EurJMedGenet2009,52:94-100. 12. SbruzziIC,PereiraAC,VasconcelosB,HonjoRS,KriegerJE,KimCA: Williams-Beurensyndrome:diagnosisbypolymorphicmarkers.Genet TestMolBiomarkers2010,14:209-214.