research article efficient gene mutation detection with...

Hindawi Publishing CorporationISRNMolecular BiologyVolume 2013 Article ID 451298 4 pageshttpdxdoiorg1011552013451298

Research ArticleEfficient IDUA Gene Mutation Detection with Combined Use ofdHPLC and Dried Blood Samples

Diogo Ribeiro1 Ana Cardoso2 Ana Joana Duarte1 Luis Vieira2 and Olga Amaral1

1 Departamento de Genetica Humana Unidade IampD-P DLS CGMJM Instituto Nacional de Saude Dr Ricardo Jorge (INSA IP)Pr Pedro Nunes 88 4099-028 Porto Portugal

2 Departamento de Genetica Humana Unidade de Tecnologia e Inovacao (UTI) Instituto Nacional de Saude Dr Ricardo Jorge(INSA IP) Avenida Padre Cruz 1649-016 Lisboa Portugal

Correspondence should be addressed to Olga Amaral olgaamaralinsamin-saudept

Received 14 March 2013 Accepted 3 April 2013

Academic Editors D Bozon E Caffarelli A D Hollenbach B L Nielsen and C-H Yuh

Copyright copy 2013 Diogo Ribeiro et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objectives Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using aneasily obtainable biological material Assessment of the feasibility of such method was tested using a GC-rich amplicon Designand Methods A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as atechnique for the detection of variants in exon 9 of the IDUA geneThe optimized method was tested in 500 genomic DNA samplesobtained from dried blood spots (DBS) Results With this dHPLC approach it was possible to detect different variants includingthe common pTrp402Ter mutation in the IDUA gene The high GC content did not interfere with the resolution and reliability ofthis technique and discrimination of G-C transversions was also achieved Conclusion This PCR-based dHPLC method is provedto be a rapid a sensitive and an excellent option for screening numerous samples obtained from DBS Furthermore it resultedin the consistent detection of clearly distinguishable profiles of the common pTrp402Ter IDUA mutation with an advantageousbalance of cost and technical requirements

1 Introduction

Mucopolysaccharidosis type I (MPS I) is a rare autoso-mal recessive disorder resulting from deficiency of theenzyme 120572-L-iduronidase (IDUA EC 32176) and leadsto the intralysosomal accumulation of undegraded gly-cosaminoglycans [1] The gene encoding 120572-L-iduronidase(IDUA OMIMno 252800) is located on chromosome4p163 and contains 14 exons [2ndash4] The most commonIDUA mutation identified in MPS I patients is pTrp402Ter(NG 0081031 g20751GgtA NM 0002033 c1205GgtA andNP 0001942 pTrp402Ter) which represents at least 45 ofthe causal alleles in Northwestern Europe North AmericaAustralia Portugal and Spain while in Russia Italy andBrazil its frequency has been estimated to be 4 11 and37 respectively [5 6]

Since pTrp402Ter is the single most common causalmutation inMPS IH (OMIM no 607014) patients ofWesternEuropean origin and accounts for 60 of the alleles in

unrelated Portuguese patients [7] we wanted to develop arapid and reliable method for the convenient detection of thisIDUAmutation (OMIMno 2528000001)

Denaturing high-performance liquid chromatography(dHPLC) was successfully applied to the screening of muta-tions involved in various diseases [8] including MPS I [9]We optimized an efficient dHPLC method and validated itsapplication to mutation screening using DBS as source ofbiological material This semiautomated technique resultsin clear profiles and facilitates the accurate detection ofthe pTrp402Ter mutation in the IDUA gene allowing thescreening of a large number of samples Conventional meth-ods for large-scale detection of mutations such as SSCPCSGE RFLP and sequencing are expensive are technicallydemanding are time consuming and sometimes have limitedresolution The application of dHPLC allows the discrimina-tion of mutations at a cost that is fourfold lower than dye-terminator sequencing The presently described approachis a cost effective sensitive and reproducible method for

2 ISRNMolecular Biology

Table 1 PCR amplification conditions

Exon 9 Amplicon size (bp) GC content () Primers (51015840 rarr 31015840) PCR program

First PCR 379 78 F-GGAGCGAGTGGTGGGAGG 97∘C5minR-GACACTCAGGCCTCGGCTC 96∘C1min

35X60∘C1min

Nested PCR 208 77 F-GGCGGCTGGGCAACGACC 72∘C1minR-GTGGGCGCGGGTGTCGTC 72∘C10min

PCR reactions were performed using PCRMasterMix (Promega) without any additives 20 pmol of forward primer and 20 pmol of reverse primer in the firstPCR 4120583L (4-5 ng) of DNAwas used in a 10120583L final volume in the nested PCR 1120583L of a 110 dilution of the first PCR product was used in a 20120583L final volume

screening large numbers of samples even from distant loca-tions with minimal technical requirements

2 Materials and Methods

21 Biological Materials A total of 500 samples fromanonymized individuals born in Portugal were randomlyselected These 500 samples consisted of surplus remaindersamples of dried blood spots (Guthrie cards DBS) from theNational Program of Neonatal Screening (Instituto Nacionalde Saude Dr Ricardo Jorge Centro de Genetica Medica DrJacinto Magalhaes) Appropriate approval for investigationaluse was obtained in accordance with national and institu-tional ethical regulations Control heterozygous (GM00799)and homozygous (GM00798) human skin fibroblast cell lineswith pTrp402Ter mutation were obtained from the CoriellInstitute for Medical Research Genomic DNAwas automati-cally extracted using a Maxwell 16 System (Promega) DNAfrom 3 punches (2mm oslash) yielded approximately 250 ng ofDNA with a purity of 18 (A260A280) and 5 ng of DNAwas used as template in the PCR reaction Differences insample quality from gDNA extracted from different sources(fibroblast cell lines andDBS)wereminimized by the dilutionof the template used for obtaining the final amplicon fordHPLC analysis

22 PCR Amplification PCR primers were based on thoseproposed by Kasper and collaborators [9] withmodificationsnamely removal of (GC) clamps and amplification of asmaller fragment for dHPLC analysis Optimization of thePCR reaction was performed by varying PCR conditionsincluding Taq mixes cycle temperaturestimes and templateconcentrations Table 1 shows the final PCR conditions PCRwith 5 ng of template DNA resulted in clear and abundantamplicons that did not require any purification procedurePrior to the dHPLC optimization the IDUA gene of controlsamples was sequenced in order to ensure its integrity

23 dHPLC Conditions Denaturing high-performance liq-uid chromatography (dHPLC) analysis was performed usingan automatedWAVENucleic Acid Fragment Analysis Systemand respective Wavemaker 34 software (Transgenomic)Melt Program from Stanford Genome Technology Center(httpinsertionstanfordedumelt) was used for the opti-mization of dHPLC conditions

Prior to dHPLC analysis PCR products were heated to96∘C for 3min and slowly cooled to 65∘C using a ramping

of 01∘C for each 6 sec (62 cycles) Five120583L of the cooled PCRproduct was applied onto a preheated C18-reversed phasecolumn (DNASep column Transgenomic) DNA was elutedat a flow rate of 09mLmin within a linear acetonitrilegradient consisting of Buffer A (01M triethylammoniumacetate (TEAA)) and Buffer B (01MTEAA 25 acetonitrile)and detected spectrophotometrically by UV absorption

The optimal percentage of Buffer B was 52 run timeand column temperature were 8min and 685∘C respectivelyAfter each run regeneration of the column was achieved bywashing with 100 Solution D (Transgenomic) for 05minfollowed by an equilibration time of 2min To test the dHPLCcapacity for detecting pTrp402Ter DNA from wild type andhomozygote were mixed in different proportions Sampleswith abnormal heteroduplex patterns as well as randomlyselected sampleswith normal profiles were amplified de novoand subjected to direct automated sequencing

3 Results

The optimal PCR conditions were as described in themethods section Optimization of dHPLC conditions andsubsequent dHPLC analysis of PCR products was carriedout using samples with and without the pTrp402Ter IDUAmutation in order to validate the technique A total of 500DNA samples plus previously characterized controls werethen amplified and analyzed by the dHPLC technique Asseen in Figure 1 a variant dHPLC profile was detected in thecase of a pTrp402Ter heterozygous carrier and four addi-tional dHPLC variant profiles were obtained correspondingto homozygosity or heterozygosity for the SNP pThr410=(NG 0081031 g20776CgtG NM 0002033 c1230CgtGand rs115790973) and homozygosity or heterozygosityfor the SNP pGly409Arg (NG 0081031 g20771GgtCNM 0002033 c1225GgtC and rs11934801) Each of thefive different dHPLC profiles was distinguishable andcharacterization was achieved by DNA sequencing

The allele frequency of SNP pGly409Arg was 4whereas SNP pThr410= had an allele frequency of 17 (forthe allelic form g20776G) similar to the one reported indatabases demonstrating the consistent detection of alter-ations and the reproducibility of the assay The dependabledetection of the pTrp402Ter mutation in heterozygous con-trol samples showed the reliability of the assay which wasfurther reinforced by the finding of a heterozygous sampleamong the 500 randomly selected samples tested

ISRNMolecular Biology 3

Heterozygous Homozygous Wild typeHeterozygousHomozygousHeterozygouspGly409ArgpThr410= pTrp402Ter

4 5 6 4 5 6 4 5 6 4 5 6 4 5 6 4 5 6

Figure 1 Different dHPLC profiles of the exon 9 of IDUA gene Presence of heteroduplexes when the absorbance was measured against timeDifferences were observed between dHPLC profiles of wild-type amplicons and amplicons carrying the pTrp402Ter mutation or the SNPspThr410= or pGly409Arg As shown retention times were 4 to 6 minutes Profiles were normalized according to their height elution timeand endpoints

The use of dried blood spots as source of DNA for dHPLCanalysis was validated by the regularity of the results

4 Discussion

The existence of multiple nucleotide variations along withhigh GC content and with mutations leading to decreasedlevels of mRNA interferes with the efficiency of mutationdetection techniques namely with SSCP allele discrimina-tion and cDNA sequencing With dHPLCrsquos high level ofdetection reproducibility sensitivity and resolution even inGC-rich regions we were able to discriminate between nor-mal mutated and polymorphic alleles The polymorphismsdetected in this study were not detected in combination withthe causal mutation pTrp402Ter As previously described byother groups DBS represent an extremely convenient sourceof biological material [10] for mutational and biochemicalscreenings This point becomes particularly important sinceunlike with enzyme assays in which differences were foundbetween DBS and leukocyte activities [11] in the case of DNAno difference was found in the results obtained from differentsources of biological materials The successful use of driedblood spots (DBS) validates the use of these samples as areliable source of biological material for the application ofthe described laboratorial approachTherefore the use of DBSis not only practical and economical but is also convenientfor the large screenings or to the study of samples fromdistant locations Since themolecular lesion tested is themostcommon causal mutation of MPS IH in Western Europeanpopulations this could be a preferable method for initialscreening in populations with that particular ancestry

According to different reports the use of dHPLC as amutation screening method is particularly advantageous notonly because of its simplicity and sensitivity [12] but alsodue to its cost which has been estimated to be about 80lower than that direct sequencing [13 14] Although thecost might vary depending on the country in our case ofit was estimated to be 350 eurosample (as opposed to 14euro per sample sequencing) Considering the use of otheralternative techniques such as SSCP or allelic discriminationthis optimized method showed a clear advantage by allowingthe efficient identification of alterations in a GC-rich regionwhile requiring very low template concentrations

In conclusion with this work we improved and devel-oped a dHPLC screening method aiming at the detection ofvariations in a region of the IDUA gene with a GC contentof 77 This dHPLC technique was found to be a very usefulscreening tool since it is a sensitive automatable economicaland highly robust method The combination of dHPLC withthe usewithDBS samplesmakes this approachmost adequatefor the screening of numerous samples

Acknowledgments

This work was financially supported by National Fundsthrough Fundacao da Ciencia e Tecnologia (FCT) MCTESPortugal under Project ldquoPICIC828222007rdquo Diogo RibeiroandAna JoanaDuarte received FCTgrants (2009) andDiogoRibeiro is MS holder at University of Minho

References

[1] E F Neufeld and J Muenzer ldquoThe mucopolysaccharidosesrdquo inThe Metabolic and Molecular Bases of Inherited Disease C RScriver A Beaudet W S Sly and D Valle Eds pp 3421ndash3452McGraw-Hill New York NY USA 8th edition 2001

[2] H S Scott L J Ashton H J Eyre et al ldquoChromosomallocalization of the human 120572-L-iduronidase gene (IDUA) to4p163rdquo The American Journal of Human Genetics vol 47 no5 pp 802ndash807 1990

[3] H S Scott D S Anson A M Orsborn et al ldquoHuman alpha-l-iduronidase cDNA isolation and expressionrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 21 pp 9695ndash9699 1991

[4] H S Scott X H Guo J J Hopwood and C P MorrisldquoStructure and sequence of the human 120572-L-iduronidase generdquoGenomics vol 13 no 4 pp 1311ndash1313 1992

[5] N J Terlato and G F Cox ldquoCan mucopolysaccharidosis type Idisease severity be predicted based on a patientrsquos genotype Acomprehensive review of the literaturerdquo Genetics in Medicinevol 5 no 4 pp 286ndash294 2003

[6] U Matte G Yogalingam D Brooks et al ldquoIdentification andcharacterization of 13 newmutations in mucopolysaccharidosistype I patientsrdquoMolecular Genetics and Metabolism vol 78 no1 pp 37ndash43 2003


[7] R Pinto C Caseiro M Lemos et al ldquoPrevalence of lysosomalstorage diseases in Portugalrdquo European Journal of HumanGenetics vol 12 no 2 pp 87ndash92 2004

[8] W Xiao and P J Oefner ldquoDenaturing high-performance liquidchromatography a reviewrdquo Human Mutation vol 17 no 6 pp439ndash474 2001

[9] D C Kasper F Iqbal L Dvorakova et al ldquoRapid and accuratedenaturating high performance liquid chromatography pro-tocol for the detection of 120572-l-iduronidase mutations causingmucopolysaccharidosis type Irdquo Clinica Chimica Acta vol 411no 5-6 pp 345ndash350 2010

[10] A J Reuser F W Verheijen D Bali et al ldquoThe use ofdried blood spot samples in the diagnosis of lysosomal storagedisordersmdashcurrent status and perspectivesrdquoMolecular Geneticsand Metabolism vol 104 no 1-2 pp 144ndash148 2011

[11] N A Chamoles M B Blanco D Gaggioli and C CasentinildquoHurler-like phenotype enzymatic diagnosis in dried bloodspots on filter paperrdquo Clinical Chemistry vol 47 no 12 pp2098ndash2102 2001

[12] P Carbonell M C Turpin D Torres-Moreno et al ldquoCompar-ison of allelic discrimination by dHPLC HRM and Taqmanin the detection of braf mutation V600Erdquo Journal of MolecularDiagnostics vol 13 no 5 pp 467ndash473 2011

[13] H Takashima C F Boerkoel and J R Lupski ldquoScreeningfor mutations in a genetically heterogeneous disorder DHPLCversus DNA sequence for mutation detection in multiplegenes causing Charcot-Marie-Tooth neuropathyrdquo Genetics inMedicine vol 3 no 5 pp 335ndash342 2001

[14] E B de Oliveira-Junior C Prando J A Lopez et al ldquoHigh-performance liquid chromatography under partially denaturingconditions (dHPLC) is a fast and cost-effective method forscreening molecular defects four novel mutations found in x-linked chronic granulomatous diseaserdquo Scandinavian Journal ofImmunology vol 76 no 2 pp 158ndash166 2012

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Stem CellsInternational



Enzyme Research



Microbiology


Table 1 PCR amplification conditions

Exon 9 Amplicon size (bp) GC content () Primers (51015840 rarr 31015840) PCR program

First PCR 379 78 F-GGAGCGAGTGGTGGGAGG 97∘C5minR-GACACTCAGGCCTCGGCTC 96∘C1min

35X60∘C1min

Nested PCR 208 77 F-GGCGGCTGGGCAACGACC 72∘C1minR-GTGGGCGCGGGTGTCGTC 72∘C10min

PCR reactions were performed using PCRMasterMix (Promega) without any additives 20 pmol of forward primer and 20 pmol of reverse primer in the firstPCR 4120583L (4-5 ng) of DNAwas used in a 10120583L final volume in the nested PCR 1120583L of a 110 dilution of the first PCR product was used in a 20120583L final volume

screening large numbers of samples even from distant loca-tions with minimal technical requirements

2 Materials and Methods

21 Biological Materials A total of 500 samples fromanonymized individuals born in Portugal were randomlyselected These 500 samples consisted of surplus remaindersamples of dried blood spots (Guthrie cards DBS) from theNational Program of Neonatal Screening (Instituto Nacionalde Saude Dr Ricardo Jorge Centro de Genetica Medica DrJacinto Magalhaes) Appropriate approval for investigationaluse was obtained in accordance with national and institu-tional ethical regulations Control heterozygous (GM00799)and homozygous (GM00798) human skin fibroblast cell lineswith pTrp402Ter mutation were obtained from the CoriellInstitute for Medical Research Genomic DNAwas automati-cally extracted using a Maxwell 16 System (Promega) DNAfrom 3 punches (2mm oslash) yielded approximately 250 ng ofDNA with a purity of 18 (A260A280) and 5 ng of DNAwas used as template in the PCR reaction Differences insample quality from gDNA extracted from different sources(fibroblast cell lines andDBS)wereminimized by the dilutionof the template used for obtaining the final amplicon fordHPLC analysis

22 PCR Amplification PCR primers were based on thoseproposed by Kasper and collaborators [9] withmodificationsnamely removal of (GC) clamps and amplification of asmaller fragment for dHPLC analysis Optimization of thePCR reaction was performed by varying PCR conditionsincluding Taq mixes cycle temperaturestimes and templateconcentrations Table 1 shows the final PCR conditions PCRwith 5 ng of template DNA resulted in clear and abundantamplicons that did not require any purification procedurePrior to the dHPLC optimization the IDUA gene of controlsamples was sequenced in order to ensure its integrity

23 dHPLC Conditions Denaturing high-performance liq-uid chromatography (dHPLC) analysis was performed usingan automatedWAVENucleic Acid Fragment Analysis Systemand respective Wavemaker 34 software (Transgenomic)Melt Program from Stanford Genome Technology Center(httpinsertionstanfordedumelt) was used for the opti-mization of dHPLC conditions

Prior to dHPLC analysis PCR products were heated to96∘C for 3min and slowly cooled to 65∘C using a ramping

of 01∘C for each 6 sec (62 cycles) Five120583L of the cooled PCRproduct was applied onto a preheated C18-reversed phasecolumn (DNASep column Transgenomic) DNA was elutedat a flow rate of 09mLmin within a linear acetonitrilegradient consisting of Buffer A (01M triethylammoniumacetate (TEAA)) and Buffer B (01MTEAA 25 acetonitrile)and detected spectrophotometrically by UV absorption

The optimal percentage of Buffer B was 52 run timeand column temperature were 8min and 685∘C respectivelyAfter each run regeneration of the column was achieved bywashing with 100 Solution D (Transgenomic) for 05minfollowed by an equilibration time of 2min To test the dHPLCcapacity for detecting pTrp402Ter DNA from wild type andhomozygote were mixed in different proportions Sampleswith abnormal heteroduplex patterns as well as randomlyselected sampleswith normal profiles were amplified de novoand subjected to direct automated sequencing

3 Results

The optimal PCR conditions were as described in themethods section Optimization of dHPLC conditions andsubsequent dHPLC analysis of PCR products was carriedout using samples with and without the pTrp402Ter IDUAmutation in order to validate the technique A total of 500DNA samples plus previously characterized controls werethen amplified and analyzed by the dHPLC technique Asseen in Figure 1 a variant dHPLC profile was detected in thecase of a pTrp402Ter heterozygous carrier and four addi-tional dHPLC variant profiles were obtained correspondingto homozygosity or heterozygosity for the SNP pThr410=(NG 0081031 g20776CgtG NM 0002033 c1230CgtGand rs115790973) and homozygosity or heterozygosityfor the SNP pGly409Arg (NG 0081031 g20771GgtCNM 0002033 c1225GgtC and rs11934801) Each of thefive different dHPLC profiles was distinguishable andcharacterization was achieved by DNA sequencing

The allele frequency of SNP pGly409Arg was 4whereas SNP pThr410= had an allele frequency of 17 (forthe allelic form g20776G) similar to the one reported indatabases demonstrating the consistent detection of alter-ations and the reproducibility of the assay The dependabledetection of the pTrp402Ter mutation in heterozygous con-trol samples showed the reliability of the assay which wasfurther reinforced by the finding of a heterozygous sampleamong the 500 randomly selected samples tested



4 5 6 4 5 6 4 5 6 4 5 6 4 5 6 4 5 6



4 Discussion




Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 5 6 4 5 6 4 5 6 4 5 6 4 5 6 4 5 6



4 Discussion




Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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