Rapid identification of HBB gene mutations by high-resolution melting analysis.pdf

Rapid identification of HBB gene mutations by high-resolutionmelting analysisHung-Chang Shiha,b,c,d,Tze-Kiong Erb,e,Tien-Jye Changc,Ya-Sian Changb,Ta-Chih Liua,b,e,Jan-Gowth Changa,b,e,f,aInstitute of Clinical Medicine,College of Medicine,Kaohsiung Medical University,Kaohsiung,TaiwanbGraduate Institute of Medicine,College of Medicine,Kaohsiung Medical University,Kaohsiung,TaiwancDepartment of Veterinary Medicine,National Chung Hsiung University,Taichung,TaiwandDepartment of Laboratory Medicine,China Medical University Hospital,Taichung,TaiwaneDepartment of Laboratory Medicine,Kaohsiung Medical University Hospital,Kaohsiung,TaiwanfCenter for Excellence in Environmental Medicine,Kaohsiung Medical University,Kaohsiung,TaiwanReceived 24 April 2009;received in revised form 3 July 2009;accepted 14 July 2009Available online 23 July 2009AbstractObjective:This study was undertaken to identify HBB gene mutation.Design and methods:Herein we evaluated high-resolution melting analysis in the identification of HBB mutations.Results:We have successfully established a diagnostic strategy for identifying HBB gene mutations including c.78ANG,c.79ANG,c.2TNG,c.79_80insT,c.84_85insC,c.123_124insT,c.125_128delTCTT,c.130 GNT,c.170GNA,c.216_217ins A and c.316197 CNT fromwild-type DNA using HRM analysis.The results of HRM analysis were confirmed by direct DNA sequencing.Conclusions:In summary,we report that HRM analysis is an appealing technique for the identification of HBB mutations.We also believe thatHRM can be used as a method for prenatal diagnosis of-thalassemia.2009 The Canadian Society of Clinical Chemists.Published by Elsevier Inc.All rights reserved.Keywords:HBB gene;Mutation;-thalassemia;High-resolution melting analysis;Single nucleotide polymorphism;Melting curveIntroductionHemoglobinopathies resulting from mutations in the-or-like globin gene clusters are the most common inheriteddisorders in humans,with around 7%of the world populationbeing carriers of a globin gene mutation 1.Molecular defectsin either regulatory or coding regions of the human-,-or-globin genes can minimally or drastically reduce theirexpression,leading to-,-or-thalassemia,respectively.Some single substitutions can lead to amino acid replacementsthat cause hemolytic anemias,such as sickle cell disease,orhemoglobins that are unstable or have altered oxygen affinity.Other sequence changes have little or no effect on hemoglobinfunction,but are useful polymorphisms for genetic studies.-thalassemia is an endemic disease in many regions of theworld.The most common genetic lesion of-thalassemia ispoint mutations.Patients of each ethnic population carry theirown specific types of mutations,consisting of a few verycommon ones and a variable number of rare ones 2,3.Inthe Southeast-Asian population,the common-thalassemiamutations include c.-78 ANG,c.2 TNA,c.52 ANT,c.84_85insC,c.125_128 delTCTT,c.130 GNT,c.216_217 insA,andc.316197 CNT 4.In Chinese,more than 20 types ofmutations have been found,and half of these types are alsofound in Taiwanese.In Orientals,it is one of the most commonhereditary diseases with carrying rates of 13%5,6.The mostcommon mutations are point mutations of-globin gene;fourAvailable online at Clinical Biochemistry 42(2009)16671676Abbreviations:HBB,Hemoglobin,beta;Hb,Hemoglobin;PCR,Polymer-ase Chain Reaction;PCR-RFLP,PCR-Restriction Fragment Length Polymorph-ism;HRM,High-Resolution Melting;SNP,Single Nucleotide Polymorphism;High performance liquid chromatography,HPLC;Capillary Electrophoresis,CE;Amplification Refracted Mutation System,ARMS;Single Base Extension,SBE.Corresponding author.Department of Laboratory Medicine,KaohsiungMedical University Hospital,100 Shih-Chuan 1st Rd.,Kaohsiung,Taiwan.E-mail address:jgchangmail.kmuh.org.tw(J.-G.Chang).0009-9120/$-see front matter 2009 The Canadian Society of Clinical Chemists.Published by Elsevier Inc.All rights reserved.doi:10.1016/j.clinbiochem.2009.07.017types of the point mutation account for over 85%of cases of-thalassemia in Taiwan.These are promoter 28 AG,codon17 AAGTAG,codon 4142 deleted 4 bases,and IVS-2 nt654 CT mutations 7.The most common-thalassemiamutation was the IVS-2 nt 654 CT mutation;and the mostcommon Hemoglobin(Hb)variant was the HbE 8.High-resolution melting(HRM)analysis is a new gene scantool that quickly performs the Polymerase Chain Reaction(PCR)and identifies sequences alterations without requiringpost-PCR treatment 912.Recently,it has also been used inthe detection of-and-thalassemia of deletion form 13,14.The authors utilized real-time gap-PCR with SYBR Green1 andHRM analysis for diagnosis of-thalassemia 3.5 kb deletion.So far,there is no thorough study on the capability of HRM toidentify HBB(hemoglobin,beta)gene point mutations.In thisstudy,we report a method for rapid detection of HBB genemutations.Materials and methodsDNA samplesDNA samples were obtained from 10 prenatal casesincluding parents,and 62 subjects including 30-thalassemiaminor,3-thalassemia major,Hb variants including 2 cases ofHbE,6 cases of Hb J-Kaohsiung and 1 cases of Hb J-Meinungand 20 normal individuals at Kaohsiung Medical UniversityHospital.Genomic DNA was collected from peripheral wholeblood using the NucleoSpin Blood Kit(Macherey-Nagel)according to the manufacturers instructions.The genotypingfrom each samples were determined by the PCR-RestrictionFragment Length Polymorphism(PCR-RFLP)previously.PrimersWe designed the primer sets on the HBB DNA sequences(NCBI Reference Sequence:NG_000007.3).Table 1 shows theprimer sets for the detection of HBB gene mutations inpromoter,exon 1,exon 2,intron 3 and exon 3.For the prenataldiagnosis of-thalassemia,we used primer P1+P3 fordetecting the promoter and initiation codon mutations,primerP2+P4 for detecting the mutations in exon 1 region except theinitiation codon,primers P5+P9 for detecting the mutation inthe exon 2 and primers P10+P11 for detecting the c.316197CNT.The primers synthesized were all of standard molecularbiology quality(Protech Technology Enterprise Co.,Ltd,Taiwan).High-resolution meltingPCR reactions were performed in duplicate in 20 L finalvolume using LightCycler 480 High-Resolution MeltingMaster(Roche Diagnostics)1contains Taq,nucleotides andthe dye ResoLight and 50 ng DNA.The primers and MgCl2were used at 2.5 mM,for HBB gene mutations.In each assay,we included DNAs with known HBB gene mutation and wild-type control DNAs.The HRM assays were performed using the LightCycler480 Instrument(Roche Diagnostics)provided with the softwareLightCycler 480 Gene Scanning Software Version 1.0(RocheDiagnostics).The PCR program requires SYBR Green I filter(533 nm)and it consists of an initial denaturationactivation step at 95 Cfor 10 min,followed by a 45-cycle program(denaturation at95 C for 15 s,annealing at 56 C or 52 C(according toTable 1)15 s and elongation at 72 C for 15 s with reading of thefluorescence;acquisition mode:single).The melting programincludes three steps:denaturalization at 95 C for 1 min,renaturation at 40 C for 1 min and then melting that consists ofa continuous fluorescent reading from 60 to 90 C at 25acquisitions per C.The shapes of difference plot curves of the duplicate of eachDNAsamplemustbereproduciblebothinshapeandpeakheight.Gene scanningThe melting curve analysis performed by the Gene ScanningSoftware comprises three steps:normalization of meltingTable 1Primers use for HRM analysis of HBB gene mutations.Detection forSequence(5 to 3)Length of PCRamplicon(bp)Nucleotides(nt)Annealingtemp.(C)Promoter and Exon 1P1 5-CCAATCTACTCCCAGGAGCA-3(forward)323nt70469nt7048852P2 5-GGCAGAGAGAGTCAGTGCCTA-3(reverse)nt70771nt70791Promoter and Initiation codonP3 5-ACTTCTCCTCAGGAGTCAGGT-3(reverse)154nt70602nt7062256Exon 1P4 5-AGACACCATGGTGCACCTGAC-3(forward)204nt70588nt7060856Exon 2P5 5-GAAGACTCTTGGGTTTCTGA-3(forward)404nt70751nt7077052P6 5-TCATTCGTCTGTTTCCCATTCTAAAC-3(reverse)nt71129nt71154Exon 2P7 5-GAGCCTTCACCTTAGGGTTT-3(reverse)164nt70895nt7091456Exon 2P8 5-CTCCTGATGCTGTTATGGGC-3(forward)193nt70876nt7089556P9 5-AGAAAACATCAAGGGTCCCA-3(reverse)nt71049nt71068Intron 2P10 5-GTGTACACATATTGACCAAATCAGGGTA-3(forward)293nt71496nt7152356P11 5-GGTAGCTGGATTGTAGCTGC-3(reverse)nt71769nt71788Intron 2P12 5-ATTTATATGCAGAAATATTG-3(reverse)223nt71699nt7171852Exon 3P13 5-CTGGATTATTCTGAGTCCAAGC-3(forward)309nt71820nt7184152P14 5-ATTAGGCAGAATCCAGATGCTC-3(reverse)nt72107nt72128PCR primers were redesigned to block SNP interference.1668H.-C.Shih et al./Clinical Biochemistry 42(2009)16671676curves,equaling to 100%the initial fluorescence and to 0%thefluorescence remnant after DNA dissociation,shifting of thetemperature axis of the normalized melting curves to the pointwhere the entire double-stranded DNA is completely denatured,and finally,the difference plot analyzes the differences inmelting curve shape by subtracting the curves from wild-typeand HBB mutations DNA,therefore the difference plot helpsclustering the samples into groups.SequencingTo confirm HRM analysis results,sequencing analysis wasalso performed in all samples.After HRM analysis,sampleswere purified with PCR-M clean up system(VIOGEN).ThePCR products generated after HRM can be sequenced directly.The sequence reaction was performed in 10 L final volumeusing 5 L of the purified PCR product,2.5 M of one of PCRprimers and 1 L of ABI PRISM terminator cycle sequencingkit v3.1(Applied Biosystems).The sequencing program is a 25-cycle PCR program(denaturation 96 C 10 s;annealing 50 C5 s and elongation 60 C 4 min).The sequence detection wasperformed in the ABI Prism 310 Genetic Analyzer(AppliedBiosystems).ResultsScreening of the HBB genes mutations:promoter and exon1 regionsIn this section,we used first-designed primer(P1+P2)toidentify HBB genes mutations in promoter and exon 1 includingc.78ANG,c.79ANG,c.2TNG,c.52ANT,c.79GNA,andc.84_85insC.Finally,we found 1 single nucleotide polymorph-ism SNP(c.9CNT)in the region and this SNP interfere theidentification of HBB genes mutations in promoter and exon 1using HRM analysis(Supplementary data 1).We were unable todifferentiate the c.78ANG,c.79ANG,c.2TNG,c.52ANT,c.79GNA,and c.84_85insC from the complicated meltingcurve.Therefore,we redesigned two new primer sets(P1+P3and P4+P2)which overlay the SNP in order to block the SNPinterference.We successfully used the new primers set toidentify c.78ANG,c.79ANG,c.2TNG,c.52ANT,c.79GNA,and c.84_85insC.Fig.1 shows that we were ableto differentiate the c.78ANG,c.79ANG,c.2TNG from themelting curve by using primers P1 and P3.Fig.2 shows that wewere able to differentiate the c.52ANT,c.79GNA,andc.84_85insC from the melting curve by using primers P4 andFig.1.Screeningof the HBB genes mutations in promoter and initiation codon.(A)is the high-resolution melting curves and(B)is the difference plots with redesignednew primer set(P1+P3).The c.78ANG,c.79ANG and c.2TNG are easily to be distinguished in the normalized and temp-shifted difference plot.1669H.-C.Shih et al./Clinical Biochemistry 42(2009)16671676P2.Those results were confirmed by direct sequencing of PCRproducts(Supplementary data 1).Screening of the HBB genes mutations:exon 2 regionIn this section,we use first-designed primers(P5+P6)toidentify HBB genes mutations in exon 2 includingc.123_124insT,c.125_128delTCTT,c.130 GNT,c.216_217insA and Hb J-Meinung.Finally,we found 3 SNPs(c.171CNG,c.315+16GNC and c.315+74TNG)in this region and the 3SNPs interfere the identification of HBB genes mutations inexon 2 using HRM analysis(Supplementary data 2).Also,we were unable to differentiate the c.123_124insT,c.125_128delTCTT,c.130 GNT,c.216_217 ins A and Hb J-Meinung from the complicated melting curve.Therefore,weredesigned anewprimer set(P5+P9)whichoverlaidthe2SNPsin order to block the SNPs interference.We successfully usedthisnewprimersettoidentifyc.123_124insT,c.125_128delTCTT,c.130 GNT,c.216_217 ins A and Hb J-Meinung(Fig.3).On the other hand,we tried to redesign twonew primers sets(P5+P7 and P8+P9)to block 3 SNPs,however,the curve of c.170GNA(Hb J-Meinung or Hb J-Bangkok)shift to the group of wild-type DNA by using theprimersetsP5andP7(Supplementarydata3).Thec.216_217insA can be easily distinguished from wild-type DNA using newprimer set(P8+P9)(Supplementary data 4).These results wereconfirmed by direct sequencing of the PCR products.Screening of the HBB genes mutations:intron 2In this section,we used first-designed primer(P10+P11)toidentify HBB genes mutations in intron 2,c.316197 C.Wediscovered 1 SNP(c.316185CNT)in intron 2,however,wewere still able to identify c.316197 C from wild-type DNA.Fig.4 shows that the SNP did not interfere with the meltingcurve.The c.316197 C could be easily seen in the normalized,temperature-shifted plot.Even though,we also redesigned anew primers set(P10+P12)in order to block the SNP.The SNPwas successfully blocked by the redesigned primer and we wereable to see the grouping in the normalized,temperature-shiftedplot(Supplementary data 5).These results were confirmed bydirect sequencing of the PCR products.Screening of the c.341TNA(Hb Kaohsiung or New York):exon 3The HRM analysis successfully differentiated the six clinicalsamples(c.341TNA)from the wild-type control DNA on theFig.2.Screening of the HBB genes mutations in exon 1.(A)is the high-resolution melting curves and(B)is the difference plots with redesigned new primer set(P4+P2).The c.79GNA,c.52ANT and c.84_85insC are easily to be distinguished in the normalized and temp-shifted difference plot.1670H.-C.Shih et al./Clinical Biochemistry 42(2009)16671676basis of the shape of normalized,temperature-shifted plot anddifference plot curves(Fig.5).These c.341TNA were clearlydistinguishable from the wild-type control DNA.HRM analysisprofiles of c.341TNA were confirmed by DNA sequencing.These results were confirmed by direct sequencing of the PCRproducts.Application of HRM analysis in prenatal diagnosis of-thalassemiaIn this section,we would like to evaluate the practicality ofthis methodology for prenatal diagnosis.Prenatal samples withknown HBB gene mutation were made by a blindedinvestigator.These clinical samples were obtained from 10families including parents and probands.Four primer sets(P1+P3,P2+P4,P5+P9 and P10+P11)were used to analyzethe samples using HRM analysis in the same PCR condition.All of the positive controls and negative controls were alsoadded in the same run.We are able to distinguish the unknownsample whether it is mutant or normal according to the meltingcurves of positive and negative controls in the normalized andtemp-shifted difference plot.For a rare case,we use the directsequencing for rechecking the result.The HRM were able to detect the mutations precisely.Fig.6showed that the representative cases were successfullyidentified by only a single run.DiscussionThere are many techniques that have been used to screen anddiagnose Hb variants and thalassemia.For example,highperformance liquid chromatography(HPLC)15,capillaryelectrophoresis(CE)16,restriction fragment length poly-morphism(RFLP)17,ampl