Genotyping of human platelet antigen-1 by gene amplification

and labelling in one system and automated fluorescence

correlation spectroscopy

Stephanie Weber,1

Stefan A. Hummel,1

Artur-Aron Weber,2

Rudolf F. Zirwes,1

Olaf H. Weiner1

and Bernadette E. Reuber1 1Evotec Technologies, EVOTEC Analytical Systems GmbH, Erkrath, and 2Institut fur

Pharmakologie und Klinische Pharmakologie, Universitatsklinikum Dusseldorf, Germany

Received 2 August 2001; accepted for publication 16 October 2001

Summary. Genotyping of human platelet antigen-1 (HPA-1) is required for the diagnosis and appropriate therapy ofalloimmunization. Recently, the HPA-1 polymorphism hasbeen identified as an inherited risk factor for thrombosis.Most currently used methods for HPA-1 genotyping havethe disadvantage of time-consuming post-polymerase chainreaction (PCR) processes such as ligation (oligonucleotideligation assay), restriction enzyme digestion (allele-specificrestriction enzyme analysis) and electrophoresis (single-strand conformation polymorphism). We present a novelmethod for HPA-1 genotyping based on a homogeneousPCR strategy (GALIOSÒ, gene amplification and labellingin one system) combined with automated fluorescencecorrelation spectroscopy (FCS). The PCR uses one pair ofgene-specific amplification primers and two allele-specific,semi-nested labelling primers. The allele-specific labelling

primers differ in a single nucleotide (T for HPA-1a/1a, C forHPA-1b/1b) and are coupled to different fluorescent dyes.The quantities of generated fluorescent PCR products areanalysed by FCS at 543 nm and 633 nm excitation wave-length respectively. The genotypes determined using thismethod were in 100% concordance with the resultsobtained by allele-specific restriction analysis (n ¼ 380samples). The assay was validated for specificity, reliabilityand the dynamic range. This innovative method of rapidHPA-1 genotyping offers a specific and robust system,which is applicable for routine HPA-1 genotyping.

Keywords: human platelet antigen-1, genotyping,GALIOSÒ, polymerase chain reaction, fluorescence cor-relation spectroscopy.

The biallelic expression of human platelet antigen-1(HPA-1, PLA) arises from a single nucleotide transition(T12548C) in the glycoprotein (GP) IIIa gene. This singlenucleotide polymorphism (SNP) causes a substitution ofleucine (HPA-1a, PLA1) to proline (HPA-1b, PLA2) at theamino acid residue 33 of GPIIIa (Newman et al, 1989).

The role of the HPA-1 polymorphism in neonatalalloimmune thrombocytopenia (Blanchette et al, 2000)and post-transfusion purpura (Waters, 1989) is well recog-nized. The altered molecular structure of GPIIIa in theHPA-1b allele may also affect the function of GPIIb/IIIa(Byzova and Plow, 2000). For example, altered fibrinogenbinding (Goodall et al, 1999) and increased outside-insignalling (Vijayan et al, 2000) have been described forthe HPA-1b allele. Although the data are not equivocal, the

HPA-1b allele, especially in combination with other inter-active risk factors, may be positively associated withthrombotic complications of atherosclerosis, such as myo-cardial infarction or ischaemic stroke (for reviews, seeNurden, 1997; Goldschmidt-Clermont et al, 1999; Byzovaand Plow, 2000). In addition, preliminary studies havedescribed a relationship between the HPA-1 genotype andplatelet sensitivity towards antiplatelet drugs such as aspirinor abciximab (Michelson et al, 2000; Szczeklik et al, 2000).If these results are confirmed by clinical studies, then HPA-1genotyping could be useful for individualized antithrombotictherapy.

The present study describes an innovative method forHPA-1 genotyping based on allele-specific polymerase chainreaction (PCR) and automated detection by fluorescencecorrelation spectroscopy (FCS) (Eigen and Rigler, 1994;Hovius et al, 2000; Foldes-Papp and Kinjo, 2001). Thesemi-nested PCR strategy, named GALIOSÒ (gene amplifi-cation and labelling in one system), uses one pair ofgene-specific amplification primers and two allele-specific

Correspondence: Stephanie Weber, Evotec Technologies, EVOTECAnalytical Systems GmbH, Max-Planck-Str. 15A, D-40699 Erkrath,

Germany. E-mail: stephanie.weber@evotecoai.com

British Journal of Haematology, 2002, 116, 839–843

Ó 2002 Blackwell Science Ltd 839

fluorescent labelling primers. After PCR, the quantities ofallele-specific fluorescent PCR products and their biophys-ical fluorescent properties are determined by FCS. Thesimultaneous analysis of these FCS parameters allowsquality control within each well. This novel method ofHPA-1 genotyping offers a homogeneous (one tube) systemwithout the need for purification of PCR products, restric-tion enzyme digestion or electrophoresis. In addition, theconfocal technology of FCS has the potential for assayminiaturization (Pope et al, 1999).

MATERIALS AND METHODS

DNA isolation. Genomic DNA was isolated from 200 ll ofcitrated whole blood using the QIAamp DNA Mini blood kit(Qiagen, Hilden, Germany).

HPA-1 genotyping by GALIOSÒ. Primer sequences werededuced from the GPIIIa gene (Zimrin et al, 1990). TheGALIOSÒ strategy uses one pair of gene-specific amplifica-tion primers (sense: 5¢-TCG CCA TAG TTC TGA TT-3¢;antisense: 5¢-CTT GTC TTC CCA CCC-3¢) and two allele-specific, semi-nested sense labelling primers (for HPA-1a:5¢-TAC AGG CCC TGC CTC T-3¢; for HPA-1b: 5¢-TAC AGGCCC TGC CTC C-3¢) in a single reaction. Labelling primerscorresponding to the target are extended, whereas3¢-mismatched primers are not (Newton et al, 1989). Eachlabelling primer type is coupled to a specific fluorochromeexcitable at 543 nm [6-carboxytetramethylrhodamine(TAMRA) for HPA-1a] or 633 nm (EVOblue 50 forHPA-1b) respectively. TAMRA (Thermo Hybaid, InteractivaDivision, Ulm, Germany) was coupled by standard phos-phoramidite chemistry at the 5¢ terminus. N-hydroxysuc-cinimide-activated EVOblue 50 (Evotec OAI, Hamburg,Germany) was 5¢ tagged via a C6-linker. All primers weresynthesized and coupled to fluorescent dyes by ThermoHybaid. Final concentrations of PCR components were:1· EVOAMP buffer (Evotec Technologies, Erkrath, Germany),4 mmol/l MgCl2, 200 lmol/l each deoxyribonucleotide,100 nmol/l sense amplification primer, 300 nmol/l anti-sense amplification primer, 1 nmol/l each labelling primer,1 U of Q-BioTaq DNA polymerase (Q-BIOgene, Heidelberg,Germany) and 100 ng of genomic DNA. After an initialdenaturation step at 94°C for 1 min, PCR was performedusing 40 cycles of denaturation (20 s at 94°C), annealing(60 s at 60°C), elongation (20 s at 72°C) and a finalelongation for 2 min at 72°C in a GeneAmp PCR System9700 (Perkin-Elmer Biosystems, Weiterstadt, Germany).PCR products were analysed by agarose (2%) gel electro-phoresis and ethidium bromide staining, as well as by FCS.

Detection by fluorescence correlation spectroscopy (FCS).Allele-specific fluorescent PCR products were detected byFCS (Eigen and Rigler, 1994; Hovius et al, 2000; Foldes-Papp and Kinjo, 2001). In FCS, temporal fluctuations in thefluorescence signal caused by the diffusion of single fluor-escent molecules into and out of a small tightly focusedconfocal element (detection volume �1 fl) are measured.These fluctuations are mathematically analysed with theautocorrelation function (Elson and Magde, 1974). The FCSautocorrelation yields information such as concentration

(n, average number of fluorescent molecules in the detectionvolume), mass-dependent translational diffusion time (s1, s2)of molecules, as well as average fluorescence intensity permolecule (counts per molecule, c.p.m.). Although any FCSinstrumentation would be suitable, in the present study,genotyping was performed with an automated instrumentfor SNP analysis (Evotec Technologies). The device isequipped with two He–Ne lasers emitting light at 543 nm(for excitation of TAMRA-labelled components) and 633 nm(for excitation of EVOblue 50-labelled components). FCSmeasurements were performed for 3 · 3 s in a microplate(UniView, 96 wells; Whatman, NJ, USA). The softwareautomatically determines the genotypes by calculating theratio of allele-specific labelled PCR product measured at543 nm (for HPA-1a) and 633 nm (for HPA-1b) respect-ively (543–633/543+633). HPA-1 genotyping was per-formed with the following ratio borders: ratios > 0Æ30represent AA (HPA-1a/HPA-1a); ratios ‡ )0Æ65 and £ 0Æ30represent AB (HPA-1a/HPA-1b); and ratios < )0Æ65 rep-resent BB (HPA-1b/HPA-1b).

HPA-1 genotyping by allele-specific restriction enzyme ana-lysis (ASRA). A modified ASRA was performed (Simseket al, 1993). A 369-bp fragment was amplified using thesense primer 5¢-TCG CCA TAG TTC TGA TT-3¢ in combina-tion with the antisense primer 5¢-CTT GTC TTC CCA CCC-3¢.The PCR product harbours two MspI cleavage sites presentin both HPA-1 alleles resulting in a 232-bp, a 131-bp and a6-bp product upon MspI digestion. The C12548T poly-morphism creates an additional cleavage site for MspI,resulting in cleavage of the 232-bp fragment into a 173-bpand a 59-bp fragment. PCR was performed as described forGALIOSÒ, except that 300 nmol/l each primer and 250 ngof genomic DNA were used. PCR products (1/10th volume)were digested with 10 U of MspI (New England Biolabs,Beverly, MA, USA) for 4 h and subsequently analysed byagarose (3%) gel electrophoresis and ethidium bromidestaining.

RESULTS

Assay principleHPA-1 genotyping was performed using GALIOSÒ, asemi-nested, allele-specific PCR strategy. Owing to theirhigh concentrations, the amplification primers dominatethe earlier PCR cycles and amplify a 181-bp fragment ofthe GPIIIa gene independently of the allele. Allele-specificfluorescent labelling primers at lower concentrations incombination with the antisense primer are extendedprimarily during later PCR cycles, resulting in thegeneration of a fluorescent 137-bp fragment. Typicalresults of HPA-1 genotyping are shown in Fig 1. Analysisof PCR products by agarose gel electrophoresis showedonly the highly amplified unlabelled 181-bp fragment(Fig 1A). However, the allele-specific fluorescent PCRproducts (137 bp) were analysed by fluorescence correla-tion spectroscopy (FCS) at 543 nm and 633 nm respect-ively (Fig 1B). FCS detection of allele-specific productsshows that, for HPA-1a/1a, only the TAMRA (543 nm)-labelled, HPA-1a-specific primers were elongated, whereas

840 S. Weber et al

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the EVOblue 50 (633 nm)-labelled HPA-1b-specific primerswere not. FCS analysis of the homozygous HPA-1b sampledemonstrated amplification of PCR products excitable at633 nm. No extension products of the TAMRA-labelledprimers were detectable at 543 nm. For the heterozygoussample, both types of allele-specific labelling primers wereelongated, resulting in PCR products detectable at bothwavelengths.

Assay reliabilityFor assay validation, 380 different DNA samples were firstgenotyped by ASRA (Fig 2). All these 380 samples werethen analysed by GALIOSÒ and FCS. Using defined ratioborders for automated genotyping (see Materials andmethods), a clear discrimination of HPA-1 genotypes waspossible in all samples studied. Results of FCS measurementson the automated instrument are shown in Fig 3. Thegenotypes determined by FCS were in 100% concordancewith the results obtained by ASRA.

Assay specificityFor validation of specificity, HPA-1 genotyping was per-formed in the presence of equivalent amounts (100 ng) ofgenomic DNA from Escherichia coli. Bacterial DNA did notaffect HPA-1 genotyping by GALIOSÒ.

Assay rangeFor evaluation of the dynamic range, HPA-1 genotypingwas performed with different quantities of genomic DNAvarying from 1 to 200 ng of DNA. These variations in targetconcentrations, e.g. as a consequence of inaccurate DNAquantification, did not affect the results of HPA-1 genotyp-ing. Reliable discrimination of all genotypes was possiblewith as little as 5 ng of DNA, indicating the high sensitivityof the assay.

DISCUSSION

In the present study, we describe a homogeneous methodfor HPA-1 genotyping based on allele-specific PCR anddetection by FCS. For HPA genotyping, several PCR-basedmethods have been described including ASRA (Newmanet al, 1989; Kuijpers et al, 1992; Simsek et al, 1993; Kalb

Fig 1. Representative example of HPA-1 genotyping performed

with allele-specific, semi-nested polymerase chain reaction (PCR)(GALIOSÒ) and detection by fluorescence correlation spectroscopy

(FCS). (A) Agarose (2%) gel electrophoresis of PCR products

(HPA1a/1a: AA, HPA1a/1b: AB, HPA1b/1b: BB, PCR negative

control without template: NC, 100-bp ladder as molecular weightmarker: M). (B) FCS detection of the same samples as in (A). Data

are means ± SD of duplicate determinations.

Fig 2. Allele-specific restriction enzyme analysis (ASRA) for HPA-1

genotyping. MspI restriction of the 369-bp PCR product resulted in

three fragments (232, 131 and 6 bp). Owing to the C12548Ttransition in the HPA-1b allele, an additional cleavage site is pre-

sent resulting in four fragments (59, 173, 131 and 6 bp) upon MspI

digestion. A representative agarose (3%) gel containing undigested

(–) and MspI digested (+) PCR products is shown (HPA1a/1a:AA, HPA1a/1b: AB, HPA1b/1b: BB, 100-bp ladder as molecular

weight marker: M).

Fig 3. Box and whisker plot of ratios of allele-specific PCR productsmeasured at 543 nm (for HPA-1a) and 633 nm (for HPA-1b)

respectively (543–633/543 + 633). N ¼ 380 different samples

were analysed. The plot shows medians, 10th, 25th, 75th and 90thpercentiles as vertical boxes with error bars and all outliers as cir-

cles. The dotted lines are ratio borders defined for automated HPA-1

genotyping.

HPA-1 Genotyping by Allele-specific PCR and Automated FCS 841

Ó 2002 Blackwell Science Ltd, British Journal of Haematology 116: 839–843

et al, 1994; Matsuo and Reid, 1996), restriction fragmentlength polymorphism (RFLP) (Andersen et al, 1993;Unkelbach et al, 1995), heteroduplex formation (O’Connoret al, 2000) and single-strand conformation polymorphism(SSCP) (Jin et al, 1993; Fujiwara et al, 1995; Peyruchaudet al, 1995; Quintanar et al, 1998). All these methodsrequire time-consuming electrophoresis and are not gener-ally applicable for large-scale routine diagnostics. Theoligonucleotide ligation assay (OLA) (Legler et al, 1996;Zotz et al, 1997) requires several post-PCR steps such asdenaturation, primer annealing and ligation. The preferen-tial homoduplex formation assay (PHFA) (Fujiwara et al,1996) and OLA are detected by enzyme-linked immuno-sorbent assay (ELISA). The need for additional enzymes (e.g.ligase for OLA, restriction enzymes for ASRA or RFLP) orantibodies (OLA, PHFA) increases costs and, because ofadditional reaction steps, increases the hands-on time. PCRwith allele-specific oligonucleotide (ASO) hybridization(McFarland et al, 1991; Bray et al, 1994) involves the riskof reduced specificity. Some studies describe HPA genotyp-ing by PCR with sequence-specific primers (SSP) (Metcalfand Waters, 1993; Merel et al, 1994; Skogen et al, 1994).In order to reduce the risk of contamination, homogeneous(one tube) genotyping methods have been developed (Boldtet al, 1997; Cavanagh et al, 1997). Homogeneous assaysystems in combination with fluorescence detection basedon fluorescence resonance energy transfer (FRET) have alsobeen used for HPA-1 genotyping (Nauck et al, 1999).However, for FRET, complex primer design is required inorder to find suitable anchor and detection primers.

This is the first study describing a homogeneous methodfor HPA-1 genotyping based on allele-specific PCR and FCS.The semi-nested PCR strategy allows highly specific HPA-1genotyping. Amplification and labelling of PCR products inone tube reduces the risk of false-positive determinations. Incontrast to HPA-1 genotyping with the LightCyclerTM

(Nauck et al, 1999), the GALIOSÒ PCR can be performedwith any standard thermocycler and reaction tubes. Inaddition, the simple primer design and assay principleenables the rapid development of further genotyping assays.The GALIOSÒ strategy has already been applied to severalSNPs, e.g. factor VLeiden, prothrombin, methylenetetra-hydrofolate reductase, and all major HPA polymorphismscan be covered. Although any other FCS device would besuitable for FCS measurements, a novel instrument wasused in our study. This instrument is an automated devicefor FCS, including adjustment, fitting of raw data andgenotype determination. This high degree of automationallows almost hands-free genotyping and makes the com-plex technology of FCS applicable for the clinical setting.The reliability of GALIOSÒ genotyping is further increasedusing FCS as the detection method, because autocorrelationof FCS raw data provides information about multiplebiophysical properties (diffusion time, counts per molecule,triplet fraction, triplet time) of fluorescent components(primers, PCR products). The software offers the possibilityof defining borders for these parameters. Measurements thatdo not fulfil the defined values are automatically excluded.This procedure offers an additional quality control. Fur-

thermore, the confocal technology of FCS is suitable forassay miniaturization, thus enabling the analysis of verysmall sample volumes (< 1 ll).

In summary, genotyping by GALIOSÒ combined withsingle molecule detection by automated FCS represents aninnovative method for HPA-1 genotyping, which is appli-cable for routine use in the clinical setting.

ACKNOWLEDGMENTS

We thank Klaus Posl, Adriane Aust, Kerstin Freidel and TimObernyer for excellent technical assistance. The expertinformatic work of Kai Diercks is gratefully acknowledged.

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