Rapid diagnosis of the French gypsy mutation in Glanzmann thrombasthenia using high- resolution melting analysis Mathieu Fiore 1 ; Alan T. Nurden 1 ; Christine Vinciguerra 2 ; Paquita Nurden 1 ; Xavier Pillois 1 1 Centre de Référence des Pathologies Plaquettaires (CRPP), Plateforme Technologique et d'Innovation Biomédicale (PTIB), Hôpital Xavier Arnozan, Pessac, France; 2 EA4174, Université Claude Bernard Lyon1 and Service d'Hématologie Biologique, Hôpital E.Herriot, Hôspices Civils de Lyon, Lyon, France Dear Sirs, Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterised by a quantitative and/or a qualitative deficiency of the αIIbβ3 inte- grin (1). It is relatively frequent in highly inbred populations such as Iraqi Jews or Jordanian Arabs, where consanguinous marriages are common (2, 3). This is also the case within the French Gypsy popu- lation of Manouche origin for whom the molecular basis of the GT trait has been identified as a G to A substitution in the ITGA2B gene at the splice donor site of in- tron 15 (4). This mutation results in abnor- mal splicing, a reading-frame shift and a premature TGA stop codon. A founder ef- fect for GT within this population is strongly indicated as most affected subjects are homozygous for the HPA-1b allele on the β3 subunit, a homozygosity that is li- mited to 2% of the normal population (5). Several genotyping techniques have been used to characterise the gypsy GT mo- lecular defect, such as restriction fragment length polymorphism (RFLP) analysis, single-strand conformation polymor- phism analysis or direct sequencing (re- viewed in [6]). All are time-consuming and labour-intensive methods although real- time PCR using a fluorescent labelled probe such as TaqMan ® or fluorescence resonance energy transfer are more cost-ef- fective. High-resolution melting (HRM) analysis has been developed as a closed- tube method for detecting DNA variations without requiring the use of labelled probes (7, 8). Specific duplex-detecting DNA dyes can be used at saturating con- centrations without inhibiting PCR. Wild- type, homozygous and heterozygous mu- tant samples are distinguished by their melting curve patterns. Heterozygous mu- tations are optimally distinguished, melt- ing at lower temperatures. But sometimes melting curves of homozygous genotypes overlay and cannot be discriminated from the wild-type pattern. In this situation, the addition of an extra specific unlabelled probe may allow the discrimination of all the genotypes (8). This unlabelled oligonu- cleotide probe is blocked at its 3’ end in order to prevent extension and is designed to hybridise on the variation of interest. We have used this approach to develop a rapid test to genotype the common mutation causing GT in the French gypsy commu- nity. A total of 26 subjects from the French Gypsy community living in the South of France were recruited by the French Refer- ence Centre for Platelet Diseases (CRPP). Eleven of the subjects fulfilled the diag- nostic criteria for GT and were confirmed to have the homozygous French gypsy mu- tation by direct sequencing or by RFLP using the procedures previously described by us (6). Likewise, seven were heterozy- gous for the French gypsy mutation. The remaining eight subjects did not possess the mutation. DNA was isolated using the QIAamp DNA blood mini-kit (Qiagen, Courtaboeuf, France). Primers were de- signed using Primer3 software (9) and Correspondence to: Alan T. Nurden IFR4, CRPP/PTIB Pessac 33604, France Tel.: +33 557 102 851, Fax: +33 557 102 864 E-mail: alan.nurden@cnrshl.u-bordeaux2.fr Received: May 3, 2010 Accepted after minor revision: July 15, 2010 Prepublished online: September 30, 2010 doi:10.1160/TH10-05-0268 Thromb Haemost 2010; 104: 1076–1077 chosen to amplify ITGA2B exon 15 and its intronic flanking regions. Sequences of the forward and reverse primers were respect- ively 5'-CCTACCCCATCACCCTATCC- CAT-3' and 5'-GCCCATGCCCTCTGCC- TCC-3'. These primers amplified a PCR product of 154-base pairs-long with a melt- ing temperature of 86.8°C. The unlabelled probe had a 3’-phosphate incorporated to prevent extension during PCR. The se- quence of the probe was 5'-AAGA- CACCCGTG AGCTGATGAGGA-P-3', in which the underlined ″A″ is specific to the French gypsy mutation and ″-P″ indicates a 3' phosphate. The asymmetric PCR reac- tion of 20 μl contained 1 μl of sample ge- nomic DNA (20–100 ng total), 10 μl of 2x LightCycler ® 480 HRM Master Mix (Roche, Meylan, France), and 2.5 mM MgCl 2 . The reaction used 150 nM final concentration for the reverse primer and the probe and 50 nM for the forward primer. PCR was performed in a Light- Cycler ® instrument (Roche) with an initial denaturing step at 95°C for 10 minutes (min); followed by 45 cycles of denatu- ration at 95°C for 10 seconds (s); annealing at 60°C for 15 s; and extension at 72°C for 15 s. Fluorescence was acquired once each cycle at the end of the extension step. After PCR, probe/double strand DNA amplicon duplexes were generated by heating samples to 95°C for 1 min, then cooling to 40°C for 1 min. The melting data were col- lected between 60°C and 75°C at 25 ac- quisitions per °C, using the “melting curves” analysis mode. Then, samples were cooled to 40°C for 10 s. The LC480 run was finished in approximately 1 hour (h) and 45 min. Melting curve profiles of conventional HRM identified all heterozygous patients with the French gypsy mutation; but as mentioned above, it was difficult to distin- guish subjects homozygous for the mu- tation from normal individuals (Fig. 1A). In contrast, as illustrated in Figure 1B, when the unlabelled probe was added to the reaction mixture, HRM displayed three types of melting curves. The 11 individuals homozygous for the French gypsy mu- tation had a higher melting temperature with an average mutated allele temperature (Tm) of 68.2 ± 0.35°C; here PCR products were perfectly paired to the unlabelled 1076 Letters to the Editor Thrombosis and Haemostasis 104.5/2010 © Schattauer 2010 For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2015-03-09 | ID: 1000518515 | IP: 193.54.110.33