open access

Vol 6, No 2 (2013)
Research paper
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The +24G > del polymorphism in the intron 3 of ITGB3 gene in the diagnostic region of HPA-1a allele genotyping: consequences and verification methods

Katarzyna Guz, Magdalena Krzemienowska, Agnieszka Orzińska, Małgorzata Uhrynowska, Krystyna Maślanka, Dorota Woźniewska, Grażyna Nowak, Ewa Brojer
Journal of Transfusion Medicine 2013;6(2):33-40.

open access

Vol 6, No 2 (2013)
ORIGINAL PAPERS

Abstract

INTRODUCTION: Genotyping of HPA-1 antigens is an important element in the diagnostics and treatment of fetal/neonatal alloimmune thrombocytopenia (FNAIT) as well as transfusion and transplantation related thrombocytopenias. Polymorphisms in the ITGB3 gene within the annealing region of primers/probes used for HPA-1 typing may affect the results of such research. The aim of this study was to explain the reason of discrepancy of HPA-1 genotype detection by PCR-SSP and the newly introduced RQ-PCR protocol; determine the frequency of HPA-1a allele mutation in the Polish population and its effect on the diagnostic procedures.

MATERIAL AND METHODS: RQ-PCR was performed in DNAs from 422 consecutive blood donors and 62 HPA-1b/b patients diagnosed for FNAIT (58) or posttransfusion complications (4), genotyped in the years 2005–2012 by PCR-SSP and from 118 individuals typed as HPA-1a negative by serological method. DNA samples detected as HPA-1b/b by PCR-SSP but HPA-1a/b by RQ-PCR were sequenced.

RESULTS: The discrepancy of the results was detected in two blood donors and in one patient with platelet refractory. In all of them sequence analysis confirmed +24G>del in the intron 3 of the ITGB3 within the region of annealing of reverse primer used for PCR-SSP. The frequency of this polymorphism in non selected blood donors was 2/422. It wasn’t present in persons diagnosed toward FNAIT or in the group selected by serological method. We have revised PCR- -SSP protocol and validated a new version of the commercial test.

CONCLUSION: Due to the mutations in the diagnostic region of ITGB3 gene each HPA-1b/b genotyping result should be confirmed using additional molecular or serological method.

Abstract

INTRODUCTION: Genotyping of HPA-1 antigens is an important element in the diagnostics and treatment of fetal/neonatal alloimmune thrombocytopenia (FNAIT) as well as transfusion and transplantation related thrombocytopenias. Polymorphisms in the ITGB3 gene within the annealing region of primers/probes used for HPA-1 typing may affect the results of such research. The aim of this study was to explain the reason of discrepancy of HPA-1 genotype detection by PCR-SSP and the newly introduced RQ-PCR protocol; determine the frequency of HPA-1a allele mutation in the Polish population and its effect on the diagnostic procedures.

MATERIAL AND METHODS: RQ-PCR was performed in DNAs from 422 consecutive blood donors and 62 HPA-1b/b patients diagnosed for FNAIT (58) or posttransfusion complications (4), genotyped in the years 2005–2012 by PCR-SSP and from 118 individuals typed as HPA-1a negative by serological method. DNA samples detected as HPA-1b/b by PCR-SSP but HPA-1a/b by RQ-PCR were sequenced.

RESULTS: The discrepancy of the results was detected in two blood donors and in one patient with platelet refractory. In all of them sequence analysis confirmed +24G>del in the intron 3 of the ITGB3 within the region of annealing of reverse primer used for PCR-SSP. The frequency of this polymorphism in non selected blood donors was 2/422. It wasn’t present in persons diagnosed toward FNAIT or in the group selected by serological method. We have revised PCR- -SSP protocol and validated a new version of the commercial test.

CONCLUSION: Due to the mutations in the diagnostic region of ITGB3 gene each HPA-1b/b genotyping result should be confirmed using additional molecular or serological method.

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Keywords

HPA, false-negative HPA-1a allele genotyping, PCR-SSP, RQ-PCR

About this article
Title

The +24G>del polymorphism in the intron 3 of ITGB3 gene in the diagnostic region of HPA-1a allele genotyping: consequences and verification methods

Journal

Journal of Transfusion Medicine

Issue

Vol 6, No 2 (2013)

Article type

Research paper

Pages

33-40

Bibliographic record

Journal of Transfusion Medicine 2013;6(2):33-40.

Keywords

HPA
false-negative HPA-1a allele genotyping
PCR-SSP
RQ-PCR

Authors

Katarzyna Guz
Magdalena Krzemienowska
Agnieszka Orzińska
Małgorzata Uhrynowska
Krystyna Maślanka
Dorota Woźniewska
Grażyna Nowak
Ewa Brojer

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