High-Resolution Melting Analysis에 의한 Duffy 혈액형군 유전형 분석 = High-Resolution Melting Analysis for Genotyping Duffy Blood Group Antigens

다국어 초록 (Multilingual Abstract)

Background:Accurate typing of Duffy blood group is important because anti-Duffy antibodies cause hemolytic transfusion reaction and hemolytic disease of the newborn. The aim of this study was to evaluate a new genotyping method using high resolution melting (HRM) analysis, a rapid and inexpensive approach for high-throughput Duffy genotyping.
Methods:A total of 20 unrelated Korean blood samples were obtained and an African-black sample was used for GATA control. Phenotyping was performed by hemagglutination (DiaMed AG, Switzerland). GATA and FYA/B PCR products were obtained by PCR-restriction fragment length polymorphism (RFLP) using Taq DNA polymerase (Promega, WI) and enzymes BanI and StyI (New England Biolab, UK). For HRM, PCR amplification was performed using LightCycler 480 ResoLight Dye (Roche, USA) and Lightcycer 480 (Roche, USA).
Results:Phenotyping and genotyping data using PCR-RFLP and HRM analysis were compared. Different types of HRM curves were obtained according to genotypes, FYA/FYA, FYB/FYB, and FYA/FYB, and to GATA mutations, homozygote FYB-33T (T/T), heterozygote FYB-33T/33C (T/C), and homozygote FYB-33C (C/C). Phenotypes 18 Fy(a＋b−), 1 Fy(a＋b＋), 1 Fy(a−b＋), and 1 Fy(a−b−) showed complete concordance with genotyping methods. Fy(a−b−) sample was found to be a FYB-33C homozygote by both genotyping methods.
Conclusion:Phenotyping and genotyping showed concordant results and both genotyping methods using PCR-RFLP and HRM analysis showed good agreement in finding mutation in GATA and FY gene coding regions. HRM analysis is suitable and reliable for high-throughput screening for Duffy genotyping.

국문 초록 (Abstract)

배경: Duffy 혈액형 항원에 대한 항체는 용혈성수혈부작용과 신생아용혈성질환을 일으키므로정확한 Duffy 혈액형 검사가 중요하다. 저자들은빠르고 경제적인 high-throughput이 가능한 새로운 high resolution melting (HRM) 분석법에 의한Duffy 유전형 검사를 평가하고자 하였다.
방법: 20개의 한국인 혈액 검체와 GATA 대조로써 흑인 검체 한 개에 대하여 다이아메드사의적혈구응집검사로 표현형을 확인하였고, Taq DNA polymerase와 BanI, StyI 효소를 사용하여 중합효소연쇄반응-제한절편길이다형성(PCR-RFLP) 검사를 시행하였다. HRM 분석을 위해 LightCycler 480 ResoLight Dye (Roche, USA)과 Lightcycer 480 (Roche, USA)으로 PCR 증폭을 시행하였다.
결과: 혈청학적 표현형과 PCR-RFLP와 HRM에의한 유전형 검사 결과를 비교하였다. HRM 분석결과, Duffy 유전형인 FYA/FYA, FYB/FYB, FYA/ FYB에 따라, 또한 GATA 유전자변이인 FYB-33T (T/T) 동형접합체, FYB-33T/33C (T/C) 이형접합체, FYB-33C (C/C) 동형접합체에 따라 HRM 곡선은 서로 다른 양상을 보였다. 표현형 검사 결과Fy(a＋b−) 18건, Fy(a＋b＋) 1건, Fy(a−b＋) 1건및 Fy(a−b−) 1건이 확인되었고 이는 두 가지 유전형 검사와 모두 일치하였다. Fy(a−b−) 검체는두 가지 유전형 검사법 모두에서 FYB-33C 동형접합체로 동일하였다.
결론: 혈청학적 표현형과 유전형 검사는 좋은일치율을 보였고 PCR-RFLP와 HRM에 의한 두가지 유전형 검사는 GATA와 Duffy 코딩부위 유전자 변이를 찾아내는 데에 동일한 결과를 보였다. HRM 분석법은 Duffy 유전자형의 high-throughput 선별검사로서 적합하며 신뢰할 수 있는 검사로 사료된다.

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