Aim Introduction Conclusion Methods Results

GENOTYPING Robert L

1 Flower ,

1Research

Genghis H

1 Lopez ,

a Di

AND GP.Mur IN AUSTRALIAN DONORS

Rhiannon S

1 McBean ,

Brett

2 Wilson ,

Yew-Wah

2 Liew ,

Catherine A

1 Hyland

& Development, 2Red Cell Reference Laboratory, Australian Red Cross Blood Service, Queensland, Australia

Introduction

Results

 Over 8% of the contemporary Australian

 HRM genotyping showed 100% concordance with serology.

population are of Asian ethnicity however, there are few reports of frequency of the GP.Mur hybrid glycophorin or the Dia single nucleotide polymorphism found in Asian ethnic groups, in Australian donors.  Antibodies to the Dia polymorphism cause transfusion reactions and haemolytic disease of the foetus and newborn.  Dia is found at a frequency of 5-10% in East Asian populations.  Antibodies to neoantigens (Mut, Mur, and Hil) found on GP.Mur can cause haemolytic transfusion reactions and haemolytic disease of the newborn.4  GP.Mur is found in 5-8% in East Asian and Southeast Asian populations.4-5

Aim To type Australian donors for these polymorphisms.

Methods  Selected-samples were retrieved from collections when Donor Mobile units were at locations with a high frequency of blood donors of East Asian background.  Dia and GP.Mur were typed using standard serological procedures.  DNA from donors that were serologypositive and serology-negative for GP.Mur and Dia were investigated to validate HRM genotyping, using primers from previously published methods, as well as noveldesigned primers to target specific polymorphic regions.  Amplification and analysis was performed using the Type-it HRM PCR kit (QIAGEN).

 GP.Mur was detected in 3 out of 100 donor samples by both serology & HRM genotyping. + Add in confidence intervals

 For all GP.Mur samples, no variant types were found (a full profile of neoantigens was detected). Figure 1. GP.Mur HRM melt profile. GP.Mur negative and homozygote control showed single peaks with Tms at 78.7 °C and 79.2°C respectively.

Colour Legend: GP.Mur negative control GP.Mur HET control GP.Mur HOM control

GP.Mur heterozygote control produced three distinct peaks.

#Putative novel allele Tm 80.1°C

# A putative new allele was detected showing a different melt profile compared to the controls – we await sequencing result to determine its significance

Table 1. Frequency of GP.Mur in selected and non-selected samples Genotype

Phenotype

GP.Mur Homozygote GP.Mur Negative GP.Mur Heterozygote TOTAL

GP.Mur positive GP.Mur negative GP.Mur positive

Selected Population 0 97 3 100

Non-selected Population 0 104 0 104

Figure 2: Dia/Dib HRM melt profile. The Diego HRM genotyping method produced melt curves characteristic of the three genotypes, DIA/DIA, DIB/DIB and DIA/DIB.

DIA/DIA

DIB/DIB

DIA/DIB

Conclusion  Procedures for HRM genotyping reliably detected the GP.Mur hybrid glycophorin and Dia SNP.

 In the selected study cohort, the antigen frequency of GP.Mur was 3% and for Dia, the frequency was surprisingly high at 11%.

 With changes in the demographics of the Australian population, this is evidence that the frequency of these phenotypes in some Australian donor cohorts is higher than previously reported. Antibodies to these ethnic-associated antigens are likely to be of increasing importance.

Table 2. Frequency of Dia/Dib antigens in selected and non-selected samples Non-selected Genotype Phenotype Selected Population Population DIA/DIA Di (a+b-) 1 0 DIA/DIB Di (a+b+) 10 1 DIB/DIB Di (a-b+) 89 99 100 100

 Dia was detected in 11 selected samples (n= 100) by both serology and HRM genotyping. Dia antigen frequency of 11% (95% CI: 4.9 - 17.1%).  Dia was detected in 1 non-selected sample (n= 100) by HRM genotyping. Dia antigen frequency of 1% (95% CI: 0 - 2.95%).  The difference of distribution of the Diego alleles between the selected and non-selected populations was found to be statistically significant by the χ2 test using a 2 x 2 contingency table (Pvalue 0.01-0.001).

References: (need to number and insert into text): Tanaka, M., Takahahi, J., Hirayama, F. and Tani, Y. (2011). High-resolution melting analysis for genotyping Duffy, Kidd and Diego blood group antigens. Legal Medicine 13: 1-6. Komatsu, F., Hasegawa, K., Yanagisawa, Y., Kawabata, T., Kaneko, Y., Watanabe, S., Miyagi, S., Sakuma, M., et al. (2004). Prevalence of diego blood group Dia antigen in mongolians: comparison with that in Japanese. Transfusion and Apheresis Science 30: 119-124. Bruce, L., Anstee, D., Spring, F. and Tanner, M. (1994). Band 3 Memphis variant II. Altered stilbene disulfonate binding and the Diego (Dia) blood group antigen are associated with the human erythrocyte band 3 mutation Pro854-->Leu. Journal of Biological Chemistry 269: 16155-16158. 4. Broadberry RE, Lin M. (1994). The incidence and significance of anti-Mia in Taiwan. Transfusion 34:349-352. 5. Prathiba, R., Lopez CG., Usin, FM. (2002) The prevalence of GP.Mur and anti-Mia in a tertiary hospital in Peninsular Malaysia. Malaysian J Pathol; 24(2) 95-98

We acknowledge Australian governments that fully fund the Australian Red Cross Blood Service for the provision of blood products and services to the Australian community.

We acknowledge Australian governments that fully fund the Australian Red Cross Blood Service for the provision of blood products and services to the Australian community.