Evaluation of Histo-Blood Group ABO Genotyping in a Danish Population: Frequency of a Novel 0 Allele Defined as O^2

Abstract

Traditional blood group ABO serology is based on immunoreactivity with the carbohydrate determinants A, B and H antigens. Recent advances at the DNA level of the ABO genes have provided a molecular genetic model for the ABO polymorphism. This genetic model has to date only been tested on a limited basis. The present study was initiated to evaluate the universality of the proposed genetic model on a larger group of serologically defined ABO phenotypes. Three hundred healthy Danish blood donors were analysed (A: 50, B: 50, AB: 50, 0:150) by PCR amplification followed by diagnostic restriction enzyme cutting. In all cases A, B, and AB at least one allele of correctly predicted status was found. However, in O phenotype individuals, 11 out of 150 carried one allele discordant to the proposed genetic model. This novel O allele (3.7% allele frequency) was further characterized by diagnostic restriction enzyme analysis in two positions divergent between A and B alleles and by DNA sequencing of the two major exons. The novel O allele is termed O2 as it typed as B in nucleotide position 526 and as A in positions 703, 796, and 803, in contrast to the most predominant O allele termed O^2, which types as A in all 4 positions. The structural defect in the O2 allele appears to be an additional substitution at nucleotide position 802. The results clearly demonstrate that with the addition of the two distinctly different O alleles, O', O2, the previously proposed molecular genetic basis of the ABO polymorphism is quite valid. More importantly the determined characteristics of these two O alleles have practical implications in ABO genotyping, because it establishes within the limits of the number of samples tested that ABO genotypes can be assessed directly by non-allele specific PCR amplification and restriction enzyme analysis.

Substrate Specificities of Three Members of the Human UDP-N-Acetyl-α-d-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase Family, GalNAc-T1, -T2, and -T3

“…Recombinant Baculovirus was obtained after two successive amplifications in Sf9 cells grown in serum-containing medium, and titers of virus were estimated by titration in 24-well plates with monitoring of enzyme activities. Controls included soluble human blood group A GalNAc-transferase (18), and the enzymatically non-functional blood group O 2 allele (19). For large scale expression in serum-free medium, Sf9 cells were adapted to growth in 2.5 expanded surface area roller bottles (in vitro) in 200 ml of 30% Grace containing 10% fetal calf serum and 70% SF-900 II medium (Life Technologies) in a 27°C roller at 0.6 rpm.…”

“…4) of GalNAc-T1, -T2, and -T3 reactions, respectively, analyzed by the same method. The monoglycosylated glycoform produced with GalNAc-T1 and -T3 have GalNAc attached in Thr in GVTSA (fragment [1][2][3][4][5][6][7][8][9][10][11][12], while the product with GalNAc-T2 has the GalNAc attached to Thr in GSTAP (fragment [13][14][15][16][17][18][19][20][21][22][23][24].…”

Rapid identification of the ABO genotypes by their single-stand conformation polymorphism

“…four nucleotide difference between the A and B alleles. In many populations the O allele has a deletion at nt 261 [7,10,23,24,30]. Many more samples with the O phenotype were analyzed than other phenotypes, but this allele was not found in the Chinese population.…”

Structural Basis for the Inactivity of Human Blood Group O2 Glycosyltransferase

“…More recently, an O 2 enzyme (O03) was discovered that was a full-length form of GTA with three substitutions, P74S, R176G, and G268R (7,8). The O 2 glycosyltransferase showed no measurable transferase activity when expressed in Sf9 cells (9).…”