ABO blood group phenotype frequency estimation using molecular phenotyping in rhesus and cynomolgus macaques

Abstract

A much larger sample (N = 2369) was used to evaluate a previously reported distribution of the A, AB and B blood group phenotypes in rhesus and cynomolgus macaques from six different regional populations. These samples, acquired from 15 different breeding and research facilities in the US, were analyzed using a real-time quantitative polymerase chain reaction (qPCR) assay that targets single nucleotide polymorphisms (SNPs) responsible for the macaque A, B and AB phenotypes. The frequency distributions of blood group phenotypes of the two species differ significantly from each other, and significant regional differentiation within the geographic ranges of each species was also observed. The B blood group phenotype was prevalent in rhesus macaques, especially those from India, while the frequencies of the A, B and AB phenotypes varied significantly among cynomolgus macaques from different geographic regions. The Mauritian cynomolgus macaques, despite having originated in Indonesia, showed significant (p ≪ 0.01) divergence from the Indonesian animals at the ABO blood group locus. Most Mauritian animals belonged to the B blood group while the Indonesian animals were mostly A. The close similarity in blood group frequency distributions between the Chinese rhesus and Indochinese cynomolgus macaques demonstrates that the introgression between these two species extends beyond the zone of intergradation in Indochina. This study underscores the importance of ABO blood group phenotyping of the domestic supply of macaques and their biospecimens.

Partial sequence analyses of exon 7 of the ABO locus of cynomolgus (Macaca fascicularis) and rhesus (M. mulatta) macaques: Indeterminate phenotypes show the presence of the O blood group

“…While these animals might carry lower titers of the A or B transferases than those homozygous for the A or B allele, presence of the O allele in these heterozygotes is not pertinent for designing donor/recipient pairs for transplantation or transfusion. The approximately 2% and 4% expected discovery rates of the O phenotype in rhesus macaques and cynomolgus macaques, respectively, are concordant with the low proportions (1%‐2% across rhesus and cynomolgus populations, respectively) of animals with indeterminate blood type estimated by Kanthaswamy et al The numbers of AO and BO heterozygous animals expected in both species may reflect that polymorphic ABO alleles have been segregating at low frequency before and throughout the 1 to 2 million year divergence of the two Macaca species . While we have identified multiple macaque O alleles within exon 7 of the ABO gene, most due to a suspected loss‐of‐function mutation at codon 268, in the future we plan to screen adjacent exon and intron sequences to characterize additional ABO phenotype subclasses not discovered in this study.…”

“…The present study's use of plasmid Sanger sequencing of a multiallelic locus suggests that primer and probe recognition site variation in some macaques might have impeded our qPCR‐based SNP probe assay from identifying A or B antigens. Moreover, a genetic cause rather than DNA degradation is probably responsible for most, if not all, of the indeterminate phenotypes observed in our previous studies . The nucleotide substitutions and deletions observed in the diagnostic sites of indeterminate alleles are projected to alter amino acids in the corresponding peptide sequences (Table ).…”

“…Using Premasuthan et al's methods, Kanthaswamy et al reported the presence of indeterminate blood types in cynomolgus and rhesus macaque populations at frequencies of 2.39% and 1.18%, respectively, and Oldt et al described an enrichment of indeterminate blood phenotypes in the hybrid zone shared by the two species as speculated previously . Multiple macaques in Oldt et al's study exhibited an indeterminate allele caused by failure of the first probe to bind with its target site coupled with the successful detection of the A or B allele with the second probe, suggesting that the indeterminate phenotypes are generated by probe‐target hybridization failures which point to an underlying genetic cause rather than DNA degradation …”

“…Premasuthan et al treated indeterminate phenotypes as artifacts of degraded DNA, and, consequently, samples that were considered not to have A, B, or AB phenotypes (hence indeterminate) were excluded from any further analysis. However, both Kanthaswamy et al and Oldt et al have argued that the indeterminate phenotypes might represent the absence of the required codons at 266 and/or 268 leading to a null O allele. Although a study by Malaivijitnond et al reported the presence of an O blood group in Thai cynomolgus macaques, both Premasuthan et al and Kanthaswamy et al have contended that reverse agglutination tests of macaque sera (using human red blood cells of known ABO type) such as those used in Malaivijitnond et al's analysis can result in false‐positive O phenotypes, as the macaque serum samples had not first been absorbed with type O human erythrocytes to remove nonspecific agglutinins.…”

“…However, both Kanthaswamy et al and Oldt et al have argued that the indeterminate phenotypes might represent the absence of the required codons at 266 and/or 268 leading to a null O allele. Although a study by Malaivijitnond et al reported the presence of an O blood group in Thai cynomolgus macaques, both Premasuthan et al and Kanthaswamy et al have contended that reverse agglutination tests of macaque sera (using human red blood cells of known ABO type) such as those used in Malaivijitnond et al's analysis can result in false‐positive O phenotypes, as the macaque serum samples had not first been absorbed with type O human erythrocytes to remove nonspecific agglutinins. Apart from Malaivijitnond et al's study, the presence of the O allele has not been conclusively showed in macaques.…”