High-throughput Kell, Kidd, and Duffy matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry-based blood group genotyping of 4000 donors shows close to full concordance with serotyping and detects new alleles.

BACKGROUND: After the ABO (ABO) and Rh (RHD and RHCE) blood group systems, Kell (KEL), Kidd (SLC14A1), and Duffy (DARC) represent the second most important clinically relevant antigens. STUDY DESIGN AND METHODS: Samples from 4000 Swiss blood donors, with serologic prevalues for K/k, Kp(a/b), Jk(a/b), and Fy(a/b), and 48 additional samples of presumptive black African origin were genotyped using high-throughput matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry, applying one single-multiplex polymerase chain reaction/primer-extension reaction simultaneously detecting 15 single-nucleotide polymorphisms. RESULTS: Genotype/phenotype concordance for K/k, Kp(a/b), Jk(a/b), and all Fy(a/b) specificities were 100, 99.98, 99.93, and 99.20%, respectively. Discrepancies were caused by erroneous serologic profiles (n = 33), mainly attributed to weakly expressed Fy(x) (n = 28). Only three discrepancies had a genetic basis. They could all be explained by newly observed silenced alleles: one KEL*02N.34 and one FY*02N.03 with predicted R700Q and G261R amino acid exchanges, respectively, and one JK*B, with an as-yet-unidentified silencing cause. According to NCBI SNP database entry for rs8176034, another new allele, KEL*02.38, had been expected, and we formally demonstrated its presence. We furthermore identified individuals with rare phenotypes, such as Js(a/b) heterozygotes among Caucasians, rare alleles, the "Swiss" JK*01N.03, and rare genotypes, such as one Fy(x) homozygote. CONCLUSION: Genotyping proved its practicability in the daily routine setting and qualitatively outperformed serology. Technology is ideal for time-insensitive donor genotyping and allows for a broad range of throughput needs. Consequently, from a technologic point of view, serotyping should be replaced by genotyping for donors' blood groups encoded by KEL, SLC14A1, and DARC.

Erythroid urea transporter deficiency due to novel JKnull alleles.

BACKGROUND: The Kidd blood group antigens Jka and Jkb are encoded by the red blood cell (RBC) urea transporter gene. Homozygosity for silent JK alleles results in the rare Jk(a-b-) phenotype. To date, seven JKnull alleles have been identified, and of these, two are more frequent in the Polynesians and Finns. This study reports the identification of other JKnull alleles in Jk(a-b-) individuals of different ethnic or geographic origins. STUDY DESIGN AND METHODS: Nine Jk(a-b-) samples and a sample from a Jk(a-b+) mother of a Jk(a+b-) baby were investigated. Polymerase chain reaction amplification and sequence analysis of the JK gene was performed. Western blotting and urea lysis were used to confirm Jk(a-b-) RBCs. RESULTS: Four novel alleles were identified: two different nonsense mutations, 202C>T (Gln68Stop) and 723delA (Ile262Stop) were identified on otherwise consensus JK*1 and JK*2 alleles, respectively. A missense mutation, 956C>T (Thr319Met), was identified in a JK*1 allele from an African-American and a JK*2 allele in two people of subcontinental Indian descent. Immunoblotting and urea lysis confirmed absence of JK glycoprotein in RBC membranes from a sample carrying the 956C>T mutation. Other previously described JKnull mutations were found in samples of origins other than in which they were first identified. CONCLUSION: The molecular bases of the Jk(a-b-) phenotype are diverse and this is the first report of JKnull alleles in individuals of African and subcontinental Indian descent. Although rare, these alleles should be taken into consideration when planning genotyping strategies for blood donors and patients.

Insights into the structure and function of membrane polypeptides carrying blood group antigens.

In recent years, advances in biochemistry and molecular genetics have contributed to establishing the structure of the genes and proteins from most of the 23 blood group systems presently known. Current investigations are focusing on genetic polymorphism analysis, tissue-specific expression, biological properties and structure-function relationships. On the basis of this information, the blood group antigens were tentatively classified into five functional categories: (i) transporters and channels, (ii) receptors for exogenous ligands, viruses, bacteria and parasites, (iii) adhesion molecules, (iv) enzymes and, (v) structural proteins. This review will focus on selected blood groups systems (RH, JK, FY, LU, LW, KEL and XK) which are representative of these classes of molecules, in order to illustrate how these studies may bring new information on common and variant phenotypes and for understanding both the mechanisms of tissue specific expression and the potential function of these antigens, particularly those expressed in nonerythroid lineage.

Photoaffinity labeling of the human red-blood-cell urea-transporter polypeptide components. Possible homology with the Kidd blood group antigen.

The tritiated urea analogue 1-(3-azido-4-chlorophenyl)-3methyl-2-thiourea ([3H]MeACPTU) was used as a probe to photolabel the human red-blood-cell membrane facilitated urea transporter. On irradiation, [3H]MeACPTU incorporated irreversibly into white ghost membranes. SDS/gel electrophoresis of membranes revealed radioactive incorporation in five major bands of 200, 110, 60, 40 and 14 kDa. The labeling of the 40-kDa and 60-kDa bands was partly prevented by the presence of a high concentration of other urea analogues such as thiourea and 1-(3,4-dichlorophenyl) 2-thiourea (DCPTU). The photolabeling pattern obtained with white ghosts of the Kidd blood-group type Jk(a-,b-) showed no labeling of the 40-kDa polypeptide. Protecting experiments carried out with anti-Jka, anti-Jkb and anti-Jk3 sera prevented radioactive incorporation in the 60-kDa band and in the 110-kDa band. Urea permeability of pink ghosts of blood type Jk(a+,b+) measured in the presence of Jk3 antibodies was 19% lower than the control values. However, urea permeability of frog urinary bladder epithelial cells was not affected by the presence of Jk-reactive antibodies. These results support the hypothesis that the Kidd antigen and the facilitated urea transporter are the same protein. Our estimation of the number of copies in each cell is close to that of the previously published value of 14000.