Stepwise partitioning of Xp21: a profiling method for XK deletions causative of the McLeod syndrome.

BACKGROUND: McLeod syndrome (MLS) is hematologically defined by the absence of the red blood cell (RBC) antigen Kx on the transmembrane RBC protein, XK, representing a highly specific diagnostic marker. Direct molecular assessment of XK therefore represents a desirable diagnostic tool. Whereas pathogenic point mutations may be simply identified, partial and complete deletions of XK on Xp21.1, eventually covering adjacent genes and causing multifaceted "continuous gene syndromes," are difficult to localize. STUDY DESIGN AND METHODS: Three different McLeod patient samples were tested using 16 initial positional polymerase chain reaction (PCR) procedures distributed over an approximately 2.8-Mbp Xp-chromosomal region, ranging telomeric from MAGEB16 to OTC, centromeric of XK. The molecular breakpoint of one sample with an apparent large Xp deletion was iteratively narrowed down by stepwise positioning further PCR procedures and sequenced. Two mutant XK genes, one previously published and serving as a positive control, were also sequenced. RESULTS: We confirmed the positive control as previously published and listed as XK*N.20 by the International Society of Blood Transfusion (ISBT). The other XK showed a novel four-nucleotide deletion in Exon 1, 195-198delCCGC (newly listed as XK*N.39 by the ISBT). The third sample had an approximately 151-kbp X-chromosomal deletion, reaching from Exon 2 of LANCL3, across XK to Exon 3 of CYBB (newly listed as XK*N.01.016 by the ISBT). Carrier status of the patients' sister was diagnosed using a diagnostic "gap-PCR." CONCLUSIONS: The stepwise partitioning of Xp21.1 is pragmatic and cost-efficient in comparison to other diagnostic techniques such as "massive parallel sequencing" given the rarity of MLS. All males with suspected MLS should be considered for molecular XK profiling.

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.