Molecular basis for the high‐incidence antigens of the Kell blood group system

Lee, S.; Naime, D; Reid, Marin E.; Redman, Colvin M.

doi:10.1046/j.1537-2995.1997.37111298088039.x

Cited by 21 publications

(16 citation statements)

References 14 publications

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“…The primers used for amplification of each exon are described in Table I. Some of the primers were described previously (33). The PCR products were separated by gel electrophoresis using 0.8% low melting agarose, and the DNA bands were purified with the Geneclean SPIN kit (Bio101, Inc., Vista, CA) as directed by the manufacturer.…”

Section: Dna Preparation Polymerase Chain Reaction and Dna Sequencingmentioning

confidence: 99%

Molecular Defects Underlying the Kell Null Phenotype

Lee¹,

Russo²,

Reiner³

et al. 2001

Journal of Biological Chemistry

121

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Expression of the Kell blood group system is dependent on two proteins, Kell and XK, that are linked by a single disulfide bond. Kell, a type II membrane glycoprotein, is a zinc endopeptidase, while XK, which has 10 transmembrane domains, is a putative membrane transporter. A rare phenotype termed Kell null (Ko) is characterized by the absence of Kell protein and Kell antigens from the red cell membrane and diminished amounts of XK protein. We determined the molecular basis of eight unrelated persons with Ko phenotypes by sequencing the coding and the intron-exon splice regions of KEL and, in some cases, analysis of mRNA transcripts and expression of mutants on the cell surface of transfected cells. Six subjects were homozygous: four with premature stop codons, one with a 5 splice site mutation, G to A, in intron 3, and one with an amino acid substitution (S676N) in exon 18. Two Ko persons with premature stop codons had identical mutations in exon 4 (R128Stop), another had a different mutation in exon 4 (C83Stop), and the fourth had a stop codon in exon 9 (Q348Stop). Two Ko persons were heterozygous for two mutations. One had a 5 splice site mutation (G to A) in intron 3 of one allele that caused aberrant splicing and exon skipping, and the other allele had an amino acid substitution in exon 10 (S363N). The other heterozygote had the same amino acid substitution in exon 10 (S363N) in one allele and a premature stop codon in exon 6 (R192Stop) in the other allele. The S363N and S676N mutants, expressed in 293T cells, were retained in a pre-Golgi compartment and were not transported to the cell surface, indicating that these mutations inhibit trafficking. We conclude that several different molecular defects cause the Kell null phenotype.

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Section: Dna Preparation Polymerase Chain Reaction and Dna Sequencingmentioning

confidence: 99%

Molecular Defects Underlying the Kell Null Phenotype

Lee¹,

Russo²,

Reiner³

et al. 2001

Journal of Biological Chemistry

121

View full text Add to dashboard Cite

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“…One or several exons were amplified from genomic DNA and sequenced using the primers also used for amplification or internal sequencing primers. For KEL, the primers of Lee et al [18] were used. …”

Section: Methodsmentioning

confidence: 99%

Extended Donor Typing by Pooled Capillary Electrophoresis: Impact in a Routine Setting

Wagner

Doescher

Bittner

et al. 2018

Transfus Med Hemother

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Background: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. Methods: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fy^a/Fy^b, Jk^a/Jk^b, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. Results: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. Conclusions: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.

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“…The coding region of KEL was amplified by PCR in 13 segments. This was performed in a PCR system (GeneAmp Models 2400, 2700, or 9700, Perkin Elmer Cetus, Norwalk, CT) with primers and conditions previously described by Lee and colleagues 11,18 . Fragments were separated electrophoretically for 25 minutes at 150 V with 3 percent agarose gel (SeaKem, FMC BioProducts, Rockland, ME) containing ethidium bromide (0.56 µg/mL).…”

Section: Methodsmentioning

confidence: 99%