Use of the Polymerase Chain Reaction–Sequence Specific Oligonucleotide Technique for the Detection of the K1/K2 Polymorphism of the Kell Blood Group System

To the Editor:

The molecular cloning and sequencing of the Kell blood group protein led to the subsequent elucidation of the KEL gene into 19 discrete exons.1 Further characterization of this gene showed that the polymorphism of K2 (k) and K1 (K) was due to a single base nucleotide substitution C701T on exon 6. This substitution disrupts the N-glycosylation motif for the K1 allele.2 Anti-K has been implicated as a major cause of hemolytic disease of the newborn (HDN). Recently, molecular methods have been described for determining KEL genotypes using polymerase chain reaction (PCR)-based techniques, including allele-specific primers3,4 and restriction fragment length polymorphism.5 In this letter, we describe a PCR–sequence specific oligonucleotide (PCR-SSO) procedure for KEL genotyping based on a previously described protocol6 and present frequency data for the K1 and K2 alleles in a white population.

A panel of serologically typed individuals, consisting of 10 K−k+ (K2K2), 8 K+k+ (K1K2), and 7 K+k− (K1K1) individuals, were initially used to establish the parameters of the K1/K2 PCR-SSO technique. Subsequently, a panel of 261 random white individuals was tested to determine the KEL genotype frequencies. Genomic DNA was extracted from peripheral blood leukocytes using a standard salting out and ethanol precipitation procedure. All primers and probes were designed in-house (Table 1) and synthesized on an Oligo 1000 (Beckman Instruments, Fullerton, CA). PCR consisted of 100 ng of genomic DNA, 25 pmol of primer KEL-6 S and KEL-6 AS, 10 mmol/L Tris-HCl pH 8.8, 50 mmol/L KCl, 1.5 mmol/L MgCl₂ , 0.1% Triton X-100, 200 μmol/L of each dNTP, and 1 U of Dynazyme DNA Polymerase (Finnzymes, Espoo, Finland) in a final volume of 25 μL. PCR conditions were 94°C for 5 minutes and then 35 cycles of 30 seconds at 94°C, 60 seconds at 58°C, and 30 seconds at 72°C, with a final cycle for 5 minutes at 72°C. This yielded a PCR-amplified product of 145 bp. Genotype identification was determined by immobilizing the PCR product on a nylon membrane (Genescreen Plus Hybridisation Transfer Membrane; DuPont, NEN Research products, Boston, MA) and hybridizing with the specific probes KEL-K1 and KEL-K2 at 42°C for at least 2 hours. An internal control probe, KEL-IC750, was used to verify that the PCR amplification and SSO procedure had been successful. The TMAC wash temperatures for each probe were 63°C, 60°C, and 57°C, respectively, followed by detection using the DIG-detection system (Boehringer Mannheim, Mannheim, Germany). Each PCR included DNA from individuals typed as K1K1, K1K2, and K2K2 to validate the specificity of the probes.

Comparison of the KEL genotypes obtained by serology and the PCR-SSO procedure described here showed complete correlation. The genotype frequencies of the random population of 261 individuals showed 88% K2K2, 12% K1K2, and 0% K1K1. This correlates with previously reported KEL genotype frequencies of a Melbourne population, taking into consideration that the sample number is approximately four times larger.7 

The PCR-SSO procedure described here enables accurate genotyping of the KEL genes, being both sensitive and reproducible. This technique is particularly useful for screening large numbers of samples and the possible application of KEL genotyping of fetal tissue for the management of HDN.