Novel multiplex PCR-SSP method for centromeric KIR allele discrimination

Abstract

Allelic diversity of the KIR2DL receptors drive differential expression and ligand-binding affinities that impact natural killer cell function and patient outcomes for diverse cancers. We have developed a global intermediate resolution amplification-refractory mutation system (ARMS) PCR-SSP method for distinguishing functionally relevant subgroups of the KIR2DL receptors, as defined by phylogenetic study of the protein sequences. Use of the ARMS design makes the method reliable and usable as a kit, with all reactions utilizing the same conditions. Six reactions define six subgroups of KIR2DL1; four reactions define three subgroups of KIR2DL2; and five reactions define four subgroups of KIR2DL3. Using KIR allele data from a cohort of 426 European-Americans, we identified the most common KIR2DL subtypes and developed the high-throughput PCR-based methodology, which was validated on a separate cohort of 260 healthy donors. Linkage disequilibrium analysis between the different KIR2DL alleles revealed that seven allelic combinations represent more than 95% of the observed population genotypes for KIR2DL1/L2/L3. In summary, our findings enable rapid typing of the most common KIR2DL receptor subtypes, allowing more accurate prediction of co-inheritance and providing a useful tool for the discrimination of observed differences in surface expression and effector function among NK cells exhibiting disparate KIR2DL allotypes.

Figure 1

KIR2DL1 allele typing method. (A) Alignment of the amino acid sequences of the 34 known KIR2DL1 allelic variants. A dash indicates identity with the consensus KIR2DL1*003, and an (*) indicates a stop codon. Structural domains are indicated: Ig-like domains (D1 and D2), stem domain (ST), transmembrane domain (TM), and cytoplasmic domain (CYT). Six PCR reactions separate the six subgroups identified by phylogenetic analysis. Four additional PCR reactions separate alleles within subgroups. Frequencies of the alleles present in the learning cohort of 426 individuals and in the testing cohort of 260 individuals are indicated. The group identification number for each allele is indicated. Alleles identified by PCR are in bold black font, and alleles that were not tested are in gray italics. (B) KIR2DL1 PCR interpretation guide. PCR profiles marked by *, #, or $ are similar and require a higher resolution of genotyping using supplemental reactions.

Figure 2

KIR2DL2 allele typing method. (A) Alignment of the amino acid sequences of the 13 known KIR2DL2 allelic variants. A dash indicates identity with the consensus KIR2DL2*003. Structural domains are indicated: Ig-like domains (D1 and D2), stem domain (ST), transmembrane domain (TM), and cytoplasmic domain (CYT). Four PCR reactions separate the three subgroups identified by phylogenetic analysis. Two additional PCR reactions separate alleles within subgroups. Frequencies of the alleles present in the learning cohort of 426 individuals and in the testing cohort of 260 individuals are indicated. The group identification of each allele is indicated. The alleles tested by PCR are in bold black font, and non-tested alleles are in gray italics. (B) KIR2DL2 PCR interpretation guide. PCR profiles marked by *, #, $ or & are similar and require a higher resolution of genotyping using supplemental reactions.

Figure 3

KIR2DL3 allele typing method. (A) Alignment of the amino acid sequences of the 34 known KIR2DL3 allelic variants. A dash indicates identity with the consensus KIR2DL3*001, an (*) indicates a stop codon. Structural domains are indicated: Ig-like domains (D1 and D2), stem domain (ST), transmembrane domain (TM), and cytoplasmic domain (CYT). Five PCR reactions separate the four subgroups identified by phylogenetic analysis. Six additional PCR reactions separate alleles within subgroups. Frequencies of the alleles present in the learning cohort of 426 individuals and in the testing cohort of 260 individuals are indicated. The group identification for each allele is indicated. The alleles tested by PCR are in bold black font, and the non-tested alleles are in gray italics. (C) KIR2DL3 PCR interpretation guide. PCR profiles marked by * or # are similar and require a higher resolution of genotyping using supplemental reactions.

Figure 4

Linkage disequilibrium between KIR2DL alleles. (A) Linkage disequilibrium analysis identifies seven common combinations of centromeric KIR2DL alleles in a cohort of 260 individuals. (B) Allelic segregation of the KIR2DL and KIR3DP1 alleles in CEPH families. Paternal KIR2DL alleles are shown in blue; maternal alleles in red. (C) KIR2DL allele typing from three generations of CEPH family individuals demonstrates Mendelian inheritance of allele combinations established by the LD study.