Facilitation of KIR genotyping by a PCR‐SSP method that amplifies short DNA fragments

Abstract

Detection of killer-cell immunoglobulin-like receptors (KIR) genes by polymerase chain reaction with sequence-specific primers (PCR-SSP) led in 1997 to the discovery that human genomes diverge largely in the KIR they encode. While only a few KIR genes are conserved in all humans, most individuals lack several those genes, which tend to associate in diverse haplotypic combinations. The PCR-SSP technique, updated to detect the more recently identified KIR genes and alleles, is still used widely to analyze the diversity of human populations, and to study the influence of KIR-gene variability on human health. Several published PCR-SSP methods for KIR genotyping, although simple and robust, have the drawback of relying on the amplification of DNA fragments spanning 0.5-2.0 kbp, which tends to fail in low-quality DNAs. Valuable collections of DNAs often include such poor quality samples, which lead to loss of data and resources. Even worse, undetected falsely negative or positive reactions may result in erroneous gene frequencies and in odd gene combinations. To address those problems, we have redesigned our previously published KIR genotyping method so that it produces short amplicons (less than 200 bp for most genes). This modification minimizes amplification failures, thus conferring greater consistency and reliability to KIR genotyping. In addition, the new PCR-SSP method detects recently described alleles of several KIR genes, and allows for discrimination between the major structural variants of KIR2DS4 and KIR3DP1 without increasing the number of reactions.

Associations of killer cell immunoglobulin-like receptor genes with complications of rheumatoid arthritis

“…Originally, our typing using primers described by Łuszczek et al 18,19 revealed relatively high representation of unusual genotype containing KIR2DL3 gene in the absence of KIR2DL1. Retyping of all KIR2DL1-negative samples with recently described primers 20 revealed the presence of KIR2DL1 gene in all KIR2DL3-positive samples, whereas KIR2DL3-negative individuals persisted negative expect for one 2DL1 þ 2DL3 À control person.…”

“…18,19 Samples typed negative for KIR2DL1 were retyped using new primers of Vilches et al 20 Statistical analysis Differences between controls, patients and patient subgroups were estimated using the two-tailed Fisher's exact test and GraphPad InStat 3 software. The correction of P-values (if o0.05) for the number of comparisons (11 genes and 7 or 3 subgroups of patients) was calculated using the formula of Svejgaard and Ryder.…”

Multiple sclerosis associates with LILRA3 deletion in Spanish patients

“…We studied by PCR 46 the presence or absence of each KIR in the genome of 224 R-MS patients and 289 healthy controls, from whom enough DNA was available. Both groups had rather similar frequencies at all KIR genes, except for KIR3DS1, under-represented among R-MS patients (OR 0.69, Po0.05, Table 4).…”

“…Presence or absence in the genome of each KIR gene was determined as described earlier. 46 The gene order is based on that of the KIR complex 21,53 -the genes that define the 'A' type of haplotype (KIR2DL3 through KIR2DS4) are represented in the middle, preceded and followed, respectively, by genes characteristic of the centromeric and the telomeric parts of 'B' haplotypes. Framework genes and pseudogenes found in all individuals are not shown.…”

Duplication, mutation and recombination of the human orphan gene KIR2DS3 contribute to the diversity of KIR haplotypes

“…Presence or absence of every KIR gene in the genome was assessed by PCR-SSP or PCR-SSOP. 25,26 20 were detected using the PCR primers Fa796 þ Ra925 (agcgt gaccttgtcctga and gcatctgtaggttcctcct) and Fc551 þ Rt682 (agagaggggacgtttaacc and ggtcactgggagctgaa), respectively, in the PCR conditions described elsewhere. 25 To discriminate KIR2DS3*001 from the novel allele KIR2DS3*004, we used a PCR-restriction fragment length polymorphism technique.…”