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Diversity across KIR haplotypes stems from differences in numbers of inhibitory and activating receptors, as well as allelic polymorphism of individual genes. The KIR locus has undergone large expansions and contractions over time and is believed to be coevolving with genes encoding its HLA class I ligands located within the MHC locus. KIR and HLA compound genotypes have been associated with susceptibility to or protection from infectious, autoimmune, reproductive, and malignant disorders. We describe here a simple and reliable multiplex PCR-SSP (sequence-specific priming) method for relatively rapid and inexpensive genotyping of 15 KIR genes using standard agarose gel electrophoresis.
Natural killer (NK) cells are central to the innate immune system and they represent the first line of defense against bacteria, parasites, viruses, and malignant cells (1). NK cells comprise 5–15% of circulating lymphocytes and are also found in tissues including the liver, peritoneal cavity, and placenta. They can mediate spontaneous killing of infected or transformed target cells and produce immunoregulatory cytokines and chemokines that stimulate the adaptive immune response (2, 3). In addition, they engage in interactions at the maternal–fetal interface that are critical for successful pregnancy (4).
NK cell activity is tightly controlled by activating and inhibitory interactions through numerous cell surface receptors. They respond to targets that express aberrant levels of MHC class I, and this involvement of MHC class I molecules in NK cell recognition of self vs nonself was originally described over two decades ago (5). MHC class I molecules are the essential signature of self that NK cell inhibitory receptors engage in order to avoid NK cell attack of normal cells (5–7). The original ‘missing self’ hypothesis (8) proposed that downregulation of class I expression on target cells leads to spontaneous destruction by NK cells. Current knowledge of NK cell biology suggests that NK cells not only patrol for abnormal cells that lack MHC class I but also for those that over-express ligands for activating receptors such as altered MHC (‘altered self’) and non-MHC ligands (‘induced self’ or ‘nonself’) (9).
The killer cell immunoglobulin-like receptors (KIR) are one of the main types of MHC class I-specific receptors utilized by human NK cells, and they are comprised of both activating and inhibitory counterparts that are encoded by highly homologous sequences. The KIR locus maps to chromosome 19q13.4 within the leukocyte receptor complex (LRC) (10). The high sequence similarity of the KIR genes may facilitate the occurrence of non-allelic homologous recombination (NAHR) (11, 12) and probably partially explains expansion and contraction of KIR haplotypes (10). KIR haplotypes have been broadly classified into two groups referred to as A and B, both of which are found in most populations but sometimes at very different frequencies. Haplotype A is invariant in terms of gene content while haplotype B is quite variable (13) (Fig. 25.1). Currently, more than 40 different B haplotypes based on gene content have been described [summarized in (14)], and further diversity is afforded by allelic polymorphism of individual KIR genes such that it is unlikely that any two randomly selected individuals have identical KIR genotypes (15). The highly polymorphic and genetically unlinked KIR and MHC class I loci appear to have coevolved to give selective advantage to higher order mammals with respect to reproduction and resistance against infections. Thus, it is not surprising that an ever-increasing number of genetic studies have shown that combinations of KIR and HLA confer either protection against or susceptibility to infectious, reproductive, autoimmune, and malignant disorders (16). KIR genotypic variation across populations is also important from an evolutionary standpoint since differences may in part reflect a response to differences in exposure to infectious agents across populations (17).
This chapter will describe a method for multiplex KIR genotyping based on our previously published methodology (18). We have used the same primers but in multiplex combinations that can be resolved by gel electrophoresis. This method detects the presence/absence of KIR genes, thus providing KIR gene profiles.
Determination of KIR genotype using this method depends on presence or absence of detectable amplification product for each gene-specific primer pair (see Notes 9–11). It is important to have a good quality gel picture for accurate judgment of the product sizes, as each well contains multiple products of varying size. The most important product is the positive internal control amplicon, which should be present in all the samples. Each KIR gene is amplified using two pairs of specific primers and products should be detected for both sets of primers. If the amplification by the two gene-specific primer pairs is discrepant for any gene, the reaction should be repeated. Detectable bands as well as absence of product can be recorded in a spreadsheet such as Microsoft Excel. Some alleles of the KIR2DS4 gene have a 22 bp deletion in exon 5 that can be detected by the KIR2DS4 primers in primer mix 8. The full-length product is 219 bp while the deletion variant is 197 bp (see Fig. 25.2B).
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
1The quality of DNA is the most important requirement for reliable PCR-SSP results. DNA extracted from heparinized blood should be avoided, as heparin is a PCR inhibitor (19).
2A total of 100–200 ng (gel electrophoresis method) DNA is required for reliable amplification using the primer mixes described here. Increasing the number of cycles is likely to increase false-positive reactions. Therefore, if the amplification is not satisfactory because of DNA quality, it is recommended that each primer mix be amplified individually, as described in (18).
3All primer mixes should be tested with positive and negative controls when new primer mixes are made from the stock primers.
4Platinum Taq (Invitrogen) polymerase gives the most reliable and consistent results for this protocol.
5A fast ramping thermal cycler with a heated lid is recommended for best results. The ‘step-down’ program was designed for this protocol to increase specificity of the amplifications. It might be necessary to alter the PCR conditions slightly depending on the PCR machine being used.
6PCR machines should be checked regularly to ensure that all wells are amplifying uniformly. It is also important to ensure that the PCR plates fit snugly into the machine.
7The quality of dNTPs is important for reliable amplifications. Avoid repeated freeze–thawing; instead freeze in small aliquots after reconstitution.
8The highly homologous sequences of the KIR loci can lead to nonspecific amplification especially with increased numbers of cycles (>30) used for PCR.
9Some primers miss a couple of alleles of a given gene as noted in Table 25.1 and others amplify one allele of another gene. Furthermore, there are likely to be yet undiscovered alleles of a gene that might not amplify with the current set of primers and alternatively some of these might cross-react with alleles of other KIR genes. We strongly recommend routine checks of the KIR database (http://www.ebi.ac.uk/ipd/kir) for new alleles which might require designing new primers. It should also be remembered that most of the available KIR gene sequences have been obtained from Caucasians and thus may not include alleles that are prevalent in other populations. This highlights the importance of using more than one pair of primers for amplification of each gene.
10The gene content of some KIR haplotypes and sequence of some KIR alleles strongly suggest that unequal crossing-over in the region accounts for a substantial amount of the diversity at this locus (20–23). This could result in the production of unusual profiles where, for example, one or more of the framework genes are missing or chimeric genes are present.
11Some of the KIR loci exhibit strong negative and positive linkage disequilibrium between them. For example, some gene pairs such as KIR2DL2/2DS2, KIR2DL1/2DP1, and KIR3DL1/2DS4 are almost always found together across different haplotypes. This information is very useful in validation of the genotyping results.