Results

In this study, we typed 14 KIR genes in 96 Japanese cases and 50 healthy controls. The frequency of each KIR is shown in Table I. Generally, three framework genes (KIR2DL4, KIR3DL2, and KIR3DL3) were detected in almost all the PV cases and controls. But in PV, KIR2DL1 corresponding to group 2 HLA-C and KIR3DL1 corresponding to HLA-Bw4 were absent in some cases (an approximately 7% loss for KIR2DL1 and 14% loss for KIR3DL1, which were not significant at the 5% level), although they were almost all detected in controls. In the respective comparison, KIR2DS1 (43 of 96 (45%) in cases vs 14 of 50 (28%) in controls) and KIR2DL5 (46 of 96 (48%) in cases vs 15 of 50 (30%) in controls) were significantly increased in PV cases at the 5% level (²-test), although the logistic analysis that was attempted to overcome the multiple testing problem was not able to distinguish them statistically, possibly because of the small sample size.

Table I - Frequency of respective KIR in 96 PV cases and 50 healthy controls.

Full table

Furthermore, some representative genotypes and their assumed haplotype combinations observed in our experiments are shown in Table II. The most frequent genotype in both cases and controls was the homozygote of the "A" haplotype (haplotype combination AA) as shown in the right half of this table. This haplotype combination AA is the Japanese predominant genotype as reported by^{Yawata et al (2002)}. Furthermore, the comparison of donors without the "B" haplotype (haplotype combination AA) and donors with at least one "B" haplotype (haplotype combination AB/BB) was focused, because significantly increased KIR2DL5 is a marker of the "B" haplotype. As shown in the right half of Table II, donors with at least one "B" haplotype (haplotype combination AB/BB) significantly increased in PV cases (53 of 96 (55%) in cases vs 18 of 50 (36%) in controls, p=0.036, two-sided Fisher's exact test) This indicated an increase in the "B" haplotype and a simultaneous decrease in the homozygotic "A" hapolotype in PV cases.

Table II - Representative KIR genotypes in 96 PV cases and 50 healthy controls and frequency of their halotype combinations.

Full table (51K)

Discussion

The results of our study showed a significant increase in donors with KIR2DS1 and/or KIR2DL5 and donors with at least one of the "B" haplotypes of PV. These phenomena would be explained by the occurrence of a substantial increase in one of the "B" haplotype groups in PV. According to the KIR haplotype model based on family studies and genomic sequencing by^{Hsu et al (2002)}, all the haplotypes with KIR2DS1 simultaneously have KIR2DL5 upstream, but KIR2DL5 does not always coexist with KIR2DS1. This haplotype structure of KIR indicates that an increase in KIR2DS1 leads to a secondary increase in KIR2DL5 in donors. Indeed, in our results, the frequencies of KIR2DS1 and KIR2DL5 were nearly identical in the respective groups (45% for 2DS1 and 48% for 2DL5 in cases; 28% for 2DS1 and 30% for 2DL5 in controls, Table I), and all the genotypes with KIR2DS1 also possessed KIR2DL5, as partly shown in Table II. Examination of these frequencies relative to the frequencies of the haplotype combination AA/AB in Table II revealed that haplotypes with both KIR2DS1 and KIR2DL5 account for a large proportion of the "B" haplotypes. This finding suggests that the increase in "B" haplotypes in PV cases was derived largely from an increase in some haplotype group with KIR2DS1. Interestingly, KIR2DS1 also showed the most significant increase in psoriatic arthritis, as described by^{Martin et al (2002)}. Our data suggest some critical significance of KIR2DS1 and/or haplotypes with KIR2DS1 in the HLA-Cw6-related disease, although PV and psoriatic arthritis may not be identical.

The relationship between the increase in "B" haplotypes and their activating ligands requires further study. KIR2DS1 is certainly an attractive candidate for exerting a direct effect on pathogenesis, as it is known to interact with HLA-Cw6 and has been independently implicated in psoriatic arthritis (^{Martin et al, 2002}). But if this were the predominant disease-causing effect, one might expect to see increases in other HLA-C alleles that interact with KIR2DS1 (i.e., Cw2, Cw4, and Cw5), and this has not proved to be the case. Additional studies will be required to determine whether KIR2DS1 is directly responsible for the effects we have observed, as opposed to one or more other features of KIR "B" haplotypes. Nevertheless, the possibility of some important functional links between activating KIRs, HLA-Cw6, and PV has emerged via the activating molecule of KIR2DS1; however, activating KIRs have not been identified as susceptible genes by genome-wide scans so far. This might be attributable to the relatively high frequency of KIR2DS1 (approximately 30%), as power to detect linkage declines as disease allele frequency increases. Also, linkage may be obscured because of requirements for other, unlinked genes and/or environmental factors that reduce the penetrance of the disease allele. Association strategies have been shown to be much more powerful than linkage studies in this setting (^{Risch and Merikangas, 1996}).

Besides direct interaction with the HLA molecule, the structurally distinctive character of "B" haplotypes is the multiplicity of activating KIRs with short cytoplasmic tails, and this character was reflected in the multiplicity of activating KIRs in donors with haplotypes AB and BB, as shown in Table II. Activating KIRs recognize not only the paired ligand HLA class I but also foreign or microbial antigens (^{Hsu et al, 2002;Martin et al, 2002}). This multiplicity of activating KIRs, if expressed functionally, might increase the chance of accidental activation of immunocytes over the threshold. Moreover, this inaccurate activation would occur especially in patients who do not possess the proper combination of inhibitory KIRs and the ligand HLA. More specifically, if there are no paired HLA, inhibitory KIRs, even if they exist, cannot inhibit the activation of immunocytes, and vice versa. This possibility had already been suggested by^{Martin et al (2002)}; however, the size of our HLA-typed samples was not large, and whether or not mismatching between HLA class I, activating KIRs, and inhibitory KIRs is predisposed to PV remains obscure.

In summary, genotypes with "B" haplotypes, especially those containing KIR2DS1 and KIR2DL5, were found to be increased in Japanese PV cases, and this phenomenon seemed to be caused by an increase in some haplotype group possessing KIR2DS1. This association of KIR2DS1 with PV is strongly concurrent with the previous implication of KIR2DS1 in psoriatic arthritis, another HLA-Cw6-associated disease (^{Martin et al, 2002}). Thus, KIR2DS1 may be a common denominator in both HLA-Cw6-associated diseases. KIR genotyping would provide a new interesting therapeutic strategy for considering the molecular basis of PV.

Materials and Methods

DNA isolation

Samples from 96 unrelated Japanese patients with PV were collected after receiving their informed consent and authorization from the Ethical Review Committee of Gene Analysis Research, Yamaguchi University, School of Medicine, and University Hospital, according to the Declaration of Helsinki principles. Fifty control samples were also obtained from unrelated healthy controls. Each genomic DNA was extracted using the QIAmp DNA Blood Maxi Kit (QIAGEN K.K., Tokyo, Japan) and stored at -20°C before use.

PCR

DNA samples were genotyped using PCR-SSP for the following KIR: 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 3DL1, 3DL2, 3DL3, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, and 3DS1. The PCR primers and conditions used were in accordance with the method described in a previous report (^{Gómez-Lozano and Vilches, 2002}). The human growth hormone (GH1) served as a positive marker of PCR. All primers were synthesized and validated by Hokkaido System Science Co., Ltd (Hokkaido, Japan). PCR was conducted by the Gene Amp PCR system 9600-R (Perkin–Elmer, Norwalk, USA.). Briefly, approximately 100 ng of genomic DNA was amplified in 20 L TaKaRa EX Taq buffer with 2 mM Mg²⁺, 200 M dNTP, and 0.5 U TaKaRa EX Taq polymerase (Takara Bio Inc., Shiga, Japan). After the initial denaturation for 2 min at 95°C, samples were amplified in 10 cycles of 20 s at 94°C, 10 s at 65°C, and 1 min 30 s at 72°C; and 20 cycles of 20 s at 94°C, 20 s at 61°C, and 1 min 30 s at 72°C. Ambiguous typing results were checked by sequencing or other PCR systems using several distinct primer sets (^{Martin et al, 2002}).

KIR genotyping

PCR products were electrophoresed on 1.2% agarose gels with ethidium bromide, and each sample was genotyped. The frequency (%) of each KIR was calculated as the percentage of positive numbers among all specimens. After the genotyping of KIR, the homozygous "A" haplotype was determined by the presence of only a set of KIRs: 2DL1, 2DL3, 2DL4, 3DL1, 3DL2, 3DL3, and 2DS4, and the heterogenous "B" haplotypes were inferred from patterns including KIR2DL2, KIR2DL5, and/or various combinations of activating KIR (^{Gómez-Lozano and Vilches, 2002;Yawata et al, 2002}). Furthermore, genotypes observed in our experiments were defined as haplotype combinations, AA, AB, and BB. Haplotype combination AA is a homozygous "A" haplotype, and haplotype combination BB has combinations of two "B" haplotypes without 3DL1 and 2DS4, which are the markers of the "A" haplotype (^{Yawata et al, 2002}). Haplotype combination AB consists of various heterogenous combinations of the "A" and "B" haplotypes. In our PCR system, KIR1D, the common variant of KIR2DS4 in the Caucasian population (^{Hsu et al, 2002}), was not distinguished from KIR2DS4. The "A" haplotype in our data contained both haplotype A-1D and haplotype A-2DS4 described by^{Hsu et al (2002)}.

Statistical analysis

Frequency differences were tested for significance by the two-sided Fisher's exact test or the ²-test. Multifactorial analysis was also attempted by logistic analysis (the SAS system).

References

References

Acknowledgments

This work was supported by grants (Nos. 12204079, 13204063, and 15591178) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.