1 Department of Clinical Immunology, Royal Perth Hospital, Perth, West Australia, 6000, 2 School of Surgery and Pathology, University of West Australia, Perth, West Australia, 6912, 3 Immunogenetics Division, Pediatrics Department, Universidade Federal de São Paulo, 4 West Australian Institute for Medical Research, Royal Perth Hospital, Perth West Australia, 6000 and 5 Obstetrics Department, Universidade Federal de Sao Paulo, Brazil
6 To whom correspondence should be addressed. Email: campbell.witt{at}health.wa.gov.au
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Abstract |
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Key words: killer cell immunoglobulin-like receptors/NK cell/recurrent spontaneous abortion
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Introduction |
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KIR2DL4 is an NK cell receptor for HLA-G (Ponte et al., 1999; Rajagopalan and Long, 1999
). KIR2DL4 has been variously reported to have activating activity for IFN-
secretion (Rajagopalan et al., 2001
; Kikuchi-Maki et al., 2003
), inhibitory activity for cytotoxicity (Ponte et al., 1999
), or activating activity for cytotoxicity (Rajagopalan et al., 2001
; Goodridge et al., 2003
; Kikuchi-Maki et al., 2003
). Part of the explanation for this apparent diversity of functions may lie in the fact that there are two functionally different alleles. One allele codes for a type I membrane receptor whereas the other codes for receptors that are poorly expressed and may be secreted (Rajagopalan et al., 2001
; Witt et al., 2002
). This polymorphism might influence the outcome of NK cell interaction with HLA-G on the trophoblast.
In this study, we determined the KIR repertoire in 52 Brazilian RSA patients. In addition we examined the frequency of the alternate KIR2DL4 genotypes in these patients.
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Materials and methods |
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A group of 51 patients with RSA and 55 controls was used for KIR repertoire. For KIR2DL4 genotyping, 52 patients with RSA and 72 controls were used. Different cohorts were evaluated because the sample amount in some cases was not enough to perform both tests. There were 19 patients and 43 controls in common between the groups. The protocol of this investigation was approved by the Medical Ethics Committee of our Institution and informed consent to participate in the study was obtained from all individuals.
KIR repertoire
The presence or absence of genes for each of the KIR receptors was determined by PCRSSP using the primers shown in Table I. Two separate reactions were included in the detection of KIR2DL1 as outlined in Table I. The 2DL1 primers detect all alleles of KIR2DL1 except those found on the B KIR haplotype. The 2DL1v primers detect all alleles of KIR2DL1 except for a rare variant found on some A haplotypes. Samples giving positive reactions with either 2DL1 or 2DL1v primers were considered to have the KIR2DL1 gene. As the majority of KIR2DS4 genes contain a 22 bp deletion and are not translated, primers that amplify only the expressed alleles of KIR2DS4 were used. Each PCR reaction contained 12.5 µl patient DNA at 20 ng/µl, 2.5 µl 10 x PCR buffer, 0.2 IU Taq polymerase (Amplitaq; Applied Biosystems, USA), 1 µl 40 mmol/l dNTP, 10 pmol of each primer and water to a final volume of 25 µl.
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KIR2DL4 genotyping
KIR2DL4 genotyping was performed as previously described (Witt et al., 2000). Briefly, a stretch of DNA including exon 6, intron 6 and exon 7 was amplified by PCR and the genotype of the PCR products was determined by capillary electrophoresis.
Statistical analysis
Differences between patients and controls in the frequency of each KIR gene were tested by the conventional 2-test. Differences in the total number of activating, inhibitory KIR, the ratio of activating to inhibitory KIR, and the distribution of KIR2DL4 genotypes was assessed by
2-test using the CLUMP program (Sham and Curtis, 1995
).
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Results |
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Discussion |
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The AA KIR2DL4 transmembrane genotype, which represents homozygosity for a membrane-bound receptor for HLA-G, was marginally increased in frequency in RSA patients. However, the other genotypes that would be expected to code for a membrane-bound receptor were not increased in RSA patients, suggesting that the increase in the AA genotype is most likely a type I error. The absence of any association of KIR2DL4 genotype with RSA or with pre-eclampsia (Witt et al., 2002) suggests that if KIR2DL4 has a critical role in pregnancy, then it must be only in rare circumstances. This is consistent with the finding of a woman who was homozygous null for KIR2DL4 but who had had multiple successful pregnancies (Gomez-Lozano et al., 2003
).
Although no association between KIR genotype and RSA was observed, and no disturbance of HLA-C allele frequencies was observed in a previous study (Christiansen et al., 1997), it may be premature to conclude that KIR receptors are not relevant to pregnancy or RSA. As the KIR receptors would be expected to interact with HLA-C on the trophoblast, it will be necessary to study the KIR repertoire in RSA cases in which the HLA-C genotype of the fetus can be established in order to determine whether the presence or absence of certain KIR receptorHLA ligand interactions are necessary. HLA-B alleles are believed not to be expressed on the trophoblast, but a lack of HLA-B alleles with the Bw4 epitope has been observerd in RSA couples (Christiansen et al., 1997
). It will therefore be important that future studies do not neglect any possible interaction between Bw4 on the fetus and the maternal NK receptors for Bw4, KIR3DL1 and KIR3DS1.
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Acknowledgements |
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References |
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Submitted on May 25, 2004; accepted on July 26, 2004.