Linkage disequilibrium organization of the human KIR superlocus: implications for KIR data analyses.

An extensive family-based study of linkage disequilibrium (LD) in the killer cell immunoglobulin-like receptors (KIR) cluster was performed. We aimed to describe the LD structure in the KIR gene cluster using a sample of 418 founder haplotypes identified by segregation in a group of 106 families from Northern Ireland. The LD was studied at two levels of polymorphism: the structural level (presence or absence of KIR genes) and the allelic level (between alleles of KIR genes). LD was further assessed using the predictive value of one KIR polymorphism for another one in order to provide an interpretative framework for the LD effect in association studies. In line with previous research, distinct patterns of KIR genetic diversity within the genomic region centromeric to KIR2DL4 (excluding KIR2DL4) and within the telomeric region including KIR2DL4 were found. In a comprehensive PPV/NPV-based LD analysis within the KIR cluster, robust tag markers were found that can be used to identify which genes are concomitantly present or absent and to further identify groups of associated KIR alleles. Several extended KIR haplotypes in the study population were identified (KIR2DS2*POS-KIR2DL2*001-KIR2DL5B*002-KIR2DS3*00103-KIR2DL1*00401; KIR2DL4*011-KIR3DL1/S1*005-KIR2DS4*003-KIR3DL2*003; KIR2DL4*00802-KIR3DL1/S1*004-KIR2DS4*006-KIR3DL2*005; KIR2DL4*00801-KIR3DL1/S1*00101-KIR2DS4*003-KIR3DL2*001; KIR2DL4*00103-KIR3DL1/S1*008-KIR2DS4*003-KIR3DL2*009; KIR2DL4*00102-KIR3DL1/S1*01502/*002-KIR2DS4*00101-KIR3DL2*002; KIR2DL4*00501-KIR3DL1/S1*013-KIR2DL5A*001-KIR2DS5*002-KIR2DS1*002-KIR3DL2*007). The present study provides a rationale for analyzing associations of KIR polymorphisms by taking into account the complex LD structure of the KIR region.

Differential RNA expression of KIR alleles.

Allelic polymorphisms dramatically influence the phenotype of human killer immunoglobulin-like receptors (KIR) by modifying their expression in cell surfaces. It is unclear though to what extent this involves transcriptional or post-transcriptional mechanisms, as quantitative RNA expression of KIR alleles has not been systematically compared. We measured RNA transcript abundance of common KIR alleles by real-time quantitative reverse transcriptase PCR (RT-PCR) in 85 PBL samples that were allele-typed in parallel. Allele type showed little influence on transcript abundance for a given KIR gene, except for: (1) KIR2DL5B*002, which consistently showed undetectable transcripts levels; (2) truncated KIR2DS4 alleles, associated with lowered expression levels; and (3) alleles of KIR2DL4 with a single-base deletion, associated with higher expression than average. Lowered levels of truncated KIR2DS4 transcripts were confirmed by dot blot of RT-PCR products, indicating imbalanced allelic RNA expression in heterozygote genotypes containing these alleles. Imbalanced expression of truncated KIR2DS4 alleles was corroborated in family samples. Gene copy number of KIR2DL1, KIR2DL3 and KIR3DL1 influenced RNA expression, genotypes with a single copy expressing on average lower transcript amounts than those with two copies. The data show that for a given KIR gene, the common allele types found in our population express comparable RNA levels, except truncated or null alleles. Thus, variation of KIR expression on cell surfaces more likely involves post-transcriptional mechanisms.

Killer cell immunoglobulin-like receptor allele discrimination by high-resolution melting.

New molecular techniques for allele discrimination such as high-resolution melting (HRM) allow fast, reliable and high-throughput typing without the use of oligonucleotide probes. HRM can also provide an alternative for problematic allele assignments caused by the presence of homopolymeric regions or ambiguous haplotyping. We show here how we have used HRM to introduce upgrades in our KIR allele level typing system for the KIR2DS4, KIR2DL4, and KIR3DL2 genes. The allele assignments obtained by HRM, confirmed by sequencing, were clear-cut and reproducible. This technique allowed for quick and easy upgrading of the KIR allele frequencies and is now validated for future updates of KIR typing. The ongoing refinement of KIR haplotype distribution in our control population will help in disease association studies involving the KIR genes.

Distinct diversity of KIR genes in three southern Indian populations: comparison with world populations revealed a link between KIR gene content and pre-historic human migrations.

By interacting with polymorphic HLA class I molecules, the killer cell immunoglobulin-like receptors (KIR) influence the innate and adaptive immune response to infection. The KIR family varies in gene content and sequence polymorphism, thereby, distinguishing individuals and populations. To investigate KIR diversity in the earliest settlers of India, we have characterized the KIR gene content in three Dravidian-speaking populations (Mollukurumba, Kanikar, and Paravar) from the state of Tamil Nadu, southern India. The activating KIR genes and putative group-B KIR haplotypes were frequent in Paravar and Kanikar, a scenario analogous to those seen previously in other populations of Indian origin, indicating that predominance of group-B KIR haplotypes is the characteristic feature of Indian populations. In contrast, the KIR gene profile of Mollukurumba was more related to Caucasian type. It is not clear whether a local-specific selection or a recent admixture from Iran is responsible for such discrete profile in Mollukurumba. Each southern Indian population had distinct KIR genotype profile. Comparative analyses with world populations revealed that group-B KIR haplotypes were frequent in the natives of India, Australia, and America, the populations associated with those involved in extensive prehistoric human migrations. Whether or not natural selection has acted to enrich group-B KIR haplotypes in these migratory descendants is an issue that requires objective testing.

Investigation of KIR gene frequencies in type 1 diabetes mellitus.

The frequency of killer immunoglobulinlike receptors (KIR) genes was examined in type 1 diabetes mellitus patients and controls from Finland. The KIR gene 2DS5 was significantly decreased in patients versus controls, but this was no longer significant after correction for the number of comparisons made.

Activating killer cell immunoglobulin-like receptor gene KIR2DS1 is associated with psoriatic arthritis.

Killer cell immunoglobulin-like receptor genotyping was performed on a cohort of American Caucasian patients with psoriasis to investigate any possible relationship between these chromosome 19 genes and autoimmune-linked disease. This patient cohort also contained a subgroup of patients who had been additionally diagnosed as positive for psoriatic arthritis (PsA). Because of the known association of human leucocyte antigen (HLA)-Cw*06 with psoriasis, the study concentrated on the five KIR genes that have HLA-C as their recognized ligand (i.e., KIR2DL1, -2DL2, -2DL3, -2DS1, and -2DS2). An increase in the frequency of the activating KIR2DS1 gene was detected in the PsA patients, compared with psoriasis patients negative for PsA and an unaffected American Caucasian control group.

Investigation of killer cell immunoglobulin-like receptor gene diversity: II. KIR2DS4.

A polymerase chain reaction sequence-specific oligonucleotide probe typing method identifying and distinguishing alleles of the KIR2DS4 gene has been established. The system is based on the specific amplification of a region of this gene, followed by hybridization with 11 sequence-specific oligonucleotide probes. The method has been applied to a healthy group of Northern Irish caucasian individuals, establishing frequencies of alleles of this locus within the local population. Furthermore, cell line DNA and families from the 13th International Histocompatibility Workshop, in addition to local families, have also been allele typed at the KIR2DS4 locus. Haplotype segregation, with respect to KIR2DS4 alleles, has been examined by using the local family data. Within all sample groups investigated, four KIR2DS4 alleles were identified, two of which are novel to this investigation.

Investigation of killer cell immunoglobulinlike receptor gene diversity: I. KIR2DL4.

Polymerase chain reaction-sequence-specific oligonucleotide probes typing procedures identifying alleles of the killer immunoglobulin-like gene (KIR2DL4) have been established. The methods, designed around the specific amplification of the D0 and D2 domains of this gene, produce discrimination of KIR2DL4 alleles. The methods have been applied to a healthy Northern Irish control group, establishing frequencies for this Caucasian population. Additionally, the KIR2DL4 allele status of cell line DNA and CEPH families, from the 13th International Histocompatibility Workshop and local families, have also been investigated.

High resolution HLA-DRB1 identification of a Caucasian population.

Polymerase chain reaction-sequence-specific oligonucleotide probes typing methods have been applied to 1000 individuals from the Northern Ireland population to give human leukocyte antigen DRB1 (HLA-DRB1) allele assignment. HLA-DRB1 allele frequencies and four-locus haplotypes (A/B/C/DR) for this Caucasian population, based on HLA class I and class II allele assignment, are now presented. No significant deviations from Hardy-Weinberg proportions were observed. The HLA-C locus exhibited marginal evidence of selection (p<0.03, uncorrected one-sided test) in the direction of balancing selection; the HLA-A, -B, and -DRB1 allele frequency distributions were compatible with expectations under a neutral model (which does not mean that selection is not operating). Evidence for selection was seen on haplotypes HLA-A*010101-B*0801-DRB1*030101 and HLA-A*290201-B*440301-DRB1*070101 based on their patterns of linkage disequilibrium.

Multiple copies of KIR 3DL/S1 and KIR 2DL4 genes identified in a number of individuals.

Multiple copies of the killer immunoglobulin-like receptor gene, 3DL/S1, have been identified in certain individuals. Additionally, allele determination of the killer immunoglobulin-like receptor gene (KIR), 2DL4, has identified three alleles of this gene present in these same individuals. This event has been confirmed by isolating three distinct KIR2DL4 allele clones in each individual, which sequenced as the alleles identified by the allele identification technique. It is our assumption that an unequal crossover event has occurred between differing KIR haplotypes resulting in the duplication of the 2DL4, 3DS1/3DL1 genes on the newly formed haplotype(s).

Study of age-association with cytokine gene polymorphisms in an aged Irish population.

The release of cytokines is of crucial importance in the regulation of the type and magnitude of the immune response in the elderly. A number of studies have shown different levels of cytokine production in the elderly. In the present study, a range of polymorphisms were chosen within the genes of cytokines (IL-2, IL-6, IL-8, IL-10, IL-12 and IFN-gamma) that have been observed at different levels within the elderly and analysed for age-association. No association was observed for the polymorphic cytokine markers and the healthy aged Irish population (or with respect to gender) examined in this study. These findings would suggest that polymorphism of cytokine genes may not play as crucial a role in healthy ageing as previously believed.

Frequency of cytokine polymorphisms in populations from western Europe, Africa, Asia, the Middle East and South America.

PCR-SSOP identification procedures for IL-2, IL-6, IL-10, TNF-alpha and TNF-beta cytokine polymorphisms have been developed. Application of the procedures to a range of diverse geographically distributed populations has identified ethnic differences within the groups studied. Five populations were investigated, Northern Ireland, South African Zulu, Omani, Singapore Chinese and Mexican Mestizos.

Molecular diversity of the HLA-C gene identified in a caucasian population.

A DNA typing procedure, based on a two stage polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) typing strategy, has been developed and applied to DNA from 1000 healthy individuals from the Northern Ireland region. The two-stage procedure involves human leukocyte antigen (HLA-C) identification through the use of a medium resolution PCR-SSOP system, followed by four secondary group specific PCR-SSOP systems, to enable allele resolution. The PCR-SSOP systems were designed for the identification of HLA-Cw alleles with possible discrimination within exons 2 and 3 of the HLA-C gene, i.e., HLA-Cw*01-Cw*16. PCR-SSP tests were designed for the resolution of HLA-Cw*17 and -Cw*18 alleles. The systems can also be used independently of each other if selective allele resolution is required. HLA-Cw allele frequencies occurring within the Northern Ireland population have been compiled, along with estimations of HLA-B/Cw haplotype frequencies.