Association of killer cell immunoglobulin-like receptor genes with Hodgkin's lymphoma in a familial study

Abstract

Background: Epstein-Barr virus (EBV) is the major environmental factor associated with Hodgkin's lymphoma (HL), a common lymphoma in young adults. Natural killer (NK) cells are key actors of the innate immune response against viruses. The regulation of NK cell function involves activating and inhibitory Killer cell Immunoglobulin-like receptors (KIRs), which are expressed in variable numbers on NK cells. Various viral and virus-related malignant disorders have been associated with the presence/absence of certain KIR genes in case/control studies. We investigated the role of the KIR cluster in HL in a family-based association study.

Methodology: We included 90 families with 90 HL index cases (age 16-35 years) and 255 first-degree relatives (parents and siblings). We developed a procedure for reconstructing full genotypic information (number of gene copies) at each KIR locus from the standard KIR gene content. Out of the 90 collected families, 84 were informative and suitable for further analysis. An association study was then carried out with specific family-based analysis methods on these 84 families.

Principal findings: Five KIR genes in strong linkage disequilibrium were found significantly associated with HL. Refined haplotype analysis showed that the association was supported by a dominant protective effect of KIR3DS1 and/or KIR2DS1, both of which are activating receptors. The odds ratios for developing HL in subjects with at least one copy of KIR3DS1 or KIR2DS1 with respect to subjects with neither of these genes were 0.44[95% confidence interval 0.23-0.85] and 0.42[0.21-0.85], respectively. No significant association was found in a tentative replication case/control study of 68 HL cases (age 18-71 years). In the familial study, the protective effect of KIR3DS1/KIR2DS1 tended to be stronger in HL patients with detectable EBV in blood or tumour cells.

Conclusions: This work defines a template for family-based association studies based on full genotypic information for the KIR cluster, and provides the first evidence that activating KIRs can have a protective role in HL.

Figure 1. Organisation of the human KIR cluster.

The Killer-cell Immunoglobulin-like receptor (KIR) genes are organised in a head-to-tail fashion on human chromosome region 19q13.4. Each gene has nine exons (illustrated for KIR3DL1) and is roughly 10–16 kb in length, with short and equally homologous intergenic sequences of about 2 kb separating each pair of genes. The organisation of the exon-intron structure of the different KIR genes is fairly consistent with the following basic arrangement: the signal sequence is encoded by the first two exons (in grey), each Ig domain (D0, D1, D2) corresponds to a single exon (exons 3–5, respectively, in blue), the linker (in yellow) and transmembrane (in white) regions are each encoded by a single exon (exons 6 and 7, respectively), and the cytoplasmic domain is encoded by two final exons (8 and 9, in red). Two systems have been generated for naming KIR genes. The first follows the CD nomenclature system as CD158a, CD158b, etc., based on an approximate centromerictelomeric order of the genes on chromosome 19 . As the CD nomenclature is not used routinely since it does not reflect structure, function, expression or localization, the Human Genome Organization (HUGO) nomenclature will be used throughout this report (www.gene.ucl.ac.uk/nomenclature/genefamily/kir.html). The HUGO nomenclature system, accounts for KIR protein structure and consists of four major subdivisions based on two features: the number of extracellular Ig domains (2D or 3D) and the length of the cytoplasmic tail (L: long or S: short). This latter information determines their functions: inhibitory (L) or activating (S). The boxes in bold indicate the activating KIR genes, and the dotted boxes correspond to the pseudogenes. The two boundary genes of the KIR cluster, KIR3DL3 and KIR3DL2, and the centrally situated KIR3DP1 pseudogene and KIR2DL4 are present in almost all individuals and are therefore considered to be framework (or anchor) genes (shown in white) . KIR3DL3 and KIR3DP1 delimit the centromeric part of the KIR locus, whereas KIR2DL4 and KIR3DL2 delimit the telomeric part. A 14 kb stretch of unique sequence separating KIR3DP1 from KIR2DL4 is the preferred site for reciprocal recombination, a mechanism resulting in the formation of new haplotypes by the reassortment of centromeric and telomeric genes. Apart from these framework genes, KIR gene content is highly variable in terms of both the number and type of genes present. Although initially considered to be separate genes, KIR2DL2 and KIR2DL3 segregate as alleles of the same locus. Similarly, KIR3DS1 segregates as an allele of the inhibitory KIR3DL1. Overall, there are nine variable genes—KIR2DS2, KIR2DL2/2DL3, KIR2DL1, KIR3DS1/3DL1, KIR2DL5, KIR2DS3, KIR2DS5, KIR2DS1, KIR2DS4—and one variable pseudogene (KIR2DP1). KIR2DL5 KIR2DS3 and KIR2DS5 may be found in both parts of the locus. We decided to represent these genes in the telomeric part of the cluster in this figure. Two major KIR haplotype groups, A and B, are classically described. The A haplotype is defined as containing the KIR3DL3, KIR2DL3, KIR2DL1, KIR2DL4, KIR3DL1, KIR2DS4 and KIR3DL2 genes. KIR gene polymorphism adds further diversity to the KIR region, with multiple alleles known for each KIR locus. Allele identification was performed for KIR2DL4, KIR2DS4 and KIR3DS1/3DL1 (indicated in red). This makes it possible to subdivide haplotype A further, according to the presence of the two common forms of KIR2DS4—the wild type KIR2DS4wt (haplotype A1) or the deletion variant KIR2DS4del, identical to KIR2DS4wt except for a 22 base-pair deletion causing a frame shift in translation (haplotype A2). B haplotypes are more variable and are characterised by the presence of more than one activating KIR gene. Genes that can be present in both group A and group B KIR haplotypes are shown in brown, and genes and/or alleles specific to group B KIR haplotypes are shown in green.

Figure 2. Phenotypic distribution of KIRs in Hodgkin's lymphoma cases.

The proportion (percentages) of present KIR genes, as determined by KIR gene content analysis are presented for the index HL cases (n = 90). Eleven KIR genes are presented in the figure. Two pairs of KIR genes are allelic: KIR3DL1/3DS1, and KIR2DL2/2DL3.

Figure 3. Genotypic reconstruction.

KIR gene content analysis provides only binary phenotyping results for each KIR (absent or present), rather than complete genotypic information. Thus, for genotype analysis (i.e. the number of copies of each KIR gene), genotypic reconstruction in two successive steps was required. The first step combined the results of KIR gene content analysis, knowledge of allelic forms of KIRs, the additional allele typing carried out for three KIR genes, and the analysis of intra-familial segregation. KIR gene content made it possible to determine genotype unambiguously only in individuals with a negative phenotypic result. For example, an individual with a negative KIR2DS2 phenotype has no copy of the gene, and its genotype can be deduced and denoted KIR2DS2(−,−). Conversely, an individual with a positive phenotype for KIR2DS2 may have either one copy of the gene, denoted KIR2DS2(+,−), or two copies of the gene, denoted KIR2DS2(+,+). Two of the nine variable KIR genes have known allelic forms: KIR2DL2/2DL3 and KIR3DS1/3DL1. We defined as (+,+) the KIR3DS1/3DL1 genotype of subjects homozygous for the presence of KIR3DS1 (phenotype KIR3DS1+, KIR3DL1−) and as (−,−) the genotype of subjects homozygous for the absence of KIR3DS1 (phenotype KIR3DS1−, KIR3DL1+). An individual with the KIR3DS1+, KIR3DL1+ phenotype has one copy of each gene and is therefore heterozygous at the locus, with genotype KIR3DS1/3DL1(+,−). The genotypes of KIR2DL2/2DL3 followed the same principle. Additional KIR allele typing was especially useful for the analysis of familial gene segregation. In particular, for KIR2DS4, it made it possible to distinguish between the two common forms, 2DS4wt and 2DS4del (Figure 1). The second step of the reconstruction was based on the KIR haplotype determination of individuals in each family. We first estimated the pairwise linkage disequilibrium (LD) pattern between KIR genes in our families, using the genotypic information obtained from the first step of reconstruction (see statistical analysis). Perfect and complete LD between pairs of KIRs made it possible to infer certain genotypes. For the few remaining unsolved KIR genotypes, haplotypic reconstruction was based on previously described haplotypes , .

Figure 4. Pairwise linkage disequilibrium between KIR genes in the family sample.

This analysis was based on the genotypes reconstructed after the first step, without using any information about LD pattern between KIR genes. Each square represents the magnitude of pairwise LD between KIR genes, as measured by D' (panel A) or r² (panel B). Red squares represent a |D'| or r² value of 1.0, corresponding to complete and perfect LD, respectively. Orange, yellow, and white squares indicate |D'| or R² values of [0.75-1[, [0.5-0.75[, and<0.5, respectively.