Molecular characterisation of KIR2DS2*005, a fusion gene associated with a shortened KIR haplotype.

KIR2DS2 is an activating homologue of KIR2DL2, an inhibitory killer-cell immunoglobulin-like receptor (KIR) that surveys expression of major histocompatibility complex-C allotypes bearing a C1 epitope. We have studied here its allele KIR2DS2*005, which shows a hybrid structure-it is identical to other KIR2DS2 alleles in the ectodomain, but has transmembrane and cytoplasmic regions identical to those of KIR2DS3(*)001, a short-tailed KIR of uncertain expression and function. Our results reveal that KIR2DS2*005 is a fusion gene-the product of an unequal crossing over by which the genes KIR2DS2 and KIR2DS3 recombined within a 400 base pair region of complete identity in intron 6. Also resulting from that recombination was a shortened KIR haplotype of the B group, in which three genes commonly linked to KIR2DS2 (KIR2DL2, KIR2DL5B and KIR2DS3) are deleted. Population studies indicate that KIR2DS2*005 is still associated to such haplotype, and it can be found in approximately 1.2% of Caucasoids. Using a combination of two monoclonal antibodies, we also demonstrate that KIR2DS2*005 encodes a molecule expressed on the surface of natural killer- and T-lymphocytes.

The cannabinoid receptor 1 gene (CNR1) and multiple sclerosis: an association study in two case-control groups from Spain.

Different studies point to the implication of the endocannabinoid system in multiple sclerosis (MS) and animal models of MS. The purpose of this study was to evaluate a possible association of MS with polymorphic markers at the CNR1 gene, encoding the cannabinoid 1 (CB(1)) receptor. We have performed a genetic analysis of an AAT repeat microsatellite localized in the downstream region of the CNR1 gene, in two case-control groups of MS patients and healthy controls (HC) from Spain (Madrid and Bilbao). MS patients with primary progressive MS (PPMS) had more commonly long ((AAT) > or = (13)) alleles and genotypes with a significant difference for genotype 7/8 in Madrid (p = 0.043) and in the sum of both groups (p = 0.016); short alleles were less frequently found in PPMS with a significant difference for allele 5 in the analysis of both groups together (p = 0.039). In patients with relapsing MS, no consistent differences in allele and genotype distribution were found. Disease severity and progression was unrelated to AAT repeat variations. In conclusion, long (AAT) > or = (13) CNR1 genotypes could behave as risk factors for PPMS.

The 5' intergenic, promoter, pseudoexon 3 and complete coding sequences of the hybrid gene KIR2DS3*002.

Genomic and mRNA sequences support the KIR2DS3*002 gene being a hybrid of KIR2DS3*00103 and KIR2DS5.

Duplication, mutation and recombination of the human orphan gene KIR2DS3 contribute to the diversity of KIR haplotypes.

The KIR2DS3 gene is an activating homologue of the inhibitory killer-cell immunoglobulin-like receptors (KIR) that recognize HLA-C molecules, enabling NK cells to survey the normal function of endogenous antigen presentation. The genetics of KIR2DS3 is complicated by the existence of alleles with similar coding sequences that map to different regions of the KIR complex in chromosome 19, or whose location in this complex is unknown. Here, by studying the family segregation of the KIR alleles 2DS3*001, *002 and *003N, and the distribution of these in unrelated individuals, we demonstrate the existence of two paralogous KIR2DS3 genes that can be inherited separately or, as it happens frequently in Caucasoids due to linkage disequilibrium, together. Each KIR2DS3 gene is almost invariably associated in its 5' end to a different copy of KIR2DL5, a gene previously shown to be duplicated in humans. KIR2DL5 and KIR2DS3 thus form two highly homologous gene clusters situated in the centromeric and the telomeric intervals of KIR haplotypes. Recombination between those clusters is the likely origin of new haplotypes, characterized in this study, which harbour further duplications or deletions of multiple KIR genes. Our results help understand the genetics of KIR2DS3 and the diversity of human KIR genotypes.

An apparent KIR2DS2-negative KIR2DL2-positive genotype discloses the novel allele KIR2DS2*00104.

KIR2DS2*00104 lacks a distinctive synonymous substitution of KIR2DS2 in nucleotide 418 that affects KIR genotyping.

Facilitation of KIR genotyping by a PCR-SSP method that amplifies short DNA fragments.

Detection of killer-cell immunoglobulin-like receptors (KIR) genes by polymerase chain reaction with sequence-specific primers (PCR-SSP) led in 1997 to the discovery that human genomes diverge largely in the KIR they encode. While only a few KIR genes are conserved in all humans, most individuals lack several those genes, which tend to associate in diverse haplotypic combinations. The PCR-SSP technique, updated to detect the more recently identified KIR genes and alleles, is still used widely to analyze the diversity of human populations, and to study the influence of KIR-gene variability on human health. Several published PCR-SSP methods for KIR genotyping, although simple and robust, have the drawback of relying on the amplification of DNA fragments spanning 0.5-2.0 kbp, which tends to fail in low-quality DNAs. Valuable collections of DNAs often include such poor quality samples, which lead to loss of data and resources. Even worse, undetected falsely negative or positive reactions may result in erroneous gene frequencies and in odd gene combinations. To address those problems, we have redesigned our previously published KIR genotyping method so that it produces short amplicons (less than 200 bp for most genes). This modification minimizes amplification failures, thus conferring greater consistency and reliability to KIR genotyping. In addition, the new PCR-SSP method detects recently described alleles of several KIR genes, and allows for discrimination between the major structural variants of KIR2DS4 and KIR3DP1 without increasing the number of reactions.

Influence of KIR gene diversity on the course of HSV-1 infection: resistance to the disease is associated with the absence of KIR2DL2 and KIR2DS2.

Herpes simplex virus type 1 (HSV-1) causes lifelong latent infections in most humans. Periodical virus reactivations from latency in the neurons of sensitive ganglia lead to transport to mucocutaneous regions and productive replication, which results in recurrent inflammatory herpetic lesions or in asymptomatic virus shedding. The medical consequences of such lesions and the frequency of recurrences vary greatly in different subjects. Furthermore, many infected individuals never suffer manifestations of the disease, even when exposed to stimuli that trigger clinical recurrences in other humans. The origin of the variability in the clinical course of HSV-1 infection remains unexplained. Herpesviruses and other pathogens sabotage the expression of major histocompatibility complex class I molecules by infected cells, thus subverting T-cell-mediated immunity. Subversion of antigen presentation is counteracted by natural killer cells, which survey the human leukocyte antigen (HLA) expression by specific receptors. These include the killer cell immunoglobulin-like receptors (KIRs), which are encoded by a complex of extremely diverse and rapidly evolving genes. Here, we analyze the contribution of KIR gene diversity to the variable clinical course of HSV-1 infection by comparing the distribution of these genes in humans with clinical manifestations of the disease with that in asymptomatically infected donors. This study provides preliminary evidence that the receptors KIR2DL2 and KIR2DS2 predispose to symptomatic HSV-1 infection and favor the frequently recurring forms of the disease. Possible contribution of the 'HLA-C1' ligand to HSV-1 disease was not statistically supported. Because of an absolute genetic linkage between KIR2DL2 and KIR2DS2, we could not determine which receptor was primarily responsible for the observed association, but our results suggest that presence in the genome of KIR2DL2 and KIR2DS2 hinders an effective cellular response to HSV-1.

Complete coding sequences and haplotypic associations of HLA-B*0707, -B*1524, -B*4405, -B*4802, -DRB1*0409, -DRB1*0411, -DRB1*1115, -DRB1*1305, and the novel allele -DRB1*0709. Group-specific amplification of cDNA from DRB1 alleles associated to DRB3 and DRB4.

We present here the characterization of the complete coding sequences, previously unavailable, of the human leukocyte antigen (HLA) alleles B*0707, B*1524, B*4405, B*4802, DRB1*0409, DRB1*0411, DRB1*1115, DRB1*1305, and that of a new allele, DRB1*0709. For the isolation of cDNA from the DRB1 gene, we designed a novel set of polymerase chain reaction (PCR) primers that makes it possible to amplify separately the groups of DRB1 alleles associated to each of the DRB3 and DRB4 loci. The primary structures, functional features, evolutionary relationships, haplotypic associations, and population distributions of each of the nine HLA-B and -DRB1 alleles reported here are reviewed.

Complementary DNA sequence of the HLA-B*3924 allele.

We have isolated the complete coding region of HLA-B*39 from a Spanish Caucasoid, using a new PCR primer for its 5' untranslated region. The cDNA matched partial genomic sequences of B*3924, an allele whose distribution appears to be restricted to Mediterranean and Arabian Caucasoids. A single amino acid change exclusive to B*3924 (threonine-98) distinguishes it from B*3903.

Genotyping of human killer-cell immunoglobulin-like receptor genes by polymerase chain reaction with sequence-specific primers: an update.

Killer-cell Immunoglobulin-like Receptors (KIR) help human natural killer (NK) cells counteract infections by pathogens that evade the immune system by inducing down-regulation of HLA class I molecules in infected cells. KIRs are structural and functionally diverse receptors encoded by a family of polymorphic genes. The most extreme aspect of KIR polymorphism is the varying content of KIR-genes in the genome of different individuals, as first demonstrated by KIR genotyping using the PCR-SSP method. Knowledge on the KIR-gene family has been recently expanded by the identification of new genes, pseudogenes and multiple gene variants, several of which escaped detection by the original genotyping technique. We present here an upgraded PCR-SSP method for KIR genotyping that integrates recent achievements in the research of the diversity of this gene family. Our method permits detection of all known KIR genes and pseudogenes in a 16-reaction set. Furthermore, an additional set of six reactions permits subtyping of KIR2DL5 variants, each of which shows well-differentiated functional and genetic features.

Complementary DNA sequence of the novel HLA-B*3704 allele.

The novel HLA-B*3704 allele was identified by polymerase chain reaction with sequence specific oligoneucletides (PCR-SSO) in a Spanish Caucasoid individual whose T-lymphocytes showed an ambiguous HLA-B phenotype. The nucleotide sequence of B*3704 was determined after reverse transcription-polymerase chain reaction (RT-PCR) amplification and molecular cloning of its complete coding region. B*3704 differs from B*3701 by a single nucleotide replacement that induces the substitution of histidine for tyrosine 171. Residue 171 is located in the alpha-helix of the alpha-2 domain, lining the A pocket of the peptide-binding site. Therefore, the His171 substitution seen in HLA-B*3704 is likely to affect its antigen-presenting properties and is probably responsible for the differentiated serological phenotype of this allele in comparison with B*3701.