Characterizing alleles with large deletions using region specific extraction.

Two novel HLA class II alleles, DRB4*03:01N and DQB1*03:276N, containing large deletions were identified during routine typing. Extraction of DNA encompassing the deletions was carried out with a panel of capture oligonucleotides followed by whole genome amplification. Next generation DNA sequencing was then used to characterize the sequences. DRB4*03:01N has a 16 kilobase pair deletion stretching upstream from intron 2 toward centromeric DRB8. DQB1*03:276N has two deletions separated by 844 nucleotides. The first deletion (3.7 kilobase pairs) is upstream of intron 1 and the second deletion removes 3.3 kilobase pairs further upstream towards centromeric DQA2.

Allelic variation of killer cell immunoglobulin-like receptor 2DS5 impacts glycosylation altering cell surface expression levels.

Natural killer cell stimulatory receptor gene, KIR2DS5, is polymorphic. While KIR2DS5*002 is most frequently observed, other alleles have also been found. The proteins encoded by these alleles (KIR2DS5*002-*009) are expressed at varying levels on the surface of NKL and Jurkat transfectants. Gel electrophoresis of all allelic products showed two isoforms which differ in the extent of maturation of N-linked glycosylation. These isoforms differed in intensity and molecular weight among the allelic products. Site-directed mutagenesis was used to identify polymorphic variation at residues 123 and 157 as key in altering glycosylation and levels of surface expression.

Extracellular domain alterations impact surface expression of stimulatory natural killer cell receptor KIR2DS5.

In the human killer cell immunoglobulin-like receptors, KIR2DL2, and KIR2DL3, a triad of amino acids in the D1 domain interact to stabilize protein structure. Substitution of any one of these residues caused significant loss of cell surface expression. Although KIR2DS4 and KIR2DS5, two homologous receptors, differ for this triad, flow cytometry analysis of NK and T cell lines transfected with stimulatory KIR genes KIR2DS4 (allele *001) and KIR2DS5 (allele *002) demonstrated cell surface expression. For KIR2DS5, restoration of the triad sequence increased surface expression. Further studies of the receptor encoded by KIR2DS5*002 showed both mature and immature protein isoforms upon gel electrophoresis coupled with surface biotinylation or deglycosylation. In contrast, the KIR2DS5*001 allelic product was not expressed on the cell surface of either NK or T cells and exhibited only a single immature isoform upon gel electrophoresis. Site-directed mutagenesis demonstrated that absence of the KIR2DS5*001-encoded protein at the cell surface was imparted primarily by two amino acid polymorphisms in the D2 domain. Analysis using molecular dynamics simulations suggested that the substitution of a proline for a serine at residue 111 or the substitution of a serine for a phenylalanine at residue 164 caused destabilization of the domain structure and intracellular retention. A third polymorphism at residue 174 impacted the level of KIR2DS5 surface expression. This is the first description at a stimulatory KIR locus of the impact of specific amino acid variations on receptor maturation and the level of surface expression.

Promoter variants of KIR2DL5 add to diversity and may impact gene expression.

Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA encompassing the putative proximal promoter and the coding region was used to identify KIR2DL5 alleles from 77 unrelated Caucasian individuals. PCR and sequencing were used to link each new allele to its neighboring KIR locus to identify 2DL5A or 2DL5B loci. Allele 2DL5A*001 was found in 24 of the 37 2DL5 positive individuals; 2DL5B*0020101 and 2DL5A*0050101 were also observed. Two new alleles, 2DL5B*008 and 2DL5B*009, contained substitutions altering the amino acid sequence of the leader and transmembrane region, respectively. Two other novel alleles, 2DL5B*0020102 and 2DL5A*0050102, contained alterations of the 5' upstream region, bringing the number of unique promoter sequences to six. Promoter activity of the alleles was compared using luciferase reporter assays. Our results support those recently published, in which the promoter of 2DL5B*0020101 was shown to be more active in vitro compared to 2DL5A*001, and also provide additional information about the transcriptional activity of the promoters of the newly characterized alleles related to two altered transcription factor binding sites.

Limited allelic diversity of stimulatory two-domain killer cell immunoglobulin-like receptors.

Genomic sequencing was used to characterize most of the coding regions of the five two-domain stimulatory killer cell immunoglobulin-like receptor (KIR) loci from 80 unrelated, primarily Caucasian, individuals. Specific loci were present in from 26% (KIR2DS3) to 98% (KIR2DS4) of individuals. The number of known alleles present varied from one (KIR2DS1, KIR2DS5) to five (KIR2DS4). The frequencies of loci and alleles were similar to observations made in populations of European and Asian ethnicities. New alleles were found at 2DS1 (*00202, *00302, *005, *006, *007) and 2DS4 (*008) loci.

Three common alleles of KIR2DL4 (CD158d) encode constitutively expressed, inducible and secreted receptors in NK cells.

Genetic polymorphism of KIR2DL4 results in alleles with either 9 or 10 consecutive adenines in exon 6, which encodes the transmembrane domain. "10A" alleles encode a membrane-expressed receptor that is constitutively expressed on resting CD56bright NK cells and on CD56dim cells after culture. However, in some individuals with the 10A allele, KIR2DL4 cannot be detected on their resting CD56bright NK cells. "9A" alleles have been predicted to encode a secreted receptor due to the splicing out of the transmembrane region. In this publication, we show that those individuals with a 10A allele who lack detectable KIR2DL4 on CD56bright NK cells express a KIR2DL4 receptor in which the D0-domain is excised. This Delta-D0 receptor cannot be detected by the available anti-KIR2DL4 monoclonal antibodies. In such individuals, KIR2DL4 becomes detectable on cultured NK cells due to up-regulation of the full-length KIR2DL4 transcript. In all individuals with 10A alleles, KIR2DL4 ceases to be expressed at the cell surface 16 days after activation, despite the maintenance of maximal levels of KIR2DL4 mRNA transcription, suggesting the existence of a negative regulator of cell surface expression. Finally, we show that the 9A allele can produce a secreted KIR2DL4 receptor.