title: Human leukocyte antigen-B (-Bw6/-Bw4 I80, T80) and human leukocyte antigen-C (-C1/-C2) subgrouping using pyrosequence analysis.
abstract: Specific combinations of killer immunoglobulin-like receptors (KIR) and human leukocyte antigen (HLA) class I molecules characterized by a particular residue 80 are significantly associated with outcomes in different pathologic conditions, such as autoimmunity, pathogenic infection, cancer, and reproductive failure. Thus, a simplified method for HLA typing used in association with the analysis of KIR genotype (Kirotype) is of particular interest to extend the analysis of larger series. Here, we describe a quick and inexpensive method that allows use of pyrosequencing, a helpful subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I(80) or -Bw4 T(80), HLA-C1, or -C2 groups and HLA-A allotypes sharing Bw4+ epitope or the rare HLA-B allotypes displaying the C1 motif. In particular, this analysis is focused on the amino acids around residue 80, known to be relevant in defining the affinity of KIR/HLA interaction and in the functional effects. This method was demonstrated to have good sensitivity, specificity, and reproducibility of detection and it was validated using a panel of HLA-typed International Histocompatibility Workshop (IHW) cell lines and clinical isolates. Using an allele quantitative acquisition mode, the method permitted us to obtain an accurate sequencing as required in heterozygous and/or homozygous sample definition.
authors.affiliation: Molecular Medicine, Istituto Giannina Gaslini, Genoa, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Ugolotti
authors.forename: Irene
authors.initials: I
authors.lastname: Vanni
authors.forename: Alessandro
authors.initials: A
authors.lastname: Raso
authors.forename: Fabio
authors.initials: F
authors.lastname: Benzi
authors.forename: Mauro
authors.initials: M
authors.lastname: Malnati
authors.forename: Roberto
authors.initials: R
authors.lastname: Biassoni
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D015551:Autoimmunity / Q000235:genetics*; D002460:Cell Line; D000939:Epitopes / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D005838:Genotype; D015235:HLA-B Antigens / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D015236:HLA-C Antigens / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D006579:Heterozygote; D006650:Histocompatibility Testing / Q000379:methods*; D006720:Homozygote; D006801:Humans; D007962:Leukocytes / Q000737:chemistry* / Q000166:cytology / Q000276:immunology; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods*; D054341:Receptors, KIR3DL1 / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D000939:Epitopes; D015235:HLA-B Antigens; D015236:HLA-C Antigens; D054341:Receptors, KIR3DL1