Identification of a new HLA-B*57 allele, HLA-B*57:71, by next-generation sequencing.

The sequence of a novel allele, HLA-B*57:71, differs from HLA-B*57:01:01 by three-nucleotide exchanges in exon 2.

Expression of Ksp-cadherin during kidney development and in renal cell carcinoma.

Cadherins are a large family of cell-cell adhesion molecules acting in a homotypic, homophilic manner that play an important role in the maintenance of tissue integrity. In the human kidney, several members of the cadherin family (including E- and N-cadherin, cadherin-6, -8 and -11) are expressed in a controlled spatiotemporal pattern. Cadherin-16, also called kidney-specific (Ksp-) cadherin, is exclusively expressed in epithelial cells of the adult kidney. In renal cell carcinomas (RCCs), which are considered to originate from epithelial kidney tubular cells, a complex pattern of cadherin expression can be observed, but Ksp-cadherin expression has not been analysed so far. In the present study, we show that the expression of Ksp-cadherin is completely abrogated in RCCs. Whereas Ksp-cadherin can be detected at later stages of tubulogenesis during human renal development and in the distal tubules of adult kidneys, no expression was found by immunohistochemistry or Western blot analysis in RCC tumour tissues and several RCC cell lines. However, despite the lack of protein expression, mRNA synthesis of Ksp-cadherin could be detected by reverse transcriptase-polymerase chain reaction analysis in all RCC tissues and most of the RCC cell lines studied, although at a reduced level. The loss of Ksp-cadherin protein was only observed in the malignant part of the tumour kidneys, whereas in the normal part of the affected kidneys Ksp-cadherin expression was clearly detected. These results indicate a downregulation of Ksp-cadherin in RCC and suggest a role for this cell adhesion molecule in tumour suppression.

Comparative analysis of the association of HLA-B*51 suballeles with Behçet's disease in patients of German and Turkish origin.

The distribution of the different HLA-B*51 suballeles among patients with Behçet's disease (BD) of German (n=33) and Turkish (n=92) origin in comparison to their presence in the respective ethnically matched healthy control groups (German: n=325, Turkish: n=93) was studied. HLA-B*51x was significantly increased in both patient groups in comparison to the controls (Germans: 58% vs. 12%, OR 9.76, P<0.001; Turkish: 75% vs. 25%, OR 9.13, P<0.001). Molecular subtyping of B*51x revealed HLA-B*51011 and B*5108 as the predominant suballeles in both patient groups and controls although with a slightly increased frequency of HLA-B*5108 in the diseased individuals. HLA-B*5105 was the only further HLA-B*51x subtype detected in one Turkish patient heterozygous also for HLA-B*5101. HLA-B*5107 although present in a Turkish as well as German control was absent in the patient groups. There was also a tendency towards a higher degree of homozygosity for HLA-B*51x in both patient groups versus the matched controls (Germans: 10% in patients vs. 2,5% in controls; Turkish: 27% in patients vs. 13% in controls). Our study further supports previous hypothesis of an association of BD with B51 suballeles which share amino-acid residues at positions 63 and 67 as well as at positions 77-83 for specific peptide binding and natural killer (NK)-cell interactions. This applies to HLA-B*5101 and B*5108, but not to HLA-B*5107 different at position 67, which could be negatively associated with BD.

New high resolution typing strategy for HLA-A locus alleles based on dye terminator sequencing of haplotypic group-specific PCR-amplicons of exon 2 and exon 3.

In this study, a new sequencing-based typing strategy for the HLA-A locus is presented which involves group-specific separate amplification of exon 2 and 3 of HLA-A alleles in a first step. Conserved HLA-A locus-specific primers of intron 1 or 3 were combined in 10 primer-mixes with group-specific primers hybridizing to the 5'- or 3'-end of exon 3 or 2 for pre-typing of the HLA-A alleles in 14 allelic groups. Maximally four overlapping short amplicons are produced under identical polymerase chain reaction (PCR) conditions with individual separate amplification of exon 2 and exon 3 of the haplotypic alleles in most heterozygous combinations. Time- and money-saving one-directional Big Dye Terminator cycle sequencing is shown to provide reliable high resolution typing of the HLA-A alleles, even in a few cases of two amplicons in one primer reaction mixture. In comparison, to other sequencing-based typing (SBT) techniques the applied typing strategy minimizes the risk of unequal amplification or of drop-outs of one of the haplotypic alleles and allows unequivocal definition of the cis/ trans linkage of polymorphic positions of the complete exon 2 and exon 3 in most heterozygous cells. This also includes detection of new alleles differing in the polymorphic template generating primer annealing sites as well as in unusual combinations of known exon 2 and 3 sequences. With 10 primer sets working under identical conditions for pre-grouping and separate amplification of the haplotypic alleles our SBT procedure also could be implemented in clinical settings of large-scale stem cell donor histocompatibility testing for fast molecular HLA-A matching.

Differential regulation of human leukocyte antigen class I genes by interferon in vivo and in vitro.

Modulation of human leukocyte antigen (HLA) class I antigens on peripheral blood lymphocytes, monocytes, and hematopoietic precursors during interferon-alpha (IFN-alpha) therapy was investigated in 18 patients with myeloproliferative syndrome. After 1 month of IFN-alpha therapy, an increased number of monocytes and hematopoietic precursor cells but not of lymphocytes expressed HLA-DQ antigens. In addition, a strong induction of HLA class I antigens was found on both hematopoietic progenitors and normal peripheral blood mononuclear cells. By daily injections of IFN in the first month of therapy, stimulation continuously increased, suggesting a major effect of IFN alpha on hematopoietic progenitors with sustained enhanced expression of HLA class I antigens during differentiation of myelomonocytic cells. HLA class I antigen expression was consistently augmented by IFN alpha in all patients irrespective of their hematologic response. Differential in vivo regulation of HLA class I antigens by IFN was confirmed by comparison of HLA-A2 with HLA-B antigen expression. In vitro expression of the HLA-B7 and -Bw64 genes had been shown earlier to be significantly more inducible by IFN than the genes coding for several other HLA class I antigens after transfection into mouse L cells. Modification of the 5' ends of the HLA-B7 and HLA-B27 genes before transfection in mouse L cells showed the presence of enhancer sequences responding to IFN treatment in the 5' untranslated region of the HLA-B7 but not of the HLA-B27 gene and suggested the presence of independently acting regulatory mechanisms independent of these enhancers.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of HLA class I genes by interferon.

Allele-specific differences in the regulation of HLA class I genes by type I interferon (IFN) were observed after transfection of eight HLA-B, -A, or -C genes into mouse L cells. HLA-B7 and -Bw64 gene expression was significantly more inducible by type I IFN than the genes coding for HLA-B27, HLA-B51, HLA-B38, HLA-B39, HLA-Cw3, and HLA-A2 antigens. Modification of the 5' end of HLA-B7 and HLA-B27 genes revealed the presence of enhancer sequences responding to IFN treatment in the 5' untranslated region of HLA-B7, but not of HLA-B27 and suggested further, independently acting enhancer elements downstream of the transcription initiation site. Comparison of 5' enhancer region sequences in correlation with type I IFN inducibility of the different HLA class I alleles indicated that the exchange of only two nucleotides in the interferon response sequence (IRS) or enhancer A region of HLA-B7 or -Bw64 could account for nonregulated promoters in all other HLA-A, -B, or -C alleles analyzed. Thus, type I IFN stimulation of HLA class I genes in mouse L cells appears to predominantly operate in most alleles by a mechanism targeted to enhancer sequences downstream of the gene's transcription initiation site.

Genetic and serological heterogeneity of the supertypic HLA-B locus specificities Bw4 and Bw6.

Gene cloning and sequencing of the HLA-B locus split antigens B38 (B16.1) and B39 (B16.2) allowed localization of their subtypic as well as their public specificities HLA-Bw4 or -Bw6 to the alpha-helical region of the alpha 1 domain flanked by the amino acid positions 74-83. Comparison of their amino acid sequences with those of other HLA-B-locus alleles established HLA-Bw6 to be distinguished by Ser at residue 77 and Asn at residue 80. In contrast, HLA-Bw4 is characterized by at least seven different patterns of amino acid exchanges at positions 77 and 80-83. Reactivity patterns of Bw4- or Bw6-specific monoclonal antibodies reveal two alloantigenic epitopes contributing to the HLA-Bw4 or -Bw6 specificity residing next to the region of highest diversity of the alpha 1 domain.

Characterization of a monoclonal anti-Bw4 antibody (Tü109): evidence for similar epitopes on the Bw4 and Bw6 antigens.

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6+ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6+ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.