Real-time PCR using fluorescent resonance emission transfer probes for HLA-B typing.

HLA genotyping by polymerase chain reaction (PCR) has some inherent labor-intensive and effort-demanding limitations. To overcome them, we have developed a real-time PCR with hybridization probes approach able to obtain a medium-low resolution HLA-B genotyping with fewer tubes and probes and with a shorter time requirement. Our strategy used 18 simultaneous reactions amplifying HLA-B alleles and an internal control. Monitorization of both amplifications in each tube is performed by the simultaneous application of two fluorescent resonance emission transfer probes: the first probe, different for each tube, is specific for the HLA-B locus and the second probe detects the control gene. A medium-low resolution (300 HLA-B allelic groups) typing is obtained for each sample by analyzing the melting curve patterns. Because some alleles may be determined without using the complete set of reactions, we present an alternative strategy: a first round of seven reactions and, according to the result, a second (or third) round of PCRs to solve the ambiguities. This method was validated in pretyped clinical samples and the results were completely concordant. Moreover, fewer ambiguous results were obtained. In summary, we present a new, faster, and more accurate method than currently used PCR techniques to type HLA-B alleles.

Insulin alleles and autoimmune regulator (AIRE) gene expression both influence insulin expression in the thymus.

It is well established that the polymorphisms at the 5' of the insulin gene (IDDM2) confers susceptibility to type 1 diabetes, probably by modifying the level of insulin expression in the thymus that in turn influences immunological tolerance to insulin as self-antigen. AIRE is a transcription regulator which controls the expression of many peripheral antigens within the thymus, among them insulin. Results presented here confirm that insulin gene copies from both parental chromosomes are expressed in human thymus and that IDDM2 class III protective alleles are indeed associated with a higher level of insulin message expression. However, differences in insulin mRNA expression among different thymi were far wider than those determined by the class I and class III insulin gene alleles and maintained a clear correlation with AIRE expression. These results confirm the effect of IDDM2 alleles on insulin expression in the thymus, but suggest that the levels of AIRE may exert a stronger influence than IDDM2 alleles themselves.

Identification of a KRAB-containing zinc finger protein, ZNF304, by AU-motif-directed display method and initial characterization in lymphocyte activation.

A novel human Krüppel-associated box (KRAB) type zinc finger protein encoding gene, ZNF304, was obtained by AU-motif-directed display and RACE. This gene, which contains a tandem AU motif in the 3' untranslated region, has an ORF 1977-bp long that codes for a putative 659 residue protein with an amino-terminal KRAB domain and 13 carboxyl-terminal C2H2 zinc finger units. The gene maps to chromosome 19q13.4, a region that contains the largest zinc finger cluster so far identified in the human genome. Structurally, ZNF304 is related to a family of repressor transcription factors. ZNF304 expression was higher in lymphoid tissues but it was also detected in the following tissues, ordered by abundance: thyroid, adrenal gland, prostate, pancreas, and skeletal muscle. Jurkat, U937, and THP1 cell lines showed a relatively low expression of ZNF304. By contrast, PBLs stimulated with PHA or PMA + ionomycin showed a biphasic expression with a sharp increase at 6 h. This induction was closely parallel to IFN-gamma expression and partially to IL-4 and IL-10. The tissue distribution and kinetics of induction suggest that ZNF304 may be involved in the regulation of lymphocyte activation.