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Summary Two partial cDNAs for von Willebrand factor (vWF) were used to investigate gene lesions and restriction fragment length polymorphisms (RFLPs) in vW disease (vWd) and normal controls. No gene alteration was detected but two TaqI RFLPs, likely to be intronic and originating from point mutations, were found in the 3’ part of vWF gene. The two TaqI RFLPs, identified by the same probe, are informative in approximately 50% of the subjects. Used in combination with two other known RFLPs, they define several haplotypes similarly distributed in vWd and normals. Linkage disequilibrium between loci identified by the RFLPs is present. In a family study the RFLP patterns demonstrate homozygosity for the affected vWF gene in a severe (type III) patient and identify several heterozygous subjects. The RFLPs analysis has been related to the haemostatic values and multimer distribution. In two of the four unrelated patients with severe vWd examined the RFLPs study indicates double heterozygosity for the affected vWF genes.

DNA from 20 Italian haemophilia B patients was analysed by the Southern blotting technique and hybridisation to a factor IX cDNA probe. A large deletion of factor IX gene was detected in one patient with antibodies to the infused factor; the EcoRI pattern of the other 19 subjects examined was normal.

The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42 6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42 6 monoclonal antibody. In addition, the 42 6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of γ-globin mRNA. The effect of the 42 6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42 6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression.

The form of alpha-thalassaemia of the Po river delta presents haematological and globin biosynthetic characteristics similar to alpha-thalassaemia-1 but never gives rise to HB H disease nor to hydrops foetalis. In alpha-thalassaemic subjects originally from this region globin mRNA translation and alpha-globin gene arrangement have been investigated. The data obtained indicate that alpha-globin synthesis and reticulocyte alpha-globin mRNA are reduced by one fourth; in addition, since the defect in alpha-globin synthesis does not change with cell ageing, a possible instability of alpha-globin mRNA is excluded. Restriction enzyme analysis of the DNA shows a normal alpha-globin gene organization. This form of alpha-thalassaemia is therefore of the non-deletion type; its molecular lesion is either at the level of alpha-globin mRNA transcription or processing. The fact that this, as well as other forms of non-deletion alpha-thalassaemia, have a phenotypic expression similar to alpha-thalassaemia 1 is discussed.

The methylation pattern of the human HLA-DR alpha gene was analyzed in normal breast tissues, breast primary tumors and lymphonodal metastases isolated from patients carrying breast carcinomas. In breast adenomas and also in normal tissues (including breast, muscle, brain, sperm and T- and B-lymphocytes), the HLA-DR alpha gene is hypermethylated at the CCGG and GCGC sites. In all tissues studied, the only constantly unmethylated region is located in the 5' portion of the gene, near the promoter sequence. Further, the results indicate that the HLA-DR alpha gene is hypomethylated in carcinomas and in the relative metastatic lymph nodes. It is suggested that hypomethylation of the human HLA-DR alpha gene could be proposed as a molecular marker of malignant breast tumors.

Six beta(+)-thalassaemic patients from the Po river delta region have been studied. Using synthetic oligonucleotides as specific hybridisation probes, the beta(+) IVS I mutation (G----A at position 108) was demonstrated. This lesion and the enzyme polymorphism pattern in the subjects examined are the same as have been described for other Mediterranean beta(+)-thalassaemias. Antenatal diagnosis through DNA analysis of beta(+)-thalassaemia is therefore possible. The production of beta globin in a beta(+), homozygote and in a beta (+), beta(0) 39 (nonsense mutation at codon 39) double heterozygote is approximately 20% and 10% respectively of total non-alpha globin synthesis. Despite some overlapping of the results, similar beta globin synthesis levels have been obtained in 43 beta(+)-thalassaemia patients. This suggests that in the Po river delta region the most common thalassaemic genes are beta(0) 39 and beta(+) IVS I.

DNA methylation at the 5'-CCGG-3' sites of the HLA-DR alpha gene and relative flanking regions has been analyzed by Msp I and Hpa II enzymatic digestion in order to determine whether a correlation exists between DNA methylation and transcription of the HLA-DR alpha gene. Unexpectedly, and in contrast to the behavior of most eukaryotic genes, no positive correlation was found between hypomethylation and expression. In fact, HLA-DR alpha appears to be fully unmethylated at Msp I/Hpa II sites in K562 cells, not expressing DR molecules, and to exhibit a high degree of methylation in Colo 38 cells, which actively transcribe the gene. The unorthodox behavior of HLA-DR alpha is not unique to melanoma or erythroid cells since a similar, positive correlation between methylation and expression also exists when this analysis is extended to cell lines belonging to other histotypes.

The methylation state of the CCGG and GCGC sites of the Ha-ras-1 oncogene was analysed in the human leukemic K562 cell line, which was found to actively transcribe this gene. The results obtained demonstrate that the Ha-ras-1 oncogene is extensively methylated in both exonic, intronic, VTR and 3′ untranslated portions, while undermethylations are present in a CG-rich island localized upstream of exon 1, near putative transcription initiation signals. Treatment of K562 cells with 5-azacytidine induces undermethylation of the Ha-ras-1 oncogene without major differences in the accumulation of Ha-ras-1 mRNA trascripts.

The methylation pattern of the human HLA-DR alpha gene was analyzed in primary tumors and lymph node metastases isolated from patients with a variety of tumors, including thyroid, pancreas, breast and gastric carcinomas and melanomas. In normal tissues (including breast, muscle, brain, sperm, T-and B-lymphocytes) the HLA-DR alpha gene is hypermethylated at CCGG and GCGC sites. In all tissues studied, the only constantly unmethylated region was located in the 5' portion of the gene. Our results indicate that the HLA-DR alpha gene is hypomethylated in metastatic lymph nodes, as well as in the carcinomas and melanomas studied. These findings lend support to the hypothesis that DNA hypomethylation of the human HLA-DR alpha gene may represent a molecular marker of malignant tumors.

International audience; Thymus leukemia antigen (TL) is an MHC class Ib molecule that is highly conserved in rats and mice with no obvious human homolog. TL is expressed in mouse small intestinal epithelial cells and is known to interact with CD8alphaalpha homodimers, which are expressed by intraepithelial lymphocytes (IELs), some other T cell subsets and some non-T cells such as a subset of dendritic cells. We show here that TL is abundantly expressed on the basolateral surface of mouse small intestinal epithelial cells and that expression is abrogated in beta2m-/- mice but unaffected in TCR-/- mice or CD8alpha chain-/- mice. We demonstrate that the interaction between TL and CD8alphaalpha is not necessary for IEL survival in vitro or in vivo and does not modulate IEL trafficking in vivo. TL co-stimulation of alpha-CD3 antibody-activated IELs resulted in modestly enhanced production of IFN-gamma in one subset of IELs. The lack of effect on IEL survival and trafficking and the modest effect on IFN-gamma production suggest that the functional consequences of TL interaction with CD8alphaalpha as well as the more general biological role of TL in mucosal immunity remains to be discovered.