Blood testing in biathlon: observation of hematocrit values during competitive periods 1994-1997.

The International Biathlon Union, through its own Medical Commission, has undertaken a three-year program aimed at identifying the hematocrit levels of athletes who participate in biathlons. The purpose was to check for hematocrit levels exceeding normal physiological values as well as for any significant modifications within individuals during the course of the competitive season. All of the athletes registered in international biathlon races were, therefore, subjected to venous blood sampling (1 cc) three times a year prior to competition, in the days preceding two World Cup races (December and January) and the World Championships (February). The blood was centrifuged, the micro-hematocrit determined, and the value (rendered anonymous) recorded. This procedure was repeated for three consecutive competitive seasons (Dec. 1994-Feb. 1995, Dec. 1995-Feb. 1996, Dec. 1996-Feb. 1997). During this three-year period, the collected data showed a significant lowering of the average hematocrit level. In fact, from the first February sampling to the third February sampling, the average hematocrit value for the male population dropped from 48.04 +/- 2.36 to 46.33 +/- 1.91, and for the female population from 44.05 +/- 2.44 to 42.52 +/- 1.92. Even the distribution of the absolute hematocrit values was modified from the first to the third competitive season, especially for the February sampling, with a > or = 50% reduction for males and a > or = 48% reduction for females.

Methylation state of cellular genes and oncogenes as a marker of malignancy in human carcinomas.

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.

Hypomethylation of the human HLA-DR alpha gene in breast carcinomas and autologous metastases.

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.

von Willebrand disease investigated by two novel RFLPs.

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.

Use of 3'HVR genomic probe for presymptomatic diagnosis of adult polycystic kidney disease in northern Italy: comparison of DNA analysis and renal ultrasonographic data.

A highly polymorphic DNA probe (3'HVR) with genetic linkage to the locus of autosomal dominant polycystic kidney disease was used for screening. Families with subjects at risk were from the Po river delta region (Northern Italy), where the disease accounts for 24% of the demands for dialysis. 3'HVR alleles were investigated in white blood cell DNA from 142 members of 18 families. The genomic marker was found informative in 88% of cases. Two recombinations between the marker and the disease locus were observed in 79 meioses. In 42 of the subjects at risk the results of DNA analysis and renal ultrasonography were compared. In 36 subjects the tests confirmed each other (18 were positive). In the other six subjects (all under 20 years of age and four under 10) only DNA analysis could diagnose the inheritance of cystic disease in the absence of demonstrable cysts. The findings indicate that in the population of the Po river delta the presymptomatic detection of adult polycystic kidney disease by 3'HVR linkage analysis is feasible in 88% of cases with approximately 95% reliability.

Clustering of undermethylated CCGG and GCGC sequences in the 5' region of the Ha-ras-1 oncogene of human leukemic K562 cells.

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 transcripts.

c-myc oncogene alterations in human thyroid carcinomas.

A possible role of the c-myc oncogene in the neoplastic transformation of the human thyroid has been investigated. The structure, the methylation status, and the copy number of this oncogene have been analyzed in normal and in tumor thyroid DNAs by Southern blotting technique and dot blot hybridization. Among six carcinomas, four presented abnormal c-myc DNA structure: Three cases showed a mutation in the 5' flanking region of the gene, originating a new EcoRI site; the other case showed a deletion of 5 kb involving the first exon of the gene. This deletion was also observed in the white blood cells of the same individual. In addition, in three carcinomas a double dose of the oncogene has been demonstrated. In all the carcinomas examined, undermethylation of the c-myc oncogene has been observed. These findings suggest that c-myc oncogene alterations might be involved in the malignant transformation of the human thyroids and can be considered as tumor markers.

Human leukemic K562 cells: suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor.

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 gamma-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.

Lack of correlation between hypomethylation and expression of the HLA-DR alpha gene.

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.

Identification of a c-myc oncogene lacking the exon 1 in the normal cells of a patient carrying a thyroid carcinoma.

In this paper we describe an alteration of the c-myc oncogene present in the white blood cells and normal as well as neoplastic thyroid cells of a subject carrying a thyroid carcinoma. Restriction enzyme mapping and hybridization to human c-myc probes specific for different regions of this gene demonstrate that this subject carries, in addition to the normal one, a c-myc oncogene lacking the first exon and part of the first intron. The levels of the c-myc mRNA in thyroid cells of this subject do not show differences with respect to thyroid cells from other subjects. Taken together, these findings indicate that the deletion of the first exon of the c-myc oncogene, in itself, does not produce overtranscription of this oncogene nor hematopoietic malignancies.

Methylation and expression of c-myc and c-abl oncogenes in human leukemic K562 cells before and after treatment with 5-azacytidine.

The correlation between expression and extent of DNA methylation of c-myc and c-abl oncogenes has been investigated in the human leukemic K-562 cell line before and after 5-azacytidine-mediated erythroid induction. RNA accumulation was analyzed by cytoplasmic dot hybridization and DNA methylation by using HpaII and MspI endonucleases, which differently cleave the CCGG sequence depending on cytosine methylation. Both the oncogenes are expressed in uninduced cells; however, whereas the c-myc expression does not change following 5-azacytidine treatment, the c-abl expression sharply decreases. The HpaII pattern shows that the c-myc DNA region is undermethylated and that the c-abl gene is hypermethylated both before and after the erythroid induction. Nevertheless, in both the genes 5-azacytidine produces variations in the MspI pattern compatible with mCmCGG to CmCGG demethylations.

Gene deletion in an Italian haemophilia B subject.

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.

beta (+)-Thalassaemia in the Po river delta region (northern Italy): genotype and beta globin synthesis.

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.

[Inhibition of heme biosynthesis in erythroid precursors (BFU-e) isolated from human peripheral blood: effects on globin synthesis]. / Inibizione della biosintesi dell'eme in precursori eritroidi (BFU-e) isolati da sangue periferico umano: effetti sulla sin tesi globinica.

We studied the relationship between heme accumulation and globin synthesis in human erythroid precursors which were stimulated by 2 I.U. of erythropoietin in semi-solid cultures (1% methyl-cellulose, 20% fetal calf serum) and treated with 6-9 micrograms/ml of desferrioxamina (DF), a potent inhibitor of heme synthesis (6). Heme accumulation was detected by specific reaction with benzidine (4), globin synthesis by CM-cellulose column chromatography. Our results demonstrate that globin gene expression occurs in DF-treated erythroid cells which do not accumulate heme molecules. As heme does affect translation and stability of globin mRNA (10) our system might be suitable for studies focused on pathological alterations of erythropoiesis associated with the presence of unstable globin mRNAs and/or unstable globins.

Molecular characteristics of a non-deletion alpha-thalassaemia of the Po River Delta.

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.