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Many transmeridian travellers, including top athletes, regularly take melatonin to reduce negative jet lag symptoms. Standard (rather than individually tailored) doses are often used. We examined the effects of a standard dose of melatonin on the body temperature rhythms of 12 elite biathletes (eight men and four women) after an eastward transmeridian flight to an international competition. The different effects on body temperature rhythms in men versus women underscore the need for personalized dosing schedules to avoid potential undesirable consequences.

Summary Three TaqI restriction fragment length polymorphisms (RFLP) detected by the central portion of von Willebrand factor cDNA, which recognizes the true gene and in addition pseudogenic sequences, were characterized and mapped. Small cDNA fragments which hybridized with DNA from families with von Willebrand disease were used. Two of the RFLP, recognized by 1.7 and 0.45 kb cDNA fragments, are not in linkage either with von Willebrand disease or with RFLP located in the von Willebrand factor (vWF) gene, which indicates their pseudogenic location. These markers located in 22q11, near to the bcr gene, provide new tools for the study of several somatic and constitutional alterations affecting this chromosomal region. The third RFLP is recognized by a cDNA fragment corresponding to the N-terminal portion of mature vWF and is localized in the true gene. Since significant linkage disequilibrium with other informative RFLP is not present, this marker contributes to the definition of family haplotypes associated with von Willebrand disease.

Increased frequency of the homozygous II ACE genotype in Italian Olympic endurance athletes

An abnormal von Willebrand factor (vWF) gene restriction pattern has been found in a patient with von Willebrand disease. Because this gene alteration is not present in his parents or in 50 normal and 25 affected subjects, and the restriction fragment length polymorphism haplotypes are inherited normally in the patient's family, we suggest that a de novo mutation is present. Bands with reduced intensity and additional fragments, observed in several restriction digests, hybridize with noncontiguous copy DNA (cDNA) portions, thus indicating the presence of a heterozygous gene deletion. The deletion removes a genomic region containing at least codons 1147 through 1854 and corresponding to the D3-A3 homologous protein domains. The extent of the vWF pseudogene on chromosome 22 is roughly similar to that of the deleted area. However, the pseudogenic nature of the deletion is excluded by the mapping of bands with reduced intensity in the patient to the true vWF gene. The vWF antigen levels are one fourth of normal and ristocetin cofactor activity is severely impaired. The reduction of high molecular weight multimers in plasma and platelets and the altered triplet morphology are compatible with the presence of a dominant variant of type II von Willebrand disease.

The map of restriction sites including and surrounding the δ- and β-globin genes has been established for three Ferrara β°-thalassaemic subjects. The fragments obtained using nine restriction enzymes do not show any differences from normal DNA. Among others, restriction enzymes giving short fragments at the 5′ and 3′ ends of the β-globin structural gene have been employed. The results obtained for the thalassaemic DNA are identical to those for control DNA, thus excluding the presence of extensive deletions in or adjacent to the coding regions of the β-globin gene in Ferrara β°-thalassaemia.

Hematological variables of 40 professional cyclists, all receiving intravenous iron supplementation, were followed during a 15-month period. Mean values for red blood cells (RBC), hemoglobin (Hb), and hematocrit (Ht) were significantly lower during the racing season (RS) than during the nonracing periods (NRP) (RBC: RS = 4.53 +/- 0.34 millions/mm3, NRP = 5.09 +/- 0.36 millions/mm3; line 7 of abstract: Hb: RS = 14.2 +/- 0.9 g/dl, MRS = 15.2 +/- 0.9 g/dl; Ht: RS = 40.7 +/- 2.7% NRP = 44.4 +/- 2.9%; P less than 0.001 for all). However, mean values for ferritin and mean corpuscular hemoglobin (MCH) were significantly higher during the racing season (ferritin: RS = 422 +/- 398 ng/ml, NRP = 311 +/- 321 ng/ml, P less than 0.05; MCH: RS = 31.5 +/- 1.3 pg, NRP = 30.0 +/- 1.4 pg; P less than 0.001). These results suggest that the reductions in RBC, Hb, and Ht found in professional cyclists during the racing season are not the consequence of a diminution of iron stores but rather of reduced erythropoiesis and increased RBC destruction.

In cases of β0- thalassaemia from Ferrara, Italy, the β-globin gene is transcribed into mRNA but no protein is synthesised. For these cases there is no hybridisation data suggesting a globin gene structural mutation. This again demonstrates the diverse molecular events which may cause this prevalent hereditary disease.

THALASSAEMIAS are inherited disorders each characterized by a slow rate of synthesis of a specific polypeptide chain of haemoglobin, the most extensively studied being beta-thalassaemia, in which beta-globin synthesis is deficient. Homozygotes for beta-thalassaemia from most of the Mediterranean region synthesize some beta-globin1,2, homozygotes from the Ferrara region synthesize none3,4. In the common Mediterranean form there is some evidence for a defect in the quantity or quality of β-globin rnRNA5,6. Failure to synthesize beta-globin could be due to defective transcription, defective translation, which could result from the absence of mechanism specific for the translation of beta-globin mRNA7–11, which probably operates at chain initiation12–14, or incomplete translation, involving a mutation in beta-globin mRNA of an amino-acid-specifying codon to an initiation or termination codon. Dreyfus et al.15 found that reticulocytes of beta-thalassaemic subjects from Ferrara incorporated no radioactive amino-acids in the tryptic peptides beta-T1 (NH2-terminal) and beta-T14 (immediately adjacent to the COOH-terminal). These findings exclude the insertion of a terminating triplet after the codon for the eighth amino-acid and of an initiating signal before the codon for the 133rd amino-acid.

The intracellular behaviour of the excess newly synthesized α-globin has been studied by incubating peripheral blood and bone marrow samples from 9 homozygous β-thalassemic subjects with labeled amino acids. The globin chains present in the cytoplasm and in association with the stromal material have been separated by column chromatography, and the incorporation of radioactivity has been determined for each globin at various incubation times. The results obtained show that the excess of α-globin is removed from the cell cytoplasm and becomes associated with the stromal material.

The authors have investigated the possibility that the Ferrara type of β-thalassemia, in which β-globulin synthesis is absent, may be due to a nonsense mutation on the gene for β-globin. This hypothesis has been tested by trying to detect the presence of newly synthesized β-globin fragments in the reticulocytes of homozygous β-thalassemic subjects. The red blood cells were incubated with [3H]leucine. The whole hemolysate was hydrolyzed with trypsin and several β-chain peptides were isolated by column chromatography and assayed for radioactivity. No incorporation of radioactive leucine could be detected in the various β-globin peptides isolated and examined. The hypothesis that a nonsense mutation is responsible for the absence of β-globin synthesis in the Ferrara β-thalassemia is not supported by these findings.

The kinetics of incorporation of 3H-labeled amino acids into α, β, and γ globin has been determined, following incubation of intact reticulocytes, in stroma-free hemolysates and in purified Hb F and Hb A from homozygous β-thalassemic subjects. The labeled globins present in the cytoplasm as well as in purified Hb F and Hb A at various incubation times, were separated by CM-cellulose chromatography, and the ratios of [3H]α to [3H]counterpart globins were determined. The results obtained show that in the initial stages of incubation, the ratios of [3H]α to [3H]-counterpart globins are higher than 1 in the total lysate and in purified hemoglobins; these ratios, after long periods of incubation, decrease to values below 1. These findings suggest that the excess of newly synthesized α molecules flows very rapidly from the polyribosomes to the hemoglobins, and then, more slowly, to a “stromal”α pool. A model is presented, showing the most likely mechanism for the overall process.

In time course experiments performed in subjects carrying haemoglobin Hasharon and beta-thalassaemia the synthesis of alphaA-, alphaHasharon-, beta- and gamma-globin has been determined. The alpha-globin molecules synthesized in excess were found to be removed from the red cell cytoplasm (phenomenon characteristic of beta-thalassaemia, described by Bargellesi et al, 1968a); in addition the alphaHasharon-globin molecules were removed at a faster rate than normal alpha-globin. The preferential removal of the mutant alpha-globin explains the reduced level of haemoglobin Hasharon found in subjects carrying the gene for beta-thalassaemia (Alberti et al, 1975). Reductions below normal levels of other haemoglobin variants occurring in the presence of the counterpart-globin thalassaemia are probably determined through a similar mechanism.

HOMOZYGOUS β-thalassaemic subjects from the Ferrara region characteristically do not synthesise β globin1,2. Some of us have presented previously data which suggest that the addition of ribosome-free supernatant isolated from red blood cells of normal adult individuals or from subjects with homozygous haemoglobin S to Ferrara thalassaemic ribosomal material induces βA globin synthesis3; these results suggested that this de novo synthesis was not associated with free normal βA globin mRNA. From these findings it has been concluded that the mutation of the Ferrara β thalassaemia does not reside inthe gene for β globin, which is in fact transcribed into normal β globin mRNA; but in a gene coding for a previously unrecognised factor, indispensable for β globin translation. Here we report data which suggest that the reticulocytes from these patients synthesise β globin following normal blood transfusion.

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.

To investigate the sporadic occurrence of hemophilia A and to estimate the sex ratio of mutation rates directly, 17 families with isolated cases of the disorder were studied by RFLP analysis and by clotting assays. Three RFLPs, one intragenic and two with close linkage to hemophilia A, were used. In eight families the RFLP study excluded the carrier status of the maternal grandmothers. Since hemostatic studies showed that the eight mothers of these propositi were hemophilia carriers, the origin of the newly mutated genes was inferred from the RFLP patterns: six hemophilic genes derived from the normal maternal grandfathers and two, from maternal grandmothers. The data indicate a higher mutation rate in males than in females, as previously suggested by segregation analysis and coagulation studies. However the sex ratio indicated by the RFLP analysis is lower than previously reported and could explain previous conflicting estimates.

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.

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.

An additional TaqI restriction site was mapped in intron 2 of the factor XII gene. The site was found only in subjects with total or partial factor XII deficiency and thus represents the true gene lesion or a very tightly linked restriction fragment length polymorphism. The altered gene identified by this marker is present in four (three heterozygotes and one homozygote) of five unrelated Hageman trait subjects from different Italian regions. In the homozygous state the altered gene gives rise to a very marked reduction of factor XII activity. No deletion was found in the deficient factor XII genes.

The cytoplasmic distribution of the excess newly synthesized α-globin, present in the red cell lysate of β-thalassemic subjects has been investigated. The labelled globins present in the cytoplasm, as well as in purified Hb A and Hb F, were separated by CM-cellulose chromatography, and the ratios 3Hα/3Hβ and 3Hα/3Hγ were determined. The results obtained show that an intracellular exchange occurs between non radioactive α-chains of existing hemoglobins and the newly synthesized ones. Since the amount of labelled α-globin chains which is found in combination with Hb A or Hb F does not account for all the newly synthesized α-globin, it is concluded that part of this α-globin must exist in a free form. A series of experimental data support the conclusion that this “free fraction” is probably composed of non heminated α-globin molecules.

Globin chain synthesis has been studied in β-thalassemia by incubating red blood cells of 10 homozygous thalassemic patients with [3H] amino acids, and by measuring the incorporation of radioactivity into the globin chains, separated by column chromatography.

THERE is disagreement about the site of the human haemoglobin structural genes, mapping of which has been attempted by in situ hybridisation1–4 and family studies5,6. After in situ hybridisation of human metaphase chromosomes with radioactive globin mRNA Price et al.1 suggested that the haemoglobin structural genes are located on the long arm of chromosome 2 and on the long arm of a B-group chromosome, the latter carrying the genes for β, γ and δ chains. Their approach was criticised because of the low specific activity of the 3H-mRNA assay used7,8, but Atwood et al.2 and Price and Hirschhorn3,4 confirmed the original assignments using human complementary DNA (cDNA). Finally it was decided that the B-group chromosome that carries haemoglobin loci is chromosome 43,4. Family studies gave results consistent with loose linkage between the locus of the MN blood group and β-thalassaemia. The MN blood group has been mapped tentatively on the long arm of chromosome 2 or on the distal end of the long arm of chromosome 4 (refs 9, 10). But the localisation of the MN system on 2q is inconsistent with data from family studies11–13, and the distal segment of 4q has been considered more probable11. For similar reasons the β-globin locus has been mapped tentatively on chromosome 4 (ref. 5). We report here a study of globin synthesis which confirms that chromosome 4 is the site of the structural gene for α-globin.

β-THALASSAEMIA is a genetically determined anaemia of man, characterized by a decrease in production1–6 or the absence7 of β-globin, and by an excess of α-globin synthesis4,6–8, which results in the α/(β + γ + δ) globin synthesis ratio largely exceeding the normal value of 1.0.