A basic tenet of the Lyon hypothesis is that X inactivation occurs randomly with respect to parental origin of the X chromosome. Yet, nonrandom patterns of X inactivation are common -often ascertained in women who manifest recessive X-linked disorders despite being heterozygous for the mutation. Usually, the cause of skewing is cell selection disfavouring one of the cell lineages created by random X inactivation. We have identified a three generation kindred, with three females who have haemophilia A because of extreme skewing of X inactivation. Although they have both normal and mutant factor VIII (FVIII) alleles, only the mutant one is transcribed; and, they share an XIST allele that is never transcribed. The skewing in this case seems to result from an abnormality in the initial choice process, which prevents the chromosome bearing the mutant FVIII allele from being an inactive X.

Summary. AC1331 cells may represent an alternative source of transplantable haemopoietic progenitor cells to CD34 1 cells. Here, we have addressed the characterization of umbilical cord blood (UCB) AC1331 cells and compared their immunophenotypic and functional features with those of UCB CD34 1 cells. UCB AC133 1 and CD34 1 cell fractions were purified by magnetic cell sorting, analysed by flow cytometry, tested for their content in blast cell colonyforming units (CFU-Bl), erythroid and granulocyte±macro-phage colony-forming units before and after expansion in the presence of various haemopoietic growth factor combinations. Median AC133 1 cell yield was 62´3%, and median AC133 1 population purity was 97´9%. AC133 1 cells were found to contain significantly more CFU-Bl than CD34 1 cells; furthermore, the replating efficiency, i.e. the number of CFU-Bl capable of generating secondary colonies, was higher in the former than in the latter cells. Both AC133 1 and CD34 1 cells displayed an increased ability to give rise to committed progenitors after 7-day expansion in liquid cultures. These data suggest that the AC133 1 cell subset is a heterogeneous pool of immature and more differentiated cells that can be maintained and expanded in well-defined culture conditions. In comparison with CD34 1 cells, UCB AC1331 cells appear to contain a higher number of early haemopoietic progenitors.

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Summary. We describe 18 novel mutations, unreported in the Haemophilia A mutation Databases, that have been identified in a cohort of unrelated, Italian patients affected with haemophilia A (HA). Screening of the factor VIII gene (FVIII) was performed using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. Eight mutations were characterized as non-missense alterations, and the remaining 10 were missense mutations. Heterozygosity for the identified mutations was observed in the female relatives of patients belonging to eight families with sporadic cases. In an attempt to understand better the causative effect of the mutations and the clinical variability of the patients, missense mutation consequences were investigated for: (1) the nature of the new amino acid; (2) the location of the substituted amino acid within crystallographic and theoretical models; and (3) the degree of conservation of the native residue in factor VIII (FVIII) protein and FVIII-related protein family aligned sequences. These research tools have provided evidence that the mutations we describe involve residues that were conserved, at least in FVIII proteins, in all the species we compared.

Deficiency or dysfunction of factor IX FIX leads to haemophilia B (HB), an X-linked, recessive, bleeding disorder. On a molecular basis, HB is due to a heterogeneous spectrum of mutations spread throughout the F9 gene. In several instances, a cause-effect relation has been elucidated, in others predicted possibilities have been offered by crystallography inspection and by software-constructed models of the protein. The aim of this study was to contribute to the understanding of HB molecular pathology. The F9 missense mutations we identified in 21 unrelated Italian HB patients by direct sequencing of the whole F9 coding regions were inspected for the causative effect they provoked on the ensuing transcript, and on the protein structure. Each alteration was studied in order to: (i) characterize the defect on the basis of the nature of the mutation; (ii) identify the predicted defect that is induced in the gene and (iii) speculate about the potential, detrimental effects which upset the protein functionality through an idealized FIX model. The resulting data may further contribute to the comprehension of the mechanisms underlying the disease.