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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.

Previous studies have shown that hemophilia B (HB) is the result of several different mutations, mostly single nucleotide substitutions, in the factor IX (FIX) gene. In order to evaluate the impact of mutation analysis on genetic counseling in sporadic and uninformative HB familial pedigrees, we re-analyzed by the conformation sensitive gel electrophoresis (CSGE) technique 14 patients, previously studied by restriction fragment length polymorphisms (RFLPs). A single mutation was present within the FIX gene of each patient: 12 mutations were single base substitutions, 1 was a base insertion, and 1 was a four nucleotide deletion; 4/12 mutations have not been described so far. By identifying the detrimental mutations in affected males, carrier status was correctly diagnosed in all the women we studied; 3/12 de novo events were found in maternal meioses with a 25% mutation rate. Identification of the genetic defect was also successfully applied to three prenatal diagnoses., (Copyright 2003 Wiley-Liss, Inc.)

Although the quality of life for haemophiliacs has clearly improved in the last few years, haemophilia still remains a serious disorder justifying prenatal diagnosis (PD) and, if necessary, termination. Because chorionic villus sampling (CVS) is performed in the first trimester of pregnancy, an increasing number of carriers are interested in this test. It has been shown that waiting for the results is particularly distressing for pregnant women, therefore decreasing the diagnostic procedure time can be psychologically helpful. Here we report on PD in a sporadic haemophilia B family based on the direct identification of the pathogenic mutation in a CVS taken at the 12th gestational week. In order to hasten the results, we recovered DNA from a single villus fragment boiled in water and used it directly for PCR reaction. Conformation-sensitive gel electrophoresis (CSGE) was used to detect the mutation in the haemophilia carrier and in the foetus. This approach allowed us to obtain a diagnosis within 24 h of CVS, thus avoiding the long-term psychological effects on the pregnant woman.

Granulocyte colony-stimulating factor (G-CSF) has had a major impact on management of "severe chronic neutropenia," a collective term referring to congenital, idiopathic, or cyclic neutropenia. Almost all patients respond to G-CSF with increased neutrophils, reduced infections, and improved survival. Some responders with congenital neutropenia have developed myelodysplastic syndrome and acute myeloblastic leukemia (MDS/AML), which raises the question of the role of G-CSF in pathogenesis. The Severe Chronic Neutropenia International Registry (SCNIR), Seattle, WA, has data on 696 neutropenic patients, including 352 patients with congenital neutropenia, treated with G-CSF from 1987 to present. Treatment and patient demographic data were analyzed. The 352 congenital patients were observed for a mean of 6 years (range, 0.1-11 years) while being treated. Of these patients, 31 developed MDS/AML, for a crude rate of malignant transformation of nearly 9%. None of the 344 patients with idiopathic or cyclic neutropenia developed MDS/AML. Transformation was associated with acquired marrow cytogenetic clonal changes: 18 patients developed a partial or complete loss of chromosome 7, and 9 patients manifested abnormalities of chromosome 21 (usually trisomy 21). For each yearly treatment interval, the annual rate of MDS/AML development was less than 2%. No significant relationships between age at onset of MDS/AML and patient gender, G-CSF dose, or treatment duration were found (P >.15). In addition to the 31 patients who developed MDS/AML, the SCNIR also has data on 9 additional neutropenic patients whose bone marrow studies show cytogenetic clonal changes but the patients are without transformation to MDS/AML. Although our data does not support a cause-and-effect relationship between development of MDS/AML and G-CSF therapy or other patient demographics, we cannot exclude a direct contribution of G-CSF in the pathogenesis of MDS/AML. This issue is unclear because MDS/AML was not seen in cyclic or idiopathic neutropenia. Improved survival of congenital neutropenia patients receiving G-CSF therapy may allow time for the expression of the leukemic predisposition that characterizes the natural history of these disorders. However, other factors related to G-CSF may also be operative in the setting of congenital neutropenia. (Blood. 2000;96:429-436)

AC133+ cells may represent an alternative source of transplantable haemopoietic progenitor cells to CD34+ cells. Here, we have addressed the characterization of umbilical cord blood (UCB) AC133+ cells and compared their immunophenotypic and functional features with those of UCB CD34+ cells. UCB AC133+ and CD34+ cell fractions were purified by magnetic cell sorting, analysed by flow cytometry, tested for their content in blast cell colony-forming units (CFU-Bl), erythroid and granulocyte-macrophage colony-forming units before and after expansion in the presence of various haemopoietic growth factor combinations. Median AC133+ cell yield was 62.3%, and median AC133+ population purity was 97.9%. AC133+ cells were found to contain significantly more CFU-Bl than CD34+ 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+ and CD34+ cells displayed an increased ability to give rise to committed progenitors after 7-day expansion in liquid cultures. These data suggest that the AC133+ 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+ cells, UCB AC133+ cells appear to contain a higher number of early haemopoietic progenitors.

We report on a girl with congenital hypoplastic anaemia, "coarse" face, generalized hypertrichosis with scalp hypotrichosis, short fifth finger, hypoplastic toenails, and mental retardation. A sister of the proposita, who died at the age of 1 year, had severe congenital anaemia, hypoplastic fingernails, low birth weight, failure to thrive, and repeated upper respiratory tract infections. Based on family history, we suspect that hypoplastic anaemia and the same multiple congenital anomalies-mental retardation syndrome (MCA/MR) were also present in this sister. To the best of our knowledge, this patient represents the first report of congenital hypoplastic anaemia and such a complex MCA/MR syndrome, probably inherited as an autosomal recessive trait., (Copyright 1999 Wiley-Liss, Inc.)

Diamond-Blackfan anaemia (DBA) is a congenital disease characterized by defective erythroid progenitor maturation: 30% of patients have congenital malformations. The link between these malformations and defective erythropoiesis is unclear: a defect in a molecule acting both on embryo development and haemopoiesis has been proposed. Inheritance is autosomal dominant in most familial cases, but recessive families have also been reported. Many cases are sporadic. A DBA locus has been mapped on chromosome 19q13.2 (Gustavsson et al, 1997), but several families unlinked to this locus have also been reported (Gustavsson et al, 1998). This paper presents clinical, epidemiological and molecular data for DBA in the Italian population. Segregation analysis of 19q markers in patients with DBA showed exclusion of this locus in 5/12 families with inherited DBA. There was evidently locus heterogeneity for DBA in this population. A new microdeletion was identified in one patient. Other families, in which DBA segregates concordantly with the 19q critical region, suggest incomplete penetrance and expressivity of the DBA gene.

Background and Objective: Since 1988 the AIEOP has used BFM-based chemotherapy for childhood ALL. Current organization and results and role of cranial irradiation in the AIEOP-ALL 91 study are reported., Design and Methods: From 1991 to 1995, 1194 children (< 15 years) with non-B ALL, were enrolled and assigned to the standard risk [SR: age > 1 year, non-T-ALL, BFM risk factor (RF) < 0.8], intermediate risk (IR: RF > or = 0.8 but < 1.7, or with RF < 0.8 and age < 1 year, or T-ALL), or high risk [HR: RF > or = 1.7, or t(9;22), or t(4;11) or prednisone poor response or late response or CNS involvement] groups. All patients received initially protocol Ia. Thereafter SR patients received HD-MTX 2 g/m2, a modified protocol II, and continuation therapy with triple intrathecal chemotherapy (TIT); IR patients received protocol Ib, HD-MTX 5 g/m2, protocol II and continuation therapy with TIT; HR patients received 9 polychemotherapy blocks, cranial irradiation and continuation therapy. Duration of treatment was 24 months. A randomized study was conducted to evaluate the impact of high-dose asparaginase in non high risk patients: the results of this study cannot be disclosed yet., Results: One thousand one hundred and fifty-two (96.5%) patients achieved CR. Overall EFS (SE) at 5-years was 71.0% (1.4), with a survival of 80.3% (1.3). Relapse occurred in 262 children (21.9%), either in the marrow (n = 192 isolated and 32 with other sites, 18.7%), in the CNS (n = 18, 1.5%), or elsewhere (n = 20, 1.7%). 5-year EFS (SE) was 83.3% (2.4) in SR, 74.7% (1.8) in IR, and 39.7% (3.5) in HR groups, respectively., Interpretation and Conclusions: Overall cure rate was higher than in the previous AIEOP-ALL 88 study. Treatment intensification with polychemotherapy blocks did not improve results in HR. Cranial irradiation can be safely omitted in over 80% of children treated with BFM based chemotherapy.

Background and Objective: The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retroviral vectors., Design and Methods: Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of Interleukin-3 (IL-3), Interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF)., Results: The results obtained indicated that the amount of transduced cells increased with the lenght of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture., Interpretation and Conclusions: These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.

The human von Willebrand factor (vWF) plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; it also binds and stabilizes factor VIII procoagulant protein. The biological functions of vWF are dependent on distinct molecular domains responsible for the specificity and affinity for ligands. The multimeric structure of vWF provides an array of binding sites that allow multivalent interactions, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. Quantitative and qualitative abnormalities of vWF cause the most common congenital bleeding disorder in humans, the von Willebrand disease (vWD). This review will provide an update on the recent advances toward the elucidation of structure-function relationships and the detection of molecular defects leading to vWD and will highlight the revised classification of vWD.

The case of a one-year-old child in whom a large catheter-related right atrial thrombus of more than a week's duration was dissolved by urokinase is presented. After one week of unsuccessful heparin treatment, urokinase, via a central venous catheter, was added at a dose of 1500 IU/kg/h. Urokinase was subsequently increased by 1000 U/kg/h every day up to a maximum of 4500 IU/kg/h on the basis of thrombus size reduction as assessed by daily cross-sectional echocardiography. One week later the thrombus was almost completely dissolved. No major bleeding occurred. Warfarin was given for the next three months and the international normalized ratio (INR) was maintanied between 2 and 3. At the end of warfarin treatment echocardiography was negative for right atrial thrombosis. This case suggests that local high dose urokinase therapy can be safely and successfully used to dissolve large right atrial thromboses of more than a week's duration.

Background and Objectives: Immune thrombocytopenic purpura (ITP) is a common hematologic disorder, two forms of which occur in children. The detection of circulating platelet antibodies is helpful in diagnosis., Materials and Methods: We evaluated three different immunological methods for detecting platelet antibodies in the serum of children with ITP. These were: a solid-phase red-cell adherence test (SPRCA), an enzyme immunoassay (EIA), and an immunofluorescence test (PSIF)., Results: The sensitivity of the methods in detecting IgG antibodies ranged from 28.1 (EIA) to 39.4% (SPRCA). We also looked for IgM antibodies by PSIF, thus raising the sensitivity of this test from 32.0 to 40.0%. A combination of two tests (SPRCA and EIA) allowed us to detect 61.8% positive samples. By doing all three tests, we obtained 71.3% positive samples. Finally, we reached 73.5% by adding PSIF for IgM. We found a higher frequency of circulating antibodies in both acute and chronic ITP at onset than in clinical remission. There were a few positive sera in chronic ITP, but not in the acute form in remission., Conclusion: The individual tests each have a relatively low sensitivity, but the combination of all three increases the diagnostic effectiveness. The finding of platelet antibodies during remission may predict evolution toward a systemic autoimmune state.

We investigated the presence of a recombinant event between the F8A gene located in intron 22 of the factor VIII gene and the two additional copies of F8A lying 500 Kb upstream of FVIII in severe hemophilic patients. The genomic DNA of 146 unrelated Italian patients with severe hemophilia A (HA) was hybridized with an F8A gene probe to detect the abnormal band patterns. A recombinant event was found in 71/146 patients, confirming the high incidence of this mutation in the Italian hemophilic population also. We conclude that the high frequency of the mutation in HA subjects allows us to make a direct and safe diagnosis in about 50% of our families without resorting to RFLP analysis.

The authors reported the outcome of a retrospective study of patients with HELLP syndrome to verify whether this pathology is a clinical entity with models of evolution, regression and well defined treatment. A total of 20 individual cases were reported at the Division of Obstetrics and Gynecology at the G. Gaslini Institute during the period from January 1990 to September 1995. All cases showed the normalisation of the various clinical and hematochemical parameters within a maximum of 8 days after birth. The rapid diagnosis, the immediate extraction of the fetus and appropriate medical treatment led to the prompt resolution of this severe pathology without the need for demolitive surgery frequently used in the past.