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Titin is a 3000 kDa large protein of vertebrate striated muscle which extends from Z discs to M lines. Within the segment of titin that locates in the I band, tissue-specific isoforms are expressed by differential splicing in correlation to the sarcomeric ultrastructure. We have now searched the M-line region of titin for differential expression. The 20 kb section from the 3' end of the gene has been sequenced and contains 23 exons. Exon/intron organization is correlated to the modular organization of the titin protein. The six exons at the 3' end of the gene encode the M-line section of titin and are referred to as Mex1 to Mex6. Analysis of the RNAs expressed in different rabbit striated muscles reveals that the exon Mex5 is either included or excluded in the titin mRNA during splicing. The levels of inclusion of Mex5 vary between different types of striated muscles. Heart expresses (Mex5+)-titin, skeletal muscles co-express tissue-specifically distinct ratios of (Mex5+) and (Mex5-)-titins. In situ hybridization of whole-mount mouse embryos with Mex5 antisense RNA provide no evidence for the exclusion of Mex5 during embryonic development. We speculate that the establishment of differential splicing pathways of M-line titin late during development may correlate with and explain the postnatal development of different M-line fine structures in the different muscles. Comparison of titin gene sequences from different vertebrates reveals that the intron sequences located upstream of Mex3 and Mex5, referred to as Min-2 and Min-4, respectively, have remained strongly conserved during evolution. While the conservation of Min-4 may be explained by its participation in the regulation of the differential skipping of Mex5, the functional significance of the conservation of the Min-2 intron located upstream of Mex3 is yet unknown.

Objective: To determine whether nuclear magnetic resonance (NMR) relaxation parameters can be used to quantify iron in tissues, the relationship between NMR spectrometric T2 relaxation measurements and tissue iron concentration were verified in a novel murine cardiac iron overload model., Methods: Congenital heterozygous thalassemic mice and controls were injected with intraperitoneal iron or saline and were sacrificed at three weeks. Samples of liver, heart and peripheral muscle were subjected to NMR relaxation measurements and continuous distribution analysis. Tissue ferritin levels were determined with immunoadsorbance techniques, and elemental iron was assayed by flame atomic absorption. Tissues were analyzed pathologically with hematoxylin and eosin and Prussian blue staining to confirm the localization of iron., Results: This murine iron loading model was uniquely successful in loading iron into the major organs, especially the heart, and produced significant reductions in T1 and T2 NMR relaxation values. There was a good correlation between soluble ferritin and total iron levels (r=0.92), indicating that there is a constant and significant fraction of total iron present in ferritin irrespective of absolute iron concentrations. Regression analysis between total iron content and T2 relaxivity showed a linear relationship (r=0.96), suggesting that the T2 relaxation parameter is related to tissue iron concentration. The regression relationship suggested that NMR can detect iron levels as low as 0.1 mg/g of tissue., Conclusions: Parenteral iron loading in mice produces unique iron overload in major organs, including the heart. Local iron deposition is detectable by NMR relaxometry at 0.1 mg/g or higher. There is a linear relationship between iron concentration and T2 relaxivity. Thus, NMR may be an important and useful clinical tool to quantify iron excess in various pathobiological states of human disease due to iron overload, including heart disease.

Data from several trials have provided direct and supportive evidence for the efficacy of deferiprone in the treatment of iron overload in thalassemia major. Deferiprone has been shown to induce sustained decreases in body iron to concentrations associated with survival free from the complications of iron overload in deferoxamine (DFO)-treated patients. Despite this evidence of efficacy, the risk of agranulocytosis mandates a careful evaluation in patients willing and able to use DFO. The incidence of agranulocytosis associated with deferiprone is under study in a prospective multicenter trial in Canada, Italy and the United States, under corporate sponsorship (Apotex Research, Canada). The results of this study should determine the risk associated with the use of this agent, and may provide the data required for an FDA decision regarding licensing of this agent for the treatment of iron overload, a goal supported by investigators worldwide.

Among individuals of Mediterranean or Middle Eastern descent, the IVS-I-6 (T-->C) mutation is one of the most common causes of beta-thalassemia. In this report, we describe the clinical phenotypes of a group of beta-thalassemia patients who are compound heterozygotes for the relatively mild IVS-I-6 (T-->C) beta-thalassemia mutation and more severe beta(+)- or beta (0)-thalassemia mutations. Although most of these patients are transfusion-dependent, the requirement for regular transfusions generally occurred late in childhood. A correlation between concomitant alpha-thalassemia and a mild transfusion-independent phenotype is not apparent, indicating the involvement of other ameliorating determinants.

Red blood cell (RBC) membranes from patients with the thalassemic and sickle hemoglobinopathies carry abnormal deposits of iron presumed to mediate a variety of oxidative-induced membrane dysfunctions. We hypothesized that the oral iron chelator deferiprone (L1), which has an enhanced capacity to permeate cell membranes, might be useful in chelating these pathologic iron deposits from intact RBCs. We tested this hypothesis in vitro by incubating L1 with RBCs from 15 patients with thalassemia intermedia and 6 patients with sickle cell anemia. We found that removal of RBC membrane free iron by L1 increased both as a function of time of incubation and L1 concentration. Thus, increasing the time of incubation of thalassemic RBCs with 0.5 mmol/L L1 from 0.5 to 6 hours, enhanced removal of their membrane free iron from 18% +/- 9% to 96% +/- 4%. Dose-response studies showed that incubating thalassemic RBC for 2 hours with L1 concentrations ranging from 0.125 to 0.5 mmol/L resulted in removal of membrane free iron from 28% +/- 15% to 68% +/- 11%. Parallel studies with sickle RBCs showed a similar pattern in time and dose responses. Deferoxamine (DFO), on the other hand, was ineffective in chelating membrane free iron from either thalassemic or sickle RBCs regardless of dose (maximum, 0.333 mmol/L) or time of incubation (maximum, 24 hours). In vivo efficacy of L1 was shown in six thalassemic patients whose RBC membrane free iron decreased by 50% +/- 29% following a 2-week course of L1 at a daily dose of 25 mg/kg. As the dose of L1 was increased to 50 mg/kg/d (n = 5), and then to 75 mg/kg/d (n = 4), 67% +/- 14% and 79% +/- 11%, respectively, of their RBC membrane free iron was removed. L1 therapy--both in vitro and in vivo--also significantly attenuated the malondialdehyde response of thalassemic RBC membranes to in vitro stimulation with peroxide. Remarkably, the heme content of RBC membranes from L1-treated thalassemic patients decreased by 28% +/- 10% during the 3-month study period. These results indicate that L1 can remove pathologic deposits of chelatable iron from thalassemic and sickle RBC membranes, a therapeutic potential not shared by DFO. Furthermore, membrane defects possibly mediated by catalytic iron, such as lipid peroxidation and hemichrome formation, may also be alleviated, at least in part, by L1.

Background: Enhanced production of fetal hemoglobin lessens the severity of beta-thalassemia and sickle cell disease. Intravenous infusion of arginine butyrate can increase the number of reticulocytes containing fetal hemoglobin in patients with these disorders, and it has induced a substantial increase in hemoglobin in one patient with thalassemia. We therefore tested the efficacy of this agent in patients with beta-hemoglobinopathies., Methods: We treated 10 patients with severe beta-thalassemia or sickle cell disease with arginine butyrate at an initial dose of 500 mg per kilogram of body weight per day (final dose, 2000 mg per kilogram per day), 6 days per week, for a mean (+/- SD) of 10 +/- 1.2 weeks (range, 9 to 13). A hematologic response was defined as an increase in the hemoglobin concentration of at least 2 g per deciliter in patients with thalassemia and as a twofold increase in the fetal hemoglobin concentration in patients with sickle cell disease., Results: Increase in gamma-globin messenger RNA and in reticulocytes containing fetal hemoglobin but not in hemoglobin were observed in the patients with thalassemia. A small, unsustained increase in fetal hemoglobin was observed in two patients with sickle cell disease. Drug toxicity was minimal at standard doses. One patient had a grand mal seizure after inadvertently receiving 2000 mg of arginine butyrate per kilogram over a period of six hours., Conclusions: Ten weeks of intravenous arginine butyrate did not produce a hematologic response in 10 patients with either severe beta-thalassemia or sickle cell disease.

Objective: To determine the effect of a transfusion program on risk of stroke recurrence in children with sickle cell disease., Design: The clinical course and experience with transfusion therapy at eight centers were reviewed for subjects whose initial stroke occurred after January 1988., Results: Sixty subjects were observed for 191.7 patient-years. Eight had a single recurrent stroke (two intracranial hemorrhages and six infarctions) for a prevalence of 13.3%, or one recurrence for each 24 patient-years of observation. Thirteen subjects had 15 transient neurologic events; two of these had subsequent strokes, but the overall risk was similar for those who did and those did not have transient events. Hemoglobin S levels were greater than the desired maximum of 30% at the time of 7 of 16 transient events and five of six recurrent infarctions. The stroke recurrence rate was similar to those in previous reports of children receiving long-term transfusion therapy but significantly less than that reported for children who did not receive transfusions (p < 0.001)., Conclusions: We conclude that maintenance of hemoglobin S at a level less than 30% appears to be effective in reducing the rate of recurrent infarction but does not prevent transient neurologic events. Transient neurologic events are common but do not appear to be related to recurrent stroke.

Hepatic non-transferrin-bound Fe (NTBI) flux and its regulation were characterized by measuring the uptake of Fe from [59Fe]/nitrilotriacetate (NTA) complexes in control and Fe-loaded cultures of human hepatocellular carcinoma cells (HepG2). Exposure to ferric ammonium citrate (FAC) for 1 to 7 days resulted in a time- and dose-dependent increase in the rate of NTBI uptake. In contrast to previous studies showing a dependence of the rate of Fe uptake on extracellular Fe, this was positively correlated with total cellular Fe content. The Fe3+ chelating agents deferoxamine (DFO), 1,2-dimethyl-3-hydroxypyrid-4-one (CP 020) and 1,2-diethyl-3-hydroxypyrid-4-one (CP 094) prevented or diminished the increase in NTBI transport when present during Fe loading and reversed the stimulation in pre-loaded cells in relation to their abilities to decrease intracellular iron. Although saturation of the Fe uptake process was not achieved in control cells, kinetic modelling to include linear diffusion-controlled processes yielded estimated parameters of Km = 4.3 microM and Vmax = 2.6 fmol/micrograms protein/min for the underlying process. There was a significant increase in the apparent Vmax (31.2 fmol/micrograms protein per min) for NTBI uptake in Fe-loaded cells, suggesting that Fe loading increases the number of a rate-limiting carrier site for Fe. Km also increased to 15.2 microM, comparable to values reported when whole liver is perfused with FeSO4. We conclude that HepG2 cells possess a transferrin-independent mechanism of Fe accumulation that responds reversibly to a regulatory intracellular Fe pool.

The efficacy of a eutectic mixture of local anesthetics (EMLA) in alleviating the pain associated with subcutaneous needle insertion for infusion of the iron-chelating agent, deferoxamine, was examined in 12 patients with homozygous beta-thalassemia. As reported by the patient using a 100-mm visual analogue scale, the pain of insertion was rated as significantly less after application of EMLA (mean +/- SD, 1.5 +/- 2.2 mm) than the pain associated with needle insertion without EMLA (34.8 +/- 33.5 mm, P = .005). Subsequently, in a double-blind randomized trial of 10 beta-thalassemia patients, EMLA was significantly better (5.7 +/- 8.2 mm) than placebo (27.0 +/- 22.8 mm, P = .01) in reducing the pain of needle insertion for deferoxamine infusion. No adverse effects were reported with the use of EMLA cream. These results suggest that EMLA may be effective in reducing the pain associated with needle insertion for subcutaneous deferoxamine infusion in beta-thalassemia patients, which may lead to improved compliance with this irritating, prolonged therapy. The safety of EMLA use in these patients, and others receiving regular parenteral therapy, should now be examined.

Permanent correction of genetic deficiencies of the hematopoietic system requires gene transfer into stem cells and long-term lineage specific expression after autologous transplantation. However, progress to develop gene therapy protocols has been hampered by the absence of in vivo assays that detect genetically deficient human hematopoietic stem cells and their diseased differentiated progeny. The establishment of systems to transplant human cells into immune-deficient SCID mice provides such an assay. We report that primitive bone marrow cells from beta-thalassemia major and sickle cell anemia patients engraft immune-deficient mice, giving rise to high levels of human erythroid and myeloid cells in response to treatment with human cytokines. The bone marrow of transplanted mice contained the entire erythroid lineage from BFU-E to mature erythrocytes expressing human gamma, beta or beta s-globin. Moreover, human erythroid cells from mice transplanted with sickle cell anemia bone marrow showed characteristic sickling under reducing conditions in an in vitro assay. This model provides a powerful in vivo system that can be used to evaluate the efficiency of globin gene transfer into primitive human hematopoietic cells, lineage-specific expression in mature erythrocytes, and ultimately correction of the cellular defect found in the erythroid lineage.

We describe a family from Bangladesh in which three children are compound heterozygotes for Hb E (alpha 2 beta 2, beta 26Glu Lys) and Hb Lepore (delta-beta fusion gene). PCR amplification and direct nucleotide sequencing established that the fusion gene is Hb LeporeHollandia, with the cross-over localized to a 40 bp window between codon 22 and IVS-1 nt 16 of the delta- and beta-globin genes. This unusual combination of mutations is associated with a relatively mild clinical phenotype, with all three affected siblings having microcytic anemia of moderate severity without the need for transfusions.

We present a case in which a Greek couple was considered not to be at risk of having children with homozygous beta-thalassaemia, an assessment based largely on the father's belief that he carried alpha-thalassaemia. After their first child was diagnosed with homozygous beta-thalassaemia, the case was re-assessed and both parents were shown to have the haematological profile of beta-thalassaemia trait. Screening for the common Mediterranean mutations demonstrated that the mother carries the IVS-1 nt 110 G-->A beta(+)-thalassaemia mutation. Direct nucleotide sequencing of PCR-amplified DNA revealed that the father carries a novel beta O-thalassaemia mutation, frameshift codons 9/10 (+T). The couple's second pregnancy was terminated after prenatal testing revealed that the fetus had inherited both parental mutations. This case illustrates the need to confirm the carrier status of individuals prior to assessing their genetic risks, and highlights the importance of being able to identify rare or novel beta-thalassaemia mutations.

We describe a Canadian infant of Jamaican descent who presented with mild anemia. Hb electrophoresis revealed Hb S and an unknown Hb variant that migrated slightly faster than Hb S on cellulose acetate. Molecular studies of the family indicated that the proband is a compound heterozygote for Hb S and Hb Caribbean. Hb Caribbean has previously been characterized as a mildly unstable hemoglobin with low oxygen affinity, due to a Leu-->Arg substitution at amino acid residue 91. The present study establishes the molecular basis for Hb Caribbean (beta 91, CTG-->CGG) and confirms that Hb S/Hb Caribbean syndrome is not associated with serious clinical manifestations.

The assessment of compliance is critical in the evaluation of the effectiveness of a new therapeutic agent. Fifteen patients with transfusion-dependent beta-thalassemia, many of whom had previously demonstrated erratic compliance with deferoxamine, were enrolled in a clinical trial of a new oral iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1). Their compliance with this medication was estimated by several existing methods and the novel Medication Event Monitoring System (MEMS). Overall compliance as assessed by the MEMS was 78.5 +/- 13.0% of prescribed doses taken, significantly lower than the corresponding rates calculated by pill counts and diaries (91.5 +/- 9.2% and 94.1 +/- 4.3%, respectively). However, several serious problems were encountered with the MEMS, mostly in the form of incorrect use of the device by the patients. Disclosure of the nature of the MEMS and the compliance monitoring process did not alter the rate of adherence with L1 therapy. Compliance as determined by pill counts did not differ between the 1st and 2nd 6-month periods. Although not reaching statistical significance, a trend towards better L1 compliance occurred in those patients in whom serum ferritin levels decreased. Patients who filled at least 50% of their diaries had significantly better compliance by pill counts than those who completed less than 50% of their diaries (95.9 +/- 4.1% and 86.5 +/- 11.1%, respectively). Steady-state L1 trough concentrations and 24-hour urinary iron excretion did not correlate with L1 compliance.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: The prognosis of patients with homozygous beta-thalassemia (thalassemia major) has been improved by transfusion and iron-chelation therapy. We analyzed outcome and prognostic factors among patients receiving transfusions and chelation therapy who had reached the age at which iron-induced cardiac disease, the most common cause of death, usually occurs., Methods: Using the duration of life without the need for either inotropic or antiarrhythmic drugs as a measure of survival without cardiac disease, we studied 97 patients born before 1976 who were treated with regular transfusions and chelation therapy. We used Cox proportional-hazards analysis to assess the effect of prognostic factors and life-table analysis to estimate freedom from cardiac disease over time., Results: Of the 97 patients, 59 (61 percent) had no cardiac disease; 36 (37 percent) had cardiac disease, and 18 of them had died. Univariate analysis demonstrated that factors affecting cardiac disease-free survival were age at the start of chelation therapy (P < 0.001), the natural log of the serum ferritin concentration before chelation therapy began (P = 0.01), the mean ferritin concentration (P < 0.001), and the proportion of ferritin measurements exceeding 2500 ng per milliliter (P < 0.001). With stepwise Cox modeling, only the proportion of ferritin measurements exceeding 2500 ng per milliliter affected cardiac disease-free survival (P < 0.001). Patients in whom less than 33 percent of the serum ferritin values exceeded 2500 ng per milliliter had estimated rates of survival without cardiac disease of 100 percent after 10 years of chelation therapy and 91 percent after 15 years., Conclusions: The prognosis for survival without cardiac disease is excellent for patients with thalassemia major who receive regular transfusions and whose serum ferritin concentrations remain below 2500 ng per milliliter with chelation therapy.

The iron chelator deferiprone (L1) reduces tissue iron stores in iron-loaded patients. Three of sixteen patients treated with deferiprone developed joint pain and swelling without evidence of systemic lupus erythematosus (SLE). Articular cartilage, synovial hypertrophy and iron deposition, and synovial lining cell proliferation, with no inflammatory or allergic reaction, were observed on synovial exploration and biopsy. Symptoms resolved partly or completely during continued drug administration. We hypothesise that deferiprone-induced shifts of iron to synovium resulted in tissue damage, accelerated by free-radical formation during incomplete complexation of iron and this bidentate chelator. This deferiprone-associated symptom complex is not associated with drug-induced SLE, and does not progress in severity during continued therapy.

Non-transferrin-bound iron (NTBI) uptake occurs in a variety of cells by a saturable, specific and temperature-sensitive process. Our previous studies indicated that NTBI uptake by cardiac myocytes and Hep G2 cells was reversibly up-regulated by iron deposition. In the present work we have characterized this up-regulation and examined its mechanism by comparing the uptake of oxidized (Fe3+) and ascorbate-reduced (Fe2+) forms of iron. Iron loading markedly enhanced the uptake of iron both in the presence and absence of ascorbate, but the increment was greater when ascorbate was absent. This up-regulation is partially inhibited by actinomycin D and cycloheximide, indicating a requirement for protein synthesis. Uptake by the iron-loaded cells was less sensitive to thiol-alkylating agents and competing metal ions, but was more sensitive to proteolysis. Iron loading causes an increase in both Km and Vmax for uptake of both Fe2+ and Fe3+, although the values differ, suggesting distinct rate-limiting steps for uptake of Fe2+ and Fe3+. Consistent with this idea, uptake of the two ions showed differential sensitivity to thiol reagents, competing metal ions and monensin. The Fe(2+)-specific chelators bathophenanthroline disulfonate and ferrozine markedly inhibited iron uptake whether ascorbate was present or not, indicating that Fe3+ uptake is dependent on reduction to the ferrous state. This requirement for reduction was independent of the iron status of the cells, demonstrating that the process of up-regulation is not due to the appearance of a new mechanism for translocation of Fe3+ without reduction. Taken together, the evidence favors a model of NTBI transport where an obligate and rate-determining reduction of Fe3+ occurs prior to or during uptake, followed by translocation through an Fe2+ carrier. The distinct translocation mechanisms of uptake in the presence and absence of ascorbate suggest that exogenous Fe2+ does not access the carrier available to the nascent ferrous ion derived from the reductase and is consistent with close coupling between the reduction and the translocation processes. In iron-loaded cells with increased rates of NTBI transport, a similar mechanism prevails.

Background: Previous studies of transfusions of newly formed red cells (neocytes) demonstrated modest extensions of transfusion interval in patients with homozygous beta-thalassemia., Study Design and Methods: The clinical benefits of a new system of neocyte preparation (Neocel, Cutter Biological, Berkeley, CA), reported to combine ease of preparation with reduction in the transfusion requirements of thalassemia patients, were evaluated. Sixteen thalassemic patients who had undergone splenectomy received eight consecutive, standard, automated, washed red cell transfusions (standard transfusions), followed by eight transfusions with the neocyte preparation (neocyte transfusions). In each arm of the study, mean pretransfusion hemoglobin and mean red cell mass transfused were carefully controlled and were similar., Results: A significant (p < 0.0001) extension of transfusion interval was observed in patients receiving neocyte transfusions (mean +/- SD; 38.7 +/- 34 days; range, 35.0-44.5), over that in those receiving standard transfusions (32.9 +/- 2.5 days; range, 29.6-38.5). The mean prolongation of transfusion interval by neocyte transfusion corresponded to a mean reduction of 25 mL in packed red cells transfused per kg of body weight per patient per year and a mean reduction in transfused iron of 15 percent per year per patient. During neocyte transfusions, blood preparation costs were considerably increased and donor exposure was significantly (p < 0.0005) higher than during the standard transfusion period., Conclusion: These data demonstrate that extension of the transfusion interval, and reduction in transfused iron, may be achieved in thalassemic patients by use of the Neocel system. These benefits are achieved, however, with substantial increases in donor exposure and in component preparation costs.

In two previous studies, we observed that recombinant human interleukin-3 (IL-3) induced an increase in marrow burst-forming unit-erythroid-derived colonies in vitro in some patients with Diamond-Blackfan anemia (DBA). To determine whether a similar erythropoietic response could be induced in vivo, we treated 13 patients with DBA (aged 4 to 19 years) with two preparations of IL-3. All patients had absent absolute reticulocyte counts and markedly reduced to absent recognizable bone marrow erythroid elements; patients with circulating reticulocytes in the previous 12 months were excluded from study. All patients except 1 had failed steroid therapy and had been transfusion-dependent since infancy; 1 patient was maintained on high-dose prednisone at the time of enrollment. On the first arm of the study, IL-3 (Immunex Corp, Seattle, WA) was administered subcutaneously using a dose escalation regimen of 125 to 500 micrograms/m2/day in divided dosage at 12-hour intervals, coadministered with 1.5 mg/kg/d of oral ferrous sulphate. Of the 13 patients that entered the trial, 4 stopped prematurely because of adverse side effects. In the other 9 evaluable cases, reticulocytes increased transiently in 1 patient from 0 to 65 x 10(9)/L after 35 days of IL-3 therapy at 250 micrograms/m2, but transfusion dependency persisted. One transient peak in absolute reticulocyte count was noted in 6 other patients, but no erythroid response was observed after completion of a full course of IL-3. Oral prednisone at 0.5 mg/kg/d was then coadministered with IL-3 at 500 micrograms/m2 to 5 of the patients without effect, and treatment was stopped. In 2 patients, a second preparation of IL-3 (Sandoz Canada Inc, Dorval, Quebec, Canada) was initiated in a dose escalation regimen of 2.5 to 10 micrograms/kg and was coadministered with ferrous sulphate. No erythroid response was observed in either patient, and in one of the two, alternate-day subcutaneous recombinant erythropoietin at 300 U/kg was administered for 3 weeks in combination with daily IL-3 at 10 micrograms/kg, but no increased erythropoiesis was seen. Significant increases in white blood cell and eosinophil counts during administration of both preparations of IL-3 were observed in all patients. These data show that the response of DBA patients to IL-3 in vivo is heterogeneous and cannot be predicted from in vitro studies. The absence of a corrective effect of IL-3 in these patients with DBA indicates that a deficiency of the cytokine is not central in the pathogenesis of the disorder.

Several life-threatening complications of the common disorder sickle cell disease require management with red blood cell transfusions and, hence, long-term iron-chelating therapy. The efficacy of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) has not previously been determined in patients with sickle cell disease. We compared the efficacy of L1 to that of standard-dose subcutaneous deferoxamine in four regularly transfused patients with homozygous sickle cell disease, who had evidence of severe iron overload and a history of poor compliance with deferoxamine. Determination of 24-hour urinary iron excretion conducted over 5 days immediately after transfusion showed that the mean daily urinary iron excretion induced by L1 at 75 mg/kg/d (0.48 +/- 0.23 mg/kg) was equivalent to that induced by deferoxamine at 50 mg/kg/d (0.39 +/- 0.06 mg/kg). In two of three patients studied, a significant (P < .025) increase in mean daily urinary iron excretion was achieved when the dose of L1 was increased to 100 mg/kg/d. Total iron balance studies, which quantitated both urinary and stool iron excretion on L1 and deferoxamine, determined that mean total daily iron excretion induced by deferoxamine (0.88 +/- 0.05 mg/kg) was significantly greater (P < .05) than that induced by L1 (0.53 +/- 0.17 mg/kg), attributable to the significantly greater stool iron excretion during deferoxamine treatment (0.50 +/- 0.16 mg/kg/d) compared with that measured during L1 treatment (0.12 +/- 0.08 mg/kg/d, P < .01). Stool iron excretion accounted for a significantly greater percentage of total iron excretion during deferoxamine treatment (59% +/- 20%) than during L1 treatment (23% +/- 14%, P < .01). These iron balance studies are the first to compare total iron excretion induced by L1 with that achieved by deferoxamine. They demonstrate that the mean total daily iron excretion during L1 treatment (0.53 +/- 0.17 mg/kg) is sufficient to maintain net negative iron balance in most regularly transfused patients with sickle cell disease. Because long-term compliance with L1 has been shown previously to be superior to that with deferoxamine in patients with homozygous beta-thalassemia, the use of L1 should increase the long-term effectiveness of iron chelation in patients with sickle cell disease.

Iron overload cardiomyopathy is an old disease that has evolved from a rare undiagnosable and untreatable condition to a now much more common, diagnosable, and potentially treatable condition. Pathologically it is due to a direct free iron effect on the myocytes, and not due to interstitial infiltration. This also implies that the disease process is reversible if the tissue iron concentration can be controlled. The advent of magnetic resonance imaging and future genetic identification can identify the population at risk. Chelation therapy, including newer forms of oral chelators, likely will be more commonly available to benefit an ever increasing number and spectrum of the population. Further active research will be needed to improve our pathophysiological understanding and clinical treatment of this increasingly common condition.

Purpose: Stimulating expression of the normal fetal globin genes is a preferred method of ameliorating sickle cell disease and beta-thalassemia for the majority of patients in North America who do not have appropriate bone marrow donors., Patients and Methods: Due to increased survival of red blood cells that contain both hemoglobin S and hemoglobin F, as little as 4-8% fetal globin synthesis in the bone marrow can produce levels of hemoglobin F of approximately 20% in the peripheral circulation. Some success has been achieved in stimulating hemoglobin F using chemotherapeutic agents (such as hydroxyurea and 5-azacytidine) and growth factors (erythropoietin) that alter erythroid growth kinetics. However, there is reluctance to treat children with chemotherapeutic agents because of possible undesirable long-term side effects., Results: Butyric acid and butyrate derivatives are generally safe compounds that stimulate the promoters of individual fetal and embryonic globin genes and thus provide a more specific therapy. An initial trial with the parent compound, given as arginine butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that can ameliorate these conditions. Phase I trials of an oral butyrate derivative with a long plasma half-life have begun., Conclusions: These agents may provide a new and specific approach for ameliorating the clinical manifestations of sickle cell disease and beta-thalassemia.

Objective: To examine the effect of frequency of oral administration of 1,2-dimethyl-3-hydroxypyrid-4-one (L1) on urinary iron excretion., Hypothesis: Sustained serum concentrations of L1 will cause more iron chelation than the same daily dose given in larger but less frequent amounts., Patients and Methods: Ten patients with thalassemia with a mean age of 20.9 +/- 4.7 years (range, 13 to 27 years), who were receiving regular treatment with 75 to 100 mg/kg/day oral L1, received 75 mg/kg/day L1 orally in equally divided doses: every 6 hours for 3 days and every 12 hours for 3 days. The two study periods occurred 1 month apart immediately after the monthly blood transfusions. Urine was collected for two consecutive 24-hour periods during each of the different schedules. Serial blood samples were collected from six patients over a 6-hour period and analyzed for total L1 and the L1 glucuronide metabolite concentrations., Results: The patient's mean hemoglobin levels (138.8 +/- 12.5 and 139.0 +/- 11.6 gm/L) and ferritin levels (2856.4 +/- 2207.8 and 2890.0 +/- 2264.4 micrograms/L) were similar during the every-6-hour and every-12-hour L1 administrations, respectively. There was significantly more urinary iron excretion when L1 was administered every 6 hours (0.59 +/- 0.29 mg/kg/day) versus every 12 hours (0.40 +/- 0.26 mg/kg/day; p = 0.0129). Calculated 24-hour area under the plasma concentration-time curve of L1 was similar during the every-6-hour (7023.9 +/- 2637.8 mg.min/L) and every-12-hour (7050.1 +/- 1668.8 mg.min/L) experiments., Conclusions: These data suggest that the sustained presence of L1 in the blood results in greater chelation of iron than that observed with larger, less frequent doses.

Given the mortality and morbidity associated with acute iron intoxication, effective iron chelation which is easily administered in an emergency situation would be ideal. The pharmacokinetics of L1 were examined in 5 healthy adult male volunteers to assess its potential for use in acute iron overload. Ferrous sulfate (600 mg), L1 (900 mg), and ferrous sulfate and L1 were administered on three separate days, each one week apart. On each test day, blood samples were collected at regular intervals for the measurement of plasma L1 and total iron. Pharmacokinetic values were calculated. The data were also compared to that obtained in 10 patients with beta-thalassemia and chronic iron overload. In the normal volunteers, a 20% decrease in the area under the concentration time curve of plasma iron and of plasma L1 was demonstrated when they were co-administered. There was no change in urinary iron excretion when L1 was given with iron (p = 0.414). The elimination half-life of L1 in the thalassemia patients (137.65 +/- 48.65 min) was significantly longer than that in the healthy volunteers (77.56 +/- 13.0) (p = 0.0047) due to larger apparent volume of distribution. In all of the iron-overloaded individuals L1 resulted in increased urinary iron excretion. None of the other pharmacokinetic variables compared were significantly different between these two groups. These studies indicate that at levels below saturation, transferrin does not allow L1 to remove absorbed iron in healthy volunteers, whereas in thalassemia patients, who are beyond saturation of their iron binding capacity, the drug binds iron and promotes its excretion.

Accumulation of Fe in the myocardium in circumstances of transferrin saturation is associated with heart failure in Fe-loaded patients. To characterize the underlying causes of this phenomenon, we measured the flux as well as the speciation of Fe in normal and Fe-loaded cultures of rat myocardiocytes. Fe loading with low-molecular-weight Fe (ferric ammonium citrate) promoted a dose- and time-dependent increase in the rate of uptake of non-transferrin-bound Fe (NTBI) that was positively correlated (R = 0.9, p < 0.005) with cellular iron content. At concentrations sufficient to produce this up-regulation, membrane integrity was unaffected but the rate of spontaneous beating of the cells was decreased by 60%. The enhanced rate of NTBI uptake in Fe-loaded cells reverted to control rates after treatment with therapeutic concentrations of Fe chelators deferoxamine, 1,2-dimethyl-3-hydroxypyrid-4-one and 1,2-diethyl-3-hydroxypyrid-4-one under conditions where approximately 80% of the cellular Fe was removed by chelation. Fe loading of cultured myocytes also induced shifts in Fe speciation. Thus the ratio of Fe bound in hemosiderin-like precipitates to ferritin-bound Fe increased twofold, from a range of 0.84 to 1.44 in control cells to 1.96 to 3.3 in iron-loaded cells. This increased ratio was similar to that measured in the heart and liver of a thalassemic patient who underwent a double transplant for the failure of both organs, even though the Fe content of the heart (mean, 5.8 mg Fe/gm dry weight) was much less than that of the liver (28.1 mg/gm dry weight). These results suggest that increased rates of uptake of NTBI may exacerbate iron loading of the heart and contribute to iron-mediated cardiotoxicity, whereas the clinical benefits of chelation therapy may be enhanced by the down-regulation of NTBI uptake.

Background: Fetal-globin (gamma-globin) chains inhibit the polymerization of hemoglobin S (sickle hemoglobin) and can functionally substitute for the beta-globin chains that are defective or absent in patients with the beta-thalassemias. Identifying safe mechanisms to stimulate fetal-hemoglobin production is therefore of great interest. Previous studies have shown that administering butyrate selectively stimulates the promoter of the human fetal-globin gene and leads to increases in gamma-globin--gene expression in the developing fetus, cultured cells, and animal models., Methods: To determine whether butyrate can stimulate fetal-globin production in humans, we treated three patients (3 to 13 years old) with sickle cell anemia and three patients (7 to 27 years old) with beta-thalassemia syndromes with a short course of intravenous infusions of arginine butyrate. The drug was infused continuously for either two or three weeks; the initial dose was 500 mg per kilogram of body weight per day. Globin-chain ratios, proportions of reticulocytes producing hemoglobin F (F reticulocytes), and levels of gamma-globin messenger RNA (mRNA) were determined before and during treatment., Results: In all six patients, fetal-globin synthesis increased by 6 to 45 percent above pretreatment levels (P < 0.01). The proportion of F reticulocytes increased about twofold, and the level of gamma-globin mRNA increased twofold to sixfold. The increase in gamma-globin synthesis led to improvement in the globin-chain ratios in the patients with thalassemia. The treatment of one patient was extended for seven weeks, and her hemoglobin level increased from 4.7 to 10.2 g per deciliter (2.9 to 6.3 mmol per liter). Side effects were minimal; one patient had a transient increase in serum aminotransferase concentrations., Conclusions: In patients with beta-hemoglobinopathies butyrate, a natural fatty acid, can significantly and rapidly increase fetal-globin production to levels that can ameliorate beta-globin disorders. Further trials of this class of compounds are warranted to determine long-term tolerance and efficacy in patients with sickle cell anemia or beta-thalassemia.

Despite the successes of deferoxamine (DFO) in the treatment and prevention of iron overload, an effective orally available iron chelating drug is needed, since erratic compliance with irritating, cumbersome parenteral infusions still results in fatal iron accumulation in many patients. Disorders of increased iron absorption should also benefit from the development of safe and effective iron chelating agents. Individuals with non-transfusion-dependent thalassemia (thalassemia "intermedia"), exhibit excessive dietary iron absorption that can lead to serious iron loading by the second or third decade of life. An orally effective iron-chelating drug would have major therapeutic advantages for all these patients.

The frequency of beta-thalassemia alleles among Filipinos is estimated to be 0.02, although little is known about the actual mutations represented in this population. Here, we describe a Filipino family in which a child has severe beta-thalassemia major. Molecular studies of the family revealed that the proband is a compound heterozygote for two previously unreported beta zero-thalassemia alleles: a frameshift mutation (-TG) at codon 67 and a deletion of the entire beta-globin gene. The 5' endpoint of this novel deletion is located approximately 3.9 kb to approximately 4.3 kb upstream of beta-globin gene, and the deletion extends 3' beyond the beta-globin gene for an undetermined distance. The occurrence of two novel beta-thalassemia alleles in a single family suggests that the Filipino population may have a unique spectrum of beta-thalassemia alleles.

Ten patients on transfusion-chelation programs were treated with bone marrow transplantation (BMT). The busulfan-cyclophosphamide conditioning regimen was inadequate in the 6 beta thalassemia major patients, resulting in marrow rejection problems for 3. Post BMT one patient remains with thalassemia major and two have mixed chimerism with a clinical status of thalassemia intermedia. Three patients have thalassemia minor from their donors, but one required a second BMT to achieve this state. Two of the four Diamond Blackfan Syndrome patients died of BMT-related complications. Two achieved normal hematopoiesis, one at the price of chronic graft vs host disease. Because of the excellent results of iron chelation in our patients, we question whether such patients with access to the state of the art transfusion-chelation programs should continue to be transplanted. This situation requires continuing evaluation.

The emulsion of lidocaine and prilocaine (EMLA) is effective in preventing the pain of venipuncture in children. It is therefore important to identify children who could benefit the most from EMLA. We studied the safety and feasibility of two methods of application of EMLA (patch and cream) in a randomized, open-label trial of EMLA patch versus EMLA cream in 160 children with chronic diseases undergoing venipuncture. EMLA patch or cream was applied 60 to 120 min before puncture. Pain was assessed by the children using a visual analogue score. Children also scored the pain of their last puncture and the pain of removing the tape. EMLA patch and cream had similar efficacy (visual analogue scores for the venipuncture were 8.5 +/- 16 and 9.5 +/- 17 out of 100, respectively). Side effects occurred in similar frequencies in the two groups. Adhesiveness of the patch was less effective than that of the cream with Tegaderm. Age was a major determinant of pain perception; younger children recalled more severe pain in their previous puncture. Children recently diagnosed had higher visual analogue scores than those with a long history of chronic disease. We concluded that EMLA patch and cream have a similar efficacy in children undergoing venipuncture. Young children recently diagnosed with chronic disease are most likely to benefit from EMLA.

The objectives of the study were to compare the efficacy and safety of a continuous infusion (CIV) of morphine and intermittent parenteral opioids (IPO) in children with sickle cell vaso-occlusive crises (VOCs); to determine whether 50% oxygen administration through a face mask can reduce the duration of severe pain in patients receiving CIV morphine; and to measure morphine concentration at steady state for pharmacokinetic and pharmacodynamic analysis in patients receiving CIV morphine. The study was designed as a prospective, controlled, "before-and-after" evaluation of two different analgesic regimens. For patients receiving CIV morphine, there was a randomized, double-blind, placebo-controlled study of O2 vs. air. The patients were 66 children with sickle cell disease, 3-18 years old, requiring opioid therapy for severe VOC (32 patients in phase A, 34 in phase B). The analgesic regimens were as follows: phase A: meperidine, morphine, or codeine IM or IV bolus every 3 or 4 hours; phase B: morphine sulfate, loading dose 0.15 mg followed by CIV 0.04 mg/kg/hr. The infusion rate was adjusted every 8 hours according to pain and/or symptoms of opioid toxicity. Pain assessment was by behavioral observation (BPS). In terms of results, the mean opioid dose (morphine equivalent) was similar in both groups (0.032 +/- 0.020 mg/kg/hr in phase A and 0.035 +/- 0.011 in phase B). However, the duration of severe pain was significantly shorter in phase B (0.9 +/- 1.0 days) than in phase A (2.0 +/- 1.8 days). No severe opioid toxicity was observed in either group. Oxygen did not shorten the duration of severe pain compared to the placebo group (0.94 +/- 1.08 and 0.95 +/- 1.19 days, respectively) nor did it prevent the appearance of new pain sites. Pharmacokinetic analysis was performed in 24 patients of phase B. Total body clearance (TBC) of morphine was greater in children before puberty than after (40.4 +/- 10 vs. 28 +/- 11 mL/kg/min; p < 0.05). In conclusion, in children with severe VOCs, continuous infusion of morphine provides better analgesia than intermittent opioid therapy. Fifty percent oxygen inhalation had no effect on the duration of pain.

Iron accumulating to excess in tissues of humans and animal models occurs mainly as complexes with transferrin, ferritin, other hemoproteins, and insoluble hemosiderin particles. To determine the distribution of Fe amongst these molecular species, we have used inductively coupled plasma-mass spectrometry as a means of on-line, isotope-specific detection for their liquid chromatographic separation. The stable isotope 57Fe is a suitable isotope for monitoring the Fe content of each fraction, and its availability at high isotopic enrichment makes it an attractive choice for tracer studies when the use of a radioisotope is undesirable, e.g., in human subjects. The detection system offers the advantages of high sensitivity (detection limits in the parts per billion range), a wide dynamic range (linearity of the calibration curve over several orders of magnitude), and on-line analysis facilitating real-time evaluation of the chromatographic separation, in addition to isotope-specific information. The Fe distributions in healthy rat livers, liver and heart tissue from Fe-loaded human subjects, and human hepatocyte cultures are reported. The ferritin:hemosiderin ratio in these samples is shown to be an indicator of the degree of Fe loading and correlates well with that determined by Zeeman-corrected electrothermal atomic absorption as an alternative means of detection.

A new regimen of 24-hr ambulatory continuous intravenous infusion of deferoxamine (CIV DFO) through central venous ports was instituted in nine patients aged (mean +/- SD) 22.4 +/- 5.8 years over a period of 15.7 +/- 7.3 months. Central venous infusion sites were changed weekly in the clinic, eliminating the necessity for reconstitution of DFO and needle insertion at home. Because CIV DFO could be interrupted only by medical personnel, patient compliance was documented accurately; patients administered 93.0% +/- 3.2% of CIV DFO prescribed. Mean urinary iron excretion on CIV DFO (66.8 +/- 50.4 mg/24 hr) was significantly greater than that quantitated during 12-hr equivalent-dose subcutaneous DFO infusions (23.4 +/- 18.3 mg/24 hr; P less than 0.025). Mean serum ferritin declined by 71% over the treatment period (P less than 0.005). This regimen confers the advantages of uninterrupted exposure to DFO, is associated with excellent patient compliance, and should be considered in any patient with severe iron overload and erratic compliance with DFO.

The effect of oxygen therapy on the number of irreversibly (ISC) and reversibly (RSC) sickled cells was studied in patients with sickle cell anemia. Inhalation of 50% oxygen in patients who were not in crisis produced a significant fall in RSCs and a lesser fall in ISCs. Twenty-five subjects in sickle cell crisis were chosen at random to receive either oxygen (15 patients) or air (10 patients). Those who received oxygen showed a significant reduction in RSCs, but not in ISCs. No significant change in RSCs or ISCs occurred in the group who received air. Despite the reduction in RSCs in the oxygen-treated group, there was no significant difference between the air and oxygen groups in the duration of severe pain, opioid administration, and hospitalization. It was also observed that crisis was associated with arterial desaturation and a reduction in the number of RSCs. We conclude that, although RSCs may be involved in the pathophysiology of sickle cell crisis, reduction in RSCs by oxygen therapy in these studies did not result in any reduction in the duration of crisis.

In patients with thalassemia intermedia in whom hyperabsorption of iron may result in serious organ dysfunction, an orally effective iron-chelating drug would have major therapeutic advantages, especially for the many patients with thalassemia intermedia in the Third World. We report reduction in tissue iron stores and normalization of serum ferritin concentration after 9-month therapy with the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in a 29-year-old man with thalassemia intermedia and clinically significant iron overload (SF 2,174 micrograms/L, transferrin saturation 100%; elevated AST and ALT, abnormal cardiac radionuclide angiogram) who was enrolled in the study with L1 75 mg/kg/day after he refused deferoxamine therapy. L1-Induced 24-hour urinary iron excretion during the first 6 months of therapy was (mean +/- SD, range) 53 +/- 30 (11 to 109) mg (0.77 mg/kg), declining during the last 3 months of L1 to 24 +/- 14 (13-40) mg (0.36 mg/kg), as serum ferritin decreased steadily to normal range (present value, 251 micrograms/L). Dramatic improvement in signal intensity of the liver and mild improvement in that of the heart was shown by comparison of T1-weighted spin echo magnetic resonance imaging with images obtained immediately before L1 administration was observed after 9 months of L1 therapy. Hepatic iron concentration decreased from 14.6 mg/g dry weight of liver before L1 therapy to 1.9 mg/g liver after 9 months of therapy. This constitutes the first report of normalization of serum ferritin concentration in parallel with demonstrated reduction in tissue iron stores as a result of treatment with L1. Use of L1 as a therapeutic option in patients with thalassemia intermedia and iron overload appears warranted.

A new hematopoietic growth factor (Steel factor) has been identified which stimulates erythroid proliferation both in vitro and in vivo. We evaluated the influence of recombinant Steel factor on hemoglobin synthesis in peripheral blood (PB) BFU-E-derived cells from normal donors by radioimmunoassay (RIA) and compared it with stimulation with GM-CSF and interleukin-3 (IL-3). Only Steel factor stimulated a significant increase in BFU-E-derived colony size and a significant increase in fetal hemoglobin (HbF) in BFU-E-derived erythroblasts from 0.49% +/- 0.27% to 6.33% +/- 1.11% in serum-deprived media and from 1.88% +/- 0.24% to 11.17% +/- 0.91% in serum. To determine whether this influence on hemoglobinization also occurred in sickle cell disease, we studied 13 patients with sickle cell disease. In serum-deprived conditions, there was a significant increase in the number and size of BFU-E-derived colonies with Steel factor that was dose-dependent. In addition, the proportion of HbF in progenitor-derived cells increased by 66% from 4.1% +/- 0.6% to 6.8% +/- 1.2% with Steel factor. In serum-containing conditions studied in 12 patients, the increase in percentage of HbF was even greater, from 10.7% +/- 0.9% in control cultures to 22.5% +/- 2.6% with Steel factor. These increases in percentage of HbF were significant and dose-dependent. An increase in percentage of HbF was observed in erythroblasts harvested on day 11, 14, and 18 of culture. A decrease in mean picograms of total Hb per cell after coculture with Steel factor was noted, suggesting that growth kinetics influenced complete hemoglobinization. In serum-deprived conditions, picograms of HbF per cell was not affected by Steel factor, and in serum-containing conditions that augment in vitro HbF production it was enhanced. Thus, Steel factor stimulated a significant increase in percentage of HbF in erythroid cells from normal donors and patients with SCA in vitro.

We have determined the beta S haplotypes in 709 patients with sickle cell anemia, 30 with SC disease, 91 with S-beta-thalassemia, and in 322 Hb S heterozygotes from different countries. The methodology concerned the detection of mutations in the promoter sequences of the G gamma- and A gamma-globin genes through dot blot analysis of amplified DNA with 32P-labeled probes, and an analysis of isolated Hb F by reversed phase high performance liquid chromatography to detect the presence of the A gamma T chain [A gamma 75(E19)Ile----Thr] that is characteristic for haplotype 17 (Cameroon). The results support previously published data obtained with conventional methodology that indicates that the beta S gene arose separately in different locations. The present methodology has the advantage of being relatively inexpensive and fast, allowing the collection of a vast body of data in a short period of time. It also offers the opportunity of identifying unusual beta S haplotypes that may be associated with a milder expression of the disease. The numerous blood samples obtained from many SS patients living in different countries made it possible to compare their hematological data. Such information is included (as average values) for 395 SS patients with haplotype 19/19, for 2 with haplotype 17/17, for 50 with haplotype 20/20, for 2 with haplotype 3/3, and for 37 with haplotype 31/31. Some information on haplotype characteristics of normal beta A chromosomes is also presented.

Two novel beta-thalassemia mutations are described. The first mutation, found in an Italian family, is a G----A substitution in nucleotide (nt) +22 relative to the beta-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3' downstream. The second mutation, found in an Irish family, is a T----C substitution in nt +1570, or 12 bp 5' upstream of the AATAAA polyadenylation signal in the 3' noncoding region. It is postulated that this mutation leads to destabilization of the encoded beta-globin mRNA.

We reviewed the linear growth and growth plate morphology in all children with homozygous beta thalassemia followed in Toronto, for whom monthly height percentiles were available before, and for a 36-month period after, the initiation of nightly subcutaneous deferoxamine therapy. All patients were less than 7 years of age when begun on deferoxamine, and had received nightly deferoxamine for a minimum of 36 months. Marked abnormalities of the metaphyseal growth plate were readily observed in the distal ulnar, radial, and tibial metaphyses in 11 of 37 patients in whom a significant decline in mean height percentile was also noted. (In 10 of these 11 patients, height was less than the 15th percentile after 36 months.) These 11 patients had received a significantly greater (p less than 0.025) initial and average daily dose of deferoxamine, and had maintained a significantly lower (p less than 0.025) mean serum ferritin concentration over the 36 months, than the remainder of the cohort. To determine whether deferoxamine played a causative role in growth failure, growth in patients who began deferoxamine before the age 2 years was compared to that of patients who began after age 5 years, for the period between 2 and 5 years of age. Only patients begun on deferoxamine prior to age 2 years demonstrated a significant (p less than 0.01) decline in height percentile by the third year, implicating deferoxamine therapy as the cause of growth failure. We conclude that both the decline in height percentile and the bony changes observed in well-chelated patients are directly related to deferoxamine therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Diamond-Blackfan anemia is a congenital disorder of erythropoiesis in humans, characterized by a macrocytic anemia often associated with physical anomalies. Mutations at either the W or Steel loci in the mouse also leads to a severe macrocytic anemia, as well as other developmental abnormalities. The W locus encodes the proto-oncogene c-kit, a member of the receptor tyrosine kinase family, while the Steel locus encodes a potent hematopoietic growth factor that is the ligand for c-kit. Growth of clonogenic marrow erythroid progenitor cells in vitro in the presence of the recombinant hematopoietic growth factors interleukin-3 (IL-3) and Steel was used to characterize this disease at the cellular level. Three patterns of in vitro marrow response to both recombinant IL-3 or Steel were observed among 10 Diamond-Blackfan patients: those that responded quantitatively and qualitatively almost as well as cells from normal marrow, those that responded at an intermediate level, and those that did not respond at all. These results provide evidence for cellular heterogeneity underlying the pathogenesis of this disorder and therefore raise the possibility that there may be more than one underlying molecular basis for the disease. No gross abnormalities in the structure of either the c-kit or Steel loci were observed in these patients. The normal response in culture of the progenitor cells from at least some patients to Steel with or without IL-3 raises the possibility of using this novel growth factor as a therapeutic agent in Diamond-Blackfan anemia.

To assess the effects of deferoxamine (DFO) on the kidneys, we studied 27 patients with thalassemia major on chronic subcutaneous (s.c.) DFO therapy. In 41% of the patients glomerular filtration rate (GFR) values were above the normal range. In a previous study similar findings were reported for thalassemia patients who did not receive DFO. The subcutaneous administration of DFO was associated with a clinically significant decrease in GFR in 40% of the patients and in a mild decrease in another 40%. In all cases of severe decreases in GFR, it tended to return to baseline values upon discontinuation of DFO. There was a significant increase in urine volume during DFO therapy. These changes are consistent with our previous observation in humans and dogs receiving high dose i.v. DFO, albeit milder.

Single-dose and steady-state pharmacokinetics of the new oral iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1) were studied in 14 patients with thalassemia and correlated with iron excretion. Food prolongs the rate of absorption of L1, but it does not affect significantly the extent of absorption measured by the area under the plasma concentration-time curve. Similarly, it does not affect the chelation potential of the drug. The mean elimination half-life of the drug is 3 hours, suggesting that a divided dose every 8 hours may assure better chelation. Our steady-state studies reveal that urinary iron excretion is independently influenced by body iron load (measured by ferritin levels) and by steady-state trough concentrations of the drug. While patients were receiving an unchanged regimen of 75 mg/kg/day, we have detected a gradual and significant decrease in trough concentrations in the presence of unchanged patients' compliance monitored by the Medication Event Monitoring System, diaries, and pill count. These findings suggest self-induction of L1 metabolism or decreased absorption during long-term therapy. Because of the concentration-dependent iron excretion, patients may need increasing doses to achieve negative iron balance.

A new beta zero-thalassemia mutation, a frameshift mutation with an insertion of a single cytosine nucleotide in codon 27-28, is described. The propositus, who is compound heterozygous for this mutation and the IVSII-654 C----T beta zero-thalassemia mutation, has the phenotype of severe beta-thalassemia major.