Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis.

Animal models indicate that the antimicrobial peptide hepcidin (HAMP; OMIM 606464) is probably a key regulator of iron absorption in mammals. Here we report the identification of two mutations (93delG and 166C-->T) in HAMP on 19q13 in two families with a new type of juvenile hemochromatosis.

Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.

Juvenile hemochromatosis is an early-onset autosomal recessive disorder of iron overload resulting in cardiomyopathy, diabetes and hypogonadism that presents in the teens and early 20s (refs. 1,2). Juvenile hemochromatosis has previously been linked to the centromeric region of chromosome 1q (refs. 3-6), a region that is incomplete in the human genome assembly. Here we report the positional cloning of the locus associated with juvenile hemochromatosis and the identification of a new gene crucial to iron metabolism. We finely mapped the recombinant interval in families of Greek descent and identified multiple deleterious mutations in a transcription unit of previously unknown function (LOC148738), now called HFE2, whose protein product we call hemojuvelin. Analysis of Greek, Canadian and French families indicated that one mutation, the amino acid substitution G320V, was observed in all three populations and accounted for two-thirds of the mutations found. HFE2 transcript expression was restricted to liver, heart and skeletal muscle, similar to that of hepcidin, a key protein implicated in iron metabolism. Urinary hepcidin levels were depressed in individuals with juvenile hemochromatosis, suggesting that hemojuvelin is probably not the hepcidin receptor. Rather, HFE2 seems to modulate hepcidin expression.

beta(S)-Globin gene cluster haplotypes in the West Bank of Palestine.

Sickle cell disease is an inherited autosomal recessive disorder of the beta-globin chain. In Palestine it is accompanied by a low level of Hb F (mean 5.14%) and a severe clinical presentation. In this study, 59 Palestinian patients, homozygotes for Hb S were studied for their haplotype background. Eight polymorphic sites in the beta-globin gene cluster were examined. The Benin haplotype was predominant with a frequency of 88.1%, followed by a frequency of 5.1% for the Bantu haplotype. One chromosome was found to carry the Cameroon haplotype (0.85%). Three atypical haplotypes were also found (5.95%). Heterogeneity was observed in Hb F production, ranging between 1.5 and 17.0%, whereas the (G)gamma ratio was homogeneous among all haplotypes with a normal amount of about 41%. Our results are in agreement with previous reports of the Benin haplotype origin in the Mediterranean.

Analysis of SLC40A1 gene at the mRNA level reveals rapidly the causative mutations in patients with hereditary hemochromatosis type IV.

Mutations in the SLC40A1 gene result in a dominant genetic disorder [ferroportin disease; hereditary hemochromatosis type (HH) IV], characterized by iron overload with two different clinical manifestations, normal transferrin saturation with macrophage iron accumulation (the most prevalent type) or high transferrin saturation with hepatocyte iron accumulation (classical hemochromatosis phenotype). In previous studies, the mutational analysis of SLC40A1 gene has been performed at the genomic DNA level by PCR amplification and direct sequencing of all coding regions and flanking intron-exon boundaries (usually in 9 PCR reactions). In this study, we analyzed the SLC40A1 gene at the mRNA level, in two RT-PCR reactions, followed by direct sequencing and/or NIRCA (non-isotopic RNase cleavage assay). This protocol turned out to be rapid, sensitive and reliable, facilitating the detection of the SLC40A1 gene mutations in two patients with hyperferritinemia, normal transferrin saturation and iron accumulation predominantly in macrophages and Kupffer cells. The first one displayed the well-described alteration V162 Delta and the second a novel mutation (R178G) that was further detected in two relatives in a pedigree analysis. The proposed procedure would facilitate the wide-range molecular analysis of the SLC40A1 gene, contributing to better understanding the pathogenesis of the ferroportin disease.

Medical students' attitudes on diversity when applying to Toronto's ophthalmology residency program.

BACKGROUND: Affirmative action is a controversial admissions policy practised by universities in the United States and other countries around the world. It is currently not used at the University of Toronto ophthalmology residency program. A survey was conducted to determine the opinions of applicants as to the role that affirmative action and quotas should play during the admissions process and to determine the current ethnic breakdown of the applicants to ophthalmology. METHODS: A survey of 14 questions was sent out to all 72 medical students applying for a residency position in our program. The response rate was 58%. The students were asked to agree or disagree on a 5-point Likert scale with statements related to ethnicity, gender, and whether affirmative action policies exist or should exist for certain groups. RESULTS: The majority of the respondents (26/42, 62%) considered themselves an ethnic minority, and 57% (24/42) considered themselves a visible minority. Most (32/42) felt that the sex of the applicant should not play a role in the selection process. Only 24% (10/42) supported affirmative action, and only 12% (5/42) supported quotas for minority applicants. INTERPRETATION: The majority of survey respondents in this study did not support affirmative action or quotas at the University of Toronto ophthalmology program. The applicants to this program represent a diverse group of individuals from a multitude of ethnic, cultural, and racial backgrounds, and, in their average opinion, affirmative action policies would not benefit our admissions program.

Treatment of Waldenström's macroglobulinemia with rituximab.

PURPOSE: Waldenström's macroglobulinemia (WM) is a low-grade lymphoplasmacytic lymphoma in which CD20 is usually expressed on tumor cells. There is evidence that patients with WM may benefit from treatment with the anti-CD20 monoclonal antibody rituximab. We performed a prospective phase II study to clearly define the activity of rituximab in patients with this disease. PATIENTS AND METHODS: Twenty-seven patients with WM were treated with rituximab 375 mg/m(2) intravenously (IV) for 4 weeks. Three months after completion of rituximab, patients without evidence of progressive disease received repeat 4-week courses of this agent. All patients were symptomatic, their median age was 72 years, and 15 patients were previously untreated. RESULTS: Twelve patients (44%; 95% confidence interval, 25.5% to 64.7%) achieved a partial response after treatment with rituximab. Median time to response was 3.3 months (range, 2.2 to 7.1 months). Responses occurred in six (40%) of 15 previously untreated patients and in six (50%) of 12 pretreated patients. Patients with a serum immunoglobulin M less than 40 g/L had a significantly higher response rate. The median time to progression for all patients was 16 months, and with a median follow-up of 15.7 months, nine of 12 responding patients remain free of progression. Treatment with rituximab was well tolerated, with approximately one fourth of patients experiencing some mild form of infusion-related toxicity, usually fever and chills. CONCLUSION: Our prospective data indicate that rituximab is well tolerated and active in patients with WM. Previously untreated and pretreated patients seem to benefit equally. Repeat 4-week courses of rituximab may prolong the duration of response of the disease, but this observation requires confirmation in prospective, randomized trials. Furthermore, studies that will combine rituximab with chemotherapy may be relevant.

Extended rituximab therapy for previously untreated patients with Waldenström's macroglobulinemia.

Waldenström's macroglobulinemia is a low-grade lymphoplasmacytoid lymphoma characterized by CD20 expression on malignant cells. Several studies have indicated that the anti-CD20 monoclonal antibody rituximab has activity against this disease. Thus, we performed a prospective study in which 17 previously untreated patients with symptomatic macroglobulinemia were treated with rituximab 375 mg/m2 intravenously for 4 weeks. Three months after completion of rituximab, patients without evidence of progressive disease received repeat 4-week courses of this agent. Six patients (35%) achieved a partial response after extended treatment with rituximab. Median time to response was 3 months. The median time to progression (TTP) for all patients was 13 months. One of 6 responding patients has progressed at 10 months, while the other 5 patients remain progression free with a follow-up range of > 22-40 months. Eight patients (47%) were rated as stable disease, and their median TTP was 9 months. Treatment with rituximab was well tolerated and was not associated with myelosuppression; one third of the patients experienced infusion-related toxicity, usually fever and chills of mild degree. Our prospective trial of extended rituximab therapy for previously untreated patients with Waldenström's macroglobulinemia indicates that this agent is active, well tolerated, and might be associated with a long period without the need for further treatment. Studies that will combine rituximab with chemotherapy or with other monoclonal antibodies might be of interest.

Role of the interview in admissions at the University of Toronto ophthalmology program.

OBJECTIVE: To investigate the role of the residency interview in admission to the University of Toronto ophthalmology program. DESIGN: Retrospective observational study. PARTICIPANTS: One hundred and sixty-nine candidates for admission to the University of Toronto ophthalmology program, 1998-2008. METHODS: Admissions scores were analyzed retrospectively to determine how the interview affected final candidate rank. Interviewers were not blinded to the candidates' applications. Candidate impressions of interview day were investigated through 2 surveys conducted in 2000 and 2005. Twenty-six of the 33 interviewed candidates responded, for a response rate of 79%. RESULTS: Pre-interview rank was found to correlate with final rank (r = 0.826, p < 0.001, n = 169, 2-tailed) and interview rank (r = 0.426, p < 0.001). Interview rank was also independently correlated with final rank (r = 0.774, p < 0.001). Half the candidates had final ranks within 1 of their pre-interview rank, and 80% were within 3 ranks. The range of rank change was from a loss of 10 ranks to a gain of 11. Survey responses demonstrated that candidates found the interview experience very valuable (14/14) and that it affected their opinion of the program positively (20/26). CONCLUSIONS: While the interview frequently does not substantially change candidate rank order, the occasional large changes suggest that it has the power to make corrections to application scores should they be necessary. The nonblinded design allows interviewers to ask candidates questions specific to their application, resulting in more profound discussion. The surveys show that candidates find the interview to be a valuable experience and likely results in them ranking Toronto's program higher in the match.

Hypochromic erythrocytes (%): a reliable marker for recognizing iron-restricted erythropoiesis and predicting response to erythropoietin in anemic patients with myeloma and lymphoma.

The aim of the study was to evaluate the role of hypochromic erythrocytes (HYPO%) compared to "traditional" and novel markers of iron status and erythropoiesis in recognizing iron-restricted erythropoiesis (IRE) and predicting response to erythropoietin (rHuEPO) in anemic patients with myeloma and lymphoma. Forty-one newly diagnosed patients who received epoetin-beta at a subcutaneous weekly dose of 30,000 IU for 6 weeks were studied. Response to rHuEPO was observed in 27 patients (65.8%). Twelve non-responders received, additionally, 200 mg of IV iron sucrose, weekly, for 4 weeks. Evaluation of markers was performed at baseline and on weeks 1, 2 and 6 for all patients and also on weeks 7-10 for non-responders to rHuEPO. Baseline HYPO%, at a cut-off value of <5%, and an increment in reticulocyte absolute number (RETICS-AB) >or= 50,000/microl and reticulocyte hematocrit (RETICS-Hct) >or= 50%, between baseline and week 2, were independent predictive factors for response to rHuEPO. We found that these markers had superior predictive value for response to rHuEPO than four widely used predictive models. Furthermore, a baseline HYPO% count of above 5% proved superior over serum ferritin < 100 ng/ml and transferrin saturation < 20% in recognizing IRE. In conclusion, baseline HYPO% either alone or in combination with RETICS-AB or RETICS-Hct after 2 weeks of rHuEPO treatment could be reliably used in predicting response to rHuEPO. Additionally, HYPO% has proved a reliable marker for recognizing IRE before rHuEPO treatment and, thus, could be used for identifying patients who will benefit from IV iron supplementation.

Evaluation of hypochromic erythrocytes in combination with sTfR-F index for predicting response to r-HuEPO in anemic patients with multiple myeloma.

The purpose of this study was to evaluate the sTfR-F index and hypochromic erythrocytes (HYPO%) as potential predictors of response to recombinant human erythropoietin (r-HuEPO) of anemic patients with multiple myeloma (MM) before treatment, as well as early in the course of treatment. Twenty-six newly diagnosed anemic MM patients received r-HuEPO 30,000 IU/wk sc, for six weeks. The sTfR-F index and HYPO% were determined at baseline and at weeks 2 and 6. Patients were classified in 1 of 4 categories of a diagnostic plot, according to erythropoietic state (ES I-IV), defined by the combination of sTfR-F index and HYPO%. Sixteen of 20 patients in ES I and II before treatment responded to r-HuEPO, whereas none of the 6 patients in ES III and IV responded (P < .001). At week 2, 44% of patients who responded and 60% of the nonresponders were in functional iron deficiency (FID) and the proportion increased to 69% and 80%, respectively, by week 6. Seven of the patients who did not respond received in addition 200 mg iron sucrose IV weekly, for the next 4 weeks, and 6 of them responded. These results suggest that combination of sTfR-F index and HYPO% in a diagnostic plot can be used as a predictive model to recognize patients who will benefit from r-HuEPO and identify FID requiring iron supplementation, before treatment and early in the course of treatment, contributing thus to optimization of r-HuEPO therapy.

Hepcidin in iron overload disorders.

Hepcidin is the principal regulator of iron absorption in humans. The peptide inhibits cellular iron efflux by binding to the iron export channel ferroportin and inducing its internalization and degradation. Either hepcidin deficiency or alterations in its target, ferroportin, would be expected to result in dysregulated iron absorption, tissue maldistribution of iron, and iron overload. Indeed, hepcidin deficiency has been reported in hereditary hemochromatosis and attributed to mutations in HFE, transferrin receptor 2, hemojuvelin, and the hepcidin gene itself. We measured urinary hepcidin in patients with other genetic causes of iron overload. Hepcidin was found to be suppressed in patients with thalassemia syndromes and congenital dyserythropoietic anemia type 1 and was undetectable in patients with juvenile hemochromatosis with HAMP mutations. Of interest, urine hepcidin levels were significantly elevated in 2 patients with hemochromatosis type 4. These findings extend the spectrum of iron disorders with hepcidin deficiency and underscore the critical importance of the hepcidin-ferroportin interaction in iron homeostasis.

On the mechanism of action of recombinant activated factor VII administered to patients with severe thrombocytopenia and life-threatening haemorrhage: focus on prothrombin activation.

We report the ex vivo effect of recombinant activated factor VII (rFVIIa) on prothrombin activation after whole blood clotting. Two patients with severe thrombocytopenia and life-threatening haemorrhage were successfully managed using a single dose of rFVIIa (90 microg/kg). Western blotting using antiprothrombin antibody showed that rFVIIa did not induce thrombin generation in citrated platelet-poor plasma. Patient sera showed significantly impaired prothrombin activation before and after rFVIIa administration. rFVIIa administration shortened the prothrombin time, activated partial thromboplastin time and Ivy bleeding time, and normalized the clot retraction. These data indicate that rFVIIa accelerated thrombin generation without significant increase of generated thrombin.

Effective hemostasis with rFVIIa treatment in two patients with severe thrombocytopenia and life-threatening hemorrhage.

We report two patients with severe thrombocytopenia and life-threatening bleeding that were successfully managed with recombinant activated factor VII (rFVIIa). The first was a 75-year-old male with Waldenström's macroglobulinemia. During a therapeutic course with fludarabine, he developed severe autoimmune thrombocytopenia resistant to conventional treatment, followed by persistent uncontrollable nasal bleeding. Platelet transfusions failed to increase the platelet count and control the hemorrhage. When hemoglobin levels fell below 8.5 g/dL and the patient's clinical condition got much worse, a single dose of 4.8 mg rFVIIa (90 microg/kg) was given as an i.v. bolus. Ten minutes after the rFVIIa injection, nasal bleeding stopped, the patient's clinical condition progressively improved, and splenectomy could be carried out uneventfully 2 days later. The second patient, a 52-year-old female, was under treatment for pre-B lymphoblastic leukemia. She developed severe thrombocytopenia, secondary to chemotherapy, complicated by massive gastrointestinal bleeding. Despite intensive treatment with platelet transfusions, hemorrhage continued and her condition deteriorated rapidly. She was then given an i.v. bolus injection of 4.8 mg rFVIIa, which resulted in cessation of hemorrhage and dramatic improvement of her clinical status. No adverse effects from the treatment with rFVIIa were observed. In conclusion, rFVIIa appears to be an attractive alternative for controlling hemorrhage in patients with severe thrombocytopenia, especially when platelet transfusions are unavailable or ineffective.

Genetic hyperferritinaemia and reticuloendothelial iron overload associated with a three base pair deletion in the coding region of the ferroportin gene (SLC11A3).

Iron overload may predominantly involve parenchymal or reticuloendothelial cells, the prototype of parenchymal iron overload being HFE-related genetic haemochromatosis. We studied a family with autosomal dominant hyperferritinaemia in whom the proband showed selective iron accumulation in the Kupffer cells on liver biopsy. Analysis of L and H ferritin genes excluded mutations responsible for hereditary hyperferritinaemia/cataract syndrome or similar translational disorders. Sequence analysis of the ferroportin gene (SLC11A3) in four individuals with hyperferritinaemia singled out a three base pair deletion in a region that contains four TTG repeats. This mutation removes a TTG unit from 780 to 791, and predicts the loss of one of three sequential valine residues 160-162. Denaturing high performance liquid chromatography can be used for its detection. SLC11A3 polymorphism analysis indicates that this probably represents a recurrent mutation due to slippage mispairing. Affected individuals may show marginally low serum iron and transferrin saturation, and young women may have marginally low haemoglobin concentration levels. Serum ferritin levels are directly related to age, but are 10-20 times higher than normal. Heterozygosity for the ferroportin Val 162 deletion represents the prototype of selective reticuloendothelial iron overload, and should be taken into account in the differential diagnosis of hereditary or congenital hyperferritinaemias.

Prolonged administration of erythropoietin increases erythroid response rate in myelodysplastic syndromes: a phase II trial in 281 patients.

Treatment with recombinant human erythropoietin (rHuEpo) improves anaemia in approximately 20% of patients with myelodysplastic syndromes (MDS). We investigated the potential advantage of a prolonged administration of rHuEpo to achieve higher erythroid response rates (RR) in 281 MDS patients: 118 with refractory anaemia (RA), 77 with refractory anaemia and ringed sideroblasts (RARS), 59 with refractory anaemia with excess of blasts and blast count < 10% (RAEB-I), and 27 with RAEB and blast count between 11-20% (RAEB-II). rHuEpo was given subcutaneously at a dose of 150 U/kg thrice weekly, for a minimum of 26 weeks. Response to treatment was evaluated after 12 and 26 weeks of therapy. The overall RR was 45.1%; the RR for RA, RARS, RAEB-I and RAEB-II were 48.3%, 58.4%, 33.8% and 13% respectively. A significant increase in RR was observed at week 26 in RA, RARS and RAEB-I patients, as the response probability increased with treatment duration. The RR was higher in the good cytogenetic prognostic group and serum Epo level of > 150 U/l at baseline predicted for non-response. The median duration of response was 68 weeks and the overall risk of leukaemic transformation was 21.7%. These results suggest that prolonged administration of rHuEpo produces high and long-lasting erythroid RR in MDS patients with low blast counts, particularly in those with pretreatment serum Epo levels of < 150 U/l and good cytogenetic prognosis.