The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia (DBA) is a constitutional erythroblastopenia characterized by absent or decreased erythroid precursors. The disease, previously mapped to human chromosome 19q13, is frequently associated with a variety of malformations. To identify the gene involved in DBA, we cloned the chromosome 19q13 breakpoint in a patient with a reciprocal X;19 chromosome translocation. The breakpoint occurred in the gene encoding ribosomal protein S19. Furthermore, we identified mutations in RPS19 in 10 of 40 unrelated DBA patients, including nonsense, frameshift, splice site and missense mutations, as well as two intragenic deletions. These mutations are associated with clinical features that suggest a function for RPS19 in erythropoiesis and embryogenesis.

Diamond-Blackfan anaemia: genetic homogeneity for a gene on chromosome 19q13 restricted to 1.8 Mb.

Diamond-Blackfan anaemia (DBA; MIM#205900) is a rare disorder manifested as a pure red-cell aplasia in the neonatal period or in infancy. The clinical hallmark of DBA is a selective decrease in erythroid precursors and anaemia. Other lineages are usually normal and the peripheral white blood cell count is normal. In approximately one-third of cases, the disease is associated with a wide variety of congenital anomalies and malformations. Most cases are sporadic, but 10-20% of them follow a recessive or a dominant inheritance pattern. A female with DBA and a chromosomal translocation involving chromosome 19q was recently identified. We undertook a linkage analysis with chromosome 19 markers in multiplex DBA families of Swedish, French, Dutch, Arabic and Italian origin. Significant linkage to chromosome 19q13 was established for dominant and recessive inherited DBA with a peak lod score at D19S197 (Zmax = 7.08, theta = 0.00). Within this region, a submicroscopic de novo deletion of 3.3 Mb was identified in a patient with DBA. The deletion coincides with the translocation break-point and, together with key recombinations, restricts the DBA gene to a 1.8-Mb region. The results suggest that, despite its clinical heterogeneity, DBA is genetically homogeneous for a gene in 19q13.

Natural history of recessive inheritance of DMT1 mutations.

DMT1 deficiency causes microcytic hypochromic anemia due to decreased erythroid iron utilization. Anemia is present from birth. Transferrin saturation is high and serum ferritin is mildly elevated, despite liver iron overload. DMT1 deficiency must be considered in the differential diagnosis of microcytic hypochromic anemia observed in the newborn period.

[Hereditary spherocytosis: guidelines for the diagnosis and management in children]. / Sphérocytose héréditaire: recommandations pour le diagnostic et la prise en charge chez l'enfant.

Hereditary spherocytosis (HS) is the commonest inherited disorder of the erythrocyte membrane in Northern Europe and North America. It is marked by a regenerative anemia which varies widely from asymptomatic patients to severe hemolysis. In 75% of HS patients, inheritance is autosomal dominant. The diagnosis of HS is easily made when there are a family history, hemolytic anemia, reticulocytosis, spherocytes and increased hyperdense cells. Specialized testing to clarify the nature of membrane disorder is required when the film appearance is atypical without a positive family history, in the absence of a family history, in the newborn and before the splenectomy, to rule out the stomatocytosis which is contraindicated. The indication for splenectomy is dependent on the degree of anemia and its clinical manifestation.

Deficiency of skeletal membrane protein band 4.1 in homozygous hereditary elliptocytosis. Implications for erythrocyte membrane stability.

Erythrocytes from three patients with severe hemolytic anemia, marked erythrocyte fragmentation, and elliptocytic poikilocytosis, were studied in terms of both their membrane protein composition and their mechanical characteristics. Erythrocytes from the patients' parents and one minimally affected and one normal sibling were also studied. Morphologic observations implied that the severely affected patients suffered from homozygous hereditary elliptocytosis because erythrocytes of both parents and the one minimally affected sibling showed moderate elliptocytosis on smear, whereas those of an unaffected sibling had normal morphology. The parallel findings of markedly reduced levels of band 4.1 in the erythrocyte membrane proteins of the patients and an intermediate reduction in the cells of the parents and the putative heterozygous sibling, suggest that the elliptocytic shape of the cells was related to the reduced levels of band 4.1. Additional studies showed marked abnormalities in cellular deformability and membrane fragility in the erythrocytes from the homozygous patients. Importantly, these changes were also closely proportional to the reduced levels of band 4.1, suggesting a central role for this protein in the maintenance of normal membrane stability and normal cell shape. It seems likely that this role for band 4.1 is intimately related to its known biochemical connection to the "membrane skeleton" through its linkage with spectrin and actin.

Restoration of normal membrane stability to unstable protein 4.1-deficient erythrocyte membranes by incorporation of purified protein 4.1.

Protein 4.1, a principal component of the erythrocyte membrane skeleton, is thought to be important in regulating membrane stability through its interaction with spectrin and actin. A key role for protein 4.1 has been indicated in studies in which deficiency of this protein was shown to result in marked instability of the membrane. In order to obtain direct evidence for the functional role of protein 4.1, we reconstituted protein 4.1-deficient membranes with purified protein 4.1 and showed restoration of membrane stability. Erythrocyte membranes totally and partially deficient in protein 4.1 were reconstituted by exchange hemolysis with various concentrations of purified protein 4.1, and their stability measured using an ektacytometer. Native erythrocyte membranes totally deficient in protein 4.1 were markedly unstable, while those partially deficient had intermediate reductions in membrane stability. Reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability. Near-normal membrane stability could be restored to both totally and partially protein 4.1-deficient membranes. In contrast, the addition of protein 4.1 to resealed membranes did not improve membrane stability. This implies that the added protein 4.1 must have access to the cell interior in order to affect membrane stability. Furthermore, in control experiments, the addition of protein 4.1 to normal membranes did not increase their stability. Also, the addition of purified spectrin and human serum albumin during resealing did not improve stability of protein 4.1-deficient membranes. These results provide direct evidence for the crucial role of protein 4.1 in regulating erythrocyte membrane stability.

An isoform-specific mutation in the protein 4.1 gene results in hereditary elliptocytosis and complete deficiency of protein 4.1 in erythrocytes but not in nonerythroid cells.

Multiple protein 4.1 isoforms are expressed in a variety of tissues through complex alternative pre-mRNA splicing events, one function of which is to regulate use of two alternative translation initiation signals. Late erythroid cells express mainly the downstream initiation site for synthesis of prototypical 80-kD isoforms; nonerythroid cells in addition use an upstream site to encode higher molecular mass isoform(s). In this study, we examined the effects of a 5' gene rearrangement in a family with hereditary elliptocytosis and complete deficiency of erythrocyte 4.1 protein on 4.1 isoform expression in erythroid vs. nonerythroid cells. Patient 4.1 mRNAs from reticulocytes, fibroblasts, and B lymphocytes were amplified by reverse transcriptase/polymerase chain reaction techniques and shown to exhibit a 318-nucleotide deletion that encompasses the downstream AUG, but leaves intact the upstream AUG. Immunoblot analysis revealed a total deficiency of 4.1 in patient red cells and a selective deficiency of 80-kD isoform(s) but not high molecular weight 4.1 in patient nonerythroid cells. Thus, the 4.1 gene mutation in this family produces an isoform-specific deficiency that is manifested clinically in tissue-specific fashion, such that red cells are affected but other cell types are unaffected because of tissue-specific differences in RNA splicing and translation initiation.

Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease.

Intracellular polymerization and sickling depend markedly on the cellular concentration of sickle hemoglobin (Hb S). A possible therapeutic strategy for sickle cell disease is based on reducing the cellular concentration of Hb S through prevention of erythrocyte dehydration. The K-Cl cotransporter is a major determinant of sickle cell dehydration and is inhibited by increasing erythrocyte Mg content. We studied 10 patients with sickle cell disease before treatment and after 2 and 4 wk of treatment with oral Mg supplements (0.6 meq/kg/d Mg pidolate). Hematological parameters, erythrocyte Na, K, and Mg content, erythrocyte density, membrane transport of Na and K, and osmotic gradient ektacytometry were measured. We found significant increases in sickle erythrocyte Mg and K content and reduction in the number of dense sickle erythrocytes. Erythrocyte K-Cl cotransport was reduced significantly. We also observed a significant reduction in the absolute reticulocyte count and in the number of immature reticulocytes. Ektacytometric analysis showed changes indicative of improved hydration of the erythrocytes. There were no laboratory or clinical signs of hypermagnesemia. Mild, transient diarrhea was the only reported side effect. We conclude that oral Mg supplementation reduces the number of dense erythrocytes and improves the erythrocyte membrane transport abnormalities of patients with sickle cell disease.

[Management of high risk pregnancy in sickle cell disease by a strategy of prophylactic red cell transfusion or automated red cell exchange]. / Prise en charge des grossesses à risque chez les femmes drépanocytaires : intérêt d'une stratégie préventive par des transfusions de globules rouges ou des échanges érythrocytaires automatisés.

OBJECTIVE: Maternal and fetal risk is often high during pregnancy in sickle cell disease. Our objective was to evaluate the benefits of a transfusion program adapted to each pregnant patient, either by red cell transfusion or by automated red cell exchange, in sickle cell patients with a history of serious obstetrical and/or sickling complications. STUDY DESIGN: We managed 18 pregnancies in 14 patients (12 SS, 1 SC, 1 S/b-thalassemia), seven of whom had a history of one or more pregnancies, with severe maternofetal complications in nine out of 10 cases. The other seven patients were pregnant for the first time and were in care because of a history of severe sickling complications. The aim was to achieve a proportion of abnormal hemoglobin (hemoglobin S or S+C) below 50% and a hemoglobin level between 9 and 11 g/dL. The choice between transfusion and red cell exchange was made in the light of the hemoglobin level. Red cell exchange was done using a Fresenius Com. Tec blood cell separator. Patients had red cell exchange in 10 cases, and transfusions in five cases. In three cases, patients had successive transfusions and red cell exchange. RESULTS: No serious maternal complication was observed. No fetal or perinatal death occurred. In one case, delivery was induced at 36 weeks of gestation because of fetal distress and hypotrophy. CONCLUSIONS: Our study suggests that women with severe sickle cell disease, even if they have a serious obstetrical history, can carry their pregnancy to term, without major obstetric complications, through a combination of early management by a multidisciplinary team and a suitable policy of prophylactic transfusion or automated red cell exchange.

[Auto-immune hemolytic anemia and dyserythropoïesis as the presenting signs of Fas-deficient condition in 3 children]. / Anémie hémolytique auto-immune et dysérythropoïèse révélatrices d'un déficit de l'apoptose Fas chez 3 enfants.

Defective apoptosis caused by mutations of the Fas gene can lead to an autoimmune lymphoproliferative syndrome (ALPS). The main autoimmune manifestations are haematological: hemolytic anemia, thrombocytopenia and neutropenia. We described 3 patients with ALPS presenting as a lymphoproliferative syndrome associated with a Coomb's negative autoimmune hemolytic anemia and dyserythropoiesis predominating on the more mature erythroblasts. Fas apoptosis deficiency was evidenced in the 3 patients by the demonstration of an increased number of CD4(-)CD8(-)TCRalphabeta(+) T cells, a decreased apoptotic response of activated T lymphocytes to anti-Apo 1-3 monoclonal antibody and the presence of a heterozygous mutation of the Fas receptor gene.

Pregnancy and paroxysmal nocturnal hemoglobinuria.

Our study concerns eight pregnancies, six of which were successful, in four patients with paroxysmal nocturnal hemoglobinuria (PNH). Several complications of PNH during pregnancy were prevented: chronic anemia, folate and iron deficiency, and deep-vein thrombosis. During puerperium, acute hemolytic crises, most probably triggered by delivery, were observed in two patients. Thrombotic complications could be prevented by early initiation of an anticoagulant therapy after delivery. The only neonatal complication, observed in two cases, was isoimmune hemolytic anemia related to the multiple blood transfusions received before and during pregnancy. These results show that successful pregnancies are possible in women with PNH provided that both the obstetricians and physicians in charge monitor the pregnancies closely.

Hemoglobin content of individual erythroblasts in hematopoietic dysplasia: marked heterogeneity at late stages of maturation.

Defective hemoglobin synthesis and gross morphologic abnormalities of erythroblasts are striking features of hematopoietic dysplasia. In order to define the effects of defective hemoglobin synthesis on hemoglobin content of erythroblasts at each stage of maturation, we measured the hemoglobin content and concentration of individual erythroblasts at various stages of maturation in the bone marrow by scanning microspectrophotometry. In contrast to normal erythroblasts, those from hematopoietic dysplasia had decreased hemoglobin content and decreased hemoglobin concentration at late stages of maturation. This observation, together with the observed near-normal hemoglobin content and concentration of circulating red cells in this disorder, suggests that polychromatophilic erythroblasts with decreased hemoglobin content fail to complete normal maturation. The abnormalities in hemoglobin content observed in vivo in hematopoietic dysplasia could be reproduced in the in vitro cultures of erythroid progenitors of bone marrow cells from the patients.

Follow-up thallium-201 scintigraphy after mantle field radiotherapy for Hodgkin's disease.

PURPOSE: Assessment of the long-term cardiac effects of mediastinal radiotherapy for Hodgkin's disease, by Thallium scintigraphy. METHODS AND MATERIALS: 32 patients (14 males and 18 females) who underwent mantle field radiotherapy for Hodgkin's disease were included in this study. Twenty patients received 4 fractions of 2.5 Gy per week and 12, five fraction of 2 Gy per week, delivered on alternate days. All the patients, except three, performed exercise testing electrocardiogram and Thallium-201 tomoscintigraphy. The average time interval from completion of treatment to the study was 7 years (range 3-13 years). No patients had clinical symptoms of cardiac disease. Mean age at the time of the study was 35 years (range 23-48 years). RESULTS: Two electrocardiograms revealed left bundle branch block and the patients were excluded from the study. Only one out of 27 exercise electrocardiograms was abnormal in a patient with mitral valve prolapse, who was also excluded from the study. Twenty-six scintigraphies were evaluable. Twenty-two (85%) were clearly abnormal with partial or complete redistribution on delayed images. The anterior region was affected in 19 of these cases (86%). Four explorations were undoubtedly normal. Coronary angiography was not performed for ethical reasons in these asymptomatic patients. CONCLUSION: Despite possible false positive tests, the high rate of abnormality (85%) in this small series is striking. These preliminary data justify larger studies and a close long-term follow-up of these patients.

Recombinant erythropoietin therapy as an alternative to blood transfusions in infants with hereditary spherocytosis.

INTRODUCTION: In hereditary spherocytosis, erythropoiesis has been described as 'sluggish' during the first months of life. The lack of appropriate erythropoietic response to compensate for increased red cell destruction necessitates blood transfusions in 70-80% of hereditary spherocytosis-affected infants during their first year of life. After this period, less than 30% require regular transfusion support. This transient requirement for transfusion led us to wonder whether anemic hereditary spherocytosis infants, like anemic premature infants, could benefit from recombinant erythropoietin therapy (rHu-Epo). MATERIAL AND METHODS: In 16 hereditary spherocytosis infants (age range 16-119 days) with severe anemia, a compassionate open preliminary study was performed. rHu-Epo treatment (1000 IU/kg/week) was instituted together with iron supplementation. Hemoglobin values and reticulocyte counts were repeatedly assessed. RESULTS: In 13 out of 16 infants, prompt increases in reticulocyte counts were noted after the first week of treatment with 1000 IU/kg/week of rHu-Epo. During treatment with Epo these infants maintained clinically acceptable levels of hemoglobin and did not require blood transfusions. As the infants grew and began to mount an adequate erythropoietic response, the rHu-Epo dose could be tapered and the treatment could be discontinued before the age of nine months. CONCLUSION: Epo treatment in most hereditary spherocytosis infants appears to be effective in the management of anemia and could serve as a valuable alternative to packed RBC transfusions.

Red blood cell abnormalities in hereditary elliptocytosis and their relevance to variable clinical expression.

Marked variations are seen in the clinical manifestations of hereditary elliptocytosis (HE). To define the cellular alteration(s) that best reflect the variable expression of the disease, we evaluated the pathobiologic features of red blood cells in a series of 18 patients with HE, 15 persons from six families with HE as a result of defects in spectrin, and 3 persons from one family with HE caused by partial or total deficiency of protein 4.1. We found that decreased cellular deformability is a distinguishing feature of red blood cells in all patients studied. Comparison of volume and hemoglobin content histograms of red blood cells and reticulocytes revealed that cell fragmentation is a feature of mature red blood cells. The extent of red blood cell fragmentation as reflected by increased percentage of microcytic red blood cells was the best indicator of the severity of hemolytic anemia. Furthermore, we found that the observed variations in cellular properties of HE red blood cells in different persons is the consequence of varying amounts of mutant protein assembled into the membrane. These findings enabled us to define the mechanistic basis for cellular changes in this red blood cell membrane disorder better and also to obtain insight into the cellular basis for variable clinical expression.

Spurious monosomy 7 in leukemia due to centromeric heteromorphism.

We report heteromorphism of the centrometric region of human chromosome 7, which was observed in the cytogenetic assessment of a complete remission of a pre-B acute lymphoblastic leukemia in the bone marrow cells of a 25-year-old woman. Classical cytogenetic study was performed, as well as metaphase and interphase fluorescence in situ hybridization (FISH) carried out with an alphoid DNA probe specific for the chromosome 7 centromere for detection of leukemic clones with monosomy 7 found at the initial diagnosis. We show an important centromeric heteromorphism of this chromosome detected by FISH and clearly visible on all metaphases and nuclei analyzed. This heteromorphism is observable as a fluorescent signal five- or sixfold larger than that on the homologue. To our knowledge, this heteromorphism of chromosome 7 has not been reported in the literature. However, with the use of FISH analysis, it could be easily mistaken for a mosaicism of monosomy 7, which can be misleading in the interpretation of the results.

Acute lymphoblastic leukemia with trisomy 21 constitutional mosaicism.

Acute lymphoblastic leukemia was diagnosed in an 11-year-old girl with mild signs of Down's syndrome. She was known since birth to have a constitutional mosaicism (46,XX/47,XX,+21c). At initial diagnosis of acute leukemia, additional chromosome changes were found in bone marrow blasts: hyperdiploidy > 50, with a structural abnormality. She was treated with a standard chemotherapeutic protocol, and has remained in complete remission for more than 3 years. The constitutional mosaicism evolved toward normalization year after year in the blood and under the effect of chemotherapy in the bone marrow.

Delivery management in a woman with thrombocytopenia of the May-Hegglin anomaly type.

Thrombocytopenia of the May-Hegglin anomaly type was diagnosed in a woman with no past history of bleeding diathesis, who had been followed during her three pregnancies. No abnormal bleeding occurred although no platelet transfusion was administered during the second and third cesarean sections. Routine platelets transfusion is unnecessary but platelets should be available for use if abnormal bleeding occurs.

[Evaluation of the Coulter S + II in neonatology]. / Evaluation du Coulter S + II en néonatologie.

The Coulter Counter S + II is one of the most frequently used automatic counter in France. The authors evaluated its performances by analysing 116 blood samples from newborn infants aged less than one month. The reliability of this counter is impaired by the numerous rejects of lymphocytes or platelets and by the failure to recognize erythroblasts or circulating immature granulocytes. Moreover, abnormal cellular populations are notified which sometimes do not appear to be linked to any clinical disorder and are not evidenced by microscope examination control. These constatations bound the utilisation of this counter at least as far as the newborn's hematology is concerned.

[Parvovirus B19 and pediatric pathology]. / Parvovirus B 19 et pathologie pédiatrique.

B 19 parvovirus is a widespread virus with primary infestation generally occurring in childhood through family and community outbreaks. Its most typical manifestation is transient erythroblastopenia with aplastic crisis, often profound, mostly affecting patients with chronic hemolytic anemia, and eventually patients with defective erythropoiesis (chronic hypoplastic anemia, iron deficiency anemia). In normal individuals the primary infestation is usually asymptomatic but may give transient hematological signs for few days: moderate reticulocytopenia, thrombopenia and neutropenia. Clinically two phases of the infection are described: 1.) a first phase of viremia of 2 to 3 days which may be accompanied by fever and myalgias; 2.) a second phase which may last for several weeks with dermatological signs, the most typical being erythema infectiosum, vasculitis, arthralgias or arthritis. In pregnant women, the primary infestation with B 19 parvovirus may lead to fetal anemia and hydrops fetalis with uneven outcomes: fetal death, chronic erythroblastopenia after birth, spontaneous resolution. Although the incidence of fetal infestation in non immunized pregnant women is still unknown, the question is raised of the recognition and protection of non immunized pregnant women at high risk of exposition to infested subjects. Long term persistence of the virus in the organism may be responsible for chronic manifestation, essentially but not exclusively in immunodeficient-patients: prolonged erythroblastopenia and chronic rheumatologic manifestations. It may be also responsible for cases of juvenile arthritis, thrombocytopenic purpura and chronic neutropenia of childhood. The diagnosis of the viral infestation is mainly based upon the detection of specific IgM, then IgG, antibodies by Elisa technique.