Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro.

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G0/G1 cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G0/G1 cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.

Diamond-Blackfan anemia, ribosome and erythropoiesis.

Diamond-Blackfan anemia is a rare inherited bone marrow failure syndrome (five to seven cases per million live births) characterized by an aregenerative, usually macrocytic anemia with an absence or less than 5% of erythroid precursors (erythroblastopenia) in an otherwise normal bone marrow. The platelet and the white cell counts are usually normal but neutropenia, thrombopenia or thrombocytosis have been noted at diagnosis. In 40 to 50% of DBA patients, congenital abnormalities mostly in the cephalic area and in thumbs and upper limbs have been described. Recent analysis did show a phenotype/genotype correlation. Congenital erythroblastopenia of DBA is the first human disease identified to result from defects in ribosomal biogenesis. The first ribosomal gene involved in DBA, ribosomal protein (RP) gene S19 (RPS19 gene), was identified in 1999. Subsequently, mutations in 12 other RP genes out of a total of 78 RP genes have been identified in DBA. All RP gene mutations described to date are heterozygous and dominant inheritance has been documented in 40 to 45% of affected individuals. As RP mutations are yet to be identified in approximately 50% of DBA cases, it is likely that other yet to be identified genes involved in ribosomal biogenesis or other pathways may be responsible for DBA phenotype.

[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.

Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia.

Spherocytic red cells with reduced membrane surface area are a feature of hereditary spherocytosis (HS) and some forms of autoimmune hemolytic anemia (AIHA). It is generally assumed that membrane loss in spherocytic red cells occurs during their sojourn in circulation. The structural basis for membrane loss in HS is improper assembly of membrane proteins, whereas in AIHA it is due to partial phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the reticulocyte stage in HS, whereas surface area loss in AIHA involves only circulating mature red cells. The validity of this hypothesis was established by documenting differences in cellular features of reticulocytes in HS and AIHA. Using a novel technique to quantitate cell surface area, the decreased membrane surface area of both reticulocytes and mature red cells in HS compared with normal cells was documented. In contrast, in AIHA only mature red cells but not reticulocytes exhibited decreased membrane surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. These findings imply that loss of cell surface area is an early event during genesis of HS red cells and challenge the existing concepts that surface area loss in HS occurs predominantly during the sojourn of mature red cells in circulation.

Autoimmune disorders of erythropoiesis.

Immune-mediated disorders of erythropoiesis can result in acquired severe anemia, low reticulocyte counts, and bone marrow exhibiting pure red cell aplasia or ineffective erythropoiesis. Erythropoiesis can be suppressed or impaired by humoral or cellular mechanisms. In vitro inhibition of erythroid colony growth by immunoglobulins or lymphocytes can be a strong argument for the immune origin of the disease. Classical etiologies are thymoma and hematologic malignancies such as chronic lymphocytic leukemia (CLL). Clonal proliferation of T cells has been incriminated. Recently, acquired circulating autoantibodies directed against erythropoietin have been detected in a case of pure red cell aplasia. Autoimmune mechanisms have also been detected or suggested in synartesis and in Fas-associated dyserythropoiesis, two distinct syndromes recently described where morphologic abnormalities specific to the erythroid lineage illustrate ineffective erythropoiesis.

Severe congenital dyserythropoietic anaemia type I: prenatal management, transfusion support and alpha-interferon therapy.

We report a case of congenital dyserythropoietic anaemia, type I, with severe pre- and postnatal manifestations. Exchange transfusions were required for fetal anaemia (3.5 g/dl) at 28 and 30 weeks of gestation. Transfusions were administered at birth (Caesarean section at week 35) and at regular intervals thereafter. At 14 months, alpha-interferon therapy was initiated (106 units three times a week). This resulted in stabilization of the haemoglobin at or above 11 g/dl and a reduction in the percentage of erythroblasts with ultrastructurally abnormal heterochromatin. After 9 months, the dose of alpha-interferon was decreased to 106 units twice a week. No relapse of anaemia was noted during an additional 4 months of follow-up.

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.

Dyserythropoiesis associated with a fas-deficient condition in childhood.

Defective lymphocyte apoptosis caused by mutations of the Fas gene can result in an autoimmune lymphoproliferative syndrome (ALPS) in humans. We report two cases of dyserythropoiesis associated with a Fas-deficient condition in childhood. In both cases, dyserythropoiesis predominated on the more mature erythroblasts, and was associated with a lymphoproliferative syndrome as well as with haemolytic anaemia, hypergammaglobulinaemia and the expansion of an unusual population of CD4- CD8- T cells that express the alpha/beta T-cell receptor. The regression of dyserythropoiesis under steroid therapy suggested that it resulted from an autoimmune mechanism, itself secondary to the lymphocyte Fas apoptosis deficiency. Fas-defective apoptosis may be a new aetiology for childhood dyserythropoiesis.

Heavy transfusions and presence of an anti-protein 4.2 antibody in 4. 2(-) hereditary spherocytosis (949delG).

BACKGROUND AND OBJECTIVE: A patient with hereditary spherocytosis (HS) was found not to have red cell membrane protein 4.2. This rare form of HS, or 4.2 (-) HS, stems from mutations within the ELB42 or the EPB3 genes. The patient had long suffered from a gastric ulcer and impaired liver function. He had had several dramatic episodes of gastrointestinal tract bleeding and had received numerous transfusions. An antibody against a high frequency, undefined antigen was found, creating a transfusional deadlock. We elucidated the responsible mutation and searched for an anti-protein 4.2 antibody. DESIGN AND METHODS: Red cell membranes were analyzed by SDS-PAGE and by Western blotting. Nucleotide sequencing was performed after reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: The not previously described mutation was a single base deletion: 949delG (CGCAECC, exon 7, codon 317) in the homozygous state. It was called protein 4.2 Nancy. The deletion placed a non-sense codon shortly downstream so that no viable polypeptide could be synthesized. The patient carried a strong antibody against protein 4.2 as shown by Western blotting. INTERPRETATION AND CONCLUSIONS: The manifestations resulting from the mutation described were compared with the picture of HS stemming from other ELB42 gene mutations. We discuss the mechanism through which the anti-protein 4.2 antibody developed. There was no way to establish or to rule out whether the antibody participated in the transfusional deadlock found in our patient.

Identification of new prognosis factors from the clinical and epidemiologic analysis of a registry of 229 Diamond-Blackfan anemia patients. DBA group of Société d'Hématologie et d'Immunologie Pédiatrique (SHIP), Gesellshaft für Pädiatrische Onkologie und Hämatologie (GPOH), and the European Society for Pediatric Hematology and Immunology (ESPHI).

Diamond-Blackfan anemia (DBA) is a constitutional disease characterized by a specific maturation defect in cells of erythroid lineage. We have assembled a registry of 229 DBA patients, which includes 151 patients from France, 70 from Germany, and eight from other countries. Presence of malformations was significantly and independently associated with familial history of DBA, short stature at presentation (before any steroid therapy), and absence of hypotrophy at birth. Two hundred twenty-two patients were available for long-term follow-up analysis (median, 111.5 mo). Of these individuals, 62.6% initially responded to steroid therapy. Initial steroid responsiveness was found significantly and independently associated with older age at presentation, familial history of DBA, and a normal platelet count at the time of diagnosis. Severe evolution of the disease (transfusion dependence or death) was significantly and independently associated with a younger age at presentation and with a history of premature birth. In contrast, patients with a familial history of the disease experienced a better outcome. Outcome analysis revealed the benefit of reassessing steroid responsiveness during the course of the disease for initially nonresponsive patients. Bone marrow transplantation was successful in 11/13 cases; HLA typing of probands and siblings should be performed early if patients are transfusion dependent, and cord blood should be preserved. Incidence of DBA (assessed for France over a 13-y period) is 7.3 cases per million live births without effect of seasonality on incidence of the disease or on malformative status. Similarly, no parental imprinting effect or anticipation phenomenon could be documented in families with dominant inheritance.

Mutations in ribosomal protein S19 gene and diamond blackfan anemia: wide variations in phenotypic expression.

Mutations of the ribosomal protein S19 (RPS19) gene were recently identified in 10 patients with Diamond Blackfan anemia (DBA). To determine the prevalence of mutations in this gene in DBA and to begin to define the molecular basis for the observed variable clinical phenotype of this disorder, the genomic sequence of the 6 exons and the 5' untranslated region of the RPS19 gene was directly assessed in DBA index cases from 172 new families. Mutations affecting the coding sequence of RPS19 or splice sites were found in 34 cases (19.7%), whereas mutations in noncoding regions were found in 8 patients (4.6%). Mutations included nonsense, missense, splice sites, and frameshift mutations. A hot spot for missense mutations was identified between codons 52 and 62 of the RPS19 gene in a new sequence consensus motif W-[YFW]-[YF]-x-R-[AT]-A-[SA]-x-[AL]-R-[HRK]-[ILV]-Y. No correlation between the nature of mutations and the different patterns of clinical expression, including age at presentation, presence of malformations, and therapeutic outcome, could be documented. Moreover, RPS19 mutations were also found in some first-degree relatives presenting only with isolated high erythrocyte adenosine deaminase activity and/or macrocytosis. The lack of a consistent relationship between the nature of the mutations and the clinical phenotype implies that yet unidentified factors modulate the phenotypic expression of the primary genetic defect in families with RPS19 mutations.

Suppression of CDA II expression in a homozygote.

The CDAN2 gene, responsible for congenital dyserythropoietic anaemia, type II (CDA II), was recently mapped to 20q11.2. We report data on an additional member of a previously studied CDA II family. This member had always been regarded as haematologically normal. Unexpectedly, she had the same microsatellite assortments around the CDAN2 alleles as her three sisters with CDA II. In particular, she was a homozygote for microsatellites D20S863 and D20S841. This prompted an analysis of all facets of her phenotype. The Ham test was negative. The bone marrow smears contained a normal proportion of binucleate erythroblasts. Electron microscopy revealed the absence of extensive stretches of cisternae beneath and parallel to the inner surface of the erythroblast plasma membrane. Proteins of the endoplasmic reticulum, which contaminate the reticulocyte plasma membrane in CDA II patients, were missing. Only the shape of the band 3 peak appeared slightly altered. This case exemplifies that homozygosity (or compound heterozygosity) for a deleterious gene may be silenced, or almost completely silenced. In recessively inherited diseases, suppressed phenotypes tend to be overlooked in siblings where both patients and unaffected individuals are expected.

Hematologic involvement in mitochondrial cytopathies in childhood: a retrospective study of bone marrow smears.

We retrospectively analyzed the bone marrow (BM) smears of 10 children with mitochondrial cytopathies. Light microscopic examination showed large and coalescent cytoplasmic vacuolization of some BM precursors in nine cases, including two children with normal peripheral blood counts and four with sideroblastic anemia. BM ultrastructural study showed abnormal mitochondria in the erythroid lineage in all three children studied. Ultrastructural studies in two cases revealed a population of giant mitochondria with abnormal ultrastructure coexisting with a population of normal mitochondria in proerythroblasts, basophil erythroblasts, and less commonly in more mature erythroblasts. In a third child, mitochondria were normal in size with cristae either absent or exhibiting abnormal longitudinal orientation. Heteroplasmic segregation of mitochondria during cell division could account for the finding of a double population of cells on ultrastructural examination. These features suggest that cytologic and ultrastructural BM examination could be useful for the diagnosis of mitochondrial disorders. That is, when large and coalescent cytoplasmic vacuoles of BM precursor cells are present, the clinician should search for mitochondrial cytopathy in a child with unexplained cytopenia(s).

Iron deficiency in pregnancy: effects on the newborn.

Iron deficiency during pregnancy affects a significant portion of women in countries with low economic wealth and is not uncommon in pregnant women in industrialized countries. Inadequate intake of iron related to diets poor in bioavailable iron is often responsible for iron deficiency before pregnancy, and metabolic adjustments (such as mobilization of iron stores and increased absorption) are insufficient to meet increasing needs during pregnancy. The effects of iron deficiency on the fetus are still controversial. Numerous measures, including the evaluation of erythrocyte ferritin, favor the hypothesis that the level of iron stores in newborns is related to maternal iron status and that the materno-fetal unit is dependent on exogenous iron, which is necessary to prevent iron deficiency in both mothers and infants. In industrialized countries, iron supplements should be prescribed for pregnant women in the third trimester, when the need for iron is prominent. In developing countries, supplementation should be initiated as soon as possible after conception because of the high prevalence of iron deficiency at the onset of pregnancy. The results of studies comparing intermittent with daily supplementation remain controversial.

Human parvovirus B19 infection in organ transplant recipients.

We report a 61-yr-old kidney transplant recipient with human Parvovirus B19 (HPV B19) infection presenting as a severe pancytopenia 1 month after transplantation. Bone marrow aspiration revealed severe erythroid hypoplasia with giant and dystrophic proerythroblasts. Bone marrow cells were positive for HPV B19 DNA detected by polymerase chain reaction (PCR). Pancytopenia resolved shortly after administration of intravenous immunoglobulins. Nineteen cases of HPV B19 infection in organ transplant recipients have been so far reported in the literature. Immunocompromised patients should be considered at risk from developing symptomatic HPV B19 infections. In such patients, specific anti-HPV B19 IgM and IgG antibodies may be absent or transient and therefore their negativity cannot rule out the diagnosis of HPV B19 infestation. Bone marrow smear morphological findings may suggest the diagnosis but testing for viral DNA by PCR is mandatory. Patients may spontaneously recover. However, since specific anti-viral therapy is not currently available, intravenous immunoglobulin administration appears to be the more efficacious treatment.

High adenosine deaminase level among healthy probands of Diamond Blackfan anemia (DBA) cosegregates with the DBA gene region on chromosome 19q13. The DBA Working Group of Société d'Immunologie Pédiatrique (SHIP).

Phenotypic characterization of Diamond Blackfan Anemia (DBA) patients and their relatives was performed in 54 families. Complete blood count, fetal hemoglobin level, erythrocyte i antigen expression, and erythrocyte adenosine deaminase (eADA) activities were quantitated in patients and relatives. eADA was elevated in 28 of 34 transfusion-independent DBA patients, whereas persistence of erythrocyte i antigen was noticed in only 10 of 20 DBA patients. High eADA activities were also found in 14 of 149 healthy family members, allowing us to identify an isolated high eADA phenotype in these families. In contrast, increase in erythrocyte i antigen expression, elevated fetal hemoglobin levels, and macrocytosis were much less frequently noted in nonaffected members of the DBA families studied. Importantly, isolated high eADA phenotype was found to be significantly associated with genetic markers on chromosome 19 that segregate with the DBA phenotype. Isolated high eADA phenotype thus seems to reflect a silent phenotype of DBA in affected families. These findings suggest that elevated eADA activity in unaffected individuals needs to be taken into account during genetic assessment of DBA families and could be used for accurate assessment of mode of inheritance.

[Pachydermoperiostosis with extramedullary hematopoiesis without myelofibrosis]. / Pachydermopériostose associée à un foyer d'hématopoïèse extra-médullaire sans myélofibrose.

BACKGROUND: In three previous reports of primary hypertrophic osteoarthropathy, an associated extramedullary hematopoiesis was related to myelofibrosis. CASE REPORT: A 44-year-old male patient with primary hypertrophic osteoarthropathy diagnosed when he was 34-year-old was referred to our hospital with an abdominal mass fortuitously detected. DISCUSSION: The present case is unique for the patient developed an extramedullary hematopoïesis without associated myelofibrosis. It suggests the possible intervention of growth factors common to the skin fibroblasts and the blood progenitor cells in the pathogenesis of primary osteoarthropathy.