Analysis of a cohort of 101 CDAII patients: description of 24 new molecular variants and genotype-phenotype correlations.

Congenital dyserythropoietic anaemia type II (CDAII) is a rare autosomal recessive disease characterized by ineffective erythropoiesis, haemolysis, erythroblast morphological abnormalities, hypoglycosylation of some red blood cell membrane proteins, particularly band 3, and mutations in the SEC23B gene. We report the analysis of 101 patients from 91 families with a median follow-up of 23 years (range 0-65); 68 patients are newly reported. Clinical and haematological parameters were separately analysed in early infancy and thereafter, when feasible. Molecular analysis of the SEC23B gene confirmed the high heterogeneity of the defect, leading to the identification of 54 different mutations, 24 of which are newly described. To evaluate the genotype-phenotype correlation, patients were grouped according to their genotype (two missense mutations vs. one missense/one drastic mutation) and assigned to two different severity gradings based on laboratory data and on therapeutic needs; by this approach only a weak genotype-phenotype correlation was observed in the analysed groups.

Toward the harmonization of result presentation for the eosin-5'-maleimide binding test in the diagnosis of hereditary spherocytosis.

BACKGROUND: The eosin-5'-maleimide (EMA) Binding test measures reduced mean channel fluorescence (MCF) reading of EMA-labeled red cells (EMA-RBCs) from patients with hereditary spherocytosis (HS). Reporting test results can be either in the actual MCF reading or as a ratio by normalization of the test MCF result to the mean MCF value of six normal controls. The latter format has potential for universal reporting. METHODS: We analyzed three years' archival MCF data from HS and non-HS patient groups for establishment of reference ranges of ratios for normal adults and HS. A prospective study used FC500 and FACS Canto II cytometers to analyze contemporaneously EMA-RBCs from several patient groups and normal donors. Statistical analyses of the prospective data determined the cut-off values, and the sensitivity and specificity for HS respectively for the MCF and the ratio result presentations. The effect of using fewer than six normal controls for the ratio denominator was explored. RESULTS: The FC500 gave a mean ratio of 0.782 (SD=0.086) in HS patients with an optimal cut-off ratio of 0.918 (98.7% specificity, 95.6% sensitivity), and gray area ratio of 0.868-0.918. The Canto II gave a mean ratio of 0.774 (SD=0.085) with an optimal cut-off ratio of 0.925 (97.1% specificity and 100% sensitivity), and gray area ratio of 0.859-0.925. CONCLUSION: Harmonization of result presentation is feasible with no apparent constraint by instrument design. Interpretation of gray-area data requires an assessment of patient's clinical presentation and family history or performing a family study.

Towards the harmonization of result presentation for the eosin-5'- maleimide (EMA) binding test in the diagnosis of hereditary spherocytosis.

Background: The EMA Binding test measures reduced mean channel fluorescence (MCF) reading of EMA-labeled red cells (EMA-RBCs) from patients with hereditary spherocytosis (HS). Reporting test results can be either in the actual MCF reading or as a ratio by normalization of the test MCF result to the mean MCF value of 6 normal controls. The latter format has potential for universal reporting. Methods: We analyzed three years' archival MCF data from HS and non-HS patient groups for establishment of reference ranges of ratios for normal adults and HS. A prospective study used FC500 and FACS Canto II cytometers to analyze contemporaneously EMA-RBCs from several patient groups and normal donors. Statistical analyses of the prospective data determined the cut-off values, and the sensitivity and specificity for HS respectively for the MCF and the ratio result presentations. The effect of using fewer than six normal controls for the ratio denominator was explored. Results:. The FC500 gave a mean ratio of 0.782 (SD = 0.086) in HS patients with an optimal cut-off ratio of 0.918 (98.7% specificity, 95.6% sensitivity), and grey area ratio of 0.868 - 0.918. The Canto II gave a mean ratio of 0.774 (SD = 0.085) with an optimal cut-off ratio of 0.925 (97.1% specificity and 100% sensitivity), and grey area ratio of 0.859 - 0.925. Conclusion: Harmonization of result presentation is feasible with no apparent constraint by instrument design. Interpretation of grey-area data requires an assessment of patient's clinical presentation and family history or performing a family study. © 2014 Clinical Cytometry Society.

Explaining survival differences between two consecutive studies with allogeneic stem cell transplantation in patients with chronic myeloid leukemia.

PURPOSE: In the two consecutive German studies III and IIIA on chronic myeloid leukemia, between 1995 and 2004, 781 patients were randomized to receive either allogeneic hematopoietic stem cell transplantation with a related donor or continued drug treatment. Despite comparable transplantation protocols and most centers participating in both studies, the post-transplant survival probabilities for patients transplanted in first chronic phase were significantly higher in study IIIA (144 patients) than in study III (113 patients). Prior to the decision on a combined analysis of both studies, reasons for this discrepancy had to be investigated. METHODS: The Cox proportional hazard cure model was used to identify prognostic factors for post-transplant survival. RESULTS: Donor-recipient matching for human leukocyte antigen, patient age, time between diagnosis and transplantation, and calendar time showed a significant influence on survival and/or the incidence of cure. Added as a further factor, affiliation to study IIIA had no significant impact any longer. CONCLUSIONS: Discrepancies in influential prognostic factors explained the different post-transplant survival probabilities between the studies. The significance of calendar time suggests a lack of consistency of transplantation practice over time. Accordingly, the prerequisite for a common assessment of overall survival in the two randomized transplantation arms was not met. Moreover, our analyses provide an independent validation of established prognostic factors and their cutoffs. The statistical approach in investigating and modeling potential prognostic factors for survival sets an example for the examination of studies with unexpected outcome differences in concurrent treatment arms.

Deep molecular response is reached by the majority of patients treated with imatinib, predicts survival, and is achieved more quickly by optimized high-dose imatinib: results from the randomized CML-study IV.

PURPOSE: Deep molecular response (MR(4.5)) defines a subgroup of patients with chronic myeloid leukemia (CML) who may stay in unmaintained remission after treatment discontinuation. It is unclear how many patients achieve MR(4.5) under different treatment modalities and whether MR(4.5) predicts survival. PATIENTS AND METHODS: Patients from the randomized CML-Study IV were analyzed for confirmed MR(4.5) which was defined as ≥ 4.5 log reduction of BCR-ABL on the international scale (IS) and determined by reverse transcriptase polymerase chain reaction in two consecutive analyses. Landmark analyses were performed to assess the impact of MR(4.5) on survival. RESULTS: Of 1,551 randomly assigned patients, 1,524 were assessable. After a median observation time of 67.5 months, 5-year overall survival (OS) was 90%, 5-year progression-free-survival was 87.5%, and 8-year OS was 86%. The cumulative incidence of MR(4.5) after 9 years was 70% (median, 4.9 years); confirmed MR(4.5) was 54%. MR(4.5) was reached more quickly with optimized high-dose imatinib than with imatinib 400 mg/day (P = .016). Independent of treatment approach, confirmed MR(4.5) at 4 years predicted significantly higher survival probabilities than 0.1% to 1% IS, which corresponds to complete cytogenetic remission (8-year OS, 92% v 83%; P = .047). High-dose imatinib and early major molecular remission predicted MR(4.5). No patient with confirmed MR(4.5) has experienced progression. CONCLUSION: MR(4.5) is a new molecular predictor of long-term outcome, is reached by a majority of patients treated with imatinib, and is achieved more quickly with optimized high-dose imatinib, which may provide an improved therapeutic basis for treatment discontinuation in CML.

Congenital dyserythropoietic anemias: molecular insights and diagnostic approach.

The congenital dyserythropoietic anemias (CDAs) are hereditary disorders characterized by distinct morphologic abnormalities of marrow erythroblasts. The unveiling of the genes mutated in the major CDA subgroups (I-CDAN1 and II-SEC23B) has now been completed with the recent identification of the CDA III gene (KIF23). KIF23 encodes mitotic kinesin-like protein 1, which plays a critical role in cytokinesis, whereas the cellular role of the proteins encoded by CDAN1 and SEC23B is still unknown. CDA variants with mutations in erythroid transcription factor genes (KLF1 and GATA-1) have been recently identified. Molecular diagnosis of CDA is now possible in most patients.

A novel type of congenital hypochromic anemia associated with a nonsense mutation in the STEAP3/TSAP6 gene.

STEAP3/TSAP6 encodes a ferrireductase that is involved in the acquisition of iron by developing erythroblasts and steap3/tsap6 null-mice display severe microcytic anemia. We report a family in which 3 siblings born to healthy parents display transfusion-dependent hypochromic anemia. A nonsense STEAP3/TSAP6 was identified in the siblings at the heterozygous state. This mutation was inherited from their father while no mutation was found in their mother. A large variability of expression was found between normal alleles in a control population, confirming a previous report that STEAP3/TSAPS6 is an expressed quantitative trait locus (e-QTL). Determination of the relative allele expression showed that the "normal" allele was expressed at a significantly higher level in the father than in the affected siblings relative to the shared mutated allele. The blood level of STEAP3/TSAP6 mRNA was severely reduced in the siblings, while both parents were in the lower range of normal controls. The STEAP3/TSAP6 protein was also reduced in lymphocytic cell lines from the patients. Collectively, our data support the hypothesis that STEAP3/TSAP6 deficiency leads to severe anemia in the affected siblings and results from the combination of a mutated allele inherited from their father and a weakly expressed allele inherited from their mother.

Growth differentiation factor 15 in patients with congenital dyserythropoietic anaemia (CDA) type II.

Congenital dyserythropoietic anaemias (CDAs) are heterogeneous, hereditary disorders hallmarked by ineffective erythropoiesis and tissue iron overload. Growth differentiation factor 15 (GDF15) was suggested to mediate iron overload in iron-loading anaemias, such as the thalassaemias and CDAI by suppressing hepcidin, the key regulator of iron absorption. Here, we show that serum GDF15 concentrations are elevated in subjects with CDAI and CDAII. Despite similar disease characteristics, CDAI patients present with significantly higher GDF15 concentrations compared to CDAII patients. Hepcidin concentrations are inappropriately low in CDAII patients considering the severe hepatic iron overload associated with this disorder. GDF15 significantly correlates with the degree of anaemia (Hb), the response of erythropoiesis (reticulocyte index) as well as with iron availability in the serum (transferrin saturation). The observation that GDF15 is elevated in CDAII patients is consistent with the proposal that GDF15 is among the erythroid factors down-regulating hepcidin and contributing to iron overload in conditions of dyserythropoiesis.

Sideroblastic anemia: molecular analysis of the ALAS2 gene in a series of 29 probands and functional studies of 10 missense mutations.

X-linked Sideroblastic Anemia (XLSA) is the most common genetic form of sideroblastic anemia, a heterogeneous group of disorders characterized by iron deposits in the mitochondria of erythroid precursors. XLSA is due to mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Thirteen different ALAS2 mutations were identified in 16 out of 29 probands with sideroblastic anemia. One third of the patients were females with a highly skewed X-chromosome inactivation. The identification of seven novel mutations in the ALAS2 gene, six missense mutations, and one deletion in the proximal promoter extends the allelic heterogeneity of XSLA. Most of the missense mutations were predicted to be deleterious, and 10 of them, without any published functional characterization, were expressed in Escherichia coli. ALAS2 activities were assayed in vitro. Five missense mutations resulted in decreased enzymatic activity under standard conditions, and two other mutated proteins had decreased activity when assayed in the absence of exogenous pyridoxal phosphate and increased thermosensitivity. Although most amino acid substitutions result in a clearly decreased enzymatic activity in vitro, a few mutations have a more subtle effect on the protein that is only revealed by in vitro tests under specific conditions.

Long-term outcome of hemizygous and heterozygous carriers of a germline GATA1 (G208R) mutation.

The transcriptional regulator GATA1 is crucially involved in megakaryocytopoiesis and erythropoiesis. Mutations of the gene which is located on the X chromosome have been associated with platelet and red blood cell abnormalities. We identified a family with a GATA1 (G208R) mutation in whom a low male birth rate and frequent miscarriages among heterozygous females suggested increased fetal death in male hemizygotes. Female mutation carriers had normal or near normal hemoglobin levels and platelet counts ranging from normal to severely reduced, probably reflecting skewed X chromosome inactivation. Platelets were dimorphous, and thrombocytopenia was associated with erythroblastosis. The only living male mutation carrier had severe macrothrombocytopenia with life-threatening bleeding episodes, moderate to severe anemia, eosinopenia, skeletal abnormalities, and abundant extramedullary hematopoiesis. Long-term sequelae in the 50-year-old patient included unilateral nephrectomy following misinterpretation of paraspinal hematopoiesis as renal cancer, spinal stenosis which was possibly favored by progressive bone marrow expansion, and severe secondary gout.

A dyserythropoietic anemia associated with homozygous Hb Plasencia [α125(H8)Leu→Arg (α2)] (HBA2:c.377T>G), a variant with an unstable α chain.

A female patient of Portuguese origin, who was born to consanguineous parents, presented with moderate anemia, mild jaundice and splenomegaly. Bone marrow aspiration showed an erythroid hyperplasia and binucleate erythroblasts, evoking a congenital dyserythropoietic anemia, type II (CDA II). Although microcytosis cast some doubt on the diagnosis, investigation was pursued along this line. The CDA II was finally ruled out as underglycosylation of band 3, remnants of endoplasmic reticulum cisternae and mutations in the SEC23B gene were all missing. On the other hand, analysis of the α-globin genes showed a base substitution at codon 125 (CTG→CGG) of the α2-globin gene, ascertaining a homozygosity for Hb Plasencia (HBA2:c.377T>G). This variant has an unstable α chain. In the absence of a thorough work-up, dyserythropoietic anemia associated with hemoglobin (Hb) variants having a moderately unstable α chain, may be mistaken for CDA II.

[Counting reticulocytes: new importance of an old method]. / Die Bestimmung der Retikulozytenzahl: Eine alte Methode gewinnt neue Bedeutung.

In clinical practice, the reticulocyte count is the most useful method to estimate red cell life span and red cell production. However, experience of hematologists as well as surveys have shown that counting of reticulocytes is often neglected in the diagnostic work-up of unclassified anemias, and many physicians have difficulties in interpreting the results. Formerly, this was partly due to the low precision of manual counts. Today, this method is largely replaced by flow cytometry, available in almost all larger systems for automated blood count analysis, at low costs and sufficient analytic precision. Interpretation is supported by calculated parameters such as the Reticulocyte Production Index. Here, the authors discuss the limits of the analytic methods and the problems of interpretation in the context of the laboratory profile and the clinical setting.

Epidemiology of rare anaemias in Europe.

Registry and epidemiological data of Rare Anaemias (RA) in Europe is in general still incomplete and/or partially documented. One important issue is the increasing prevalence of haemoglobin disorders (HD) due to migrations from high prevalence areas. The size of the problem, particularly for sickle cell disease (SCD), is already having an impact on health services in many European countries. The best known cause of rare anaemias associated with congenital haemolytic anaemia (CHA) in Europe is Hereditary Spherocytosis (HS) a red blood cell (RBC) membrane defect with a prevalence of 1 to 5 cases per 10.000 individuals. Some other causes of CHA are extremely rare and only few individual cases have been described worldwide (i.e. some RBC enzymopathies). Congenital defects of erythropoiesis are less frequent Diamond-Blackfan Anaemia (DBA) and Fanconi Anaemia (FA) exhibit a very low prevalence ranging from 4 to 7 per million live births. Congenital Dyserythropoietic Anaemia (CDA), a genetically heterogenous group, is still less frequent and exhibits a large variability of frequency depending on the European region: 0.1-3.0 cases per million births In addition many cases are known from a large autosomal dominant family in Sweden. Although incidence of Paroxysmal Nocturnal Haemoglobinuria (PNH) in Europe is still unknown, data collection from different sources has given quotes of 1 case per 100,000 individuals to 5 cases per million births.

Frequency of congenital dyserythropoietic anemias in Europe.

Congenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and striking abnormalities of erythroblast morphology. The mutated genes are known for the most frequent types, CDA I and II, but data about their frequency do not exist. The objective of this retrospective study was to estimate the frequency of CDA I and II, based on all cases reported in the last 42 yr in publications and identified registries or surveys. Reports were collected of 124 and 377 confirmed cases of CDA I and CDA II cases, respectively. The cumulated incidence of both types combined varied widely between European regions, with minimal values of 0.08 cases/million in Scandinavia and 2.60 cases/million in Italy. CDA II is more frequent than CDA I, with an overall ratio of approximately 3.2, but the ratio also varied between different regions. The most likely explanations for the differences are both differences in the availability of advanced diagnostic procedures and different levels of the awareness for the diagnosis of the CDAs. The estimations reported here are most probably below the true incidence rates, because of failure to make the correct diagnosis and to underreporting. Limited data do not suggest differing levels of risk in identified ethnic groups.

Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.

Bulky extramedullary hematopoiesis is not a rare complication of congenital dyserythropoietic anemia.

Bulky extramedullary hematopoiesis, usually detected in the thorax by imaging techniques, is a well-known complication in many types of congenital anemias. Here, we describe 12 cases of congenital dyserythropoietic anemia with extramedullary hematopoiesis which was always located in the paravertebral space of the thoracic spine and in other paraspinal regions in a few cases. All bulks were originally detected in chest radiographs and confirmed by imaging techniques such as computed tomography and/or magnetic resonance imaging. In some cases, thoracotomy was performed for suspected malignancy. Although the true prevalence is not known, paravertebral masses in patients with CDA of any type are not uncommon and should be the first differential diagnosis considered when masses adjacent to the spine are detected in this disorder.