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ObjectiveMajeed syndrome (MJS) is an autosomal recessive, systemic autoinflammatory disease (SAID) caused by biallelic loss-of-function variants in theLPIN2gene. It is characterized by early-onset chronic recurrent multifocal osteomyelitis (CRMO), dyserythropoietic anemia, and neutrophilic dermatosis. We analyzed a cohort of uncharacterized Indian patients for pathogenic variants inLPIN2and other genes associated with SAIDs.MethodsWe performed whole-exome sequencing (WES) for 1 patient and next-generation sequencing (NGS) targeted gene panel for SAIDs in 3 patients. One patient was a referral from neurology after clinical exome sequencing identified a novel variant inLPIN2. We reviewed the literature for all published studies of mutation-positive MJS patients and have summarized their clinical features and disease-causing variants.ResultsWe describe the largest series of patients with MJS outside of the Middle East. All 5 patients are homozygous for novel, possibly pathogenic variants in theLPIN2gene. Two of these variants are missense substitutions, and 3 are predicted to alter transcript splicing and create a truncated protein. In addition to the classical features of CRMO and anemia, patients exhibited previously unreported features, including abdominal pain, recurrent diarrhea/ear discharge, and erythema nodosum.ConclusionPatients with MJS may present initially to different specialists, and thus it is important to create awareness in the medical community. In India, consanguinity is a common sociocultural factor in many ethnic communities and an abbreviated NGS gene panel for autoinflammatory diseases should include MJS. The unavailability of interleukin 1 inhibitors in some countries poses a treatment challenge.

Infantile pyknocytosis is a rare and self-limiting cause of hemolytic anemia in neonates. It can result in severe anemia and hyperbilirubinemia. The pathogenesis is unknown: a genetic origin has been discussed; however, based on the current literature it is not clear which genetic mutations should be considered. We present a case of a premature twin, in whom genetic screening was performed. Genetic mutations in 46 genes associated with hereditary hemolytic anemia and dyserythropoietic anemia were tested. No mutations were found. In infantile pyknocytosis, a genetic defect in these genes is unlikely.

Southeast Asian Ovalocytosis results from a heterozygous deletion of 9 amino acids in the erythrocyte anion exchange protein AE1 (band 3). The report of the first successful birth of an individual homozygous for this mutation showed an association with severe dyserythropoietic anemia. Imaging of the proband’s erythrocytes revealed the presence of band 3 at their surface, a reduction in Wr(b) antigen expression, and increases in glycophorin C, CD44, and CD147 immunoreactivity. Immunoblotting of membranes from heterozygous Southeast Asian Ovalocytosis red cells showed a quantitative increase in CD44, CD147, and calreticulin suggesting a defect in reticulocyte maturation, as well as an increase in phosphorylation at residue Tyr359 of band 3, and peroxiredoxin-2 at the membrane, suggesting altered band 3 trafficking and oxidative stress, respectively. In vitro culture of homozygous and heterozygous Southeast Asian Ovalocytosis erythroid progenitor cells produced bi- and multi-nucleated cells. Enucleation was severely impaired in the homozygous cells and reduced in the heterozygous cells. Large internal vesicular accumulations of band 3 formed, which co-localized with other plasma membrane proteins and with the autophagosome marker, LC3, but not with ER, Golgi or recycling endosome markers. Immunoprecipitation of band 3 from erythroblast cell lysates at the orthochromatic stage showed increased interaction of the mutant band 3 with heat shock proteins, ubiquitin and cytoskeleton proteins, ankyrin, spectrin and actin. We also found that the mutant band 3 forms a strong interaction with non-muscle myosins IIA and IIB, while this interaction could not be detected in wild type erythroblasts. Consistent with this, the localization of non-muscle myosin IIA and actin was perturbed in some Southeast Asian Ovalocytosis erythroblasts. These findings provide new insights toward understanding in vivo dyserythropoiesis caused by the expression of mutant membrane proteins.

Anemia is a hematological disorder that adversely affects the health of millions of people worldwide. Although many variables influence the development and exacerbation of anemia, one major contributing factor is the impairment of erythropoiesis. Normal erythropoiesis is highly regulated by the zinc finger transcription factor GATA-1. Disruption of the zinc finger motifs in GATA-1, such as produced by germline mutations, compromises the function of this critical transcription factor and causes dyserythropoietic anemia. Herein, we utilize a combination of in vitro and in vivo studies to provide evidence that arsenic, a widespread environmental toxicant, inhibits erythropoiesis likely through replacing zinc within the zinc fingers of the critical transcription factor GATA-1. We found that arsenic interacts with the N- and C-terminal zinc finger motifs of GATA-1, causing zinc loss and inhibition of DNA and protein binding activities, leading to dyserythropoiesis and an imbalance of hematopoietic differentiation. For the first time, we show that exposures to a prevalent environmental contaminant compromises the function of a key regulatory factor in erythropoiesis, producing effects functionally similar to inherited GATA-1 mutations. These findings highlight a novel molecular mechanism by which arsenic exposure may cause anemia and provide critical insights into potential prevention and intervention for arsenic-related anemias.

Purpose BiallelicCADvariants underlie CAD deficiency (or early infantile epileptic encephalopathy-50, [EIEE-50]), an error of pyrimidine de novo biosynthesis amenable to treatment via the uridine salvage pathway. We further define the genotype and phenotype with a focus on treatment. Methods Retrospective case series of 20 patients. Results Our study confirms CAD deficiency as a progressive EIEE with recurrent status epilepticus, loss of skills, and dyserythropoietic anemia. We further refine the phenotype by reporting a movement disorder as a frequent feature, and add that milder courses with isolated developmental delay/intellectual disability can occur as well as onset with neonatal seizures. With no biomarker available, the diagnosis relies on genetic testing and functional validation in patient-derived fibroblasts. Underlying pathogenic variants are often rated as variants of unknown significance, which could lead to underrecognition of this treatable disorder. Supplementation with uridine, uridine monophosphate, or uridine triacetate in ten patients was safe and led to significant clinical improvement in most patients. Conclusion We advise a trial with uridine (monophosphate) in all patients with developmental delay/intellectual disability, epilepsy, and anemia; all patients with status epilepticus; and all patients with neonatal seizures until (genetically) proven otherwise or proven unsuccessful after 6 months. CAD deficiency might represent a condition for genetic newborn screening.

Krüppel-like factor 1 (KLF1/EKLF) is a transcription factor that globally activates genes involved in erythroid cell development. Various mutations are identified in the human KLF1 gene. The E325K mutation causes congenital dyserythropoietic anemia (CDA) type IV, characterized by severe anemia and non-erythroid-related symptoms. The CDA mutation is in the second zinc finger of KLF1 at a position functionally involved in its interactions with DNA. The molecular parameters of how CDA-KLF1 exerts its biological effects have not been addressed. Here, using an in vitro selection strategy we determined the preferred DNA-binding site for CDA-KLF1. Binding to the deduced consensus sequence is supported by in vitro gel shifts and by in vivo functional reporter gene studies. Two significant changes compared to WT binding are observed: G is selected as the middle nucleotide and the 3’-portion of the consensus sequence is more degenerate. As a consequence CDA-KLF1 did not bind the WT consensus sequence. However, activation of ectopic sites is promoted. Continuous activation of WT target genes occurs if they fortuitously contain the novel CDA site nearby. Our findings provide a molecular understanding of how a single mutation in the KLF1 zinc finger exerts an effects on erythroid physiology in CDA type IV.

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five–amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.

Congenital Dyserythropoietic Anemia (CDA) is a heterogeneous group of hematological disorders characterized by chronic hyporegenerative anemia and distinct morphological abnormalities of erythroid precursors in the bone marrow. In many cases, a final diagnosis is not achieved due to different levels of awareness for the diagnosis of CDAs and lack of use of advanced diagnostic procedures. Researchers have identified five major types of CDA: types I, II, III, IV, and X-linked dyserythropoietic anemia and thrombocytopenia (XLDAT). Proper management in CDA is still unsatisfactory, as the different subtypes of CDA have different genetic causes and different but overlapping patterns of signs and symptoms. For this reason, we developed a new telemedicine tool that will help doctors to achieve a faster diagnostic for this disease. Using open access code, we have created a responsive webpage named CoDysAn (Congenital Dyserythropoietic Anemia) that includes practical information for CDA awareness and a step-by-step diagnostic tool based on a CDA algorithm. The site is currently available in four languages (Catalan, Spanish, Italian, and English). This telemedicine webpage is available at http://www.codysan.eu.

Aim We aim to report the clinical manifestations, genetic testing results, magnetic resonance imaging (MRI) findings and biologics used in the management of non-bacterial osteomyelitis in our center. Methods We conducted a retrospective review of medical records. A previously proposed classification was implemented as follows: chronic recurrent multifocal osteomyelitis (CRMO), chronic non-bacterial osteomyelitis (CNBO) and acute non-bacterial osteomyelitis. Results Four females and three males with a median age at presentation of 6 years (6 months–14 years) presented with arthralgia (7/7), back pain (4/7), arthritis (4/7) and bone pain (2/7). Six patients had CRMO and one patient had CNBO. Genetic testing revealed an apparent homozygote p.S734L LPIN2 mutation in two siblings, a heterozygote p.M694V MEFV mutation in one patient with familial Mediterranean fever and heterozygote p.Q219H PSTPIPI variant of unknown significance in one patient. The most common lesions on MRI involved the tibia (6/7), talar bones (5/7), fibula (4/7) and sacroiliac joints (4/7). Three patients received infliximab. Two are in remission after 2 and 5 years, and the third was advanced after 5 years to canakinumab. Two other patients received canakinumab first. One patient with Majeed syndrome and dyserythropoietic anemia exhibited evidence of improvement, and one had partial improvement and was then treated with infliximab. Conclusion Non-bacterial osteomyelitis may coexist with other autoinflammatory diseases. MRI remains a favorable diagnostic tool and genetic testing may have a limited role in selected cases. Infliximab and canakinumab are associated with variable outcomes, and 6-week or less dosing intervals for both medications may be more effective.

The article presents the current data on the pathogenesis, features of clinical manifestations, and diagnostics of different variants of chronic non-bacterial («sterile») osteomyelitis (CNO). The inherited forms of the disease, such as syndromes of deficiency of the interleukin-1 receptor antagonist (DIRA), sterile pyarthrosis with pyoderma gangrenosum and acne (PAPA-syndrome), dyserythropoietic anemia with chronic non-bacterial osteomyelitis (Majeed-syndrome) are described. The pathogenesis of CNO and related diseases is based on hyperproduction of proinflammatory cytokines, particularly of interleukin-1 β and tumor necrosis factor- α . Based on the literature data and our own observations, the recommendations for diagnostics and treatment of CNO are presented.

Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5′-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5′-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease.

The dyserythropoietic anemia disease is a genetic disorder of erythropoiesis characterized by morphological abnormalities of erythroblasts. This is caused by human gene C15orf41 mutation. The uncharacterized C15orf41 protein is involved in the formation of a functional complex structure. The uncharacterized C15orf41 protein is thermostable, unstable and acidic. This is associated with TPD (Treponema Pallidum) domain (135 to 265 residue position) and three PTM sites such as K50 (Acetylation), T114 (Phosphorylation) and K176 (Ubiquitination). C15orf41 is paralogous to isoform-1 (gi|194018542|) and open reading frame isoform-CRA_c (gi|119612744|) of Homo sapiens located at chromosome 15. It interacts with the human ATP (Adenosine Triphosphate) binding domain 4 (ATPBD4) having similarity score 0.725 as per protein-protein interaction (PPI) network analysis. This data provides valuable insights towards the functional characterization of human gene C15orf41.

We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.

Background: Iron overload is an emerging and underestimated problem in management of patients with hereditary anemia characterized by chronic hemolysis or ineffective erythropoiesis in the absence of regular red cell transfusions. Liver iron overload was present in 65% of the patients with hereditary hemolytic anemia who were never transfused. (Van Straaten et al., Br J Haematol. 2018) Iron-loading results from inadequately high intestinal iron absorption in response to low hepcidin and ultimately leads to iron overload in liver and other organs. Iron chelation treatment is recommended, however drug-related toxicity is considerable, and treatment with iron chelators is expensive. Alternative treatment options for anemic patients not tolerating iron chelating agents are not available. For adequate iron absorption, a low luminal pH is required to solubilize dietary iron salts, and to promote the reduction of dietary Fe(III) to Fe(II) by ferriredutases as only Fe(II) can be absorbed. Proton pump inhibitors (PPI) block gastric acid secretion and thereby impede iron absorption. No studies are available that quantify effects of PPIs on dietary iron absorption in human. However, PPIs have shown to minimize phlebotomy requirements in patients with hereditary hemochromatosis. (Vanclooster et al., Gastroenterology. 2017) Here we report the design of the currently ongoing PPI SHINE AGAIN study that evaluates the efficacy and safety of PPI treatment in patients with non-transfusion dependent hereditary anemias. Methods: The PPI SHINE AGAIN is a phase 3, multicentre, randomized, placebo-controlled, cross-over clinical trial (Netherlands Trial Register, identifier [NL6659]). Transfusion independent adults with a form of hemolytic or dyserythropoietic anemia and mild to moderate iron overload are randomized in a 1:1 ratio to start with either esomeprazole 40 mg (administered orally BID) or placebo. Mild to moderate iron overload is defined as baseline liver iron content (LIC) of 3-15 mg Fe/g dry weight as measured by T2* MRI (MRQuantif Software Université de Rennes, https://imagemed.univ-rennes1.fr/en/mrquantif/quantif.php) without or on stable iron chelation therapy with no expected dose adjustments over the next 2 years. Additional criteria included baseline Hb ≤ 11.3 g/dL, expected to receive less than 4 red cell transfusions in the following 12 months, and no phlebotomies. The trial consists of two treatment periods of 12 months each (Figure), directly starting after baseline MRI. The primary study end-point is effectiveness of PPI treatment defined as difference in delta LIC after one-year treatment with esomeprazole compared to one-year treatment with placebo. Key secondary endpoints include tolerability, quality of life (assessed by EQ5D-5L questionnaire) and cost-effectiveness (assessed by iMTA Productivity Cost Questionnaire and iMTA Medical Consumption Questionnaire). Inclusion in the PPI SHINE AGAIN was completed in April 2019. Thirty patients are enrolled (non-transfusion dependent β-thalassemia n=10; pyruvate kinase deficiency n=8; congenital dyserythropoietic anemia n=3; sideroblastic anemia n=3; SCD n=2; HbH disease n=2; hereditary elliptocytosis n=1; G6PD deficiency n=1) with a median age of 44 years (IQR 28, 54). Results are expected Q3 2020. The trial is funded by ZonMW, The Netherlands Organization for Health and Research Development, and by the Innovatiefonds Zorgverzekeraars. Disclosures Nur: Novartis Pharmaceuticals: Consultancy. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding.

Introduction Congenital dyserythropoietic anemias (CDA) are rare inherited red cell disorders characterized by ineffective erythropoiesis and inappropriate reticulocytopenia. CDAs are usually difficult to diagnose due to variable phenotypes and overlapping bone marrow (BM) morphology with other disorders. Numerous implicated causal genes make Sanger sequencing a less likely approach and hence, the use of targeted resequencing can expedite molecular diagnosis. This study aimed at determining the genetic spectrum of CDAs and translating the results into patient care. Methods Twenty nine patients with clinical and laboratory evidence suggestive of CDA and 1 patient suggestive of CDA with thrombocytopenia by BM morphology were studied. Various biochemical and molecular tests were done to exclude common hemolytic anemias. Common SEC23B: p.Tyr462Cys variant in our patients with CDA was screened by Sanger sequencing. DNA libraries were prepared using TruSight One Sequencing Panel and TruSeq Custom Amplicon Panel and sequenced on Illumina platform. After data analysis variants were classified and the most likely disease-causing variants were validated by Sanger sequencing followed by pedigree analysis. Results Out of 27 patients of suspected CDA, SEC23B: p.Tyr462Cys variant was found in 10 patients. Rest of the remaining 17 patients were subjected to targeted resequencing. Data analysis revealed novel potentially pathogenic variants in compound heterozygosity in SEC23B in 4 patients and 1 patient had a heterozygous variant in SEC23B. There could be the possibility of intronic or large indel in her. The variants were distributed throughout the SEC23B gene. Notably, in 7 patients with suspected CDA, the final molecular diagnosis were hemolytic anemias. Of them, 4 showed likely pathogenic variants in PKLR gene and 1 each had probably causal variant in MTRR, SPTB and PIEZO1 genes. In the patient's with pyruvate kinase deficiency, screening by enzyme assays were normal. Except for the patient with MTRR gene defect all 6 had transfusion dependent anemia and BM showed dyserythropoiesis. One patient each of GATA1 gene variant (novel) and a known pathogenic variant p.Glu325Lys in KLF1 gene (CDA type IV) was detected. Of 17 cases subjected to targeted resequencing the diagnosis was achieved in ~76% (13/17) of cases. The phenotypes correlated with the genetic defects found in the SEC23B gene. The homozygous and compound heterozygous defects in this gene cause CDA type II. As anticipated GATA1 gene defect (p.Val205Leu) was found in a patient of X-linked thrombocytopenia with dyserythropoietic anemia. Patient with KLF1 had high levels of fetal hemoglobin along with features of dyserythropoiesis in BM compatible with the phenotype of variant p.Glu325Lys causing CDA type IV. Phenotype-genotype correlation was discrepant in 7 cases of CDA. In 4 cases pyruvate kinase deficiency (PKLR) was found and each case of hereditary xerocytosis (PIEZO1), membrane defect (SPTB) and MTRR defect was found. Conclusion(s) CDA showed a highly varied etiology. Our experience demonstrates a high diagnostic yield (~76%) of targeted resequencing for molecular diagnosis of suspected CDAs. Discrepancy was noted in 41% (7/17) cases with suspected CDA which were diagnosed as hemolytic anemia after molecular analysis. Establishing the correct diagnosis of pyruvate kinase deficiency led to an evidence-based decision of splenectomy that eliminated transfusion dependence. In the patient with MTRR defect change in therapy was suggested. Prenatal diagnosis was done for 2 families, where in 1 of the family both the SEC23B variants were novel and in compound heterozygosity. This study highlights the importance of genetic testing in patients under frequent blood transfusions and suspected CDAs, to provide accurate diagnosis and therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Congenital dyserythropoietic anemia type I (CDA I) is associated, as other anemic noninflammatory states, with ineffective erythropoiesis and increased iron absorption, which may lead to complication of iron overload. The latter complication requires iron-chelating therapy, which may be associated with adverse effects and toxicity. Gastric acid production is known to be an important factor that facilitates non-heme iron absorption. The purpose of this study was to examine whether treatment with proton pump inhibitors (PPIs) can decrease iron absorption in patients with CDA I. Eight CDA I patients (4 boys) aged 12-18 years with mild iron overload (not yet requiring chelating therapy) received 20 mg/d omeprazole for 6 months. Blood samples were obtained for ferritin, C-reactive protein, hemoglobin, calcium, and magnesium at baseline, at the end of months intervention and 6 months after its cessation. The mean ferritin level decreased from 585 ± 180 ng/ml at baseline to 522 ± 172 ng/ml at the end of 6-month treatment and 660 ± 256 ng/ml 6 months after cessation of omeprazole treatment (p = 0.009). Omeprazole treatment caused a nonsignificant reduction in the mean iron level (iron 159 ± 42, 136 ± 54,167 ± 34 µg/dl, p = 0.302). However, mean hemoglobin level was mildly but significantly reduced (Hg 10.0 ± 0.8, 9.55 ± 1.0, 10.4 ± 10.7 g/dl, p = 0.002). No adverse effects were reported. Our investigation suggests that administration of PPI to patients with CDA I may reduce iron absorption and may lower iron overload and the need for chelation therapy.

Myelolipoma (ML) is an uncommon benign mesenchymal neoplasia composed of mature adipose and hematopoietic tissues of uncertain etiology. Less than 3% of MLs occur in the mediastinal topography. The main differential diagnosis involves extramedullary hematopoiesis; therefore, pathological evaluation is essential for the definitive diagnosis. The authors report the case of a 50-year-old man diagnosed with congenital dyserythropoiesis and secondary hemosiderosis, who presented a posterior mediastinal tumor. The tumor was resected. It was macroscopically characterized by mature fat tissue with fibrous areas and soft consistency, which was yellowish at the cut surface. Histology revealed a well-defined nodule composed of adipocytes and hematopoietic tissue represented by erythroid, granulocytic, and megakaryocytic series, which was consistent with the diagnosis of ML located in the posterior mediastinum. There was no recurrence of the lesion during the 3-year follow-up. The aim of this report is to show the diagnosis of an unusual mediastinal lesion in the context of a chronic hematologic disease.

To the Editor: Majeed syndrome (OMIM #609628) is a syndromic form of chronic recurrent multifocal osteomyelitis (CRMO) that presents with early onset, severe CRMO, and a microcytic dyserythropoietic anemia of variable severity. Onset in all reported cases has been in the first 2 years of life1,2,3. A minority of patients develop a neutrophilic dermatosis. The disease is due to mutations in LPIN2 , which encodes LIPIN2, a phosphatidate phosphatase important in lipid metabolism2,3,4,5,6. Here we present the case histories of 2 male cousins of Indian heritage with Majeed syndrome. Both born to parents with consanguineous marriages, the proband presented with typical Majeed syndrome (onset of CRMO by 2 yrs of age, significant microcytic dyserythropoietic anemia, and failure to thrive), but the cousin had later onset, milder disease more reminiscent of nonsyndromic CRMO (onset at 8 yrs of age and minimal anemia). Both were found to be homozygous for the same novel LPIN2 mutation. Our report details the natural history of the disease in these 2 boys, expands the at-risk ethnic and racial group of Majeed syndrome, and demonstrates that the disease can present … Address correspondence to Dr. P.J. Ferguson, Department of Pediatrics, University of Iowa Carver College of Medicine, 200 Hawkins Drive, Iowa City, Iowa 52242, USA. E-mail: polly-ferguson{at}uiowa.edu

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cKOMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches.

To the Editor: Chronic recurrent multifocal osteomyelitis (CRMO) is hypothesized to be an autoimmune disorder because of its association with multiple autoimmune diseases, including inflammatory bowel disease, psoriasis, acne, pustulosis, Sweet syndrome, dyserythropoietic anemia, pyoderma gangrenosum (PG), sclerosing cholangitis, inflammatory arthritis, Still disease, Takayasu arteritis (TA), Ollier disease, and dermatomyositis1,2. PG association with TA is rare; however, Ujiie, et al showed that PG is associated with TA in 33% of patients3. Occurrence of all 3 conditions together (CRMO, PG, and TA) is very rarely reported. A 10-year-old girl born to nonconsanguineous parents presented with a history of intermittent swelling over the right side of the mandible, associated with pain and claudication pain over legs and back with breathlessness on exertion for the past 2 years. She also developed ulcerative cauliflower-like skin lesions over the dorsum of left foot over the past 2 months (Figure 1). On examination, her pulses were absent … Address correspondence to Dr. S. Kumar, Christian Medical College, Department of Pediatrics, Christian Medical College, Vellore, Tamil Nadu 632004, India. E-mail: sathishkumar{at}cmcvellore.ac.in

Defects in mevalonate kinase, a critical rate-limiting enzyme in cholesterol and isoprene metabolism, have been associated with 2 clinical phenotypes: mevalonic aciduria, which presents in infancy or early childhood with growth failure, dysmorphic features, and neurologic disease; and hyperimmunoglobulinemia D and periodic fever syndrome, which usually presents outside the neonatal period as an autoinflammatory periodic fever syndrome. This report describes a kindred with 2 siblings affected by severe mevalonate kinase deficiency (mevalonic aciduria) with perinatal onset. Dysmorphic and central nervous system abnormalities, anemia, and cholestasis were prominent features in 1 sibling. Both cases were fatal, 1 in the immediate neonatal period and 1 in utero. The small number of cases of mevalonate kinase deficiency presenting in the perinatal period have typically been severely affected, with signs and symptoms of a severe multisystem disorder. Predominant features of perinatal onset mevalonate kinase deficiency include intrauterine growth restriction, cerebral ventriculomegaly, dysmorphic features, skeletal abnormalities, dyserythropoietic anemia with extramedullary erythropoiesis, thrombocytopenia, cholestatic liver disease, persistent diarrhea, renal failure, recurrent sepsis-like episodes, and failure to thrive. Clinical findings may mimic severe intrauterine viral infection, a chromosomal abnormality, or an acute sepsis syndrome, potentially contributing to delays in diagnosis of this rare condition. Perinatal onset mevalonate kinase deficiency is associated with a very poor prognosis, with death in utero or in early infancy. Detailed autopsy findings in mevalonate kinase deficiency have rarely been reported.

To the editor: Southeast Asian ovalocytosis (SAO) is caused by a heterozygous 27-nucleotide deletion in SLC4A1 coding for band 3, the anion-exchange protein of the red cell membrane.[1][1][⇓][2]-[3][3] This asymptomatic dominant trait is considered as a host genetic adaptation to malaria in

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.

Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of rare hereditary disorders of erythropoiesis characterized by morphologic abnormal erythroblasts in the bone marrow. Three types of the disease are known as type I, II and III, and the variant type of CDA and several minor subgroups of CDA have been also reported since the first classification. Recently, responsible genes for type I (CDAN1) and type II (SEC23B) have been identified and the molecular pathogenesis of the disease is currently being explored. Although CDAs rarely transform to myelodysplastic syndrome or leukemia, the disease is important to understand the mechanism of hemopoiesis in humans.

Mevalonate kinase deficiency (MKD) is a rare inborn error of metabolism caused by mutations in the mevalonate kinase (MVK) gene. The clinical phenotype is variable, ranging from the hyperimmunoglobulinemia D and periodic fever syndrome (HIDS) to mevalonic aciduria (MA), a severe metabolic disease. We report here for the first time (to our knowledge) the case of a patient with MKD and congenital dyserythropoietic anemia. Clinical and laboratory characteristics of inflammatory attacks were compatible with HIDS, but mild dysmorphic features and elevated urinary mevalonic acid levels in the absence of an inflammatory attack suggested an intermediate phenotype between HIDS and MA. Genomic sequencing of the MVK gene revealed compound heterozygosity for a missense mutation previously described in MA (V310M) and a novel missense mutation (Y116H). By contrast, sequencing of the novel CDAII (SEC23B) gene revealed no mutations, suggesting that the bone marrow abnormalities were causally related to the MKD. Treatment with corticosteroids and colchicine directed at controlling the autoinflammatory disease resulted in improvement of the anemia.

SEC23B gene encodes an essential component of the coat protein complex II (COPII)-coated vesicles. Mutations in this gene cause the vast majority the congenital dyserythropoietic anemia Type II (CDA II), a rare disorder resulting from impaired erythropoiesis. Here, we investigated 28 CDA II patients from 21 unrelated families enrolled in the CDA II International Registry. Overall, we found 19 novel variants [c.2270 A>C p.H757P; c.2149−2 A>G; c.1109+1 G>A; c.387(delG) p.L129LfsX26; c.1858 A>G p.M620V; c.1832 G>C p.R611P; c.1735 T>A p.Y579N; c.1254 T>G p.I418M; c.1015 C>T p.R339X; c.1603 C>T p.R535X; c.1654 C>T p.L552F; c.1307 C>T p.S436L; c.279+3 A>G; c. 2150(delC) p.A717VfsX7; c.1733 T>C p.L578P; c.1109+5 G>A; c.221+31 A>G; c.367 C>T p.R123X; c.1857_1859delCAT; p.I619del] in the homozygous or the compound heterozygous state. Homozygosity or compound heterozygosity for two nonsense mutations was never found. In four cases the sequencing analysis has failed to find two mutations. To discuss the putative functional consequences of missense mutations, computational analysis and sequence alignment were performed. Our data underscore the high allelic heterogeneity of CDA II, as the most of SEC23B variations are inherited as private mutations. In this mutation update, we also provided a tool to improve and facilitate the molecular diagnosis of CDA II by defining the frequency of mutations in each exon. Am. J. Hematol., 2010. © 2010 Wiley-Liss, Inc.

A family with congenital dyserythropoietic anaemia type III was studied. Twenty patients and 10 of their healthy siblings were clinically examined and questioned about their medical history. Blood sampling and bone marrow aspirations were also performed. Forty-five percent of the patients reported symptoms of anaemia and 35% regularly felt weakness, fatigue, or headache. However, the majority of the patients regarded themselves as healthy. The bone marrow showed a uniform picture of erythroid hyperplasia with multinuclear erythroblasts and gigantoblasts with up to 12 nuclei. There was laboratory evidence of intravascular haemolysis and mild anaemia. We also observed a high prevalence of monoclonal gammopathy of undetermined significance (3 cases) and myeloma (1 case) among the patients.

Two affected individuals of the Swedish family with CDA, type III, in which the disease is transmitted as an autosomal dominant character, were studied. Both cases displayed features hitherto undescribed in this family but described in patients with CDA, type III, in whom the inheritance may have been as an autosomal recessive character. Such features were: (a) haemosiderinuria, (b) grossly disorganised erythroblast nuclei, (c) differences in the ultrastructural appearances of individual nuclei within the same multinucleate erythroblast and (d) intraerythroblastic inclusions resembling precipitated globin chains. In both cases the giant mononucleate erythroblasts and the multinucleate erythroblasts had total DNA contents up to 28c (1c = haploid DNA content) and 48c respectively, and some DNA synthesising bi- and multinucleate erythroblasts contained one or more nuclei which were unlabelled with 3H-thymidine. These findings are similar to those in patients with the autosomal recessive type of disease. Thus no major phenotypic differences are yet apparent between cases of CDA, type III, with different patterns of inheritance. Analysis of the surface erythrocyte proteins of the 2 Swedish CDA, type III, patients with monoclonal antibodies recognising Band 3, glycophorins A, B, C and D, Rh, CD44, CD47, CD55, CD58, CD59, Lutheran, Kell, LW and acetylcholinesterase did not reveal any gross abnormality of expression of these proteins. A slightly altered expression of blood group antigens A and H was revealed by the lectins Dolichos biflorus and Ulex europaeus and the Mr of Band 3 as judged by SDS polyacrylamide gel electrophoresis was also slightly reduced, suggesting that there may be minor alterations in the degree of N-glycosylation of some red cell membrane constituents.

Contains fulltext : 70405.pdf (Publisher’s version ) (Closed access) Congenital dyserythropoietic anemia (CDA) is a rare group of red blood cell disorders characterized by ineffective erythropoiesis and increased iron absorption. To determine whether growth differentation factor 15 (GDF15) hyper-expression is associated with the ineffective erythropoiesis and iron-loading complications of CDA type I (CDA I), GDF15 levels and other markers of erythropoiesis and iron overload were studied in blood from 17 CDA I patients. Significantly higher levels of GDF15 were detected among the CDA I patients (10 239 +/- 3049 pg/mL) compared with healthy volunteers (269 +/- 238 pg/mL). In addition, GDF15 correlated significantly with several erythropoietic and iron parameters including Hepcidin-25, Ferritin, and Hepcidin-25/Ferritin ratios. These novel results suggest that CDA I patients express very high levels of serum GDF15, and that GDF15 contributes to the inappropriate suppression of hepcidin with subsequent secondary hemochromatosis.

Anemia is a commonly encountered problem in the fetal and neonatal period, and can lead to significant morbidity and mortality. Intrinsic disorders of the erythrocyte, such as the hemoglobinopathies, enzyme deficiencies, and membrane defects are common causes of neonatal anemia. Genetic diseases that lead to decreased erythrocyte production, such as Diamond-Blackfan anemia, Schwachman-Diamond syndrome, and Congential Dyserythropoietic Anemia, are rare causes of perinatal anemia, but are important to recognize as they are often associated with other congenital abnormalities and require specialized treatment. This review focuses on the perinatal presentation and management of intrinsic erythrocyte disorders, as well as on the diagnosis and management of genetic conditions leading to erythrocyte underproduction.

Hypocholesterolemia of unknown etiology has been previously described in various chronic anemias. Few small studies also suggested that those patients have a lower incidence of atherosclerotic events. The aim of our study was to determine the extent of hypocholesterolemia in various types of anemias. We studied 59 patients with chronic anemias associated with high-erythropoietic activity (thalassemia intermedia, congenital dyserythropoietic anemia type I, congenital spherocytosis), 8 patients with low-erythropoietic activity anemias (acquired aplastic anemia, Fanconi anemia, and Diamond Blackfan anemia), and 20 healthy controls. Mean serum cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, hemoglobin, serum ferritin, soluble transferrin receptor (STR), and serum erythropoietin levels were determined in each patient. All patients with chronic anemia and increased erythropoietic activity had hypocholesterolemia, whereas none of those with low erythropoietic activity was hypocholesterolemic. Mean serum cholesterol, HDL cholesterol, and LDL cholesterol levels were found to be significantly lower in the high-erythropoietic activity group (80+/-19 mg/dl; 31+/-10 mg/dl; 35+/-14 mg/dl, respectively) compared with the control group (P

Majeed syndrome is an autoinflammatory disorder consisting of chronic recurrent multifocal osteomyelitis, congenital dyserythropoietic anemia, and neutrophilic dermatosis. To date, 2 unrelated families with Majeed syndrome have been reported. Mutations in LPIN2 have been found in both families. Here we report a third consanguineous family with Majeed syndrome with a novel mutation. The patient, a 3-year-old Arabic girl, had hepatosplenomegaly and anemia as a neonate. At age 15 months, she developed recurrent episodes of fever and multifocal osteomyelitis. In addition, bone marrow aspiration demonstrated significant dyserythropoiesis, suggesting Majeed syndrome. Coding sequences and splice sites of LPIN2 were sequenced in the patient and her mother. A homozygous single-basepair change was detected in the donor splice site of exon 17 (c.2327+1G>C) in the patient; her mother was heterozygous at this site. These data confirm the role of LPIN2 mutations in the etiology of Majeed syndrome.

Although acute lymphoblastic leukemia (ALL) is the most common childhood cancer, the etiology is usually unknown. However, genetic studies suggest that in some cases, inherited mutations in genes that regulate hematopoiesis contribute to leukemogenesis [1-3]. In a recent example, two independent studies of families with autosomal dominant thrombocytopenia and high incidence of lymphoblastic leukemia revealed multiple germline, single-nucleotide mutations in the gene encoding the ETS family transcriptional repressor ETV6 [2,3]. While ETV6 is involved in the most common translocation found in childhood ALL (ETV6-RUNX1), and somatic ETV6 mutations have also been previously described in B cell ALL [4], myelodysplastic syndrome [5] and T cell leukemias [6], these reports were the first to describe disease-causing germline ETV6 mutations. ETV6 contains three functional domains: pointed (PNT), involved in protein-protein interactions including homo-oligomerization; central, which promotes DNA binding and is essential for repressive function in vitro; and the ETS domain, which binds DNA. The two independent groups identified, four distinct mutations occurring in the ETS domain (R369Q, R399C, R418G, and 385_418del), and both groups identified the same mutation in the central domain (P214L). Functionally, each of these mutations appears to abrogate ETV6 nuclear localization, resulting in loss of transcriptional repression normally conferred by ETV6. Importantly, addition of WT ETV6 was unable to restore transcriptional repression lost by variant ETV6, suggesting that germline variant ETV6 acts in a dominant negative fashion (Figure ​(Figure1).1). Impaired nuclear localization is common to all of the studied mutants, as well as ETV6-RUNX1 [7], implying a shared mechanism of failed tumor suppression. Interestingly, while in ETV6-RUNX1+ ALL the allele not involved in the translocation is usually deleted or mutated, the “wild-type” ETV6 allele remained intact in each of the four tumors studied by these groups, again supporting the hypothesis of dominant negative function of the inherited variant proteins. Figure 1 Mutation of ETV6 impairs nuclear localization and transcriptional repression Affected families studied by both groups displayed mild to moderate thrombocytopenia, some of them red cell macrocytosis, and predisposition not only for leukemia but also a number of other cancers. Malignancies developed in individuals inheriting ETV6 mutations included skin cancer, MDS, CMML, stage IV colon cancer, and, most commonly, pre-B cell ALL. Out of 17 patients carrying ETV6 variants studied by the University of Washington group, nine developed malignancies, including three leukemias. In 10 affected individuals studied by the University of Colorado group, three developed B cell leukemia. ETV6 mutations identified in both studies segregated completely with thrombocytopenia and predisposition toward cancer development, demonstrating a dominant transmission pattern. Interestingly, all patients with malignancies carried ETV6 mutations, but several individuals with mutations did not have cancer. Many somatic ETV6 mutations have been described previously in cancer, including somatic alterations affecting the same amino acids disrupted by recently described germline mutations. However, all five germline mutations identified were absent from public databases, and family members with inherited ETV6 variants did not exhibit mutations in other known leukemia predisposition genes, such as TP53, RUNX1 or ANKRD26. The consistent features of thrombocytopenia and cancer risk displayed by families in both studies highlights the functional relevance of germline ETV6 mutations, but the incomplete penetrance of malignancies indicates that other factors are required for oncogenesis. The discovery of germline ETV6 variants is an exciting new finding with clinical relevance both for individuals inheriting these mutations as well as patients who may develop leukemia after acquiring mutations in ETV6 or related pathways. Little is currently known about what other oncogenic mediators may act in cooperation with mutated ETV6, but these factors likely play an important role in determining clinical outcome and may even represent targets for novel therapeutic approaches. Experiments to identify cooperating mutations using unbiased or targeted next generation sequencing identified alterations in a number of genes that are implicated in tumorigenesis, including RUNX1 and KRAS in a case of MDS, and a novel PAX5-SHB fusion in ALL. These data and those from large scale sequencing efforts such as TARGET, suggest that mutation in a variety of genes can cooperate with ETV6 dysfunction to promote leukemogenesis, particularly in those with critical roles in hematopoiesis. In summary, these reports have established that ETV6 germline mutations impair its nuclear localization and transcriptional repression and result in thrombocytopenia and predisposition to cancer, but much remains to be explored in how ETV6 dysfunction initiates leukemogenesis, and what cooperating mutations are necessary for progression to clinical disease. The high incidence of B cell leukemias in these families, as well as the high rate of ETV6 translocation and/or deletion in sporadic precursor B cell ALL, suggests that ETV6 may play an unappreciated role in B cell development. The identification of dominant negative ETV6 variants will allow some of these questions to be addressed experimentally. Better understanding of aberrant ETV6 activity, as it relates to hematopoietic differentiation and leukemogenesis, may guide the development of new treatment strategies benefitting patients with both heritable and acquired forms of cancer.

Congenital erythropoietic porphyria (CEP) is a rare genetic disease that is characterized by a severe cutaneous photosensitivity causing unrecoverable deformities, chronic hemolytic anemia requiring blood transfusion program, and by fatal systemic complications. A correct and early diagnosis is required to develop a management plan that is appropriate to the patient's needs. Recently only one case of X-linked CEP had been reported, describing the trans-acting GATA1-R216W mutation. Here, we have characterized two novel X-linked CEP patients, both with misleading hematological phenotypes that include dyserythropoietic anemia, thrombocytopenia, and hereditary persistence of fetal hemoglobin. We compare the previously reported case to ours and propose a diagnostic paradigm for this variant of CEP. Finally, a correlation between phenotype variability and the presence of modifier mutations in loci related to disease-causing gene is described.

Congenital dyserythropoietic anemia type I (CDA I) is a rare autosomal recessive disorder with ineffective erythropoiesis and iron overloading. More than 100 cases have been described, but with the exception of a report on a large Bedouin tribe, these reports include only small numbers of cases, and no data on the lifetime evolution of the disease are available. Since 1967, we have been able to follow 21 cases from 19 families for up to 37 years. Twenty-one patients with a confirmed diagnosis of CDA I exhibited chronic macrocytic anemia of variable severity, requiring regular red cell transfusions only in 2 individuals. Four developed gallstones before the age of 30 years. Fifteen of 16 cases alive at the time of analysis showed mutations of at least one allele from exons 6 to 28 within CDAN1. Iron overloading is to be expected in all patients. In 9 patients, iron depletion was started between the ages of 7 and 36 years. Splenectomy, which was performed in 7 patients, did not result in improvement of hemoglobin values. Five patients were treated with interferon alpha-2a, and all responded with a rise in hemoglobin concentration of between 25 and 35 g/L (2.5 and 3.5 g/dL) starting within 4 weeks.

GATA1 is mutated in patients with 2 different disorders. First, individuals with a GATA1 mutation that blocks the interaction between GATA-1 and its cofactor Friend of GATA-1 (FOG-1) suffer from dyserythropoietic anemia and thrombocytopenia. Second, children with Down syndrome who develop acute megakaryoblastic leukemia harbor mutations in GATA1 that lead to the exclusive expression of a shorter isoform named GATA-1s. To determine the effect of these patient-specific mutations on GATA-1 function, we first compared the gene expression profile between wild-type and GATA-1–deficient megakaryocytes. Next, we introduced either GATA-1s or a FOG-binding mutant (V205G) into GATA-1–deficient megakaryocytes and assessed the effect on differentiation and gene expression. Whereas GATA-1–deficient megakaryocytes failed to undergo terminal differentiation and proliferated excessively in vitro, GATA-1s–expressing cells displayed proplatelet formation and other features of terminal maturation, but continued to proliferate aberrantly. In contrast, megakaryocytes that expressed V205G GATA-1 exhibited reduced proliferation, but failed to undergo maturation. Examination of the expression of megakaryocyte-specific genes in the various rescued cells correlated with the observed phenotypic differences. These studies show that GATA-1 is required for both normal regulation of proliferation and terminal maturation of megakaryocytes, and further, that these functions can be uncoupled by mutations in GATA1.

Congenital dyserythropoietic anaemia (CDA) type I is a rare, inherited disorder characterised by ineffective erythropoiesis and macrocytic anaemia. Complex bone disease has only occasionally been associated with this disease. CDA I is caused by mutations in the CDAN1 gene encoding for codanin-1. Our aim was to characterise the CDAN1 mutation in eight unrelated patients with sporadic CDA I, three of whom had complex bone disease. Six novel mutations in the CDAN1 gene were identified. In two patients, one mutation and in another, both mutations were elusive. No patient was homozygous for a null-type mutation. However, one patient with complex bone disease was homozygous for a splice-site mutation (IVS-12+5G>A). Western blotting revealed that codanin-1 synthesis was 65% less than the control. Five single nucleotide polymorphisms (SNPs) previously unreported in the literature or the SNP database were also identified. Although the absence of codanin-1 is probably lethal, the presence of 35% of the protein was compatible with life but was associated with severe clinical manifestations. However, in most patients studied, no correlation could be established between the expected levels of codanin-1 or the nature of the mutation and the severity of the clinical manifestations.

We studied two related patients (III.4 and III.10) with spontaneous clinical bleeding problems and a family history suggestive of X-linked inherited thrombocytopenia (Figure 1A). Both patients presented with hematomas, ecchymosis, epistaxis, and gingival bleeding symptoms since childhood and developed splenomegaly. Hematological investigations (after the patient was splenectomized) showed the presence of macrothrombocytopenia combined with mild features of dyserythropoiesis including poikilocytosis, anisocytosis, and schizocytes, Howell–Jolly bodies in erythrocytes and stomatocytes but no anemia (Table I). Both patients did not respond to immune thrombocytopenic purpura-specific therapy such as intravenous immunoglobulins, steroids, or splenectomy. Bone marrow analysis showed hypermegakaryopoiesis with an increased number of immature megakaryoblasts but no features of dysmyelopoiesis. Platelet functional studies for patient III:4 showed normal aggregations for collagen and ristocetin (amplitudes of 84% and 86%, respectively) and mildly reduced for ADP (amplitude of 40%). The PFA100 closure time with collagen/epinephrine for patient III:4 was prolonged (4300 s; nl: 82–142 s) while normal with collagen/ADP. X-linked macrothrombocytopenia with platelet dysfunction has been described by others and us for patients with mutations in the gene for the transcription factor GATA1 [1–10]. The GATA family of nuclear regulatory proteins serves as a prototype for the action of lineage-restricted transcription factors. The GATA1 protein is typically present in the more differentiated cells of the erythroid, megakaryocytic, mast, and eosinophilic lineages, whereas GATA2 expression is limited to HSCs and early haematopoietic progenitors [11]. Remarkably since its first description in 2001, still only a limited number of families are presently reported with germline GATA1 missense mutations and these presented with closely related but still somewhat altered hematological disorders. They comprise severe dyserythropoietic anemia with macrothrombocytopenia (V205M, G208R, and D218Y), macrothrombocytopenia with mild dyserythropoietic features (G208S, D218G), macrothrombocytopenia with mild b-thalassemia (R216Q), and congenital erythropoietic porphyria (R216W) [1–10]. Figure 1(B) shows the position of these mutations that are all present in the N-terminal zinc finger of GATA1. Males with these mutations have enlarged platelets with a paucity of alpha granules and a reduced function. Some patients also present with splenomegaly [7, 9] or myelodysplasia with marked organomegaly [10], likely due to ineffective and consequential extramedullary hematopoiesis. Genotype–phenotype correlations are hampered by the low numbers of families with a GATA1 mutation to date, with only two mutations (R216Q [6–8] and G208R [5, 10]) being identified in more than one family. Sanger sequencing of the different GATA1 exons using leukocyte gDNA of patient III:4 showed the presence of a missense mutation gac (D) to aac (N) at codon 218 (Figure 1C). Interestingly, we already have described two other missense mutations in this residue for two unrelated families for which patients presented with mild macrothrombocytopenia with dyserythropoiesis (D218G) [2] and severe macrothrombocytopenia with anemia (D218Y) [3]. A genomic DNA sample was not available from subject III:10 to confirm the presence to the GATA1 D218N variant. The D218N GATA1 variant was not present in the dbSNP or 1000 genomes databases (www.1000genomes.org). In addition, SIFT (sift.jcvi.org) predicted GATA1 variants D218G/Y and N to be damaging. The platelet defect for propositus III:4 and his relative III:10 seems milder than found for subjects with the D218G or D218Y mutations as the degree of thrombocytopenia is less severe and especially their platelets are large but not giant (Table I and Figure 1D). Actually, without having evidence of an X-linked inheritance patterns in this family, a single patient with this type of platelet defect might escape the genetic diagnosis of GATA1. This could be a reason why still only few patients have been described with GATA1 mutations still giving us no idea about the Correspondence: Kathleen Freson, Center for Molecular and Vascular Biology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. Tel: +32-16346017. Fax: +32-16345990. E-mail: kathleen.freson@ med.kuleuven.be Keywords

Parvovirus B19 (PVB19) infection can induce transient anaemia in patients with increased erythropoiesis. However, the dynamic change within the bone marrow after PVB19 infection is not well understood. Increased erythropoiesis is a physiological phenomenon in puerperital women. Nevertheless, anaemia as a result of PVB19 infection in puerperital women has never been reported. We report one patient with eclampsia and two patients with pre-eclampsia who had transient, severe anaemia during the puerperital period because of PVB19 infection. Viral genomes were detected in the peripheral blood during the anaemic period by polymerase chain reaction and became undetectable after the anaemia was resolved. Viral genomes and protein could also be detected in bone marrow by in situ hybridization and immunohistochemical staining, respectively. Serial aspiration cytology of bone marrow showed severe dysplastic change involving erythroid precursors with a few apoptotic cells at the initial onset of anaemia, markedly increased apoptotic cells that was confirmed by the increased expression of activated caspase 3, around the nadir of anaemia, and a normal marrow picture without features of apoptosis after recovery from anaemia. Our data indicates that PVB19 infection can induce transient, severe dyserythropoietic anaemia in puerperital women with pre-eclampsia/eclampsia and the pathogenetic mechanism may probably involve the induction of apoptosis following PVB19 infection.

Congenital dyserythropoietic anemia type II (CDA II) is the most frequent type of congenital dyserythropoietic anemia. More than 200 cases have been described, but with the exception of a report by the International CDA II Registry, these reports include only small numbers of cases and no data on the lifetime evolution of the disease. Since 1967, we were able to follow 48 cases of CDA II from 43 families for up to 35 years. All patients exhibit chronic anemia of variable severity requiring regular red cell transfusions only in a minority of children; 60% developed gallstones before the age of 30 years, and 16 patients had cholecystectomy between 8 and 34 years of age. Iron overload was a frequent complication. In 16 cases, iron depletion started between 7 and 36 years. Three patients died from secondary hemochromatosis. Splenectomy, performed in 22 cases, led to moderate increases in hemoglobin values and eliminated the need for transfusions but did not prevent further iron loading. The current recommendation is to consider splenectomy if the anemia compromises patients' performance, and to manage iron overload according to the guidelines derived from patients with thalassemia. (Blood. 2003;102:4576-4581)

Here, we report the case of a child who, since birth, showed persistent macrocytosis and elevated mean corpuscular volume of the erythrocytes. Bone marrow biopsy revealed gross disorganisation of the erythroblastic series both at the light and electron microscopic examination, with complete absence of dysplastic features in the granulocytic and megakaryocytic series. Common causes of macrocytosis were excluded. The spectrum of morphological findings were not consistent with any of the classical types of congenital dyserythropoietic anaemias (CDAs) and serological findings of CDA type II were absent. The most outstanding feature was a marked irregularity of the nuclear outline of the late erythroblasts that presented thick-ending finger-like projections. The combination of macrocytosis without anaemia and these morphologic erythroblastic changes have not been previously reported in the setting of classical and variant forms of CDAs.

Congenital dyserythropoietic anemias (CDAs) constitute a rare group of inherited red-blood-cell disorders associated with dysplastic changes in late erythroid precursors. CDA type I (CDAI [MIM 224120], gene symbol CDAN1) is characterized by erythroid pathological features such as internuclear chromatin bridges, spongy heterochromatin, and invagination of the nuclear membrane, carrying cytoplasmic organelles into the nucleus. A cluster of 45 highly inbred Israeli Bedouin with CDAI enabled the mapping of the CDAN1 disease gene to a 2-Mb interval, now refined to 1.2 Mb, containing 15 candidate genes on human chromosome 15q15 (Tamary et al. 1998). After the characterization and exclusion of 13 of these genes, we identified the CDAN1 gene through 12 different mutations in 9 families with CDAI. This 28-exon gene, which is transcribed ubiquitously into 4738 nt mRNA, was reconstructed on the basis of gene prediction and homology searches. It encodes codanin-1, a putative o-glycosylated protein of 1,226 amino acids, with no obvious transmembrane domains. Codanin-1 has a 150-residue amino-terminal domain with sequence similarity to collagens and two shorter segments that show weak similarities to the microtubule-associated proteins, MAP1B (neuraxin) and synapsin. These findings, and the cellular phenotype, suggest that codanin-1 may be involved in nuclear envelope integrity, conceivably related to microtubule attachments. The specific mechanisms by which codanin-1 underlies normal erythropoiesis remain to be elucidated.

We describe a new case of congenital dyserythropoietic anemia (CDA) type III. This least common type of CDA was diagnosed at the age of 59 in a 70-year-old woman who suffered from a young age from mild macrocytic anemia, while the long follow up since diagnosis documented a benign clinical course. No family history of blood diseases was obtained and no anemia was documented in the medical records of any of her four children. The bone marrow (BM) examination on light microscopy revealed a severe erythroid hyperplasia with the presence of giant multinucleated erythroblasts. Ultrastructural examination of the BM disclosed the presence of many large multinucleated erythroblasts bearing a variety of ultrastructural findings: nuclear clefts, autophagic vacuoles, iron-loaded mitochondria, and intracytoplasmic myelin figures. In addition, extensive hyperlobulation of the nucleus and partial loss of nuclear membrane with “spilling” of nuclear material to the adjacent cytoplasm was also noted in some of the erythroblasts. These last two findings have not been previously described in CDA III. Am. J. Hematol. 70:72–76, 2002. © 2002 Wiley-Liss, Inc.

We report, for the first time, an unusual case of congenital anaemia with the clinical diagnosis of haemoglobin H disease complicated by morphological features at the light and electron microscopy level very similar to those of CDA-I. The red cell indices and the globin chain biosynthetic ratio were not characteristic of the defective haemoglobin genotype. The haematological, clinical and morphological data strongly suggest the novel coexistence of the two defects in a patient. The disease is characterised by a unique dyserythropoietic phenotype of diagnostic importance, which possibly brings new data regarding the reciprocal interaction between the two diseases, especially concerning a specific abnormality in globin chain synthesis in CDA-I, as previously suggested.

The discovery of the gene responsible for most cases of hereditary haemochromatosis has made it possible to evaluate the role of this gene in iron accumulation in other conditions. Previous observations in patients with various inherited anaemias are suggestive of a role of the haemochromatosis gene in the noniatrogenic form of iron overload that may occur in these conditions.We studied two brothers with congenital dyserythropoietic anaemia type II, presenting with marked noniatrogenic iron overload. Young age, familial occurrence, concordance of iron overload, and histology were all suggestive of a genetic basis for the iron overload. A search for C282Y, H63D, S65C, and IVS3 + 1G?T mutations was performed by polymerase chain reaction after extraction of genomic DNA from whole blood.A search for the C282Y, H63D, S65C, and IVS3 + 1G?T mutations in the HFE gene proved to be negative. Despite the existence of ineffective erythropoiesis with mild anaemia, both patients were treated with regular phlebotomies in order to prevent long-term complications of their iron-overload. This treatment was well tolerated.1. C282Y, H63D, S65C, and IVS3 + 1G?T mutations were negative in two brothers with congenital dyserythropoietic anaemia, in whom young age, familial occurrence, concordance of iron overload, and the histological pattern were all suggestive of hereditary haemochromatosis. 2. Treatment with regular phlebotomies, in order to prevent complications of iron overload, was well tolerated despite the existence of mild haemolytic anaemia.

Objectives Congenital dyserythropoietic anemia (CDA) type I is a rare autosomal recessive macrocytic anemia whose natural history is not well documented. The aim of the present study was to evaluate the clinical picture of the disease in young adults. Methods The study sample consisted of 17 patients of mean age 11.9 +/- 5.4 yr (range 18-33 yr) and one older patient (age 44 yr), all Israeli Bedouins. The degree of anemia was evaluated as well as the extent of development of gallstones and iron overload. In each subject we determined the hemochromatosis gene mutations and the uridine dyphosphate-glucoronosyltransferase (UGT-1A) gene polymorphism associated with Gilbert's syndrome. Results The patients were found to have moderate anemia, with the women displaying lower mean hemoglobin levels than the men (8.2 +/- 0.9 g dL(-1) vs. 10 +/- 1.3 g dL(-1); P=0.0059). The majority of patients (59%) had received at least one blood transfusion, with the women having a significantly higher transfusion requirement. Although delayed puberty was noted, final height and weight were within normal limits, and eight patients had progeny. Biliary stones were found in three of 16 patients, two of whom were homozygous for UGT-1A gene polymorphism. None of the patients carried the common hemochromatosis gene mutation, although serum ferritin levels were moderately elevated (788 +/- 332 ng mL(-1)). Conclusions CDA type I in young adults is characterized by moderate macrocytic anemia, more severe in women, and a tendency to cholelithiasis and secondary progressive iron overload. We suggest that iron overload in this patient population should be monitored and chelation therapy initiated when indicated to prevent organ damage

Congenital dyserythropoietic anemias (CDAs) are a group of relatively rare inherited anemias. They are characterized by ineffective erythropoiesis and classified as three major groups and a number of variants. CDA type II, also known as hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS), is the most frequent one. A number of associations with CDA II have been reported, although each described only one or a few patients. Here we presented a piebald woman with vaginal atresia who was tested for anemia and diagnosed as CDA type II. Piebaldism and anemia association were previously described in the mouse. Our case was the first that shows the features of both piebaldism and CDA in the same patient. This association may suggest a stem cell defect to cause both hematopoietic and cutaneous manifestations.

Background: Peripheral blood smear examination and other routine laboratory assays are not always sufficient to diagnose various diseases which affect the blood and bone marrow. A bone marrow aspirate examination is essential in most of the cases.Methods: This work was a hospital-based cross sectional analytical observational study carried out in the department of pathology at the Chitwan Medical College, Teaching Hospital, Nepal, over a period of 3 years from January, 2013 to December 2015. Bone marrow was aspirated from posterior superior iliac crest under local anesthesia; sternum was the alternative site in obese patients. Univariate analysis was performed for each variable using frequency distribution and means with the help of Statistical Package for Social Sciences 20.0 (SPSS 20.0) software.Results: One hundred and fifty-nine patients were included in the study. Eight cases of 'dry tap' were excluded, for whom bone marrow biopsy was advised. Anemia was the largest group followed by malignancy, infection and miscellaneous diseases. Among anemia, megaloblastic anemia was the most common, followed by aplastic anemia, dyserythropoietic anemia and myelopthisic anemia. The category 'malignancy' consisted of leukemia, multiple myeloma and myelodysplastic syndrome. Regarding individual diseases, megaloblastic anemia was the most common haematological disorder followed by immune thrombocytopenic purpura, leukemia and aplastic anemia.Conclusions: Bone marrow aspiration cytology is a mildly invasive technique which can diagnose many haematological and non-hematologic diseases that can be confirmed by more advanced investigations, if needed: serological, biochemical or molecular. However, bone marrow sample cannot be obtained (dry tap) in a proportion of cases. In such cases, a bone marrow biopsy needs to be performed.

Congenital dyserythropoietic anemia (CDA) represents a genotypically and phenotypically heterogeneous group of disorders. CDA type II, the most frequent variant, was recently shown to be caused by mutations in the gene encoding the secretory COPII component SEC23B. We report two siblings hailing from Punjab in northern India with classical CDA type II where this mutation was demonstrated.A 7-year-old girl presented with transfusion-dependent anemia, splenomegaly, and progressive growth failure since 1 year of age. Her 5-year-old brother was similarly afflicted, but there was no other family history. Extensive prior work-up for hemolytic anemia, storage and metabolic disorders, and infectious diseases was negative. Hemoglobin was 71 g/l with normal leukocyte, platelet, and corrected reticulocyte counts. Bone marrow examination revealed marked normoblastic erythroid hyperplasia with dyserythropoiesis (36%) and the presence of bi- and multinucleated erythroblasts with equal-sized nuclei. Many pseudo-Gaucher cells were also seen. Iron stores were increased although ring sideroblasts were absent. Hereditary erythrocyte multinuclearity with positive acidified serum (HEMPAS) test revealed lysis of the red cells in four out of five control sera.Genomic DNA sequencing of the SEC23B exon 12 revealed homozygosity for c.1385 A → G; Y462C mutations in both siblings.CDA has traditionally been a difficult diagnosis to establish, since it requires exclusion of other causes of dyserythropoiesis and the performance of complex tests including HEMPAS and electron microscopy for confirmation. The availability of molecular genetic testing for SEC23B promises to streamline and hasten the diagnostic process for this rare and intriguing disease.