Haemoglobinopathies and other rare anemias in Spain: ten years of a nationwide registry (REHem-AR).

REHem-AR was created in 2013. The progressive implementation of neonatal screening for haemoglobinopathies in Spanish autonomous communities where the registry had not been implemented, as well as the addition of new centres during this period, has considerably increased the sample of patients covered. In this study, we update our previous publication in this area, after a follow-up of more than 5 years. An observational, descriptive, multicentre and ambispective study of adult and paediatric patients with haemoglobinopathies and rare anaemias registered in REHem was performed. The data are from a cross-sectional analysis performed on 1 June, 2023. The study population comprised 1,756 patients, of whom 1,317 had SCD, 214 had thalassaemia and 224 were diagnosed with another condition. Slightly more than one third of SCD patients (37%) were diagnosed based on neonatal bloodspot screening, and the mean age at diagnosis was 2.5 years; 71% of thalassaemia patients were diagnosed based on the presence of anaemia. Vaso-occlusive crisis and acute chest syndrome continue to be the most frequent complications in SCD. HSCT was performed in 83 patients with SCD and in 50 patients with thalassaemia. Since the previous publication, REHem-AR has grown in size by more than 500 cases. SCD and TM are less frequent in Spain than in other European countries, although the data show that rare anaemias are frequent within rare diseases. REHem-AR constitutes an important structure for following the natural history of rare anaemias and enables us to calculate investment needs for current and future treatments.

New Cases and Mutations in SEC23B Gene Causing Congenital Dyserythropoietic Anemia Type II.

Congenital dyserythropoietic anemia type II (CDA II) is an inherited autosomal recessive blood disorder which belongs to the wide group of ineffective erythropoiesis conditions. It is characterized by mild to severe normocytic anemia, jaundice, and splenomegaly owing to the hemolytic component. This often leads to liver iron overload and gallstones. CDA II is caused by biallelic mutations in the SEC23B gene. In this study, we report 9 new CDA II cases and identify 16 pathogenic variants, 6 of which are novel. The newly reported variants in SEC23B include three missenses (p.Thr445Arg, p.Tyr579Cys, and p.Arg701His), one frameshift (p.Asp693GlyfsTer2), and two splicing variants (c.1512-2A>G, and the complex intronic variant c.1512-3delinsTT linked to c.1512-16_1512-7delACTCTGGAAT in the same allele). Computational analyses of the missense variants indicated a loss of key residue interactions within the beta sheet and the helical and gelsolin domains, respectively. Analysis of SEC23B protein levels done in patient-derived lymphoblastoid cell lines (LCLs) showed a significant decrease in SEC23B protein expression, in the absence of SEC23A compensation. Reduced SEC23B mRNA expression was only detected in two probands carrying nonsense and frameshift variants; the remaining patients showed either higher gene expression levels or no expression changes at all. The skipping of exons 13 and 14 in the newly reported complex variant c.1512-3delinsTT/c.1512-16_1512-7delACTCTGGAAT results in a shorter protein isoform, as assessed by RT-PCR followed by Sanger sequencing. In this work, we summarize a comprehensive spectrum of SEC23B variants, describe nine new CDA II cases accounting for six previously unreported variants, and discuss innovative therapeutic approaches for CDA II.

Rabbit antithymocyte globulin versus horse antithymocyte globulin for treatment of acquired aplastic anemia: a retrospective analysis.

Studies comparing rabbit antithymocyte globulin (rATG) and horse ATG (hATG) in patients with aplastic anemia (AA) have shown conflicting results. These studies included fewer than 60 subjects in the rATG arm with relatively short follow-up. A total of 169 patients treated with rATG and 62 treated with hATG were included in this retrospective analysis, across 33 centers. Patients were treated with rATG or hATG plus cyclosporine A. Over half were classified, as having severe AA (SAA) or very severe AA (VSAA), and the mean follow-up was 45 months. There was no significant difference detected in cumulative response to treatment or survival between the rATG and hATG groups. The response to treatment was 63 % in the rATG group versus 66 % in the hATG group at 3 months. By 12 months, this pattern had reversed, and 84 % of rATG patients had responded to treatment versus 76 % in the hATG group (n.s.). Early mortality due to infection tended to be higher with rATG compared to hATG (n.s). rATG and hATG would seem to be therapeutically equivalent in SAA and VSAA. However, patients treated with rATG may take longer to respond than those treated with hATG and may also require more active prevention of early infections.

Women with Gaucher Disease.

Gaucher disease is an inherited disorder in which there is a deficiency of the enzyme glucocerebrosidase, which leads to the accumulation of glucosylceramide. Although much scientific evidence is now available, there is still limited data on the impact on the different life stages of women with this disease. Among other alterations, a delay in menarche has been described, although it has not been related to fertility problems. Menorrhagia is relatively frequent, being related to the presence of thrombocytopenia, thrombocytopathies or coagulation disorders. On the other hand, pregnancy planning is an increasingly frequent concern. All patients should undergo genetic counseling, and it is important to monitor the appearance or worsening of organomegaly, bone and hematologic abnormalities to establish clinical and therapeutic recommendations. Management during the puerperium will depend on the evolution of gestation, and, during the lactation period, the potential appearance of bone complications should be assessed. An early onset of menopause, compared to the general population, has also been described, which may accelerate the development of osteopenia. Finally, although the usual screening protocols for neoplasms are currently being performed, it is recommended to watch for early signs of liver or renal neoplasms when examining the results of imaging tests performed during evaluations for this disease.

Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation.

Inhibition of the heterodimeric amino acid carrier SLC7A5/SLC3A2 (LAT1/CD98) has been widely studied in tumor biology but its role in physiological conditions remains largely unknown. Here we show that the SLC7A5/SLC3A2 heterodimer is constitutively present at different stages of erythroid differentiation but absent in mature erythrocytes. Administration of erythropoietin (EPO) further induces SLC7A5/SLC3A2 expression in circulating reticulocytes, as it also occurs in anemic conditions. Although Slc7a5 gene inactivation in the erythrocyte lineage does not compromise the total number of circulating red blood cells (RBCs), their size and hemoglobin content are significantly reduced accompanied by a diminished erythroblast mTORC1 activity. Furthermore circulating Slc7a5-deficient reticulocytes are characterized by lower transferrin receptor (CD71) expression as well as mitochondrial activity, suggesting a premature transition to mature RBCs. These data reveal that SLC7A5/SLC3A2 ensures adequate maturation of reticulocytes as well as the proper size and hemoglobin content of circulating RBCs.

Next generation sequencing for diagnosis of hereditary anemia: Experience in a Spanish reference center.

BACKGROUND AND AIMS: Hereditary anemia (HA) encloses a wide group of rare inherited disorders with clinical and hematologic overlaps that complicate diagnosis. MATERIALS AND METHODS: A 48-gene panel was developed to diagnose HA by Next Generation Sequencing (NGS) in a large cohort of 165 patients from 160 unrelated families. RESULTS: Patients were divided in: A) patients who had a suspicion of a specific type of HA (n = 109), and B) patients who had a suspicion of HA but with no clear type (n = 56). Diagnostic performance was 83.5% in group A and a change of the initial diagnosis occurred in 11% of these patients. In group B, 35.7% of patients achieved a genetic diagnosis. NGS identified 6 cases of xerocytosis, 6 of pyruvate kinase (PK) deficiency, 4 of G6PD, and 1 case of phytosterolemia with no initial suspicion of these pathologies, which is clinically relevant since they have specific treatment. Five patients were found to carry variants associated to two different pathologies (4 of them combining a metabolic deficiency and a membrane defect), and 44 new variants were identified in 41 patients. CONCLUSION: The use of NGS is a sensitive technique to diagnose HA and it shows better performance when patients are better characterized.

[Consensus document for the diagnosis and treatment of pyruvate kinase deficiency]. / Documento de consenso para el diagnóstico y tratamiento del déficit de piruvato quinasa.

Pyruvate kinase (PK) deficiency is the second most frequent enzymopathy and the most common cause of chronic hereditary non-spherocytic haemolytic anaemia. Its global prevalence is underestimated due to low clinical suspicion of mild cases, associated with difficulties in the performance and interpretation of PK enzymatic activity assays. With the advent of next generation sequencing techniques, a better diagnostic approach is achieved. Treatment remains based on red blood cell transfusions and splenectomy, with special attention to iron overload, not only in transfusion-dependent patients. Nowadays, allogeneic hematopoietic stem cell transplantation is the only curative treatment, recommended only in selected cases of severely affected patients with an HLA-identical donor. Novel pharmacological and gene therapies are in clinical trials, with promising results. In this article, the Spanish Erythropathology Group reviews the current situation of PK deficiency, paying special attention to the usefulness of different diagnostic techniques and to actual and emerging treatments.

Clinical significance of occult cerebrospinal fluid involvement assessed by flow cytometry in non-Hodgkin's lymphoma patients at high risk of central nervous system disease in the rituximab era.

BACKGROUND AND AIM: Flow cytometry (FCM) analysis of cerebrospinal fluid (CSF) is more sensitive than conventional cytology (CC) for diagnosis of lymphomatous meningeosis, but the clinical significance of occult central nervous system (CNS) disease (positive FCM with negative CC) remains unknown. PATIENTS AND METHODS: CSF samples from 105 patients with newly diagnosed aggressive lymphomas at high risk of CNS involvement were prospectively studied by both CC and FCM, and results were correlated with cumulative incidence of CNS relapse and overall survival (OS). Patients were divided into three groups: 1) patients without CNS involvement (CC-/FCM-; n=83); 2) individuals with occult CNS disease (FCM+/CC-; n=15); and 3) cases with CNS disease (CC+/FCM+; n=7). RESULTS: Six cases showed CNS relapse or progression: two in Group 1 (2.4%), two in Group 2 (13%) and two in Group 3 (28.5%) (Group 2 vs. 1, P=0.04; Group 3 vs. 1, P<0.001). Patients from Groups 2 (P=0.05) and 3 (P<0.001) also showed a higher cumulative incidence of CNS relapse than those from Group 1. Significant differences were observed in OS between FCM-/CC- and FCM+/CC+ cases (P=0.02), while patients with occult CNS disease (FCM+/CC-) displayed intermediate OS rates, although differences did not reach statistical significance. CONCLUSIONS: The presence of occult CNS involvement at diagnosis in patients with NHL at high risk of CNS disease is associated with a higher probability of CNS relapse.

Early detection of lysosomal diseases by screening of cases of idiopathic splenomegaly and/or thrombocytopenia with a next-generation sequencing gene panel.

Lysosomal diseases (LD) are a group of about 70 rare hereditary disorders (combined incidence 1:5000) in which diverse lysosomal functions are impaired, impacting multiple organs and systems. The first clinical signs and symptoms are usually unspecific and shared by hundreds of other disorders. Diagnosis of LD traditionally relies on performing specific enzymatic assays, if available, upon clinical suspicion of the disorder. However, the combination of the insidious onset of LD and the lack of awareness on these rare diseases among medical personnel results in undesirable diagnostic delays, with unchecked disease progression, appearance of complications and a worsened prognosis. We tested the usefulness of a next-generation sequencing-based gene panel for quick, early detection of LD among cases of idiopathic splenomegaly and/or thrombocytopenia, two of the earliest clinical signs observed in most LD. Our 73-gene panel interrogated 28 genes for LD, 1 biomarker and 44 genes underlying non-LD differential diagnoses. Among 38 unrelated patients, we elucidated eight cases (21%), five with LD (GM1 gangliosidosis, Sanfilippo disease A and B, Niemann-Pick disease B, Gaucher disease) and three with non-LD conditions. Interestingly, we identified three LD patients harboring pathogenic mutations in two LD genes each, which may result in unusual disease presentations and impact treatment. Turnaround time for panel screening and genetic validation was 1 month. Our results underline the usefulness of resequencing gene panels for quick and cost-effective screening of LDs and disorders sharing with them early clinical signs.

Multicenter comparison of CD34+ myeloid cell count by flow cytometry in low-risk myelodysplastic syndrome. Is it feasible?

BACKGROUND: Accuracy of bone marrow (BM) blast count in low-risk myelodysplastic syndromes (MDS) still remains a challenge though it is essential for prognosis. We investigated whether the enumeration of CD34+ myeloid cells by flow cytometry immunophenotyping (FCI) could be used as a consistent parameter for clinical MDS studies. METHODS: Six clinical centers entered the study and information on their FCI protocols was recorded. Sixty-seven flow cytometry listmodes from BM samples of patients with low-risk MDS with <5% BM blasts were exchanged among participants in two different rounds. Interlaboratory variations on the quantification of CD34+ myeloid cells were calculated and strategies to solve differences were evaluated. RESULTS: An overall "very good" agreement on CD34+ cell count among participants (intraclass correlation coefficient = 0.720) was observed, but agreement was "low" in 22 files. No single parameter could fully explain all discrepancies, but 3 technical issues were identified as relevant: the use of the CD34/CD45/CD117/HLA-DR mAb combination, acquisition of ≥50,000 events and a low percentage of debris/aggregates. The frequency of discordant results increased with the accumulation of pitfalls (none, 16%; 1 pitfall, 40%; 2 pitfalls, 83%; P = 0.006). Finally, the use of a common gating strategy for analysis increased the percentage of files with "very good" agreement to 100%. CONCLUSIONS: Prevention of specific technical pitfalls is mandatory to reach a good reproducibility of CD34+ cell count among centers. These recommendations set the basis for laboratory standardization and enable the use of CD34+ cell enumeration as additional information in low-risk MDS patients. © 2017 International Clinical Cytometry Society.

CD5 provides viability signals to B cells from a subset of B-CLL patients by a mechanism that involves PKC.

B-chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease characterized by an accumulation of B lymphocytes expressing CD5. To date, the biological significance of this molecule in B-CLL B cells remains to be elucidated. In this study, we have analysed the functional consequences of the binding of an anti-CD5 antibody on B-CLL B cells. To this purpose, we have measured the percentage of viability of B-CLL B cells in the presence or in the absence of anti-CD5 antibodies and also examined some of the biochemical events downstream the CD5-signalling. We demonstrate that anti-CD5 induces phosphorylation of protein tyrosine kinases and protein kinase C (PKC), while no activation of Akt/PKB and MAPKs is detected. This signalling cascade results in viability in a group of patients in which we observe an increase of Mcl-1 levels, whereas the levels of bcl-2, bcl-x(L) and XIAP do not change. We also report that this pathway leads to IL-10 production, an immunoregulatory cytokine that might act as an autocrine growth factor for leukaemic B cells. Inhibition of PKC prevents the induction of Mcl-1 and IL-10, suggesting that the activation of PKC plays an important role in the CD5-mediated survival signals in B cells from a subset of B-CLL patients.

Diagnostic screening of paroxysmal nocturnal hemoglobinuria: Prospective multicentric evaluation of the current medical indications.

BACKGROUND: Although consensus guidelines have been proposed in 2010 for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometry (FCM), so far no study has investigated the efficiency of such medical indications in multicentric vs. reference laboratory settings. METHODS: Here we evaluate the efficiency of consensus medical indications for PNH testing in 3,938 peripheral blood samples submitted to FCM testing in 24 laboratories in Spain and one reference center in Brazil. RESULTS: Overall, diagnostic screening based on consensus medical indications was highly efficient (14% of PNH+ samples) both in the multicenter setting in Spain (10%) and the reference laboratory in Brazil (16%). The highest frequency of PNH+ cases was observed among patients screened because of bone marrow (BM) failure syndrome (33%), particularly among those with aplastic anemia (AA; 45%) and to a less extent also a myelodysplastic syndrome (MDS; 10%). Among the other individuals studied, the most efficient medical indications for PNH screening included: hemolytic anemia (19%), hemoglobinuria (48%) and unexplained cytopenias (9%). In contrast, only a minor fraction of the patients who had been submitted for PNH testing because of unexplained thrombosis in the absence of cytopenia, were positive (0.4%). CONCLUSIONS: In summary, our results demonstrate that the current medical indications for PNH screening by FCM are highly efficient, although improved screening algorithms are needed for patients presenting with thrombosis and normal blood cell counts. © 2016 International Clinical Cytometry Society.

Synergistic Activity of Deguelin and Fludarabine in Cells from Chronic Lymphocytic Leukemia Patients and in the New Zealand Black Murine Model.

B-cell chronic lymphocytic leukemia (CLL) remains an incurable disease, and despite the improvement achieved by therapeutic regimes developed over the last years still a subset of patients face a rather poor prognosis and will eventually relapse and become refractory to therapy. The natural rotenoid deguelin has been shown to induce apoptosis in several cancer cells and cell lines, including primary human CLL cells, and to act as a chemopreventive agent in animal models of induced carcinogenesis. In this work, we show that deguelin induces apoptosis in vitro in primary human CLL cells and in CLL-like cells from the New Zealand Black (NZB) mouse strain. In both of them, deguelin dowregulates AKT, NFκB and several downstream antiapoptotic proteins (XIAP, cIAP, BCL2, BCL-XL and survivin), activating the mitochondrial pathway of apoptosis. Moreover, deguelin inhibits stromal cell-mediated c-Myc upregulation and resistance to fludarabine, increasing fludarabine induced DNA damage. We further show that deguelin has activity in vivo against NZB CLL-like cells in an experimental model of CLL in young NZB mice transplanted with spleen cells from aged NZB mice with lymphoproliferation. Moreover, the combination of deguelin and fludarabine in this model prolonged the survival of transplanted mice at doses of both compounds that were ineffective when administered individually. These results suggest deguelin could have potential for the treatment of human CLL.