Severe pulmonary arterial hypertension in congenital sideroblastic anemia from PUS1 mutation - a case report.

BACKGROUND: Myopathy, lactic acidosis and inherited sideroblastic anemia (MLASA) are a group of rare intriguing disorders with wider pathophysiological implications. One of the causes of MLASA is the mutation in PUS1 gene that encodes for pseudouridine synthase. This PUS1 mutation results in MLASA in which anemia and myopathy predominate. Severe pulmonary arterial hypertension has not been previously reported in patients with PUS1 gene mutation. CASE REPORT: A 17 year old girl with congenital sideroblastic anemia presented with worsening of breathlessness. Severe pulmonary artery hypertension was documented on investigations. A homozygous variant in exon 3 of gene PUS1,( chromosome 12:g.131932301 C > T c.430 C > T) was found on sanger sequencing. CONCLUSION: We document severe pulmonary arterial hypertension in a patient of congenital sideroblastic anemia from PUS1 gene. We hypothesis that cross talk with TGFb pathways might occur in PUS1 mutation, and that might cause severe PAH. This observation might have therapeutic implications.

Elucidating the Role of Human ALAS2 C-terminal Mutations Resulting in Loss of Function and Disease.

The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.

Hematologic Manifestations in Primary Mitochondrial Diseases.

Primary mitochondrial disorders (PMDs) are known for their pleiotropic manifestations in humans, affecting almost any organ or system at any time. Hematologic manifestations, such as cytopenias and sideroblastic anemia, occur in 10% to 30% of patients with confirmed PMDs. These can be the initial presenting features or complications that develop over time. Surveillance for these manifestations allows for prompt identification and treatment. This article provides an overview of the pathophysiology underpinning the hematologic effects of mitochondrial dysfunction, discussing the 3 key roles of the mitochondria in hematopoiesis: providing energy for cell differentiation and function, synthesizing heme, and generating iron-sulfur clusters. Subsequently, the diagnosis and management of mitochondrial disorders are discussed, focusing on hematologic manifestations and the specific conditions commonly associated with them. Through this, we aimed to provide a concise point of reference for those considering a mitochondrial cause for a patient's hematologic abnormality, or for those considering a hematologic manifestation in a patient with known or suspected mitochondrial disease.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

ABSTRACT: Among the most common genetic alterations in myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with a more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here, we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct comutation pattern, and a lack of favorable survival seen with other SF3B1 mutations. Moreover, compared with other hot spot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS.

Mitochondrial tRNA pseudouridylation governs erythropoiesis.

ABSTRACT: Pseudouridine is the most prevalent RNA modification, and its aberrant function is implicated in various human diseases. However, the specific impact of pseudouridylation on hematopoiesis remains poorly understood. Here, we investigated the role of transfer RNA (tRNA) pseudouridylation in erythropoiesis and its association with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA) pathogenesis. By using patient-specific induced pluripotent stem cells (iPSCs) carrying a genetic pseudouridine synthase 1 (PUS1) mutation and a corresponding mutant mouse model, we demonstrated impaired erythropoiesis in MLASA-iPSCs and anemia in the MLASA mouse model. Both MLASA-iPSCs and mouse erythroblasts exhibited compromised mitochondrial function and impaired protein synthesis. Mechanistically, we revealed that PUS1 deficiency resulted in reduced mitochondrial tRNA levels because of pseudouridylation loss, leading to aberrant mitochondrial translation. Screening of mitochondrial supplements aimed at enhancing respiration or heme synthesis showed limited effect in promoting erythroid differentiation. Interestingly, the mammalian target of rapamycin (mTOR) inhibitor rapamycin facilitated erythroid differentiation in MLASA-iPSCs by suppressing mTOR signaling and protein synthesis, and consistent results were observed in the MLASA mouse model. Importantly, rapamycin treatment partially ameliorated anemia phenotypes in a patient with MLASA. Our findings provide novel insights into the crucial role of mitochondrial tRNA pseudouridylation in governing erythropoiesis and present potential therapeutic strategies for patients with anemia facing challenges related to protein translation.

Myelodysplastic Neoplasms (MDS) with Ring Sideroblasts or SF3B1 Mutations: The Improved Clinical Utility of World Health Organization and International Consensus Classification 2022 Definitions, a Single-Centre Retrospective Chart Review.

Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an SF3B1 mutation. In the MEDALIST trial and in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with SF3B1 mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated SF3B1 mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.

Prognostic impact of SF3B1 mutation and multilineage dysplasia in myelodysplastic syndromes with ring sideroblasts: a Mayo Clinic study of 170 informative cases.

The revised 4th edition of the World Health Organization (WHO4R) classification lists myelodysplastic syndromes with ring sideroblasts (MDS-RS) as a separate entity with single lineage (MDS-RS-SLD) or multilineage (MDS-RS-MLD) dysplasia. The more recent International Consensus Classification (ICC) distinguishes between MDS with SF3B1 mutation (MDS-SF3B1) and MDS-RS without SF3B1 mutation; the latter is instead included under the category of MDS not otherwise specified. The current study includes 170 Mayo Clinic patients with WHO4R-defined MDS-RS, including MDS-RS-SLD (N=83) and MDS-RSMLD (N=87); a subset of 145 patients were also evaluable for the presence of SF3B1 and other mutations, including 126 with (87%) and 19 (13%) without SF3B1 mutation. Median overall survival for all 170 patients was 6.6 years with 5- and 10-year survival rates of 59% and 25%, respectively. A significant difference in overall survival was apparent between MDS-RS-MLD and MDS-RS-SLD (P<0.01) but not between MDS-RS with and without SF3B1 mutation (P=0.36). Multivariable analysis confirmed the independent prognostic contribution of MLD (hazard ratio=1.8, 95% confidence interval: 1.1-2.8; P=0.01) and also identified age (P<0.01), transfusion need at diagnosis (P<0.01), and abnormal karyotype (P<0.01), as additional risk factors; the impact from SF3B1 or other mutations was not significant. Leukemia-free survival was independently affected by abnormal karyotype (P<0.01), RUNX1 (P=0.02) and IDH1 (P=0.01) mutations, but not by MLD or SF3B1 mutation. Exclusion of patients not meeting ICC-criteria for MDS-SF3B1 did not change the observations on overall survival. MLD-based, as opposed to SF3B1 mutation-based, disease classification for MDS-RS might be prognostically more relevant.

Comprehensive Genomic Analysis Identifies a Diverse Landscape of Sideroblastic and Nonsideroblastic Iron-Related Anemias with Novel and Pathogenic Variants in an Iron-Deficient Endemic Setting.

Inherited iron metabolism defects are possibly missed or underdiagnosed in iron-deficient endemic settings because of a lack of awareness or a methodical screening approach. Hence, we systematically evaluated anemia cases (2019 to 2021) based on clinical phenotype, normal screening tests (high-performance liquid chromatography, α gene sequencing, erythrocyte sedimentation rate, C-reactive protein, and tissue transglutaminase), and abnormal iron profile by targeted next-generation sequencing (26-gene panel) supplemented with whole-exome sequencing, multiplex ligation probe amplification/mitochondrial DNA sequencing, and chromosomal microarray. Novel variants in ALAS2, STEAP3, and HSPA9 genes were functionally validated. A total of 290 anemia cases were screened, and 41 (14%) enrolled for genomic testing as per inclusion criteria. Comprehensive genomic testing revealed pathogenic variants in 23 of 41 cases (56%). Congenital sideroblastic anemia was the most common diagnosis (14/23; 61%), with pathogenic variations in ALAS2 (n = 6), SLC25A38 (n = 3), HSPA9 (n = 2) and HSCB, SLC19A2, and mitochondrial DNA deletion (n = 1 each). Nonsideroblastic iron defects included STEAP3-related microcytic anemia (2/23; 8.7%) and hypotransferrenemia (1/23; 4.3%). A total of 6 of 22 cases (27%) revealed a non-iron metabolism gene defect on whole-exome sequencing. Eleven novel variants (including variants of uncertain significance) were noted in 13 cases. Genotype-phenotype correlation revealed a significant association of frameshift/nonsense/splice variants with lower presentation age (0.8 months versus 9 years; P < 0.01) compared with missense variants. The systematic evaluation helped uncover an inherited iron defect in 41% (17/41) of cases, suggesting the need for active screening and awareness for these rare diseases in an iron-deficient endemic population.

Luspatercept for the treatment of congenital sideroblastic anemia: Two case reports.

Congenital sideroblastic anemia (CSA) is a group of disorders caused by different genetic mutations that result in low iron utilization and ineffective erythropoiesis. Current treatments are limited, and some patients do not respond to vitamin B6 therapy. Luspatercept is a novel erythropoietic maturation agent approved for adult ß-thalassemia and Myelodysplastic syndromes with ring sideroblasts (MDS-RS) associated with ineffective erythropoiesis. Here we report 2 patients with CSA due to mutations in ALAS2 and SLC25A38 genes who became unresponsive after a period of treatment with vitamin B6 and iron chelators but achieved transfusion independence and a markedly reduced spleen after combination with luspatercept.

A stepwise diagnostic approach for undiagnosed Anemia in children: A model for low-middle income country.

BACKGROUND: Reaching a precise diagnosis in rare inherited anemia is extremely difficult and challenging, especially in areas with limited use of genetic studies, which makes undiagnosed anemia a unique clinical entity in tertiary hematology centers. In this study, we aim at plotting a stepwise diagnostic approach in children with undiagnosed anemia while identifying indications for genetic testing. PATIENTS AND METHODS: A one-year cross-sectional study involved 44 children and adolescents with undiagnosed anemia after undergoing an initial routine panel of investigations. They were classified based on mean corpuscular volume (MCV) into 3 groups: microcytic (n = 19), normocytic (n = 14) and macrocytic (n = 11). An algorithm that included four levels of investigations was devised for each category. RESULTS: After applying a systematic diagnostic approach, 33 patients (75 %) were diagnosed of whom 7 (15 %) had combined diagnoses, while 11 (25 %) patients remained undiagnosed. Based on the first, second, third and fourth levels of investigations, patients were diagnosed, respectively, as follows: of the 11 patients, 7 were microcytic, 3 normocytic and 1 macrocytic; of the 7 patients, 2 were microcytic, 2 normocytic, and 3 macrocytic; of 10 patients, 5 were microcytic, 4 normocytic and 1 macrocytic; finally, of the 16 patients, 8 were microcytic, 6 normocytic and 2 macrocytic. Numbers recorded appear higher than the actual number of the patients because some of them were diagnosed by more than one level of investigation. The diagnoses obtained in the microcytic group showed hemoglobinopathies, iron refractory iron deficiency anemia (IRIDA), membrane defects, sideroblastic anemia, hypo-transferrinemia, a combined diagnosis of sickle cell trait and pyropoikilocytosis. The diagnoses also showed a combined diagnosis of hereditary spherocytosis (HS) and alpha thalassemia minor, and a combined diagnosis of iron deficiency anemia and beta thalassemia minor, while 15 % remained undiagnosed. In the normocytic group, the diagnosis revealed autosomal recessive (AR) HS, vitamin B12 deficiency, pyruvate kinase deficiency (PKD), congenital dyserythropoietic anemia (CDA) type I, Diamond Blackfan anemia and beta thalassemia major. In addition, it showed a combined diagnosis of AR HS and CDA type II, a combined diagnosis of AR HS and PKD, and a combined diagnosis of dehydrated stomatocytosis (DHS) and G6PD carrier, meanwhile 20 % remained undiagnosed. Finally, the macrocytic group was diagnosed by vitamin B12 deficiency, sideroblastic anemia, PKD, a combined diagnosis of PKD and G6PD deficiency carrier, while 45 % remained undiagnosed. CONCLUSION: Conducting a stepwise approach with different levels of investigations may help reach the diagnosis of difficult anemia without having to resort to unnecessary investigations. Combined diagnosis is an important cause of undiagnosed anemia, especially in countries with high frequency of consanguinity. The remaining 25 % of the patients continued to be undiagnosed, requiring more sophisticated investigations.

Spontaneous, simultaneous bilateral osteonecrosis of the femoral heads in a patient with sideroblastic anaemia with B-cell immunodeficiency, periodic fever and developmental delay syndrome.

Sideroblastic anaemia with B-cell immunodeficiency, periodic fever and developmental delay is a recently described, rare syndrome characterised by numerous manifestations underpinned by mutations in transfer RNA nucleotidyltransferase. The pathogenesis arises from mitochondrial dysfunction, with impaired intracellular stress response, deficient metabolism and cellular and systemic inflammation. This yields multiorgan dysfunction and early death in many patients with survivors suffering significant disability and morbidity. New cases, often youths, are still being described, expanding the horizon of recognisable phenotypes. We present a mature patient with spontaneous bilateral hip osteonecrosis that likely arises from the impaired RNA quality control and inflammation caused by this syndrome.

Clinical characteristics of Japanese patients with myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis.

Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a rare disease, which presents with features of myelodysplastic syndromes with ring sideroblasts and essential thrombocythemia, as well as anemia and marked thrombocytosis. SF3B1 and JAK2 mutations are often found in patients, and are associated with their specific clinical features. This study was a retrospective analysis of 34 Japanese patients with MDS/MPN-RS-T. Median age at diagnosis was 77 (range, 51-88) years, and patients had anemia (median hemoglobin: 9.0 g/dL) and thrombocytosis (median platelet count: 642 × 109/L). Median overall survival was 70 (95% confidence interval: 68-not applicable) months during the median follow-up period of 26 (range: 0-91) months. A JAK2V617F mutation was detected in 46.2% (n = 12) of analyzed patients (n = 26), while an SF3B1 mutation was detected in 87.5% (n = 7) of analyzed patients (n = 8). Like those with myelodysplastic syndromes or myeloproliferative neoplasms, patients often received erythropoiesis-stimulating agents and aspirin to improve anemia and prevent thrombosis. This study, which was the largest to describe the real-world characteristics of Japanese patients with MDS/MPN-RS-T, showed that the patients had similar characteristics to those in western countries.