Rapid detection of blood using a novel application of RT-RPA integrated with CRISPR-Cas: ALAS2 detection as a model.

A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (27 dilution) or 0.0218 µL (26 dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.

ALAS2 overexpression alleviates oxidative stress-induced ferroptosis in aortic aneurysms via GATA1 activation.

Background: Aortic aneurysm, characterized by abnormal dilation of the aorta, poses significant health risks. This study aims to investigate the interaction between 5-aminolevulinate synthase 2 (ALAS2) and GATA-binding protein 1 (GATA1) in ferroptosis and oxidative stress responses in aortic aneurysm. Methods: A weighted gene co-expression network analysis (WGCNA) was performed on the differentially expressed genes (DEGs) within the GSE9106 dataset to identify the key module. Subsequently, protein-protein interaction (PPI) network analysis was performed on the key module. Mouse aortic vascular smooth muscle cells (MOVAS) were treated with hydrogen peroxide (H2O2) to induce oxidative stress, and ferroptosis inducers and inhibitors were added to evaluate their effects on iron content and oxidative stress markers. Through a series of in vitro cellular experiments, we assessed cell viability, expression levels of GATA1 and iron mutation-associated proteins, as well as cellular phenotypes such as inflammatory responses and apoptosis rates. Results: Three candidate genes (ALAS2, GYPA, and GYPB) were upregulated in the thoracic aortic aneurysm (TAA) samples of the GSE9106 dataset. The H2O2 treatment increased the MOVAS cells' iron content and oxidative stress, upregulated ALAS2 protein levels, and decreased the ferroptosis-related protein levels. ALAS2 overexpression reversed H2O2-induced apoptosis and increased the inflammatory cytokine levels. Additionally, the knockdown of GATA1 partially reversed the protective mechanism of overexpressed ALAS2 on H2O2-induced ferroptosis. Conclusions: ALAS2 overexpression reduced H2O2-induced oxidative damage and iron-induced apoptosis in MOVAS cells, and GATA1 knockdown partially reversed this protective effect. These findings suggested that the ALAS2 and GATA1 regulatory pathways may be potential therapeutic targets in aortic aneurysms.

Amplifying T cell-mediated antitumor immune responses in nonsmall cell lung cancer through photodynamic therapy and anti-PD1.

Photodynamic therapy (PDT) is nowadays widely employed in cancer treatment. We sought to assess the efficacy of combining PDT with anti-programmed cell death protein 1 (PD1) and to investigate the associated mechanisms in nonsmall cell lung cancer (NSCLC). We established a xenograft tumor model in C57BL/6J mice using Lewis lung carcinoma (LLC) cells, recorded tumor growth, and quantified reactive oxygen species (ROS) levels using a ROS detection kit. Pathological changes were assessed through H&E staining, while immunofluorescence (IF) was used to determine the expression of CD8 and Foxp3. Transcriptomic analysis was conducted, analyzing differential expressed genes (DEGs) among control, PDT, and PDT combined with anti-PD1 (PDT+anti-PD1) groups. Functional enrichment analysis via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed. The Cancer Genome Atlas (TCGA) database was utilized to analyze the expression of aminolevulinate synthase gene (ALAS2), integrin alpha10 (ITGA10), ATP1A2, a disintegrin and metalloprotease 12 (ADAM12), and Lox1 in lung adenocarcinoma and adjacent tissues, with concurrent immune infiltration analysis. Quantitative real-time polymerase chain reaction and western blot were employed to measure mRNA and protein expression levels. Treatment with PDT combined with anti-PD1 significantly inhibited tumor growth and increased the number of CD8+ cells while decreasing Foxp3+ cells. Immune infiltration results presented ALAS2, ADAM12, and ITGA10 were associated with various types of T cells or macrophages. Additionally, the expression levels of EGFR, ERK, and PI3K/Akt were suppressed after PDT with anti-PD1 treatment. Our findings collectively suggest that PDT combined with anti-PD1 treatment could enhance the infiltration of CD8+ T cells, suppressing tumor growth, and this effect was associated with ALAS2, ITGA10, and ADAM12. The underlying mechanism might be linked to EGFR, ERK, and PI3K/Akt signaling. Overall, this study provides valuable insights into the application of PDT combined with anti-PD1 treatment in NSCLC.

Protective effects of electroacupuncture on polycystic ovary syndrome in rats: Down-regulating Alas2 to inhibit apoptosis, oxidative stress, and mitochondrial dysfunction in ovarian granulosa cells.

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder affecting women at reproductive age. The therapeutic effect of electroacupuncture (EA) on PCOS has been revealed, while the anti-PCOS mechanisms of EA have not been fully explored. In this study, PCOS were induced in rats by daily injection with dehydroepiandrosterone (DHEA) for 20 days and EA treatment was performed for 5 weeks. The mRNA expression profiles in ovarian tissues from control, PCOS, and EA-treated rats were examined by high-throughput mRNA sequencing. 5'-aminolevulinate synthase 2 (Alas2), a vital rate-limiting enzyme of the heme synthesis pathway, was selected to be further studied. PCOS led to the upregulation of Alas2 mRNA, whereas EA treatment restored this change. In vitro, primary ovarian granulosa cells (GCs) were challenged with H2O2 to mimic the oxidative stress (OS) state in PCOS. H2O2 induced apoptosis, OS, mitochondrial dysfunction, as well as Alas2 overexpression in GCs, while lentivirus-mediated Alas2 knockdown evidently restrained the above impairments. In summary, this study highlights the crucial role of Alas2 in cell apoptosis, OS, and mitochondrial dysfunction of PCOS GCs and provides potential therapeutic candidates for further investigation on PCOS treatment.

METTL3-modified lncRNA-SNHG8 binds to PTBP1 to regulate ALAS2 expression to increase oxidative stress and promote myocardial infarction.

Myocardial infarction (MI) is one of the important factors leading to death in today's society. Therefore, to study the related mechanism of MI and reduce myocardial ischemia-reperfusion injury is an important link to reduce MI injury. MI mice in vivo and cell model in vitro were constructed. The cardiac function and MI area of mice were detected, and myocardial tissue injury was detected by HE staining. ALAS2 expression in mice myocardial tissue was detected by IHC. The expressions of lncRNA-SNHG8, METTL3, PTBP1 and ALAS2 in myocardial tissue or cardiomyocytes were detected by qRT-PCR assay. MTT assay was used to measured viability of cardiomyocytes. The oxidative stress level in myocardial tissue or cardiomyocytes was detected by ELISA assay and ROS assay. RIP-qPCR and RNA pulldown assays determined the interaction between METTL3 and lncRNA-SNHG8, as well as PTBP1 and ALAS2. lncRNA-SNHG8 knockdown in MI mice was reduced myocardial infarction size, alleviated myocardial tissue injury and oxidative stress, and inhibited ALAS2 expression in myocardial tissue. RNA pulldown and RIP assays showed that lncRNA-SNHG8 binged with PTBP1 and PTBP1 interacted with ALAS2 mRNA. Knockdown of lncRNA-SNHG8, METTL3 or PTBP1 in MI cells enhanced viability of myocardial cells, attenuated ROS release and MDA level, increased SOD level, alleviated oxidative stress. ALAS overexpression attenuated the corresponding effect of knockdown of lncRNA-SNHG8 and/or PTBP1 on MI cells. In sum, our paper is demonstrated for the first time that METTL3 can promote lncRNA-SNHG8 through m6A modification, thereby regulating ALAS2 to induce oxidative stress and aggravate myocardial injury.

Case report: An infant boy with X-linked sideroblastic anaemia successfully treated by umbilical cord blood haematopoietic stem cell transplantation.

X-linked sideroblastic anaemia (XLSA) is an inherited disorder caused by mutations in genes encoding proteins involved in the biosynthesis of haem. The pathogenic gene, as well as the pathogenesis and diagnosis of XLSA, have been fully elucidated in previous studies. However, only a few new advances have been made in managing XLSA in recent years, and blood transfusion remains the primary treatment. We report a case of umbilical cord blood haematopoietic stem cell transplantation in a male infant diagnosed with XLSA who was born with asphyxia due to severe anaemia. Early hepatic vein occlusion occurred after transplantation. However, this complication was rapidly controlled after active treatment, and the child's quality of life improved significantly. Haematopoietic stem cell transplantation is a promising alternative treatment for XLSA.

A pilot study of oral iron therapy in erythropoietic protoporphyria and X-linked protoporphyria.

The use of iron supplementation for anemia in erythropoietic protoporphyria (EPP) is controversial with both benefit and deterioration reported in single case reports. There is no systematic study to evaluate the benefits or risks of iron supplementation in these patients. We assessed the potential efficacy of oral iron therapy in decreasing erythrocyte protoporphyrin (ePPIX) levels in patients with EPP or X-linked protoporphyria (XLP) and low ferritin in an open-label, single-arm, interventional study. Sixteen patients (≥18 years) with EPP or XLP confirmed by biochemical and/or genetic testing, and serum ferritin ≤30 ng/mL were enrolled. Baseline testing included iron studies, normal hepatic function, and elevated plasma porphyrins and ePPIX levels. Oral ferrous sulfate 325 mg twice daily was administered for 12 months. The primary efficacy outcome was the relative difference in total ePPIX level between baseline and 12 months after starting treatment with iron. Secondary measures included improvement in serum ferritin, plasma porphyrins, and clinical symptoms. Thirteen patients had EPP (8 females, 5 males) and 3 had XLP (all females) and the mean age of participants was 38.8 years (SD 14.5). Ten patients completed all study visits limiting interpretation of results. In EPP patients, a transient increase in ePPIX levels was observed at 3 months in 9 of 12 (75%) patients. Iron was discontinued in 2 of these patients after meeting the protocol stopping rule of a 35% increase in ePPIX. Seven patients withdrew before study end. Ferritin levels increased on iron replacement indicating an improvement in iron status. A decrease in ePPIX was seen in both XLP patients who completed the study (relative difference of 0.67 and 0.5 respectively). No substantial changes in ePPIX were seen in EPP patients at the end of the study (n = 8; median relative difference: -0.21 (IQR: -0.44, 0.05). The most common side effects of iron treatment were gastrointestinal symptoms. Hepatic function remained normal throughout the study. Our study showed that oral iron therapy repletes iron stores and transiently increases ePPIX in some EPP patients, perhaps due to a transient increase in erythropoiesis, and may decrease ePPIX in XLP patients. Further studies are needed to better define the role of iron repletion in EPP. Trial registration: NCT02979249.

Heme Biosynthetic Gene Expression Analysis With dPCR in Erythropoietic Protoporphyria Patients.

Background: The heme biosynthesis (HB) involves eight subsequent enzymatic steps. Erythropoietic protoporphyria (EPP) is caused by loss-of-function mutations in the ferrochelatase (FECH) gene, which in the last HB step inserts ferrous iron into protoporphyrin IX (PPIX) to form heme. Aim and method: The aim of this work was to for the first time analyze the mRNA expression of all HB genes in peripheral blood samples of patients with EPP having the same genotype FECH c.[215dupT]; [315-48T > C] as compared to healthy controls by highly sensitive and specific digital PCR assays (dPCR). Results: We confirmed a decreased FECH mRNA expression in patients with EPP. Further, we found increased ALAS2 and decreased ALAS1, CPOX, PPOX and HMBS mRNA expression in patients with EPP compared to healthy controls. ALAS2 correlated with FECH mRNA expression (EPP: r = 0.63, p = 0.03 and controls: r = 0.68, p = 0.02) and blood parameters like PPIX (EPP: r = 0.58 p = 0.06). Conclusion: Our method is the first that accurately quantifies HB mRNA from blood samples with potential applications in the monitoring of treatment effects of mRNA modifying therapies in vivo, or investigation of the HB pathway and its regulation. However, our findings should be studied in separated blood cell fractions and on the enzymatic level.

ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation.

Iron­sulfur (Fe-S) clusters have been shown to play important roles in various cellular physiological process. Iron­sulfur cluster assembly 2 (ISCA2) is a vital component of the [4Fe-4S] cluster assembly machine. Several studies have shown that ISCA2 is highly expressed during erythroid differentiation. However, the role and specific regulatory mechanisms of ISCA2 in erythroid differentiation and erythroid cell growth remain unclear. RNA interference was used to deplete ISCA2 expression in human erythroid leukemia K562 cells. The proliferation, apoptosis, and erythroid differentiation ability of the cells were assessed. We show that knockdown of ISCA2 has profound effects on [4Fe-4S] cluster formation, diminishing mitochondrial respiratory chain complexes, leading to reactive oxygen species (ROS) accumulation and mitochondrial damage, inhibiting cell proliferation. Excessive ROS can inhibit the activity of cytoplasmic aconitase (ACO1) and promote ACO1, a bifunctional protein, to perform its iron-regulating protein 1(IRP1) function, thus inhibiting the expression of 5'-aminolevulinate synthase 2 (ALAS2), which is a key enzyme in heme synthesis. Deficiency of ISCA2 results in the accumulation of iron divalent. In addition, the combination of excessive ferrous iron and ROS may lead to damage of the ACO1 cluster and higher IRP1 function. In brief, ISCA2 deficiency inhibits heme synthesis and erythroid differentiation by double indirect downregulation of ALAS2 expression. We conclude that ISCA2 is essential for normal functioning of mitochondria, and is necessary for erythroid differentiation and cell proliferation.

Iron, Heme Synthesis and Erythropoietic Porphyrias: A Complex Interplay.

Erythropoietic porphyrias are caused by enzymatic dysfunctions in the heme biosynthetic pathway, resulting in porphyrins accumulation in red blood cells. The porphyrins deposition in tissues, including the skin, leads to photosensitivity that is present in all erythropoietic porphyrias. In the bone marrow, heme synthesis is mainly controlled by intracellular labile iron by post-transcriptional regulation: translation of ALAS2 mRNA, the first and rate-limiting enzyme of the pathway, is inhibited when iron availability is low. Moreover, it has been shown that the expression of ferrochelatase (FECH, an iron-sulfur cluster enzyme that inserts iron into protoporphyrin IX to form heme), is regulated by intracellular iron level. Accordingly, there is accumulating evidence that iron status can mitigate disease expression in patients with erythropoietic porphyrias. This article will review the available clinical data on how iron status can modify the symptoms of erythropoietic porphyrias. We will then review the modulation of heme biosynthesis pathway by iron availability in the erythron and its role in erythropoietic porphyrias physiopathology. Finally, we will summarize what is known of FECH interactions with other proteins involved in iron metabolism in the mitochondria.

X-linked sideroblastic anaemia in a female fetus: a case report and a literature review.

BACKGROUND: X-linked sideroblastic anaemia (XLSA) is commonly due to mutations in the ALAS2 gene and predominantly affects hemizygous males. Heterozygous female carriers of the ALAS2 gene mutation are often asymptomatic or only mildly anaemic. XLSA is usually characterized by microcytic erythrocytes (reduced mean corpuscular volume (MCV)) and hypochromia, along with increased red cell distribution width. However, in females with XLSA the characteristic laboratory findings can be dimorphic and present with macrocytic (elevated MCV) in addition to microcytic red cells. CASE PRESENTATION: We report a case of fetal anaemia, presenting in the early third trimester of pregnancy, in a female fetus. Ultrasound findings at 29 weeks were of cardiomegaly, prominent umbilical veins, a small rim of ascites, and mean cerebral artery peak systolic velocity (PSV) value above 1.5 Multiples of the Median (MoM). She underwent non-invasive prenatal testing that determined the rhesus genotype of the fetus to be rhesus B negative. No red blood cell antibodies were reported. Other investigations to determine the underlying cause of fetal anaemia included microarray comparative genomic hybridization, serology to exclude congenital infection and a peripheral blood film and fetal bilirubin to detect haemolysis. The maternal grandmother had a history of sideroblastic anaemia diagnosed at the age of 17 years. The mother had mild macrocytic anaemia with haemoglobin of 10.4 g/dl and MCV of 104 fl. The fetal anaemia was successfully treated with two in utero transfusions (IUTs), and delivery occurred via caesarean section at 37 weeks of gestation. The red cell gene sequencing in both the mother and fetus were heterozygous for an ALAS2 mutation causing in utero manifestations of XLSA. The haemoglobin on discharge to the local hospital at five days of age was 19.1 g/dl. Subsequently, the infant became anaemic, requiring regular 3-4 monthly blood transfusions and demonstrating overall normal development. Her anaemia was unresponsive to pyridoxine. CONCLUSIONS: This is one of four cases reporting multiple female members presenting with discordant clinical features of XLSA from being entirely asymptomatic to hydropic in utero. Our report is novel in that there are no previous cases in the literature of anaemia in a female fetus heterozygous for ALAS2 mutation.

A hemizygous p.R204Q mutation in the ALAS2 gene underlies X-linked sideroblastic anemia in an adult Chinese Han man.

BACKGROUND: X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia (CSA), and is associated with the mutations in the 5-aminolevulinate synthase 2 (ALAS2). The genetic basis of more than 40% of CSA cases remains unknown. METHODS: A two-generation Chinese family with XLSA was studied by next-generation sequencing to identify the underlying CSA-related mutations. RESULTS: In the study, we identified a missense ALAS2 R204Q mutation in a hemizygous Chinese Han man and in his heterozygous daughter. The male proband presented clinical manifestations at 38 years old and had a good response to pyridoxine. CONCLUSIONS: XLSA, as a hereditary disease, can present clinical manifestations later in lives, for adult male patients with ringed sideroblasts and hypochromic anemia, it should be evaluated with gene analyses to exclude CSA.

Identification of novel UROS mutations in a patient with congenital erythropoietic porphyria and efficient treatment by phlebotomy.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder of the heme biosynthetic pathway that is characterized by uroporphyrinogen III synthase (UROS) deficiency and the accumulation of non-physiological isomer I porphyrins. These phototoxic metabolites predominantly produced by the erythron result in ineffective erythropoiesis, chronic hemolysis and splenomegaly, but they also disseminate in tissues causing bullous photosensitivity to UV light and skin fragility that may progress to scarring with photo mutilation. Therapeutic management is currently limited to supportive care and bone marrow transplantation is reserved for the most severe cases. We describe here a 26-year-old women previously diagnosed with CEP harbouring two novel UROS gene mutations whose pathogenic mechanism was investigated by extensive molecular analysis. Clinical features included disabling hypertrichosis and skin photosensitivity without hemolysis. The first and rate-limiting 5-aminolevulinate synthase 2 (ALAS2) enzyme controls heme synthesis and porphyrin production in erythroid cells, while iron availability modulates its expression through a post-transcriptional mechanism. We performed iterative phlebotomies over 26 months to induce iron depletion in the patient and investigated the effectiveness and tolerance of this cost-effective approach. We observed a progressive decrease in plasma ferritin and urinary porphyrins upon treatment without inducing anemia. The patient reported improved quality of life and photosensitivity. Our data confirm recent reports highlighting the benefit of iron restriction on the disease phenotype through a reduction in porphyrin accumulation. This new strategy may represent an efficient and well-tolerated treatment for CEP patients with skin involvement and limited hematological component if iron restriction is carefully monitored.

A Novel ALAS2 Missense Mutation in Two Brothers With Iron Overload and Associated Alterations in Serum Hepcidin/Erythroferrone Levels.

Iron loading anemias are characterized by ineffective erythropoiesis and iron overload. The prototype is non-transfusion dependent ß-thalassemia (NTDT), with other entities including congenital sideroblastic anemias, congenital dyserythropoietic anemias, some hemolytic anemias, and myelodysplastic syndromes. Differential diagnosis of iron loading anemias may be challenging due to heterogeneous genotype and phenotype. Notwithstanding the recent advances in linking ineffective erythropoiesis to iron overload, many pathophysiologic aspects are still unclear. Moreover, measurement of hepcidin and erythroferrone (ERFE), two key molecules in iron homeostasis and erythropoiesis, is scarcely used in clinical practice and of uncertain utility. Here, we describe a comprehensive diagnostic approach, including next-generation sequencing (NGS), in silico modeling, and measurement of hepcidin and erythroferrone (ERFE), in two brothers eventually diagnosed as X-linked sideroblastic anemia (XLSA). A novel pathogenic ALAS2 missense mutation (c.1382T>A, p.Leu461His) is described. Hyperferritinemia with high hepcidin-25 levels (but decreased hepcidin:ferritin ratio) and mild-to-moderate iron overload were detected in both patients. ERFE levels were markedly elevated in both patients, especially in the proband, who had a more expressed phenotype. Our study illustrates how new technologies, such as NGS, in silico modeling, and measurement of serum hepcidin-25 and ERFE, may help in diagnosing and studying iron loading anemias. Further studies on the hepcidin-25/ERFE axis in additional patients with XLSA and other iron loading anemias may help in establishing its usefulness in differential diagnosis, and it may also aid our understanding of the pathophysiology of these genetically and phenotypically heterogeneous entities.

Regulation and tissue-specific expression of δ-aminolevulinic acid synthases in non-syndromic sideroblastic anemias and porphyrias.

Recently, new genes and molecular mechanisms have been identified in patients with porphyrias and sideroblastic anemias (SA). They all modulate either directly or indirectly the δ-aminolevulinic acid synthase (ALAS) activity. ALAS, is encoded by two genes: the erythroid-specific (ALAS2), and the ubiquitously expressed (ALAS1). In the liver, ALAS1 controls the rate-limiting step in the production of heme and hemoproteins that are rapidly turned over in response to metabolic needs. Several heme regulatory targets have been identified as regulators of ALAS1 activity: 1) transcriptional repression via a heme-responsive element, 2) post-transcriptional destabilization of ALAS1 mRNA, 3) post-translational inhibition via a heme regulatory motif, 4) direct inhibition of the activity of the enzyme and 5) breakdown of ALAS1 protein via heme-mediated induction of the protease Lon peptidase 1. In erythroid cells, ALAS2 is a gatekeeper of production of very large amounts of heme necessary for hemoglobin synthesis. The rate of ALAS2 synthesis is transiently increased during the period of active heme synthesis. Its gene expression is determined by trans-activation of nuclear factor GATA1, CACC box and NF-E2-binding sites in the promoter areas. ALAS2 mRNA translation is also regulated by the iron-responsive element (IRE)/iron regulatory proteins (IRP) binding system. In patients, ALAS enzyme activity is affected in most of the mutations causing non-syndromic SA and in several porphyrias. Decreased ALAS2 activity results either directly from loss-of-function ALAS2 mutations as seen in X-linked sideroblastic anemia (XLSA) or from defect in the availability of one of its two mitochondrial substrates: glycine in SLC25A38 mutations and succinyl CoA in GLRX5 mutations. Moreover, ALAS2 gain of function mutations is responsible for X-linked protoporphyria and increased ALAS1 activity lead to acute attacks of hepatic porphyrias. A missense dominant mutation in the Walker A motif of the ATPase binding site in the gene coding for the mitochondrial protein unfoldase CLPX also contributes to increasing ALAS and subsequently protoporphyrinemia. Altogether, these recent data on human ALAS have informed our understanding of porphyrias and sideroblastic anemias pathogeneses and may contribute to new therapeutic strategies.

Molecular pathophysiology and genetic mutations in congenital sideroblastic anemia.

Sideroblastic anemia is a heterogeneous congenital and acquired disorder characterized by anemia and the presence of ring sideroblasts in the bone marrow. Congenital sideroblastic anemia (CSA) is a rare disease caused by mutations in genes involved in the heme biosynthesis, iron-sulfur [Fe-S] cluster biosynthesis, and mitochondrial protein synthesis. The most prevalent form of CSA is X-linked sideroblastic anemia, caused by mutations in the erythroid-specific δ-aminolevulinate synthase (ALAS2), which is the first enzyme of the heme biosynthesis pathway in erythroid cells. To date, a remarkable number of genetically undefined CSA cases remain, but a recent application of the next-generation sequencing technology has recognized novel causative genes for CSA. However, in most instances, the detailed molecular mechanisms of how defects of each gene result in the abnormal mitochondrial iron accumulation remain unclear. This review aims to cover the current understanding of the molecular pathophysiology of CSA.

Clinical and genetic features of congenital dyserythropoietic anemia (CDA).

INTRODUCTION: Congenital dyserythropoietic anemias (CDA) are characterized by hyporegenerative anemia with inadequate reticulocyte values, ineffective erythropoiesis, and hemolysis. Distinctive morphology of bone marrow erythroblasts and identification of causative genes allow classification into 4 types caused by variants in CDAN1, c15orf41, SEC23B, KIF23, and KLF1 genes. OBJECTIVE: Identify pathogenic variants in CDA patients. METHODS: Massive parallel sequencing with a targeted gene panel, Sanger sequencing, Comparative Genome Hybridization (CGH), and in silico predictive analysis of pathogenicity. RESULTS: Pathogenic variants were found in 21 of 53 patients studied from 44 unrelated families. Six variants were found in CDAN1: two reported, p.Arg714Trp and p.Arg725Trp and, four novel, p.Arg623Trp, p.Arg946Trp, p.Phe1125Ser and p.Ser1227Gly. Twelve variants were found in SEC23B: seven reported, p.Arg14Trp, p.Glu109Lys, p.Arg217Ter, c.835-2A>G, p.Arg535Ter, p.Arg550Ter and p.Arg718Ter and, five novel, p.Val164Leu, p.Arg190Gln, p.Gln521Ter, p.Arg546Trp, and p.Arg611Gln. The variant p.Glu325Lys in KLF1 was found in one patient and p.Tyr365Cys in ALAS2 in an other. Moreover, we identified genomic rearrangements by CGH in some SEC23B-monoallelic patients. CONCLUSIONS: New technologies for genetic studies will help to find variants in other genes, in addition to those known, that contribute to or modulate the CDA phenotype or support the correct diagnosis.

[Successful treatment of X-linked sideroblastic anemia with ALAS2 R452H mutation using vitamin B6].

A 45-year-old man presented with fatigue and pain in the finger joints. Despite having a history of suspected sideroblastic anemia since the age of 18 years, he had not been followed up for years. Upon presentation, laboratory data revealed microcytic anemia and elevated serum ferritin levels. In addition, ringed sideroblasts were increased in the bone marrow. A liver biopsy revealed hemochromatosis and cirrhosis. Furthermore, genetic analysis revealed that he harbored the ALAS2 R452H mutation, leading to the diagnosis of X-linked sideroblastic anemia (XLSA). Accordingly, oral folate or vitamin (Vit) B12 was administered, but his anemia did not respond. However, his hemoglobin level increased from 7 to 11 g/dl with an additional prescription of oral VitB6, which facilitated the patient to undergo phlebotomy to ameliorate organ dysfunctions caused by iron overload. Previous research has revealed that ALAS2 R452 mutations confer poor responses to VitB6 therapy. Hence, accrual of patients with an unexpectedly better response, which was observed in our case, may help elucidate the pathogenesis of and therapies for XLSA.