Current Status of Molecular Diagnosis of Hereditary Hemolytic Anemia in Korea.

Hereditary hemolytic anemia (HHA) is considered a group of rare hematological diseases in Korea, primarily because of its unique ethnic characteristics and diagnostic challenges. Recently, the prevalence of HHA has increased in Korea, reflecting the increasing number of international marriages and increased awareness of the disease. In particular, the diagnosis of red blood cell (RBC) enzymopathy experienced a resurgence, given the advances in diagnostic techniques. In 2007, the RBC Disorder Working Party of the Korean Society of Hematology developed the Korean Standard Operating Procedure for the Diagnosis of Hereditary Hemolytic Anemia, which has been continuously updated since then. The latest Korean clinical practice guidelines for diagnosing HHA recommends performing next-generation sequencing as a preliminary step before analyzing RBC membrane proteins and enzymes. Recent breakthroughs in molecular genetic testing methods, particularly next-generation sequencing, are proving critical in identifying and providing insight into cases of HHA with previously unknown diagnoses. These innovative molecular genetic testing methods have now become important tools for the management and care planning of patients with HHA. This review aims to provide a comprehensive overview of recent advances in molecular genetic testing for the diagnosis of HHA, with particular emphasis on the Korean context.

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Background: Anemia in children is common and finding the underlying cause is often uncomplicated. However, in some cases, the underlying diagnosis is rare and difficult to diagnose. Case presentation: A toddler presented with severe anemia with normal red cell indices and a low reticulocyte count. The remaining hematological parameters were normal, bar a slight thrombocytosis. At this point a diagnosis of transient erythroblastopenia of childhood (TEC) was made. The child continued to have slight anemia with intermittent macrocytosis and reticulocytopenia throughout childhood. Growth and development was normal, and there were no signs of congenital abnormalities in the heart or kidneys nor any craniofacial or phalangeal defects. Repeated bone marrow examinations showed no significant abnormal findings. As a teenager the patient was diagnosed with Diamond-Blackfan anemia through an exome-based gene panel which revealed a mutation in the RPL11 gene. Interpretation: Congenital bone marrow failure syndromes do not always present in the classical way, leading to a delayed diagnosis. The increasing availability of different gene panels for patients with persistent abnormal hematological laboratory parameters offers the possibility of a more accurate diagnostic pathway, which is important for adequate follow-up and genetic counselling.

Identification of the molecular etiology in rare congenital hemolytic anemias using next-generation sequencing with exome-based copy number variant analysis.

OBJECTIVES: In congenital hemolytic anemias (CHA), it is not always possible to determine the specific diagnosis by evaluating clinical findings and conventional laboratory tests. The aim of this study is to evaluate the utility of next-generation sequencing (NGS) and clinical-exome-based copy number variant (CNV) analysis in patients with CHA. METHODS: One hundred and forty-three CHA cases from 115 unrelated families referred for molecular analysis were enrolled in the study. Molecular analysis was performed using two different clinical exome panels in 130 patients, and whole-exome sequencing in nine patients. Exome-based CNV calling was incorporated into the traditional single-nucleotide variant and small insertion/deletion analysis pipeline for NGS data in 92 cases. In four patients from the same family, the PK Gypsy variant was investigated using long-range polymerase chain reaction. RESULTS: Molecular diagnosis was established in 86% of the study group. The most frequently mutated genes were SPTB (31.7%) and PKLR (28.5%). CNV analysis of 92 cases revealed that three patients had different sizes of large deletions in the SPTB and six patients had a deletion in the PKLR. CONCLUSIONS: In this study, NGS provided a high molecular diagnostic rate in cases with rare CHA. Analysis of the CNVs contributed to the diagnostic success.

Early Onset of Severe Anemia Caused by Hb Calgary (HBB: C.194G > T): Another Case Report.

Unstable hemoglobin (Hb) variants are a rare cause of congenital hemolytic anemia. We describe a Chinese girl who presented with transfusion-dependent anemia in early infancy. Her diagnosis of Hb Calgary [ß64(E8)Gly > Val; HBB:c.194G > T] was not made until molecular testing was performed at the age of 5 years. Our case highlights the importance of early genetic testing in order to make the diagnosis, which may not only be useful for patient management and family counseling, but also for avoiding further unnecessary investigative attempts.

PIEZO Ion Channels in Cardiovascular Functions and Diseases.

The cardiovascular system provides blood supply throughout the body and as such is perpetually applying mechanical forces to cells and tissues. Thus, this system is primed with mechanosensory structures that respond and adapt to changes in mechanical stimuli. Since their discovery in 2010, PIEZO ion channels have dominated the field of mechanobiology. These have been proposed as the long-sought-after mechanosensitive excitatory channels involved in touch and proprioception in mammals. However, more and more pieces of evidence point to the importance of PIEZO channels in cardiovascular activities and disease development. PIEZO channel-related cardiac functions include transducing hemodynamic forces in endothelial and vascular cells, red blood cell homeostasis, platelet aggregation, and arterial blood pressure regulation, among others. PIEZO channels contribute to pathological conditions including cardiac hypertrophy and pulmonary hypertension and congenital syndromes such as generalized lymphatic dysplasia and xerocytosis. In this review, we highlight recent advances in understanding the role of PIEZO channels in cardiovascular functions and diseases. Achievements in this quickly expanding field should open a new road for efficient control of PIEZO-related diseases in cardiovascular functions.

Diagnosing Transient Erythroblastopenia of Childhood: A Review for Pediatricians.

Transient erythroblastopenia of childhood is a rare, benign, self-limited condition seen in infants and young children. Laboratory studies will show moderate or severe normochromic normocytic anemia accompanied by absent or low reticulocytes, neutropenia, and mild thrombocytosis or thrombocytopenia. The etiology is unclear, but it has been associated with clinical or laboratory evidence of a recent viral syndrome. Initial diagnostic studies should be aimed at identifying potential causes of anemia, but a confirmed diagnosis is usually obtained once the hemoglobin level has normalized spontaneously. Differentiation from Diamond-Blackfan anemia is critical, especially in infants. Once the diagnosis is established, treatment is supportive, but red blood cell transfusion is indicated in severe cases. High clinical suspicion is imperative to avoid needless diagnostic and therapeutic measures. [Pediatr Ann. 2024;53(1):e34-e36.].

Vesiculation in irradiated and cation-leaky-stored red blood cells.

BACKGROUND: Extracellular vesicles (EVs) are released by red blood cells (RBCs) throughout their life-span and also during hypothermic storage when they accumulate in the blood bag. We queried whether stored RBCs with increased cation permeability, either from donors with familial pseudohyperkalaemia (FP) or caused by irradiation, vesiculate more readily. STUDY DESIGN AND METHODS: Recent technical advances have revealed at least two sub-populations of MVs in RBC storage units: macrovesicles (2-6 µm) and microvesicles (1-2 µm). Using nanoparticle tracking analysis, imaging flow cytometry, and protein quantification methods, we measured and characterized vesicles released by RBCs from control and FP individuals at three different storage time-points (day 4, day 17, and day 29). The RBCs had either been stored untreated or irradiated on either day 1 or day 14 of storage. RESULTS: We found no difference in the number or size of vesicles released between cation-leaky FP RBCs and non-FP controls. Similarly, irradiated and non-irradiated RBCs showed very similar patterns of vesicle release to during cold-storage. The only significant difference in vesicle release was the increase in accumulated vesicles with length of storage time which has been reported previously. DISCUSSION: EVs in stored blood are potential contributors to adverse transfusion reactions. The number of vesicles released during 35-day hypothermic storage varies between donors and increases with storage duration. However, increased cation permeability and irradiation do not appear to affect vesicle formation during RBC cold-storage.

Deciphering and disrupting PIEZO1-TMEM16F interplay in hereditary xerocytosis.

ABSTRACT: Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

Transient erythroblastopenia due to a GATA1 variant in an infant female.

Diamond-Blackfan anemia (DBA) is a congenital anemia with erythroid cell aplasia. Most of the causative genes are ribosomal proteins. GATA1, a hematopoietic master transcription factor required for erythropoiesis, also causes DBA. GATA1 is located on Xp11.23; therefore, DBA develops only in males in an X-linked inheritance pattern. Here, we report a case of transient erythroblastopenia and moderate anemia in a female newborn infant with a de novo GATA1 variant. In this patient, increased methylation of the GATA1 wild-type allele was observed in erythroid cells. Skewed lyonization of GATA1 may cause mild transient erythroblastopenia in a female patient.

Transient presence of stomatocytes: A clue to the diagnosis of overhydrated hereditary stomatocytosis in a child with beta-thalassemia.

BACKGROUND: Overhydrated hereditary stomatocytosis (OHSt) is a rare disorder characterized by abnormalities in erythrocytic volume homeostasis. Early and accurate diagnosis is essential for appropriate management and genetic counseling. METHODS: We present the case of a child with beta-thalassemia and a history of multiple blood transfusions. Clinical presentation, laboratory findings, and genetic testing were reviewed. Peripheral blood smear examination and genetic analysis were performed. RESULTS: The patient was admitted with severe anemia, and peripheral blood smear examination revealed the presence of up to 50% stomatocytes. Laboratory investigations showed abnormalities in red blood cell parameters, including decreased hemoglobin levels and increased mean corpuscular volume. Genetic testing identified a heterozygous mutation in the RHAG gene, confirming the diagnosis of OHSt. The presence of stomatocytes in the peripheral blood smear was transient, correlating with episodes of hemolysis and its control.

The cation-leaky hereditary stomatocytosis syndromes: A tale of six proteins.

This review concerns a series of dominantly inherited haemolytic anaemias in which the membrane of the erythrocyte 'leaks' the univalent cations, compromising the osmotic stability of the cell. The majority of the conditions are explained by mutations in one of six genes, coding for multispanning membrane proteins of different structure and function. These are: RhAG, coding for an ammonium carrier; SLC4A1, coding for the band 3 anion exchanger; PIEZO1, coding for a mechanosensitive cation channel; GLUT1, coding for a glucose transporter; KCNN4, coding for an internal-calcium-activated potassium channel; and ABCB6, coding for a porphyrin transporter. This review describes the five clinical syndromes associated with genetic defects in these genes and their variable genotype/phenotype relationships.

Diagnostic yield of targeted next-generation sequencing for pediatric hereditary hemolytic anemia.

BACKGROUND: Hereditary hemolytic anemia (HHA) refers to a heterogeneous group of genetic disorders that share one common feature: destruction of circulating red blood cells (RBCs). The destruction of RBCs may be due to membranopathies, enzymopathies, or hemoglobinopathies. Because these are genetic disorders, incorporation of next-generation sequencing (NGS) has facilitated the diagnostic process of HHA. METHOD: Genetic data from 29 patients with suspected hereditary anemia in a tertiary hospital were retrospectively reviewed to evaluate the efficacy of NGS on hereditary anemia diagnosis. Targeted NGS was performed with custom probes for 497 genes associated with hematologic disorders. After genomic DNA was extracted from peripheral blood, prepared libraries were hybridized with capture probes and sequenced using NextSeq 550Dx (Illumina, San Diego, CA, USA). RESULT: Among the 29 patients, ANK1 variants were detected in five, four of which were pathogenic or likely pathogenic variants. SPTB variants were detected in six patients, five of which were classified as pathogenic or likely pathogenic variants. We detected g6pd pathogenic and spta1 likely pathogenic variants in two patients and one patient, respectively. Whole-gene deletions in both HBA1 and HBA2 were detected in two patients, while only HBA2 deletion was detected in one patient. One likely pathogenic variant in PLKR was detected in one patient, and one likely pathogenic variant in ALAS2 was detected in another. CONCLUSION: Here, NGS played a critical role in definitive diagnosis in 18 out of 29 patients (62.07%) with suspected HHA. Thus, its incorporation into the diagnostic workflow is crucial.

A novel missense variant in ATP11C is associated with reduced red blood cell phosphatidylserine flippase activity and mild hereditary hemolytic anemia.

Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient-derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS-stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C-deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs.

Molecular diagnosis of hereditary hemolytic anemias: Recent updates.

Hereditary hemolytic anemia (HHA) is a heterogeneous group of disorders due to genetically caused defects in red blood cell membrane structure, enzymes, heme and globin synthesis, erythroid proliferation, and differentiation. Traditionally, the diagnostic process is complex and includes a plethora of tests from routine to highly specialized ones. The inclusion of molecular testing has significantly improved the diagnostic yield. The value of molecular testing is broader than just rendering the correct diagnosis, as it may also guide therapeutic decisions. As more molecular modalities become available for clinical use, it is imperative to understand their benefits and disadvantages pertaining to the HHA diagnostics. Re-evaluation of the traditional diagnostic workflow may also bring forth additional benefits. This review focuses on the current state of molecular testing for HHA.

Activation of pyruvate kinase as therapeutic option for rare hemolytic anemias: Shedding new light on an old enzyme.

Novel developments in therapies for various hereditary hemolytic anemias reflect the pivotal role of pyruvate kinase (PK), a key enzyme of glycolysis, in red blood cell (RBC) health. Without PK catalyzing one of the final steps of the Embden-Meyerhof pathway, there is no net yield of adenosine triphosphate (ATP) during glycolysis, the sole source of energy production required for proper RBC function and survival. In hereditary hemolytic anemias, RBC health is compromised and therefore lifespan is shortened. Although our knowledge on glycolysis in general and PK function in particular is solid, recent advances in genetic, molecular, biochemical, and metabolic aspects of hereditary anemias have improved our understanding of these diseases. These advances provide a rationale for targeting PK as therapeutic option in hereditary hemolytic anemias other than PK deficiency. This review summarizes the knowledge, rationale, (pre)clinical trials, and future advances of PK activators for this important group of rare diseases.

Usefulness of HbA1c Measurements by HPLC- and Immuno-Assay for Screening Unstable Hemoglobinopathy.

OBJECTIVE: Unstable hemoglobinopathy (UH), red blood cell membrane disease (MD), and red blood cell enzymopathy are known as major congenital hemolytic anemias. Specialized examinations are needed for their differential diagnosis. We hypothesized that simultaneous measurements of HbA1c levels using high-performance liquid chromatography (HPLC) by fast mode (FM) and immunoassay [HPLC (FM)-HbA1c and IA-HbA1c, respectively] are useful for the differential diagnosis of UH from other congenital hemolytic anemias and verified this hypothesis in this study. METHODS: HPLC (FM)-HbA1c and IA-HbA1c levels were simultaneously measured in 5 variant hemoglobinopathy (VH) patients with ß-chain heterozygous mutation, 8 MD patients, 6 UH patients, and 10 healthy controls. None of the patients had diabetes mellitus. RESULTS: In VH patients, HPLC-HbA1c levels were low, whereas IA-HbA1c levels were within the reference range. In MD patients, HPLC-HbA1c and IA-HbA1c levels were similarly low. In UH patients, both HPLC-HbA1c and IA-HbA1c levels were low, but HPLC-HbA1c levels were significantly lower than IA-HbA1c levels. The HPLC-HbA1c/IA-HbA1c ratio was 90% or more in all MD patients and control subjects. This ratio was, however, less than 90% in all VH patients and UH patients. CONCLUSION: The HPLC (FM)-HbA1c/IA-HbA1c ratio calculated using simultaneous measurements of HPLC (FM)-HbA1c and IA-HbA1c levels is useful for the differential diagnosis of VH, MD, and UH.