First Report of Hb Youngstown in Capillary Electrophoresis and Overlapping Hb Analysis Findings with Hb Rush.

Hb Youngstown [HBB:c.305A > C] is a rare unstable hemoglobin caused by the substitution of glutamic acid with alanine at codon 101 of the Beta globin chain. It causes hemolytic anemia in the heterozygous state. This is a case of a six-year-old Chinese-Javanese girl with heterozygous Hb Youngstown and clinical features of chronic hemolysis and iron overload. Hb Youngstown appears at the S window near to 4.6 minutes on high-performance liquid chromatography (HPLC) and can form a hybrid tetramer on alkaline gel electrophoresis seen as two distinct bands cathodal to A and close to F. For the first time, Hb Youngstown is captured with capillary electrophoresis (CE) and shown to be eluted at zone 8. Clinical presentation and Hb analysis results of this heterozygous Hb Youngstown overlap with heterozygous Hb Rush. They can only be differentiated at molecular level by Beta globin gene sequencing or intact mass spectrometry.

Targeted next-generation sequencing identifies eighteen novel mutations expanding the molecular and clinical spectrum of PKLR gene disorders in the Indian population.

Pyruvate kinase deficiency (PKD) is an autosomal recessive condition, caused due to homozygous or compound heterozygous mutation in the PKLR gene resulting in non-spherocytic hereditary hemolytic anemia. Clinical manifestations in PKD patients vary from moderate to severe lifelong hemolytic anemia either requiring neonatal exchange transfusion or blood transfusion support. Measuring PK enzyme activity is the gold standard approach for diagnosis but residual activity must be related to the increased reticulocyte count. The confirmatory diagnosis is provided by PKLR gene sequencing by conventional as well as targeted next-generation sequencing involving genes associated with enzymopathies, membranopathies, hemoglobinopathies, and bone marrow failure disorders. In this study, we report the mutational landscape of 45 unrelated PK deficiency cases from India. The genetic sequencing of PKLR revealed 40 variants comprising 34 Missense Mutations (MM), 2 Nonsense Mutations (NM), 1 Splice site, 1 Intronic, 1 Insertion, and 1 Large Base Deletion. The 17 novel variants identified in this study are A115E, R116P, A423G, K313I, E315G, E318K, L327P, M377L, A423E, R449G, H507Q, E538K, G563S, c.507 + 1 G > C, c.801_802 ins A (p.Asp268ArgfsTer48), IVS9dsA-T + 3, and one large base deletion. In combination with previous reports on PK deficiency, we suggest c.880G > A, c.943G > A, c.994G > A, c.1456C > T, c.1529G > A are the most frequently observed mutations in India. This study expands the phenotypic and molecular spectrum of PKLR gene disorders and also emphasizes the importance of combining both targeted next-generation sequencing with bioinformatics analysis and detailed clinical evaluation to elaborate a more accurate diagnosis and correct diagnosis for transfusion dependant hemolytic anemia in a cohort of the Indian population.

Transient neonatal hemolytic anemia due to the novel gamma globin gene mutation HBG2:C.290T>C, p.Leu97Pro (hemoglobin Wareham).

Unstable gamma globin variants can cause transient neonatal hemolytic anemia. We have identified a novel variant in a newborn who presented with jaundice and anemia requiring phototherapy and red blood cell transfusion. The patient was found to be heterozygous for the mutation HGB2:c.290T>C, p.Leu97Pro, which we have termed hemoglobin (Hb) Wareham. This substitution is expected to generate an unstable hemoglobin with increased oxygen affinity based on the homologous mutation previously described in the beta globin gene, which is termed as Hb Debrousse. The patient fully recovered by 9 months of age as expected with the transition from fetal to adult hemoglobin.

Glucose 6 Phosphate Isomerase Deficiency, a Rare Hemolytic Anemia Misdiagnosed as Hereditary Spherocytosis.

Hereditary hemolytic anemias are a heterogenous group of disorders that include membranopathies, enzymopathies, and hemoglobinopathies. Genetic testing is helpful in the diagnostic workup when the clinical and laboratory workup is not conclusive. Here, we present a case of a 21-month-old female who was initially diagnosed with hereditary spherocytosis based on the presence of a variant of unknown significance in the SPTB gene. Further genetic workup revealed a homozygous glucose 6 phosphate isomerase mutation and the patient was ultimately diagnosed with glucose 6 phosphate isomerase deficiency.

The first Chinese with Hb Chile leading to chronic anemia and methemoglobinemia: a case report.

BACKGROUND: Hemoglobin (Hb) Chile [ß28(B10) Leu > Met; HBB: c.85 C > A] is a rare hemoglobin variant caused by a missense mutation in the HBB gene. Only one case of Hb Chile has been reported worldwide so far. It is an unstable hemoglobin, characterized by cyanosis associated with chronic methemoglobinemia and hemolytic anemia induced by sulfonamides or methylene blue. CASE PRESENTATION: A 9-year-3-month-old girl had mild anemia of unknown etiology for more than 6 years. She had a slight pallor without other symptoms or signs. The complete blood count revealed normocytic normochromic anemia with a sometimes-elevated reticulocyte count, and the bone marrow cytology showed marked erythroid hyperplasia, but the tests related to hemolysis were normal. Therefore, the whole exome sequencing was performed and showed a heterozygous mutation for HBB: c.85 C > A. With asymptomatic methemoglobinemia confirmed later, she was eventually diagnosed with Hb Chile. CONCLUSIONS: This is the first report of Hb Chile in China and the second worldwide. This case shows that Hb Chile is clinically heterogeneous and difficult to diagnose and expands our understanding on the clinical and hematological traits of the disease.

Recognition of a novel variant of phosphoglycerate kinase 1 deficiency PGK1 Galveston (c.472G > C) in a child with hemolytic anemia, neurologic dysfunction and myopathy.

The enzyme phosphoglycerate kinase 1 (PGK1) catalyzes the first ATP producing reaction in the glycolysis pathway. Certain mutations to the coding gene of PGK1 present clinically with varying manifestations including hemolytic anemia, central nervous system (CNS) dysfunction and myopathy. Various PGK1 mutations have been described in the literature at the clinical and molecular level. Herein, we describe a novel case PGK1 mutation (PGK1 Galveston) in a 4-year-old boy who presented with all three manifestations. We discuss the characteristic hematopathology findings from this patient as well as provide a comparison with previously described neuroimaging findings. The variable clinical presentation of this condition along with its inherent uniqueness provide a diagnostic challenge for physicians. This presentation will add to the current body of knowledge for this condition and help guide future investigation and management.

Successful Treatment of a Child with Hemoglobin Hammersmith with Hematopoietic Stem Cell Transplantation.

Hemoglobin (Hb) Hammersmith, formed by serine substitution for phenylalanine at residue 42 in the beta-globin chain, is a very rare variant of unstable hemoglobin with low oxygen affinity. For patients with hemoglobinopathies, it is well-established that hematopoietic stem cell transplantation provides a complete cure, but the literature on its role for those with Hb Hammersmith is limited. A seven-month-old girl who was examined for anemia and splenomegaly was followed up for congenital hemolytic anemia. The patient with visible cyanosis of the lips and whose p50 was low in blood gas was diagnosed with Hb Hammersmith through the DNA sequence analysis. During the follow-up, frequent blood transfusions had to be given due to anemia aggravated by infections. Following a successful hematopoietic stem cell transplant from an HLA-matched sibling, the patient completely recovered from Hb Hammersmith. The case is presented because of its rarity.

Hb Ryazan: An Elongated C-Terminal ß-Chain Due to a New Frameshift Mutation, HBB: c.396delG p.Val133Trpfs*25.

We identified a novel abnormal hemoglobin variant caused by a frameshift mutation at nucleotide position 396 in exon 3 of the ß-globin gene (HBB): NM_000518:c.396delG. This variant causes an emergence of alternative amino acid sequence starting at codon 133 and a new stop codon formed in the 3' untranslated region (3'UTR) of the HBB gene at amino acid position 158. This ß-globin gene variant was identified in a woman with a long history of hemolytic anemia. We named this variant Hb Ryazan after the proband's city of origin.

Erythropoietin and IGF-1 signaling synchronize cell proliferation and maturation during erythropoiesis.

Tight coordination of cell proliferation and differentiation is central to red blood cell formation. Erythropoietin controls the proliferation and survival of red blood cell precursors, while variations in GATA-1/FOG-1 complex composition and concentrations drive their maturation. However, clear evidence of cross-talk between molecular pathways is lacking. Here, we show that erythropoietin activates AKT, which phosphorylates GATA-1 at Ser310, thereby increasing GATA-1 affinity for FOG-1. In turn, FOG-1 displaces pRb/E2F-2 from GATA-1, ultimately releasing free, proproliferative E2F-2. Mice bearing a Gata-1(S310A) mutation suffer from fatal anemia when a compensatory pathway for E2F-2 production involving insulin-like growth factor-1 (IGF-1) signaling is simultaneously abolished. In the context of the GATA-1(V205G) mutation resulting in lethal anemia, we show that the Ser310 cannot be phosphorylated and that constitutive phosphorylation at this position restores partial erythroid differentiation. This study sheds light on the GATA-1 pathways that synchronize cell proliferation and differentiation for tissue homeostasis.

Inherited CD59 deficiency, where neurology and genetics intertwine.

OBJECTIVES: To describe the clinical phenotype of eight children diagnosed with CD59 deficiency and their ultimate neurological outcome. METHODS: The data of our cases were extensively reviewed both clinical and ancillary tests; investigations included: neuroimaging, neurophysiological studies, and laboratory tests. RESULTS: All patients presented during early infancy with Guillain-Barre syndrome later they suffered repeated relapses leading to the diagnosis of chronic axonal neuropathy. Recurrent stroke and acute necrotizing encephalopathy were described, 2 patients in each group. One girl developed acute disseminated encephalomyelitis while one boy developed acute transverse myelitis. Overt hemolytic anemia requiring blood transfusion reported in six patients. CONCLUSION: Inherited CD59 deficiency is an autosomal recessive disorder which can have devastating neurological consequences. First line immunotherapy including intravenous immunoglobin, corticosteroids, and plasma exchange may have transient beneficial effect. Reports of targeted therapy with eculizumab might be lifesaving. Genetic counseling is crucial.

Nmnat3 deficiency in hemolytic anemia exacerbates malaria infection.

Malaria is an infectious disease caused by Plasmodium parasites and has high mortality rates, especially among children in African and Southeast Asian countries. Patients with hemolytic anemia are suggested to adapt protective measures against malarial infection. Nicotinamide adenine dinucleotide (NAD+) is a crucial cofactor associated with numerous biological processes that maintain homeostasis in all living organisms. In a previous study, we had demonstrated that the deficiency of nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3), an enzyme catalyzing NAD+ synthesis, causes hemolytic anemia accompanied by a drastic decline in the NAD+ levels in the erythrocytes. It is well known that hemolytic anemia is linked to a reduced risk of malarial infections. In the present study, we investigated whether hemolytic anemia caused by Nmnat3 deficiency is beneficial against malarial infections. We found that Nmnat3 deficiency exacerbated malarial infection and subsequently caused death. Moreover, we demonstrated that the NAD+ levels in malaria-infected Nmnat3 red blood cells significantly increased and the glycolytic flow was largely enhanced to support the rapid growth of malarial parasites. Our results revealed that hemolytic anemia induced by the deletion of Nmnat3 was harmful rather than protective against malaria.

HK1 haemolytic anaemia in association with a neurological phenotype and co-existing CEP290 Meckel-Gruber in a Romani family.

HK1 deficient Haemolytic Anaemia in association with a Neurological Phenotype & co-existing Meckel-Gruber due to CEP290 in a Romani family.

Red cell membrane disorders: structure meets function.

The mature red blood cell (RBC) lacks a nucleus and organelles characteristic of most cells, but it is elegantly structured to perform the essential function of delivering oxygen and removing carbon dioxide from all other cells while enduring the shear stress imposed by navigating small vessels and sinusoids. Over the past several decades, the efforts of biochemists, cell and molecular biologists, and hematologists have provided an appreciation of the complexity of RBC membrane structure, while studies of the RBC membrane disorders have offered valuable insights into structure-function relationships. Within the last decade, advances in genetic testing and its increased availability have made it possible to substantially build upon this foundational knowledge. Although disorders of the RBC membrane due to altered structural organization or altered transport function are heterogeneous, they often present with common clinical findings of hemolytic anemia. However, they may require substantially different management depending on the underlying pathophysiology. Accurate diagnosis is essential to avoid emergence of complications or inappropriate interventions. We propose an algorithm for laboratory evaluation of patients presenting with symptoms and signs of hemolytic anemia with a focus on RBC membrane disorders. Here, we review the genotypic and phenotypic variability of the RBC membrane disorders in order to raise the index of suspicion and highlight the need for correct and timely diagnosis.

Acute Hemolytic Anemia Caused by G6PD Deficiency in Children in Mayotte: A Frequent and Severe Complication.

Severe hemolytic anemia is a rare complication of glucose-6-phosphate dehydrogenase (G6PD) deficiency. It occurs with the Mediterranean (Med) variant corresponding to a class 2 deficiency according to the World Health Organization (WHO) classification, and it correlates with a severe deficiency in G6PD activity. In Mayotte, the majority of patients have the African (A-) variant as a WHO class 3 deficiency. Yet we have observed numerous cases of severe hemolytic anemia defined by a hemoglobin level of <6 g/dL. In this study, we aimed to describe the epidemiological, clinical, and biological features as well as the treatment modalities of children presenting with a severe hemolytic crisis secondary to G6PD deficiency in Mayotte. The secondary objective was to study the disease genotype when this information was available. Between April 2013 and September 2020, 73 children presented with severe anemia because of G6PD deficiency in Mayotte. The median hemoglobin level during the hemolytic crises was 3.9 g/dL. All of the patients underwent a transfusion and hospitalization. Twenty patients had a disease genotype: 11 had the African mutation and 9 had the Med mutation. Although they are among the most common triggers of G6PD acute hemolytic anemia, drugs were found to not be present and fava bean ingestion was found in only 1 child. One of the specific triggers was traditional medicine, including Acalypha indica . Severe hemolytic crisis in children because of G6PD deficiency is a frequent occurrence in Mayotte. The patients had severe disease symptoms, but the severity did not correlate with the genotype: the African (A-) variant and the Med variant resulted in the same level of disease severity.

A rare mutation (p.F149del) of the NT5C3A gene is associated with pyrimidine 5'-nucleotidase deficiency.

Pyrimidine 5'-nucleotidase deficiency is a rare erythrocyte enzymopathy. Here we report two cases of hemolytic anemia in brothers of Polish origin that are associated with a very rare mutation. Heterozygous deletion in the NT5C3A gene (c.444_446delGTT), inherited most likely from their asymptomatic mother, resulted in a single amino acid residue deletion (p.F149del) in cytosolic pyrimidine 5'-nucleotidase. However, only the mutated transcript was present in the reticulocyte transcriptome of both patients. Only residual activity of pyrimidine 5'-nucleotidase in the brothers' erythrocytes could be observed when compared with the controls, including their asymptomatic father and sister. Western blot showed no sign of the presence of 5'-nucleotidase protein in the erythrocytes of both studied patients. The 2.5-fold reduction of the purine/pyrimidine ratio observed only in the brothers' erythrocytes confirms the correlation of the results of molecular analysis, including whole-exome sequencing, with the phenotype of the pyrimidine 5'-nucleotidase deficiency. Altogether, our results may substantiate the hypothesis of the heterogeneity of the molecular basis of the defect involving both the mutation presented here and negative regulation of expression of the "normal" allele.

Hereditary nonspherocytic hemolytic anemia caused by glucose-6-phosphate isomerase (GPI) deficiency in a Chinese patient: a case report.

BACKGROUND: Glucose phosphate isomerase (GPI) deficiency is a rare autosomal recessive disorder that causes hereditary nonspherocytic hemolytic anemia (HNSHA). Homozygous or compound heterozygous mutation of the GPI gene on chromosome 19q13 is the cause of GPI deficiency. Fifty-seven GPI mutations have been reported at the molecular level. CASE PRESENTATION: A 5-month-old boy was presented with repeated episodes of jaundice after birth. He suffered from moderate hemolytic anemia (hemoglobin levels ranging from 62 to 91 g/L) associated with macrocytosis, reticulocytosis, neutropenia, and hyperbilirubinemia. Whole-exome sequencing showed that he has a missense mutation c.301G > A (p.Val101Met) in exon 4 and a frameshift mutation c.812delG (p.Gly271Glufs*131) in exon 10. Mutation p.Gly271Glufs*131 is a novel frameshift null mutation in GPI deficiency. CONCLUSION: In a patient with recurrent jaundice since birth, mutations in the GPI gene associated with HNSHA should be evaluated. The c.812delG (p.Gly271Glufs*131) variant may be a novel mutation of the GPI gene. Compound heterozygous mutations c.301G > A (p.Val101Met) and c.812delG (p.Gly271Glufs*131) are not relevant to neurological impairment.

Sterile α-motif domain requirement for cellular signaling and survival.

Hundreds of sterile α-motif (SAM) domains have predicted structural similarities and are reported to bind proteins, lipids, or RNAs. However, the majority of these domains have not been analyzed functionally. Previously, we demonstrated that a SAM domain-containing protein, SAMD14, promotes SCF/proto-oncogene c-Kit (c-Kit) signaling, erythroid progenitor function, and erythrocyte regeneration. Deletion of a Samd14 enhancer (Samd14-Enh), occupied by GATA2 and SCL/TAL1 transcription factors, reduces SAMD14 expression in bone marrow and spleen and is lethal in a hemolytic anemia mouse model. To rigorously establish whether Samd14-Enh deletion reduces anemia-dependent c-Kit signaling by lowering SAMD14 levels, we developed a genetic rescue assay in murine Samd14-Enh-/- primary erythroid precursor cells. SAMD14 expression at endogenous levels rescued c-Kit signaling. The conserved SAM domain was required for SAMD14 to increase colony-forming activity, c-Kit signaling, and progenitor survival. To elucidate the molecular determinants of SAM domain function in SAMD14, we substituted its SAM domain with distinct SAM domains predicted to be structurally similar. The chimeras were less effective than SAMD14 itself in rescuing signaling, survival, and colony-forming activities. Thus, the SAMD14 SAM domain has attributes that are distinct from other SAM domains and underlie SAMD14 function as a regulator of cellular signaling and erythrocyte regeneration.

Severe neonatal haemolytic anaemia caused by compound heterozygous KLF1 mutations: report of four families and literature review.

Mutations in the KLF1 gene, which encodes a transcription factor playing a role in erythropoiesis, have recently been demonstrated to be a rare cause of hereditary haemolytic anaemia. We described the genotypic and phenotypic spectra of four unrelated families with compound heterozygous class 2/class 3 KLF1 mutations. All patients had p.G176RfsX179 on one allele and either p.A298P, p.R301H or p.G335R on the other allele. All presented on the first day of life with severe haemolytic anaemia with abnormal red blood cell morphology, markedly increased nucleated red blood cells and hyperbilirubinaemia. Three patients later became transfusion-dependent. All parents with heterozygous KLF1 mutation without co-inherited thalassaemia had normal to borderline mean corpuscular volume (MCV) and normal to slightly elevated Hb F. Fifteen previously reported cases of biallelic KLF1 mutations were identified from a literature review. All except one presented with severe haemolytic anaemia in the neonatal period. Our finding substantiates that compound heterozygous KLF1 mutations are associated with severe neonatal haemolytic anaemia and expands the haematologic phenotypic spectrum. In carriers, the previously suggested findings of low MCV, high Hb A2 and high Hb F are inconsistent; thus this necessitates molecular studies for the identification of carriers.

The rare hemoglobin variant Hb Mizuho: report of a Swiss family and literature review.

Hb Mizuho is a very rare unstable hemoglobin; here, we describe the clinical history of three Swiss family members with Hb Mizuho together with a systematic review of the previously six published cases. The clinical history of the adult woman we report here is unique since this is the first Hb Mizuho presenting with Moyamoya complications and the first case reported with long-term erythrocyte exchange. The literature review showed that Hb Mizuho was mainly reported as a de novo mutation, with the exception of children descended from known cases. All published patients with this unstable hemoglobin showed severe hemolytic anemia with the exception of one; all were regularly transfused. Patients with higher HbF levels might require fewer transfusions. All patients underwent splenectomy at a median age of 4 years and had variable clinical improvement; some achieved complete resolution of transfusion dependency after splenectomy. Iron overload in Hb Mizuho patients seems to be mainly attributed to transfusions and has less to do with ineffective erythropoiesis. Diagnosis might be challenging; a normal hemoglobin electrophoresis should not rule out the diagnosis of unstable hemoglobin in patients with otherwise unexplained hemolytic anemia. This series shows the enormous utility of using molecular techniques for diagnosis.

Coinherited Hemoglobin H/Constant Spring Disease and Heterozygous Hemoglobin Tak Causing Severe Hemolytic Anemia in a Thai Boy.

Hemoglobin (Hb) H/Constant Spring disease is a common nondeletional Hb H disease, typically causing a more severe phenotype than the deletional Hb H disease counterpart. Hb Tak, resulting from a dinucleotide insertion (+AC) at codon 146 of beta-globin gene, has an increased oxygen affinity and usually presents with polycythemia. We studied a case of a 4-year-old Thai boy with a severe, early-onset anemia. To our knowledge, he is the first reported patient with Hb H/Constant Spring disease and heterozygous Hb Tak. Trio-whole-exome sequencing does not identify other genetic variants that may contribute to the severity of anemia. The observation suggests that coinherited Hb H/Constant Spring and heterozygous Hb Tak lead to severe hemolytic anemia.