Severe Chronic Hemolytic Anemia Research Articles

Ex Vivo Treatment By Mitapivat, an Allosteric Pyruvate Kinase Activator, Reduced Oxidative Stress and Promoted Terminal Erythropoiesis in a Severe Hemolytic Anemia Patient Due to Krϋppel-like Factor 1 Mutations

Introduction: Human erythroid Krüppel-like Factor 1 (EKLF or KLF1), a hematopoietic-specific transcription factor encoded from KLF1 plays roles as the master regulator of erythroid development, including globin switching and expression, pyruvate kinase ( PKLR) function, heme synthesis, structural membrane, minor blood group, and cell cycle regulation. Most patients carrying KLF1 mutations present with severe chronic hemolytic anemia with low pyruvate kinase-red blood cell (PKR) activity and require regular transfusion. Previously, our group described the effects of ex vivo mitapivat (AG-348), an allosteric activator of PKR, treatment on the peripheral RBCs of two non-regular transfused chronic hemolytic anemia patients due to KLF1 mutations by increased PKR activity and ATP production of treated RBCs (Suksangpleng et al, Blood 2019; 134 (Supplement_1): 2220). We are currently studying the erythropoietic characteristics and investigating the effects of treatment with mitapivat to improve the severe anemia observed in these KLF1 patients. Objective: To evaluate the effects of ex vivo mitapivat treatment on the terminal erythropoiesis characteristics, including cell viability, oxidative stress, necrosis, and erythroid differentiation, of one severe hemolytic anemia patient due to KLF1 mutations. Methods: The 20-mL EDTA whole blood sample was collected from one regular transfused severe hemolytic anemia patient who was identified with KLF1 alleles mutations. The CD34 +-peripheral blood mononuclear cells (PBMCs) were separated by the specific anti-CD34 antibody-labelled magnetic beads and cultured by 3 phases of primary erythroid culture system, including phase 1: erythroblast transformation, phase 2: erythroblast expansion, and phase 3: erythroid differentiation. Cell viability was monitored in all 3 phases. The erythroid differentiation was monitored by stage-specific erythroid surface marker antibodies using flow cytometry. The ex vivo treatment was performed in phase 3 by incubating the cultured erythroid cells with the completed tissue cultured medium containing 40 nM mitapivat (Agios Pharmaceuticals, Inc.). Intracellular reactive oxygen species (ROS) were measured by fluorescent detection and necrotic cells were stained with Annexin V/PI and numbered by flow cytometry. Results: One index patient with regular transfused chronic hemolytic anemia showed low level of Hb (< 11 g/dL), high reticulocyte count (> 2%), high Hb F level (> 2%), and no PKLR mutation identified. The allele genotypes of KLF1 were identified as compound heterozygous mutation including unusual amino acid (Q58X) and missense mutation (A298P) on allele 1 and 2, respectively (Table 1). Proerythroblasts were transformed from the separated CD34 +-PBMCs in the Phase 1 culture period (Day 1 - Day 6) and highly expanded in Phase 2 (Day 7 - Day 13). After being induced to erythroid differentiation in Phase 3, the viability of erythroblasts gradually decreased while increased intracellular ROS was found. These phenomena corresponded to the increased numbers of necrotic cells during erythroid stage differentiation. Interestingly, the ex vivo treatment with mitapivat decreased intracellular ROS and necrotic cell numbers of the stage-differentiated erythroblasts, with more viable cell counts compared with the untreated condition in terminal erythropoiesis (Figure 1A, 1B, 1C). Moreover, the ex vivo treatment with mitapivat supported the effective stage differentiation shown by higher FITC- and lower PE-mean fluorescent intensity (MFI) that represented two stage-specific erythroid markers, including CD235a (glycophorin A) and CD71 (transferrin receptor), respectively, than those of untreated differentiated erythroblasts (Figure 1D). Conclusion: This study showed the effects of ex vivo treatment with mitapivat which could reduce oxidative stress, promote cell viability and stage differentiation in terminal erythropoiesis of one regular transfused hemolytic anemia patient due to KLF1 mutations. Mitapivat is being studied in clinical trials in PK deficiency (NCT03853798, NCT05144256, and NCT05175105), thalassemia (NCT04770753 and NCT04770779) and sickle cell disease (NCT05031780). Further evaluation of mitapivat in patients with severe hemolytic anemia due to KLF1 mutations is warranted as a potential therapeutic option for patients with KLF1 disease.

Beneficial effects of endurance exercise training on skeletal muscle microvasculature in sickle cell disease patients

AbstractSickle cell disease (SCD) is a genetic hemoglobinopathy leading to 2 major clinical manifestations: severe chronic hemolytic anemia and iterative vaso-occlusive crises. SCD is also accompanied by profound muscle microvascular remodeling. The beneficial effects of endurance training on microvasculature are widely known. The aim of this study was to evaluate the effects of an endurance training program on microvasculature of skeletal muscle in SCD patients. A biopsy of the vastus lateralis muscle and submaximal incremental exercise was performed before and after the training period. Of the 40 randomized SCD patients, complete data sets from 32 patients were obtained. The training group (n = 15) followed a personalized moderate-intensity endurance training program, while the nontraining (n = 17) group maintained a normal lifestyle. Training consisted of three 40-minute cycle ergometer exercise sessions per week for 8 weeks. Histological analysis highlighted microvascular benefits in the training SCD patients compared with nontraining patients, including increases in capillary density (P = .003), number of capillaries around a fiber (P = .015), and functional exchange surface (P < .0001). Conversely, no significant between-group difference was found in the morphology of capillaries. Indexes of physical ability also improved in the training patients. The moderate-intensity endurance exercise training program improved the muscle capillary network and partly reversed the microvascular defects commonly observed in skeletal muscle of SCD patients. This trial was registered at www.clinicaltrials.gov as #NCT02571088.

PS1575 USE OF THE NEW DEFERASIROX FORMULATION IN PEDIATRIC THALASSEMIA PATIENTS: ONE YEAR RESULTS

Background:Thalassemia is the most common and severe chronic hemolytic anemia in Greece, the National Registry recently reporting more than 2000 transfusion‐dependent patients. As a combined result of disease pathophysiology and transfusion treatment, thalassemic patients present with iron overload from an early age. Deferasirox (DFX), the newest of chelators used to excrete iron, was until recently administered in the form of dispersible tablets. However, the same active substance was licensed as film coated tablets (FCT) in an effort to overcome issues related to drug intolerance. Because of the same active substance being used, no clinical trials in iron overloaded patients were required from regulatory authorities.Aims:Aim of the present study was to evaluate safety and efficacy of the new DFX formulation in pediatric patients with transfusion‐dependent thalassemic syndromes.Methods:Pediatric patients with transfusion‐dependent thalassemia, followed at a single pediatric center, were enrolled. Patients were prospectively evaluated as to hepatic function, glomerular and tubular renal function, and liver and cardiac iron overload over a twelve‐month period following initiation of DFX FCT. Drug related adverse events were recorded. Statistical analysis was performed by SPSS 23, Pearson Correlation and Wilcoxon test.Results:Out of 19 patients enrolled 12 (63%) were males. Mean patient age was 13.8 years (7–18 years). Prior to DFX FCT administration 14/19 patients (73%) were on the previous DFX formulation (dispersible tablet, DT) and 5/19 patients on a different iron chelator. At the end of study, mean ferritin values showed no statistically significant change. With regards to renal function, 18/19 patients (94.7%) presented with a reduction in glomerular filtration rate (GFR) at some time point during follow up, however, in no case was reduction found to be more than &gt; 33% compared to baseline. Transient increase of spot urinary protein: creatinine ratio was reported in 4/19 patients (21%), and of spot urinary calcium: creatinine ratio in 18/19 patients (94.7%) – in no case increase reaching statistical significance. Mild, transient elevation of alanine transaminase (ALT) and of alkaline phosphatase was reported in 3/19 patients (15.7%) and of aspartate transaminase (AST) in 2 patients (10.5%). No statistically significant correlation was found between DFX FCT dose and laboratory parameters studied, i.e ferritin, GFR, protein: creatinine and calcium: creatinine ratio, AST, ALT or ALP. With regards to cardiac and liver magnetic resonance imaging (MRI), assessment at the beginning and end of study was performed in 8 out of 19 patients (42%). All patients presented with a reduction in liver iron, which reached the limit of statistical significance, and a statistically significant reduction in cardiac iron. Reported drug‐related adverse events were mild gastrointestinal symptoms in 3/19 patients (15.8%) and skin rash in 1 patient (5.2%).Summary/Conclusion:Study results demonstrated that the new DFX formulation is effective in reducing iron overload in thalassemic children, while maintaining a satisfactory safety profile. Improvements in formulation's mode of administration can increase treatment adherence and minimize iron toxicity. That, in combination with documented safety and efficacy, may improve quality of life for patients with transfusion‐dependent thalassemia.

PB1973 CONTRIBUTION OF NGS IN THE DIAGNOSIS OF HEREDITARY SPHEROCYTOSIS: CASE REPORT

Background:Hereditary spherocytosis (HS) is the most frequent cause of non‐immune haemolytic anaemia (HA) among individuals of Northern European ancestry; its estimated prevalence is 1:2000. HS results from various molecular defects leading to decreased production or abnormality of one or more red blood cell (RBC) membrane proteins i.e., ankyrin, protein 4.2, band 3 and spectrin. The majority of HS cases (75%) are inherited in an autosomal dominant manner. Especially because of the spherical shape, RBCs are trapped in the spleen and have a shortened life span. Among other factors, according to the importance of the deficiency or protein abnormality it conducts to a clinical expression that may range from asymptomatic to severe chronic HA. Most often a phenotypic diagnosis can be made but next‐generation gene sequencing (NGS) has a place in difficult cases i.e. unsolved cases of HA, transfusion prior to diagnosis and discrepancy between clinical phenotype and diagnosis.Aims:We report here the case of a 18‐year‐old patient with a suspected HS, prominent splenomegaly and unknown personal or family history.Methods:Case report:The patient complained of fatigue for several months and grade 2 dyspnea. In the past, he repeatedly presented with biliary colic pain. He apparently had no personal or family medical history. Two years ago, he nevertheless had jaundice secondary to haemolytic anaemia. The evolution was spontaneously favourable and no further investigation was undertaken. He has a twin brother and a 20‐year‐old sister, both in good health. The clinical examination found a prominent splenomegaly. Haemoglobinopathy and most frequent RBC enzyme deficiencies were excluded. Given the high clinical suspicion of HS, his parents and siblings (although asymptomatic) were screened for HS.Results:The main biological data are reported in the Table 1.Molecular genetic analyses were performed: NGS targeted sequencing panel, containing panel 40 genes associated to haemolytic anemia, and confirmation by Sanger identified a nonsense mutation on the cytoplasmic domain of Band3. The mutation is novel and drastic; it causes a stop codon at 75 residues. The mutation is transmitted by the mother. NGS targeted sequencing analysis did not demonstrate any other abnormalities that could explain the phenotypic variability observed in the propositus and his mother.Given the profound anaemia associated to the voluminous spleen (main diameter of 25 cm by CT‐scan evaluation) and given the presence of symptomatic gallstones, the patient underwent a splenectomy and a cholecystectomy. After the procedure, he recovered from fatigue and the haemoglobin level returned to normal with no signs of residual haemolysis.Summary/Conclusion:This case reports a HS related to a new nonsense mutation in SLC4A1. The transmission is autosomal dominant but the penetrance is heterogeneous within the same family.image

Coinheritance of High Oxygen Affinity Hb Helsinki [HBB: c.248A>T; β82(EF6)Lys→Met] with Hb H Disease

Hb Helsinki [HBB: c.248A>T; β82(EF6)Lys→Met] is a high oxygen affinity hemoglobin (Hb) causing polycythemia, whereas Hb H (β4) disease causes mild to severe chronic hemolytic anemia. The clinical characteristics, gel electrophoresis, capillary electrophoresis (CE) and molecular genotyping of a case of Hb Helsinki coinherited with Hb H disease in an ethnic Malay is described, illustrating the interaction between the β-globin variant and coinheritance of three α gene deletions. The proband was asymptomatic, exhibited microcytosis and a normal with Hb value.

Molecular characterization of six new cases of red blood cell hexokinase deficiency yields four novel mutations in HK1

Hexokinase (HK) is a key enzyme of glycolysis, the only metabolic pathway able to provide the red blood cell with ATP. HK deficiency is a very rare hereditary disorder with severe chronic nonspherocytic hemolytic anemia (HNSHA) as a major clinical feature. To date, only 24 patients with HK deficiency have been identified. Here, we report the molecular analysis of six new cases of HK deficiency. A total of six different mutations were detected in HK1, four of them described here for the first time: c.2599C>T p.(His867Tyr), c.1799C>T p.(Thr600Met), c.873-2A>G and c.493-1G>A. The pathogenic nature of the identified missense mutations was confirmed by biochemical and 3-dimensional structural analysis. The effects of the novel splice site mutation c.873-2A>G were studied at the level of pre-mRNA processing, and confirmed at the protein level. All together, these results provide a better insight into the pathogenesis of this rare red cell disorder, and contribute to a better understanding of the genotype-phenotype correlation in HK deficiency.

Alu-Element Insertion in Pklr Gene As a Novel Cause of Severe Hereditary Nonspherocytic Hemolytic Anemia

Red blood cell pyruvate kinase deficiency (PKD) is the most frequent enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Its incidence has been estimated to be 1 in 20,000, especially in regions where consanguinity is common. It is inherited in autosomal recessive manner occurring as result of homozygous or compound heterozygous mutations affecting both alleles in PKLR gene. Over 200 mutations have been described in patients with PKD causing various severity of hemolysis. Most cases are due to missense mutations, but small deletions, insertions, splice site alterations, frame shifts, disruption of erythroid specific promoters, and nonsense mutations have been reported.Alu elements are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. These mobile elements have contributed significantly to evolution and human diversity by causing gene rearrangements. Alu insertions particularly have been associated with a number of human diseases either by disrupting a coding region or a splice signal. In this abstract, we report an Alu-element insertion in the coding region of PKLR gene as a novel cause of PKD causing severe hereditary nonspherocytic hemolytic anemia.The proband was a 16-month-old middle eastern male born to consanguineous parents. He was diagnosed with severe chronic hemolytic anemia and neonatal jaundice requiring regular blood transfusion since birth. There were no known family members with anemia requiring transfusions. The patient's RBC phenotypic analysis was challenging since most of the circulating RBCs were donor cells. Using a Next-Generation sequencing panel for 27 hemolytic anemia associated genes, we identified and characterized a novel homozygous insertional mutation in exon 6 of PKLR gene along with heterozygosity for ANK1 c.1404+15C>T variant. The mutation in exon 6 was identified as Alu element insertion. The inserted Alu element (~370 bp in size), belonging to the youngest Yb8 subfamily, disrupts the reading frame at isoleucine 314 in exon 6 of PKLR gene, leading to a premature stop codon within the inserted sequence. Quantitative reverse transcription-PCR (qRT-PCR) in the patient's reticulocyte RNA using primers for the sequence before the Alu insertion revealed a transcript present but decreased by 80% compared to the average of controls' reticulocyte RNA. This truncated transcript even if translated is expected to cause severe enzyme deficiency.Both parents had a normal hemoglobin level with mild reticulocytosis. The mother had a PK enzyme activity of 4.5 units/g Hb (normal range 6.7-14.3 units/g Hb) indicating that she is heterozygous for PKD. Paternal PK activity level was not determined due to insufficient sample. Carrier testing for mutation in PKLR gene in both parents is currently in process at the time of this submission.The variant in ANK1 gene was predicted to be damaging by affecting the splice site in ankyrin gene. However the father was also heterozygous for this variant and his ektacytometry was normal suggesting a benign nature of ANK1 c.1404+15C>T.This case represents, to our knowledge, the first report of a pathogenic PKLR mutation due to an Alu element insertion and demonstrates a novel mechanism causing severe hereditary nonspherocytic hemolytic anemia. This report also illustrates the challenge of diagnosing congenital hemolytic anemias in chronically transfused patients. The patients often present with a complex clinical picture and ambiguous laboratory findings and may have several potentially damaging genetics variants identified by next generation sequencing. A critical evaluation of the clinical symptoms, laboratory findings and genetic data from other family members and bioinformatics analysis are required to clarify the contribution of these variants and to arrive at an accurate diagnosis, which will guide the institution of targeted interventions and appropriate genetic counseling. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hb Southampton [B106(G8)Leu→PRO, CTG→CCG] in a Uruguayan woman

Hemoglobin Southampton (also known as hemoglobin Casper) is a rare hemoglobin structural variant resulting from a substitution of a leucine residue for proline at codon beta106 [beta106(G8)Leu→Pro, CTG→CCG]. It is very unstable and associated with severe hemolytic anemia. We detected this mutation in a 37-year-old Uruguayan woman with a history of severe chronic hemolytic anemia since her childhood. According to our knowledge this is the first time that this variant has been found in the Uruguayan population.

Novel Haemoglobin Variant [β42 Phe → Cys]: Hb Little Venice

Abstract 1013We report a novel haemoglobin (Hb) variant at position [β42 Phe → Cys] found in a Caucasian male infant with severe chronic haemolytic anaemia. He presented at 5 weeks of age with fever and vomiting and was found to have apparent oxygen saturations of 82% in air by pulse oximetry and Hb 6.7 g/dl. Absolute reticulocyte count 353 × 109/l, LDH 872 u/L, bilirubin 23 mmol/L, direct antiglobulin test (DAT) negative. Blood film showed marked anisopoikilocytosis, keratocytes, irregularly contracted cells, basophilic stippling, nucleated red blood cells, and increased polychromasia consistent with haemolysis and oxidative damage. Echocardiogram, electrocardiogram and chest radiograph were normal. Arterial blood gas revealed normal PaO2. Haptoglobins were absent. Required regular blood transfusions until 6 months of age when he achieved a 3 months period without transfusion and could be further investigated. Hb electrophoresis showed a normal pattern. No evidence of glucose 6 phosphate deficiency, pyruvate kinase or 5’ pyrimindine nucleotide deficiency. Heinz preparation was positive, with positive heat stability and isopropanol test suggesting the presence of variant haemoglobin. Snap frozen mass spectrometry was performed which demonstrated a low abundance (4%) of a variant haemoglobin. This was further characterised by DNA sequencing of the beta-globin gene which revealed the presence of a novel heterozygous mutation of the beta chain. A single amino acid substitution at the 42 amnio acid of the beta chain was identified resulting in Phenylalanine being replaced by cysteine [β42 Phe → Cys]. Both parents were studied for the mutation and were found to be negative. At 2 years of age the level of variant haemoglobin was 12% and oxygen dissociation curve showed oxygen P50 of 29.5 mmHg (reference range: 29.5 – 32.0) indicating no evidence of altered oxygen affinity. He continues with severe chronic haemolytic anaemia exacerbated by infections and requires a monthly regular transfusion regime.Phenylalanine in position 42 (the first position of the region between the C and D helices - CD1) of the β chain participates in the contacts with haem. It is a critical amino acid in the haem pocket, maintaining solubility and stability. Substitution of this phenylalanine by cysteine removes a contact with haem leaving a gap at the surface of the haem pocket and results in instability. There are three other Hb variants resulting from substitutions in position 42: Hb Hammersmith, Hb Louisville and Hb Sendagi, substitutions for serine, leucine and valine respectively. Hb Hammersmith being more severe and leading to transfusion dependence in some of the cases, whilst the others are mild to moderate, only requiring transfusions for infective episodes. There are over 800 variant Hb are described with the majority not being clinically significant. Of those which are significant, greater then 95% are due to single amino acid substitutions (as in our case) which changes the structure of the Hb. The clinical abnormalities are attributable to the changes in Hb solubility, stability and capacity to carry and deliver oxygen. Disclosures:No relevant conflicts of interest to declare.

A case study of a child with chronic hemolytic anemia due to a Donath–Landsteiner positive, IgM anti‐I autoantibody

In children, paroxysmal cold hemoglobinuria (PCH) is generally considered an acute self-limited autoimmune hemolytic anemia caused by an IgG biphasic auto-anti-P antibody identified by the Donath-Landsteiner (D-L) test. We report a case of a 5-year-old female with a chronic hemolytic anemia. The etiology of the hemolysis appears to be an unusual D-L positive, IgM antibody with specificity for the I antigen. The clinical course is described and a discussion of PCH and the D-L antibody is presented. We also discuss intravenous immunoglobulin infusions as a therapy for children with this form of severe chronic autoimmune hemolytic anemia.

Hemolytic anemia due to hemoglobin Evans in an Argentinean family

Unstable hemoglobins are structural variants of the hemoglobin molecule, mostly originated by single amino-acid replacement in some globin chains. These changes affect molecule stability, leading to loss of solubility, precipitation, and cellular lysis. Patients carrying these unstable hemoglobins may present mild to severe chronic hemolytic anemia. Hemoglobin Evans is an unstable variant originated by replacement of valine with methionine at position 62 of the α-globin chain. We have identified this variant in a girl with an acute hemolytic crisis associated to pharyngitis, as well as in two of her family members. This is the third case of hemolytic anemia due to hemoglobin Evans reported in the literature.

Severe Thalassemia Intermedia Resulting From Coinheritance of IVSI-110 G>A Beta Gene Mutation and Duplication of Alpha-Globin Gene Cluster in Homozygosis.

Abstract 2011Poster Board I-1033 IntroductionThe clinical severity of thalassemia intermedia depends on the degree of a/non-a-chains iimbalance. Among the molecular mechanisms responsible for thalassemia intermedia is the coinheritance of excessive a-globin gene production with a defective beta-globin gene. Materials and Methods:we describe an Italian family where thalassemia intermedia apparently segregates as a dominant form but it tourned out to be due to the coinheritance of a beta-globin mutation and a duplication of the alpha-globin gene cluster. The father (aged 51yrs) showed a well tolerated severe chronic hemolytic anemia (Hb 7.5-8.5 g/dL) not transfusion dependent, jaundice, splenomegaly and leg ulcers:The mother (aged 46yrs) has a completely normal hematological and hemoglobin pattern. Two sons (19 and 14 yrs) showed more severe clinical manifestations than the father. They underwent splenectomy at 12 and 13 years respectively without any benefit and afterwards they become transfusion dependent. Results:The hemoglobin analysis revealed that the father and the sons were heterozygotes for the beta mutation IVSI-110 G>A. MLPA analysis of the alpha-globin gene cluster disclosed a full duplication of the alpha-globin locus, spanning a 175 kb from the telomere to the 3'HVR downstream of the alpha-globin gene and including the upstream regulatory element HS-40. This rearrangement increases the number of the active alpha-globin genes in cis from 2 to 4.Surprisengly it was found in heterozygosis in both parents and in homozygosis in both sons. The hematological and molecular data of the family are reported in the table.In the father the 6 alpha-globin genes led to increased synthesis of alpha-chains; the coinheritance with a beta-thalassemia mutation causes a moderate/severe thalassemia intermedia phenotype. The presence of 8 alpha-globin genes in the sons raises further the degree of globin-chains imbalance and exacerbates the clinical phenotype. It is important to note that splenectomy worsened the clinical course.in the 2 homozygotes for the alpha duplication. Conclusions:Based also on previous experience we suggest that splenectomy in patients with a real excess of alphaa chain production is unconvenient since a large amount of circulating red cells with precipitated alpha chains may be responsible for increased hemolysis as well as increased risk of thrombosis This family moreover raises concerns regarding genetic counselling, suggesting that whenever one of the partner is affected by TI it is advisable a complete molecular screening of the couple in order to exclude any possible alpha gene defects interactionAge/genderHb g/dLRBC X1012/LMCV fLMCH pgHbA2 %HbF %Retics %SpleenTransfusionChelationa genesβ genesMother46/F12.84.8781.726.32.50.91.29normalnonoaa,aa/aaβN/βNFather51/M8.54.4764.019.05.03.72.3520 cmnonoaa,aa/aaβT/βNSon19/M6.03.8060.016.54.37.57.57splenectomyyesyesaa,aa/aa,aaβT/βNSon14/F5.93.6959.916.04.07.710.61splenectomyyesyesaa,aa/aa,aaβT/βN Disclosures:No relevant conflicts of interest to declare.

Three-Dimensional Transesophageal Echocardiogram Provides Real-Time Guidance During Percutaneous Paravalvular Mitral Repair

A 65-year-old woman with severe paravalvular mitral regurgitation and chronic hemolytic anemia was referred for surgical consultation. Her medical history included rheumatic heart disease with mitral valve replacement in 1977, aortic valve replacement in 1988, and repeat mechanical mitral valve replacement in 2002. Predominant symptoms were New York Heart Association function class III dyspnea and fatigue. The patient was considered at high risk for surgical complication because of the 3 previous valve surgeries and significant pulmonary hypertension (systolic pulmonary pressure, 70 mm Hg). The Figure demonstrates repair of the significant paravalvular …

A novel G6PD mutation leading to chronic hemolytic anemia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an important cause of hemolytic anemia worldwide. Severely affected patients have chronic hemolysis with exacerbations following oxidative stress. Mutations causing severe chronic non-spherocytic hemolytic anemia (CNSHA) commonly cluster in Exon 10, a region important for protein dimerization. An African-American male presented at age 2 weeks with pallor and jaundice, and was found to have hemolytic anemia with G6PD deficiency. His severe clinical course was inconsistent with the expected G6PD A(-) variant. DNA sequencing revealed two common mutations (A(-)) and a third novel Exon 10 mutation. This inherited haplotype represents a novel triple G6PD coding mutation causing chronic hemolysis.

Coexistence of Congenital Red Cell Pyruvate Kinase Deficiency and Hereditary Stomatocytosis.

We describe a case of chronic hemolytic anemia due to the co-presence of pyruvate kinase (PK) deficiency and Hereditary Stomatocytosis (HSto). The propositus was a 30 years old adopted male with no known family history; he had severe neonatal jaundice requiring exchange transfusion, followed by a life-long history of moderate to severe chronic hemolytic anemia (Hb 7–10 g/dL), with jaundice and splenomegaly. At the age of 6 months hemoglobin screening was made and a beta trait was found. At the age of 20 splenectomy and cholecystectomy were performed. Surgery resulted in an increase of 1.5 g/dL in haemoglobin, and in a conspicuous rise of reticulocytes (from 125×109/L to 562×109/L). Two thrombotic events occurred thereafter, the former 6 days after surgery, and the latter two years later, during a toxoplasmosis infection. At the time of the study Hb was 10.8 g/dL, MCV 82.2 fL, reticulocytes 562×109/L, unconjugated bilirubin 2.19 mg/dL, LDH 335 U/L, haptoglobin <20 mg/dL, serum ferritin 342 ng/mL and transferrin saturation 71%. The peripheral blood smear examination showed the presence of echinocytes (13%), stomatocytes (11%), acantocytes (10%), schistocytes (7%), elliptocytes (6%), spherocytes (4%), target cells (4%) and a few erythroblasts. Erythrocyte osmotic fragility was decreased; screening test for unstable hemoglobins and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of red cell membrane gave normal results. The study of the most important red cell enzymes revealed reduced PK activity (6.0 UI/gHb, normal range 11.1–15.59 UI/gHb) and thermal stability (43%, normal range 57–100%). Direct sequencing of PK-LR gene showed a compound heterozygosity for mutation 1456T (Arg486Trp) and the new variant −73g>c. Mutation −73g>c occurs in the most proximal of the four GATA motifs in the R-type promoter region and possibly result in a decrease of mRNA synthesis, as already reported for the variant −72a>g (Manco et al, 2000). Molecular analysis of HFE gene showed heterozygosity for H63D mutation. The history of post splenectomy thrombosis and the presence of stomatocytes in peripheral blood smear prompted us to investigate for the coexistence of hereditary stomatocytosis. The determination of plasma potassium and sodium concentration revealed an increase in intracellular sodium (16.3 mmol/LRBC, reference range 5.0–12.0) and a decrease in intracellular potassium (74.73 mmol/LRBC, reference range 90–103), suggestive for a diagnosis of dehydrated HSto, or hereditary xerocytosis. This defect likely accounts for the thrombophilic state in this case, since HSto is known to be associated with hypercoagulability, particulary after splenectomy.

What is new in iron overload?

Children with severe chronic hemolytic anemia or congenital erythroblastopenia are transfusion dependent. Long-term transfusion therapy prolongs life but results in a toxic accumulation of iron in the organs. The human body cannot actively eliminate excess iron. Therefore, the use of a chelating agent is required to promote excretion of iron. So far, iron chelation has been done by subcutaneous infusion of deferoxamine given over 10 h, 5–6 days per week. Compliance is poor and chelation often insufficient. Ferritin measurements and sometimes liver biopsies are used to evaluate the iron burden in the body. At the present time, new iron chelators that can be given orally are available. Furthermore, magnetic resonance imaging (MRI) assessment of tissue iron is a noninvasive and highly reproducible method, which is able to quantitate organ iron burden. In conclusion, iron overload can be measured more accurately with noninvasive methods such as MRI. Deferasirox is a once-daily oral therapy for treating transfusional iron overload, which improves patient compliance and quality of life.

Pulmonary Hypertension in a Case of Hb-Mainz Hemolytic Anemia

The development of pulmonary arterial hypertension (PAH) is the leading cause of mortality in patients with thalassemia and sickle cell anemia and was reported to occur in hemolytic anemias such as hereditary stomatocytosis, and paroxysmal nocturnal hemoglobinuria. Here, we report for the first time on the development of PAH in a patient with Hb-Mainz hemolytic anemia. Hb-Mainz is an unstable hemoglobin variant resulting from mutations at codon 98 of the beta chain gene (Val>Glu) characterized by severe chronic hemolytic anemia. The development of PAH in this patient further supports the contention that there is a clinical syndrome of hemolysis-associated development of PAH.

Two new phosphoglycerate kinase mutations associated with chronic haemolytic anaemia and neurological dysfunction in two patients from Spain

We report two previously undescribed mutations of the phosphoglycerate kinase gene (PGK1) leading to enzyme deficiency. In both cases, the patients were of Spanish origin and they exhibited a severe life-long chronic haemolytic anaemia associated with progressive neurological impairment. Sequence analysis of the first patient's entire PGK1 gene found a novel missense mutation (140T > A). This mutation caused an amino acid change of Ile to Asn at 46th position from the NH(2)-terminal serine residue (Ile46Asn), which has been called PGK-Barcelona based on the place of origin of the patient. In the second patient, a G to A transversion was discovered at nucleotide 958 (958G > A). This caused a Ser319Asn amino acid substitution. Since this mutation had not been previously described, the provisional name of PGK-Murcia was given to this deficient enzyme. The crystal structure of porcine PGK was used as a molecular model to investigate how these mutations may affect enzyme structure and function. In both cases, the mutations did not modify any of the PGK binding sites for ATP or 3PG, so their consequence is related to a loss of enzyme stability rather than a decrease of enzyme catalytic function.

Pregnancy outcome of hemoglobin H disease

Hemoglobin H (Hb H) disease is one of the major thalassemic diseases and it predominantly affects Southeast Asia the Middle East Greece and Cyprus. The loss of 3 a-genes results in a severe imbalance in the production of globin chains and the globins produced in excess form Hb H an unstable hemoglobin with a high oxygen affinity. The clinical manifestations of Hb H disease vary from no clinical symptoms to severe chronic hemolytic anemia. The physiologic changes during pregnancy increase the severity of anemia in the mother which could affect pregnancy outcome as well as clinical symptoms. There has been no large-series report on this disease in pregnant women. (authors)

G6PD deficiency in Vataliya prajapati community settled in Surat

A Study on Vataliya Prajapati was published earlier but heterozygous females were not identified. To compare incidence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in random and unrelated subjects, present and previous study and as per their original habitat. Incidence of heterozygous deficiency and clinical implication of deficiency was also determined. Camps were organized in Katargaon and Amroli regions. Blood specimens, with relevant demographic information, were collected from those who attended the camp. A total of 1644 random blood samples were collected from 404 families participating in the camps. Nitroblue tetrazolium dye test was used for G6PD deficiency screening and quantitative assay for measurement of G6PD enzyme activity. Chi2 test was used to evaluate significance and mean values were compared by the Student's ''t'' test. Incidence of G6PD deficiency was found as 22% among all the random samples tested. However, the G6PD deficiency among unrelated members was 27.9% in males and 12.4% (P< 0.001,df 1). The 13.9% of the females with heterozygous G6PD deficient status, together with the homozygous deficient phenotype makes the incidence comparable with males. Incidence of deficiency was comparable with previous study, in Katargam and Amroli and in Amerli and Bhavganar districts. Deficient subjects had mild anemia and hemolytic crisis rarely occurred. Vataliya Prajapatis have high incidence of G6PD deficiency without severe chronic hemolytic anemia. However before prescribing medicines physician should know the G6PD status of a Vataliya Prajapati patient.