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Hye Lim Jung - One of the best experts on this subject based on the ideXlab platform.
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Epidemiological Study of Hereditary Hemolytic Anemia in the Korean Pediatric Population during 1997–2016: a Nationwide Retrospective Cohort Study
Journal of Korean medical science, 2020Co-Authors: Ye Jee Shim, Hye Lim Jung, Hee Young Shin, Young Tak Lim, Jeong-ok Hah, Hyoung Jin Kang, Jung Yoon Choi, Jae Min Lee, Eu Jeen Yang, Hee Jo BaekAbstract:BACKGROUND Hereditary Hemolytic Anemia (HHA) is a rare disease characterized by premature red blood cell (RBC) destruction due to intrinsic RBC defects. The RBC Disorder Working Party of the Korean Society of Hematology established and updated the standard operating procedure for making an accurate diagnosis of HHA since 2007. The aim of this study was to investigate a nationwide epidemiology of Korean HHA. METHODS We collected the data of a newly diagnosed pediatric HHA cohort (2007-2016) and compared this cohort's characteristics with those of a previously surveyed pediatric HHA cohort (1997-2006) in Korea. Each participant's information was retrospectively collected by a questionnaire survey. RESULTS A total of 369 children with HHA from 38 hospitals distributed in 16 of 17 districts of Korea were investigated. RBC membranopathies, hemoglobinopathies, RBC enzymopathies, and unknown etiologies accounted for 263 (71.3%), 59 (16.0%), 23 (6.2%), and 24 (6.5%) of the cases, respectively. Compared to the cohort from the previous decade, the proportions of hemoglobinopathies and RBC enzymopathies significantly increased (P < 0.001 and P = 0.008, respectively). Twenty-three of the 59 hemoglobinopathy patients had immigrant mothers, mostly from South-East Asia. CONCLUSION In Korea, thalassemia traits have increased over the past 10 years, reflecting both increased awareness of this disease and increased international marriages. The enhanced recognition of RBC enzymopathies is due to advances in diagnostic technique; however, 6.5% of HHA patients still do not have a clear diagnosis. It is necessary to improve accessibility of diagnosing HHA.
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epidemiological study of Hereditary Hemolytic Anemia in the korean pediatric population during 1997 2016 a nationwide retrospective cohort study
Journal of Korean Medical Science, 2020Co-Authors: Ye Jee Shim, Hye Lim Jung, Hee Young Shin, Young Tak Lim, Jeong-ok Hah, Hyoung Jin Kang, Jung Yoon Choi, Jae Min Lee, Eu Jeen Yang, Hee Jo BaekAbstract:BACKGROUND Hereditary Hemolytic Anemia (HHA) is a rare disease characterized by premature red blood cell (RBC) destruction due to intrinsic RBC defects. The RBC Disorder Working Party of the Korean Society of Hematology established and updated the standard operating procedure for making an accurate diagnosis of HHA since 2007. The aim of this study was to investigate a nationwide epidemiology of Korean HHA. METHODS We collected the data of a newly diagnosed pediatric HHA cohort (2007-2016) and compared this cohort's characteristics with those of a previously surveyed pediatric HHA cohort (1997-2006) in Korea. Each participant's information was retrospectively collected by a questionnaire survey. RESULTS A total of 369 children with HHA from 38 hospitals distributed in 16 of 17 districts of Korea were investigated. RBC membranopathies, hemoglobinopathies, RBC enzymopathies, and unknown etiologies accounted for 263 (71.3%), 59 (16.0%), 23 (6.2%), and 24 (6.5%) of the cases, respectively. Compared to the cohort from the previous decade, the proportions of hemoglobinopathies and RBC enzymopathies significantly increased (P < 0.001 and P = 0.008, respectively). Twenty-three of the 59 hemoglobinopathy patients had immigrant mothers, mostly from South-East Asia. CONCLUSION In Korea, thalassemia traits have increased over the past 10 years, reflecting both increased awareness of this disease and increased international marriages. The enhanced recognition of RBC enzymopathies is due to advances in diagnostic technique; however, 6.5% of HHA patients still do not have a clear diagnosis. It is necessary to improve accessibility of diagnosing HHA.
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Hereditary Hemolytic Anemia in Korea from 2007 to 2011: A study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology.
Blood research, 2013Co-Authors: Eun Sil Park, Hye Lim Jung, Hee Jin Kim, Sung Sup Park, Soon Hwan Bae, Hee Young Shin, Sang Hoon Song, Kyung Nam Koh, Chuhl Joo Lyu, Young Tak LimAbstract:Background The number of patients diagnosed with Hereditary Hemolytic Anemia (HHA) has increased since the advent of novel diagnostic techniques that accurately identify this disorder. Here, we report data from a survey on the prevalence and characteristics of patients diagnosed with HHA in Korea from 2007 to 2011.
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A new paradigm in the diagnosis of Hereditary Hemolytic Anemia.
Blood research, 2013Co-Authors: Hye Lim JungAbstract:Hereditary Hemolytic Anemia (HHA) is a very rare disease entity characterized by premature red blood cell (RBC) destruction and Anemia due to intrinsic RBC defects. The 3 main etiologies causing HHA, in order of frequency, are RBC membrane disorders, hemoglobin disorders, and RBC enzyme disorders. The prevalence of HHA in Korea is very low, because Hereditary spherocytosis (HS) is less common in Asians than in Caucasians-with an incidence of 1 in 5000 births-and because Korea is not located in the thalassemia belt [1]. After the establishment of the HHA Working Party (WP) in the Korean Society of Hematology, 2 nationwide epidemiologic studies of HHA were conducted by the HHA WP: a 10-year retrospective survey carried out from 1997 to 2006 and a 5-year prospective survey carried out from 2007 to 2011 after the introduction of a standard operating procedure (SOP) for the diagnosis of HHA [2, 3]. According to the results of a recent survey performed from 2007 to 2011 in Korea, 198 (122 men and 74 women) patients were diagnosed with HHA. The median age of the patients was 32 months (range: 0-187 months), and there were 127 (64.8%) patients with RBC membranopathies, 39 (19.9%) with hemoglobinopathies, 26 (13.3%) with RBC enzymopathies, and 3 (1.5%) patients had HHA of unknown etiology [3]. Data comparison between this study and studies performed during 1997-2006 and 1981-1990 [1-3] revealed that the proportion of hemoglobinopathy and enzymopathy has been gradually increasing (Fig. 1). This finding is probably due to an improvement in the diagnostic techniques for HHA, especially that of globin gene sequencing and RBC membrane protein and enzyme analysis, and an increase in multiracial marriages especially with South East Asians. Fig. 1 Survey results of Hereditary Hemolytic Anemia incidence in Korea showing the increasing proportions of hemoglobinopathy and enzymopathy. Nationwide epidemiologic studies have highlighted the need for an update of the SOP with newly developed, more accurate diagnostic methods for HHA, as well as the nationwide standardization of the methods and reporting of laboratory tests. HS, the most common HHA characterized by abnormal RBC morphology and shortened RBC life span owing to extravascular hemolysis, is caused by a deficiency or dysfunction of membrane proteins associated with the RBC cytoskeleton: spectrin, ankyrin, band 3, protein 4.1, protein 4.2, and glycophorin. The genes encoding RBC membrane proteins and their respective chromosomal locations are known [4, 5]. The osmotic fragility (OF) test has been the most commonly used laboratory test for the screening of HS. However, the OF test has low sensitivity and specificity, and can give false negative results in 10-20% of HS cases, and false positive results in autoimmune Hemolytic Anemia. Furthermore, the OF test may be normal in cases of mild HS, in the presence of iron deficiency and obstructive jaundice, and in the recovery phase from an aplastic crisis [4]. In recently published guidelines for the diagnosis of HS, the eosin 5-maleimide (EMA) binding test and hypertonic cryohemolysis (HCH) test have been proposed as more reliable screening tests for HS. EMA interacts with the e-NH2 group of Lys-430 on band 3 protein of the RBC membrane, and the EMA binding test uses flow cytometry to determine the amount of fluorescence derived from the EMA-bound band 3 protein. EMA-labeled HS RBCs with or without band 3 deficiency produce 25-30% lower mean channel fluorescence than normal RBCs. The HCH test measures % cryohemolysis at 540 nm after transfer of RBCs from 37℃ to 0℃ for 10 minutes. The EMA binding test and HCH test have a higher predictive value in the diagnosis of HS because there have been no reports of positive results in cases of immune or non-membrane-associated Hemolytic Anemia [4-6]. In addition, the flow cytometric OF (FC OF) test was recently developed as an assay that can replace the classical OF test [7]. The HHA WP compared the sensitivity, specificity, and predictive values of 3 screening tests (EMA binding test, FC OF test, and HCH test) in the diagnosis of HS. The results showed that both the EMA binding test and FC OF test had high sensitivity, specificity, and predictive values for the diagnosis of HS, whereas the HCH test had low sensitivity, specificity, and predictive values, and failed to discriminate between patients with HS and those with iron deficiency Anemia. For the confirmatory diagnosis of RBC membranopathy, RBC membrane protein analysis is usually performed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method [4, 5]. However, the SDS-PAGE method requires multiple step reactions and manual manipulations, which are labor-intensive and time consuming (about 1 week), and carry a greater risk of error owing to their complexity. Moreover, the SDS-PAGE method may be inaccurate in band 3 deficiency, because it yields results in the form of ratios of protein to band 3. The HHA WP is introducing quadruple-time of flight liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a new method of RBC membrane protein analysis. LC-MS/MS is a very accurate method for measuring the mass of metabolites, drugs, proteins, lipids, and carbohydrates after ionizing the substrate. LC-MS/MS measures each protein separately, is not influenced by band 3 deficiency, and is known to be a simple, less time consuming (2-3 days), sensitive, and reproducible method for protein quantification. Among the 20 or more RBC enzyme deficiencies known to cause HHA, deficiencies involving 3 enzymes such as glucose-6-phosphate dehydrogenase, pyruvate kinase, and pyrimidine 5'-nucleotidase are relatively common [8]. The Mayo Clinic measures 10 RBC enzymes to diagnose enzymopathy, including phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, pyruvate kinase, glucose phosphate isomerase and hexokinase of the Embden-Meyerhof pathway; glucose-6-phosphate dehydrogenase of the hexose monophosphate shunt; adenylate kinase, pyrimidine 5'-nucleotidase and adenosine deaminase of the purine-pyrimidine pathway. For the confirmatory diagnosis of RBC enzymopathy, the RBC enzyme analysis method based on kinetic spectrophotometry has been used. Nevertheless, this method requires multiple step reactions and manual manipulations, which are labor intensive and time consuming, and carry a greater risk of error owing to their complexity. Therefore, the HHA WP is developing the multiplex RBC enzyme analysis using the ultra-performance liquid chromatography-tandem mass spectrometry method, which is expected to be a simple, rapid, sensitive, and reproducible quantification method for RBC enzymes [9]. Current HHA diagnostic guidelines recommend RBC membrane protein gene analysis when RBC protein analysis does not explain the clinical outcome and mode of inheritance in the patient with HS [4, 5]. Recently, the HHA WP started performing RBC membrane protein and enzyme gene analysis with the next generation sequencing method to define the common RBC membrane protein and enzyme gene abnormalities in Koreans. Thirteen genes encoding RBC membrane proteins (spectrin α and β, ankyrin, band 3, protein 4.1, protein 4.2, and glycophorins), and 15 genes encoding RBC enzymes (phosphofructokinase, triose phosphate isomerase, phosphoglycerate kinase, pyruvate kinase, glucose phosphate isomerase, hexokinase, phosphoglycerate kinase, acetyl cholinesterase, enolase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, adenylate kinase, pyrimidine 5'-nucleotidase, adenosine deaminase, glutathione reductase) will be the target of next generation sequencing. As the prevalence and etiologies of HHA are changing in Korea, nationwide epidemiologic studies will be carried out continuously with updated standardized SOP for HHA (Fig. 2). Fig. 2 Updated standard operating procedure for the diagnosis of Hereditary Hemolytic Anemia. Abbreviations: HHA, Hereditary Hemolytic Anemia; CBC, complete blood cell count; PBS, peripheral blood smear; TIBC, total iron binding capacity; LDH, lactate dehydrogenase; ...
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Hereditary Hemolytic Anemia in Korea From 1997 to 2011: A Study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology
Blood, 2012Co-Authors: Eun Sil Park, Hye Lim Jung, Sung Sup Park, Hee Soon Cho, Jeong-ok Hah, Hong Hoe Koo, Moon Kyu Kim, Yeo-kyeoung Kim, Young Dae Kim, Hee Jin KimAbstract:Abstract 5157 Background: With the development of diagnostic technique, an accurate diagnosis of Hereditary Hemolytic Anemia (HHA)- red blood cell (RBC) membranopathy, hemoglobinopathy, RBC enzymopahty – have been made. Therefore, we surveyed the prevalence and characteristics of patients diagnosed as HHA during recent five years in Korea. Methods: Through the use of questionnaires, information on the clinical and laboratory findings of HHA diagnosed from 2007 to 2011 in Korea was collected. The globin gene analysis (direct sequencing) and RBC enzyme analysis was performed at the representative laboratories. A total of 203 cases were collected in this study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology. Results: Patients number of RBC membranopathy, hemoglobinopathy, and RBC enzymopahty was 125, 47, and 31, respectively. Percentage of patients with dominant family history was 57% in patients with Hereditary spherocytosis (n=116) and dominant symptoms were Anemia, jaundice, splenomegaly and gallstones. Osmotic fragility test and flow cytometric method for detection of RBC membrane defect were performed about 60% of patients. RBC membrane protein analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on 59 patients. Of the 47 cases of hemoglobinopathies, 36 cases (77%) were β-thalassemia minor, 10 cases (21%) were α-thalassemia minor and one case (2%) was unstable Hb, Hb M-Saskatoon (beta 64 His-→Tyr). Median age at diagnosis was 7 years (range: 6 months–58 years). Eleven of 47 cases (23%) had family history of HHA. As all thalassemia patients were thalassemia minor, they presented with mild jaundice or pallor. Of the 31 patients were diagnosed as RBC membranopathy, pyruvate kinase deficiency was 3 cases and glucose-6 phosphate dehydrogenase deficiency was 2, and other various forms were reported. Conclusions: We could confirm that accurate diagnosis has been made in more patients using elegant diagnostic technique. However, more defined diagnostic approaches were needed in this rare disease and further systematic supporting systems for patients and their families were warranted in public health aspect. Disclosures: No relevant conflicts of interest to declare.
Shiro Miwa - One of the best experts on this subject based on the ideXlab platform.
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Red blood cell enzymes and their clinical application.
Advances in clinical chemistry, 1998Co-Authors: Hisaichi Fujii, Shiro MiwaAbstract:Publisher Summary The chapter discusses red blood cell enzymes and their clinical application. Structure and functions of major red blood cell enzymes are discussed in the chapter wherein hexokinase, glucose phosphate isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, diphosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, adenylate kinase, pyrimidine 5'-nucleotidase, adenosine deaminase are described. The chapter also discusses Hereditary Hemolytic Anemia associated with red blood cell enzyme deficiency in detail. The various defects in the Embden-Meyerhof pathway, hexose monophosphate pathway and glutathione metabolism and synthesis, and nucleotide metabolism are presented. Hereditary nonHemolytic blood disorders associated with red blood cell enzyme deficiency are discussed next in the chapter wherein diphosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, and NADH cytochrome b5 reductase deficiency are described. The chapter discusses Hereditary nonhematologic disorders that can be diagnosed by the determination of red blood cell enzyme activity. The various topics covered are enzyme deficiencies associated with immunological disorders; enzyme deficiencies in the metabolism of purine; prolidase deficiency; acatalasemia; galactosemia; porphyria; and carbonic anhydrase deficiency.
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Molecular analysis of glucose phosphate isomerase deficiency associated with Hereditary Hemolytic Anemia.
Blood, 1996Co-Authors: Hitoshi Kanno, Hisaichi Fujii, Akira Hirono, Yoji Ishida, Shoichi Ohga, Yasuhiko Fukumoto, Kenji Matsuzawa, Satoru Ogawa, Shiro MiwaAbstract:We report here two new cases of glucose phosphate isomerase (GPI) deficiency associated with Hemolytic Anemia and present the results of molecular analysis of the five Japanese GPI variants. A Japanese girl (GPI Fukuoka) had an episode of prolonged neonatal jaundice and at 3 years of age was admitted due to acute Hemolytic crisis occurring with upper respiratory tract infection. Red blood cell (RBC) GPI activity was decreased to 11.8% of normal and the reduced glutathione (GSH) level of RBCs was slightly decreased. A 54-year-old Japanese man (GPI Iwate) was hospitalized due to chronic active hepatitis, and compensated hemolysis was noted. RBC GPI activity of the proband was decreased to 18.8%, and the GSH content was about half of the normal mean value. Sequencing of the reticulocyte GPIcDNA showed homozygous missense mutations 1028CAG-->CGG (343Gln-->Arg), 14ACC-->A7C (5Thr-- >lle), 671ACG-->A7G (224Thr-->Met), and 1615GAC-->AAC (539Asp-->Asn) in GPI Narita, GPI Matsumoto, GPI Iwate, and GPI Fukuoka, respectively. We also identified GPI Kinki as a compound heterozygote of 1124ACA-- >AGA(375Thr-->Arg)/ 1615GAC-->AAC(539Asp-->Asn). Our findings, together with the previous results of other investigators, showed that the GPI gene mutations so far identified were heterogeneous, although most GPI variants had common biochemical characteristics such as heat instability and normal kinetics. Several amino acid substitutions were identified in the proximity of the catalytically important amino acid residues such as Ser/Asp 159/160, Asp341, and Lys518, which have been identified in the structural analysis of the pig GPI. The molecular characterization of human GPI variants, therefore, may provide new insights into the genotype-phenotype correlation of GPI deficiency as well as the structure-function relationship of this enzyme.
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Molecular basis of erythroenzymopathies associated with Hereditary Hemolytic Anemia: Tabulation of mutant enzymes
American journal of hematology, 1996Co-Authors: Shiro Miwa, Hisaichi FujiiAbstract:Molecular abnormalities of erythroenzymopathies associated with Hereditary Hemolytic Anemia have been determined by means of molecular biology. Pyruvate kinase (PK) deficiency is the most common and well-characterized enzyme deficiency in the glycolytic pathway, and it causes Hereditary Hemolytic Anemia. To date, 47 gene mutations have been identified. We identified one base deletion, one splicing mutation, and six distinct missense mutations in 12 unrelated families with a homozygous PK deficiency. Mutations located near the substrate or fructose-1,6-diphosphate binding site may change the conformation of the active site, resulting in a drastic loss of activity and severe clinical symptoms. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common metabolic disorder, and it is associated with chronic Hemolytic Anemia and/or drug-or infection-induced acute Hemolytic attack. An estimated 400 million people are affected worldwide. The mutations responsible for about 78 variants have been determined. Some have polymorphic frequencies in different populations. Most variants are produced by one or two nucleotide substitutions. Molecular studies have disclosed that most of the class 1 G6PD variants associated with chronic hemolysis have the mutations surrounding either the substrate or the NADP binding site. Among rare enzymopathies, missense mutations have been determined in deficiencies of glucosephosphate isomerase, phosphofructokinase (PFK), aldolase, triosephosphate isomerase (TPI), phosphoglycerate kinase, and adenylate kinase. Compound heterozygosity with missense mutation and base deletion has been determined in deficiencies of hexokinase and diphosphoglyceromutase. Compound heterozygosity with missense and nonsense mutations has been identified in TPI deficiency. One base deletion resulting in a frameshift and the premature termination of translation and splice junction mutations resulting in abnormally spliced PFK-M mRNA have been identified in homozygous PFK deficiency. An exception is Hemolytic Anemia due to increased adenosine deaminase activity. The basic abnormality appears to result from the overproduction of a structurally normal enzyme. © 1996 Wiley-Liss, Inc.
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cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with Hereditary Hemolytic Anemia.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Hitoshi Kanno, Hisaichi Fujii, Akira Hirono, Shiro MiwaAbstract:Abstract cDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-specific primer. The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino acids, the same number as that by rat R-type PK. Compared with human L-type PK, R-type PK was 31 amino acids longer at the amino terminus. We also cloned and characterized R-type PK cDNA clones from patients with Hereditary Hemolytic Anemia from a PK deficiency, PK Tokyo. A single nucleotide substitution (ACG to ATG) was found at nucleotide 1151 of the coding sequence of the R-type PK, which caused an amino acid substitution, Thr384----Met. Dot blot hybridization of PCR-amplified genomic DNA from patients and their parents by allele-specific oligonucleotide probes showed that the parents, who were second cousins, were heterozygous. To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coli and characterized the enzyme. The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris.HCl, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK. From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene.
Hisaichi Fujii - One of the best experts on this subject based on the ideXlab platform.
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Red blood cell enzymes and their clinical application.
Advances in clinical chemistry, 1998Co-Authors: Hisaichi Fujii, Shiro MiwaAbstract:Publisher Summary The chapter discusses red blood cell enzymes and their clinical application. Structure and functions of major red blood cell enzymes are discussed in the chapter wherein hexokinase, glucose phosphate isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, diphosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, adenylate kinase, pyrimidine 5'-nucleotidase, adenosine deaminase are described. The chapter also discusses Hereditary Hemolytic Anemia associated with red blood cell enzyme deficiency in detail. The various defects in the Embden-Meyerhof pathway, hexose monophosphate pathway and glutathione metabolism and synthesis, and nucleotide metabolism are presented. Hereditary nonHemolytic blood disorders associated with red blood cell enzyme deficiency are discussed next in the chapter wherein diphosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, and NADH cytochrome b5 reductase deficiency are described. The chapter discusses Hereditary nonhematologic disorders that can be diagnosed by the determination of red blood cell enzyme activity. The various topics covered are enzyme deficiencies associated with immunological disorders; enzyme deficiencies in the metabolism of purine; prolidase deficiency; acatalasemia; galactosemia; porphyria; and carbonic anhydrase deficiency.
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Molecular analysis of glucose phosphate isomerase deficiency associated with Hereditary Hemolytic Anemia.
Blood, 1996Co-Authors: Hitoshi Kanno, Hisaichi Fujii, Akira Hirono, Yoji Ishida, Shoichi Ohga, Yasuhiko Fukumoto, Kenji Matsuzawa, Satoru Ogawa, Shiro MiwaAbstract:We report here two new cases of glucose phosphate isomerase (GPI) deficiency associated with Hemolytic Anemia and present the results of molecular analysis of the five Japanese GPI variants. A Japanese girl (GPI Fukuoka) had an episode of prolonged neonatal jaundice and at 3 years of age was admitted due to acute Hemolytic crisis occurring with upper respiratory tract infection. Red blood cell (RBC) GPI activity was decreased to 11.8% of normal and the reduced glutathione (GSH) level of RBCs was slightly decreased. A 54-year-old Japanese man (GPI Iwate) was hospitalized due to chronic active hepatitis, and compensated hemolysis was noted. RBC GPI activity of the proband was decreased to 18.8%, and the GSH content was about half of the normal mean value. Sequencing of the reticulocyte GPIcDNA showed homozygous missense mutations 1028CAG-->CGG (343Gln-->Arg), 14ACC-->A7C (5Thr-- >lle), 671ACG-->A7G (224Thr-->Met), and 1615GAC-->AAC (539Asp-->Asn) in GPI Narita, GPI Matsumoto, GPI Iwate, and GPI Fukuoka, respectively. We also identified GPI Kinki as a compound heterozygote of 1124ACA-- >AGA(375Thr-->Arg)/ 1615GAC-->AAC(539Asp-->Asn). Our findings, together with the previous results of other investigators, showed that the GPI gene mutations so far identified were heterogeneous, although most GPI variants had common biochemical characteristics such as heat instability and normal kinetics. Several amino acid substitutions were identified in the proximity of the catalytically important amino acid residues such as Ser/Asp 159/160, Asp341, and Lys518, which have been identified in the structural analysis of the pig GPI. The molecular characterization of human GPI variants, therefore, may provide new insights into the genotype-phenotype correlation of GPI deficiency as well as the structure-function relationship of this enzyme.
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Molecular basis of erythroenzymopathies associated with Hereditary Hemolytic Anemia: Tabulation of mutant enzymes
American journal of hematology, 1996Co-Authors: Shiro Miwa, Hisaichi FujiiAbstract:Molecular abnormalities of erythroenzymopathies associated with Hereditary Hemolytic Anemia have been determined by means of molecular biology. Pyruvate kinase (PK) deficiency is the most common and well-characterized enzyme deficiency in the glycolytic pathway, and it causes Hereditary Hemolytic Anemia. To date, 47 gene mutations have been identified. We identified one base deletion, one splicing mutation, and six distinct missense mutations in 12 unrelated families with a homozygous PK deficiency. Mutations located near the substrate or fructose-1,6-diphosphate binding site may change the conformation of the active site, resulting in a drastic loss of activity and severe clinical symptoms. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common metabolic disorder, and it is associated with chronic Hemolytic Anemia and/or drug-or infection-induced acute Hemolytic attack. An estimated 400 million people are affected worldwide. The mutations responsible for about 78 variants have been determined. Some have polymorphic frequencies in different populations. Most variants are produced by one or two nucleotide substitutions. Molecular studies have disclosed that most of the class 1 G6PD variants associated with chronic hemolysis have the mutations surrounding either the substrate or the NADP binding site. Among rare enzymopathies, missense mutations have been determined in deficiencies of glucosephosphate isomerase, phosphofructokinase (PFK), aldolase, triosephosphate isomerase (TPI), phosphoglycerate kinase, and adenylate kinase. Compound heterozygosity with missense mutation and base deletion has been determined in deficiencies of hexokinase and diphosphoglyceromutase. Compound heterozygosity with missense and nonsense mutations has been identified in TPI deficiency. One base deletion resulting in a frameshift and the premature termination of translation and splice junction mutations resulting in abnormally spliced PFK-M mRNA have been identified in homozygous PFK deficiency. An exception is Hemolytic Anemia due to increased adenosine deaminase activity. The basic abnormality appears to result from the overproduction of a structurally normal enzyme. © 1996 Wiley-Liss, Inc.
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cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with Hereditary Hemolytic Anemia.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Hitoshi Kanno, Hisaichi Fujii, Akira Hirono, Shiro MiwaAbstract:Abstract cDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-specific primer. The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino acids, the same number as that by rat R-type PK. Compared with human L-type PK, R-type PK was 31 amino acids longer at the amino terminus. We also cloned and characterized R-type PK cDNA clones from patients with Hereditary Hemolytic Anemia from a PK deficiency, PK Tokyo. A single nucleotide substitution (ACG to ATG) was found at nucleotide 1151 of the coding sequence of the R-type PK, which caused an amino acid substitution, Thr384----Met. Dot blot hybridization of PCR-amplified genomic DNA from patients and their parents by allele-specific oligonucleotide probes showed that the parents, who were second cousins, were heterozygous. To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coli and characterized the enzyme. The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris.HCl, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK. From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene.
Richard Van Wijk - One of the best experts on this subject based on the ideXlab platform.
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Infantile Pyknocytosis in a Premature Dichorionic Diamniotic Twin.
Journal of pediatric hematology oncology, 2020Co-Authors: Mandy E Spoorenberg, Richard Van Wijk, Renate E Wachters-hagedoorn, Jacques B De KokAbstract:Infantile pyknocytosis is a rare and self-limiting cause of Hemolytic Anemia in neonates. It can result in severe Anemia and hyperbilirubinemia. The pathogenesis is unknown: a genetic origin has been discussed; however, based on the current literature it is not clear which genetic mutations should be considered. We present a case of a premature twin, in whom genetic screening was performed. Genetic mutations in 46 genes associated with Hereditary Hemolytic Anemia and dyserythropoietic Anemia were tested. No mutations were found. In infantile pyknocytosis, a genetic defect in these genes is unlikely.
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Interplay of erythropoietin, fibroblast growth factor 23, and erythroferrone in patients with Hereditary Hemolytic Anemia
Blood advances, 2020Co-Authors: Annelies J. Van Vuren, Richard Van Wijk, Michele F. Eisenga, Stephanie Van Straaten, Andreas Glenthøj, Carlo A. J. M. Gaillard, Stephan J. L. Bakker, Martin H. De Borst, Eduard J. Van BeersAbstract:Recently, erythropoietin (EPO) was identified as regulator of fibroblast growth factor 23 (FGF23). Proteolytic cleavage of biologically active intact FGF23 (iFGF23) results in the formation of C-terminal fragments (cFGF23). An increase in cFGF23 relative to iFGF23 suppresses FGF receptor signaling by competitive inhibition. EPO lowers the i:cFGF23 ratio, thereby overcoming iFGF23-mediated suppression of erythropoiesis. We investigated EPO-FGF23 signaling and levels of erythroferrone (ERFE) in 90 patients with Hereditary Hemolytic Anemia (www.trialregister.nl [NL5189]). We show, for the first time, the importance of EPO-FGF23 signaling in Hereditary Hemolytic Anemia: there was a clear correlation between total FGF23 and EPO levels (r = +0.64; 95% confidence interval [CI], 0.09-0.89), which persisted after adjustment for iron load, inflammation, and kidney function. There was no correlation between iFGF23 and EPO. Data are consistent with a low i:cFGF23 ratio. Therefore, as expected, we report a correlation between EPO and ERFE in a diverse set of Hereditary Hemolytic Anemias (r = +0.47; 95% CI, 0.14-0.69). There was no association between ERFE and total FGF23 or iFGF23, which suggests that ERFE does not contribute to the connection between FGF23 and EPO. These findings open a new area of research and might provide potentially new druggable targets with the opportunity to ameliorate ineffective erythropoiesis and the development of disease complications in Hereditary Hemolytic Anemias.
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Decreased Activity and Stability of Pyruvate Kinase in Hereditary Hemolytic Anemia: A Potential Target for Therapy By AG-348 (Mitapivat), an Allosteric Activator of Red Blood Cell Pyruvate Kinase
Blood, 2019Co-Authors: Minke A.e. Rab, Stephanie Van Straaten, Eduard J. Van Beers, Brigitte A. Van Oirschot, Bart J. Biemond, Jennifer Bos, Penelope A. Kosinski, Charles Kung, Richard Van WijkAbstract:Background: Reactive oxygen species (ROS) play an important role in the complex and multifactorial pathophysiology of Hereditary Hemolytic Anemia like sickle cell disease (SCD), β-thalassemia and Hereditary xerocytosis (HX). Increased intracellular levels of oxidative stress disrupt normal cell functioning and may contribute to premature red blood cell (RBC) clearance from the circulation. Pyruvate kinase (PK) is a key regulatory enzyme of glycolysis, the cell's main source of energy. Because PK is very sensitive to redox balance we hypothesized that increased levels of oxidative stress in SCD, β-thalassemia and HX impairs proper enzyme function, thereby compromizing RBC energy metabolism. This may contribute to disease pathophysiology. Aims: To investigate if secondary deficiency of PK is common in SCD, thalassemia, and HX, and to investigate if PK in these disorders is able to respond to treatment with the allosteric PK activator AG-348 (mitapivat). Methods: Enzymatic activities of red cell PK and hexokinase (HK) were measured together with PK-thermostability in order to assess relative PK activity and enzyme stability. Purified RBCs were incubated with AG-348 (3.33μM) for 24 hours after which PK activity and ATP response was measured. RBCs of SCD patients were also analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry (Rab et al, Am J Hematol, 2019). Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO2 at which RBCs start to sickle during deoxygenation under shear stress. Results: Thirty-eight patients and 21 healthy controls (HC) were included. The patient cohort consisted of patients homozygous for HbS (HbSS, n=26), patients compound heterozygous for HbS and HbC (HbSC, n=4), β-thalassemia major (regularly transfused, n=3), and Hereditary xerocytosis (n=5). Patients showed reticulocytosis and, in line with this, a concomitant increase in HK activity. In contrast however, relative PK activity was decreased significantly compared to HK in HbSS, β-thalassemia and HX patients, but not in HbSC patients (Figure 1A). PK thermostability was significantly decreased compared to healthy controls in HbSS patients and patients with HX (Figure 1B). In HbSC and β-thalassemia patients, PK-thermostability was comparable to HC. PK thermostability strongly correlated with absolute reticulocyte count (ARC), indicating that patients displaying the highest degree of PK instability had the highest reticulocyte count (Figure 1C). This suggests that in general, a higher degree of PK instability is associated with more severe Anemia due to a high Hemolytic rate. In SCD patients, PK-thermostability inversely correlated with PoS, indicating that decreased PK stability is associated with sickling at higher pO2 (r=-0.646, p<0.001, Figure 1F). When purified RBCs were incubated with 3.33μM of the allosteric PK-activator AG-348, an increase in PK activity was seen in all patients and HCs, with a mean increase of 122% in HbSS (range 111-139%, n=6), 137% in β-thalassemia (n=1), 163% in HX (range 152-174%, n=2) and 143% in HC (range 113-173%, n=9, Figure 1E). Accordingly, ATP-levels increased in all patients and HCs, with a mean increase of 133% in HbSS (range 125-141%, n=5), 144% patient with β-thalassemia (n=1), 121% in HX (range 112-129, n=3), and 132% in HCs (range 101-149%, n=9, Figure 1E). Conclusion: PK enzyme activity and stability is compromised in patients with various forms of Hereditary Hemolytic Anemia. This implies that PK stability and, hence, compromised red cell metabolism could contribute to the complex pathophysiology of these diseases. In SCD patients, reduced PK-thermostability is associated with higher PoS, which we previously have shown to be associated with more severe disease (Rab et al, Am J Hematol, 2019, ASH 2019 abstract ID128870). This is confirmed by the correlation of decreased PK-thermostability with increased reticulocyte count as presented in this study. Current studies are in progress to further substantiate the underlying mechanism(s) involved, and to investigate whether AG-348 may ameliorate clinical features such as hemolysis, sickling tendency and iron overload. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Kosinski:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. Kung:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding.
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Phosphatidylserine-Exposing Extracellular Vesicles after Splenectomy Are Associated with Increased D-Dimers and Fibrin Generation in Hereditary Hemolytic Anemia
Blood, 2018Co-Authors: Stephanie Van Straaten, Wouter W. Van Solinge, Richard Van Wijk, Chi Hau, Najat Hajji, Jill Verhoeven, Roger E.g. Schutgens, Rienk Nieuwland, Eduard J. Van BeersAbstract:Background Thrombosis is a common complication of Hereditary Hemolytic Anemia (HHA). Etiology of a hypercoagulable state in patients with HHA involves inflammation and splenectomy, although the etiology of the latter is insufficiently established. Because the concentration of circulating extracellular vesicles (EVs) has been reported to increase after splenectomy, and because in patients with sickle cell disease (SCD) circulating EVs are associated with coagulation, we analyzed the concentration of circulating EVs and their procoagulant activity in plasma from splenectomized and non-splenectomized patients with HHA. Methods This is a cross sectional, observational study in adult patients with HHA (SCD, other hemoglobin disorders, red cell enzyme disorders, red cell membrane disorders). Blood samples were collected with a 21-gauge butterfly needle and collected in 9 mL citrate phosphate dextrose adenine (CPDA) vacutainers, without use of a tourniquet. The tubes were mixed gently and the time between blood collection and centrifugation was maximum one hour. EVs in platelet-depleted plasma were labeled for Heat Shock Protein 70, CD14 (monocyte-derived EVs), CD61 (platelet EVs), CD62e (endothelial EVs), CD62p (P-selectin-exposing platelet EVs), CD71 (reticulocyte EVs), CD144 (endothelial EVs), CD235a (erythrocyte EVs) and lactadherin (phosphatidylserine (PS)-exposing EVs), and measured with a dedicated flow cytometer for EVs (A60-micro, Apogee Flow systems; lower limit of detection 170-180 nm single EVs). The coagulant activity of EVs was studied by a fibrin generation test, which measures the EV-dependent clotting time of plasma. The time to fibrin formation (1/2max) was measured using optical densitometry (λ = 405 nm) and an arbitrary cut off of V1/2max 25% difference between duplicates or from patients that used anticoagulant medication were excluded from analysis Results Ninety seven patients were included in the study. Baseline characteristics are shown in Table 1. FGT of 63 patients were included. Thirteen patients (21%) had a positive FGT. Patients with positive FGT had increased concentrations of circulating EVs (CD61, CD71, lactadherin: p= Conclusion In this study we show that in HHA patients the plasma concentration of lactadherin-binding and thus PS-exposing EVs correlates with fibrin generation in vitro and plasma D-dimer concentration, indicating that EVs may be associated with the hypercoagulable state that is observed in patients with HHA. Splenectomized patients had higher concentrations of lactadherin-binding EVs, and their plasma samples were prone to clot, as shown by fibrin generation in vitro. As the spleen is the main organ removing PS-exposing cells, higher levels of PS-exposing EVs in such patients may be due to reduced clearance, which in turn may contribute to the increased risk of thrombosis in patients after splenectomy. Disclosures Schutgens:Novo Nordisk: Research Funding; Uniqure BV: Research Funding; Pfizer: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding; Baxalta/Shire: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding. van Beers:RR Mechatronics: Research Funding; Bayer: Research Funding; Pfizer: Research Funding; Agios: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding.
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Squeezing for Life – Properties of Red Blood Cell Deformability
Frontiers Media S.A., 2018Co-Authors: Rick Huisjes, Anna Bogdanova, Raymond M Schiffelers, Lars Kaestner, Wouter W. Van Solinge, Richard Van WijkAbstract:Deformability is an essential feature of blood cells (RBCs) that enables them to travel through even the smallest capillaries of the human body. Deformability is a function of (i) structural elements of cytoskeletal proteins, (ii) processes controlling intracellular ion and water handling and (iii) membrane surface-to-volume ratio. All these factors may be altered in various forms of Hereditary Hemolytic Anemia, such as sickle cell disease, thalassemia, Hereditary spherocytosis and Hereditary xerocytosis. Although mutations are known as the primary causes of these congenital Anemias, little is known about the resulting secondary processes that affect RBC deformability (such as secondary changes in RBC hydration, membrane protein phosphorylation, and RBC vesiculation). These secondary processes could, however, play an important role in the premature removal of the aberrant RBCs by the spleen. Altered RBC deformability could contribute to disease pathophysiology in various disorders of the RBC. Here we review the current knowledge on RBC deformability in different forms of Hereditary Hemolytic Anemia and describe secondary mechanisms involved in RBC deformability
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Screening tools for Hereditary Hemolytic Anemia: new concepts and strategies.
Expert review of hematology, 2021Co-Authors: Elisa Fermo, Cristina Vercellati, Paola BianchiAbstract:INTRODUCTION Hereditary Hemolytic Anemias are a group of rare and heterogeneous disorders due to abnormalities in structure, metabolism, and transport functions of erythrocytes; they may overlap in clinical and hematological features making differential diagnosis difficult, particularly in mild and atypical forms. AREAS COVERED In the present review, the main tools currently adopted in routine hematologic investigation for the diagnosis of Hereditary Hemolytic Anemias are described, together with the new diagnostic approaches that are being to be developed in the next future. Available recommendations in this field together with a systematic review through MEDLINE, EMBASE, and PubMED for publications in English from 2000 to 2020 in regards to diagnostic aspects of Hereditary Hemolytic Anemias have been considered. EXPERT OPINION The recent development of specific molecules and treatments for Hereditary Hemolytic Anemias and the increased interest in translational research raised the attention on differential diagnosis and the demand for novel diagnostic assays and devices. Automatic blood cell analyzers, omic-approaches including NGS technologies, and development of new automated tools based on artificial neural networks definitely represent the future strategies in this field.
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molecular characterization of three new mutant enzymes of pyrimidine 5 nucleotidase causing Hereditary Hemolytic Anemia
Blood, 2007Co-Authors: Laurent R Chiarelli, Elisa Fermo, Paola Bianchi, Simone M Morera, Paola Rognoni, Alessandro Galizzi, Alberto Zanella, Giovanna ValentiniAbstract:Pyrimidine 5′-nucleotidase (P5′N-1) is a dephosphorylating enzyme that catalyzes the hydrolysis of various pyrimidine nucleoside 5′-monophosphates, particularly UMP and CMP, to produce the corresponding nucleosides. In RBC the reaction is essential for the removal of the nucleotides mainly arising from ribosomal RNA degradation during final erythroid maturation. Hereditary P5′N-1 deficiency is the third most common enzymopathy causing Hereditary non-spherocytic Hemolytic Anemia. The disorder is transmitted as an autosomal recessive trait and is usually characterized by mild-to-moderate Hemolytic Anemia and accumulation of pyrimidine nucleotides within the erythrocyte. The enzyme is strongly inactivated by heavy metals; thus P5′N-1 deficiency can be acquired as a result of lead poisoning. The P5′N-1 gene is localized on 7p15-p14 and the cDNA has been cloned and sequenced. 24 different mutations have been identified so far, most of them at the homozygous level. Recently, five pathological variants of P5′N-1 have been in-depth characterized, and the molecular bases of the P5′N-1 deficiency has been elucidated. To unravel the cause of the P5′N-1 deficiency found in patients with Hemolytic Anemia and homozygous for 3 newly identified missense mutations (c.187T>C, c.469G>C, c.740T>C; Balta et al, Blood ASH2006, 108:3743; Manco et al, Haematologica2006, 91:266–267), we have undertaken a functional analysis of the 3 mutant enzymatic forms. The C63R, G157R and I247T proteins were produced as recombinant forms, purified and biochemically characterized. All enzymes were altered, although to a different extent, either in their catalytic efficiency or in thermal stability, the G157R being the most impaired enzyme. Catalytic efficiency of all mutants turned expecially towards UMP (about 50 to 200 times), owing to the increased Km values (about 10–25 times higher). The kinetic behaviour vs CMP was partly affected, the catalytic activity being moderately reduced (Kcat lowered to 5–20%). The G157R protein was highly heat unstable, halving the activity in about 23 min at 37°C, whereas C63R and I247T mutants at the same temperature maintained fully activity for more than 2 hours. However, at higher temperature also C63R and I247T mutants resulted less stable than the wild-type enzyme losing the activity in few minutes (t1/2 at 46°C, about 5 min vs 2 hours of the wild-type enzyme). Therefore, although mutations targeted different regions of the P5′N-1 structure, unexpectedly they produced similar aberrant effects on the molecular properties of the enzyme. Gly157 is a conserved amino acid, located close to the substrate binding site. Very likely, position 157 cannot tolerate the large and charged arginine side-chain introduced by c.469G>C mutation. Thus, it is conceivable that the drastic G157R substitution not only indirectly affects the binding of the substrate(s), but also weakens the protein stability. Cys63 and Ile247 are located far away from the catalytic site. Nevertheless, our biochemical data indicate that they are functionally and structurally important for preserving the enzyme activity. Thus, as in other cases, the decreased catalytic efficiency of C63R and I247T enzymes seems to result from secondary effects related to propagating conformational changes.