scholarly journals Biochemical, Cellular, and Proteomic Characterization of Hereditary Spherocytosis Among Tunisians

2021 ◽  
Vol 55 (1) ◽  
pp. 117-129
Keyword(s):  
Membrane Protein ◽  
Hemolytic Anemia ◽  
Erythrocyte Membrane ◽  
Hereditary Spherocytosis ◽  
Osmotic Fragility ◽  
High Specificity ◽  
Protein Deficiency ◽  
Screening Tests ◽  
Tunisian Population ◽  
Tunisian Patients

Background/Aims: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. Methods: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5’-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. Results: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. Conclusion: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

Clinical and Haematologic Features of 300 Patients Affected by Hereditary Spherocytosis as a Function of the Type of the Membrane Protein Defect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1724-1724
Author(s):  
Wilma Barcellini ◽  
Mariagabriella Mariani ◽  
Cristina Vercellati ◽  
Anna P. Marcello ◽  
Elisa Fermo ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Hereditary Spherocytosis ◽  
Osmotic Fragility ◽  
Band 3 ◽  
Screening Tests ◽  
Molecular Defect ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Sds Page ◽  
Ankyrin Deficiency

Abstract Hereditary Spherocytosis (HS) is caused by defects of red cell membrane proteins (spectrin, ankyrin, band 3 and band 4.2). The aim of this study is to analyse a large database of 300 HS patients grouped according to SDS-PAGE, 1) to ascertain whether the clinical/haematological features and response to splenectomy are related to the type of molecular defect, and 2) to compare the sensitivity of the most common laboratory screening tests for HS in the various subsets of patients. Three hundred consecutive patients were investigated; 41 patients had been splenectomised before the time of the study, and 21 thereafter. In not splenectomised subjects, anaemia was severe in 7% of cases, mild to moderate in 52% and compensated in the remaining cases. The median number of spherocytes at the peripheral blood smear examination was 7%. The most frequent protein abnormalities revealed by SDS-PAGE were band 3 (54%) and spectrin and/or ankyrin (34%) deficiency. The membrane protein defect was undetectable in 3% of splenectomised versus 11% of not splenectomised patients. In patients evaluated by SDS-PAGE before and after splenectomy surgery allowed the identification of the defect (one band 3 and seven spectrin/ankyrin deficiency) in all the 8 previously unclassified cases. No significant differences were observed among clinical and haematological parameters of patients grouped according to the type of biochemical defect, although the degree of anaemia, haemolysis markers and median spherocyte number were higher in spectrin deficient than in the other groups of patients. Splenectomy was clinically effective in correcting both anaemia and haemolysis, but splenectomised spectrin/ankyrin deficient patients showed after splenectomy a slightly lower median rise in haemoglobin, and a higher reticulocyte number and unconjugated bilirubin level than band 3 deficient patients. The red cell osmotic fragility tests’ sensitivity varies greatly and ranged from 48 to 95%. The sensitivity was similar in patients with band 3 and spectrin/ankyrin deficiency and also in patients without detectable membrane defect. Furthermore, the sensitivity of all the methods investigated increased in splenectomised cases. AGLT displayed the highest sensitivity, and the association of AGLT and NaCl test on incubated blood reached a sensitivity of 99%, enabling the diagnosis of the atypical HS cases, such as those with rare or no spherocytes in blood smears, normal MCHC and reticulocyte counts.

Erythrocyte membrane protein defects in hereditary spherocytosis patients in Turkish population

Hematology ◽  
2012 ◽  
Vol 17 (4) ◽  
pp. 232-236 ◽  
Author(s):  
Aylin Canbolat Ayhan ◽  
Inci Yildiz ◽  
Sedef Yüzbaşıoğlu ◽  
Tiraje Celkan ◽  
Hilmi Apak ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Hereditary Spherocytosis ◽  
Turkish Population ◽  
Erythrocyte Membrane Protein

Molecular Diagnosis of Hereditary Spherocytosis By Multi-Gene Target Sequencing in Korea

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3347-3347
Author(s):  
Qute Choi ◽  
Jung Ah Kim ◽  
Kyong Ok Im ◽  
Si Nae Park ◽  
Yoomi Park ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Membrane Protein ◽  
Hemolytic Anemia ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Hereditary Spherocytosis ◽  
Conflicts Of Interest ◽  
Gene Target ◽  
Protein Encoding ◽  
Encoding Genes

Abstract Background: Hereditary spherocytosis (HS) is the most common cause of hereditary hemolytic anemia. Spherocytes formed by defective membrane proteins are selectively captured in the spleen and destroyed, leading to hemolytic anemia. Current tests used to diagnose HS focus on the detection of hemolysis or indirectly assess protein defects. Direct methods to detect protein defects are complicated and difficult to implement. Recent next-generation sequencing (NGS) methods enable large-scale gene mutation analyses to be used for such diagnoses. In this study, we investigated the patterns of genetic variation associated with HS to determine the molecular mechanisms underlying the condition. Specifically, we analyzed mutations in red blood cell membrane protein-encoding genes in Korean HS patients using NGS. Methods: In total, 60 patients with HS were enrolled in this study. Targeted sequencing of 43 genes (17 membrane protein-encoding genes, 20 enzyme-encoding genes, and 6 additional candidate genes) was performed using the Illumina HiSeq platform and variants were called according to a data-processing pipeline. Results: Of the 60 patients, 50 (83%) had one or more significant variants in a membrane protein gene. A total of 54 significant variants (8 previously reported and 46 novel) were detected in 6 membrane protein-encoding genes, i.e., SPTB, ANK1, SPTA1, SLC4A1, EPB41, and EPB42. The most variants (28) were detected in SPTB. Four significant variants, all of which were previously reported, were detected in genes encoding enzymes (ALDOB, G6PD, GAPDH, and GSR). Additionally, 5 previously reported variants were detected in UGT1A1. These results suggest 35 primer sets that can be used to diagnose HS. Conclusion: This was the first large-scaled genetic study of Korean HS patients. These results clarify the pattern of genetic variation associated with HS in Korean patients. They will enable easier, more rapid diagnosis of HS. Disclosures No relevant conflicts of interest to declare.

A Case of Congenital Red Cell Pyruvate Kinase Deficiency Associated with Hereditary Spherocytosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5272-5272
Author(s):  
Cristina Vercellati ◽  
Anna Paola Maria Luisa Marcello ◽  
Elisa Fermo ◽  
Paola Bianchi ◽  
Carla Boschetti ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Peripheral Blood ◽  
Blood Smear ◽  
Hereditary Spherocytosis ◽  
Osmotic Fragility ◽  
Peripheral Blood Smear ◽  
Red Cell ◽  
Red Cell Membrane

Abstract Abstract 5272 Pyruvate kinase (PK) deficiency, transmitted as an autosomal recessive trait, is the most common erythroenzymopathy of glycolytic pathway (prevalence of 1:20,000) associated with chronic non spherocytic hemolytic anemia from mild to severe. More than 180 mutations in the PK-LR gene have been so far reported, and genotype-phenotype correlation has been established for some of them. Hereditary Spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The main clinical features are hemolytic anemia from compensated to severe, variable jaundice, splenomegaly and cholelythiasis. The molecular defect is highly heterogeneous, caused by proteins involved in the attachment of cytoskeleton to the membrane integral domain (spectrin, ankyrin, band 3 and protein 4.2). We describe a case of PK deficiency associated with HS. The propositus was a 13 years-old Italian male with neonatal jaundice and need of blood transfusion (Hb 5.8 g/dL) during an infectious episode. At the time of the study Hb was 13.9 g/dL, MCV 81.8 fL, reticulocytes 207×109/L, unconjugated bilirubin 2.16 mg/dL, LDH 605 U/L, haptoglobin <20 mg/dL. The peripheral blood smear examination showed the presence of spherocytes (16%) and some ovalocytes (2%). The study of the most important red cell enzymes revealed reduced PK activity (59% of normal). Direct sequencing of PK-LR gene showed compound heterozygosity for the 994A mutation (Gly332Ser) and the −148T variant localized the erythroid specific promoter region. The presence of spherocytes in peripheral blood smear prompted us to investigate for the coexistence of HS. Erythrocyte osmotic fragility was decreased and SDS–PAGE analysis of red cell membrane proteins revealed a 30% spectrin reduction. Family study demonstrated a heterozygous condition for the 994A mutation in the father, who also displayed comparable enzyme deficiency, whereas promoter variant −148T was detected in the mother and in the brother. No red cell membrane abnormalities were present in the family members, although positive EMA binding test and increased osmotic fragility were found in the father and brother. The co-existence of HS and PK deficiency is very rare event, only few cases are described to date. Clinical, family and molecular studies allowed the determination of the interrelationship between the two RBC abnormalities in the patient and his relatives. The reduced PK activity in the propositus and his father is justified by heterozygous 994A mutation. The more severe clinical picture in the propositus could be caused by the coexistence of HS and by the presence of −148T mutation, that although it seems not to have effects on PK-LR mRNA expression, is often detected in PK deficient subjects with heterozygous PK mutations. Disclosures: No relevant conflicts of interest to declare.

Erythrocyte membrane protein alterations underlying clinical heterogeneity in hereditary spherocytosis

1994 ◽  
Vol 88 (1) ◽  
pp. 52-55 ◽  
Author(s):  
Emanuele Miraglla Giudice ◽  
Achille Iolascon ◽  
Luciano Pinto ◽  
Bruno Nobili ◽  
Silverio Perrotta
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Hereditary Spherocytosis ◽  
Clinical Heterogeneity ◽  
Erythrocyte Membrane Protein ◽  
Protein Alterations
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