Anemia Advances in diagnosis and treatments. II. Practice of diagnosis and treatments. 4. Hemolytic anemia. 1) Membrane protein defect and genetic analysis in hereditary spherocytosis.

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.

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

Cellular Physiology and Biochemistry ◽

10.33594/000000333 ◽

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.

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Clinical and genetic diagnosis of thirteen Japanese patients with hereditary spherocytosis

Human Genome Variation ◽

10.1038/s41439-021-00179-1 ◽

2022 ◽

Vol 9 (1) ◽

Author(s):

Keiko Shimojima Yamamoto ◽

Taiju Utshigisawa ◽

Hiromi Ogura ◽

Takako Aoki ◽

Takahiro Kawakami ◽

...

Keyword(s):

Hemolytic Anemia ◽

Hereditary Spherocytosis ◽

Genetic Diagnosis ◽

Genomic Analysis ◽

Japanese Patients ◽

Asian Countries ◽

Target Capture ◽

Detailed Family History ◽

Target Capture Sequencing ◽

Chronic Hemolytic Anemia

AbstractHereditary spherocytosis is the most frequent cause of hereditary hemolytic anemia and is classified into five subtypes (SPH1-5) according to OMIM. Because the clinical and laboratory features of patients with SPH1-5 are variable, it is difficult to classify these patients into the five subtypes based only on these features. We performed target capture sequencing in 51 patients with hemolytic anemia associated with/without morphological abnormalities in red blood cells. Thirteen variants were identified in five hereditary spherocytosis-related genes (six in ANK1 [SPH1]; four in SPTB [SPH2]; and one in each of SPTA1 [SPH3], SLC4A1 [SPH4], and EPB42 [SPH5]). Among these variants, seven were novel. The distribution pattern of the variants was different from that reported previously in Japan but similar to those reported in other Asian countries. Comprehensive genomic analysis would be useful and recommended, especially for patients without a detailed family history and those receiving frequent blood transfusions due to chronic hemolytic anemia.

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