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Stepwise process for genetic-based diagnosis of hereditary spherocytosis. Abbreviations: CBC, complete blood cell counting; HS, hereditary spherocytosis; LDH, lactate dehydrogenase; NGS, next-generation sequencing; RBC, red blood cell.
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Inherited hemolytic anemias (IHAs) are genetic diseases that present with anemia due to the increased destruction of circulating abnormal RBCs. The RBC abnormalities are classified into the three major disorders of membranopathies, hemoglobinopathies, and enzymopathies. Traditional diagnosis of IHA has been performed via a step-wise process combini...
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... is an inherited disorder characterized by the presence of spherical-shaped RBCs on peripheral blood smears and is most commonly associated with autosomal dominant in- heritance [23]. HS RBC membranes in HS patients show qualitative and/or quantitative abnormalities of proteins in- cluding isolated ankyrin or combined ankyrin and spectrin, isolated spectrin, band 3, and protein 4.2. The defects in the membrane components increase membrane fragility and induce vesiculation with or without the band 3 protein. HS is diagnosed through laboratory tests including RBC mor- phology and osmotic fragility test as well as family history (Fig. 3). The osmotic fragility test has been considered the gold standard screening test for HS but provides false-neg- ative findings in about 25% of patients [24]. Eosin-5'-mal- eimide measurement and SDS-polyacrylamide gel electro- phoresis of erythrocyte membrane proteins are also useful for screening HS, but standardization of these methods is currently lacking [25,26]. None of the HS screening tests can detect all patients because the clinical phenotypes are widely variable, ranging from asymptomatic to severely af- fected [27]. A few patients with mild hemolysis may develop marked anemia if their bone marrow erythrocyte production is transiently halted by viral (parvovirus B19) or other in- fections [28]. This scenario would be an aplastic crisis since the bone marrow can no longer compensate for ongoing hemolysis. In neonates or transfused individuals, diagnosis can be difficult due to unclear morphological features and screening tests may be unreliable [23]. Molecular testing is useful for primary differential diagnosis or confirming HS and defining the genotype-phenotype correlation [24,29,30]. The defects in RBC membrane components in HS are typically caused by their corresponding gene ...
Similar publications
Hereditary hemolytic anemias present a unique diagnostic challenge due to their wide phenotypic and genotypic spectrum. Accurate diagnosis is essential to ensure appropriate treatment. We report two cases, which presented as hemolytic anemias, but initial workup was inconclusive and they were finally diagnosed with the help of Next Generation Seque...
Citations
... The study revealed that autosomal dominant HE was the most common red blood cell membrane disorder, accounting for 33% of cases, followed by autosomal recessive HPP at 28%. Although HE can result from mutations in the SPTA1, SPTB, or EPB41 genes [23], the majority of HEcausing mutations in Thai patients (28 out of 29) were found to be recurrent mutations in the SPTB gene. These include the SPTB Providence (SPTB c.6055T>C), SPTB Buffalo (SPTB c.6074T>G) and SPTB Chiang Mai (SPTB c.6224A>G) mutations, which lie within the exons 29/30 of the SPTB gene and result in defective β-spectrin tetramerization [2]. ...
... However, unlike HPP, most affected patients with HS required infrequent or did not require transfusions, with only 32% (6 out of 19) becoming transfusion-dependent. Among the five genes associated with HS, namely ANK1, SPTB, SPTA1, SLC4A1 and EPB42 [23], mutations in ANK1 and SPTB genes were identified in the seven HS patients who received a molecular diagnosis. Although being derived from a small sample size, the finding that ANK1 and SPTB are the predominant mutated genes causing HS is consistent with findings observed in the large North American cohort [27], and Chinese [28,29] and Korean patients with HS [30]. ...
Red blood cell (RBC) membrane disorders represent a significant category of hereditary hemolytic anemia; however, information from Southeast Asia is limited. We established a national registry aiming to characterize RBC membrane disorders and their molecular features in Thailand. A total of 100 patients (99 kindreds) diagnosed with RBC membrane disorders between 2010 and 2021 from six university hospitals were enrolled. The most prevalent disorders observed were hereditary elliptocytosis (HE; n=33), hereditary pyropoikilocytosis (HPP; n=28), hereditary spherocytosis (HS; n=19), Southeast Asian ovalocytosis (SAO; n=10 of 9 kindreds), and two cases of homozygous SAO. The remaining cases were grouped as unclassified membrane disorder. Seventy-six patients (76%) were molecularly confirmed by PCR, direct DNA sequencing, or hi-throughput sequencing. The primary causative gene for HE and HPP was SPTB, accounting for 28 out of 29 studied alleles for HE and 56 of 56 studied alleles for HPP. In the case of HS, dominant sporadic mutations in the ANK1 gene (n=4) and SPTB gene (n=3) were identified as the underlying cause. Notably, the four most common variants causing HE and HPP were SPTB Providence (c.6055 T>C), SPTB Buffalo (c.6074 T>G), SPTB Chiang Mai (c.6224 A>G), and SPTB c.6171__82delins TGCCCAGCT. These recurrent SPTB mutations accounted for 79 out of 84 mutated SPTB alleles (94%). In summary, HE and hereditary HPP associated with recurrent SPTB mutations are the predominant types of RBC membrane disorders observed in Thailand. These findings have significant implications for the clinical management and future research of RBC membrane disorders in the region.
... Elliptocytosis has been shown to result from autosomal dominant mutations in the α-spectrin gene (SPTA1), β-spectrin gene (SPTB), and the EPB41 gene encoding protein 4.1R [6]. Mutations in SPTA1 are most frequent and could account for about 65% of HE cases, followed by SPTB in 30% and EPB41 in ~ 5% of cases [7,8]. Inheritance of a homozygous SPTA1 mutation or of compound heterozygous mutations can lead to the more severe clinical phenotype of hereditary pyropoikilocytosis (HPP) that is characterized by an overt hemolytic anemia [9]. ...
Hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP) are considered a group of hemolytic anemias (HE/HPP) due to inherited abnormalities of erythrocyte membrane proteins with a worldwide distribution. Most cases are associated with molecular abnormalities linked to spectrin, band 4.1, and ankyrin. The present study aimed to identify significant molecular signatures on a target panel of 8 genes using whole exome sequencing (WES) in 9 Bahraini patients with elliptocytosis. Case selection was based on presence of anemia not associated with iron deficiency or hemoglobinopathy and demonstrating > 50% elliptocytes in blood smears. The c.779 T > C mutation of SPTA1 (Spectrin alpha), which is a known deleterious missense mutation that inhibits normal association of spectrin molecules to form tetramers, was seen in 4 patients in homozygous (n = 1) and heterozygous (n = 3) states. The αLELY abnormality in association with compound heterozygous mutations in SPTA1 was present in 5 patients (2 associated with the SPTA1 c.779 T > C variant; 3 with c.3487 T > G and various other SPTA1 mutations of uncertain/unknown significance). Seven patients had SPTB (Spectrin beta) mutations, predicted as likely benign by in silico analysis. A novel EPB41 (Erythrocyte Membrane Protein Band 4.1) mutation with potential deleterious impact was also seen. Finally, 2 cases showed an InDel (insertion–deletion mutations) abnormality in the gene that codes for the mechanosensitive ion-channel PIEZO (Piezo Type Mechanosensitive Ion Channel Component 1). PIEZO mutations are reported to cause red cell dehydration but have not been previously described in HE/HPP. Results of this study confirm the involvement of previously reported abnormalities in SPTA1 and suggest possible involvement of other candidate genes in a disorder involving polygenic interactions.
... The advantages of using gene panels as a filtration search scenario include shorter turnaround times, easier data processing, more coverage in the regions of interest, and fewer incidental results. [16][17][18] A homozygous missense mutation was found on exon 7 of PKLR gene that had high potential to correlate and explain her symptoms and phenotype. This missense mutation leads to a change of amino acid number 339 both forms translated protein from aspartic acid to asparagine (D339N). ...
Red cell pyruvate kinase deficiency is one of the most common erythrocytic glycolytic pathway defects connected with congenital non-spherocytic anemia. The condition inherited as an autosomal recessive Mendelian trait is caused by mutations in the PKLR gene located on chromosome 1q21. Pyruvate kinase enzyme is crucial in the energy-producing glycolysis pathway that provides red blood cells with the primary source of energy (ATP). We report here a case of a Saudi female patient that was initially diagnosed at a few months old with beta-thalassemia major and was treated with regular blood transfusions and iron overload management. At the time of our sample collection, the patient was recently transferred to King Abdul Aziz University Hospital. Genetic testing was performed to identify the disease-contributing variant of beta-thalassemia using TaqMan genotyping of six common beta-thalassemia variants (negative results). NGS targeted HBB gene sequencing which did not reveal any related variants. MLPA was performed to rule out alpha thalassemia diagnosis. The use of whole genome sequencing revealed a rare missense variant in the PKLR gene c.1015G>A (D339N) in a homozygous state that correlates to her severe phenotype. Documenting this incident will aid medical staff in providing appropriate care to similar cases and highlights the importance of following up with the diagnosis investigation process to minimize misdiagnosis incidences.
... On the contrary, membranopathies, despite the morphological examination of stained blood smear, allow the diagnosis in a relatively important number of cases; it is frequently hampered by several interferences. Examples of these interferences are the following: (a) the coinheritance of more than one RBC defect [18], (b) the existence of de novo mutations [53][54][55][56][57], (c) the overlapping of clinical variability and (d), the degree of reticulocytosis and/or to the frequent blood transfusion requirements especially in newborns and children [58][59][60]. ...
The red blood cells (RBCs) carry oxygen from the lungs to the tissues, and for this, they must be able to deform. Accordingly, an impairment of RBC deformability is the cause of RBCs trapping and removal by the spleen and haemolysis. The most common causes for the decline in red cell deformability are the RBC membrane defects (abnormal shape or ionic transport imbalance), haemoglobinopathies (increased rigidity), or enzyme deficiencies (decreased anti-oxidant defences or ATP content). The most common cause of hereditary anaemia in childhood is hereditary spherocytosis (HS), characterised by a marked RBC deformabiity. A decreased RBC deformability has been found in hereditary haemolytic anaemias (HHAs) using the new-generation osmotic gradient ektacytometry (OGE), probably due to a combination of membrane protein defects and ionic imbalance. Therefore, OGE is currently considered the gold standard for the measurement of RBC deformability and the most useful complementary tool for the differential diagnosis of HHAs. Moreover, since several new forms of treatment are currently developed for hereditary RBC defects, the clinical interest of OGE is increasing. The aim of this chapter is to provide further information about the use of RBC deformability in clinical diagnosis and the OGE as a new challenge to decrease the frequency of undiagnosed rare anaemias.
... A wide range of genetically and phenotypically variable illnesses that are brought on by an increase in the rate of RBC oxidation are collectively referred to as hereditary hemolytic anaemia (HHA) 1,2 . The degree of this destruction determines the severity of the anaemia or the timing of the onset of hemolysis. ...
Background: Hereditary hemolytic anemia is a frequent genetic condition that can have varied levels of morbidity and death. These genetic diseases raise the burden on the patient as well as society and pose a serious threat to public health. Aim: To determine the frequency of various causes of hereditary hemolytic anemia among patients in a tertiary care hospital. Methodology: It was a descriptive cross sectional study. Patients (n=252) were enrolled through probability convenient sampling. The study was conducted at Pathology Laboratory, Jinnah Hospital, Lahore. Blood samples were taken from all patients for complete blood count, peripheral smear, reticulocyte count, indirect bilirubin, serum lactate dehydrogenase, and direct antiglobulin test. Median corpuscular fragility (MCF) >4.45g/l NaCl was considered positive. Quantitative enzyme-based assays of Glucose 6 phosphate dehydrogenase were determined spectrophotometrically by using Pointe scientific G6PD reagent. Data were entered and analyzed using SPSS v25.0. A p-value of 0.05 was used to define statistical significance after post-stratification analysis using the chi-square test. Results: age range in this study was from 1 to 60 years with a mean age of 29.3±9.5 years. According to causes of heredity hemolytic anemia distribution, Thalassaemia major 137(54.4%) was the most common cause followed by Thalassaemia intermedia at 41(16.3%), Sickle Cell Anemia at 34(13.5%), Heredity spherocytosis as 31(12.3%) and Glucose-6 phosphatase deficiency as 9(3.6%). Practical Implication: As there is a high incidence of anemia among our population and there is lack of local data that specifically addresses this health issue thus current study was planned. This study highlighted that educating the entire medical community could help lower the prevalence of thalassemia major and emphasise the need of diagnosing the thalassemia trait and provide premarital and prenatal counselling. Conclusion: It was concluded that majority of hereditary hemolytic anaemias were hemoglobinopathies, which place a heavy strain on families and society. The occurrence of these conditions can be reduced in large part by prevention. Keywords: Heredity Hemolytic Anemia, Heredity Spherocytosis, Thalassaemia Trait and Prention.
... 1 These conditions are commonly associated with mutated ANK1 (ankyrin-1) (40%-65%), SPTB (beta spectrin) (15%-30%), SLC4A1 (band 3) (20%-35%), EPB42 (protein 4.2) (<5%), and SPTA1 (alpha spectrin) (<5%). [2][3][4] RBC volume disorders are broadly divided into overhydrated and dehydrated stomatocytosis. These disorders have been extensively reviewed in the past and will not be discussed in detail here. ...
Introduction
Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia.
Materials and Methods
Our hemolytic anemia diagnostic next‐generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty‐six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019.
Results
We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha‐LELY and alpha‐LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients.
Conclusions
Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha‐LELY and alpha‐LEPRA should be included in all targeted HHA panels.
... Hereditary hemolytic anemia (HHA) is defined as a group of heterogeneous and rare diseases caused by defects of red blood cell (RBC) metabolism and/or defects of the plasma membrane, which leads to the lysis or premature clearance of these cells [1]. Representative laboratory findings comprise decreased haptoglobin, increased lactate dehydrogenase (LDH) and unconjugated bilirubin, reticulocytosis, abnormal RBC morphology, and often iron overload. ...
Background:
Hereditary hemolytic anemia (HHA) is defined as a group of heterogeneous and rare diseases caused by defects of red blood cell (RBC) metabolism and RBC membrane, which leads to lysis or premature clearance. The aim of this study was to investigate individuals with HHA for potential disease-causing variants in 33 genes reported to be associated with HHA.
Methods:
A total of 14 independent individuals or families diagnosed with suspected HHA, and in particular, RBC membranopathy, RBC enzymopathy, and hemoglobinopathy, were collected after routine peripheral blood smear testing. A custom designed panel, including the 33 genes, was performed using gene panel sequencing on the Ion Torrent PGM™ Dx System. The best candidate disease-causing variants were confirmed by Sanger sequencing.
Results:
Several variants of the HHA-associated genes were detected in 10 out of 14 suspected HHA individuals. After excluding those variants predicted to be benign, 10 pathogenic variants and 1 variant of uncertain significance (VUS) were confirmed in 10 individuals with suspected HHA. Of these variants, the p.Trp704Ter nonsense variant of EPB41 and missense p.Gly151Asp variant of SPTA1 were identified in two out of four hereditary elliptocytoses. The frameshift p.Leu884GlyfsTer27 variant of ANK1, nonsense p.Trp652Ter variant of the SPTB, and missense p.Arg490Trp variant of PKLR were detected in all four hereditary spherocytosis cases. Missense p.Glu27Lys, nonsense p.Lys18Ter variants, and splicing errors such as c.92 + 1G > T and c.315 + 1G > A within HBB were identified in four beta thalassemia cases.
Conclusions:
This study provides a snapshot of the genetic alterations in a cohort of Korean HHA individuals and demonstrates the clinical utility of using gene panels in HHA. Genetic results can provide precise clinical diagnosis and guidance regarding medical treatment and management for some individuals.
... The arrival of recent advancements in next-generation sequencing (NGS) technology has proven to be highly reliable, faster, and cost-effective, allowing us to monitor multiple genes simultaneously. Moreover, NGS can be helpful in diagnosis where systematic laboratory studies seem to be inconclusive in newborn infants and transfusiondependent cases (Del Orbe Barreto et al. 2016;Agarwal et al. 2016;Kim and Kim 2017;He et al. 2017;Aggarwal et al. 2020;Andolfo et al. 2021). In this study, we present 26 HS patients detected with 21 ANK1 variants, including 13 novel variants by NGS technology, and studied the spectrum of those ANK1 variants. ...
Hereditary Spherocytosis (HS) is a common cause of hemolytic anemia varying from mild to severe hemolysis due to defects in red cell membrane protein genes, namely ANK1, SPTB, SPTA1, SLC4A1, and EPB42. These genes are considerably very large spaning 40–50 exons making gene-by-gene analysis costly and laborious by conventional methods. In this study, we explored 26 HS patients harboring 21 ANK1 variants identified by next-generation sequencing (NGS), characteristics and spectrum of the detected ANK1variants were analyzed in this study. Clinically, all the HS patients showed moderate to severe transfusion-dependent hemolytic anemia, some requiring splenectomy. We identified 13 novel and 8 reported variants, mainly 9 frameshifts, 2 missense, 6 nonsense, and 4 splice site ANK1 variants, using NGS technology. Frameshifts were remarkably the most common variant type seen in Indian HS patients with ANK1 gene defects. We have also explored expression levels of red cell membrane ankyrin protein by flow cytometry in 14 HS patients with ANK1 gene defects and a significant reduction in ankyrin protein expression has been found. This report mainly illustrates the molecular and phenotypic heterogeneity of ANK1 variants causing HS in Indian patients. Ankyrin-1 mutations are a significant cause of loss of function in dominant HS in the Indian population. Comprehensive genetic and phenotypic evaluation assists in implementing the knowledge of genetic patterns and spectrum of ANK1 gene variants, providing molecular support for HS diagnosis.
... hereditary spherocytosis (HS) is the most common in the Caucasian population [4,5]. This group of diseases includes erythrocyte membranopathies, hemoglobinopathies, and enzymopathies [5][6][7]. The molecular defects are highly heterogeneous involving many different genes, and the severity of the disease varies widely, from fully compensated to transfusion-dependent anemia [4,5,8]. ...
... nonsense, splicing site, and frameshift), which allowed us to select 2533 variants (Additional file 1:Table S1.7). Particularly interesting for us were genes associated with clinical phenotypes of known inherited hemolytic anemia[6], including erythrocyte membranopathies (ANK1, SPTB, SPTA1, SLC4A1, EPB42, EPB41, PIEZO1, KCNN4, RHAG), erythrocyte enzymopathies(G6PD, PKLR, ENO1, AK1, GPI, NT5C3A, GCLC, GPX1, GSR, GSS, HK1, BPGM, PGK1, TPI1), and erythrocyte hemoglobinopathies(HBB, HBA1, HBA2). Known polymorphisms have been detected in 14 genes involved in known erythrocyte pathologies, including 8 functional variants with potentially large pathological effects (Additional file 1:Table S1.8). ...
Pyrimidine 5′-nucleotidase deficiency is a rare erythrocyte enzymopathy. Here we report two cases of hemolytic anemia in brothers of Polish origin that are associated with a very rare mutation. Heterozygous deletion in the NT5C3A gene (c.444_446delGTT), inherited most likely from their asymptomatic mother, resulted in a single amino acid residue deletion (p.F149del) in cytosolic pyrimidine 5′-nucleotidase. However, only the mutated transcript was present in the reticulocyte transcriptome of both patients. Only residual activity of pyrimidine 5′-nucleotidase in the brothers’ erythrocytes could be observed when compared with the controls, including their asymptomatic father and sister. Western blot showed no sign of the presence of 5′-nucleotidase protein in the erythrocytes of both studied patients. The 2.5-fold reduction of the purine/pyrimidine ratio observed only in the brothers’ erythrocytes confirms the correlation of the results of molecular analysis, including whole-exome sequencing, with the phenotype of the pyrimidine 5′-nucleotidase deficiency. Altogether, our results may substantiate the hypothesis of the heterogeneity of the molecular basis of the defect involving both the mutation presented here and negative regulation of expression of the “normal” allele.
... The present study describes an HS pedigree with four family members involved. According to related guidelines (21,22), whole-exome sequencing was performed in the patient and a novel ANK1 mutation, c.4707G>A (p.Trp1569*), was identified. In recent years, an increasing number of novel mutations related to HS have been identified with second-generation sequencing (23). ...
... However, it is not widely available for the clinical diagnosis of HS in mainland China. Therefore, the combination of EMA testing and second-generation sequencing is recommended in the Chinese and South Korean guidelines for HS diagnosis (21,22). In the present case, EMA testing and second-generation sequencing helped to diagnose HS. ...
The present study describes the clinical profile and ankyrin 1 (ANK1) mutation status of a Chinese family with hereditary spherocytosis (HS). A young male patient (proband) was diagnosed with HS after presenting with anaemia and jaundice. The Coombs test was negative and spherocytes were found in peripheral blood smears. Magnetic resonance imaging showed splenomegaly and splenic iron depositions. The red blood cell osmotic fragility test was positive. The eosin-5'-maleimide binding test showed reduced mean channel fluorescence. Whole-exome sequencing revealed a novel ANK1 mutation (c.4707G>A), resulting in a nonsense mutation (p.Trp1569*). The patient's father, paternal aunt and paternal grandmother exhibited comparable clinical symptoms and Sanger sequencing confirmed the same mutation in these family members. To the best of our knowledge, an HS pedigree with this novel ANK1 nonsense mutation has not been previously reported. At the same time, the unique clinical presentation of this pedigree helps our understanding of the heterogeneity of clinical manifestations of HS.
