Could the 14q23.2 microdeletion or AKAP5 haploinsufficiency be a potential cause of intellectual disability?

Intellectual disability is characterized by impairment in at least two of the following areas: social skills, communication skills, self-care tasks, and academic skills. These impairments are evaluated in relation to the expected standards based on the individual's age and cultural levels. Additionally, intellectual disability is typically defined by a measurable level of intellectual functioning, represented by an intelligence quotients core of 70 or below. Autism spectrum disorder is a developmental disability resulting from differences in the brain, often characterized by problems in social communication and interaction, and limited or repetitive behaviors or interests. Hereditary spherocytosis is a disease characterized by anemia, jaundice, and splenomegaly as a result of increased tendency to hemolysis with morphological transformation of erythrocytes from biconcave disc-shaped cells with central pallor to spherocytes lacking central pallor due to hereditary injury of cellular membrane proteins. An 11-year-old female patient was referred to Pediatric Genetics Subdivision due to the presence of growth retardation and a diagnosis of hereditary spherocytosis. Since she also had dysmorphic facial features, such as frontal bossing, broad and prominent forehead, tubular nasal structure, and thin vermillion, genetic tests were performed. Chromosomal microarray analysis revealed a 2.5 Mb deletion in the 14q23.2q23.3 region. Deletion was also identified in the same region in her father, who had the same phenotypic characteristics, including hereditary spherocytosis and learning difficulties. We propose that the PLEKHG3 and AKAP5 genes, which are located in this region, may contribute to the development of intellectual disability.

Reticulocyte Antioxidant Enzymes mRNA Levels versus Reticulocyte Maturity Indices in Hereditary Spherocytosis, ß-Thalassemia and Sickle Cell Disease.

The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and peroxiredoxin 2 (Prx2) are particularly important in erythroid cells. Reticulocytes and other erythroid precursors may adapt their biosynthetic mechanisms to cell defects or to changes in the bone marrow environment. Our aim was to perform a comparative study of the mRNA levels of CAT, GPX1, PRDX2 and SOD1 in reticulocytes from healthy individuals and from patients with hereditary spherocytosis (HS), sickle cell disease (SCD) and ß-thalassemia (ß-thal), and to study the association between their transcript levels and the reticulocyte maturity indices. In controls, the enzyme mRNA levels were significantly correlated with reticulocyte maturity indices for all genes except for SOD1. HS, SCD and ß-thal patients showed younger reticulocytes, with higher transcript levels of all enzymes, although with different patterns. ß-thal and HS showed similar reticulocyte maturity, with different enzyme mRNA levels; SCD and HS, with different reticulocyte maturity, presented similar enzyme mRNA levels. Our data suggest that the transcript profile for these antioxidant enzymes is not entirely related to reticulocyte maturity; it appears to also reflect adaptive mechanisms to abnormal erythropoiesis and/or to altered erythropoietic environments, leading to reticulocytes with distinct antioxidant potential according to each anemia.

Coinheritance of hereditary spherocytosis with haemochromatosis: next-generation sequencing reveals.

We report the case of a man in his 50s with extravascular haemolysis, fluctuating indirect hyperbilirubinaemia, elevated transferrin saturation with hyperferritinaemia and normal liver enzymes. Spherocytes were detected in a blood smear and a mutation of unknown significance, c.1626+1G>A p.?, in intron 13 of the SLC4A1 gene, was identified by next-generation sequencing (NGS). The same mutation was found in his daughter, who presented with similar laboratory changes, confirming the diagnosis of hereditary spherocytosis. Abdominal MRI showed hepatosplenomegaly with hepatic iron overload. In this context of haemolysis (without anaemia) and iron overload, a diagnosis of haemochromatosis was presumed. NGS confirmed the presence of the variants p.(His63Asp) and p.(Cys282Tyr) in heterozygosity in the HFE gene. We report this case for the rarity of co-existing two haematological diseases counteracting each other.

The Correlation Between Clinical Phenotype and Genotype of Hereditary Spherocytosis.

Objective: Hereditary spherocytosis (HS) is a common hereditary hemolytic disease. This study aimed to explore the correlation between the phenotype and mutant genotype of HS to improve the clinical understanding of HS. Methods: This study reported a case of spontaneous mutation of the ANK1 gene in HS, reviewed previous studies on the genotype-phenotype correlation of HS, statistically analyzed the main types of gene mutations in HS, and summarized the clinical data of patients. Results: This patient had clinical manifestations of anemia, splenomegaly, peripheral blood smear with increased spherocytosis, and bilirubin, confirmed as ANK1 gene mutant HS by gene detection. In addition, this study included 14 previous studies on genotype-phenotype correlation, collected data, and determined that the ANK1 and SPTB genes were the most common types of gene mutations in HS patients. The mutant HS of the ANK1 gene would lead to lower hemoglobin levels. Conclusion: The results of this study showed that ANK1 and SPTB were the most common types of gene mutations in HS patients. Compared with patients with the SPTB genotype HS, patients with ANK1 mutant HS had more severe extravascular hemolysis, and a higher proportion needed splenectomy in early childhood.

Molecular characteristics of hereditary red blood cell membrane disorders in Thailand: a multi-center registry.

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 2011 and 2020 from seven 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.

Hereditary Spherocytosis: Can Next-Generation Sequencing of the Five Most Frequently Affected Genes Replace Time-Consuming Functional Investigations?

Congenital defects of the erythrocyte membrane are common in northern Europe and all over the world. The resulting diseases, for example, hereditary spherocytosis (HS), are often underdiagnosed, partly due to their sometimes mild and asymptomatic courses. In addition to a broad clinical spectrum, this is also due to the occasionally complex diagnostics that are not available to every patient. To test whether next-generation sequencing (NGS) could replace time-consuming spherocytosis-specific functional tests, 22 consecutive patients with suspected red cell membranopathy underwent functional blood tests. We were able to identify the causative genetic defect in all patients with suspected HS who underwent genetic testing (n = 17). The sensitivity of the NGS approach, which tests five genes (ANK1 (gene product: ankyrin1), EPB42 (erythrocyte membrane protein band4.2), SLC4A1 (band3), SPTA1 (α-spectrin), and SPTB (ß-spectrin)), was 100% (95% confidence interval: 81.5-100.0%). The major advantage of genetic testing in the paediatric setting is the small amount of blood required (<200 µL), and compared to functional assays, sample stability is not an issue. The combination of medical history, basic laboratory parameters, and an NGS panel with five genes is sufficient for diagnosis in most cases. Only in rare cases, a more comprehensive functional screening is required.

[Clinical and genotypic analysis of hereditary spherocytosis combined with cholestasis among pediatric patients].

Objective: To understand the clinical and genetic characteristics of hereditary spherocytosis (HS) combined with cholestasis among pediatric patients. Methods: 12 cases of HS children accompanied by cholestasis at Hunan Children's Hospital were selected as the research subjects between January 2013 and December 2022. Clinical data were collected. Whole-exome sequencing was performed by second-generation sequencing. Suspected pathogenic mutation sites were verified by Sanger sequencing. Results: All pediatric patients were admitted to the hospital due to their yellow skin tone. Eight cases (66.67%) had a positive family history. The clinical manifestations were jaundice, splenomegaly (12/12), abdominal pain, anemia (4/12), and hepatomegaly (5/12). All pediatric patients had decreased hemoglobin, an increased reticulocyte ratio, total bilirubin and direct bilirubin, a positive erythrocyte fragility test, and remarkable spherical erythrocytes in their peripheral blood. Seven cases had elevated aminotransferase; four cases had severely elevated aminotransferase and bilirubin; eight cases had biliary calculi; and two cases had a dilated biliary tract. Liver pathological examination showed mild damage to the liver cells (G1S1) in three pediatric cases. Five children had a total of six unreported mutations: SPTB gene c.2431_2450del, c.4974-2A > G, c.2575G > A, and exon 22-35 deletion; ANK1 gene: c.2379-2380delC; and c .6dupC. Children still had abnormal bilirubin levels following treatment. Two pediatric cases underwent splenectomy. Bilirubin and hemoglobin levels returned to normal after surgery. Conclusion: Children with HS may experience cholestasis, and those with poor treatment results may consider undergoing a splenectomy. Six new types of variants have expanded the HS gene mutation spectrum.

Mitapivat reprograms the RBC metabolome and improves anemia in a mouse model of hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common, nonimmune, hereditary, chronic hemolytic anemia after hemoglobinopathies. The genetic defects in membrane function causing HS lead to perturbation of the RBC metabolome, with altered glycolysis. In mice genetically lacking protein 4.2 (4.2-/-; Epb42), a murine model of HS, we showed increased expression of pyruvate kinase (PK) isoforms in whole and fractioned RBCs in conjunction with abnormalities in the glycolytic pathway and in the glutathione (GSH) system. Mitapivat, a PK activator, metabolically reprogrammed 4.2-/- mouse RBCs with amelioration of glycolysis and the GSH cycle. This resulted in improved osmotic fragility, reduced phosphatidylserine positivity, amelioration of RBC cation content, reduction of Na/K/Cl cotransport and Na/H-exchange overactivation, and decrease in erythroid vesicles release in vitro. Mitapivat treatment significantly decreased erythrophagocytosis and beneficially affected iron homeostasis. In mild-to-moderate HS, the beneficial effect of splenectomy is still controversial. Here, we showed that splenectomy improves anemia in 4.2-/- mice and that mitapivat is noninferior to splenectomy. An additional benefit of mitapivat treatment was lower expression of markers of inflammatory vasculopathy in 4.2-/- mice with or without splenectomy, indicating a multisystemic action of mitapivat. These findings support the notion that mitapivat treatment should be considered for symptomatic HS.

Next generation sequencing (NGS) interest in deciphering erythrocyte molecular defects' association in red cell disorders: Clinical and erythrocyte phenotypes of patients with mutations inheritance in PIEZO1, Spectrin ß1, RhAG and SLC4A1.

We report here an instructive case referred at 16 months-old for exploration of hemolysis without anemia (compensated anemia with reticulocytosis). The biology tests confirmed the hemolysis with increased total and indirect bilirubin. The usual hemolysis diagnosis tests were normal (DAT, G6PD, PK, Hb electrophoresis) except cytology and ektacytometry suggesting an association of multiple red blood cell (RBC) membrane disorders. This led us to propose a molecular screening analysis using targeted-Next Generation Sequencing (t-NGS) with a capture technique on 93 genes involved in RBC and erythropoiesis defects. We identified 4 missense heterozygous allelic variations, all of them were described without any significance (VUS) in the SLC4A1, RhAG, PIEZO1 and SPTB genes. The study of the familial cosegregation and research functional tests allowed to decipher the role of at least two by two genes in the phenotype and the hemolytic disease of this young patient. Specialized t-NGS panel (or virtual exome/genome sequencing) in a disease-referent laboratory and the motivated collaboration of clinicians, biologists and scientists should be the gold standard for improving the diagnosis of the patients affected with RBC diseases or rare inherited anemias.

Genotype-degree of hemolysis correlation in hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is a common inherited hemolytic anemia, caused by mutations in five genes that encode erythrocyte membrane skeleton proteins. The red blood cell (RBC) lifespan could directly reflect the degree of hemolysis. In the present cohort of 23 patients with HS, we performed next-generation sequencing (NGS) and Levitt's carbon monoxide (CO) breath test to investigate the potential genotype-degree of hemolysis correlation. RESULTS: In the present cohort, we identified 8 ANK1,9 SPTB,5 SLC4A1 and 1 SPTA1 mutations in 23 patients with HS, and the median RBC lifespan was 14(8-48) days. The median RBC lifespan of patients with ANK1, SPTB and SLC4A1 mutations was 13 (8-23), 13 (8-48) and 14 (12-39) days, respectively, with no statistically significant difference (P = 0.618). The median RBC lifespan of patients with missense, splice and nonsense/insertion/deletion mutations was 16.5 (8-48), 14 (11-40) and 13 (8-20) days, respectively, with no significant difference (P = 0.514). Similarly, we found no significant difference in the RBC lifespan of patients with mutations located in the spectrin-binding domain and the nonspectrin-binding domain [14 (8-18) vs. 12.5 (8-48) days, P = 0.959]. In terms of the composition of mutated genes, 25% of patients with mild hemolysis carried ANK1 or SPTA1 mutations, while 75% of patients with mild hemolysis carried SPTB or SLC4A1 mutations. In contrast, 46.7% of patients with severe hemolysis had ANK1 or SPTA1 mutations and 53.3% of patients with severe hemolysis had SPTB or SLC4A1 mutations. However, there was no statistically significant difference in the distribution of mutated genes between the two groups (P = 0.400). CONCLUSION: The present study is the first to investigate the potential association between genotype and degree of hemolysis in HS. The present findings indicated that there is no significant correlation between genotype and degree of hemolysis in HS.

[Clinical and gene mutation characteristics of patients with hereditary ellipsocytosis: nine cases report and literature review].

Objective: To report gene mutations in nine patients with hereditary elliptocytosis (HE) and analyze the characteristics of pathogenic gene mutations in HE. Methods: The clinical and gene mutations of nine patients clinically diagnosed with HE at Institute of Hematology & Blood Diseases Hospital from June 2018 to February 2022 were reported and verified by next-generation sequencing to analyze the relationship between gene mutations and clinical phenotypes. Results: Erythrocyte membrane protein gene mutations were detected among nine patients with HE, including six with SPTA1 mutation, one with SPTB mutation, one with EPB41 mutation, and one with chromosome 20 copy deletion. A total of 11 gene mutation sites were involved, including 6 known mutations and 5 novel mutations. The five novel mutations included SPTA1: c.1247A>C (p. K416T) in exon 9, c.1891delG (p. A631fs*17) in exon 15, E6-E12 Del; SPTB: c.154C>T (p. R52W) ; and EPB41: c.1636A>G (p. I546V) . Three of the six patients with the SPTA1 mutation were SPTA1 exon 9 mutation. Conclusion: SPTA1 is the most common mutant gene in patients with HE.

A de novo ANK1 mutation in a childhood hereditary spherocytosis: a case report.

BACKGROUND: Due to the heterogeneity of the phenotype of Hereditary spherocytosis (HS) patients, some patients may have rare clinical complications such as biliary obstruction and ultra-high bilirubinemia. CASE PRESENTATION: A 8-y-old boy presented to the emergency with complaints of anemia for 6 years and worsened abdominal pain and scleral yellowing of the skin for 2 days. Physical examination showed tenderness in the middle and upper abdomen and splenomegaly. Abdominal CT revealed biliary obstruction. Genetic analysis revealed a de novo mutation in the gene ANK1, HS with biliary obstruction was diagnosed. The surgery of bile duct exploration and T-tube drainage, and splenectomy were performed successively. This patient was followed up for 13 months after splenectomy, and his condition was stable. CONCLUSION: The diagnosis of HS is not clinically difficult, and once a patient with HS is diagnosed, regular follow-up management and standardized treatment are required. Genetic testing is also needed to screen for other genetic disorders that may co-exist in patients with HS who do not have a good efficacy or who have a long-term chronic onset of jaundice.

Splenectomy improves erythrocyte functionality in spherocytosis based on septin abundance, but not maturation defects.

Splenectomy improves the clinical parameters of patients with hereditary spherocytosis, but its potential benefit to red blood cell (RBC) functionality and the mechanism behind this benefit remain largely overlooked. Here, we compared 7 nonsplenectomized and 13 splenectomized patients with mutations in the ß-spectrin or the ankyrin gene. We showed that hematological parameters, spherocyte abundance, osmotic fragility, intracellular calcium, and extracellular vesicle release were largely but not completely restored by splenectomy, whereas cryohemolysis was not. Affected RBCs exhibited decreases in ß-spectrin and/or ankyrin contents and slight alterations in spectrin membrane distribution, depending on the mutation. These modifications were found in both splenectomized and nonsplenectomized patients and poorly correlated with RBC functionality alteration, suggesting additional impairments. Accordingly, we found an increased abundance of septins, small guanosine triphosphate-binding cytoskeletal proteins. Septins-2, -7, and -8 but not -11 were less abundant upon splenectomy and correlated with the disease severity. Septin-2 membrane association was confirmed by immunolabeling. Except for cryohemolysis, all parameters of RBC morphology and functionality correlated with septin abundance. The increased septin content might result from RBC maturation defects, as evidenced by (1) the decreased protein 4.2 and Rh-associated glycoprotein content in all patient RBCs, (2) increased endoplasmic reticulum remnants and endocytosis proteins in nonsplenectomized patients, and (3) increased lysosomal and mitochondrial remnants in splenectomized patients. Our study paves the way for a better understanding of the involvement of septins in RBC membrane biophysical properties. In addition, the lack of restoration of septin-independent cryohemolysis by splenectomy may call into question its recommendation in specific cases.

[Genetic Analysis of a Chinese Pedigree with Hereditary Spherocytosis Caused by Copy Number Variation Deletion of SPTB Gene].

OBJECTIVE: To investigate the molecular mechanism of the disease based on the clinical characterization and genetic mutation analysis in a family with hereditary spherocytosis. METHODS: The proband with jaundice and anemia was referred to Yidu Central Hospital of Weifang in May 2021. Peripheral blood samples were collected from six members of the family. Second-generation sequencing was used to screen the pathological mutations, and the clinically significant variant sites were selected. Then the relevant databases were used to analyze the variant sites, and RT-qPCR was used to detect the relative mRNA levels of candidate gene. The structure and function of SPTB protein were analyzed by UniProt and SMART databases. RESULTS: We infer that the SPTB gene copy number variation (CNV) deletion was co-segregated with the phenotype of the patients in this family based on the results of second-generation sequencing (about 700 target genes). The UCSC Genome Browser demonstrated that the deleted region was mainly located in exon2-3 of SPTB gene. The results of RT-qPCR showed that the relative SPTB mRNA levels of all patients were lower than the healthy control. UniProt and SMART databases analysis showed that SPTB protein without CH1 and CH2 domains could not bind to erythrocyte membrane actin. CONCLUSION: The CNV deletion of SPTB gene may be the reason for the hereditary spherocytosis in this family.

Clinical utility of targeted next-generation sequencing panel in routine diagnosis of hereditary hemolytic anemia: A national reference laboratory experience.

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)7 TAA (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.

[Analysis of the characteristics of SPTB gene variants among 16 children with Hereditary spherocytosis].

OBJECTIVE: To analyze the clinical characteristics and spectrum of SPTB gene variants among 16 Chinese children with Hereditary spherocytosis (HS) and explore their genotype-phenotype correlation. METHODS: Sixteen children who were diagnosed with HS at the Affiliated Hospital of Capital Institute of Pediatrics from November 2018 to July 2022 were selected as the research subjects. Genetic testing was carried out by whole exome sequencing. Candidate variants were verified by Sanger sequencing and subjected to bioinformatic analysis and prediction of 3D structure of the protein. Correlation between the SPTB genotypes and clinical phenotypes was analyzed using Chi-squared test. RESULTS: The male-to-female ratio of the HS patients was 6 : 10, with the median age being 7-year-and-10-month. Clinical features of the patients have included anemia, reticulocytosis and gradual onset of splenomegaly. Mild, moderate and severe anemia have respectively occurred in 56.25% (9/16), 31.25% (5/16) and 12.50% (2/16) of the patients. SPTB gene variants were detected in all patients, among which 10 were unreported previously and 7 were de novo in origin. Loss of function (LOF) variants accounted for 93.75% (15/16). Only one missense variant was detected. Eleven, 4 and 1 of the variants had occurred in the repeat domain, CH1 domain, and dimerization domain, respectively. There was no significant correlation between the type or domain of the SPTB gene variants with the clinical features such as severity of anemia (x² = 3.345, P > 0.05). All of the variants were predicted to be pathogenic or likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION: Mild to moderate anemia are predominant clinical features of the HS children harboring a SPTB gene variant, for which LOF variants are the main mutational type. The clinical feature of HS is unaffected by the type of the variants.

De novo variations of ANK1 gene caused hereditary spherocytosis in two Chinese children by affecting pre-mRNA splicing.

BACKGROUND AND AIMS: Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic disorders. Here, two unrelated families with the probands displaying typical manifestations of HS were enrolled. Our study aimed to characterize the effect of two novel variants in HS patients on gene splicing to help minimize the rate of misdiagnosis of HS and enhance clinicians' understanding of the disease. PARTICIPANTS AND METHODS: A retrospective review was conducted. Peripheral blood samples were collected from all the family members, and genomic DNA was extracted for genetic diagnostics. First, high-throughput sequencing technology was used for the preliminary screening of candidate causative variants. Thereafter, the variants were verified via Sanger sequencing. Furthermore, a pathogenicity analysis of the detected variants was performed including in silico prediction and in vitro experiments. We constructed matched wild-type and mutant-type minigene plasmid of ANK1 based on HEK293T cells to address the effects of variants on mRNA splicing. RESULTS: The c.1305 + 2 T > A (family1) and c.1305 + 2del (family2) variants were detected in the ANK1 gene. These two de novo mutations described by us which have not been reported prior to this study. Moreover, the validation results of splicing reporter systems revealed that the intronic mutations resulted in abnormal pre-mRNA splicing. Specifically, the minigene plasmid expressing the c.1305 + 2 T > A variant transcribed the two aberrant transcripts: r.1305_1306ins1305 + 1_1305 + 229 and r.1305_1306ins1305 + 1_1305 + 552. The minigene plasmid expressing c.1305 + 2del transcribed the two aberrant transcripts: r.1305_1306ins1305 + 1_1305 + 228 and r.1305_1306ins1305 + 1_1305 + 551. CONCLUSION: The two de novo variants identified in the ANK1 gene were the genetic etiology of the probands with HS in our study. Our findings further enrich the HS genotype database and provide a basis for genetic counselling and molecular diagnosis.

Targeted next-generation sequencing identifies novel deleterious variants in ANK1 gene causing severe hereditary spherocytosis in Indian patients: expanding the molecular and clinical spectrum.

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.

A large family of hereditary spherocytosis and a rare case of hereditary elliptocytosis with a novel SPTA1 mutation underdiagnosed in Taiwan: A case report and literature review.

RATIONALE: Hereditary spherocytosis (HS) has a defect in the vertically connected proteins on the cell membrane of red blood cells (RBC). Hereditary elliptocytosis (HE) has a defect in proteins that connect the cell membrane horizontally. We reported two families of RBC membrane disorders in Taiwanese, one was HS and the other was HE. PATIENT CONCERNS: Case 1. A 19-year-old male student with chronic jaundice and splenomegaly. His mother, maternal uncle, grandmother, and many members of older generations also had splenomegaly and underwent splenectomy. Case 2. A 40-year-old man has experienced pallor and jaundice since the age of 20 and was found to have splenomegaly, and gall bladder stones in the older age. His younger sister also had pallor and jaundice for a long time. DIAGNOSES: In case 1, a peripheral blood smear showed 20% spherocytes. Eosin-5-maleimide labeled RBC by flow cytometry showed a result of 30.6 MCF (cutoff value: 45.5 MCF). He was diagnosed with HS. The gene analysis identified a heterozygous mutation with c.166A > G (p.Lys56Glu) in the SLC4A1 gene in this proband, his mother, and maternal uncle. In case 2, more than 40% of ellipsoid RBC present in the peripheral blood smear. He was diagnosed with HE. Genetic analysis of the SPTA1 gene identified a novel heterozygous exon2, c.86A > C, p.Gln29Prol mutation. INTERVENTIONS: The two patients had compensated anemia, clinical follow-up instead of splenectomy was done. OUTCOMES: The two patients had normal daily activities and lives. LESSONS: We reported two Taiwanese families, one was hereditary spherocytosis affected by a heterozygous mutation with c.166A > G (p.Lys56Glu) in SLC4A1, and the other was hereditary elliptocytosis caused by a novel heterozygous SPTA1 gene mutation, c. 86A > C, p.Gln29Prol. These 2 seemingly common hereditary red blood cell membrane protein defects induced by hemolysis are usually underdiagnosed or misdiagnosed.