Dominant Hereditary Spherocytosis Research Articles

Autosomal Dominant Hereditary Spherocytosis In An Elderly Patient

Hereditary spherocytosis (HS) is an autosomal dominant inherited hemolytic anemia that usually manifests in early adolescence. A very small proportion (5%) may undergo de novo mutations and present at an elderly age without any positive family history. The disease is characterized by anemia, jaundice, splenomegaly, and the presence of spherocytes in peripheral blood, which are osmotically fragile. A 55-year-old elderly male presented with generalized weakness and a history of repeated blood transfusions for 5 months. He had recurrent jaundice for 8 years. There was no relevant family or drug history. He was anemic, icteric, and had mild splenomegaly. Investigation revealed persistently low Hb with normal red cell indices. There were a lot of spherocytes with polychromasia on the peripheral blood film. There was also evidence of hemolysis with a high reticulocyte count and higher levels of total and indirect bilirubin. Autoimmune hemolysis was excluded by negative direct and indirect Coomb’s tests, and Hb defect was excluded by normal Hb electrophoresis. We also ruled out other infectious causes. The ultrasound confirmed splenomegaly. The osmotic fragility test showed increased osmotic fragility of the patient’s red cells. History, symptoms, and test results strongly point to autosomal dominant hereditary spherocytosis with new mutations. An eosin-5-Maleimide binding test was advised, along with family screening of the patient. Bangladesh J Medicine 2024; 35: 110-113

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.

Hereditary Spherocytosis Due to a Novel Variant, P.Q1034X, in the Beta Subunit of the Spectrin Gene

Introduction: Hereditary Spherocytosis (HS) is the most common red cell membrane disorder. 25-30% of cases involve the SPTB gene which encodes for β-Spectrin, a protein that maintains red blood cell shape. Heterozygous variants in SPTB are associated with autosomal dominant HS and elliptocytosis. While genetic testing is not routinely done to confirm HS, it is useful in atypical presentations. Case Description: A 1 week old male presented to the pediatric hematology/oncology clinic for anemia. He was born late preterm and had a history of hyperbilirubinemia requiring phototherapy, failure to thrive, and developmental delay. On examination, he was noted to have hypotonia. There was no known family history of hematologic problems. Based on this constellation of signs and symptoms, he had a comprehensive hematologic and genetic workup. On lab evaluation, his peripheral blood smear showed normocytic normochromic red blood cells with some spherocytes, significant polychromasia, normal WBC and normal platelet morphology. His newborn screen was normal, direct coombs' negative, osmotic fragility test was positive, and protein band 3 reduction was abnormal. His abdominal ultrasound was normal. Whole exome sequencing with variant segregation analysis was significant for heterozygosity of the p.Q1034X variant of the SPTB gene. This variant in the SPTB gene has not been previously reported. Discussion: We found a novel, de novo variant in an infant with HS through whole exome sequencing. This variant is predicted to cause loss of normal protein function either through protein truncation or non-mediated mRNA decay resulting in fragile red blood cells. While neither parent was found to carry this mutation, germline mosaicism should not be excluded. Physicians should be aware that prenatal diagnosis is available to address the risk of recurrence in future pregnancies.

Targeted next-generation sequencing identified novel mutations associated with hereditary anemias in Brazil

Hereditary anemias are a group of heterogeneous disorders including hemolytic anemias and hyporegenerative anemias, as congenital dyserythropoietic anemia (CDA). Causative mutations occur in a wide range of genes leading to deficiencies in red cell production, structure, or function. The genetic screening of the main genes is important for timely diagnosis, since routine laboratory tests fail in a percentage of the cases, appropriate treatment decisions, and genetic counseling purposes. A conventional gene-by-gene sequencing approach is expensive and highly time-consuming, due to the genetic complexity of these diseases. To overcome this problem, we customized a targeted sequencing panel covering 35 genes previously associated to red cell disorders. We analyzed 36 patients, and potentially pathogenic variants were identified in 26 cases (72%). Twenty variants were novel. Remarkably, mutations in the SPTB gene (β-spectrin) were found in 34.6% of the patients with hereditary spherocytosis (HS), suggesting that SPTB is a major HS gene in the Southeast of Brazil. We also identified two cases with dominant HS presenting null mutations in trans with α-LELY in SPTA1 gene. This is the first comprehensive genetic analysis for hereditary anemias in the Brazilian population, contributing to a better understanding of the genetic basis and phenotypic consequences of these rare conditions in our population.

Rapid Identification of Biallelic SPTB Mutation in a Neonate with Severe Congenital Hemolytic Anemia and Liver Failure

Heterozygous pathogenic variants in SPTB cause autosomal dominant hereditary spherocytosis, an important cause of neonatal nonimmune hemolytic anemia. Biallelic mutations are rarely reported, all with severe neonatal presentation. We describe rapid (68 h) genomic diagnosis of homozygous β-spectrin deficiency in a newborn with severe transfusion-dependent hemolytic anemia, conjugated hyperbilirubinemia, and progressive liver failure. Trio whole-exome sequencing identified a novel biallelic SPTB variant (c.6119C>T; p.Thr2040Ile) located in the critical spectrin repeat region. Pretransfusion blood film showed marked spherocytosis including microspherocytes and nucleated erythrocytes, and eosin-5-maleimide (E5M) staining was markedly reduced, supporting pathogenicity. Both asymptomatic heterozygous parents demonstrated mildly reduced E5M staining, with occasional spherocytes and elliptocytes. Early molecular diagnosis facilitated hypertransfusion to suppress ineffective erythropoiesis and reverse hepatic dysfunction. This report broadens the genotypic and phenotypic spectrum of spectrin deficiency and highlights the utility of rapid genomic testing in facilitating early diagnosis and informing targeted therapy in critically ill patients.

Altered Splicing from a Mutated Alternate Branch Point Is Common in Severe Alpha-Spectrin Linked Inherited Anemia

Hereditary spherocytosis (HS), the most common inherited hemolytic anemia in Northern Europeans, is dominantly inherited in ~two third of cases. Clinically, patients with nondominant HS (ndHS) are more severely affected than those with typical, dominant HS. Biochemical and genetic studies implicate defects of α-spectrin in most ndHS patients and in patients with the related disorder hereditary pyropoikilocytosis (HPP). However, in most cases, neither the precise genetic basis nor the mechanism of disease are known. We studied individuals from 23 kindreds: 10 ndHS kindreds, 3 HPP kindreds, and 10 kindreds with transfusion-dependent (TD) anemia, using whole exome sequencing. A variety of novel mutant SPTA1 alleles were identified, including nonsense, splicing, and insertion/deletion mutations, frequently in trans to missense mutations. One patient had no SPTA1 mutations and 14 patients had only one SPTA1 mutation. Patients with 0 or 1 mutation all carried the common ndHS-linked α-spectrinBug Hill allele. We hypothesized that a production-defective SPTA1 allele is shared by these patients and is in linkage disequilibrium with the αBug Hill allele. Whole genome sequencing was performed on 2 ndHS patients heterozygous for the αBug Hill variant. Data were compared to samples in the 1000 Genomes database with the αBug Hill variant. A series of genetic analyses revealed a single common SPTA1 variant, the αLEPRA allele. This variant, described in a ndHS patient in trans to an SPTA1 nonsense mutation (JCI 98:2300, 1996), was associated with an elongated α-spectrin mRNA transcript. The αLEPRA allele was only present on 4 of 4610 alleles in 1000 Genomes. In all 4 cases, it was heterozygous and in cis to the αBugHill allele. Analysis of αBugHill haplotypes revealed 3 predominant patterns with the haplotype of the ndHS patients identical to the 4 heterozygous αLEPRA individuals. Genotyping the mutation-negative patient alleles revealed all carried the αLEPRA mutation. In the original report, RT-PCR of reticulocyte RNA demonstrated the αLEPRA allele was associated with an elongated α-spectrin mRNA transcript originating 70nt from the 3' end of intron 30. It was unclear if or how the αLEPRA mutation influenced α-spectrin mRNA splicing, as identical elongated α-spectrin mRNA transcripts are observed in erythroid cells from patients who do not carry the αLEPRA allele. Splicing analysis of intron 30 using SCROOGLE predicted: 1) a branch point at the expected location 31bp 5' of exon 31 (branch point 1, BP1); 2) an alternate upstream branch point centered on an “A” 2bp 3' of the αLEPRA mutation (BP2) 98bp 5' of exon 31; 3) identified a novel alternate 3' acceptor site downstream of BP2; and 4) the αLEPRA mutation significantly improves BP2. To determine if BP2 is a functional BP, we studied SPTA1 splicing using minigene assays in K562 cells. Wild type (WT) minigenes produced a small amount of elongated α-spectrin mRNA, while mutation of the obligate “A” of BP2 completely eliminated elongated transcript production. Mutation of the BP2 “A” using CRISPRCas9-based gene editing on a WT background in K562 cells eliminated the elongated α-spectrin mRNA transcripts. Minigene assays also revealed deletion of the alternate 3' acceptor splice site or improvement of BP1 to a U2 consensus site both eliminated the elongated transcript. Thus the αLEPRA mutation in intron 30, located upstream of an alternate 3' acceptor site, changes a weak alternate BP to a strong BP in the context of a poor primary BP. These changes lead to increased utilization of an alternate 3'acceptor site, creating an elongated transcript that leads to frameshift with a novel termination codon. This termination codon is in a position predicted to activate nonsense mediated decay (NMD). To address whether NMD of the elongated transcript is the mechanism of α-spectrin deficiency, we created K562 cells homozygous for the αLEPRA allele using gene editing and treated them with emetine or cycloheximide, NMD inhibitors. In both WT and homozygous αLEPRA cells, the total amount of elongated transcript increased relative to WT. These studies resolve an important unanswered question by demonstrating a novel mechanism of genetic disease is responsible for many cases of ndHS, HPP, and TD anemia. These data will facilitate disease diagnosis, as most current diagnostic gene panels do not include this intronic region, and they identify a new target for therapeutic gene manipulation. DisclosuresNo relevant conflicts of interest to declare.

Genotype/Phenotype Correlations in 103 Children from 87 Families with Hereditary Spherocytosis

Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians. Patients with HS show a high degree of phenotypic variability from asymptomatic to transfusion dependence. Much of this variability stems from HS not being a single uniform disorder but instead being a collection of disorders involving mutations in 5 different genes (ANK1, SPTB, SPTA1, SLC4A1, and EPB42 ) encoding for the 5 major cytoskeletal proteins in red blood cells (ankyrin, β and α spectrin, band 3, and protein 4.2 respectively). These proteins are responsible for maintaining the biconcave disk morphology of red blood cells.Traditionally the diagnosis of HS has been made without genetic testing. We believe that knowledge of the HS subtype may impact future clinical management decisions, e.g. what type of splenectomy to perform-partial vs total and impact on genetic counseling needs. As a result we are now pursuing genetic testing on all our patients with HS.Over the past 16 years at Sick Kids Hospital (Toronto, Canada), we have followed 257 children with HS. In the past year we have also been offering genetic testing (following informed consent) to all children (<18 years old) still being followed in our center. DNA is sent to Prevention Genetics (Wisconsin, USA) where Next Generation sequencing is performed. Mutations are verified by Sanger sequencing.In total, we have genetic test results on 103 children from 87 families to date. Mutations in ANK1 were most common (50 children/43 families); followed by mutations in SPTB (32 children/26 families); SLC4A1 (7 children/7 families) and finally SPTA1 (6 children/3 families) EPB42 mutations were not found to be causative for HS in our cohort. In 8 children (8 families) no causative mutation for HS was found. In 23% of children multiple mutations were found -particularly heterozygous mutations of SPTA1 in combination with ANK1 or SPTB mutations.The majority of mutations in our patients were nonsense, frameshift, or splice site mutations. Most were novel - not previously described mutations, and unique to families. There were 5 exceptions to this; 1) c.4339-99C>T in SPTA1 (referred to as the αLEPRA mutation) in 4 children/2 families with an α spectrin form of HS, and in another 2 children (1 family) with an ankyrin form of HS; 2) an ANK1 c.5097-33G>A (8 children/6 families); 3) an ANK1 c.1405-9G>A (4 children/3 families); 4) SPTB c.6037C>T (3 children/2 families); and 5) SPTB c.5266C>T (3 children/2 families). The ANK1 c.5097-33G>A mutation had not been previously identified and yet was the most frequently detected mutation in our HS population.Most children (68/103) were found to have autosomal dominant (AD) HS: 33 children with the ankyrin subtype, 26 with the β spectrin subtype, 4 with the band 3 subtype and 5 in whom no mutation could be found but where there was a clear history of AD inheritance. Autosomal recessive (AR) inheritance was confirmed in 7 children - all 6 with α spectrin form of HS and 1 with a β spectrin form of HS. Twenty-eight children were spontaneous new mutations for HS: ANK (n=17), β spectrin (n=5), band 3 (n=3) and 3 in whom no mutation could be found. In all AR forms of HS, the index case had initially been thought to be a spontaneous new mutation for an AD form of HS; genetic testing resolved the inheritance pattern.Patients were categorized according to disease severity, primarily on the basis of their need for transfusions (a reflection of baseline hemoglobin) and splenectomy. The proportion of children that have required transfusions and needed splenectomies was: 83%/83% (α spectrin); 48%/18% (ankyrin), 23%/10% (β spectrin) and 29%/0% (band 3).Most children in our center undergoing splenectomy have undergone partial splenectomy; few have required subsequent total splenectomy. However, of the 5/6 children with α spectrin form of HS that underwent partial splenectomy 3 have subsequently needed, or are being considered for, total splenectomy. This suggests that due to the extreme severity of the α spectrin subtype of HS, children with this form of HS may not do well long-term with partial splenectomy and are likely to eventually require a total splenectomy.Our study to date represents one of the largest and most comprehensive genetic analyses of a cohort of HS patients. Our findings will add to the growing understanding of the disease, and will be important to provide comprehensive genetic counseling and possibly in the future to guide management of selected cases. DisclosuresDrury:Prevention Genetics: Employment.

Development Of a Comprehensive Rapid Next-Generation Sequencing Assay For The Diagnosis Of Inherited Hemolytic Anemia

Hereditary hemolytic anemia is caused by defects in hemoglobin, in the red blood cell (RBC) cytoskeleton proteins, or by deficiencies in RBC enzymes. RBC cytoskeletal disorders include hereditary spherocytosis (HS), elliptocytosis (HE), pyropoikilocytosis (HPP), and stomatocytosis (HSt), which are typically inherited as autosomal dominant disorders but can also present as recessive forms, frequently severe. The RBC enzymopathies, such as glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiency, are X-linked or recessively inherited disorders causing hemolytic anemia. Although rare, congenital dyserythropoietic anemias (CDA) are inherited red cell lineage disorders which occasionally, especially CDA-II, can be misdiagnosed as HS. Diagnosis of an inherited hemolytic anemia not due to a hemoglobin disorder is based upon morphology of the RBCs, functional analysis of the RBCs through ektacytometry or osmotic fragility, and enzymatic assays. The diagnosis of severely affected patients is complicated by transfusion dependence. Diagnosis based on genetic mutations is attractive, especially in transfused patients, and provides additional insight into the mechanisms of disease. Despite these advantages, genetic diagnoses have been limited by expense and long turn-around times for clinical results. To address these issues, we have developed a rapid comprehensive clinical next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. The protein-coding exons plus 25 bases of exon/intron junction as well as promoter sequences with known relationship to clinical phenotypes were included in the design. Genomic DNA was digested with a panel of 8 restriction enzymes and oligonucleotide probes were used to enrich the target regions. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Enrichment and sequencing were completed within 48 hours. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software.Initial validation included 5 affected probands, 1 affected sibling of a proband, 4 parental samples, and 2 unrelated control individuals with no history of hemolytic anemia. Overall, > 99% of all nucleotides in the regions of interest had at least 20X sequencing coverage. Our assay confirmed a previously identified maternally inherited nonsense mutation, Y1089X, and a paternally inherited A970D missense variant in SPTA1 in a patient (SPCA) with severe HS. The A970D SPTA1 missense change was also seen in two unaffected parents and an unaffected control, further validating that this missense variant alone does not cause dominant HS. The common SPTA1 allele, αLELY was seen in the clinically unaffected mother of SPCA, but was not found in the affected child, suggesting that this allele must be inherited in trans with a pathologic SPTA1 mutation to have clinical effect. Analysis of additional probands revealed both dominant and recessive forms of HS due to mutations in ANK1. Patient AAHS2 presented with typical dominant HS, and was found to have a novel ANK1 frameshift mutation, c.3464delG. Patient HEEM presented with severe transfusion-dependent anemia and was found to have one reported ANK1 missense mutation, T1075I (ankyrin Tubarao) in the spectrin-binding domain, and one novel nonsense mutation, Y735X. In addition, we identified novel amino acid changes in the newly identified dehydrated HSt gene PIEZO1. HEGR, a patient with hemolytic anemia, splenomegaly and portal vein thrombosis in infancy was found to have a novel 6 base pair insertion at R1462 in PIEZO1. Thal1 was previously diagnosed with dominant β-thalassemia, however had a more severe presentation than expected. We found a PIEZO1missense mutation R2302H in Thal1 that likely explains the severe phenotype.The initial validation of this comprehensive sequencing assay has demonstrated that this is a robust and rapid diagnostic tool for patients with severe hereditary hemolytic anemia. Simultaneous investigation of the key protein-coding genes involved in proper RBC function and survival, provides new insight into the variable phenotypes of patients with hemolytic anemia and ultimately will improve the management of patients with severe disease. Disclosures:No relevant conflicts of interest to declare.

Linkage Analysis of Hereditary Spherocytosis in Four Generations of a Family with SPTB Gene Deficiency

Hereditary Spherocytosis (HS) is an inherited hemolytic anemia caused by the defects on membrane proteins and characterized by icterus, anemia and splenomegaly. Hereditary Spherocytosis clinically and genetically comprises a heterogeneous group of hemolytic anemias. In about 75% of the cases, the inheritance follows an autosomal dominant pattern and about 25% of cases occur sporadically. The aim of this study was to find which gene was responsible for the autosomal dominant HS in a large family of four generations. The linkage to the major HS genes (including SPTA1, SPTB, ANK1 ve SLC4A1) was searched by using LINKAGE and MERLIN analysis and LOD-score statistical calculations. As a result of using these analyses, we confirmed that the HS in this family was an autosomal dominant form of the disease and the incidence was linked to the SPTB gene which maps to chromosome 14. Linkage analysis is an effective prescreening method in genetically heterogeneous diseases and a new mutation analysis study is in the planning stage to detect the mutation in the SPTB gene.

Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient

Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient

Band 3 Edmonton I, a novel mutant of the anion exchanger 1 causing spherocytosis and distal renal tubular acidosis

dRTA (distal renal tubular acidosis) and HS (hereditary spherocytosis) are two diseases that can be caused by mutations in the gene encoding the AE1 (anion exchanger 1; Band 3). dRTA is characterized by defective urinary acidification, leading to metabolic acidosis, renal stones and failure to thrive. HS results in anaemia, which may require regular blood transfusions and splenectomy. Mutations in the gene encoding AE1 rarely cause both HS and dRTA. In the present paper, we describe a novel AE1 mutation, Band 3 Edmonton I, which causes dominant HS and recessive dRTA. The patient is a compound heterozygote with the new mutation C479W and the previously described mutation G701D. Red blood cells from the patient presented a reduced amount of AE1. Expression in a kidney cell line showed that kAE1 (kidney AE1) C479W is retained intracellularly. As kAE1 is a dimer, we performed co-expression studies and found that, in kidney cells, kAE1 C479W and G701D proteins traffic independently from each other despite their ability to form heterodimers. Therefore the patient carries one kAE1 mutant that is retained in the Golgi (G701D) and another kAE1 mutant (C479W) located in the endoplasmic reticulum of kidney cells, and is thus probably unable to reabsorb bicarbonate into the blood. We conclude that the C479W mutant is a novel trafficking mutant of AE1, which causes HS due to a decreased cell-surface AE1 protein and results in dRTA due to its intracellular retention in kidney.

A complex splicing defect associated with homozygous ankyrin-deficient hereditary spherocytosis

AbstractDefects in erythrocyte ankyrin are the most common cause of typical, dominant hereditary spherocytosis (HS). Detection of ankyrin gene mutations has been complicated by allelic heterogeneity, large gene size, frequent de novo mutations, and associated mRNA instability. Using denaturing high-performance liquid chromatography (DHPLC)–based mutation detection, a mutation in the splice acceptor of exon 17 was discovered in a Turkish family. Reticulocyte RNA and functional minigene splicing assays in heterologous cells revealed that this mutation was associated with a complex pattern of aberrant splicing, suggesting that removal of intron 16 is important for ordered ankyrin mRNA splicing. As predicted by clinical, laboratory, and biochemical studies, the parents were heterozygous and the proband was homozygous for this mutation. These data indicate that DHPLC offers a highly sensitive, economic, and rapid method for mutation detection and, unlike previously suggested, homozygosity for a mutation associated with dominant ankyrin-linked HS may be compatible with life.

Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle

Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1.

Hematologically important mutations: Ankyrin variants in hereditary spherocytosis

The primary defect in the hereditary spherocytosis (HS) syndromes is a qualitative or quantitative alteration in one or more erythrocyte membrane proteins. Mutation of the erythrocyte membrane protein ankyrin are the most common cause of typical, dominant HS. Ankyrin mutations also cause nondominant spherocytosis due to ankyrin gene promoter or de novo mutations. In most cases, HS-related ankyrin mutations are private. A summary of reported HS-associated ankyrin gene mutations is provided in this report.

Hereditary spherocytosis—defects in proteins that connect the membrane skeleton to the lipid bilayer

The molecular causes of hereditary spherocytosis (HS) have been unraveled in the past decade. No frequent defect is found, and nearly every family has a unique mutation. In dominant HS, nonsense and frameshift mutations of ankyrin, band 3, and β-spectrin predominate. Recessive HS is most often due to compound heterozygosity of defects in ankyrin, α-spectrin, or protein 4.2. Common combinations include a defect in the promoter or 5′-untranslated region of ankyrin paired with a missense mutation, a low expression allele of α-spectrin plus a missense mutation, and various mutations in the gene for protein 4.2. In most patients’ red cells, no abnormal protein is present. Only rare missense mutations, like ankyrin Walsrode (V463I) or β-spectrin Kissimmee (W202R), have given any insight into the functional domains of the respective proteins. Although the eminent role of the spleen in the premature hemolysis of red cells in HS is unquestioned, the molecular events that cause splenic conditioning of spherocytes are unclear. Electron micrographs show that small membrane vesicles are shed during the formation of spherocytes. Animal models give further insight into the pathogenetic consequences of membrane protein defects as well as the causes of the variability of disease severity.

Simultaneous (AC)n microsatellite polymorphism analysis and single-stranded conformation polymorphism screening is an efficient strategy for detecting ankyrin-1 mutations in dominant hereditary spherocytosis.

Nonsense/stop mutations in the ankyrin-1 gene (ANK1) are a major cause of dominant HS (dHS) (frequency of 23% in German dHS patients). To date, no common mutation has been found and therefore a simple mutation screening is not feasible. The reduced expression of one cDNA allele in the (AC)n microsatellite polymorphism of the ankyrin-1 gene, as seen in about 20% of Czech patients with dHS, may identify candidates with a possible frameshift/nonsense mutation. In order to verify the efficiency of this screening we screened the ankyrin-1 gene of 22 Czech dHS patients for both the reduced cDNA allele expression in the frequent (AC)n and the common exonic 26/39 polymorphisms, as well as for polymerase chain reaction (PCR) single-stranded conformation polymorphisms in any one of the 42 exons of ANK1. Anomalous PCR products were sequenced. We found seven new ANK1 frameshift/nonsense mutations in nine patients with, but in none of six patients without, a reduced cDNA allele expression (efficiency of 78%). We conclude that screening of dHS patients for such a reduced allele expression in common ANK1 polymorphisms is an efficient procedure for the identification of candidates for frameshift/nonsense mutations in the ankyrin-1 gene.

A recurrent frameshift mutation of the ankyrin gene associated with severe hereditary spherocytosis.

During a survey of typical, dominant hereditary spherocytosis (HS) patients, we identified a frameshift mutation of the ankyrin gene in three unrelated probands. All three probands, who were from different ethnic backgrounds, suffered from severe HS requiring splenectomy. Analysis of both intragenic and flanking polymorphisms revealed that these probands did not share a common ankyrin allele, providing evidence that these mutations arose independently on separate chromosomal backgrounds. This frameshift mutation associated with severe HS, Ankyrin Florianópolis, is the first description of a recurrent mutation in the ankyrin gene.

Red blood cell membrane disorders.

The recent discovery of the specific molecular defects in many patients with hereditary spherocytosis and hereditary elliptocytosis/pyropoikilocytosis partially clarifies the molecular pathology of these diseases. HE and HPP are caused by defects in the horizontal interactions that hold the membrane skeleton together, particularly the critical spectrin self-association reaction. Single gene defects cause red cells to elongate as they circulate, by a unknown mechanism, and are clinically harmless. The combination of two defective genes or one severe alpha spectrin defect and a thalassaemia-like defect in the opposite allele (alphaLELY) results in fragile cells that fragment into bizarre shapes in the circulation, with haemolysis and sometimes life-threatening anaemia. A few of the alpha spectrin defects are common, suggesting they provide an advantage against malaria or some other threat. HS, in contrast, is nearly always caused by family-specific private mutations. These involve the five proteins that link the membrane skeleton to the overlying lipid bilayer: alpha and beta spectrin, ankyrin, band 3 and protein 4.2. Somehow, perhaps through loss of the anchorage band 3 provides its lipid neighbours (Peters et al, 1996), microvesiculation of the membrane surface ensues, leading to spherocytosis, splenic sequestration and haemolysis. Future research will need to focus on how each type of defect causes its associated disease, how the spleen aggravates membrane skeleton defects (a process termed 'conditioning'), how defective red, cells are recognized and removed in the spleen, and why patients with similar or even identical defects can have different clinical severity. Emphasis also needs to be given to improving diagnostic tests, particularly for HS, and exploring new options for therapy, like partial splenectomy, which can ameliorate symptoms while better protecting patients from bacterial sepsis and red cell parasites, and perhaps from atherosclerosis (Robinette & Franmeni, 1977) and venous thrombosis (Stewart et al, 1996).

The red blood cell band 3 variant (band 3Biceêtrel:R490C) associated with dominant hereditary spherocytosis causes defective membrane targeting of the molecule and a dominant negative effect.

Hereditary spherocytosis (HS), a common human inherited haemolytic anaemia, is associated with partial deficiency of different erythrocyte membrane proteins. In a subset of dominant HS, a partial membrane expression deficiency of band 3, the erythrocyte anion exchanger (AE1), have previously been characterized, and several mutations in the band 3 gene have been found: amino acid substitutions at conserved positions in the membrane domain, nonsense and frameshift mutations. In HS patients bearing missense mutations, the mutated transcript was present, whereas only the normal transcript was found in HS patients with frameshift mutations. In the former group, the membrane expression deficiency of band 3 was significantly more important than that observed in the latter group of HS patients with frameshift mutations, suggesting that missense mutations may have a dominant negative effect. In the present study, transient and stable transfections of K562 and COS-7 cells were used to demonstrate, by immunoblots of cell lysates and immunofluorescence studies, that the band 3 membrane domain bearing the R490C mutation (band 3Bicetrel) is not targeted to the plasma membrane and is retained in the endoplasmic reticulum. Transient cotransfections of K562 cells with plasmid coding for the normal membrane domain of band 3, together with increasing amounts of plasmid coding for the mutated R490C membrane domain, demonstrated that the band 3 mutant polypeptide exerts a dominant negative effect on the plasma membrane targeting of the normal band 3.

A 5′ splice region G → C mutation in exon 3 of the human β‐spectrin gene leads to decreased levels of β‐spectrin mRNA and is responsible for dominant hereditary spherocytosis (spectrin Guemene‐Penfao)

We studied a family with autosomal dominant hereditary spherocytosis (HS) associated with a mild spectrin deficiency. Linkage analysis using two microsatellite markers (D14S63 and D14S271) very close to the beta-spectrin gene (SPTB) showed that HS co-segregated with alleles of these microsatellite markers and the linkage between the marker and HS was statistically significant. The presence of a beta-spectrin protein polymorphism (beta-spectrin Vay; A1880V) in trans of the HS allele was not itself deleterious, but allowed the detection of decreased membrane expression of the spherocytic beta-spectrin allele in two HS-affected subjects. Direct sequencing of the coding exons of the beta-spectrin gene in one affected subject showed the presence of a G-->C transversion at the terminal nucleotide of exon 3, which did not change the leucine codon 100 (CTG-->CTC). The presence of the mutation was confirmed by restriction enzyme digestion at the DNA level in all affected SH members of the family. The G-->C mutation severely reduced the utilization of the 5' splice site and resulted in aberrant mRNA splicing with intron 3 retention.