Combination of two mutant alpha spectrin alleles underlies a severe spherocytic hemolytic anemia.

H Wichterle; M Hanspal; J Palek; P Jarolim

doi:10.1172/jci119041

A family affair—Severe fetal and neonatal hemolytic anemia due to novel alpha‐spectrin mutations in two siblings

American Journal of Medical Genetics Part A ◽

10.1002/ajmg.a.61455 ◽

2019 ◽

Vol 182 (3) ◽

pp. 561-564

Author(s):

Roopali Donepudi ◽

Lauren Westerfield ◽

Ashley Stonecipher ◽

Ahmed A. Nassr ◽

Magdalena S. Cortes ◽

...

Keyword(s):

Hemolytic Anemia ◽

Alpha Spectrin

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Structural and functional heterogeneity of alpha spectrin mutations involving the spectrin heterodimer self-association site: relationships to hematologic expression of homozygous hereditary elliptocytosis and hereditary pyropoikilocytosis

Blood ◽

10.1182/blood.v75.11.2235.2235 ◽

1990 ◽

Vol 75 (11) ◽

pp. 2235-2244 ◽

Cited By ~ 29

Author(s):

T Coetzer ◽

J Palek ◽

J Lawler ◽

SC Liu ◽

P Jarolim ◽

...

Keyword(s):

Hemolytic Anemia ◽

Hexagonal Structure ◽

Clinical Severity ◽

Related Disorder ◽

Virtual Absence ◽

Hereditary Elliptocytosis ◽

Alpha Spectrin ◽

Self Association ◽

Partial Deficiency ◽

Membrane Skeletons

Abstract Defects involving alpha spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various alpha Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four alpha Sp mutations: alpha 1/74, alpha 1/46, alpha 1/65, and alpha 1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp alpha 1/74, Sp alpha 1/46, and alpha 1/65 homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp alpha 1/74 had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD alpha 1/46 were not as severely affected. The Sp alpha 1/65 homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp alpha 1/65 had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various alpha Sp mutants that underlie differences in clinical expression.

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Structural and functional heterogeneity of alpha spectrin mutations involving the spectrin heterodimer self-association site: relationships to hematologic expression of homozygous hereditary elliptocytosis and hereditary pyropoikilocytosis

Blood ◽

10.1182/blood.v75.11.2235.bloodjournal75112235 ◽

1990 ◽

Vol 75 (11) ◽

pp. 2235-2244

Author(s):

T Coetzer ◽

J Palek ◽

J Lawler ◽

SC Liu ◽

P Jarolim ◽

...

Keyword(s):

Hemolytic Anemia ◽

Hexagonal Structure ◽

Clinical Severity ◽

Related Disorder ◽

Virtual Absence ◽

Hereditary Elliptocytosis ◽

Alpha Spectrin ◽

Self Association ◽

Partial Deficiency ◽

Membrane Skeletons

Defects involving alpha spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various alpha Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four alpha Sp mutations: alpha 1/74, alpha 1/46, alpha 1/65, and alpha 1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp alpha 1/74, Sp alpha 1/46, and alpha 1/65 homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp alpha 1/74 had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD alpha 1/46 were not as severely affected. The Sp alpha 1/65 homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp alpha 1/65 had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various alpha Sp mutants that underlie differences in clinical expression.

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Transfusion-Dependent Alpha-Spectrin Deficient Hemolytic Anemia Due to Maternal Uniparental Disomy

Blood ◽

10.1182/blood.v118.21.1030.1030 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1030-1030

Author(s):

Hannah Bogardus ◽

Vincent P Schulz ◽

Yelena Maksimova ◽

Barbara A Miller ◽

Bernard G. Forget ◽

...

Keyword(s):

Hemolytic Anemia ◽

Nonsense Mutation ◽

Genomic Dna ◽

Gene Locus ◽

Uniparental Disomy ◽

Chromosome 1 ◽

Single Chromosome ◽

Maternal Uniparental Disomy ◽

Alpha Spectrin ◽

Exon 19

Abstract Abstract 1030 We studied a girl with hereditary spherocytosis who required in utero transfusions and remained transfusion-dependent after birth. Initial clinical, laboratory, and biochemical characteristics of this patient have been described (Blood 78:3043, 1991). Severe alpha-spectrin deficiency was observed in membranes prepared from her marrow-derived cultured erythroid cells. Since this report, despite splenectomy at age 2 years, the proband remains transfusion-dependent. Our goal was to determine the molecular basis of the patient's transfusion-dependent anemia. Typically, alpha-spectrin deficiency is recessively inherited due to homozygous or compound heterozygous inactivating mutations of the alpha-spectrin gene. Genomic DNA from the proband was amplified using primers flanking the 52 coding exons and promoter region of the alpha-spectrin gene. Capillary electrophoresis-based nucleotide sequencing identified a homozygous nonsense mutation in exon 19 of the alpha-spectrin gene. The mother was heterozygous for this mutation; the father did not carry the mutation. To determine if a deletion involving the alpha-spectrin gene locus leading to homozygosity of the nonsense mutation in the proband was present, array comparative genomic hybridization (aCGH) was performed using the 44K CGH microarray (G4426A; Agilent Technologies, Santa Clara, CA). This array includes 44,000 60-mer oligonucleotides covering the entire human genome at a density of ∼14–15 oligonucleotides/MB. No deletions or rearrangements were identified by aCGH. To interrogate the alpha-spectrin locus at higher resolution, SNP typing of proband and parental genomic DNA was performed using the Illumina HumanHap 550 BeadChip. This array contains >550,000 SNPs and includes twenty intragenic SNPs in the alpha-spectrin gene locus. These studies identified a large region of homozygosity at 1q21, approximately 10 megabases in length, extending from rs6657293 (154,995,473, hg18) to rs6670426 (165,730,530) including the PRCC gene (155,003,897) to the CD247 gene (165,888,000) on chromosome 1q in genomic DNA from the proband. This region of homozygosity includes the alpha-spectrin gene locus and 158 other genes. Analyses of the SNP data also confirmed paternity and maternity, with a P-P-C heritability of 0.9997 and no inheritance violations in the alpha-spectrin gene region. Finally, to exclude the possibility of an intragenic microdeletion in the alpha-spectrin gene locus, copy number profiling using quantitative, real-time PCR was performed. Amplicons included exon 19, the location of the nonsense mutation, as well as exons 2, 17, 40, 52 and the 3'UTR. The proband and both parents had 2 copies of the corresponding alpha-spectrin region at all sites examined. No deletions were identified, excluding the possibility that a small deletion of the SPTA1 gene is the cause of homozygosity of exon 19 in the proband. Together, these data indicate there is partial maternal uniparental disomy of chromosome 1 in the proband and that reduction to homozygosity of the 1q region containing the maternal SPTA nonsense mutation is responsible for the alpha-spectrin deficient hemolytic anemia phenotype. This is the first case of uniparental disomy leading to an erythrocyte membrane-associated hemolytic anemia. The homozygosity associated with uniparental disomy consists of duplicated copies of alleles from a single chromosome, leading to increased risk of homozygosity for deleterious recessive mutations. Uniparental disomy should be considered when unexpected results are obtained during carrier testing in a recessive disorder. Disclosures: No relevant conflicts of interest to declare.

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Hereditary poikilocytic anemia associated with the co-inheritance of two alpha spectrin abnormalities

Blood ◽

10.1182/blood.v71.5.1390.1390 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1390-1396 ◽

Cited By ~ 8

Author(s):

TA Iarocci ◽

GM Wagner ◽

N Mohandas ◽

PA Lane ◽

WC Mentzer

Keyword(s):

Hemolytic Anemia ◽

Mechanical Stability ◽

Black Family ◽

Family Members ◽

Electrophoretic Analysis ◽

Clinical Severity ◽

Structural Defects ◽

Prolonged Incubation ◽

Functional Consequences ◽

Alpha Spectrin

Abstract This report describes a black family in which two distinct structural defects of alpha spectrin were inherited singly and in combination. The propositus, who has a poikilocytic hemolytic anemia that shares many of the features of hereditary pyropoikilocytosis (HPP) or homozygous elliptocytosis, is a compound heterozygote for both the spectrin alpha 1/65 and spectrin alpha 1/50a defects as demonstrated by electrophoretic analysis of spectrin tryptic fragments. The spectrin alpha 1/65 defect alone was found in his mother and sibling, while the spectrin alpha 1/50a defect was present in the father and another sibling. The red cell spectrin content was normal in all family members. The functional consequences of inheritance of these two spectrin defects were compared with those found in an unrelated patient with classic HPP who had the alpha 1/50a spectrin defect and was spectrin deficient as well. Prolonged incubation at 37 degrees C resulted in striking budding, fragmentation, and sphering of classic HPP red cells but only minimal changes in propositus cells. The percentage of spectrin dimers was increased tenfold in classic HPP, sevenfold in the propositus, and threefold in other family members. Mechanical stability of erythrocyte ghosts, measured by ektacytometry, was reduced severely in both classic HPP and in the propositus, but only moderately in other family members. Thus, co-inheritance of two alpha spectrin defects can result in a poikilocytic hemolytic anemia milder than that usually found in HPP. The greater clinical severity of HPP may be a consequence of the presence of spectrin deficiency, a finding absent in the propositus.

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Hereditary poikilocytic anemia associated with the co-inheritance of two alpha spectrin abnormalities

Blood ◽

10.1182/blood.v71.5.1390.bloodjournal7151390 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1390-1396

Author(s):

TA Iarocci ◽

GM Wagner ◽

N Mohandas ◽

PA Lane ◽

WC Mentzer

Keyword(s):

Hemolytic Anemia ◽

Mechanical Stability ◽

Black Family ◽

Family Members ◽

Electrophoretic Analysis ◽

Clinical Severity ◽

Structural Defects ◽

Prolonged Incubation ◽

Functional Consequences ◽

Alpha Spectrin

This report describes a black family in which two distinct structural defects of alpha spectrin were inherited singly and in combination. The propositus, who has a poikilocytic hemolytic anemia that shares many of the features of hereditary pyropoikilocytosis (HPP) or homozygous elliptocytosis, is a compound heterozygote for both the spectrin alpha 1/65 and spectrin alpha 1/50a defects as demonstrated by electrophoretic analysis of spectrin tryptic fragments. The spectrin alpha 1/65 defect alone was found in his mother and sibling, while the spectrin alpha 1/50a defect was present in the father and another sibling. The red cell spectrin content was normal in all family members. The functional consequences of inheritance of these two spectrin defects were compared with those found in an unrelated patient with classic HPP who had the alpha 1/50a spectrin defect and was spectrin deficient as well. Prolonged incubation at 37 degrees C resulted in striking budding, fragmentation, and sphering of classic HPP red cells but only minimal changes in propositus cells. The percentage of spectrin dimers was increased tenfold in classic HPP, sevenfold in the propositus, and threefold in other family members. Mechanical stability of erythrocyte ghosts, measured by ektacytometry, was reduced severely in both classic HPP and in the propositus, but only moderately in other family members. Thus, co-inheritance of two alpha spectrin defects can result in a poikilocytic hemolytic anemia milder than that usually found in HPP. The greater clinical severity of HPP may be a consequence of the presence of spectrin deficiency, a finding absent in the propositus.

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The Spectrum of Alpha-Spectrin Associated Hereditary Spherocytosis

Blood ◽

10.1182/blood.v126.23.941.941 ◽

2015 ◽

Vol 126 (23) ◽

pp. 941-941 ◽

Cited By ~ 1

Author(s):

Satheesh Chonat ◽

Mary Risinger ◽

Neha Dagaonkar ◽

Tamara Maghathe ◽

Jennifer Rothman ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hemolytic Anemia ◽

Premature Termination ◽

Hereditary Spherocytosis ◽

Compound Heterozygous ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Alpha Spectrin ◽

In Trans ◽

Generation Sequencing

Abstract Hereditary spherocytosis (HS) is a genetically and phenotypically heterogeneous hemolytic anemia caused by deficiency in red blood cell (RBC) cytoskeleton proteins leading to disruptions in the vertical association of the cytoskeleton with the RBC lipid bilayer. Monoallelic mutations in the genes encoding ankyrin (ANK1), beta-spectrin (SPTB) and band 3 (SLC4A1) or biallelic mutations in the genes encoding alpha-spectrin (SPTA1), ankyrin, and protein 4.2 (EPB42) result in HS. Autosomal recessive HS due to compound heterozygous defects in SPTA1 is typically severe and diagnosis based on phenotypic assays like RBC morphology, osmotic fragility or ektacytometry is complicated by transfusion dependence resulting in most of the circulating RBCs to be of donor origin. We have developed a rapid comprehensive next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. We describe here patients with hemolytic anemia due to SPTA1 mutations, identified utilizing this assay, and their phenotype-genotype correlation. Each of these cases, when possible, has been also evaluated with ektacytometry and immunoblotting of RBC ghosts for alpha-spectrin quantitation. Wichterle et al in 1996 had estimated that alphaLEPRA(Low Expression PRAgue) mutation (c.4339-99C>T) occurs in SPTA1 gene in about 5% of Caucasians. This mutation leads to activation of an alternate acceptor splice site at position -70 of intron 30, causing frame shift and premature termination, thereby leading to decrease in alpha-spectrin production in this allele to about 16% of normal. We have found a cohort of three transfusion-dependent hereditary hemolytic anemia cases where a nonsense mutation in SPTA1 gene has occurred in trans to alphaLEPRA mutation, resulting in premature termination (see Table 1). Transfusion dependence was alleviated in two of these patients after splenectomy; the third one did not have splenectomy yet. RBC phenotype explored after splenectomy revealed an ektacytometry curve indicating spherocytosis (Figure 1A) and severely decreased alpha-spectrin on immunoblotting along with significant decrease of the associated beta-spectrin (Figure 1B). A patient with moderately severe form of HS, maintaining a hemoglobin value greater than 7 g/dL and requiring only occasional transfusions during periods of illness or stress, was found to have alphaLEPRA occurring in trans to an intronic splicing mutation c.1351-1G>TG where there is substitution at nucleotide-1 of intron positioned between nucleotides 1350 and 1351 of the SPTA1 mRNA. This splicing mutation may allow for some expression of functional alpha-spectrin protein from this allele in contrast to no protein expression in the previous cases of premature termination. Alternatively, other gene mutations, not identified by the next-generation sequencing panel we used, may contribute to this patient's milder phenotype. A couple with history of two fetal losses associated with hydrops fetalis seeked genetic counseling and gave consent to have diagnostic evaluation of genes associated with non-immune hemolytic anemia using targeted next-generation sequencing. Results of the panel revealed a heterozygous frameshift SPTA1 mutation in each of the parents (c.4206delG in the father and c.4180delT in the mother). These mutations in compound heterozygous state in the offspring likely caused total absence of alpha spectrin and fatal hemolytic anemia by the time of birth. Hereditary Spherocytosis is characterized by wide phenotypic variability that will be better understood with studies of genotype-phenotype association. While complete absence of alpha-spectrin expression due to null mutations of both SPTA1 alleles is incompatible with life, a nonsense or splicing SPTA1 mutation in trans to an alphaLEPRA low expression allele causes severe or moderately severe recessive HS, respectively. Targeted next-generation sequencing can be an effective diagnostic tool particularly for patients requiring frequent transfusions that preclude meaningful phenotypical testing of their red blood cells. Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 2. Figure 2. Disclosures Begtrup: GeneDx: Employment.

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