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

ABSTRACT: Defects 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.

SUBMITTER: Edelman EJ

PROVIDER: S-EPMC1890827 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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

Edelman E Jennifer EJ Maksimova Yelena Y Duru Feride F Altay Cigdem C Gallagher Patrick G PG

Blood 20070227 12

Defects 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 ...[more]

PMID: 17327413

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Project description:Hereditary spherocytosis (HS) is the most common red blood cell (RBC) membrane disorder causing hereditary hemolytic anemia. Patients with HS have defects in the genes coding for ankyrin (ANK1), band 3 (SLC4A1), protein 4.2 (EPB42), and α (SPTA1) or β-spectrin (SPTB). Severe recessive HS is most commonly due to biallelic SPTA1 mutations. α-spectrin is produced in excess in normal erythroid cells, therefore SPTA1-associated HS ensues with mutations causing significant decrease of normal protein expression from both alleles. In this study, we systematically compared genetic, rheological, and protein expression data to the varying clinical presentation in eleven patients with SPTA1-associated HS. The phenotype of HS in this group of patients ranged from moderately severe to severe transfusion-dependent anemia and up to hydrops fetalis which is typically fatal if transfusions are not initiated before term delivery. The pathogenicity of the mutations could be corroborated by reduced SPTA1 mRNA expression in the patients' reticulocytes. The disease severity correlated to the level of α-spectrin protein in their RBC cytoskeleton but was also affected by other factors. Patients carrying the low expression αLEPRA allele in trans to a null SPTA1 mutation were not all transfusion dependent and their anemia improved or resolved with partial or total splenectomy, respectively. In contrast, patients with near-complete or complete α-spectrin deficiency have a history of having been salvaged from fatal hydrops fetalis, either because they were born prematurely and started transfusions early or because they had intrauterine transfusions. They have suboptimal reticulocytosis or reticulocytopenia and remain transfusion dependent even after splenectomy; these patients require either lifetime transfusions and iron chelation or stem cell transplant. Comprehensive genetic and phenotypic evaluation is critical to provide accurate diagnosis in patients with SPTA1-associated HS and guide toward appropriate management.

Project description:Background and aimsHereditary 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 methodsA 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.ResultsThe 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.ConclusionThe 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.

Dataset Information

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

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

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