Aberrant splicing contributes to severe α-spectrin–linked congenital hemolytic anemia
Patrick G. Gallagher, … , Susan J. Baserga, Vincent P. Schulz
Patrick G. Gallagher, … , Susan J. Baserga, Vincent P. Schulz
Published July 1, 2019; First published April 30, 2019
Citation Information: J Clin Invest. 2019;129(7):2878-2887. https://doi.org/10.1172/JCI127195.
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Research Article Genetics Hematology

Aberrant splicing contributes to severe α-spectrin–linked congenital hemolytic anemia

Abstract

The etiology of severe hemolytic anemia in most patients with recessive hereditary spherocytosis (rHS) and the related disorder hereditary pyropoikilocytosis (HPP) is unknown. Whole-exome sequencing of DNA from probands of 24 rHS or HPP kindreds identified numerous mutations in erythrocyte membrane α-spectrin (SPTA1). Twenty-eight mutations were novel, with null alleles frequently found in trans to missense mutations. No mutations were identified in a third of SPTA1 alleles (17/48). WGS revealed linkage disequilibrium between the common rHS-linked αBH polymorphism and a rare intron 30 variant in all 17 mutation-negative alleles. In vitro minigene studies and in vivo splicing analyses revealed the intron 30 variant changes a weak alternate branch point (BP) to a strong BP. This change leads to increased utilization of an alternate 3′ splice acceptor site, perturbing normal α-spectrin mRNA splicing and creating an elongated mRNA transcript. In vivo mRNA stability studies revealed the newly created termination codon in the elongated transcript activates nonsense-mediated decay leading to spectrin deficiency. These results demonstrate that a unique mechanism of human genetic disease contributes to the etiology of a third of rHS cases, facilitating diagnosis and treatment of severe anemia and identifying a new target for therapeutic manipulation.

Authors

Patrick G. Gallagher, Yelena Maksimova, Kimberly Lezon-Geyda, Peter E. Newburger, Desiree Medeiros, Robin D. Hanson, Jennifer Rothman, Sara Israels, Donna A. Wall, Robert F. Sidonio Jr., Colin Sieff, L. Kate Gowans, Nupur Mittal, Roland Rivera-Santiago, David W. Speicher, Susan J. Baserga, Vincent P. Schulz

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Figure 5

Analyses of nonsense-mediated decay.

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Analyses of nonsense-mediated decay. The influence of NMD on the stabili...
The influence of NMD on the stability of the elongated α-spectrin transcript in WT K562 cells and in K562 cells rendered homozygous for the αLEPRA allele. (A) After treatment with the NMD inhibitor emetine (E) or cycloheximide (C), or both, the amounts of total α-spectrin and the elongated α-spectrin transcript were determined by real-time RT-PCR with fluorescent Taqman probes. After NMD inhibition, amounts of elongated α-spectrin transcript were increased in αLEPRA cells (adjusted P values 0.0014, 0.0027, and 0.0014 for WT vs. cycloheximide, emetine, and cycloheximide + emetine, respectively) and to a lesser extent in WT cells (adjusted P values 0.084, 0.012, and 0.0047, respectively). (B) Minigene assay of NMD. Two nucleotides were inserted into the polypyrimidine tract of the primary 3′ acceptor site of exon 30 in the minigene model to place the elongated transcript in-frame. As predicted, NMD was abrogated and the amounts of elongated α-spectrin transcript from both the WT and the αLEPRA minigene constructs significantly increased. Three biologic samples each were tested in 3 independent experiments with the mean calculated from 9 values. Statistical significance was determined by 2-tailed Student’s t test.