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

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.503-503
• Main Authors: Lezon-Geyda, Kimberly, Schulz, Vincent P, Maksimova, Yelena, Gallagher, Patrick G.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-117752

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'