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 39 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 59 of exon 31 (branch point 1, BP1); 2) an alternate upstream branch point centered on an "A" 2bp 39 of the αLEPRA mutation (BP2) 98bp 59 of exon 31; 3) identified a novel alternate 39 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 39 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 39 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 39acceptor 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. Disclosures No relevant conflicts of interest to declare.