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.

Figure 1

Splicing analyses of the ankyrin^Ankara mutation in reticulocyte RNA. (A) To determine if the ankyrin^Ankara mutation was associated with altered mRNA splicing, reticulocyte RNA was reverse transcribed with oligo d(T). RT products were PCR amplified with primers in exons 15 and 17. M indicates markers. Control (C) reticulocyte RT-PCR product yielded the expected cDNA fragment of 248 bp. In the father (F), the expected band of 248 bp and 3 additional bands of higher molecular weight were visualized. In the proband (P), the normal 248-bp cDNA product was not seen and only the 3 additional higher molecular weight bands visualized in the father's cDNA were found. (B) Determination of the nucleotide sequence of these higher molecular weight bands yielded novel ankyrin cDNA isoforms containing additional sequence of +15 bp, +172 bp, +257 bp and +260 bp, respectively, from the 3′ end of intron 16. The corresponding predicted splicing patterns of these isoforms are shown.

Figure 2

Minigene analyses of ankyrin mRNA splicing. Wild-type and mutant ankyrin^Ankara minigenes from exon 15 to exon 20 were prepared and transfected into COS and K562 cells. The sequence of the RT-PCR products derived from RNA of transfected cells was analyzed. RNA from cells transfected with the wild-type minigene contained the expected normally sized and spliced product (wild-type [WT], top line). RT-PCR products derived from RNA of cells transfected with the mutant ankyrin^Ankara minigene yielded 11 mutant spliceoforms, shown schematically, but no wild-type product. Similar studies with reticulocyte RNA from the proband yielded the same results, except that the +26-bp and +144-bp spliceoforms were not found.