Diagram of α-spectrin gene exons (not to scale) and strategy used to obtain overlapping cDNA fragments, indicated by the black bars. (A) The + indicates the L207P mutation and the asterisks indicate single nucleotide polymorphisms (SNPs) used as linkage markers to distinguish between the 2 α-spectrin alleles in the subcloned amplified cDNA fragments. (B) Reamplification of PCR products from different subclones of the exon 15 to 32 cDNA fragment, using primers to encompass exons 22 and 23. All subclones of the L207P allele had a normal-sized band (no. 1). However, subclones of the non-L207P allele demonstrated 3 different higher molecular weight bands corresponding to fragments with incremental sizes of 36, 260, or 620 bp (nos. 2, 3, and 4, respectively). M indicates size markers. To the right of the gel are diagrams of the different splicing isoforms, with insertions derived from the 5′ end of IVS 22 (open bar). The arrow indicates the in-frame stop codon located at the very 5′ end of the retained intronic sequence. (C) Nucleotide sequence at the junction of exon 22 and IVS 22 in the cDNAs corresponding to the non-L207P allele of the HPP proband. The encoded amino acid sequence is shown below the sequence, and the exon/intron junction is indicated by the arrowhead above the sequence. The in-frame termination codon is shown in the box.