Genetic characterization of band 3 Neapolis mutation. (A) Genomic analysis. Sequence of PCR-amplified genomic DNA of proband band 3. The sequence shown encompasses the donor splice site of intron 2. Note that the second nucleotide of intron 2 was changed from T to C. (B) RT-PCR analysis. Reticulocyte cDNAs were PCR amplified using primers localized in exons 1 and 3. cDNA of a healthy subject generated a 215-bp band. Two bands of 340 bp and 132 bp were noted in the cDNA of the proband. In the heterozygous parent (the mother), all 3 amplified products were seen. A similar pattern also was seen using cDNA from the father (data not shown). (C) Sequencing of PCR products. The 2 abnormal amplified products obtained in the proband (panel 1B, 340 bp and 132 bp) were sequenced. The 340-bp fragment corresponds to mRNA in which intron 2 is retained, and the 132-bp fragment corresponds to mRNA in which exon 2 is skipped. (D) Schematic diagram of the different patterns of splicing. In contrast to normal splicing shown in the top panel, 2 aberrant splicing patterns are seen in band 3 Neapolis. Retaining of intron 2 leads to a premature termination of translation after the 19th triplet (i), and skipping of exon 2 leads to a loss of the normal translation start site for erythroid AE1 protein (ii).