Exclusion of AE1 gene as cause of CDA-II. (A) Pedigrees of 7 families with CDA-II: haplotype analysis of 3 markers tightly linked to the SLC4A1 gene in 7 CDA-II families unlinked to CDAN2 locus on chromosome 20. White symbols indicate healthy subjects; black symbols, patients. (B) SDS-PAGE of red blood cell membrane proteins. SDS-PAGE was performed using the discontinuous buffer system of Laemmli with acrylamide linear gradient from 5% to 15%. The proteins were stained with Coomassie brilliant blue and gels were scanned using a laser densitometer (Ultroscan; Pharmacia, Uppsala, Sweden). The amount of the major membrane proteins was then expressed as the value of the relative surface area present under the densitometric curve, and the results were normalized to the protein 4.1. Erythrocyte membrane analysis of each patient and control was performed 3 times using different gels. The reproducibility of AE1 and protein 4.1 measurements was within a range of ± 4%. The amount of AE1 and protein 4.1 was compared with normal values obtained from controls. ^* indicates AE1; 1 and 10, controls; 2, HS due to AE1 deficiency; 3, 4, and 5, “typical” CDA-II; and 6, 7, 8, and 9, severe CDA-II.