A VARIANT OF THE EPB3 GENE OF THE ANTI-LEPORE TYPE IN HEREDITARY SPHEROCYTOSIS

The EPB3 gene encodes band 3 (anion exchanger 1) of the red cell membr ane, A subset of hereditary spherocytosis (HS) is associated with EPB3 gene mutations and band 3 deficiency, We report a large Italian famil y in which 10 of the 27 members investigated displayed an autosomal do minant HS, SDS-PAGE revealed a reduction in band 3 in the patients, Sc reening of the Pst I polymorphic site confirmed the linkage of HS with the EPB3 gene. Analysis of complementary and genomic DNA showed a lar ge additional segment. Nucleotide sequencing disclosed an in-frame dup lication of 69 nucleotides (nt) including a triplet of intronic origin and a genuine exonic duplication of 66 nt. Two CCTGC sequences occurr ed close to one another, one near the intron 12 acceptor splice site ( nt -7 to -3), and the other within exon 13 (nt 1494-1498). We assumed that the abnormal allele arose from an unequal recombination event of the anti-Lepore type between the two CCTGC sequences, At the level of the mutated protein, termed band 3 Milano, the additional segment (Gln plus duplication of residues 478-499) corresponded to the last part o f the third transmembrane domain (TM3), the entire second outer loop a nd part of TM4 as it is currently defined in hydropathy analysis, Afte r deglycosylation of band 3, only the normal band was detected, suppor ting the view that band 3 Milano is probably not incorporated into the membrane.