Electron microscopic evidence of impaired intramembrane particles and instability of the cytoskeletal network in band 4.2 deficiency in human red cells.

To obtain direct evidence of impaired intramembrane particles (IMPs) and a deranged cytoskeletal network in situ in human red cells of band 4.2 deficiency, electron microscopic studies were performed utilizing the freeze fracture method for IMPs and the quick-freeze deep-etching method for the cytoskeletal network. Three patients with three different previously identified mutations of the band 4.2 gene, i.e., band 4.2 Komatsu (homozygous; codon 175 GAT --> TAT), band 4.2 Nippon (homozygous; codon 142 GCT --> ACT), and band 4.2 Shiga (compound heterozygous; codon 317 CGC --> TGC and codon 142 GCT --> ACT), were selected for this study. The decrease in the number of IMPs with increase in their size was most marked in band 4.2 Komatsu, which was clinically most severe with no band 4.2 protein. In this regard, in band 4.2 Nippon, which showed moderate severity in clinical hematology with a nearly missing band 4.2 protein, increased sizing was less marked. The abnormalities in IMPs were the least in band 4.2 Shiga, which demonstrated compensated hemolysis with band 4.2 protein in a trace amount. The extent of the impairment of IMPs may be reflected by the total absence or the presence of band 4.2 protein even in a trace amount and/or by the specific site(s) of the mutation of the band 4.2 gene. Derangement of the cytoskeletal network was also observed in these three patients. It was most abnormal in band 4.2 Komatsu, and less so in band 4.2 Nippon and in band 4.2 Shiga. These results clearly indicate that 1) band 4.2 plays an important role not only in its binding to band 3 but also to the skeletal network (mostly to spectrins) vertically, and 2) its deficiency produces critical abnormality in maintenance of the structural and functional integrity of the integral proteins (such as band 3), as well as the cytoskeletal network.