Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency

Abstract

Hereditary spherocytosis (HS) is a common hemolytic anemia of variable clinical expression. Pathogenesis of HS has been associated with defects of several red cell membrane proteins including erythroid band 3. We have studied erythrocyte membrane proteins in 166 families with autosomal dominant HS. We have detected relative deficiency of band 3 in 38 kindred (23%). Band 3 deficiency was invariably associated with mild autosomal dominant spherocytosis and with the presence of pincered red cells in the peripheral blood smears of unsplenectomized patients. We hypothesized that this phenotype is caused by band 3 gene defects. Therefore, we screened band 3 DNA from these 38 kindred for single strand conformational polymorphisms (SSCP). In addition to five mutations detected previously by SSCP screening of cDNA, we detected 13 new band 3 gene mutations in 14 kindred coinherited with HS. These novel mutations consisted of two distinct subsets. The first subset included seven nonsense and frameshift mutations that were all associated with the absence of the mutant mRNA allele from reticulocyte RNA, implicating decreased production and/or stability of mutant mRNA as the cause of decreased band 3 synthesis. The second group included five substitutions of highly conserved amino acids and one in-frame deletion. These six mutations were associated with the presence of comparable levels of normal and mutant band 3 mRNA. We suggest that these mutations interfere with band 3 biosynthesis leading thus to the decreased accumulation of the mutant band 3 allele in the plasma membrane.

Heterogenous band 3 deficiency in hereditary spherocytosis related to different band 3 gene defects

“…In three HS families in which linkage analyses selected the band 3 gene as candidate, DGGE and subsequent sequencing led to identify band 3 mutations which were related to the HS phenotype and were not found in the normal family members: 1475delG mutation in family HSB3-1 (band 3 Bicêtre II ); R490C mutation in family HSB3-3 (band 3 Bicêtre I ); T837M mutation in family HSB3-4 (band 3 Philadelphia already identified in two other unrelated HS kindreds ( Jarolim et al, 1996)).…”

“…Several mutations associated with band 3 deficient HS have been found in the band 3 gene. Amino acid substitutions at conserved positions have been identified (Alloisio et al, 1993;Jarolim et al, 1995Jarolim et al, , 1996Maillet et al, 1995;Eber et al, 1996) but nonsense and frameshift mutations appear to be the most common, and can lead to mRNA instability (Miraglia del Giudice et al, 1997;Jenkins et al, 1996;Alloisio et al, 1996;Jarolim et al, 1996) or abnormal band 3 which is not recovered into the membrane ( Jarolim et al, 1994;Bianchi et al, 1996).…”

Disorders of red cell membrane

“…Patients with b-spectrin defects typically have mild to moderately severe HS while patients with a-spectrin suffer from severe HS. Band 3 deficiency is found in approximately 15-20% of HS patients, presenting with a phenotype of a mild to moderate anaemia although a few cases of severe HS have also been reported (Jarolim et al, 1996;Dhermy et al, 1997;Tanner, 2002). Band 3 mutations are dominantly inherited.…”

Trafficking and Folding Defects in Hereditary Spherocytosis Mutants of the Human Red Cell Anion Exchanger

“…Approximately 20% of HS cases are due to mutations in AE1. The mutations cause a decrease (20-40%) in total red cell AE1 content due to absence of the mutant protein and an associated decrease in band 4.2 content (3)(4)(5)(6)(7)(8)(9)(10). Studies have been unable to detect either the Prague (10) or the Prague II (5) mutant proteins in mature red cells suggesting that they are unstable and degraded during erythroid development.…”

“…The results imply that the HS mutant AE1 is not present in mature erythrocytes because the protein is not targeted properly to the plasma membrane and is therefore lost during erythroid development. Figure 1A shows a folding model of the membrane domain of human AE1 (22) and the location of the HS mutations (L707P, R760Q, R760W, R808C, H834P, T837M and R870W) examined in this study (3,5). The R760Q mutant was chosen for detailed characterization since it has been shown to be absent from mature red cells (5).…”