Erythrocyte Ankyrin Promoter Mutations Associated with Recessive Hereditary Spherocytosis Cause Significant Abnormalities in Ankyrin Expression

Abstract

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, ؊108T to C and ؊108T to C in cis with ؊153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a human A ␥-globin gene expressed ␥-globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant ؊108 promoter demonstrated variegated ␥-globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked ؊108/؊153 mutations. Three transgenic lines had undetectable levels of A ␥-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of A ␥-globin mRNA than wild type. Two of four expressing lines showed variegated ␥-globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy numberindependent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations. Hereditary spherocytosis (HS)1 is a common hemolytic anemia characterized by the presence of spherically shaped erythrocytes on peripheral blood smear. The principal cellular defect in HS is loss of erythrocyte membrane surface area relative to intracellular volume, accounting for the spherical shape as well as decreased deformability of the erythrocyte (1). The primary biochemical defects in HS reside in the proteins of the erythrocyte membrane, particularly those proteins involved in the interactions between the membrane skeleton and the lipid bilayer: ankyrin, ␣ and ␤ spectrin, band 3, and protein 4.2 (2, 3). In two thirds to three quarters of cases, HS is inherited in an autosomal dominant fashion (1-4). In the remaining patients, HS is inherited in a recessive fashion or is the result of a de novo mutation.Ankyrin-1 (ANK1, Mendelian Inheritance in Man 182900) deficiency is one of the most common abnormalities found in the erythrocyte membranes of HS patients (5, 6). First identified in preparations of erythrocyte membranes, ankyrin provides the primary linkage between the spectrin-actin-based erythrocyte membrane skeleton and the plasma membrane by attaching tetramers of spectrin to the cytoplasmic domain of band 3 (7, 8). Studies have revealed that abnormalities of the ankyrin gene, primarily frameshift or nonsense mutations, are the most common cause of typical, dominant HS (3, 9 -11).Ankyrin-1 is transcribed in erythroid cells from a compact, erythroid-specific promoter (12). One molecular mechanism that could lead to ankyrin deficiency is a mutation in the ankyrin erythroid promoter leading to decr...

Clinical and hematologic features of 300 patients affected by hereditary spherocytosis grouped according to the type of the membrane protein defect

“…8,9 The molecular defect is highly heterogeneous, involving the genes encoding for spectrin, ankyrin, band 3 and protein 4.2. [10][11][12] The deficiency or dysfunction of any of these proteins, which are involved in the attachment of the cytoskeleton to the membrane integral domain, results in a loss of surface area and leads to spheroidal, osmotically fragile cells that are selectively trapped in the spleen. 13,14 The defective protein can be detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), which allows the identification of different subsets of patients; 3,15,16 although some HS subjects remain unclassified by this technique; 16,[17][18][19] efforts to identify the protein defect by genetic analysis in unclassified cases have been unsuccessful.…”

Selective neutrality analysis of 17 STRs in Mediterranean populations

“…30 Following a candidate gene approach, we choose short tandem repeat loci localized in coding and noncoding regions of genes for enzymes of oxidative metabolism, immunity system and erythrocyte membrane components, 31 which are frequent targets of natural selection. 32,33 Erythrocyte membrane components such as spectrin and ankyrin have been associated with spherocytosis, ovalocitosis [34][35][36][37] and interactions with different species of plasmodium. [38][39][40] TNF genes encode for proteins belonging to the group of inflammatory cytokines.…”

Surgeons' and Hematologists' Decisions on Splenectomy in the Treatment of Congenital Spherocytosis

“…Presently available techniques are not sufficient to detect the anomaly in parents, hence this phenomenon can be attributed to spontaneous mutation or expression of an odd gene, which remains below the threshold of defect detectability (2)(3)(4)(5).…”

“…It is characterized by an erythrocyte structure disorder, which may result in premature hemolysis. Congenital spherocytosis (CS) may depend not only on a genetic mutation, but also on recessive mutation transfer, which may provide a satisfactory explanation for the atypical cases (2)(3)(4)(5)(6).…”

“…Congenital spherocytosis (MinkowskyChauffard Syndrome) is one of the most common hemolytic anemias in Poland, occurring with a frequency of 1/5000 to 1/2000 inhabitants (1,3,4). It is characterized by an erythrocyte structure disorder, which may result in premature hemolysis.…”