Hereditary spherocytosis due to band 3 deficiency: 15 novel mutations in <i>SLC4A1</i>
Authors: Rob Van Zwieten, Jerney J.J.M. François, Karin Van Leeuwen, Annet C.W. Van Wesel, Robin Van Bruggen, Wouter W. Van Solinge, Dirk Roos, Arthur J. Verhoeven, Richard Van Wijk
Journal: American Journal of Hematology (2012)

Hereditary spherocytosis (HS) is the most frequently encountered disorder of the red blood cell membrane. It is the most common cause of hereditary hemolytic anemia in the Western population and is inherited in an autosomal dominant fashion with an estimated incidence of 1:2,000. The clinical severity of HS varies considerably even within one family, reflecting the genetic heterogeneity of the disorder. We studied a group of 190 patients diagnosed with HS based on morphological examination of the blood smear (i.e., the detection of spherocytes), decreased binding of eosino-5′-maleimide (EMA), and increased osmotic fragility of red blood cells (RBCs). Approximately two-thirds of our HS patients displayed, in addition, a partial deficiency of spectrin, whereas spectrin was normal in the remainder of patients. Because of these differences in spectrin content and the fact that EMA mainly binds to the extracellular part of the RBC transmembrane protein Band 3, we inferred a higher occurrence of Band 3 mutations in the group of patients showing only decreased EMA binding. We therefore performed DNA sequence analysis of SLC4A1, the gene that encodes Band 3, in a cohort of 70 randomly selected unrelated patients from both groups of HS patients. The results revealed a total of 29 mutations (22 different ones) in SLC4A1, all of which were likely to significantly affect Band 3 protein structure and/or function (Table 1). As expected, most mutations were detected in the group of patients without spectrin deficiency (n = 42, 26 mutations), whereas in the group of spectrin-deficient HS patients (n = 28) only three patients carried such a mutation. All patients were heterozygous for the particular mutation in SLC4A1. Fifteen of the listed mutations have not been previously reported in the literature. These mutations were not detected among 100 normal control alleles. Interestingly, approximately one-third of the mutations found in this study were likely to affect correct processing of SLC4A1 pre-mRNA. In case of the c.1,432-2A>T and c.2655+1G>A mutations, aberrant processing was confirmed by studying erythroid-specific RNA isolated from ex vivo generated patient erythroblasts (Table 1). In case of the c.106+1G>A, c.1,890+2T>A, and c.2,655+1G>A mutations, the deleterious effects on proper Band 3 protein expression can be inferred from the fact that only the in trans Band 3 Memphis allele was visualized by polyacrylamide gel electrophoresis of RBC membrane proteins. Notably, the public version of the Human Gene Mutation Database (HGMD—http://www.hgmd.cf.ac.uk) currently lists only 4% of mutations in SLC4A1 as affecting correct processing of pre-mRNA. The results of our study suggest that this particular type of mutations may be more common than currently considered. Rob Van Zwieten1 Jerney J.J.M. François2 Karin Van Leeuwen1 Annet C.W. Van Wesel2 Robin Van Bruggen1 Wouter W. Van Solinge2 Dirk Roos1 Arthur J. Verhoeven3 Richard Van Wijk2 1Department of Blood Cell Research, Laboratory of Red Blood Cell Diagnostics, Sanquin Blood Supply Foundation, Amsterdam The Netherlands; 2Department of Clinical Chemistry and Haematology, Laboratory for Red Blood Cell Research, University Medical Center, Utrecht, The Netherlands; 3Department of Medical Biochemistry, Academic Medical Centre, University of Amsterdam, The Netherlands