Absence of Band 3 in Severe Dyserythropoietic/Hemolytic Anemia with Complete Distal Renal Acidosis and a Novel Homozygous Exon 12 C.1430C>a (p.Ser477X) SLC4A1 Gene Mutation

The solute carrier 4A1 gene (SLC4A1) encodes theband 3 or bicarbonate anionic exchanger 1 (AE1). It is not only the major glycoprotein of the red blood cell (RBC) membrane but also expressed in acid secreting alpha-intercalated kidney cells. Functionally impairing SLC4A1 mutations reduce the expression and/or activity of AE1 thereby causing a unique combination of hemolytic anemia and distal renal tubular acidosis (dRTA). So far, only four such particular homozygote mutations have been documented in humans: an exon 11 p.400-408 deletion in Southeast Asian ovalocytosis (SAO) with transfusion-dependent dyserythropoietic anemia and dRTA (Picard et al, Blood 123:1963;2014), an exon 13 p.V488M mutation in transfusion-dependent hereditary spherocytosis (HS) and dRTA lacking band 3 (Ribeiro et al, Blood 96:1602;2000), an exon 16 p.S667F mutation in transfusion-dependent HS and incomplete dRTA (Toye et al, Blood 111:5380;2008), and finally an exon 19 p.Ala858Asp in a compensated hemolytic anemia with marked acanthocytosis, echinocytosis and dRTA (Fawaz et al, Europ J Haematol 88:350;2012). We report herein a novel homozygote variant in exon 12 in a patient with a transfusion-dependent dyserythropoietic/hemolytic anemia and complete dRTA.

The now 4-years old Turkish boy was born after 32 weeks of gestation and presented with a severe hemolytic anemia (Hb 40 g/L) that required exchange transfusions and a complete dRTA that was treated with oral bicarbonate. He also suffered from delayed psychomotoric developmental with failure to thrive, trigonocephalus and strabismus convergens. Bone marrow smears showed marked dyserythropoiesis but normal myeloid and megakaryocytic lineages. Although necessary monthly transfusions impeded the patient's direct diagnostic work-up, a flow cytometric eosin-5-maleimide assay eventually revealed a reduced staining of his consanguine parents' and his two siblings' erythrocytes, who all had subclinical signs of spherocytosis despite normal RBC counts.

Based on these findings, we analyzed the SLC4A1 gene and found two homozygous sequence variants in the patient, namely a novel disease-relevant exon 12 nonsense mutation c.1430C>A (p.Ser477X) and a disease-unrelated c.2312-48T>G (rs13306780). The ensuing stop codon of the former truncates the protein, prevents band 3 formation and reduces glycophorin A expression. Bright field imaging uncovered few phenotypic spherocytic band 3 null RBCs even in the peripheral blood.

In accordance with the autosomal recessive inheritance pattern, both healthy parents as well as his healthy siblings were found to be heterozygous carriers. The band 3 protein was reduced to 50-60% in the parents' erythrocytes. An increased approximately 22 kDa-sized band was evident in Coomassie stained gels of the heterozygous mutation carriers' membrane preparations and classified by immunoblotting as peroxiredoxin 2 (PRDX2), which plays a major role in protecting RBCs from oxidative stress.

Taken together, the provided data clearly confirm the relevance of this particular c.1430C>A (p.Ser477X) SLC4A1 mutation in the disease process. Of note, such a severe dyserythropoietic anemia and complete dRTA was also recently reported in a patient with Southeast Asian ovalocytosis and another form of homozygous SLC4A1 mutation (Picard et al, Blood 123:1963;2014).