Regulators of Erythrocyte Volume as Modifiers in Sickle Cell Disease: The Gardos Channel.

Sickle cell disease (SCD) is a monogenic disorder manifest by clinical heterogeneity, indicating that other genetic variants and/or environmental factors influence disease severity. Erythrocyte hydration plays a critical role in SCD pathophysiology, as dehydration contributes directly to HbS polymerization. Changes in the function of transporters associated with erythrocyte dehydration have been observed in SCD, suggesting that these transporters and their regulators act as modifier genes in SCD. Three pathways mediate cation loss and cellular dehydration in the erythrocyte: the KCl cotransport pathway (KCC), a Ca ++ -activated K + channel (Gardos channel), and a sickling induced, nonselective leak pathway (P sickle ) whose molecular identity is unknown. Trials of pharmacologic blockade of KCC and Gardos pathways in the treatment of SCD have been performed or are ongoing. The goal of our studies is to identify genetic variants in modifier genes in patients with SCD that influence erythrocyte hydration, clinical severity, and response to therapy. DNA from 80 sickle cell patients, 67 SS, 12 SC, and 1 Sβ o thal, was prepared. PCR-based strategies, either denaturing high performance liquid chromatography (DHPLC) and/or direct capillary nucleotide sequence analysis, were used to screen the sequence of the coding region exons, splice junctions, and core promoter regions of the KCC3 gene, the Gardos channel gene (KCNN4), the aquaporin 1 gene (AQP1), the aquaporin 3 gene (AQP3), and exons 16, 17 and 18 of the band 3 gene (SLC4A1). These 3 SLC4A1 exons were selected because band 3 has been suggested as a candidate for P sickle and missense mutations that convert band 3 from an anion exchanger to a nonselective cation leak channel have recently been described. Beyond the identification of numerous nonfunctional SNPs, no functional SNPs/missense mutations or other disease-associated mutations were identified in the KCC3 gene (26 exons, 26 amplicons), the AQP1 gene (4 exons, 4 amplicons), the AQP3 gene (6 exons, 6 amplicons), and the band 3 exons. In the Gardos channel gene (9 exons, 11 amplicons), a missense mutation, Ser66Gly, was discovered in exon 2 of 3 patients. This mutation, in a highly conserved residue, is located in the S2 loop near the cytoplasmic junction and is predicted to influence channel function and/or localization. In a fourth patient with mild SC disease, an exon 5 splice junction mutation was discovered. Reverse-transcription PCR analyses of the Gardos channel mRNA in reticulocyte RNA from this patient demonstrated a significant decrease in wild type Gardos channel mRNA and an increase in an alternately spliced smaller mRNA species. Nucleotide sequence analyses demonstrated that aberrant mRNA splicing in this smaller transcript eliminated several exons, including exon 5, and predicted a nonfunctional channel protein. Testing of the functional significance of these Gardos channel variants is in progress. These data indicate that the Gardos channel may be a modifier gene in SCD. Candidate modifier gene studies of pathways influencing erythrocyte hydration have the potential to directly impact management of patients with SCD, allowing prediction of outcome, response to treatment with agents that influence erythrocyte hydration, and identification of targets for therapeutic intervention.