The Gardos channel (KCNN4) is a Ca2+ sensitive, intermediate conductance, K+ selective channel abundant in several tissues including red blood cells (RBC) where it is involved in cell volume regulation (Hoffman et al. 2003, Cahalan et al. 2015).
Hereditary dehydrate stomatocytosis (DHSt) is characterized by mild to moderate congenital hemolytic anemia with reticulocytosis and splenomegaly associated with RBC dehydration. Affected RBC are characterized by a nonspecific cation leak, reflected in elevated Na+ content, decreased K+, elevated MCHC and MCV, and decreased osmotic fragility. The definitive diagnosis of DHSt is made by osmotic gradient ektacytometry, which shows a leftward shift of the bell-shaped curve.
Heterozygous mutations associated with delayed channel inactivation have been identified for PIEZO1 (Zarychanski et al. 2012), although there are patients lacking a mutation in this gene. Differential diagnosis in hereditary stomatocytosis is important because in DHSt patients, splenectomy is contraindicated due to increased risk of thromboembolic complications.
We studied a 40 year-old Italian man, affected since infancy by chronic hemolyitc anemia (Hb 7-9 g/dL, reticulocytes 10%). The unrelated parents and three siblings were healthy with normal hematologic parameters. He was occasionally transfused until splenectomy, performed at the age of 11 years. After splenectomy Hb levels maintained around 10 g/dL, thrombotic events did not occur. Increased ferritin levels (>1000 ng/ml) and moderate iron overload required chelation therapy. Peripheral blood smear showed anysopoikilocytosis with stomatocytes (13%) echinocytes (7%) schistocytes (6%) ellyptocytes (5%) spherocytes (4%). Bone marrow evaluation revealed erythroid hyperplasia with some dyserytrhopoietc changes, in particular binucleated erythroblasts. Eosin 5 maleimide (EMA) binding test was negative; despite the high number of stomatocytes in peripheral blood, the osmotic gradient ektacytometry curve was not suggestive for hereditary xerocytosis.
The proband's first daughter, born at term after an uneventful pregnancy, presented with severe anemia (Hb 6.1 g/dL) requiring RBC transfusion at birth and at 4 months; at the age of 1 year she displayed a clinical pattern similar to the one of the father, with median Hb levels of 9 g/dL and no need of transfusions.
To uncover the underlying etiologies, all the family members underwent whole exome sequencing. Only one candidate gene, the KCNN4 could be identified: a heterozygous missense mutation (c.1055G>A, p.R352H) was detected as a de novo mutation in the proband, and dominantly transmitted to the daughter. The mutation falling in exon 7 involved in the calmodulin binding domain was predicted to be pathogenic by in silico analysis. Sanger sequencing confirmed the presence of the mutation.
Very recently (July 2015), the same mutation in the KCNN4 gene was described by other groups (Rapetti-Mauss et al. 2015; Andolfo et al. 2015). In heterologous expression systems it could be shown that the Ca2+ sensitivity of the mutated Gardos channel was increased.
We performed single cell experiments, including patch-clamp recordings in RBCs and precursor cells, Ca2+ fluorescence imaging as well as tracer flux experiments in RBCs of the above described patients and healthy controls to get a mechanistic link between the Gardos channel mutation and the cellular symptoms. Despite the presence of a single mutation in Gardos channel alone, pathological alterations in function of multiple ion transport systems were revealed during these studies.
Our findings together with recent reports (Rapetti-Mauss et al. 2015; Andolfo et al. 2015; Glogowska et al. 2015) suggest that the prevalence of mutations in the Gardos channel is higher than previously thought. Therefore it is important to understand the mechanisms implicated by the mutation and to develop reliable diagnostic tools and treatment strategies.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.