Abstract 684

Genome-wide phenotype-driven studies have emerged as a powerful tool for the identification of novel genes in a biological process of interest, new functions for existing genes and the establishment of mouse models of human disease. Recent advances in exomic and whole genome sequencing has simplified mutation detection, making genetic screens in the mouse more facile. We have employed a large-scale mutagenesis screen in mice using the chemical mutagen N-ethyl-N-nitrosourea (ENU) to identify genes regulating erythropoiesis. In this dominant screen, G1 progeny were screened at 7 weeks of age by automated hematological analysis for isolated abnormalities of red cell indices (HCT, RCC and MCV) defined as greater than three standard deviations from the wild type population. Here, we describe the RBC10 mouse line, in which homozygotes had mild anemia with microcytosis, target cells, spherocytes, reticulocytosis and increased resistance to osmotic stress. A combination of SSLP and SNP mapping isolated the genetic mutation to a 3.65 Mb region on chromosome 8. Whole genome Next Generation Sequencing of the mapped interval identified an A to T nucleotide substitution within the coding region of the potassium chloride (KCl) co-transporter, Slc12a4 (KCC1), resulting in a non-conservative amino acid substitution from methionine to lysine at position 935 within the C-terminal cytoplasmic domain. KCC1 and KCC3 function are the major erythroid KCl co-transporters that export KCl in response to hypotonic swelling. We hypothesized that the M935K was an activating mutation of KCC1, based upon the microcytosis and resistance to osmotic stress, which contrasts with the macrocytic phenotype of red cells lacking both KCC1 and KCC3. 86Rb efflux assay confirmed increased KCC1 activity of RBC mutant red cells. We propose that the M935K mutation increases activity of KCC1 by preventing phosphorylation of the nearby threonine reside, a critical regulatory target of the serine-threonine kinase, WNK1.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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