The Xla (X-linked anemia) Mouse: A Transient Neonatal Anemia Caused by a Gata1 Splicing Mutation,
Authors: Miller Kyle , Silvey Michael , Logsdon Derek , Balch Frederick , Nsumu Ndona , Sokolovsky Inna , Gibson Margaret , Bi Chengpeng , Heruth Daniel P. , White Robert A 
Published: October 2019

Abstract 3162

The Xla (X-linked anemia) mutant mouse was generated by N-ethyl-N-nitrosourea (ENU) mutagenesis and results in a severe and transient neonatal anemia. Xla/+ females exhibit severe anemia with 50% the level of red blood cell number, hematocrit and hemoglobin. Male Xla mice die in utero at 10.5 days gestation. The neonatal anemia observed in Xla/+ female pups is resolved by weaning age at 3 weeks by which time the mice present with a normal hematological phenotype. It is unknown how the neonatal anemia in Xla/+ females is alleviated. Previously, we mapped the Xla locus to the proximal end of the X chromosome near candidate gene Gata1 which showed no change in the coding sequence of GATA1 protein. Now we report the identification of a Gata1 mutation in Xla mice that results in an mRNA splicing defect. A nucleotide change (G to A) was identified 5 base pairs downstream of Exon 1E in intron 1 of the Xla Gata1 gene and results in the lack of incorporation of Exon 1E in the Gata1 mRNA expressed from the mutant locus. Therefore, in some erythroid lineage cells in Xla/+ mice, the normal 1E exon of Gata1 mRNA is replaced by Exon 1Eb/c which is known not to impact erythropoeisis since no GATA1 protein is made by this mRNA due to its inability to bind to ribosomes. These data show the Xla mouse results from a single nucleotide change impacting the normal splicing of the Gata1 gene. A second goal of this study was to understand why Xla/+ mice exhibit the neonatal transient anemia. A contributing factor is X chromosome inactivation which occurs in female mice during development. The short-term anemia in Xla mice was thought to be due to clonal selection of erythroid lineage cells characterized by the expression of GATA1 protein from the active X chromosome expressing only from the wild type Gata1 locus. Using an X-linked gene expressed in red blood cells (Pgk1, phosphoglycerate kinase 1) that varies between Xla mice and a wild derived strain, CAST/Ei, we examined the active state of the X chromosomes based on the expression of Pgk1 RNA in reticulocytes from hybrid Xla mice generated by breeding of these different strains. Examining expression of the X-linked Pgk1 SNP variant in the RNA of reticulocytes from hybrid Xla/+ mice reveals red blood cells are generated from two types of erythroid lineage cells. Pgk1 SNP RT-PCR analysis reveals that red blood cells not only derive from erythroid progenitors with the active X chromosome carrying the wild type Gata1 gene but also red blood cells are produced by erythroid lineage cells expressing the Xla mutant Gata1 mRNA on the active X chromosome (which does not make GATA1 protein). Therefore, some Xla erythroid cells derive from progenitors which express Gata1 transcripts using Exon 1Eb/c that does not stimulate erythropoiesis due to lack of GATA1 protein. The question is how these erythroid precursors generate normal red blood cells without the production of GATA1 protein. We hypothesize there is a developmentally expressed compensatory gene or pathway replacing GATA1 expression in GATA1-lacking erythroid precursors and required for the production of red blood cells in Xla mice. Analysis is underway to identify a potential novel gene or pathway impacting erythropoiesis in these mutant mice.

Disclosures:

No relevant conflicts of interest to declare.