Inherited compound heterozygous mutations in KLF1. (A) FACS histogram overlay for CD44 expression on red blood cells for the mother, father, and proband compared with an isotype (negative) and a normal (positive) control. There is reduced expression of CD44 in both parents and 2 populations of CD44 red blood cells in the proband: CD44 high and CD44 absent. The CD44 high cells are transfused. (B) FACS histogram for CD235 (GPA) expression. There are no differences in CD235 surface expression among the normal control, the parents, and the proband. (C) Sanger sequencing traces for the parents and proband for the 2 KLF1 gene positions indicated: (i) at the start of exon 2, and (ii) the middle of exon 3. Asterisks indicate sequence variations that lead to W30X or R319EfX34 coding changes. (D) Subsampling of RNA-seq reads from the proband, filtered for those with a mapq score of ≥20 and map location to >1 exon. From a total of >100 e1-e2 splice junction tags, there were no examples of alternative mutation-skipping splice junctions. (E) Western blot of COS7 cells transfected with expression vectors for wild-type and W30X mutant KLF1 cDNA fused to a V5 tag. The bottom (anti-V5) blot shows robust expression of 2 KLF1-V5 bands in the wild-type transduced cells and no protein in the W30X-mutant transduced cells. Lane 3 is a mock-transfected control. The top panel is an antiactin blot to confirm equal protein loading.