MODELING DIAMOND-BLACKFAN ANEMIA

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DESCRIPTION (provided by applicant): One-quarter of families with Diamond-Blackfan anemia (DBA) have heterozygous mutations in RPS19, which encodes the 40S ribosomal subunit protein S19 (Willig et at. 1999). Children with DBA have a nonregenerative red blood cell aplasia with variable birth defects, including growth retardation, craniofacial malformations, triphalangeal thumbs and preaxial polydactyly. In view of the universal cellular requirement for ribosomes, the tissue specificity of RPS19 phenotypes is puzzling. The mechanism of erythroid marrow failure in DBA is unknown. In Drosophila, riboprotein mutations, termed Minutes, are abundant and dispersed across the genome (Lambertsson 1998). They are homozygous lethal and have similar heterozygous phenotypes consisting of smaller, thinner bristles and profoundly delayed development (Schultz 1929). Minutes were instrumental in early studies that defined concepts of cell autonomy, clonal lineage and developmental compartmentation, as Minute cells compete poorly with wild type clones in somatic mosaics. Apart from human DBA, little is known about ribosomal protein (RP) gene mutations in vertebrates. Recently, my laboratory discovered that the spontaneous mouse mutation Bst (belly spot and tail) is caused by an intragenic deletion in Rpl24, which encodes the 60S ribosomal subunit protein L24 (Oliver et al. 2004). Bst/+ mice have vertebral defects (tail kinks), preaxial polydactyly, midventral spotting and a deficiency of retinal ganglion cells. Homozygotes die before implantation. The Bst mutation causes aberrant splicing in 80% of Rpl24 transcripts, truncation of L24 protein, and delayed ribosome biogenesis in vivo. Bst/+ fibroblasts grow slower than wild type, remain longer in G1 phase, and exhibit decreased overall rates of protein synthesis. Mouse BAG and human cDNA transgenes correct this phenotype. The hematopoietic capacity of Bst/+ mice has not been explored in detail. In this proposal, we aim to: (1) generate mice with null and hypomorphic mutations in Rps19, using selected gene trap ES cell lines; (2) characterize hematopoiesis in Rps19 knockout mice and the spontaneous riboprotein mutants Bst/+ (Rpl24) and Ts/+ (Rpl38), under normal and erythroid stress conditions; and (3) test two alternative mechanisms for the marrow specificity of DBA mutations - that normal erythroid progenitors express relatively low levels of Rps19 mRNA, making them particularly sensitive to reduced gene dosage, and that S19 has an extraribosomal function to promote red cell differentiation.