Ribosomal Protein S24 Gene Is Mutated in Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with marked clinical heterogeneity, an increased risk of malignancy and mutations in ribosomal protein (RP) S19 in 25% of probands. To identify other gene(s) mutated in DBA and investigate their expression and function, we performed a genome-wide screen using a 10,000 single nucleotide polymorphism mapping set (Affymetrix) on a large family comprising 10 informative meioses. We found linkage of the DBA phenotype to regions on chromosome 8q, 10 and 6. The RP gene RPS24, is located in the linked region on chromosome 10; we sequenced exons, intron-exon boundaries and the promoter regions in this family, and found a nonsense mutation (316C>T) in exon 4 of RPS24 in five affected individuals, and a wild type sequence in five unaffected family members. This mutation causes the change of Gln106STOP and is predicted to result in formation of a truncated RPS24 protein. Subsequently, we sequenced DNA from 215 unrelated DBA probands, 30 with RPS19 mutations and 185 without. We found another nonsense mutation in exon 2 in a sporadic case, and a splice site deletion resulting in skipped exon 2 in another proband and in his father; over 200 control individuals did not have any of the above sequence changes, indicating that they are pathogenic mutations. To explore the normal role of RPS24 and consider how its dysfunction might result in DBA we performed real time RT-PCR (rt-PCR) and western blotting experiments on 20 normal human tissues and on lymphoblastoid cell lines from diseased and control individuals. Interestingly, rt-PCR of total human RPS24 and RPS19 mRNA revealed a tissue-specific variation in expression level. We found co-ordinate expression of both genes in the majority of studied tissues. Lymphoblastoid cell lines from both probands with nonsense mutations showed a reduced level of RPS24 mRNA, suggesting degradation of mutated transcripts due to nonsense mediated decay, while the RPS19 mRNA level in these patients was normal or elevated. Western blot experiments revealed a reduction of RPS24 protein in lymphoblastoid cell lines from all three mutated probands compared to control samples. Interestingly, co-ordinate expression of RPS24 and RPS19 protein was found in these patients as well as in other patients with RPS19 mutations or without any mutations, suggesting co-regulation of RP expression. To determine whether recruitment of mRNA to polysomes was impaired in DBA patients, we separated lymphoblast cell line lysates from nine diseased and four control individuals on sucrose gradients. We did not detect any significant difference in the RNA ratio of polysome-bound/free ribosomal subunits between diseased and control samples (p<0.3). It is likely that lymphoblasts, which are not defective in DBA, have sufficient ribosomes to mask an abnormality in translation.

In summary, our results suggest that in addition to RPS24 and RPS19, which are mutated in ~ 27% of index cases, other DBA genes are also RP genes or genes involved in ribosome biogenesis or translation, and reinforce the notion that DBA is a ribosomal disease. This study opens new avenues for studying and understanding the pathogenesis of DBA.