Ips Cell Lines From Members Of a Family With Variable Penetrance Of An RPS19 Mutation Recapitulate The Diamond Blackfan Anemia (DBA) Phenotype and Show Differences In Ribosome Biogenesis and TGF Beta Signaling

DBA is an inherited bone marrow failure syndrome that usually presents in the first year of life with red cell aplasia and variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations in one of 11 genes encoding small or large subunit ribosomal proteins (RPs). It is not known how RP haploinsufficiency causes failed erythropoiesis. The penetrance of DBA varies widely, even between members of the same family. This is presumably due to modifying factors but the nature of these remains enigmatic. We have previously derived induced pluripotent stem cells (iPSCs) from fibroblasts of a DBA patient with a RPS19 mutation and the mutant line showed abnormal ribosome biogenesis and altered erythropoiesis, thus recapitulating the DBA phenotype.

To investigate the mechanisms underlying variable penetrance in DBA, we generated induced pluripotent stem cells (iPSCs) from members of a family with DBA caused by a nonsense mutation in RPS19 (p.Q126X). We analyzed iPSCs from two DBA patients and an asymptomatic individual, all of whom carry the RPS19 mutation. iPSC lines from the DBA affected individuals showed faulty 40S (small) ribosomal subunit assembly, reduced processing of 18S rRNA, and impaired hematopoiesis, with about 2.5 fold decreased expansion of mutant erythroblasts compared to those derived from iPSCs from normal individuals or the asymptomatic carrier. In contrast, iPSCs from the asymptomatic carrier showed milder defects in ribosome biogenesis and hematopoiesis. Genetic correction of ribosomal protein deficiency via cDNA transfer into the “safe harbor” AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis in the DBA iPSCs.

Using an Affymetrix human exon array to investigate the pathways that are affected in these iPSCs, we observed striking over-activation of the TGF beta pathway in iPSCs of the DBA affected individuals compared to the normal control lines. For example, TGF beta downstream genes, such as DKK1, BAMBI, IGFBP5, COL3A1, COLA1A1, SERPINE1, TGFBI and RGS5 were significantly increased in the DBA iPSCs compared to wild type control cells (Table 1 

We conclude that the variable penetrance of the RPS19 mutation in this family is due to modulation of its effect on ribosome biogenesis and that TGF beta signaling may be important in DBA pathogenesis. Whether the genetic factors responsible for the variable penetrance are inherited or acquired remains an important question.