Hal E. Schneider

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17 are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35A as a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35A in DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35A wild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.

CTLA-4 is a T cell antigen that is structurally related to CD28 and serves as a high affinity ligand for the B cell antigen B7-1/2. Unlike CD28, the function of CTLA-4 is unclear, although reports have implicated the antigen in the costimulation of T cells. Recently, phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the costimulatory function of CD28 by virtue of its ability to bind to a pYMNM motif within the cytoplasmic tail of the antigen. In this study, we show that CTLA-4 can also associate with PI 3-kinase as detected by lipid kinase analysis and immunoblotting with anti-p85 antiserum. High pressure liquid chromatographic separation of deacylated lipids showed the presence of a peak corresponding to PI-3-P. Anti-CTLA-4 ligation of the receptor induced a significant increase in the levels of precipitable PI 3-kinase activity. Peptide binding studies revealed that the NH2- and COOH-terminal SH2 domains of p85 bind the CTLA-4 cytoplasmic pYVKM motif with an affinity (ID50: 0.6 and 0.04 microM), that is similar to CD28. CTLA-4 binding to PI 3-kinase provides further evidence that CTLA-4 is not an inert counterreceptor, but rather is coupled to an intracellular signaling molecule with the capacity to regulate cell growth.