Diamond Blackfan anemia is an inherited bone marrow failure syndrome characterized by a severe deficiency of red blood cells, despite the fact that all other hematopoietic lineages differentiate normally and are present in normal numbers. Mutations in 13 different ribosomal protein genes have been identified in multiple unrelated families, most resulting in haploinsufficiency. Project 1. The goal of this project is to provide a molecular diagnosis to each DBA patient. We previously showed that deletions of ribosomal protein genes was responsible for about 10% of all DBA mutations (Farrar JE, Vlachos A, Atsidaftos E, Carlson-Donohoe H, Markello TC, Arceci RJ, Ellis SR, Lipton JM, Bodine DM. Ribosomal protein gene deletions in Diamond-Blackfan Anemia. Blood. 118 (26): 6943-51, 2011). To identify novel DBA mutations, we have initiated whole exome sequencing analysis of 4 unrelated family groups consisting of parents, a DBA proband and either an affected or unaffected sibling. The inclusion of the parents and sibling added a great deal of power to our analysis allowing us to identify 3 strong candidate genes (MCM-2, FLNB and SEMA7A in our first 4 families. We are currently validating these mutations in vitro and we are screening a pool of 96 undiagnosed DBA patients to see if they have any of the newly identified mutation. We have enrolled additional sets of families whose sequence is proceeding through the pipeline. Project 2: Attempts to generate mouse models of DBA by knocking out the mouse Rps19 gene have been unsuccessful. Preliminary data suggests that the output of the normal mouse Rps19 allele increases to compensate for the loss of the second allele. RPS19's only function appears to be in the assembly of the small ribosomal subunit. We hypothesized that mild deficiencies in the levels of ribosomal proteins that serve additional functions might be better candidates for a mouse model of DBA. Mutations in the RPL11 gene account for 10% of known DBA mutations. In addition to its role in the assembly of the large ribosomal subunit, RPL11 also interacts with MDM2, p53 and the rRNA processing pathways, which may account for the significantly higher frequency of craniofacial and physical abnormalities seen in patients with RPL11 mutations. We have generated CRISPR guide RNAs to use for Cas9 mediated introduction of deletions into exon 1 of the Rpl11 gene. Obviously a realistic mouse model would be useful to to provide an in vivo system to study the mechanism of erythroid failure and to test potentially useful drugs.Project 3. The majority of DBA patients are heterozygous for a ribosomal protein gene carrying a deleterious mutation and a normal healthy gene. Haploinsufficeincy results because of decreased translation of functional RP protein. The RP mRNAs are among those RNAs with Terminal Oligo pyrimidine sequence in the 5UTR that regulates the rate of translation. In this project we will analyze a reporter cell line we have developed that has a luciferase gene with a TOP UTR. In collaboration with NCATS we will screen small molecules to see which ones can increase the translation of TOP mRNA. These compounds will be further tested in patient cells and our mouse model.
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