Mutations in RPL17 expand the molecular basis of Diamond-Blackfan anemia and guide insights into unique biochemical signatures underscoring ribosomopathies. E. E. Davis1, D. W. Reid2, J. Liang3, J. R. Willer1, L. Fievet1, Z. A. Bhuiyan4, A. L. Wall1, J. S. Beckmann5, N. Katsanis1, C. V. Nicchitta2,6, F. Fellmann4 1) Center for Human Disease Modeling, Duke University Medical Center, Durham, NC, USA; 2) Department of Biochemistry, Duke University Medical Center, Durham, NC, USA; 3) BGI-Shenzhen, Shenzhen, China; 4) Service de Génétique Médicale, CHUV, Lausanne, Switzerland; 5) Swiss Institute of Bioinformatics, Lausanne, Switzerland; 6) Department of Cell Biology, Duke University Medical Center, Durham, NC, USA.

   Diamond-Blackfan anemia (DBA) is a rare, clinically heterogeneous disorder hallmarked by red blood cell aplasia and incompletely penetrant defects in facio-skeletal development. Associated typically with loss-of-function mutations in at least ten ribosomal components, the extensive genetic heterogeneity poses significant molecular challenges. However, the biochemically tractable organellar basis of DBA, a clinical entity within the ribosomopathies, offers the unique opportunity to investigate the mechanisms underlying disease pathology. Here, we report the genetic, functional, and biochemical dissection of a multigenerational Swiss family with anemia, neutropenia and variable craniofacial and limb abnormalities segregating under a dominant inheritance paradigm. We conducted whole exome sequencing in three affected individuals and identified a novel single nucleotide change in a splice acceptor region of the 60S ribosomal protein L17 encoding gene, RPL17; this variant segregated with all seven affected family members, and mRNA splicing studies showed that the mutation resulted in an in-frame deletion of exon 5. To investigate the physiological relevance and pathogenicity of this variant, we used in vivo complementation studies in zebrafish. First, rpl17 suppression results in micrognathia and anemia phenotypes in developing zebrafish embryos that are orthologous to those observed in patients. Subsequently, we used these relevant phenotypic readouts in zebrafish to determine that the deletion of exon 5 is pathogenic. To explore the mechanistic basis for the ribosomal dysfunction in affected individuals in this pedigree, and to elucidate further the precise roles of RPL17 in the large 60S ribosomal subunit, we conducted a series of biochemical assays using patient-derived cell lines and rpl17 zebrafish morphants. Whereas ribosome maturation was not significantly altered in mutants versus controls, ribosome profiling studies demonstrated reductions in the translation of mRNAs encoding proteins functioning in key developmental pathways relevant to craniofacial development and red cell generation and homeostasis. Strikingly, ribosome profiling of both mutant cells and morphant embryos revealed a distinct translation profile consistent with selective elongational pausing. Together, these studies highlight the power of a multidisciplinary approach to inform both disease architecture and pathogenesis, and also the molecular mechanisms of ribosome function.

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