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Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multi-lineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/- HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome-editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

Long thought to be too big and too ubiquitous to fail, we now know that human cells can fail to make sufficient amounts of ribosomes, causing a number of diseases collectively known as ribosomopathies. The best characterized ribosomopathies, with the exception of Treacher Collins syndrome, are inherited bone marrow failure syndromes, each of which has a marked increase in cancer predisposition relative to the general population. Although rare, emerging data reveal that the inherited bone marrow failure syndromes may be underdiagnosed on the basis of classical symptomology, leaving undiagnosed patients with these syndromes at an elevated risk of cancer without adequate counselling and surveillance. The link between the inherited ribosomopathies and cancer has led to greater awareness that somatic mutations in factors involved in ribosome biogenesis may also be drivers in sporadic cancers. Our goal here is to compare and contrast the pathophysiological mechanisms underpinning ribosomopathies to gain a better understanding of the mechanisms that predispose these disorders to cancer.

Diamond–Blackfan anemia (DBA) is a rare genetic hypoplasia of erythroid progenitors characterized by mild to severe anemia and associated with congenital malformations. Clinical manifestations in DBA patients are quite variable and genetic testing has become a critical factor in establishing a diagnosis of DBA. The majority of DBA cases are due to heterozygous loss‐of‐function mutations in ribosomal protein (RP) genes. Causative mutations are fairly straightforward to identify in the case of large deletions and frameshift and nonsense mutations found early in a protein coding sequence, but diagnosis becomes more challenging in the case of missense mutations and small in‐frame indels. Our group recently characterized the phenotype of lymphoblastoid cell lines established from DBA patients with pathogenic lesions in RPS19 and observed that defective pre‐rRNA processing, a hallmark of the disease, was rescued by lentiviral vectors expressing wild‐type RPS19. Here, we use this complementation assay to determine whether RPS19 variants of unknown significance are capable of rescuing pre‐rRNA processing defects in these lymphoblastoid cells as a means of assessing the effects of these sequence changes on the function of the RPS19 protein. This approach will be useful in differentiating pathogenic mutations from benign polymorphisms in identifying causative genes in DBA patients.

Diamond Blackfan anemia syndrome (DBAS) is a rare, heritable bone marrow failure syndrome characterized by severe macrocytic anemia, congenital anomalies and predisposition to cancer, most often diagnosed during infancy. More than 98% of DBAS patients with a molecular diagnosis have mutations in a gene encoding one of the ~80 ribosomal proteins (RP) leading to haploinsufficiency. A molecular diagnosis in a patient with DBAS is critical for a definitive diagnosis, the identification of compatible related transplant donors, and developing reproductive strategies for families. Targeted sequencing of RP genes, single nucleotide polymorphism comparative genome hybridization (SNP array) to detect >30 kb deletions (Farrar et al. Blood. 2011) and exome sequencing (WES) (Ulrisch et al. Am J Hum Genet. 2018) has identified RP mutations in ~80% of patients, leaving ~20% of patients with DBAS without a molecular diagnosis. Targeted sequencing and WES focus on only coding sequences. We hypothesized that remaining 20% of DBAS mutations were in the non-coding regions of RP genes, such as promoters or introns. To test this hypothesis, we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 14 patients with no molecular diagnosis after targeted sequencing, SNP array or WES. On average, we aligned ~3.2x10 7 paired end reads of 250 base pairs for each patient (~65X coverage). We focused our analysis on the sequences in and around the RP genes. To identify deletions, we used a suite of detection tools: DELLY, GRIDSS, MANTA, and LUMPY. More than 90% of deletions identified by any 2 of these tools were confirmed by PCR. We identified 5 deletions in the introns of RP genes, ranging from 11 to 467 base pairs in length, which we hypothesized disrupted splicing of the nascent RNA transcript. To test this, we created minigenes in which we replaced exon 2 of a gamma globin gene with either the WT or mutant RP exon. All wild type exons spliced normally. A 467 base pair deletion in RPL27 exon 3 was sufficient to prevent the correct splicing of that intron. Examination of the eCLIP data for RNA binding proteins revealed that spliceosome complex proteins (including SF3B1, SF3B4 and EFTUD2) and Dead-box RNA helicases bind in the deleted region. A 28 base pair deletion in exon 3 of RPL6 removes a polypyrimidine tract that is a critical part of the 3' splice junction consensus sequence, which we presume is also deleterious. The other 3 intronic deletions did not disrupt splicing. We also identified 2 causative point mutations. A point mutation 5 bases into intron 1 of the RPS26 gene changes a base in the 5' splice donor consensus sequence, which activated a cryptic splice donor in the 5' untranslated region. This aberrant splice removes the ATG initiation codon causing an untranslatable RNA. In another patient, we identified a mutation in exon 1 of the RPS27 gene, judged to be a benign amino acid change. This mutation disrupted splicing.by activating a cryptic splice donor site in the 5' untranslated region which removes the ATG initiation codon and causes a frame shift. We were referred two patients with possible duplications of the RPL35a gene. To identify duplications, we employed MinION long read single molecule sequencing. We had an average read length of ~ 6-10kb with the longest read being 1.3Mb. Overall coverage was >85X. We used minimap2 to align the reads to the reference human genome and used SNIFFLES to call the variants. One patient was the parent of DBAS-affected patient with no history of anemia. In this patient, we identified a duplication of 400 kb that included the entire RPL35a region along with genes on either side. We conclude that this duplication is not likely to cause DBA. The second patient was diagnosed with DBAS. In this patient, we identified a duplication of 4 kb including exons 1 and 2 of RPL35a We conclude that this duplication disrupts the RPL35a gene and is a likely cause of DBA. Whole genome sequencing of 15 DBAS patients identified 5 likely causative mutations in RP genes, confirming that most genetically undiagnosed cases of DBAS will involve known genes encoding RP. We conclude that the pipeline for obtaining a molecular diagnosis for DBAS from targeted sequencing, SNP array, and exome sequencing to whole genome sequencing. Disclosures Vlachos: Novartis: Membership on an entity's Board of Directors or advisory committees. Lipton: Celgene: Membership on an entity's Board of Directors or advisory committees.

IKZF1 encodes Ikaros, a zinc finger–containing transcription factor crucial to the development of the hematopoietic system. Germline pathogenic variants in IKZF1 have been reported in patients with acute lymphocytic leukemia and immunodeficiency syndromes. Diamond–Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by erythroid hypoplasia, associated with a spectrum of congenital anomalies and an elevated risk of certain cancers. DBA is usually caused by heterozygous pathogenic variants in genes that function in ribosomal biogenesis; however, in many cases the genetic etiology is unknown. We identified a germline IKZF1 variant, rs757907717 C > T, in identical twins with DBA-like features and autoimmune gastrointestinal disease. rs757907717 C > T results in a p.R381C amino acid change in the IKZF1 Ik-x isoform (p.R423C on isoform Ik-1), which we show is associated with altered global gene expression and perturbation of transcriptional networks involved in hematopoietic system development. These data suggest that this missense substitution caused a DBA-like syndrome in this family because of alterations in hematopoiesis, including dysregulation of networks essential for normal erythropoiesis and the immune system.

Heterozygous inactivating mutations in ribosomal protein genes (RPGs) are associated with hematopoietic and developmental abnormalities, activation of p53, and altered risk of cancer in humans and model organisms. Here we performed a large‐scale analysis of cancer genome data to examine the frequency and selective pressure of RPG lesions across human cancers. We found that hemizygous RPG deletions are common, occurring in about 43% of 10,744 cancer specimens and cell lines. Consistent with p53‐dependent negative selection, such lesions are underrepresented in TP53 ‐intact tumors ( P ≪ 10−10), and shRNA‐mediated knockdown of RPGs activated p53 in TP53 ‐wild‐type cells. In contrast, we did not see negative selection of RPG deletions in TP53 ‐mutant tumors. RPGs are conserved with respect to homozygous deletions, and shRNA screening data from 174 cell lines demonstrate that further suppression of hemizygously deleted RPGs inhibits cell growth. Our results establish RPG haploinsufficiency as a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically. ![][1] Hemizygous deletion of ribosomal protein genes (RPGs) is a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically. [1]: /embed/graphic-1.gif

While impaired ribosomal biogenesis is observed in neurodegenerative diseases, its pathogenic contributions are not clear. For instance, it is well established that in rodent neurons, genetic inhibition of RNA-polymerase 1 that transcribes rRNA results in structural disruption of the nucleolus, neuronal apoptosis, and neurodegeneration. However, in most neurodegenerative diseases, nucleolar morphology is unaffected. It is reported here that in primary cortical neurons from newborn rats, inhibition of ribosomal biogenesis by shRNA-mediated knockdowns of several ribosomal proteins including S6, S14, or L4 resulted in p53-mediated apoptosis despite absence of structural disruption of the nucleolus. Conversely, knockdown of the RP L11, which in nonneuronal systems mediates p53 activation downstream of ribosomal stress, protected neurons against inhibition of ribosomal biogenesis but not staurosporine. Moreover, overexpression of L11 enhanced p53-driven transcription and increased neuronal apoptosis. In addition, inhibition of p53, or L11 knockdown, blocked apoptosis in response to the RNA analog 5-fluorouridine which perturbed nucleolar structure, inhibited ribosomal synthesis, and activated p53. Although the DNA double-strand break (DSB) inducer etoposide activated p53, nucleolar structure appeared intact. However, by activating the DNA damage response kinase ATM, etoposide increased 47S pre-rRNA levels, and enhanced nucleolar accumulation of nascent RNA, suggesting slower rRNA processing and/or increased Pol1 activity. In addition, shL11 reduced etoposide-induced apoptosis. Therefore, seemingly normal morphology of the neuronal nucleolus does not exclude presence of ribosomal stress. Conversely, targeting the ribosomal stress-specific signaling mediators including L11 offers a novel approach to uncover neurodegenerative contributions of deregulated ribosomal synthesis as exemplified in DSB-challenged neurons.

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Diamond Blackfan Anemia Syndrome (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe macrocytic anemia, most often diagnosed during infancy. Congenital anomalies and predisposition to cancer are also important features of DBA. Establishment of a molecular diagnosis in a patient with DBA is critical to determine treatment strategies (i.e. the identification of compatible related transplant donors), as well as developing reproductive strategies for genetically at risk families. The overwhelming majority (>98.75%) of DBA patients with a molecular diagnosis have mutations in a Ribosomal Protein (RP) gene. Targeted and exome sequencing (WES) strategies can identify RP mutations in >70% of DBA patients (Ulrisch et al. Am J Hum Genet. 2018). Single Nucleotide Polymorphism Comparative Genome Hybridization (SNP array) detects >30 kb deletions of RP genes (which cannot be identified by sequencing) in ~10% of DBA patients (Farrar et al. Blood. 2011), leaving ~20% of DBA patients without a molecular diagnosis. We hypothesized that smaller copy number variants (CNVs - either insertions or deletions) in RP genes that are below the limit of detection of SNP array are responsible for the remaining 20%. To test this hypothesis we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 6 patients who had no mutations detected by WES or SNP array. On average, we aligned ~1x1010paired end reads of 250 base pairs for each patient (~83X coverage of the genome). The aligned sequences were analyzed for CNVs using two independent software packages. Delly analyzes the two ends of each sequence read and maps them to the current human reference genome. Read ends that map further apart than expected are flagged as potential CNVs. CNVkit estimates regions of copy loss by changes in average sequencing depth. Using relatively relaxed thresholds in Delly and CNVkit we identified ~100 candidate CNVs in each patient. We filtered out CNVs present in public databases and focused on those CNVs in the region of the RP genes. This analysis identified 2-5 potential RP gene associated CNVs in each patient. We designed PCR primers that flanked each putative CNV and confirmed at least one RP CNV in all 6 patient DNAs. At this time, the CNVs in two patients are in the process of evaluation. We have validated causative RP CNVs in the other 4 patients, representing one known and three novel DBA genes. One patient had a 464 bp deletion in 3rdintron of the RPL27 gene, which is mutated in other DBA patients. We hypothesized that the deletion caused a splicing defect. Using a mini gene in which the second intron of the gamma globin gene was replaced with the 3rdintron of either the wild type or mutant RPL27 gene, we showed that the mutant exon was not spliced. An alternative hypothesis, that the deletion removed an enhancer element, was also tested, but no enhancer activity was detected. We conclude that the RPL27exon deletion causes aberrant splicing leading to an unstable RPL27 mRNA and haploinsufficiency of RPL27. A second patient had a 3.5 kb deletion at the 3' end of the RPS5 gene, including the stop codon and poly A addition site. We hypothesized that the lack of the 3' processing signals would lead to an unstable mRNA. To test this hypothesis we generated MYC and FLAG tagged wildtype and 3' deleted RPS5 genes and co-transfected them into 293T cells. Regardless of the tag used, RT-PCR analysis showed a severe reduction in the mutant mRNA levels. Western Blot analysis demonstrated that only the wild type protein was expressed, leading to the conclusion that the RPS5 truncation led to an unstable RPS5 mRNA and haploinsufficiency of RPS5. A third patient had a 28 kb deletion that removes the RPS9 gene. shRNA knockdown of RPS9 mRNA in normal CD34+ cells inhibited erythroid differentiation, leading to the conclusion that RPS9 deficiency causes DBA. Finally, we observed a 3 bp insertion in exon 6 of the RPL14 gene. The deletion adds an alanine residue to a string of 10 alanines in the wild type allele. We confirmed the insertion by targeted sequence analysis of patient DNA. Our data show that WGS can identify small CNVs that cause DBA in at least 2/3 of patients who do not have a mutation detectable by other methods. We believe that WGS analysis following targeted sequencing, SNP array and WES can identify virtually all DBA mutations. With declining WGS costs, we recommend adding WGS to the molecular diagnostic pipeline for genetic testing of DBA. Disclosures Farrar: Novartis: Research Funding. Vlachos:Novartis: Other: Steering committee member.

Diamond-Blackfan anemia (DBA) is a congenital ribosomopathy and bone marrow failure syndrome manifesting typically in infancy with erythroid hypoplasia. Approximately half of affected individuals also have developmental anomalies. Over time, additional cytopenias can develop, including reduced hematopoietic stem and progenitor cells (HSPC). Heterozygous loss-of-function mutations in over 20 ribosomal protein (RP) genes cause approximately 70% of DBA cases, although only 7 genes (RPS19, RPL5, RPS26, RPL11, RPL35a, RPS24 and RPS7) account for over 90% of patients with a known DBA genotype. Medical therapies including steroids, chronic transfusions are partially effective but have considerable side effects. Hematopoietic stem cell transplantation (HSCT) from matched related or unrelated donors is curative with recently reported good outcomes, although many patients lack a suitable donor and/or have serious treatment-related comorbidities that increase HSCT-related toxicities. Case reports of spontaneous genetic reversion in DBA suggest that RP gene-corrected HSPC have competitive advantage over RP-deficient cells, thus providing the rationale for gene therapy as a feasible therapeutic approach. Induced pluripotent stem cell (iPSC) technology provides a robust model of human disease and can recapitulate hematopoietic defects encountered in bone marrow failure syndromes. The goals of this study was to establish a culture system from patient-derived iPSCs that can recapitulate key aspects of DBA pathophysiology and provide a preclinical model for gene manipulation to correct the abnormal phenotype (Figure 1). We developed iPSCs from individuals with DBA who were enrolled on INSIGHT (NCT02720679), an IRB-approved, prospective study that includes biobanking of peripheral blood mononuclear cells (PBMNC) from patients with bone marrow failure syndromes. We first reprogrammed these DBA PBMNC into iPSCs using non-integrating Sendai virus to establish lines with pathogenic mutations in RPS19 (c.191>T, p.Leu64Pro), RPS19 (c.184C>T, p.Arg62Trp), RPL11 (c.61dupT, p.Cys21Leufs*13), and a variant of uncertain significance (VUS) in RPS7 (c.277_279delGTC, pVal93del). Undifferentiated iPSC lines exhibited abnormal ribosomal biogenesis revealed by polysome profiling and pre-rRNA analysis. Upon in vitrodifferentiation to hematopoietic lineages, the mutant iPSCs recapitulated DBA phenotypes with reduced CD34+ HSPCs, near absence of erythroid colonies (BFU-E and CFU-E) colonies and failure to produce erythroid cells in liquid culture. We used two methods to correct single nucleotide RP mutations in DBA iPSCs (Figure 1): i) CRISPR/Cas9-mediated homology-directed repair, and ii) base-editing, which utilizes catalytically inactive Cas9 fused to a deaminase that interconverts nucleotides directly in the absence of double-stranded DNA breaks. Corrected "isogenic" lines showed phenotype similar to wild type controls, with restored erythroid differentiation, and normal polysome maturation and pre-rRNA ratios. Because some patients carry large intragenic or whole RP gene deletions that are not amenable to gene correction, we also explored the feasibility of gene rescue by inserting a wild type copy of the defective gene (Figure 1). Using zinc-finger nuclease (ZFN), we inserted wild type RP cDNA constructs into the "safe harbor" AAVS1 locus on chromosome 19, thereby rescuing abnormal phenotypes of patient-derived iPSC lines with RPS19(p.Arg62Trp) and RPL11(p.Cys21Leufs*34) mutations. Additionally, we explored lentiviral gene delivery as an alternative method for RP gene replacement. We compared different promoters including MND, PGK and EF1a and found that the latter was most effective at rescuing RP gene expression in iPSC cells. Transduction of lentiviral vectors with wild type RPS19or RPL11fused to the EF1a promoter into three iPSC lines with RPS19or RPL11mutations resulted in stable transgene expression of RPS19or RPL11genes and phenotypic rescue. This study supports the feasibility of establishing iPSCs from DBA subjects with different genotypes. These iPSC lines provide a useful resource for numerous studies of DBA including preclinical approaches to gene therapy, evaluating the pathogenicity of RP gene variants of unknown significance and examining the pathophysiology of RP haploinsufficiency. Disclosures Estepp: Esperion: Consultancy; Forma Therapeutics: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Eli Lilly and Co: Research Funding; Daiichi Sankyo: Consultancy. Weiss:GlaxoSmithKline: Consultancy; Rubius Inc.: Consultancy; Cellarity Inc.: Consultancy; Beam Therapeutics: Consultancy; Esperion: Consultancy.

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. A global reduction in ribosome levels in Diamond-Blackfan anemia profoundly alters translation of a select subset of transcripts, thereby impeding erythroid lineage commitment. Keyword: hematopoiesis; lineage commitment; GATA1; genetics; ribosome; translation; Diamond-Blackman anemia; erythropoiesis, National Institutes of Health (U.S.) (Grants R01 DK103794), National Institutes of Health (U.S.) (Grant R33 HL120791)

There are an increasing number of human disorders linked to defects in ribosome synthesis collectively known as ribosomopathies. Here we use the prototypical ribosomopathy, Diamond-Blackfan anemia, to explore relationships between the structure of the ribosome, its biogenesis, and the molecular mechanisms that contribute to disease pathology. Other ribosomopathies are discussed as they relate to the genes affected and pathophysiological mechanisms involved in Diamond-Blackfan anemia. The recent finding that several genes affecting ribosome biogenesis are somatically mutated in human tumors implies that understanding the molecular mechanisms underlying this rare group of disorders will likely have much broader implications.

Heterozygous inactivating mutations in ribosomal protein genes (RPGs) are associated with hematopoietic and developmental abnormalities, activation of p53, and altered risk of cancer in humans and model organisms. Here we performed a large‐scale analysis of cancer genome data to examine the frequency and selective pressure of RPG lesions across human cancers. We found that hemizygous RPG deletions are common, occurring in about 43% of 10,744 cancer specimens and cell lines. Consistent with p53‐dependent negative selection, such lesions are underrepresented in TP53‐intact tumors (P ≪ 10−10), and shRNA‐mediated knockdown of RPGs activated p53 in TP53‐wild‐type cells. In contrast, we did not see negative selection of RPG deletions in TP53‐mutant tumors. RPGs are conserved with respect to homozygous deletions, and shRNA screening data from 174 cell lines demonstrate that further suppression of hemizygously deleted RPGs inhibits cell growth. Our results establish RPG haploinsufficiency as a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically.

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while

Diamond Blackfan anaemia (DBA) is a congenital bone marrow failure syndrome characterised by selective red cell hypoplasia. DBA is most often due to heterozygous mutations in ribosomal protein (RP) genes that lead to defects in ribosome biogenesis and function and result in ribosomal stress and p53 activation. The molecular mechanisms underlying this pathology are still poorly understood and studies on patient erythroid cells are hampered by their paucity. Here we report that RP-mutated lymphoblastoid cell lines (LCLs) established from DBA patients show defective rRNA processing and ribosomal stress features such as reduced proliferation, decreased protein synthesis, and activation of p53 and its target p21. These phenotypic alterations were corrected by gene complementation. Our data indicate that DBA LCLs could be a useful model for molecular and pharmacological investigations.

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene.

A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as ‘new system’ names.

BackgroundDiamond-Blackfan anaemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterised by erythroid hypoplasia. It is associated with congenital anomalies and a high risk of developing specific cancers. DBA is caused predominantly by autosomal dominant pathogenic variants in at least 15 genes affecting ribosomal biogenesis and function. Two X-linked recessive genes have been identified.ObjectivesWe aim to identify the genetic aetiology of DBA.MethodsOf 87 families with DBA enrolled in an institutional review board-approved cohort study (ClinicalTrials.gov Identifier:NCT00027274), 61 had genetic testing information available. Thirty-five families did not have a known genetic cause and thus underwent comprehensive genomic evaluation with whole exome sequencing, deletion and CNV analyses to identify their disease-associated pathogenic variant. Controls for functional studies were healthy mutation-negative individuals enrolled in the same study.ResultsOur analyses uncovered heterozygous pathogenic variants in two previously undescribed genes in two families. One family had a non-synonymous variant (p.K77N) inRPL35; the second family had a non-synonymous variant (p. L51S) inRPL18. Both of these variants result in pre-rRNA processing defects. We identified heterozygous pathogenic variants in previously known DBA genes in 16 of 35 families. Seventeen families who underwent genetic analyses are yet to have a genetic cause of disease identified.ConclusionsOverall, heterozygous pathogenic variants in ribosomal genes were identified in 44 of the 61 families (72%). De novo pathogenic variants were observed in 57% of patients with DBA. Ongoing studies of DBA genomics will be important to understand this complex disorder.

Calcium/calmodulin-dependent protein kinase 4 (gene and transcript: CaMK4; protein: CaMKIV) is the nuclear effector of the Ca(2+) /calmodulin kinase (CaMK) pathway where it coordinates transcriptional responses. However, CaMKIV is present in the cytoplasm and axons of subpopulations of neurons, including some sensory neurons of the dorsal root ganglia (DRG), suggesting an extranuclear role for this protein. We observed that CaMKIV was expressed strongly in the cytoplasm and axons of a subpopulation of small-diameter DRG neurons, most likely cutaneous nociceptors by virtue of their binding the isolectin IB4. In IB4+ spinal nerve axons, 20% of CaMKIV was colocalized with the endocytic marker Rab7 in axons that highly expressed CAM-kinase-kinase (CAMKK), an upstream activator of CaMKIV, suggesting a role for CaMKIV in signaling though signaling endosomes. Using fluorescent in situ hybridization (FISH) with riboprobes, we also observed that small-diameter neurons expressed high levels of a novel 3' untranslated region (UTR) variant of CaMK4 mRNA. Using rapid amplification of cDNA ends (RACE), reverse-transcription polymerase chain reaction (RT-PCR) with gene-specific primers, and cDNA sequencing analyses we determined that the novel transcript contains an additional 10 kb beyond the annotated gene terminus to a highly conserved alternate polyadenylation site. Quantitative PCR (qPCR) analyses of fluorescent-activated cell sorted (FACS) DRG neurons confirmed that this 3'-UTR-extended variant was preferentially expressed in IB4-binding neurons. Computational analyses of the 3'-UTR sequence predict that UTR-extension introduces consensus sites for RNA-binding proteins (RBPs) including the embryonic lethal abnormal vision (ELAV)/Hu family proteins. We consider the possible implications of axonal CaMKIV in the context of the unique properties of IB4-binding DRG neurons.

Classical 5q- syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q- syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q- deletions.

Background Shwachman–Diamond syndrome (SDS), associated with SBDS mutations, is characterized by pancreatic exocrine dysfunction and marrow failure. Sdo1, the yeast ortholog of SBDS, is implicated in maturation of the 60S ribosomal subunit, with delayed export of 60S-like particles from the nucleoplasm when depleted. Sdo1 is needed for release of the anti-subunit association factor Tif6 from 60S subunits, and Tif6 may not be recycled to the nucleus when Sdo1 is absent. Methods To clarify the role of SBDS in human ribosome function, TF-1 erythroleukemia and A549 lung carcinoma cells were transfected with vectors expressing RNAi against SBDS. Results Growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to controls. To analyze the effect of SBDS on 60S subunit maturation in A549 cells, subunit localization was assessed by transfection with a vector expressing a fusion between human RPL29 and GFP: we found a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits. Polysome analysis of TF-1 knockdown cells showed a decrease in free 60S and 80S subunits. We also analyzed the levels of eIF6 (human ortholog of Tif6) following near-complete knockdown of SBDS in TF-1 cells and found an approximately 20% increase in the amount of eIF6 associated with the 60S subunit. Conclusions We conclude that knockdown of SBDS leads to growth inhibition and defects in ribosome maturation, suggesting a role for wild-type SBDS in nuclear export of pre-60S subunits. Furthermore, knockdown of SBDS may interfere with eIF6 recycling. Pediatr Blood Cancer 2013;60:281–286. © 2012 Wiley Periodicals, Inc.

Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform.

Diamond Blackfan Anemia (DBA) is a congenital red blood cell aplasia usually caused by haploinsufficiency of selected ribosomal protein genes. Approximately 10% of DBA patients are anemic at birth, and 80% of DBA patients present by 6 months. These and other data suggest that the DBA erythroid defect is exacerbated by the switch from fetal to adult hematopoiesis. We hypothesize that unique molecular or cellular features initiate DBA pathogenesis during fetal hematopoiesis that manifests after the switch to adult erythropoiesis as severe anemia. To test this hypothesis, we developed a mouse model with constitutive Rpl11 haploinsufficiency in fetal and adult hematopoietic tissues driven by VavCre. Rpl11 is a commonly mutated ribosomal gene in DBA. In adult mice, reduced Rpl11 expression results in severe anemia by 6 weeks, with markedly reduced hemoglobin, increased red cell volume and elevated erythroid adenosine deaminase. Flow cytometric analysis of the bone marrow (BM) revealed no change in the frequency of megakaryocyte-erythroid progenitors (MEP) but a significant increase in both BFU-E and CFU-E, with a corresponding decrease in maturing Ter119+ cells. Functional progenitor assays showed reduced and delayed proliferation with an impaired ability to produce viable CD71+ Ter119+ cells. Treatment of purified Rpl11+/- BFU-E with dexamethasone rescued this proliferative defect. Thus, our data indicate that a differentiation block and functional impairment at the BFU-E stage of adult erythropoiesis contribute to Rpl11 haploinsufficient anemia. At the molecular level, Rpl11 haploinsufficiency in BM resulted in the accumulation of 32S and 12S pre-rRNA. Polysome profiling of total BM suggested reduced polysome assembly and impaired translation. Transcriptional profiling of normal and Rpl11+/- MEP, BFU-E, CFU-E and Ter119+ cells identified 13 differentially expressed genes across DBA erythropoiesis including known and novel targets such as CDKN1A, TNFSF4 and TSPAN14 . Enrichment of the p53 pathway was unique to BFU-E and CFU-E progenitors. Interestingly, TGFB1 and EGF pathways were also selectively affected within Rpl11+/- BFU-E and CFU-E progenitors, suggesting p53-independent self-renewal and proliferative defects in these populations. In contrast to adult hematopoiesis, fetal hematopoiesis defects are less severe. For example, fetal BFU-E are not significantly increased within E14 fetal livers and functionally generate a similar number of maturing erythroblasts in vitro . Lodish and colleagues have established that fetal liver BFU-E comprise at least two distinct subpopulations, termed "early" and "late". Early BFU-E have high self-renewing capacity whereas late BFU-E have low self-renewing capacity. To determine whether the balance between early and late BFU-E is affected by Rpl11 haploinsufficiency, and to identify gene expression changes in early and late fetal Rpl11+/- BFU-E, we performed single cell RNA-sequencing. Normal fetal livers contained similar proportions of early and late BFU-E, whereas normal adult bone marrows only contained late BFU-E. Surprisingly, Rpl11+/- fetal livers primarily contained late BFU-E. One explanation for this observation is that early BFU-E are depleted from Rpl11+/- fetal livers as this population compensates for reduced function of non-renewing late BFU-E. Interestingly, late BFU-E from Rpl11+/- BM have a stronger "late" signature than their normal counterparts suggesting a reduced self-renewing capacity of BM BFU-E in DBA. We are currently testing this hypothesis by overexpressing early genes associated with BFU-E self-renewal in Rpl11+/- BFU-E and reassessing erythroid phenotypes. In conclusion, our studies show that erythropoiesis is defective in Rpl11+/- fetal liver at the cellular and molecular level, although functional impairment is not as marked as in adult mice. Moreover, single cell RNA-sequencing of BM and fetal BFU-E in our Rpl11+/- mouse model has uncovered a novel hypothesis for DBA pathogenesis that the loss of early, self-renewing BFU-E in fetal liver is a harbinger of adult erythroid failure. Disclosures Glader: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Neuberg: Synta Pharmaceuticals: Other: Stock shares.

Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous disorder characterized by erythroid failure, congenital anomalies and a predisposition to cancer. Faulty ribosome biogenesis, resulting in pro-apoptotic erythropoiesis leading to erythroid failure, is hypothesized to be the underlying defect. The genes identified to date that are mutated in DBA all encode ribosomal proteins associated with either the small (RPS) or large (RPL) subunit and in these cases haploinsufficiency gives rise to the disease. Extraordinarily robust laboratory and clinical investigations have recently led to demonstrable improvements in clinical care for patients with DBA.

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.

While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in decreased transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including Diamond-Blackfan anemia (DBA) and 5q− syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies. Stem Cells 2015;33:925–938

Diamond-Blackfan Anaemia (DBA) is a rare inherited anaemia caused by heterozygous mutations in one of 13 ribosomal protein genes. Erythroid progenitors (BFU-E and CFU-E) in bone marrow (BM) show a proapoptotic phenotype. Suspicion of DBA is reached after exclusion of other forms of BM failure syndromes. To improve DBA diagnosis, which is confirmed by mutation analysis, we tested a new approach based on the study of extracellular vesicles (EVs) isolated from plasma by differential centrifugations and analysed by flow cytometry. We chose CD34, CD71 and CD235a markers to study erythroid EVs. We characterised the EVs immunophentoypic profiles of 13 DBA patients, 22 healthy controls and 16 patients with other haematological diseases. Among the three EVs clusters we found, only the CD34+/CD71low population showed statistically significant differences between DBA patients and controls (p< 0.05). The absence of this cluster is in agreement with the low levels of BFU-E found in DBA patients. The assessment of ROC curves demonstrated the potential diagnostic value of this population. We suggest that this assay may be useful to improve DBA diagnosis as a quicker and less invasive alternative to BM BFU-E culture analysis.

Diamond-Blackfan anemia (DBA) typically presents with red blood cell aplasia that usually manifests in the first year of life. The only gene currently known to be mutated in DBA encodes ribosomal protein S19 (RPS19). Previous studies have shown that the yeast RPS19 protein is required for a specific step in the maturation of 40S ribosomal subunits. Our objective here was to determine whether the human RPS19 protein functions at a similar step in 40S subunit maturation. Studies where RPS19 expression is reduced by siRNA in the hematopoietic cell line, TF-1, show that human RPS19 is also required for a specific step in the maturation of 40S ribosomal subunits. This maturation defect can be monitored by studying rRNA-processing intermediates along the ribosome synthesis pathway. Analysis of these intermediates in CD34− cells from the bone marrow of patients with DBA harboring mutations in RPS19 revealed a pre-rRNA–processing defect similar to that observed in TF-1 cells where RPS19 expression was reduced. This defect was observed to a lesser extent in CD34+ cells from patients with DBA who have mutations in RPS19.

Gene products mutated in the inherited bone marrow failure syndromes dyskeratosis congenita (DC), cartilage-hair hypoplasia (CHH), Diamond-Blackfan anemia (DBA), and Shwachman-Diamond syndrome (SDS) are all predicted to be involved in different aspects of ribosome synthesis. At this moment, however, it is unclear whether this link indicates a causal relationship. Although defective ribosome synthesis may contribute to each of these bone marrow failure syndromes (and perhaps others), precisely which feature of each disease is a consequence of failure to produce adequate amounts of ribosomes is obscured by the tendency of each gene product to have extraribosomal functions. Delineation of the precise role of each gene product in ribosomal biogenesis and in hematopoietic development may have both therapeutic and prognostic importance and perhaps even direct the search for new bone marrow failure genes.

The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3′ end of 18S rRNA. Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable. Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size. Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects. The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects. Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3′ end of 18S rRNA and formation of active 40S ribosomal subunits.

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA., Highlights • Ribosomopathies such as DBA are caused by ribosome dysfunction that activates p53. • p53-independent pathways may suggest possible treatments for DBA. • Expression analysis was performed in three p53-null models of DBA. • Genes involved in apoptosis and cell redox homeostasis were especially affected. • DBA is due to cumulative effects of p53-dependent and independent pathways.

Diamond-Blackfan anemia (DBA) is a cancer-prone inherited bone marrow failure syndrome. Approximately half of DBA patients have a germ-line mutation in a ribosomal protein gene. We used whole-exome sequencing to identify disease-causing genes in 2 large DBA families. After filtering, 1 nonsynonymous mutation (p.I31F) in the ribosomal protein S29 (RPS29[AUQ1]) gene was present in all 5 DBA-affected individuals and the obligate carrier, and absent from the unaffected noncarrier parent in 1 DBA family. A second DBA family was found to have a different nonsynonymous mutation (p.I50T) in RPS29. Both mutations are amino acid substitutions in exon 2 predicted to be deleterious and resulted in haploinsufficiency of RPS29 expression compared with wild-type RPS29 expression from an unaffected control. The DBA proband with the p.I31F RPS29 mutation had a pre-ribosomal RNA (rRNA) processing defect compared with the healthy control. We demonstrated that both RPS29 mutations failed to rescue the defective erythropoiesis in the rps29(-/-) mutant zebra fish DBA model. RPS29 is a component of the small 40S ribosomal subunit and essential for rRNA processing and ribosome biogenesis. We uncovered a novel DBA causative gene, RPS29, and showed that germ-line mutations in RPS29 can cause a defective erythropoiesis phenotype using a zebra fish model.

The first several months of life are a critical period for neuronal plasticity in the visual cortex, during which anatomical and physiological development depends upon visual experience. Rearing in darkness slows the time course of this critical period, such that at 5 weeks normal cats are more plastic than dark-reared cats, while at 20 weeks dark-reared cats are more plastic. This study reports the identification of a subset of mitochondrial genes that are regulated in this manner. Opponent patterns of bidirectional expression were found: several genes (ATPase 6, cytochrome b, NADH dehydrogenase subunits 4 and 2) showed elevation in normal cats at 5 weeks and in dark-reared cats at 20 weeks ("plasticity" genes); others (NADH dehydrogenase subunits 3 and 5) showed the opposite ("anti-plasticity" genes). These findings add a new dimension to the growing evidence that changes in mitochondrial gene expression are involved in the neuroplastic response.

RPM2 is a Saccharomyces cerevisiae nuclear gene that encodes the protein subunit of mitochondrial RNase P and has an unknown function essential for fermentative growth. Cells lacking mitochondrial RNase P cannot respire and accumulate lesions in their mitochondrial DNA. The effects of a new RPM2 allele, rpm2-100, reveal a novel function of RPM2 in mitochondrial biogenesis. Cells with rpm2-100 as their only source of Rpm2p have correctly processed mitochondrial tRNAs but are still respiratory deficient. Mitochondrial mRNA and rRNA levels are reduced in rpm2-100 cells compared to wild type. The general reduction in mRNA is not reflected in a similar reduction in mitochondrial protein synthesis. Incorporation of labeled precursors into mitochondrially encoded Atp6, Atp8, Atp9, and Cytb protein was enhanced in the mutant relative to wild type, while incorporation into Cox1p, Cox2p, Cox3p, and Var1p was reduced. Pulse-chase analysis of mitochondrial translation revealed decreased rates of translation of COX1, COX2, and COX3 mRNAs. This decrease leads to low steady-state levels of Cox1p, Cox2p, and Cox3p, loss of visible spectra of aa3 cytochromes, and low cytochrome c oxidase activity in mutant mitochondria. Thus, RPM2 has a previously unrecognized role in mitochondrial biogenesis, in addition to its role as a subunit of mitochondrial RNase P. Moreover, there is a synthetic lethal interaction between the disruption of this novel respiratory function and the loss of wild-type mtDNA. This synthetic interaction explains why a complete deletion of RPM2 is lethal.

The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.

Introduction: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by erythroid hypoplasia. It is associated with a number of congenital anomalies and a high risk of developing specific cancers. DBA is caused by germline mutations or deletions in genes affecting ribosomal biogenesis and function, with autosomal dominant or X-linked recessive patterns of inheritance. The most commonly mutated gene is RPS19, seen in approximately 25% of patients. About 45% of DBA families have no known disease-causing pathogenic variant. Methods: Affected and unaffected individuals from families with DBA were ascertained through the IRB-approved NCI IBMFS retrospective/prospective cohort study (ClinicalTrials.gov Identifier: NCT00027274). Study participants completed detailed family and medical history questionnaires, medical records were reviewed, and a subset of families underwent clinical evaluations at the NIH Clinical Center. DBA patients enrolled prior to 2014 underwent routine clinical mutation testing for the established DBA genes; beginning in 2014, DBA patient samples (buccal and blood DNA) were evaluated by whole exome sequencing (WES) for mutation identification. We incorporated WES with deletion analyses and copy number variant (CNV) assessment to uncover the genetic changes causative of DBA. Deletion analyses performed included SNP genotyping and array comparative genomic hybridization. Functional effects of the genetic variants were proven by pre-rRNA processing defect analysis by Northern blot. Controls for functional studies were healthy mutation-negative individuals from the IBMFS study. Results: Genetic testing information was available in 61 of the 87 families with DBA enrolled in the IBMFS study. Thirty-five of the 61 families did not have a known genetic cause at enrollment. Our combined approach of WES, deletion and CNV analyses identified the causative pathogenic variant in 18 of the 35 (51%) uncharacterized DBA families. We discovered pathogenic variants in two previously undescribed genes in two DBA families. One family had a nonsynonymous variant (p.K77N) in RPL35; the second family had a nonsynonymous variant (p. L51S) in RPL18. Both of these variants result in characteristic pre-rRNA processing defects. Our analyses also uncovered germline mosaic deletions in known DBA genes in both buccal and blood cells of two patients from two different families. One was a 1.8 Mb mosaic deletion in chromosome 15 including RPS17; the other was a large 2.5 Mb mosaic deletion on chromosome 3 including RPL35A. In addition to these findings, we found variants in previously known DBA-associated ribosomal genes in 14 of the 35 families. We further evaluated the genomic characteristics of the entire DBA cohort. Pathogenic variants in ribosomal DBA genes were found in a total of 44 of the 61 families (72%) on whom genetic testing information and/or biospecimens were available. RPS19 was the most frequently mutated gene and accounted for 36% of families, followed by RPL35A and RPS26, accounting for 14% and 11% each, respectively. Notably, 30% of the variation in disease-causing genes in our cohort was due to a single copy or mosaic gene deletion. We had complete parental testing and inheritance information on 23 (52%) of the 44 families whose gene was identified. Ten of the 23 (43%) had an inherited mutation and 13 (57%) had a de novo change in the causative gene (both parents were negative for the affected child's disease-associated mutation). At this time, 17 of 61 families tested (28%) do not have a characterized disease-associated mutation. Conclusion: This efficient comprehensive genomic approach was the basis for our discovery of two novel causes of DBA, characterization of ribosomal gene deletions not previously described to be disease-associated, and of DBA-associated germline mosaicism. We identified the disease-associated mutations in 51% (18 of 35) of our families without a known genetic cause of DBA. A total of 74% (44 of 61) of our families are now genetically characterized. Our comprehensive approach appears to provide more genomic information than other methods since pathogenic variants of DBA genes have been reported previously in about 55% of DBA patients. Disclosures No relevant conflicts of interest to declare.

In this issue of Blood, Devlin and colleagues use a new strategy to create a mouse model for the inherited bone marrow failure syndrome, DBA. The result, while recapitulating certain aspects of the disease and representing a positive step forward, also demonstrates that significant hurdles remain in faithfully creating a mammalian model for DBA.

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of ~9 × 10−6. The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the β-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Δrpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.

Autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defects decreasing Fas function and is characterized by lymphadenopathy/splenomegaly and expansion of CD4/CD8 double-negative T cells. This latter expansion is absent in the ALPS variant named Dianzani Autoimmune/lymphoproliferative Disease (DALD). In addition to the causative mutations, the genetic background influences ALPS and DALD development. We previously suggested a disease-modifying role for the perforin gene involved in familial hemophagocytic lymphohistiocytosis (FHL). The UNC13D gene codes for Munc13-4, which is involved in perforin secretion and FHL development, and thus, another candidate for a disease-modifying role in ALPS and DALD. In this work, we sequenced UNC13D in 21 ALPS and 20 DALD patients and compared these results with sequences obtained from 61 healthy subjects and 38 multiple sclerosis (MS) patients. We detected four rare missense variations in three heterozygous ALPS patients carrying p.Cys112Ser, p.Val781Ile, and a haplotype comprising both p.Ile848Leu and p.Ala995Pro. Transfection of the mutant cDNAs into HMC-1 cells showed that they decreased granule exocytosis, compared to the wild-type construct. An additional rare missense variation, p.Pro271Ser, was detected in a healthy subject, but this variation did not decrease Munc13-4 function. These data suggest that rare loss-of-function variations of UND13D are risk factors for ALPS development.

Shwachman Diamond syndrome (SDS) is an inherited bone marrow failure syndrome typically characterized by neutropenia, exocrine pancreas dysfunction, metaphyseal chondrodysplasia, and predisposition to myelodysplastic syndrome and leukemia. SBDS, the gene affected in most cases of SDS, encodes a protein known to influence many cellular processes including ribosome biogenesis, mitotic spindle assembly, chemotaxis, and the regulation of reactive oxygen species production. The best characterized role for the SBDS protein is in the production of functional 60S ribosomal subunits. Given that a reduction in functional 60S subunits could impact on the translational output of cells depleted of SBDS we analyzed protein synthesis in yeast cells lacking SDO1, the ortholog of SBDS. Cells lacking SDO1 selectively increased the synthesis of POR1, the ortholog of mammalian VDAC1 a major anion channel of the mitochondrial outer membrane. Further studies revealed the cells lacking SDO1 were compromised in growth on non-fermentable carbon sources suggesting mitochondrial function was impaired. These observations prompted us to examine mitochondrial function in human cells where SBDS expression was reduced. Our studies indicate that reduced expression of SBDS decreases mitochondrial membrane potential and oxygen consumption and increases the production of reactive oxygen species. These studies indicate that mitochondrial function is also perturbed in cells expressing reduced amounts of SBDS and indicate that disruption of mitochondrial function may also contribute to SDS pathophysiology.

Ribosomopathies refer collectively to a group of disorders characterised by disruption of ribosome synthesis. Classically, these are rare genetic syndromes with developmental abnormalities, often involving abnormal blood cell production, as seen in Diamond Blackfan anaemia, the 5q-myelodysplastic syndrome and Shwachman-Diamond syndrome or causing craniofacial anomalies (Treacher Collins syndrome). However, research has recognised several new putative ribosomopathies (Bowen-Conradi syndrome and North American Indian childhood cirrhosis), and new revelations may force the reclassification of some older disorders (dyskeratosis congenita and cartilage hair hypoplasia). Finally, some, but not all, ribosomopathies predispose patients to solid tumours or haematologic malignancies. Here, we critically review the underlying genetics in old and new ribosomopathies and examine the pathophysiologic consequences of aberrant ribosome biogenesis. Key Concepts: Ribosomopathies refer collectively to a group of disorders characterised by disruption of ribosome synthesis. Most ribosomopathies are rare genetic syndromes with developmental abnormalities, often involving abnormal blood cell production or causing craniofacial anomalies. There are several new putative ribosomopathies and the classification of some disorders is in flux. Diamond-Blackfan anemia and Treacher Collins syndrome are two well-described ribosomopathies that share pathophysiologic similarities but have different clinical manifestations for the most part. Some, but not all, ribosomopathies predispose patients to solid tumours or haematologic malignancies. Keywords: ribosomopathies; developmental disorders; ribosomal stress; p53; bone marrow failure syndromes

Before we can explore large-scale implementations of the digital research library, we need to know more about how new technology initiatives fit into local organizational contexts to meet the needs and expectations of local users. We must look at the conditions that have preexisted local initiatives, how organizations have responded, and be aware of the rationale behind those responses. How we explain what we do in these contexts will play a major role in determining the shape of the humanities research library of the future. The electronic text initiative at the Pennsylvania State University Libraries will be our case in point.