Anna Aspesi

The ribosomal basis of diamond‐blackfan anemia: mutation and database update

Diamond‐Blackfan Anemia (DBA) is characterized by a defect of erythroid progenitors and, clinically, by anemia and malformations. DBA exhibits an autosomal dominant pattern of inheritance with incomplete penetrance. Currently nine genes, all encoding ribosomal proteins (RP), have been found mutated in approximately 50% of patients. Experimental evidence supports the hypothesis that DBA is primarily the result of defective ribosome synthesis. By means of a large collaboration among six centers, we report here a mutation update that includes nine genes and 220 distinct mutations, 56 of which are new. The DBA Mutation Database now includes data from 355 patients. Of those where inheritance has been examined, 125 patients carry a de novo mutation and 72 an inherited mutation. Mutagenesis may be ascribed to slippage in 65.5% of indels, whereas CpG dinucleotides are involved in 23% of transitions. Using bioinformatic tools we show that gene conversion mechanism is not common in RP genes mutagenesis, notwithstanding the abundance of RP pseudogenes. Genotype–phenotype analysis reveals that malformations are more frequently associated with mutations in RPL5 and RPL11 than in the other genes. All currently reported DBA mutations together with their functional and clinical data are included in the DBA Mutation Database. Hum Mutat 31:1269–1279, 2010.

Diamond-Blackfan anemia: genotype-phenotype correlations in Italian patients with RPL5 and RPL11 mutations

Background Diamond-Blackfan anemia is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors. About 40% of patients display various malformations. Anemia is corrected by steroid treatment in more than 50% of cases; non-responders need chronic transfusions or stem cell transplantation. Defects in the RPS19 gene, encoding the ribosomal protein S19, are the main known cause of Diamond-Blackfan anemia and account for more than 25% of cases. Mutations in RPS24, RPS17, and RPL35A described in a minority of patients show that Diamond-Blackfan anemia is a disorder of ribosome biogenesis. Two new genes (RPL5, RPL11), encoding for ribosomal proteins of the large subunit, have been reported to be involved in a considerable percentage of patients. Design and Methods In this genotype-phenotype analysis we screened the coding sequence and intron-exon boundaries of RPS14, RPS16, RPS24, RPL5, RPL11, and RPL35A in 92 Italian patients with Diamond-Blackfan anemia who were negative for RPS19 mutations. Results About 20% of the patients screened had mutations in RPL5 or RPL11, and only 1.6% in RPS24. All but three mutations that we report here are new mutations. No mutations were found in RPS14, RPS16, or RPL35A. Remarkably, we observed a higher percentage of somatic malformations in patients with RPL5 and RPL11 mutations. A close association was evident between RPL5 mutations and craniofacial malformations, and between hand malformations and RPL11 mutations. Conclusions Mutations in four ribosomal proteins account for around 50% of all cases of Diamond-Blackfan anemia in Italian patients. Genotype-phenotype data suggest that mutation screening should begin with RPL5 and RPL11 in patients with Diamond-Blackfan anemia with malformations.

Analysis of the Ribosomal Protein S19 Interactome

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Loss of GATA-1 Full Length as a Cause of Diamond–Blackfan Anemia Phenotype

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. Pediatr Blood Cancer 2014;61:1319–1321.

A new database for ribosomal protein genes which are mutated in Diamond‐Blackfan Anemia

Mutations in ribosomal proteins RPS19, RPS24 and RPS17 have been reported in Diamond‐Blackfan Anemia (DBA), an autosomal dominant disease characterised by pure red cell aplasia. DBA is the prototype of ribosomapathies: a protein synthesis defect in a tissue with a high cellular turnover is considered the cause of the erythroid progenitor failure. We have created the Diamond‐Blackfan Anemia mutation database to curate and record DBA gene mutations, together with their functional consequences and clinical phenotypes. This locus‐specific resource is open to future submissions and is available online (http://www.dbagenes.unito.it). It is founded on the Leiden Open (source) Variation Database (LOVD) system and includes data from sequence and structure analysis tools, genomic database resources and published reports. It lists all identified variants and background genomic information. Phenotypic data are accessed by selecting a particular mutation. The database includes 219 unique variants of which 86 are disease‐causing mutations. The database will be supplemented with other DBA genes as soon as they are reported and their mutations are identified and it should be of assistance to clinicians and investigators involved in DBA research and care.

XRCC1 and ERCC1 variants modify malignant mesothelioma risk: A case–control study

Malignant pleural mesothelioma (MPM) is a rare aggressive tumor associated with asbestos exposure. The possible role of genetic factors has also been suggested and MPM has been associated with single nucleotide polymorphisms (SNPs) of xenobiotic and oxidative metabolism enzymes. We have identified an association of the DNA repair gene XRCC1 with MPM in the population of Casale Monferrato, a town exposed to high asbestos pollution. To extend this observation we examined 35 SNPs in 15 genes that could be involved in MPM carcinogenicity in 220 MPM patients and 296 controls from two case-control studies conducted in Casale (151 patients, 252 controls) and Turin (69 patients, 44 controls), respectively. Unconditional multivariate logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). Two DNA repair genes were associated with MPM, i.e. XRCC1 and ERCC1. Considering asbestos-exposed only, the risk increased with the increasing number of XRCC1-399Q alleles (Casale: OR=1.44, 95%CI 1.02-2.03; Casale+Turin: OR=1.34, 95%CI 0.98-1.84) or XRCC1 -77T alleles (Casale+Turin: OR=1.33, 95%CI 0.97-1.81). The XRCC1-TGGGGGAACAGA haplotype was significantly associated with MPM (Casale: OR=1.76, 95%CI 1.04-2.96). Patients heterozygotes for ERCC1 N118N showed an increased OR in all subjects (OR=1.66, 95%CI 1.06-2.60) and in asbestos-exposed only (OR=1.59, 95%CI 1.01-2.50). When the dominant model was considered (i.e. ERCC1 heterozygotes CT plus homozygotes CC versus homozygotes TT) the risk was statistically significant both in all subjects (OR=1.61, 95%CI 1.06-2.47) and in asbestos-exposed only (OR=1.56, 95%CI 1.02-2.40). The combination of ERCC1 N118N and XRCC1 R399Q was statistically significant (Casale: OR=2.02, 95%CI 1.01-4.05; Casale+Turin: OR=2.39, 95%CI 1.29-4.43). The association of MPM with DNA repair genes support the hypothesis that an increased susceptibility to DNA damage may favour asbestos carcinogenicity.

Exploiting Pre-rRNA Processing in Diamond Blackfan Anemia Gene Discovery and Diagnosis

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. Am. J. Hematol. 89:985–991, 2014.

Genetic Variants Associated with Increased Risk of Malignant Pleural Mesothelioma: A Genome-Wide Association Study

Asbestos exposure is the main risk factor for malignant pleural mesothelioma (MPM), a rare aggressive tumor. Nevertheless, only 5–17% of those exposed to asbestos develop MPM, suggesting the involvement of other environmental and genetic risk factors. To identify the genetic risk factors that may contribute to the development of MPM, we conducted a genome-wide association study (GWAS; 370,000 genotyped SNPs, 5 million imputed SNPs) in Italy, among 407 MPM cases and 389 controls with a complete history of asbestos exposure. A replication study was also undertaken and included 428 MPM cases and 1269 controls from Australia. Although no single marker reached the genome-wide significance threshold, several associations were supported by haplotype-, chromosomal region-, gene- and gene-ontology process-based analyses. Most of these SNPs were located in regions reported to harbor aberrant alterations in mesothelioma (SLC7A14, THRB, CEBP350, ADAMTS2, ETV1, PVT1 and MMP14 genes), causing at most a 2–3-fold increase in MPM risk. The Australian replication study showed significant associations in five of these chromosomal regions (3q26.2, 4q32.1, 7p22.2, 14q11.2, 15q14). Multivariate analysis suggested an independent contribution of 10 genetic variants, with an Area Under the ROC Curve (AUC) of 0.76 when only exposure and covariates were included in the model, and of 0.86 when the genetic component was also included, with a substantial increase of asbestos exposure risk estimation (odds ratio, OR: 45.28, 95% confidence interval, CI: 21.52–95.28). These results showed that genetic risk factors may play an additional role in the development of MPM, and that these should be taken into account to better estimate individual MPM risk in individuals who have been exposed to asbestos.

Analysis of telomeres in peripheral blood cells from patients with bone marrow failure

The determination of telomere length is useful for the characterization of dyskeratosis congenita (DC) and of aplastic anemias (AA) as well as hematological malignancies. Short telomeres result from a specific defect of telomere maintenance in DC and likely from higher cellular turnover in AA and hematological malignancies. Data are not conclusive for Diamond‐Blackfan anemia (DBA), a pure erythroid aplasia due to defects of ribosomal proteins. Our aim was to evaluate the utility of a qPCR method for telomere length assessment to evaluate the diagnostic contribution of telomere measurement in bone marrow failure syndromes (BMFS).

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

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.