RPS19 mutations in patients with Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is an inherited disease characterized by pure erythroid aplasia. Thirty percent (30%) of patients display malformations, especially of the hands, face, heart, and urogenital tract. DBA has an autosomal dominant pattern of inheritance. De novo mutations are common and familial cases display wide clinical heterogeneity. Twenty-five percent (25%) of patients carry a mutation in the ribosomal protein (RP) S19 gene, whereas mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 are rare. These genes encode for structural proteins of the ribosome. A link between ribosomal functions and erythroid aplasia is apparent in DBA, but its etiology is not clear. Most authors agree that a defect in protein synthesis in a rapidly proliferating tissue, such as the erythroid bone marrow, may explain the defective erythropoiesis. A total of 77 RPS19 mutations have been described. Most are whole gene deletions, translocations, or truncating mutations (nonsense or frameshift), suggesting that haploinsufficiency is the basis of DBA pathology. A total of 22 missense mutations have also been described and several works have provided in vitro functional data for the mutant proteins. This review looks at the data on all these mutations, proposes a functional classification, and describes six new mutations. It is shown that patients with RPS19 mutations display a poorer response to steroids and a worse long-term prognosis compared to other DBA patients.     

Nonsense-mediated and nonstop decay of ribosomal protein S19 mRNA in Diamond-Blackfan anemia

Mutations in the ribosomal protein (RP)S19 gene have been found in about 25% of the cases of Diamond-Blackfan anemia (DBA), a rare congenital hypoplastic anemia that includes variable physical malformations. Various mutations have been identified in the RPS19 gene, but no investigations regarding the effect of these alterations on RPS19 mRNA levels have been performed. It is well established that mutated mRNA containing a premature stop codon (PTC) or lacking a stop codon can be rapidly degraded by specific mechanisms called nonsense mediated decay (NMD) and nonstop decay. To study the involvement of such mechanisms in DBA, we analyzed immortalized lymphoblastoid cells and primary fibroblasts from patients presenting different kinds of mutations in the RPS19 gene, generating allelic deletion, missense, nonsense, and nonstop messengers. We found that RPS19 mRNA levels are decreased in the cells with allelic deletion and, to a variable extent, also in all the cell lines with PTC or nonstop mutations. Further analysis showed that translation inhibition causes a stabilization of the mutated RPS19 mRNA. Our findings indicate that NMD and nonstop decay affect the expression of mutated RPS19 genes; this may help to clarify genotype-phenotype correlations in DBA.     

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.     

The functions of RPS19 and their relationship to Diamond-Blackfan anemia: a review

The relatively new study of ribosomal proteins has allowed for greater understanding of protein synthesis; however the connection between ribosomal proteins' roles and that of disease pathophysiology has not yet been established. RPS19 is a ribosomal protein linked to Diamond-Blackfan anemia whose functions have begun to be elucidated. We review here the known roles of RPS19 in both ribosome construction and other extra-ribosomal functions and discuss their relationship to Diamond-Blackfan anemia.     

Identification of mutations in the ribosomal protein L5 (RPL5) and ribosomal protein L11 (RPL11) genes in Czech patients with Diamond‐Blackfan anemia

Diamond‐Blackfan anemia (DBA) is a congenital red blood cell aplasia that is usually diagnosed during early infancy. Apart from defects in red blood cell maturation, the disorder is also associated with various physical anomalies in 40% of patients. Mutations in the ribosomal protein (RP) S19 are found in 25% of patients, while mutations in other proteins of the small ribosomal subunit—RPS17 and RPS24—have been found in a fraction of patients. Recently, mutations in RPL5, RPL11, and RPL35a of the large ribosomal subunit have also been reported in several DBA patients. Here, we present the identification of mutations in the RPL5 and RPL11 genes in patients from the Czech DBA Registry. Mutations in RPL5 were identified in eight patients from 6 out of 28 families (21.4%), and mutations in RPL11 in two patients from 2 out of 28 families (7.1%). Interestingly, all 10 patients with either an RPL5 or RPL11 mutation exhibited one or more physical anomalies; specifically, thumb anomalies (flat thenar) were always present, while no such anomaly was observed in seven patients with an RPS19 mutation. Moreover, 9 out of 10 patients with either an RPL5 or RPL11 mutation were born small for gestational age (SGA) compared to 3 out of 7 patients from the RPS19‐mutated group. These observations may suggest that mutations, at least in RPL5, seem to generally have more profound impact on fetal development than mutations in RPS19. Since RPL5 and RPL11, together with RPL23, are also involved in the MDM2‐mediated p53 pathway regulation, we also screened the RPL23 gene for mutations; however, no mutations were identified. Hum Mutat 0, 1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Bilateral microtia and cleft palate in cousins with Diamond‐Blackfan anemia

We report on maternal first cousins with bilateral microtia, micrognathia, cleft palate and hematologic findings of Diamond‐Blackfan anemia (DBA). The similarity of findings shared between our cases and a female reported by Hasan and Inoue [ 1993 ] suggests that this is a distinctive syndrome, rather than a chance association. DBA is a heterogeneous disorder, caused in about 25% of cases by heterozygous mutations in the RPS19 gene (DBA1). Mutation analysis in our cases did not show an RPS19 mutation, and 2 alleles were present in each. Segregation analysis for DBA1 on chromosome 19 and DBA2 on 8p23 was not consistent with linkage. We conclude that this syndrome of microtia, cleft palate and DBA is not allelic to known DBA loci. © 2001 Wiley‐Liss, Inc.     

A functional assay for the clinical annotation of genetic variants of uncertain significance in Diamond–Blackfan anemia

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.     

Frameshift mutation in p53 regulator RPL26 is associated with multiple physical abnormalities and a specific pre-ribosomal RNA processing defect in diamond-blackfan anemia

Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ～30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large-scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth RP regulating p53 activity that is linked to DBA.     

Specific Interaction of a 25-kilodalton Cellular Protein,a 40S Ribosomal Subunit Protein,with the Internal Ribosome Entry Site of Hepatitis C Virus Genome

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5 noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5 NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662–1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5- or 3- deleted mutants of the HCV 5 NCR as competitor RNAs for binding of p25 to wild-type HCV 5 NCR. Competitor RNAs lacking nucleotides (nt) 47–74 or nt 279–331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.     

Role of blood ribosomal protein S19 in coagulum resorption: A study using Gln137Glu‐ribosomal protein S19 gene knock‐in mouse

Sera of human, guinea pig or mouse contain a strong monocyte chemoattractant capacity that is attributed to the ribosomal protein S19 (RP S19) oligomers generated during blood coagulation. In contrast, sera prepared from Gln137Glu‐RP S19 gene knock‐in mice contained negligible chemoattractant capacity. When coagula that had been pre‐formed from the blood of both the wild type and knock‐in mice were intraperitoneally inserted into host mice, after 3 days of recovery, the knock‐in mouse coagula remained larger than the wild type mouse coagula. The wild type mouse coagula were covered by multiple macrophage layers at the surface and were infiltrated inside by macrophages. Knock‐in mouse coagula exhibited less macrophage involvement. When coagula of knock‐in mice and coagula of knock‐in mice containing C5a/RP S19, an artificial substitute of the RP S19 oligomers, were intraperitoneally inserted as pairs, the C5a/RP S19 containing coagulum was more rapidly absorbed, concomitant with increased macrophage involvement. Finally, when the knock‐in mouse and wild type mouse coagula pairs were inserted into mice in which macrophages had been depleted using clodronate liposome, the size difference of recovered coagula was reversed. These results indicate the importance of the RP S19 oligomer‐induced macrophage recruitment in coagulum resorption.     

2S methionine-rich protein (SSA) from sunflower seed is an IgE-binding protein

BACKGROUND: Sunflower seed contains 2S albumins that in other crops have been associated with allergenicity. The sunflower seed methionine-rich 2S albumin (SSA) may be an IgE-binding protein responsible for anaphylactic reactions in some sunflower seed-sensitive subjects. The objective was to demonstrate that SSA is an IgE-binding protein. METHODS: SSA was purified and the amino-acid sequence determined. The degree of purity of SSA was evaluated by silver staining, and its IgE-binding capacity by immunoblotting with serum from a subject with a convincing clinical history of anaphylaxis to sunflower seed. RESULTS: The amino-acid sequence confirmed that the purified protein was the mature form of the methionine-rich storage protein SSA from sunflower seed (Helianthus annuus). The SSA was specifically recognized by IgE from the serum of the sunflower seed-allergic subject. CONCLUSIONS: SSA is an IgE-binding protein, and subjects allergic to sunflower seed whose IgE binds to SSA are at risk of developing allergic reactions if they consume SSA.     

Human corticotropin-releasing hormone receptor gene (CRHR) is located on the long arm of chromosome 17 (17q12—qter)

Using polymerase chain reaction amplification of commercially available DNA templates, we have mapped the human corticotropin-c releasing hormone receptor gene (CRHR) to the long arm of chromosome 17 (17q12—qter)     

Novel coding-region polymorphisms in mitochondrial seryl-tRNA synthetase (SARSM) and mitoribosomal protein S12 (RPMS12) genes in DFNA4 autosomal dominant deafness families

Two genes for components of the mitochondrial translational apparatus, mitochondrial seryl-tRNA synthetase (SARSM) and mitoribosomal protein S12 (RPMS12) lie adjacent to one another on human chromosome 19, within the critical interval for the autosomal dominant deafness locus DFNA4. Both genes are plausible candidates for DFNA4, based on the fact that deafness mutations in mtDNA have been mapped both to tRNA-ser(UCN) and to the accuracy domain of the small subunit rRNA. We have sequenced the coding regions, proximal promoters, 5' and 3' UTR and splice junctional regions of both genes in two families with DFNA4-linked deafness and in controls. Novel polymorphisms 84425C>T, 83907A>G, 79485T>G, 79406C>T, 71755A>C and 68686C>G (numbered as in GenBank AC011455) were found in one or both families, but none is a plausible disease-causing mutation. Although regulatory mutations affecting either gene could still be involved in the phenotype, structural gene mutations affecting SARSM or RPMS12 can be excluded from consideration as the cause of DFNA4-linked deafness, at least in the families identified thus far.     

Congenital dyserythropoietic anemia type II (CDAII) is caused by mutations in the SEC23B gene

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease characterized by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypoglycosylation of some red blood cell (RBC) membrane proteins. Recent studies indicated that CDAII is caused by a defect disturbing Golgi processing in erythroblasts. A linkage analysis located a candidate region on chromosome 20, termed the CDAN2 locus, in the majority of CDAII patients but the aberrant gene has not so far been elucidated. We used a proteomic‐genomic approach to identify SEC23B as the candidate gene for CDAII by matching the recently published data on the cytoplasmic proteome of human RBCs with the chromosomic localization of CDAN2 locus. Sequencing analysis of SEC23B gene in 13 CDAII patients from 10 families revealed 12 different mutations: six missense (c.40C>T, c.325G>A, c.1043A>C, c.1489C>T, c.1808C>T, and c.2101C>T), two frameshift (c.428_428delAinsCG and c.1821delT), one splicing (c.689+1G>A), and three nonsense (c.568C>T, c.649C>T, and c.1660C>T). Mutations c.40C>T and c.325G>A were detected in unrelated patients. SEC23B is a member of the Sec23/Sec24 family, a component of the COPII coat protein complex involved in protein transport through membrane vesicles. Abnormalities in this gene are likely to disturb endoplasmic reticulum (ER)‐to‐Golgi trafficking, affecting different glycosylation pathways and ultimately accounting for the cellular phenotype observed in CDAII. Hum Mutat 30:1–7, 2009. © 2009 Wiley‐Liss, Inc.     

The IL-17F signaling pathway is involved in the induction of IFN-gamma-inducible protein 10 in bronchial epithelial cells

BACKGROUND: IL-17F is involved in airway inflammation, but its biologic activity and signaling pathway remain incompletely defined. Interferon-gamma-inducible protein 10 (IP-10) is widely expressed and plays a role in airway inflammatory diseases. OBJECTIVE: We sought to investigate the functional linkage between IL-17F and IP-10 expression in bronchial epithelial cells. METHODS: Bronchial epithelial cells were cultured in the presence or absence of IL-17F, and/or a T(H)1 cytokine, T(H)2 cytokines, proinflammatory cytokines, various kinase inhibitors, or a Raf1 dominant-negative mutant to analyze the expression of IP-10. Moreover, the involvement of p90 ribosomal S6 kinase (p90RSK) and cyclic AMP response element-binding protein (CREB) in IL-17F-induced IP-10 expression were investigated. RESULTS: IL-17F induces the gene and protein expression of IP-10. The addition of IFN-gamma, IL-1beta, and TNF-alpha augmented IL-17F-induced IP-10 expression. The mitogen-activated protein kinase kinase (MEK) inhibitors PD98059, U0126, and Raf1 kinase inhibitor I significantly inhibited its production. In contrast, a p38 inhibitor, a JNK inhibitor, protein kinase C inhibitors, and a phosphatidylinositol 3-kinase inhibitor, showed no inhibitory effect. Furthermore, overexpression of a Raf1 dominant-negative mutant inhibited its expression. Of interest, IL-17F phosphorylated p90RSK and CREB, and transfection of the cells with a short interfering RNA for p90RSK or CREB inhibited its expression, suggesting p90RSK and CREB as novel signaling molecules of IL-17F. CONCLUSION: IL-17F is a potent inducer of IP-10 in bronchial epithelial cells through the activation of the Raf1-MEK1/2-extracellular signal-regulated kinase 1/2-p90RSK-CREB pathway, supporting its regulatory role in airway inflammation. CLINICAL IMPLICATIONS: The IL-17F-IP-10 axis might be a novel and critical therapeutic target for airway inflammatory diseases.     

Involvement of regional neutrophil apoptosis promotion by ribosomal protein S19 oligomers in resolution of experimental acute inflammation

Isolated peripheral neutrophils spontaneously underwent apoptosis in association with extra‐cellular liberation of the monocyte‐attracting ribosomal protein S19 (RP S19) oligomers. This apoptosis was prevented by the simultaneous presence of anti‐RP S19 antibodies or of a C5a receptor antagonist, but was promoted by supplementing extrinsic RP S19 oligomers. Transformed HL‐60 cells to over‐produce Gln137Asn‐mutant RP S19 were differentiated to neutrophil‐like cells. The neutrophil‐like cells gained resistance against the spontaneous apoptosis concomitant with the generation of non‐functional RP S19 oligomers. When the neutrophil‐like cells were intradermally transplanted into mice, the mutant RP S19‐producing neutrophils persisted for a long period of time, whereas wild‐type RP S19‐producing neutrophils underwent apoptosis and were promptly cleared by infiltrated macrophages. When an experimental pleurisy was introduced by injecting carrageenan into the pleural cavity of mice, the inflammation spread slightly to lung parenchyma. When antibodies neutralizing the RP S19 oligomers were simultaneously administrated with carrageenan, the neutrophil infiltration in the lung parenchymal lesion become more severe, occurring as alveolar septal destruction and hemorrhage concomitant with an augmented neutrophil number in the pleural exudate. These results indicate the importance of the RP S19 oligomers and the C5a receptor in neutrophil clearance and acute inflammation resolution.     

Monocyte chemotactic S19 ribosomal protein dimer in atherosclerotic vascular lesion

To elucidate the molecular mechanism inducing monocyte/macrophage infiltration in the atherosclerotic lesion, we measured the monocyte chemotactic capacity in the extracts of aortic lesions. Five out of seven extracts exhibited significant chemotactic activities. Immunohistochemical examination with an anti-CD68 monoclonal antibody demonstrated that the five positive lesions possessed obvious monocyte/macrophage infiltrations at the intima, whereas the two negative lesions did so at significantly lower intensities. We subjected the chemotactic extracts to immunological analyses to identify the monocyte chemoattractant in them. The monocyte chemotactic capacities of all positive extracts were removed with anti-S19 ribosomal protein (RP S19) antibody beads and antimonocyte chemoattractant protein-1 (MCP-1) antibody beads. In three of the five extracts, the anti-RP S19 antibody beads were more effective than the anti-MCP-1 antibody beads for removal, while in the remaining two extracts, the opposite was observed. A combined immunoabsorption with these beads depleted the monocyte chemotactic capacity of a representative sample of each group. Consistently, the chemotactic capacity of an apparently RP S19 dimer-predominant extract was strongly inhibited by the presence of a C5a receptor antagonist. These results suggest that the RP S19 dimer and MCP-1 play a major role in the monocyte/macrophage infiltration of the atherosclerotic vascular lesion. L. Shi and S. Tsurusaki contributed equally to this work.