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1
Kuramitsu, Madoka, Aiko Sato-Otsubo, Tomohiro Morio, Masatoshi Takagi, Tsutomu Toki, Kiminori Terui, RuNan Wang, et al. "Extensive gene deletions in Japanese patients with Diamond-Blackfan anemia." Blood 119, no. 10 (March 8, 2012): 2376–84. http://dx.doi.org/10.1182/blood-2011-07-368662.
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Abstract Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.
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Gazda, Hanna, Michael Landowski, Christopher Buros, Adrianna Vlachos, Colin A. Sieff, Peter E. Newburger, Edyta Niewiadomska, et al. "Array Comparative Genomic Hybridization of Ribosomal Protein Genes In Diamond-Blackfan Anemia Patients; Evidence for Three New DBA Genes, RPS8, RPS14 and RPL15, with Large Deletion or Duplication." Blood 116, no. 21 (November 19, 2010): 1007. http://dx.doi.org/10.1182/blood.v116.21.1007.1007.
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Abstract Abstract 1007 Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, usually presenting during infancy or in early childhood. Although anemia is the most prominent feature of DBA, the disease is also characterized by cancer predisposition, growth retardation and congenital malformations, in particular craniofacial, upper limb, heart and urinary system defects, which are present in ∼30-50% of patients. We completed our large scale sequencing of 80 ribosomal protein (RP) genes and found eight of them mutated in DBA. In total, together with three RP genes identified by others, there are 11 genes mutated in ∼54% of DBA patients; RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, RPL19 and RPL26. To search for moderate and large RP gene deletions and duplications we performed high resolution array comparative genomic hybridization on 80 DNA samples from DBA patients who did not have mutations in the 11 known RP genes. We found a deletion of exon 2 and 3 (4800 bp), deletion of the coding region, and duplication of exons 2 and 3 (488 bp) in RPS19 gene in three probands; three deletions of exons 1, 2 and 3 in RPS17 in three probands (2920 bp, 2886 bp and 3018 bp); and deletion of exons 1, 2 and 3 of the RPS26 gene. We also identified two deletions and a duplication in three RP genes previously not found mutated in DBA; RPS8 duplication of exon 3 (764 bp), RPS14 deletion of exons 2, 3, 4 and 5 (2568 bp) and RPS15 deletion of exon 4 (1995 bp). The deletions and duplications are being confirmed by multiplex PCRs. Interestingly, RPS14 was previously identified as a 5q- syndrome gene demonstrating that abnormality of this protein can cause both DBA and 5q- syndrome. These data bring to 14 the total number of RP genes mutated in DBA. Disclosures: No relevant conflicts of interest to declare.
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Kuramitsu, Madoka, Tomohiro Morio, Masatoshi Takagi, Tsutomu Toki, Kiminori Terui, RuNan Wang, Atsuko Masumi, et al. "New Determination Method for Extensive Gene Deletions In Diamond–Blackfan Anemia." Blood 116, no. 21 (November 19, 2010): 4231. http://dx.doi.org/10.1182/blood.v116.21.4231.4231.
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Abstract Abstract 4231 Introduction: Fifty percent of Diamond–Blackfan anemia (DBA) patients possess mutations in ribosomal protein genes. Although several ribosomal protein genes, RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26, have been reported to be mutated in some DBA patients, including point mutations, nonsense mutations, deletions, splice site mutations, and translocations, other DBA patients appear to have intact ribosomal protein genes. To identify new mutations in ribosomal protein genes from a different aspect, we focused on extensive deletions in these genes, such as mutations involving loss of a whole allele. In this study, we applied quantitative genomic PCR, and successfully developed a convenient method for detecting extensive deletions designated the “DBA gene copy number assay”. Methods: DBA patients should have an intact allele and a mutated allele for the responsible ribosomal protein gene, meaning that they will have an abnormal karyotype (gene copy number of N) if they have an extensive deletion. We attempted to clarify the copy numbers of ribosomal protein genes by the difference in a 1-cycle delay of threshold in a quantitative PCR (q-PCR) assay. To detect extensive deletions, at least 2 sets of gene-specific primers for each DBA responsible gene (RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26) were prepared. Appropriate primers to fit the setting that the threshold cycle (Ct) of the q-PCR should occur within 1 cycle of the Ct scores of other primer sets were selected. After validation, we identified 6, 3, 4, 3, 3, 6, 9, 3, and 2 specific primer sets for RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26, respectively. By simply looking at the q-PCR amplification curves by eye, we were easily able to judge the copy numbers of 2N (normal) or N (abnormal) for the ribosomal protein genes. Results: We performed the DBA gene copy number assay for 14 randomly selected undiagnosed patients from the Japanese DBA genomic resource at the University of Hirosaki, who had no mutations by genomic sequencing analyses. For each case, all the DBA responsible genes were confirmed using the diagnostic primers. The results of the DBA gene copy number assays revealed that 5 of the 14 probands (36%) had an extensive deletion in one of the DBA responsible genes. As an interesting case among the 5 positive cases, we confirmed an extensive deletion in the RPS19 gene. The Ct scores for 4 of the 9 primer sets for RPS19 demonstrated a 1-cycle delay, while the scores for the other 5 primer sets were normal. By genomic PCR amplification analyses, we identified a deletion from nt. -1400 to +5757 (7157 nucleotides) in the RPS19 gene. The deleted region included the promoter region, and exons 1, 2, and 3 of the RPS19 gene. The remaining 4 cases were 1 proband with an RPL5 deletion, 1 with an RPL35A deletion and 2 with RPS17 deletions. In particular, the extensive deletions in the RPL5 and RPS17 alleles are the first such cases reported. Discussion: Since it has been difficult to address the loss of a whole allele in DBA, such mutations have not been precisely examined within the DBA responsible genes. Our data suggest that extensive deletions in ribosomal protein genes comprise a significant proportion of DBA cases in Japan. Our novel method could become a useful tool for screening the gene copy numbers of ribosomal protein genes, and for identifying new pathological mutations. Disclosures: No relevant conflicts of interest to declare.
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Gazda, Hanna T., Mee Rie Sheen, Adrianna Vlachos, Valerie Choesmel, Marie-Francoise O’Donohue, Hal Schneider, Natasha Darras, et al. "Identification of New Rare Sequence Changes in RP Genes in Diamond-Blackfan Anemia and Association of the RPL5 and RPL11 Mutations with Craniofacial and Thumb Malformations." Blood 112, no. 11 (November 16, 2008): 39. http://dx.doi.org/10.1182/blood.v112.11.39.39.
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Abstract Diamond-Blackfan anemia (DBA), a congenital bone marrow failure syndrome, is characterized by red blood cell aplasia, macrocytic anemia, clinical heterogeneity, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital anomalies, present in ~30–47% of patients. The disease is associated with mutations in six ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, and L11, in about 40–45% of patients. To continue our large scale screen of RP genes in a DBA population, we sequenced 12 RP genes, S15, L36, L31, L37A, S7, S27A, S14, S23, L3, L23, S17, and L27A in our DBA patient cohort of 200 families. We identified the second known mutation in RPS17 and possible pathogenic single mutations in four more RP genes, S7, L36, S15, and S27A. These are a donor splice site mutation (intron 2) in RPS7, a deletion of two nucleotides causing frameshift in RPS17 and RPL36, and two missense changes in RPS15 and RPS27A. Northern blot analysis demonstrated that lymphoblastoid cells from the patient with RPS7 mutation displayed higher levels of 45S and 30S pre-rRNAs compared to normal cells, similar to results in HeLa cells with siRNA-based knock-down of RPS7. There is a strong defect in 5′-ETS processing, resulting in accumulation of 45S and 30S pre-rRNAs, and a strong drop of levels of the 41S, 21S and 18S-E intermediates, whereas the amount of precursors to the large ribosomal subunit RNAs were unchanged. These results suggest that mutation of RPS7 in this DBA patient directly affects maturation of pre-rRNA. In addition, review of available medical data of 20 patients with mutations in RPL5 revealed that majority of them (14/20) have physical malformations including craniofacial, thumb and heart anomalies. Similarly, 12/18 patients with RPL11 mutations presented with physical malformations, while among 76 reported DBA patients with RPS19 mutations, only 35 (46%) had physical abnormalities. Remarkably, 9 of 14 patients with RPL5 mutations and physical abnormalities have cleft lip/ palate or cleft soft palate, isolated or in combination with other facial malformations and/or with other physical abnormalities such as heart or thumb anomalies. In contrast, none of 12 patients with RPL11 mutations and malformations have craniofacial abnormalities (p=0.007, Fisher’s exact test [FET]). Moreover, none of the 35 reported patients with RPS19 mutations and malformations presented with cleft lip and/or palate (p=9.745×10−7for RPL5 vs RPS19, FET). We conclude craniofacial clefting is associated with mutations in RPL5. In addition, 8/20 patients with mutated RPL5 and 8/18 patients with mutated RPL11 have thumb abnormalities, compared to only 9% of patients with RPS19 mutations. Moreover, congenital heart defects were found more often among patients with RPL5 mutations (5/20) compared with RPL11 (3/18) and RPS19 (4/76) (p=0.017 for RPL5 vs RPS19, FET). Strikingly, the majority (11/20) of patients with RPL5 mutations presented with multiple, severe abnormalities, including craniofacial, heart and/or thumb malformations. In contrast, patients with RPL11 and RPS19 mutations who presented with multiple physical abnormalities were uncommon, three patients out of 18, and 16 out of 76, respectively (p=0.02 for RPL5 vs RPL11 and p=0.0047 for RPL5 vs RPS19, FET). In summary, we identified single mutations in four genes as well as the second mutation in RPS17, suggesting that sequence changes in RPS7, RPS17, RPL36, RPS15, and RPS27A are rare events in DBA. Mutations in RPL5 are associated with multiple physical abnormalities including craniofacial, thumbs and heart anomalies, while thumb malformations are predominantly present in patients carrying mutations in RPL11.
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Moetter, Jessica, Mutlu Kartal, Joerg Meerpohl, Alexandra Fischer, Thorsten Simon, Sandra Urbaniak, Shinsuke Hirabayashi, et al. "Analysis of Ribosomal Protein Genes Associated with Diamond Blackfan Anemia (DBA) In German DBA Patients and Their Relatives." Blood 118, no. 21 (November 18, 2011): 729. http://dx.doi.org/10.1182/blood.v118.21.729.729.
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Abstract Abstract 729 On behalf of the German DBA registry. The first two authors contributed equally. Mutations of ribosomal protein (RP) genes cause diamond blackfan anemia (DBA), a congenital bone marrow failure syndrome characterized by erythroid failure associated with diverse malformations of multiple organ systems. Familial dominant inheritance has been established in at least one third of all cases. Some affected family members present only with history of transient anemia during childhood without additonal clinical abnormalities. Laboratory parameters supporting the diagnosis of DBA and pointing towards a non-penetrant carrier are macrocytosis, elevated hemoglobin F (HbF) and adenosine deaminase (ADA). The presence of a more severe disease phenotype in DBA offsprings affected by the disease raised the question of potential genetic anticipation. However, the insufficient longitudinal datasets, the non-prospective nature of investigations and other complex issues such as fecundity bias make it difficult to evaluate this problem in DBA patients. The purpose of the current study was to investigate the incidence of mutations in ribosomal protein genes in family members of DBA patients, irrespective of family history, specific symptoms or laboratory parameters. Based on the results from the ongoing DBA re-sequencing study we determined the mutational status of German DBA patients for the following 12 RP genes: RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPL5, RPL9, RPL11, RPL19, RPL26, RPL35a. Out of 179 index patients with DBA, 94 patients carried an exclusive mutation or deletion of the following 9 genes: RPS19, n=46 (25,7%); RPL5, n=15 (8,4%); RPS26, n=12 (6,7%); RPL11, n=8(4,5%), RPL35a, n=6 (3,4%), RPS17, n=3 (1,7%), RPS24, n=2 (1,1%), RPS10, n=1 (0,6%), RPS7, n=1 (0,6%). In two patients, large genomic deletions were discovered using Affymetrix 6.0 SNP-array karyotyping, which encompass RPL5 and both copies of RPS17, respectively. No aberrations were found in RPL9, RPL19 and RPL26. A total of 85 patients (47%) were negative for all 12 tested genes. In the next step, RP genes with nonsynonymous changes identified in index patients were interrogated in patients` relatives. The investigation of parent-offspring trios revealed de novo gene aberrations in 32/55 (58%) of families (RPS19, n=14; RPL5, n=8; RPS26, n=4; RPL11, n=3, RPL35a, n=2; RPS17, n=1). Maternal inheritance pattern was identified in a total of 17/55 families (RPS19, n=10; RPS24, n=2; RPL5, n=2; RPS10, n=1; RPS26, n=1; RPL11, n=1), whereas a paternal transmission of the mutation was observed in 6/55 families (RPS19, n=3; RPL5, n=1; RPL35a, n=1 and RPS26, n=1). Among parents with identified mutation, 43% were silent carriers (presenting with normal phenotype and no history of anemia). Strikingly, 4/10 silent carriers with mutation (RPS10 Lys24Arg; RPS26 Arg87X; RPS19 Val30SerfsX46; RPS19 Arg94X) had normal HbF and ADA values. Taken together, spontaneous remission and silent clinical phenotype accounted for 87% of parental cases with identified RP mutations as opposed to 24% of offsprings with respective RP mutations. In summary, in German DBA cohort, RP genetic defects arise de novo at a frequency of 58% and are inherited over at least one generation in 42% of cases, with no significant clustering of RP gene mutations. Maternal inheritance pattern predominates at 74% of all inherited cases. The unequal cumulative frequency of spontaneous remission and silent phenotype among parental subjects versus offsprings with mutated RP genes might indicate increasing severity of the disease in each generation. This observation and the unexpectedly high frequency of silent carriers with normal HbF and ADA values warrants further studies with a large number of longitudinal datasets and known RP gene status. Disclosures: No relevant conflicts of interest to declare.
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Pospisilova, Dagmar, Jana Cmejlova, Jan Stary, Zdena Cerna, Jiri Hak, Radek Cmejla, and Barbora Ludikova. "Phenotype / Genotype Correlations in Diamond-Blackfan Anaemia – An Update From the Czech National DBA Registry." Blood 118, no. 21 (November 18, 2011): 5269. http://dx.doi.org/10.1182/blood.v118.21.5269.5269.
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Abstract Abstract 5269 Introduction Diamond-Blackfan anaemia (DBA) is a rare congenital red cell aplasia that is also associated with various physical anomalies in 40% of patients. Haploinsufficiency of ribosomal proteins (RPs) production due to various mutations in RPs is believed to be the cause of DBA. However, the precise mechanism of erythroid failure and development of anomalies remains under debate. Here we report a summary of clinical and laboratory data from the Czech National DBA Registry. Patients and Methods The Czech DBA registry has been created in the period 1991–2011. All patients were examined by experienced haematologists and detailed history was obtained. The following analyses were done: bone marrow analysis, eADA levels and clonogenic assays as previously published. PCR and direct sequencing was used to identify mutations in the genes coding for the following 21 RPs: RPS2, RPS3, RPS3a, RPS10, RPS12, RPS13, RPS14, RPS16, RPS17, RPS19, RPS24, RPS25, RPS30, RPL5, RPL11, RPL13, RPL23, RPL26, RPL27, RPL35a and RPL36. Results The Czech DBA Registry currently comprises 39 patients (14 males and 25 females; 1:1.79 ratio) aged 6 months-53 years from 34 families. Seventeen (28.8 %) patients were born small for gestational age (SGA), which is significantly higher in comparison with the population of Czech healthy newborns (p>0,001). In 27 (69.2%) patients, one or more anomalies were found (thumb anomalies, high-arched palate, craniofacial dysmorphism, Klippel-Feil syndrome, Sprengel`s deformity, neck, heart and kidney anomalies, microcephaly, micropthalmia). Nineteen (48.7%) patients have short stature. Only 6 (15%) patients have neither anomalies nor short stature. Two patients (5.1%) have developed malignancy. The first one died at the age of 5 due to AML, the second patient with an RPL11 mutation developed non-Hodgkin lymphoma at the age of 36. eADA levels were increased in 16/18 (88.8%) of non-transfused patients. Eighteen (46.2%) patients are transfusion dependent, while 10 (24.6%) are in remission and 11(28.2%) are on steroid treatment. So far, 23 different heterozygous mutations in five different RPs – RPS17, RPS19, RPS26, RPL5, and RPL11 – have been identified in 28 patients (71.8%) from 23 families (67.6%). Most mutations were found in the RPS19 gene – 10 patients (25.6%) from 8 families (23.5%), followed by the RPL5 gene (8 patients (20.5%) from 6 families (17.6%)); the RPS26 gene (5 patients (12.8%) from 5 families (14.7%)); the RPL11 gene (4 patients (10.3%) from 3 families (8.8%)); and RPS17 (1 patient (2.6%) from 1 family (2.9%). We identified three new mutations in the RPS19 gene (c.58G>C, p.Ala20Pro; c.195C>G, p.Tyr65X; and c.356dupG, p.Gly120ArgfsX34), one new mutation in the RPS26 gene (c.6_9delAAAG, p.Lys4GlufsX40), and one familial mutation in the RPL11 gene (c.281T>G, p.Leu94X). The comparison of the group of patients with RPS19 mutations (n=10) with the group of patients with RPL5 and 11 mutations (n=12) showed two significant differences. Firstly, 11/12 (92%) patients with RPL5 or RPL11 mutations were born SGA, secondly, all patients with an RPL5 or RPL11 mutation have a thumb defect with one or more other anomalies, while in the RPS19-mutated group the frequency of both markers was lower (30%). RPL5 and RPL11 mutations seem to have more profound impact on fetal development than mutations in RPS19. Patients with an RPS26 mutation have no thumb anomalies, but they show other skeletal (ribs and neck) anomalies. Conclusions The incidence of DBA in the Czech Republic is calculated to be 8.1/1 million live births. We observed a higher frequency of associated anomalies (70%) than was previously reported. Mutations in RPs of the small ribosomal subunit were found in 14 families (60.9%), while 9 mutations in proteins of the large ribosomal subunit represented 39.1%, together amounting to 72% of resolved DBA cases in the Czech Republic. Four new mutations have not been described yet. We found significant genotype-phenotype correlations. National registries are therefore the important tool for better understanding of several aspects of the disease. The work was supported by grants 00023736 (RC, JC) and NT 11059 (DP) from the Ministry of Health, and MSM 6198959205 from the Ministry of Education, Czech Republic. Disclosures: No relevant conflicts of interest to declare.
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Slaminko, T. L., C. R. Bowen, and G. L. Hartman. "Multi-Year Evaluation of Commercial Soybean Cultivars for Resistance to Phytophthora sojae." Plant Disease 94, no. 3 (March 2010): 368–71. http://dx.doi.org/10.1094/pdis-94-3-0368.
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Phytophthora sojae causes damping-off, root rot, and stem rot of soybean, particularly in poorly drained soils. Soybean cultivar resistance is one of the primary methods to control this disease, with Rps1c, Rps1k, and Rps1a being the most commonly used genes. The Varietal Information Program for Soybeans (VIPS) at the University of Illinois evaluates soybean cultivars for resistance to a number of diseases including Phytophthora root rot (PRR). The objectives of this research were to evaluate PRR resistance among commercial cultivars or advanced lines, and to compare these results with the information on PRR resistance provided by the company that entered the cultivar in VIPS. Each year from 2004 to 2008, between 600 and 900 cultivars were evaluated for resistance to either race 17 or 26 of P. sojae using the hypocotyl inoculation method. P. sojae single resistance genes were reported in 1,808 or 51% of the entries based on company information. Of these, the most commonly reported resistance genes were Rps1c (50%), Rps1k (40%), and Rps1a (10%). To a much smaller degree, companies reported using Rps3a (0.3%), Rps1b (0.2%), and Rps7 (0.2%). For the duration of the 5-year testing period, almost half of the cultivars (46%) were entered in VIPS with no reported resistance genes, and only nine out of a total of 3,533 entries (less than 0.3%) reported a stacked combination of resistance genes. Agreement between company-reported genes and any resistance found in the VIPS PRR evaluation was highest for those cultivars claiming to have Rps1c (90%) and Rps1k (83%), followed by Rps1a (70%). On average, 54% of the cultivars submitted to VIPS each year were new, reflecting the rapid development and turnover of soybean cultivars provided by the soybean seed companies.
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Konno, Yuki, Tsutomu Toki, Satoru Tandai, Gang Xu, Kiminori Terui, Shouichi Ohga, Seiji Kojima, et al. "Mutations in Ribosomal Protein Genes of Diamond-Blackfan Anemia Patients in Japan." Blood 114, no. 22 (November 20, 2009): 3204. http://dx.doi.org/10.1182/blood.v114.22.3204.3204.
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Abstract Abstract 3204 Poster Board III-141 Diamond-Blackfan anemia (DBA) is an inherited congenital bone marrow failure syndrome, characterized by red blood cell aplasia, macrocytic anemia, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, which occur in about 40% of patients. Approximately 90% of patients present during the first year of life or in early childhood. Recent studies have shown that the disease is associated with heterozygous mutations in the ribosomal protein (RP) genes RPS19, RPS24, and RPS17, encoding small ribosomal subunit proteins, and in RPL5, RPL11 and RPL35a, encoding large ribosomal subunit proteins, in about 50% of patients with DBA in Western countries. There have been no studies to determine the incidence of these mutations in Asian patients with DBA. In this study, 44 probands (46 patients) with DBA in Japan were screened for mutations of the 6 known DBA genes RPS19, RPS24, RPS17, RPL5, RPL11, and RPL35a, in addition to RPS14, which is implicated in the 5q- syndrome, a subtype of myelodysplastic syndrome characterized by a defect in erythroid differentiation. Mutations in RPS19, which have been found in 25% of patients in Western countries, were detected in 6 probands (13.6%). Missense mutations were noted in 5 of these probands, and a frameshift mutation caused by a single-nucleotide insertion was found in 1 case. Three of 7 patients had multiple malformations. Novel mutations in RPL5 were identified in 3 probands (6.8%). Insertion of 2 nucleotides was found in 1 case, affecting the reading frame. Two cases had point mutations, which resulted in a loss of the first initiation codon. All 3 patients with RPL5 mutations had multiple physical anomalies. Remarkably, 2 of 3 patients with RPL5 mutations had cleft palate, whereas no other DBA patients presented with cleft palate. Mutations in RPL11 were identified in 2 patients (4.5%). Deletion of 1 or 2 nucleotides was found in each case, leading to a shift in the reading frame. In contrast to previous reports on patients with RPL11 mutations, thumb anomalies were not seen. Deletion of 1 nucleotide in RPS17 was identified in 1 patient (2.3%), resulting in introduction of a premature stop codon. RPS17 mutations are rare and have been only reported in 2 patients with DBA. Anomalies were not seen in our patient. In summary, RP gene mutations were identified in 27.3% of DBA index cases in Japan. No mutations were detected in RPS14, RPS24 and RPL35a. In Japan, the frequency of mutations in the RP genes appears to be lower than in Western countries. Mutations in RPL5 are associated with multiple physical abnormalities, including cleft palate. Disclosures No relevant conflicts of interest to declare.
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Farrar, Jason E., Adrianna Vlachos, Eva Atsidaftos, Hannah Carlson-Donohoe, Thomas C. Markello, Robert J. Arceci, Steven R. Ellis, Jeffrey M. Lipton, and David M. Bodine. "Ribosomal protein gene deletions in Diamond-Blackfan anemia." Blood 118, no. 26 (December 22, 2011): 6943–51. http://dx.doi.org/10.1182/blood-2011-08-375170.
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Abstract 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.
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Dorrance, A. E., H. Jia, and T. S. Abney. "Evaluation of Soybean Differentials for Their Interaction with Phytophthora sojae." Plant Health Progress 5, no. 1 (January 2004): 9. http://dx.doi.org/10.1094/php-2004-0309-01-rs.
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Soybean lines, each containing a different resistance gene (Rps), are used as differentials to characterize isolates of Phytophthora sojae as physiologic races. Surveys in different soybean production regions have used various sets of soybean differentials thought to carry the same Rps genes. In some instances, isolates of P. sojae have been reported to have different reactions when evaluated in labs using different sets of differentials that were believed to have the same Rps gene. The objective of this study was to compare the consistency of racial classification when three different sets of soybean differentials were challenged with a common set of five races of P. sojae from Ohio and Indiana. Three soybean differential sets (USDA Soybean Germplasm Collection, The Ohio State University, and USDA-ARS Purdue University) were challenged with P. sojae using the hypocotyl inoculation test at OSU and USDA-ARS Purdue. Isolates of races 1, 3, 4, 7, and 25 from Ohio and Indiana had the same reaction on all three sets of soybean differentials for Rps1b, Rps1c, Rps1k, Rps3a, Rps3b, Rps3c, Rps6, Rps7, and on differentials Harlon, Harosoy 12xx, L59-731, and Union for Rps1a. L88-8470 used as a differential for Rps1a and L93-3312 used for Rps1d did not have the expected response. Isolates of races 4 and 25 from Ohio and Indiana responded differently on differentials with the Rps2 gene because this gene was not used previously to characterize races of P. sojae. A similar reaction occurred when differentials with Rps4 and Rps5 were inoculated with isolates of races 1 and 7, respectively. A standardized set of soybean differentials, corresponding to different maturity groups, for thirteen of the fourteen Rps genes is recommended. Accepted for publication 5 February 2004. Published 9 March 2004.
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Du, Hong-Yan, M. Tarek Elghetany, Blanche P. Alter, and Akiko Shimamura. "P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,." Blood 118, no. 21 (November 18, 2011): 3432. http://dx.doi.org/10.1182/blood.v118.21.3432.3432.
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Abstract Abstract 3432 Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation. Disclosures: No relevant conflicts of interest to declare.
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Yang, Jin, Sujiao Zheng, Xiaomen Wang, Wenwu Ye, Xiaobo Zheng, and Yuanchao Wang. "Identification of Resistance Genes to Phytophthora sojae in Domestic Soybean Cultivars from China Using Particle Bombardment." Plant Disease 104, no. 7 (July 2020): 1888–93. http://dx.doi.org/10.1094/pdis-10-19-2201-re.
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Phytophthora root and stem rot caused by Phytophthora sojae is a destructive disease that afflicts soybean plants throughout the world. The use of resistant soybean cultivars is the primary means of managing this disease, as well as the most effective and economical approach. There are abundant soybean germplasm resources in China that could be deployed for breeding programs; however, the resistance genes (Rps genes) in most cultivars are unknown, leading to uncertainty concerning which are resistant cultivars for use. The resistance genes Rps1a, Rps1c, and Rps1k prevent root and stem rot caused by most P. sojae isolates within a Chinese field population. This study identified three Rps genes in Chinese domestic soybean cultivars using three related avirulence genes by particle bombardment. The complex genetic diversity of soybean cultivars and P. sojae strains has made it difficult to define single Rps genes without molecular involvement. Gene cobombardment is a method for identifying Rps genes quickly and specifically. We showed that cultivars Dongnong 60 and Henong 72 contained Rps1a, while Hedou 19, Henong 76, 75-3, Wandou 21020, Zheng 196, Wandou 28, Heinong 71, and Wandou 29 all contained Rps1c. The cultivars Jidou 12, Henong 72, Heinong 71, and Wandou 29 contained Rps1k. The cultivar Henong 72 contained both Rps1a and Rps1k, while Wandou 29 and Heinong 71 contained both Rps1c and Rps1k. We then evaluated the phenotype of 11 domestic soybean cultivars reacting to P. sojae using the isolates P6497 and Ps1. The 11 domestic cultivars were all resistant to P6497 and Ps1. This research provides source materials and parent plant strains containing Rps1a, Rps1c, and Rps1k for soybean breeding programs.
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Kuang, Gangqiao, Wenjing Tao, Shuqing Zheng, Xiaoshuang Wang, and Deshou Wang. "Genome-Wide Identification, Evolution and Expression of the Complete Set of Cytoplasmic Ribosomal Protein Genes in Nile Tilapia." International Journal of Molecular Sciences 21, no. 4 (February 12, 2020): 1230. http://dx.doi.org/10.3390/ijms21041230.
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Ribosomal proteins (RPs) are indispensable in ribosome biogenesis and protein synthesis, and play a crucial role in diverse developmental processes. In the present study, we carried out a comprehensive analysis of RPs in chordates and examined the expression profiles of the complete set of 92 cytoplasmic RP genes in Nile tilapia. The RP genes were randomly distributed throughout the tilapia genome. Phylogenetic and syntenic analyses revealed the existence of duplicated RP genes from 2R (RPL3, RPL7, RPL22 and RPS27) and 3R (RPL5, RPL19, RPL22, RPL41, RPLP2, RPS17, RPS19 and RPS27) in tilapia and even more from 4R in common carp and Atlantic salmon. The RP genes were found to be expressed in all tissues examined, but their expression levels differed among different tissues. Gonadal transcriptome analysis revealed that almost all RP genes were highly expressed, and their expression levels were highly variable between ovaries and testes at different developmental stages in tilapia. No sex- and stage-specific RP genes were found. Eleven RP genes displayed sexually dimorphic expression with nine higher in XY gonad and two higher in XX gonad at all stages examined, which were proved to be phenotypic sex dependent. Quantitative real-time PCR and immunohistochemistry ofRPL5b and RPL24 were performed to validate the transcriptome data. The genomic resources and expression data obtained in this study will contribute to a better understanding of RPs evolution and functions in chordates.
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Ito, Etsuro, Kenichi Yoshida, Yusuke Okuno, Aiko Sato-Otsubo, Tsutomu Toki, Satoru Miyano, Yuichi Shiraishi, et al. "Identification of Two New DBA Genes, RPS27 and RPL27, by Whole-Exome Sequencing in Diamond-Blackfan Anemia Patients." Blood 120, no. 21 (November 16, 2012): 984. http://dx.doi.org/10.1182/blood.v120.21.984.984.
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Abstract Abstract 984 Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome, characterized by red blood cell aplasia, macrocytic anemia, and increased risk of malignancy. Approximately 90% of patients present during the first year of life or in early childhood. About 40–50% of DBA cases are familial with autosomal dominant, while the remainder is sporadic cases whose mode of inheritance is largely unknown. Although anemia is the most prominent feature of DBA, up to 40% of patients also accompany other symptoms including growth retardation and/or a variety of congenital malformations. Recent studies have shown that the disease could be associated with heterozygous mutations in ribosomal protein (RP) genes, including six small subunit RP genes RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26 as well as four large subunit RP genes RPL5, RPL11, RPL26, and RPL35A, which collectively account for about 50% of patients with DBA. In addition, germline mutations in the GATA1 gene encoding a hematopoietic transcription factor, have been also reported in two DBA families. However, it is clear that the molecular etiology of many DBA cases remains to be covered. To identify new mutations that are responsible for DBA, we performed whole-exome sequencing on 40 DBA patients with no documented mutations/deletions involving known DBA genes. After excluding all variants registered in the 1000 Genomes Project, or dbSNP131, or found in our inhouse SNP database, we searched for non-synonymous mutations involving RP genes as possible candidate for novel DBA genes. In this study, we identified probable pathogenic mutations in two novel RP genes, RPS27 and RPL27 in two patients. The first case was a 1-year-old girl who harbored a single nucleotide substitution at the splice acceptor site in intron 1 of RPL27 (c.-2–1G>A), which results in splicing error. She had atrial septal defect and pulmonary stenosis, and responded to steroid treatment. The second case was a 2-year-old girl carrying a frameshift deletion of RPS27 (c.90delC, p.Tyr31ThrfsX5), leading to a premature truncation. This patient had no abnormalities and responded to steroid treatment. An additional five missense SNVs affecting single cases was identified in five genes, including RPL3L, RPL8, RPL13, RPL18A, and RPL31, together with two in-frame deletions of RPL6 and RPL14 in two patients, which cause deletion of a single amino-acid. However, the pathological significance in these 7 cases is uncertain. In the remaining 31 patients, no mutations were detected in RP genes. In conclusion, we identified novel germline mutations of RP genes that could be responsible for DBA, further confirming the concept that the RP genes are common targets of germline mutations in DBA patients and also suggested the presence of non-RP gene targets for DNA. Disclosures: No relevant conflicts of interest to declare.
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Wilkes, Mark, Elena Bibikova, Minyoung Youn, Alex Gia Lee, Ascia Eskin, Stanley Nelson, Bertil Glader, Anupama Narla, and Kathleen M. Sakamoto. "Role of Mir-34 Upregulation in Disruption of c-Myc, c-Myb and NOTCH Signaling in Diamond-Blackfan Anemia." Blood 128, no. 22 (December 2, 2016): 3895. http://dx.doi.org/10.1182/blood.v128.22.3895.3895.
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Abstract Approximately 53% of patients with Diamond-Blackfan Anemia (DBA) have mutations in one of nine ribosomal protein components (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPL5, RPL11 and RPL35A), with RPS19 being the most commonly affected (25% of all patients). Mutations found in patients result in protein haploinsufficiency and subsequent ribosomal stress, which increases the expression and activity of p53, leading to a failure of HSC differentiation during erythropoiesis. While RPS19-/- mice are not viable, we have generated a model system using different shRNA constructs to generate varying degrees of RPS19 knockdown in cultured cells and animal models. Cultured cells (including CD34+ HSCs derived from fetal liver or cord blood) recapitulate features of DBA erythropoietic failure in vitro and in vivo, including p53 upregulation and defects in erythrocyte differentiation, while anemias and skeletal defects are observed in zebrafish models. Using next-generation miRNA sequencing on the Illumina HiSeq2000 platform, we identified a number of miRNAs differentially expressed between human fetal liver CD34+ HSCs expressing normal or reduced RPS19. Preprocessing, alignment, and miRNA quantification were carried out using miRDeep2 [1] software. Differential expression analysis was performed with DESeq2 [2], which tests for differential expression based on a model using negative binomial distribution for count data from high-throughput sequencing assays. Candidates underwent a second round of screening based on robustness of expression differential (fold increase/decrease, variation of fold increase/decrease across replicates, and increase/decrease relative to RPS19 expression) and possible links to hematopoiesis and erythropoiesis, as reported in the scientific record. All three miR-34 isoforms (a, b and c) are robustly upregulated upon RPS19 reduction, with the extent of miR-34 upregulation being inversely proportional to RPS19 expression. When RPS19 protein expression is suppressed by approximately 50% (as observed in DBA patients), miR-34a, b and c isoforms are upregulated by 3.2, 2.2 and 2.6 fold respectively. In other systems, increased miR-34 results in modulation of a number of factors linked to erythropoiesis, including downregulation of c-Myc, Myb and NOTCH signaling. Genome-wide transcriptional analysis using next-generation sequencing on the Illumina HiSeq platform in CD34+ HSCs indicates c-Myc (4.2 fold), c-Myb (2.8 fold) and a number of NOTCH effectors [SIRT1 (1.8 fold) andHES1 (1.9 fold)] are indeed downregulated at the transcriptional level. Results were validated with qRT-PCR and western blot analysis, however, while transcriptional modulation fully accounts for c-Myc downregulation at the mRNA and protein level, we propose that c-Myb protein levels are modulated both transcriptionally and post-translationally, as protein expression is significantly further downregulated than mRNA message (6.3 fold). We propose that miR-34 upregulation in RPS19-depleted cells occurs through p53 and initiates genomic changes incompatible with erythropoiesis through downregulation of transcription factors c-Myc, c-Myb and the NOTCH signaling pathway. Disclosures No relevant conflicts of interest to declare.
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Liu, Mi-Li, Wei-Bing Fan, Ning Wang, Peng-Bin Dong, Ting-Ting Zhang, Ming Yue, and Zhong-Hu Li. "Evolutionary Analysis of Plastid Genomes of Seven Lonicera L. Species: Implications for Sequence Divergence and Phylogenetic Relationships." International Journal of Molecular Sciences 19, no. 12 (December 14, 2018): 4039. http://dx.doi.org/10.3390/ijms19124039.
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Plant plastomes play crucial roles in species evolution and phylogenetic reconstruction studies due to being maternally inherited and due to the moderate evolutionary rate of genomes. However, patterns of sequence divergence and molecular evolution of the plastid genomes in the horticulturally- and economically-important Lonicera L. species are poorly understood. In this study, we collected the complete plastomes of seven Lonicera species and determined the various repeat sequence variations and protein sequence evolution by comparative genomic analysis. A total of 498 repeats were identified in plastid genomes, which included tandem (130), dispersed (277), and palindromic (91) types of repeat variations. Simple sequence repeat (SSR) elements analysis indicated the enriched SSRs in seven genomes to be mononucleotides, followed by tetra-nucleotides, dinucleotides, tri-nucleotides, hex-nucleotides, and penta-nucleotides. We identified 18 divergence hotspot regions (rps15, rps16, rps18, rpl23, psaJ, infA, ycf1, trnN-GUU-ndhF, rpoC2-rpoC1, rbcL-psaI, trnI-CAU-ycf2, psbZ-trnG-UCC, trnK-UUU-rps16, infA-rps8, rpl14-rpl16, trnV-GAC-rrn16, trnL-UAA intron, and rps12-clpP) that could be used as the potential molecular genetic markers for the further study of population genetics and phylogenetic evolution of Lonicera species. We found that a large number of repeat sequences were distributed in the divergence hotspots of plastid genomes. Interestingly, 16 genes were determined under positive selection, which included four genes for the subunits of ribosome proteins (rps7, rpl2, rpl16, and rpl22), three genes for the subunits of photosystem proteins (psaJ, psbC, and ycf4), three NADH oxidoreductase genes (ndhB, ndhH, and ndhK), two subunits of ATP genes (atpA and atpB), and four other genes (infA, rbcL, ycf1, and ycf2). Phylogenetic analysis based on the whole plastome demonstrated that the seven Lonicera species form a highly-supported monophyletic clade. The availability of these plastid genomes provides important genetic information for further species identification and biological research on Lonicera.
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Kenney, Scott P., and Xiang-Jin Meng. "The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication." Journal of Virology 89, no. 7 (January 21, 2015): 3793–803. http://dx.doi.org/10.1128/jvi.03582-14.
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ABSTRACTHepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that contains an insertion of a 171-nucleotide sequence encoding amino acids 21 to 76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of the HEV ORF1 protein. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range of the virus, allowing it to infect cell lines from multiple animal species, including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics to attempt to define which aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV and that lysine residues within the RPS17 insertion, but not nuclear localization of the ORF1 protein, correlate with the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV.IMPORTANCEHEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in immunocompromised populations. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems in which to propagate the virus. Recently, insertions and rearrangements of the hypervariable region (HVR) within the HEV genome, allowing for cell culture adaptation and expansion of the host range, have been reported. We utilized these cell culture-adapted HEV strains to assess how the HVR may be involved in virus replication and host range. We provide evidence that insertion of the RPS17 sequence in HEV likely confers nuclear trafficking capabilities to the nonstructural protein of the virus and that lysine residues within the RPS17 insertion are important for enhanced replication of the virus. These data will help to elucidate the mechanism of cross-species infection of HEV in the future.
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Leitz, R. A., G. L. Hartman, W. L. Pedersen, and C. D. Nickell. "Races of Phytophthora sojae on Soybean in Illinois." Plant Disease 84, no. 4 (April 2000): 487. http://dx.doi.org/10.1094/pdis.2000.84.4.487d.
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Phytophthora root rot of soybean (Glycine max (L.) Merr.), caused by Phytophthora sojae M. J. Kauffmann & J. W. Gerdemann, has been isolated throughout the soybean-producing regions of the United States. There are more than 39 identified races of P. sojae pathogenic on soybean, and 13 host resistance alleles have been identified at 7 loci (1). None of these alleles confers resistance to all races of P. sojae. The most commonly used resistance allele, Rps1k, confers resistance to the greatest number of races (2). The objective of this study was to identify races of P. sojae in Illinois soybean fields to determine whether the currently used resistance alleles are effective against the P. sojae races found in Illinois. Soybean breeders must be aware of the existence and distribution of races to incorporate appropriate sources of genetic resistance into cultivars. From 192 soil samples collected throughout Illinois in 1997, 33 isolates were obtained and identified to race by inoculating Rps isolines of soybean cv. Williams. A new race with virulence to the Rps1d and Rps7 alleles, designated as race 54, accounted for 48% of the isolates. Another new race with virulence to Rps1d, Rps3a, Rps3c, Rps4, Rps5, Rps6, and Rps7 alleles, designated race 55, was identified in one sample. One isolate, identified as race 41, was obtained from a diseased plant with the Rps1k allele. Another isolate, identified as race 43, was obtained from a diseased plant with the Rps1c allele. Based on virulence patterns of P. sojae, most of the isolates obtained from Illinois soils were races 1, 3, and 4 or variants of these races, such as race 54, with added virulence to the Rps1d allele. References: (1) A. F. Schmitthenner. 1999. Compendium of Soybean Diseases. 4th ed. G. L. Hartman, J. B. Sinclair, and J. C. Rupe, eds. The American Phytopathological Society, St. Paul, MN. pp. 39‐42. (2) A. F. Schmitthenner, M. Hobe, and R. G. Bhat. Plant Dis. 78:269, 1994
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Gazda, Hanna T., Mee Rie Sheen, Natasha Darras, Hal Shneider, Colin A. Sieff, Sarah E. Ball, Edyta Niewiadomska, et al. "Mutations of the Genes for Ribosomal Proteins L5 and L11 Are a Common Cause of Diamond-Blackfan Anemia." Blood 110, no. 11 (November 16, 2007): 421. http://dx.doi.org/10.1182/blood.v110.11.421.421.
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Abstract Diamond-Blackfan anemia (DBA), a form of congenital red cell aplasia with marked clinical heterogeneity and increased risk of malignancy, has been associated with mutations in ribosomal protein (RP) gene RPS19 in 25% of probands and in RPS24 or RPS17 in ∼2% of patients. Thus, DBA appears to be a disorder of ribosome synthesis. To test the hypothesis that mutations in other RP genes may also cause DBA, we carried out direct sequencing of candidate RP genes. Genomic DNA samples from 96 unrelated DBA probands (14 familial and 82 sporadic cases) without RPS19 or RPS24 mutations were screened for mutations in RPS3a, RPS13, and RPS16 (previous studies revealed that RPs S19, S24, S3a, S13, and S16 are involved in binding of eIF-2 to the 40S subunit); RP genes L18, L13A, L36, L28, L18A, L40, S5, S9, S11, and S28 (located on chromosome 19); and RP genes, L5, L11, L22, S8, and S27 (on chromosome 1). PCR primers were designed to amplify the coding exons and intron/exon boundaries. We found multiple mutations in two RP genes, L5 and L11. Subsequently we sequenced these two genes in 42 additional DNA samples from DBA probands. In total, we screened 5′UTR, promoter and coding regions, and exon/intron boundaries of RPL5 and RPL11 in 138 DBA unrelated probands. We identified 14 mutations in RPL5 in 138 probands (∼10%), 13 of which are nonsense mutations, deletions or insertions of 1–5 nucleotides causing frameshift and premature termination. One missense mutation, 418G>A, results in a G140S substitution. We found nine mutations in RPL11 in138 DBA probands (6.5%), including five acceptor or donor splice site mutations (introns 1–4) and four deletions or insertions of 1–4 nucleotides causing frameshifts (codons 32-120). None of these sequence changes were found on the NCBI (http://www.ncbi.nlm.nih.gov/SNP/) or the HapMap (http://www.hapmap.org/) SNP lists. Both genes, as well as RPL23 have recently been demonstrated by others to activate the p53 tumor suppressor protein by inhibiting MDM2-mediated p53 ubiquitination and degradation. Moreover, knockdown of any of these genes by siRNAs markedly reduced p53 induction by the ribosomal biogenesis stressor, actinomycin-D. These findings suggest that DBA patients with mutated L5 and L11 proteins may have inadequate p53 pathway activation and (consistent with clinical observations) be at increased risk for neoplasia. We are currently investigating the role of RPL5 and RPL11 mutations in ribosomal biogenesis and in the p53-mediated cell cycle arrest and apoptosis in DBA patients.
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Pospisilova, Dagmar, Jana Cmejlova, Tomas Adam, and Radek Cmejla. "Effects of Leucine Administration in Diamond-Blackfan Anemia Patients." Blood 110, no. 11 (November 16, 2007): 1686. http://dx.doi.org/10.1182/blood.v110.11.1686.1686.
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Abstract Diamond-Blackfan anemia (DBA) attracts much attention, since the symptoms of the disease are associated with mutations in ribosomal protein (RP) S19 in 25% of patients and in RPS17 and RPS24 in other DBA patients, indicating a possible relationship between ribosomal function, translation level and erythropoiesis. Indeed, translational efficiency has been found to be lowered in most DBA patients, and the amino acid leucine was tested in vitro as a potential modulator of protein synthesis with promising results. We therefore decided to evaluate the effects of leucine administration in several DBA patients. For leucine therapy, 4 patients with the lowest levels of translation (patients 1, 2, 4 and 6; see Table) and 2 others were selected from the Czech DBA registry. Due to iron overload, all patients were receiving iron chelation therapy at the start of the leucine therapy. A total dose of 2000 mg/m2/day of L-leucine was administered orally in three subdoses in the form of a capsule prepared by the hospital pharmacy. The doses were based on the leucine content in sports dietary protein supplements, and reduced according to each patient’s body surface area. Two and 4 hours after administration, serum leucine levels doubled, but did not exceed normal values. Changes in other amino acids serum levels were not observed. After 8 weeks of leucine supplementation, all patients reported a noticeable increase in appetite and weight gain. Over a period of 6 months of follow-up, a gradual improvement in reticulocyte counts, hemoglobin levels and a reduction of serum ferritin levels were observed in all patients (see Table). One patient became transfusion independent, and is currently still in remission (>5 months); in two other transfusion dependent patients, the inter-transfusion period doubled; in steroid-dependent patients, the steroid dose could be reduced. The patient with the RPS17 mutation significantly improved in weight and well-being, and the iron chelation therapy was stopped. Our results thus show for the first time that leucine administration can greatly improve the quality of life of DBA patients in at least two ways - it can reduce the need for iron chelation; and it can gradually enhance erythropoiesis, reducing the steroid dose or the frequency of transfusions. Patients’ characteristics Patient No. Age (y) / Sex Status before Leu Level of translation (% of controls)* Duration of Leu administration (mo) Serum ferritin level before Leu/current (μg/l) Reticulocyte count before Leu/current (%) Effect of Leu administration MUT: mutation in RPS17; NM: no mutation in RPS17, RPS19 or RPS24; TD: transfusion dependent; HDS: high dose steroid treatment; LDS: low dose steroid treatment; ND: not done; PTP: prolongation of the transfusion period (before Leu/ current); *: Haematologica91:1456(2006) 1 NM 7 / F TD 21 12 1220 / 381 0.1 / 3.3 Remission 2 NM 8 / M TD 47 9 1311 / 492 0.1 / 0.4 PTP (3 / 6 weeks) 3 NM 11 / F TD ND 2 1950 / ND 0.1 / ND Increased appetite 4 MUT 31 / M LDS 39 8 860 / 496 1.1 / 1.5 Steroid dose reduction 5 NM 13 / M TD 77 6 1427 / 1110 0.6 / 1.4 PTP (4 / 8 weeks) 6 NM 18 / M HDS 42 12 1605 / 862 0.4 / 0.8 Steroid dose reduction
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Maki, Carl, Douglas D. Rhoads, Mary J. Stewart, Barbara Van Slyke, and Donald J. Roufa. "The Drosophila melanogaster RPS17 gene encoding ribosomal protein S17." Gene 79, no. 2 (July 1989): 289–98. http://dx.doi.org/10.1016/0378-1119(89)90211-4.
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Quarello, Paola, Emanuela Garelli, Adriana Carando, Patrizia Pappi, Alfredo Brusco, Irma Dianzani, and Ugo Ramenghi. "High Frequency of Large Gene Deletions Detected by Multiplex Ligation-Dependent Probe Amplification in Diamond Blackfan Anemia,." Blood 118, no. 21 (November 18, 2011): 3428. http://dx.doi.org/10.1182/blood.v118.21.3428.3428.
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Abstract Abstract 3428 Background: Diamond-Blackfan anemia (DBA,#MIM105650) is a rare congenital pure red cell aplasia characterized by normochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. Although mutations of eleven ribosomal protein (RP) genes have been detected in more than 50% of DBA patients the remaining patients appear to have intact ribosomal protein genes using stadard sequencing methods (Boria et al. Hum Mut 2010). We previously described the detection of three large RPS19 deletions using the MLPA (Multiplex Ligation-dependent Probe Amplification) technique (Quarello et al. Haematologica 2008). As MLPA is an efficient and rapid technique that detects gene dosage alterations, we thus decided to apply this approach also to other RP genes. Aim: To search for unidentified RP large deletions we applied the MLPA technique in Italian DBA patients who have been found mutation-negative by sequencing. Methods: Italian DBA patients without RP genes mutations (73/156, 47%) were included in this study. The analysis was performed using a homemade MLPA kit following the recommendations provided by MRC Holland (Amsterdam, The Netherlands, www.mlpa.com). The probes were designed to detect deletions of six RP genes (RPS17, RPS19, RPS26, RPL5, RPL11, RPL35A). Deletions of probe recognition sequences were apparent by a 35–50% reduced relative peak area of the amplification product of that probe. Results: The results of the MLPA assay revealed that 13 out of the 73 probands (18%) had a multi-exonic deletion in one of the six DBA genes analyzed. We identified four deletions of the RPS17 gene, three of the RPS26 gene, three of the RPL35A, two of the RPL11 gene and one of the RPL5 gene. No additional RPS19 deletions were found. DBA patients with deletions showed a severe phenotype with a very high percentage of transfusion dependence (85%). Somatic malformations were observed only in two patients. Conclusion: We detected a high percentage of deletions of known DBA genes in a cohort of patients in whom no mutations were found by RP sequencing. Mutation screening of the RP genes with a combination of sequencing and MLPA reached an overall detection rate of 61.5% (96/156). In our cohort, large genomic deletions represent up to 18% of all mutations detected. In conclusion, we stress the high percentage of identified RP genes deletions in DBA patients. We also highlight that a gene-dosage technique, such as MLPA, should complement sequencing in a clinical environment since only a combined approach of this kind permits the comprehensive detection of all mutations in the DBA RP genes. Disclosures: No relevant conflicts of interest to declare.
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Zhu, Mengjia, and Liqun Wang. "Whole Blood mRNA Expression Pattern Differentiates AD Patients and Healthy Controls Through Bioinformatics Analysis." Journal of Biology and Life Science 10, no. 2 (April 28, 2019): 46. http://dx.doi.org/10.5296/jbls.v10i2.14491.
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Background: Gene chip has a wide range of applications in screening disease markers.Methods: GSE63063 dataset including 238 healthy controls and 285 patients with Alzheimer’s disease (AD) was downloaded to investigate the whole blood mRNA expression pattern. Lumi and LIMMA packages of R software were used to screening differential-expressed genes (DEGs). We functionally annotate DEGs through DAVID database. Then STRING database and Cytoscape software were used to construct protein-protein interaction models for hub genes.Results: Our results indicated that 51 DEGs altered in AD patients compared with healthy controls. These DEGs was associated with transcription (BP), RNA binding (MF) and ribosome (CC) terms and the ribosome signaling pathway. In addition, Ribosomal protein S17 (RPS17) was identified as the top 1 in hub genes using maximal clique centrality. RPS17 mutations reduced erythrocyte production and impaired brain development. Finally, the expression levels of the three genes (NDUFA1, RPL36AL, and NDUFS5) showed a good predictive effect.Conclusion: In conclusion, we explored the expression of genes in the AD blood and NDUFA1 may be a potential biomarker for predicting AD.
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Cmejla, Radek, Jana Cmejlova, Helena Handrkova, Jiri Petrak, and Dagmar Pospisilova. "Ribosomal protein S17 gene (RPS17) is mutated in Diamond-Blackfan anemia." Human Mutation 28, no. 12 (2007): 1178–82. http://dx.doi.org/10.1002/humu.20608.
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Singh, Sharon, Sehba Dsilva, Jeffrey Michael Lipton, Steven Ellis, and Johnson M. Liu. "Embryoid Body Defect in Mouse Rps19-Haploinsufficient Embryonic Stem Cell Model of Diamond Blackfan Anemia." Blood 112, no. 11 (November 16, 2008): 3093. http://dx.doi.org/10.1182/blood.v112.11.3093.3093.
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Abstract Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that is characterized by erythroid hypoplasia, risk of other cytopenias, congenital anomalies and a cancer predisposition. Thus far, all the genes identified as mutated in DBA encode ribosomal proteins (RPS19, RPS17, RPS24, RPL5, RPL11, and RPL35a). In the 25% of DBA patients with RPS19 mutations, haploinsufficiency of RPS19 has been linked to faulty ribosome biogenesis, which ultimately predisposes erythroid precursors to apoptosis through as yet unknown mechanisms. Previous attempts by others to apply targeted mutagenesis to Rps19 were unsuccessful because of compensatory Rps19 expression from the non-targeted allele. We have concentrated our efforts on characterizing the murine Rps19-mutated embryonic stem (ES) cell, S17-10H1, which was generated using a genetrap strategy. The gene-trap vector contains a strong splice acceptor-β-geo cassette-poly A termination, and following insertion, it should cause splicing with the exon upstream and termination at the poly A signal, effectively cutting Rps19 in half. S17-10H1 was sequenced using 3′ RACE (rapid amplification of cDNA ends) to confirm insertion of the vector between exons 2 and 3 of Rps19. PCR with primers against the β-geo sequence was also used to confirm insertion of the gene trap vector into the mutant ES cells. Western blot analysis of two different ES cell samples confirmed at least 50% less Rps19 protein than found in the wild-type parental ES cell line, AK7. The ES cells were subsequently induced to undergo primary differentiation into embryoid bodies (EBs). Although there was no significant difference in the EB size or shape at day 5 of culture, the number of EBs that formed in the mutant cultures was decreased by at least three-fold. Preliminary experiments indicated no obvious morphological differences in day 13 EBs derived from parental or mutant ES cells. We attempted to create chimeric mice by microinjection of the S17-10H1 cell line into 36 blastocysts. Six chimeric mice were set up in mating pairs with C57BL/6J partners. Analysis of more than 60 pups from the 60% chimeric male revealed a lack of germline transmission, possibly indicating that this mutation leads to embryonic lethality or inability to complete gametogenesis. We conclude that this ES cell differentiation model mimics the human disease in leading to Rps19 haploinsufficiency and provides a new and potentially powerful tool that can be used to elucidate molecular mechanisms and test potential therapies in DBA.
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Farrar, Jason E., Michelle Nater, Emi Caywood, Michael A. McDevitt, Jeanne Kowalski, Clifford M. Takemoto, C. Conover Talbot, et al. "Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia." Blood 112, no. 5 (September 1, 2008): 1582–92. http://dx.doi.org/10.1182/blood-2008-02-140012.
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Abstract 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.
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Utsugisawa, Taiju, Toshitaka Uchiyama, Hiromi Ogura, Takako Aoki, Isao Hamaguchi, Akira Ishiguro, Akira Ohara, et al. "Elevated Red Cell Reduced Glutathione Is a Novel Biomarker of Diamond-Blackfan Anemia." Blood 124, no. 21 (December 6, 2014): 1342. http://dx.doi.org/10.1182/blood.v124.21.1342.1342.
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Abstract Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia characterized by congenital anomalies and predisposition to cancer. Recent observation disclosed that heterogeneous mutations in ribosomal protein (RP) genes are present in approximately 50% of patients, suggesting that diagnosis should be made by clinical phenotypes such as age, hematological findings or positive family history. Although elevated activity of red cell adenosine deaminase (eADA) has been utilized as a useful biomarker for differential diagnosis of DBA, approximately 20% of DBA patients are eADA-negative. Recent observations suggested that ribosomal haploinsufficiency increases oxidative stress, leading to p53 gene activation and premature death of erythroid cells. We hypothesized that reduced glutathione (GSH), an essential antioxidant of erythroid cells, might be upregulated in red cells of DBA subjects. In order to test this hypothesis, we examined red cell GSH as well as eADA of 22 patients in 18 DBA families, in whom we had identified gene mutations in RPS19, RPL5, RPL11, RPS10, RPS17 or RPS35a. All except one DBA patients showed elevated GSH (>88.6 mg/dl RBC, M+SD), whereas 17 out of 22 patients exhibited elevated eADA (>2.31 IU/g Hb, M+3SD). We also examined 14 unaffected members of the DBA families, with 1 out of 14 subjects showing elevated GSH and none showing elevated eADA. We performed linear discriminant analysis between DBA and non-DBA subjects with both eADA and GSH using the Support Vector Machine (SVM) from 36 subjects, and successfully obtained a formula to discriminate DBA from unaffected subjects: 0.937*eADA+0.0702*GSH-7.9044 >0. By using this formula, all DBA examined can be diagnosed and unaffected family members can be excluded. Since approximately 50% of clinically diagnosed DBA cases have no causative RP gene mutations, the combined assessment of eADA and GSH might be quite useful for biochemical diagnosis of DBA. Disclosures No relevant conflicts of interest to declare.
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Dorrance, A. E., S. A. McClure, and A. deSilva. "Pathogenic Diversity of Phytophthora sojae in Ohio Soybean Fields." Plant Disease 87, no. 2 (February 2003): 139–46. http://dx.doi.org/10.1094/pdis.2003.87.2.139.
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Problems with early season soybean stand establishment, and an increase in incidence of Phytophthora root and stem rot caused by Phytophthora sojae, prompted a reassessment of the pathogen population in Ohio. Earlier studies had indicated a potential for pathogen adaptation to commonly deployed Rps genes in soybeans. Fifty-seven fields, part of an earlier study in 1990 and 1991, along with 29 additional fields were sampled in either 1997 or 1999. Two soybean cultivars, Sloan (rps) and Resnik (Rps1k), were used as bait in a seedling bioassay to isolate P. sojae from the soil samples. P. sojae was recovered from 82 of the 86 fields sampled. Of the 429 isolates recovered from these soils, 325 and 104 were baited with soybean cultivars Sloan and Resnik, respectively. The P. sojae population in Ohio increased in the number of pathotypes (races) as well as in complexity since the earlier surveys. There were 72 and 202 pathotypes identified on 8 and 13 Rps gene differentials, respectively, in the current study. When the data were compared by location, 96, 65, 73, 78, 51, and 52% of the locations had at least one isolate with virulences to Rps1a, Rps1b, Rps1c, Rps1k, Rps3a, and Rps6, respectively. The mean complexity, the number of susceptible interactions on 8 differentials, increased from 3.01 to 4.06 between 1991 and 1997/1999. In addition, the pathogenic diversity as measured by the Shannon index increased from 2.71 to 3.28 for isolates recovered from the 57 fields sampled in both surveys. Producers whose fields were sampled were surveyed to determine if changes in the P. sojae population could be linked with production practices. There was a significant association between (P ≤ 0.05) reduced tillage practices and the presence of isolates that had virulence to Rps1k; reduced tillage fields also had isolates with virulence to a greater number of differentials. Due to the percentage of isolates that have virulence to many of the Rps genes, it is questionable how long a single Rps gene or several stacked Rps genes will remain viable disease management tools for P. sojae, unless a novel Rps gene is identified.
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Payne, Elspeth M., Maria Virgilio, Anupama Narla, Hong Sun, Michelle Levine, Barry H. Paw, Nancy Berliner, A. Thomas Look, Benjamin L. Ebert, and Arati Khanna-Gupta. "L-leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway." Blood 120, no. 11 (September 13, 2012): 2214–24. http://dx.doi.org/10.1182/blood-2011-10-382986.
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Abstract Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34+ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.
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Ellis, Steven R., Carlos Arce-Lara, Jacqueline M. Caffrey, and Diana A. Alvarez-Arias. "Ribosomal Protein S19 and Diamond Blackfan Anemia." Blood 104, no. 11 (November 16, 2004): 2839. http://dx.doi.org/10.1182/blood.v104.11.2839.2839.
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Abstract Diamond Blackfan Anemia (DBA) is one of several bone marrow failures that have been linked to defects in ribosome synthesis. 25% of DBA cases are linked to mutations in ribosomal protein S19 (Rps19). The etiology of the remaining cases is unknown. To gain a better understanding of the function of the Rps19 family of proteins we have characterized members of this protein family in the yeast, Saccharomyces cerevisiae. In yeast, Rps19 is encoded by duplicated genes, RPS19A and RPS19B. Yeast cells lacking both RPS19 genes are not viable, whereas those lacking a single gene are viable but have growth defects. These latter strains are defective in a specific step in rRNA processing that preferentially affects the maturation of 40S ribosomal subunits. We scanned other yeast strains with mutations in genes for 40S subunit proteins for processing phenotypes similar to RPS19 mutants. Several have phenotypes that overlap with RPS19 mutants, but only RPS18 stands out as being virtually identical to RPS19 mutants. The human RPS18 gene is therefore a candidate locus for pathogenic mutations in DBA patients with normal RPS19. We are currently developing strategies to sequence RPS18 genes from DBA patients with normal RPS19 to determine if mutations in RPS18 are associated with DBA. We have also developed a yeast system for the functional testing of mutant alleles of RPS19 found in DBA patients. In general, a mutation is considered pathogenic if it is not found in unaffected family members and in the general population. We have found, however, that several missense mutations classified as pathogenic in DBA patients do not affect Rps19 function in the yeast system. The failure of these mutations to affect Rps19 function in yeast points to a need for functional testing of RPS19 mutant alleles in human cells.
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Chen, Chaoyi, Jianrui Yuan, Guangdong Ji, Shicui Zhang, and Zhan Gao. "Amphioxus ribosomal proteins RPS15, RPS18, RPS19 and RPS30-precursor act as immune effectors via killing or agglutinating bacteria." Fish & Shellfish Immunology 118 (November 2021): 147–54. http://dx.doi.org/10.1016/j.fsi.2021.09.001.
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Virgilio, Maria, Grzegorz Pietka, and Elspeth M. Payne. "Ribosomal Proteins Rps19 and Rps14 Cooperate As Tumor Suppressor Genes with p53." Blood 124, no. 21 (December 6, 2014): 2943. http://dx.doi.org/10.1182/blood.v124.21.2943.2943.
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Abstract Diamond-Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome that manifests as a profound macrocytic anemia and classically presents within the first year of life. Heterozygous mutations in, or genomic loss of one of several Ribosomal Protein (RP) genes have been identified in over 50% of DBA patients, most commonly RPS19, accounting for 25% of all cases. DBA shares a similar erythroid phenotype to the 5q- subtype of myelodysplastic syndrome in which anemia is thought to arise from heterozygous loss of RPS14. Anemia in these conditions is at least partially due to p53-mediated apoptosis and cell cycle arrest of erythroid progenitors. To further study the role of p53 in the pathogenesis of DBA and 5q- syndrome, we employed genome editing tools to generate stable Rps14 and Rps19 knockout zebrafish lines. We generated Transcription Activator-Like Effector Nucleases (TALENs) targeting exon 1 of rps19 and exon 1 of rps14 as well as Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) single guide RNAs (sgRNA) targeting exon 2 of rps19. TALENs or CRISPRs were injected into p53m214k/m214k zebrafish embryos at the single-cell stage. This zebrafish line carries a mutated p53 that is insensitive to DNA damage and hence prone to tumor formation. rps19 CRISPR sgRNAs were injected with mRNAs encoding Cas9, Cas9D10A nickase, and a ssDNA guide with a human DBA mutation. For each cohort of embryos injected, genomic DNA analysis from 20 phenotypically normal embryos from each clutch was screened to determine the efficacy of cleavage by TALEN and CRISPR using MiSeq. Mutations were identified in 30% (rps14 TALEN) 29% (rps19 TALEN), 27% (rps19 Crispr Cas9) and 12% (rps19 Crispr Cas9D10A) of reads. None of the rps19Crispr Cas9D10A carried the ssDNA guide mutation, rather single nucleotide variants and indels similar to those observed with Cas9. The remaining embryos from each F0 clutch were raised in order to generate stable mutant lines in the F1 generation; however, early, overt tumor growth was noted in all RP injected lines. Tumors were observed from 4 months post fertilization compared with 9 months for uninjected controls. F0 RP mosaic fish continued to develop tumors earlier than uninjected counterparts. At 10 months of age tumor development was statistically significantly higher in rps19 and rps14 TALEN and rps19 Cas9D10A and trended towards significance in rps19 Cas9 injected fish. Overall survival was significantly reduced in each of the cohorts compared to p53m214k/m214k uninjected controls (p<0.0001). Preliminary histology of grown tumors has shown melanomas and malignant peripheral nerve sheath tumors. Zebrafish injected with an unrelated TALEN targeting a zinc transporter (SLC30A10), into p53m214k mutant embryos do not show any increase in tumor formation compared to uninjected controls. Notably several RP’s have been shown to be haploinsufficient tumor suppressor genes in their own right in zebrafish and drosophila models. To determine if the early tumor development in p53m214k zebrafish was simply additive to a potential tumor suppressor effect of Rps14 or Rps19 alone, we injected WT embryos with the rps14 and rps19 TALENS. High mortality in rps14 and rps19 TALEN injected WT embryos impeded this analysis; however recent published reports on stable Rps19 mutant zebrafish do not report an increase in tumor incidence. Interestingly, embryo survival was not affected when TALENs were injected into p53m214/+. Analysis of these zebrafish is ongoing. Our results show that loss of Rps14 or Rps19 accelerates the development of tumors in the p53m214k/m214k mutant line. This effect is independent of the RP or the method of mutation (TALEN vs CRISPR), indicating that off target effects are unlikely to be responsible for this observation. As these are mosaic F0 fish, it is possible that tumors may arise from cells with homozygous RP mutations. Further molecular analysis will reveal this. We have now identified 2 stable Rps19 mutant lines, and tumor analysis of F1 fish from these lines is ongoing. In conclusion, we have shown that loss of Rps14 or Rps19 cooperates with a loss of function p53 mutation to accelerate tumor formation and death. Our results highlight the importance of caution in using p53 suppressors as a therapeutic option in RP deficient patients. Disclosures No relevant conflicts of interest to declare.
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Thompson, Michael D., Colleen M. Jacks, Todd R. Lenvik, and J. Stephen Gantt. "Characterization of rps17, rpl9 and rpl15: three nucleus-encoded plastid ribosomal protein genes." Plant Molecular Biology 18, no. 5 (March 1992): 931–44. http://dx.doi.org/10.1007/bf00019207.
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Dutt, Shilpee, Anupama Narla, Jeffery Lorne Kutok, and Benjamin L. Ebert. "Lineage-Specific Activation of p53 in Response to Ribosomal Haploinsufficiency in Human Bone Marrow Cells." Blood 114, no. 22 (November 20, 2009): 948. http://dx.doi.org/10.1182/blood.v114.22.948.948.
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Abstract Abstract 948 Haploinsufficiency for the ribosomal protein genes RPS14 and RPS19 have been implicated in the erythroid defect in the 5q- syndrome and Diamond Blackfan Anemia, respectively. However, the mechanism by which defective ribosome biogenesis causes erythroid failure is unknown. In this study, we found that shRNA mediated knockdown of RPS14 or RPS19 in primary human CD34+ cells stabilize TP53 by day 4 after infection with concomitant arrest of these cells at G1 stage of cell cycle. The levels of TP53 attained are comparable to the levels observed following gamma irradiation (5Gy) of the CD34+ cells. Using quantitative PCR, we confirmed that stabilized TP53 activates expression of downstream target genes MDM2, p21, Bax and Wig-1. Furthermore, treatment of the CD34+ cells with Nutlin-3 phenocopies RPS14 or RPS19 knockdown, suggesting that the mechanism of TP53 activation is mediated by MDM2 pathway. Conversely, treatment with pifithrin-alpha, which inhibits the transactivation activity of TP53, rescues the effects of RPS14 or RPS19 knockdown. The in vitro activation of TP53 in CD34+ cells was restricted to erythroid cell lineage, consistent with the clinical phenotype of RPS14 or RPS19 haploinsufficiency. Moreover, immunohistochemical analysis of bone marrow biopsies from patient with the 5q- syndrome demonstrated intense staining of TP53 that was restricted to erythroid progenitor cells. Taken together our study indicates that inhibition of ribosomal biogenesis causes TP53 activation selectively in erythroid progenitor cells. Clinically, TP53 staining of patient samples could be used as a diagnostic marker for some types of MDS. Disclosures: No relevant conflicts of interest to declare.
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Pali, Mihaela, Jessica Moetter, Joerg Meerpohl, Mutlu Kartal, Alexandra Fischer, Sandra Urbaniak, Peter Noellke, et al. "Identification of Novel Mutations In Ribosomal Genes In Patients with Diamond Blackfan Anemia (DBA) In Germany and Genotype-Phenotype Correlation Analysis." Blood 116, no. 21 (November 19, 2010): 2244. http://dx.doi.org/10.1182/blood.v116.21.2244.2244.
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Abstract Abstract 2244 In the current study, 122 index patients in addition to parents with DBA registered in the observational DBA study were screened for mutations in the aforementioned 11 genes. Overall, we detected mutations in 51% (n=62) of index patients in the following genes: RPS19 (33.6%, n=41), RPL5 (10.7%, n=13), RPL11 (3.3%, n=4), RPS26 (1.6%, n=2), RPS24 (0.8%, n=1) and RPS10 (0.8%, n=1). Only mutations altering the amino acid composition or resulting in splice site disruption (+/−1bp intronic) were included. Putatively pathogenic mutations localized in the 5‘UTR or intronic regions, (although not present as SNPs in healthy controls) were excluded from this study. In a high number of patients multiple genes were sequenced but no mutual mutations were found. RPS26 mutations detected in two patients included c.1A>G (previously reported by Doherty et al, 2010) and a novel initiation codon mutation, c.3+1G>A. Mutations detected in RPS10 and RPS24 were not previously described: c.71A>G (p.Lys24Arg) and c.2T>G. Various physical abnormalities were present in 57%, 75%, and 100% of patients with RPS19, RPL5/11, RPS26 mutations, respectively. Interestingly, no malformations were exhibited by probands with affected RPS10 and RPS24 genes. It is well known, that imbalances of ribosomal proteins can activate the p53 pathway: RPL5 and RPL11 (in addition to RPL23, RPL26, RPS3, RPS7) were reported to inhibit the activity of MDM2 thus influencing p53 activation. To assess if mutations in RPL5 and RPL11 might have different impact on physical development than RPS19, we compared the frequency of congenital abnormalities in both groups. The following organ systems were affected in patients with mutated RPL5/11 and RPS19 genes and physical anomalies, respectively: craniofacial region: 83% and 50% thumb: 33% and 12%, kidney 17% and 4%, heart: 67% and 46%, skeletal system: 17% and 25%, neurobehavioral deficits: 33% and 12%, skin pigment changes: 17% and 8%. Cleft palate was exclusive to RPL5/11 patients (p= .007; Fisher's exact test), and growth retardation was present in 60% of RPL5 vs. 8% of RPS19 patients (p= 0.008). While none of the patients with growth retardation and RPS19 mutation were small for gestational age (SGA), all but one RPL5 patients proved to be SGA. We next focused our analysis on hematological presentation and treatment modalities of these two subgroups. There were no differences regarding lowest and median platelet, white blood and absolute neutrophil count. Further evaluation of treatment outcome did not reveal any statistical asymmetry with regard to spontaneous remission, steroid or transfusion dependency and hematopoietic stem cell transplantation. In summary, we detected 62 distinct mutations in 6 ribosomal genes in 51% of German DBA patients. Growth retardation predominates in RPL5 patients and cleft palate is solely present in patients with RPL5 and RPL11 mutations. Mutated RPL5/11 vs. RPS19 genes do not seem to have a divergent influence on hematopoiesis in vivo. ***On behalf of the German DBA registry. Disclosures: No relevant conflicts of interest to declare.
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Horos, Rastislav, Hanna IJspeert, Erdogan Taskesen, Ruud H. Delwel, Dagmar Pospisilova, and Marieke M. von Lindern. "Ribosomal Deficiencies Cause Translational Deregulation of Genes Crucial for Erythropoiesis." Blood 114, no. 22 (November 20, 2009): 179. http://dx.doi.org/10.1182/blood.v114.22.179.179.
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Abstract Abstract 179 Diamond Blackfan anemia (DBA) is a rare congenital pure red cell aplasia in which mutations in distinct ribosomal proteins (RPs) have been identified in 50% of patients. Mutated genes include Ribosomal Protein Small 19 (RPS19), RPS24 and RPS17, components of the 40S ribosomal subunit, and Ribosomal Protein Large 11 (RPL11), RPL5 and RPL35A, components of the 60S subunit. It is not known why mutations in such ubiquitously expressed proteins cause such a strong erythroid phenotype. Previously, we showed that selective translation of transcripts with a complex RNA structure in the 5'untranslated region (5'UTR) is crucial in Stem Cell Factor (SCF) dependent expansion of the early erythroid compartment. Transcripts such as Immunoglobulin binding protein 1 (Igbp1) are actively recruited into translating polyribosomes upon SCF signaling which activates the PI3K-mTOR pathway leading to release of eukaryote initiation factor 4E (eIF4E), a limiting factor for assembly of the scanning complex. The scanning complex also involves the ribosomal subunits. We therefore investigated if polysome recruitment of specific mRNAs is affected in erythroblasts deficient for RPs. Mouse primary erythroblasts derived from p53 deficient and wild-type (wt) fetal livers were cultured in presence of erythropoietin, SCF and dexamethasone under serum free conditions. Downregulation of either Rps19 or Rpl11 by lentivirus-delivered shRNA resulted in severely reduced proliferation and inhibition of differentiation in comparison to nontransduced cells or cells expressing a scrambled control shRNA. Analysis of subpolysomal and polysome-bound RNA by sucrose gradient centrifugation showed a specific reduction of the 40S and 60S ribosomal subunit upon knock down of Rps19 and Rpl11, respectively. Subpolysomal and polysome-bound RNA fractions from 3 independent experiments were used for expression profiling. Surprisingly, the polysome recruitment of transcripts that require SCF and increased eIF4E availability to be translated was not affected by RP deficiency. The ratio of polysome association was calculated per gene for each experiment and datasets from wt, scrambled treated, Rps19 and Rpl11 deficient conditions were compared using F-test with random variance model (p<0.0005). This revealed a distinct set of mRNAs, including Fxc1 (fractured callus expressed-1), Siva1 (apoptosis inducing gene), Csde1 (cold shock domain containing E1) and Cdc25B (cell dividion cycle homolog B) as being down regulated from translating polyribosomes upon RP deficiency. This downregulation was independent from the presence of p53, or glucocorticoids and also occurred in cells deficient for other ribosomal proteins (Rps14, Rpl8). Currently we are analyzing selective translation of these genes in erythroblasts of DBA patients. Erythroblasts could be cultured from peripheral blood of DBA patients although expansion potential of the cultures was decreased compared to control cultures. Protein expression of several identified target genes (CSDE1, CDC25B) was down regulated in erythroblasts derived from DBA patients as compared to controls, whereas their transcript level on total mRNA remained unchanged, indicating a specific translation defect. Further investigation showed that loss of Csde1 perturbed cell cycle progression and erythroid differentiation. In conclusion, we identified transcripts whose translation is selectively affected in RP deficient erythroblasts cultured from mouse fetal livers or DBA patients. We propose that the erythroid phenotype in DBA patients is caused by defective translation of specific set of mRNAs, which are essential for erythroid development. Disclosures: Pospisilova: Grant NS9935-3 from Ministry of Health, Czech Republic: Research Funding.
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Kazerounian, Shideh, Pedro Ciarlini, Roxanne Ghazvinian, Meritxell Alberich-Jorda, Daniel Yuan, Mugdha Joshi, Hong Zhang, Alan Beggs, and Hanna T. Gazda. "Increased Tumorigenesis In Ribosomal Proteins L5 and S24 Heterozygous Mice." Blood 122, no. 21 (November 15, 2013): 1227. http://dx.doi.org/10.1182/blood.v122.21.1227.1227.
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Abstract Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia inherited in an autosomal dominant pattern caused by mutations in ribosomal protein (RP) genes and in an X-linked recessive pattern by GATA1 mutations. Heterozygous mutations and large deletions in 11 RP genes, RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A, RPS7, RPS10, RPS26, RPL26, and RPL15, are present in ∼65% of DBA patients. DBA is associated with congenital abnormalities in ∼50% of patients and with increased risk of malignancy. To investigate the molecular pathogenesis of RPL5 and RPS24 gene mutations, we generated two murine lines of heterozygous mice, Rpl5 and Rps24, by knocking-out exons 1-8 in the Rpl5 gene and exons 2-3 in the Rps24 gene in C57BL/6 mice. Knock-out of both alleles of Rpl5 and Rps24 genes are embryonic lethal. In contrast, heterozygous mice exhibited normal hematological phenotype, as well as normal Rpl5 and Rps24 RNA and protein levels in their tissues, suggesting that the presence of one allele was sufficient to support the normal function of ribosomal proteins L5 and S24 in mice. To evaluate the risk of cancer development in Rpl5 +/- and Rps24 +/- mice, we monitored these mice and wild type mice until late age. Out of 21 Rpl5 +/- mice (between the ages of 14 and 26 months), two mice developed tumors at 22 months of age and two mice were euthanized due to severe dermatitis at the same age. Similarly, we have been monitoring 23 Rps24 +/- mice between 15 and 26 months of age. One of these mice developed a tumor at 17 months of age, five mice were euthanized due to severe dermatitis between the ages of 17 and 19 months, and two mice were euthanized due to injuries at ages 15 and 29 months. We also monitored 20 control wild-type mice ranging from 13 to 26 months of age. To this date, no tumors have been detected in wild-type mice, although nine of these mice developed severe dermatitis and were euthanized. Histological and immunohistochemical studies were performed to determine the nature of tumors in Rpl5 +/- and Rps24 +/- mice. Comparison of tumor tissues with normal skin from wild-type or Rpl5 +/- and Rps24 +/- with no detected tumors showed that all tissues had normal epidermis and underlying dermis, but connective tissues from tumor sections consisted of a densely cellular neoplasms composed of predominantly atypical spindle shaped cells arranged in intersecting fascicles. The tumor cells had strong cytoplasmic reactivity for vimentin and negative staining for S100, CD45, and pan-keratin, consistent with a high-grade spindle cell sarcoma. Recent studies conducted by the DBA Registry of North America revealed that out of 608 DBA patients, 18 with median age of 41 years developed various types of cancer including sarcomas, colon cancer, and acute myeloid leukemia. The relative risk of cancer in DBA was increased 5.4 fold compared to general population (Vlachos, et al., 2012). Our studies also suggest the correlation between ribosomal protein gene mutations and cancer. However, further studies are required to better understand the underlying molecular mechanism. Disclosures: No relevant conflicts of interest to declare.
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Maceckova, Zuzana, Pavla Koralkova, Jana Volejnikova, Petr Vojta, Alexandra Jungova, Zuzana Saxova, Renata Mojzikova, et al. "Two Novel Mutations in Ribosomal Proteins in the Czech National Diamond-Blackfan Anemia Registry." Blood 128, no. 22 (December 2, 2016): 3903. http://dx.doi.org/10.1182/blood.v128.22.3903.3903.
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Abstract The Czech National Diamond-Blackfan Anemia (DBA) Registry currently consists of 51 patients; in 36 of them ribosomal protein (RP) mutations have previously been identified. Targeted sequencing of genes encoding RP, exomes and transcriptomic sequencing and/or array CGH were performed in newly diagnosed DBA patients and in patients without a causative mutation. Two novel mutations in RPS7 and RPL11 were discovered in 5 individuals from two families. Aheterozygous mutation g[3580153G>T] in exon 6 of a gene coding for RPS7, leading to V134F substitution, was found in an 18-year-old female who was transfusion dependent during infancy, responded to steroids and is currently in remission. The identical mutation was detected in the patient's mother and older sister, who both are asymptomatic with borderline hemoglobin (Hb) levels, mild macrocytosis with no active treatment. A functional study using cellular models revealed that RPS7-deficient MRC-5 fibroblasts transiently expressing RPS7-V134F exhibit altered protein synthesis, defective ribosomal RNA processing, increased nucleolar and ribosomal stress. This was evidenced by overexpression and translocation of p53 protein to the nucleus in RPS7-V134F but not RPS7 wild type transfected cells. These data are consistent with previously reported cellular phenotypes in DBA. In addition, increased levels of erythrocyte adenosine deaminase (e-ADA) were detected in all three family members with V134F RPS7 mutation (patient: 4.2±0.8 IU/g Hb; mother: 3.5±0.2 IU/g Hb; sister: 4.6±0.3 IU/g Hb; reference range: 0.8-2.5 IU/g Hb). Recently it was shown that ribosomal insufficiency increases oxidative stress in red blood cells (RBC) of DBA patients. In this family, increased levels of reactive oxygen species (ROS) were detected by flow cytometry only in patient's RBC, but not in RBC of her asymptomatic mother and sister. Consistently, only the patient showed increased anti-oxidative defense parameters compared to the controls and her mother and sister. Markedly elevated levels of reduced glutathione (GSH) and glucose 6-phosphate dehydrogenase (G6PD), two essential antioxidants, were detected in patient's RBC (GSH: 4356±44 mM; G6PD: 10.3±0.6 IU/g Hb) compared to controls (GSH: 2434±454 mM; G6PD: 5.4-7.0 IU/g Hb) and patient's mother (GSH: 2125 mM; G6PD: 5.3±0.2 IU/g Hb) and sister (GSH: 2450±35 mM; G6PD: 6.5±0.1 IU/g Hb). Concomitant increase (approximately 2.5 times) in the activity of hexokinase (HK) and pyruvate kinase (PK) and in the levels of ATP in patient's RBC indicates augmented energy metabolism to sustain the integrity of RBC. Flow cytometry of Annexin V binding revealed increased exposure of phosphatidylserines on RBC membrane of the patient compared to controls and her mother and sister suggesting that the tendency towards redox balance restoration and membrane integrity does not completely prevent an enhanced recognition and destruction of patient's RBC by reticuloendothelial macrophages. The second c.281T>G point mutation in RPL11 was found in a 34-year-old woman with unspecified macrocytic anemia, who developed diffuse large B-cell lymphoma (DLBCL). She was transfusion dependent in infancy, responded to steroids and is currently in the anemia remission and second remission of DLBCL. Identical mutation was detected in her 56-year-old mother, followed for macrocytic anemia and gammopathy. In 10 DBA cases without the causative mutation an array CGH was performed, but no large deletions in RPS19, RPS17, RPS26, RPL5 and RPL11 genes were detected. In summary, we present the first case of a missense mutation in RPS7 as the cause of DBA; all to date published RPS7 mutations clinically associated with DBA were located in splice sites. Similarly, the co-occurrence of DBA and DLBCL has not been previously published. We also emphasize that the phenotype of family members with the same mutations could be different, including silent carriers. All family members should therefore be examined even if asymptomatic. Testing of eADA levels could serve as a sensitive screening method in apparently unaffected family members. Finally we suppose that altered RBC metabolism may affect the lifespan of RBC in DBA and thus contribute to the worsening of the blood condition, especially during infections. The analyses in a large cohort of DBA patients are ongoing. Grant Support: AZV16-32105A, GA15-13732S, MH CZ-DRO FNOL 00098892, NPU LO 1304, IGA UP LF_2016_014. Disclosures No relevant conflicts of interest to declare.
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Abayasekara, Nirmalee, Anupama Narla, Slater Hurst, Hong Sun, Maria Virgilio, Elspeth Payne, Benjamin L. Ebert, A. Thomas Look, Nancy Berliner, and Arati Khanna-Gupta. "L-Leucine Improves the Anemia of DBA and the 5q- Syndrome Via Activation of the mTOR Pathway in a p53-Independent Manner." Blood 120, no. 21 (November 16, 2012): 1257. http://dx.doi.org/10.1182/blood.v120.21.1257.1257.
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Abstract Abstract 1257 Haploinsufficiency of ribosomal proteins (RP) has been shown to be the common basis for the anemia observed in ribosomopathies such as Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q (del(5q) MDS). DBA is a congenital bone marrow failure syndrome characterized by a profound macrocytic anemia. More than half the patients with DBA have been shown to have a heterozygous loss of an RP gene, with RPS19 being the most frequently mutated. The “5q- syndrome” is a subtype of myelodysplastic syndrome (MDS) also characterized by severe anemia that is caused by heterozygous loss of the RPS14 gene on chromosome 5q. The p53 pathway is known to play a critical role in the pathophysiology of the ribosomopathies. The leading hypothesis is that ribosomal haploinsufficiency leads to disrupted ribosome biogenesis with an accumulation of free ribosomal proteins that bind MDM2. MDM2 is an E3 Ubiquitin ligase that normally binds to and targets p53 for proteosomal degradation. The consequent accumulation of p53 leads to cell cycle arrest and apoptosis, which ultimately results in anemia. Several animal models have shown that the anemia associated with RP haploinsufficiency is almost completely alleviated in a p53 null background. However, we and others have shown that p53-independent pathway(s) also contribute to the anemia associated with RP haploinsufficiency. We have previously modeled DBA and del (5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14 respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. We have further demonstrated that treatment of Rps19 and Rps14 deficient embryos with the amino acid L-Leucine, a known activator of mRNA translation, results in a marked improvement in anemia. This observation was confirmed in primary human CD34+ cells, following shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that L-leucine treatment activates the mTOR pathway in zebrafish embryos deficient in Rps19 or Rps14. In order to determine if the effect of L-Leucine on RP deficient erythroid cells is p53 dependent, we injected rps19 and rps14 morpholinos into zebrafish embryos and treated them with L-Leucine. Total RNA was collected 48hpf and evaluated for expression of p53 by qPCR analysis. As expected, the expression of p53 and its downstream targets (p21 and PUMA) were upregulated in Rps14 and Rps19 deficient embryos. P53 expression levels remained elevated even after L-Leucine treatment. Levels of p21, a direct transcriptional target of p53, remained unchanged in L-Leucine treated RP deficient zebrafish embryos; however, expression of PUMA increased following L-Leucine treatment. The expression of the PUMA gene has previously been shown to have a p53-independent regulatory component. Preliminary studies in the A549 cell line, which harbors wild type p53, also showed increased levels of p53 expression upon shRNA mediated downregulation of both RPS19 and RPS14, which remained unaltered following L-Leucine treatment. These observations are currently being confirmed in primary human CD34+ cells, following shRNA knockdown of RPS19 and RPS14. Our preliminary studies show that the effect of L-Leucine in improving the anemia in models of DBA and del(5q) MDS occurs independently of p53. This supports our hypothesis that the erythroid phenotype in these disorders has a p53-independent component. Our finding that L-Leucine treated RP deficient cells are likely to express elevated rather than diminished levels of p53 in spite of improved anemia also has important implications for the clinical management of patients, since p53 inactivation is associated with tumor growth. A trial using L-Leucine for patients with DBA will be opening soon in the United States. Disclosures: Ebert: Celgene: Consultancy; Genoptix: Consultancy.
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Goldberg, Tracie A., Adrianna Henson, Sharon Singh, Abdallah Nihrane, Jeffrey Michael Lipton, Steven Ellis, and Johnson M. Liu. "Specific Hematopoietic and Erythroid Differentiation Defects in Mouse Embryonic Stem (ES) Cells with Abortive Ribosome Assembly." Blood 114, no. 22 (November 20, 2009): 1088. http://dx.doi.org/10.1182/blood.v114.22.1088.1088.
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Abstract Abstract 1088 Poster Board I-110 Background Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes, characterized by erythroid hypoplasia, congenital anomalies and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS19, RPS17, RPS24, RPL5, RPL11, RPL35a), which somehow triggers apoptosis of erythroid precursors. There is a marked variation in phenotype among members of the same family and also between subsets of patients with different mutations. Methods We studied primary and secondary in vitro differentiation of two murine ES gene trap cell lines with mutations in Rps19: S17-10H1, in which Rps19 is disrupted by insertion of the ROSAFARY gene trap vector between exons 2 and 3; and YHC074, in which the pGT0Lxf gene trap vector is inserted between exons 3 and 4 and whose growth is feeder cell-independent. For primary differentiation and generation of embryoid bodies (EBs), the ES cells were cultured in a serum-supplemented methylcellulose-based medium containing stem cell factor (SCF). After 7 days, the cultures were fed with a medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 13 for hematopoietic percentage. Secondary (hematopoietic) differentiation was performed on day 9 EBs. EBs were harvested and disrupted with collagenase, and the disrupted cells were suspended in a serum-supplemented methylcellulose-based medium with SCF, IL-3, IL-6 and epo. Hematopoietic colonies were counted on day 10. Results Decreased expression of Rps19 protein was confirmed by Western blot analysis in both S17-10H1 and YHC074 gene trap cell lines. We focused on YHC074 because its growth is feeder-independent, and it expresses approximately 50% of normal Rps19 levels. By polysome analysis, we found a selective reduction in the 40S subunit peak in mutant YHC074 cells as compared to parental controls. By Northern blot assays, we also found a relative increase in the 21S pre-rRNA to 18S rRNA ratio in mutant YHC074 cells. The viability of undifferentiated ES cells was not significantly different from parental control cells in the first 72 hours of culture; however, there was a significantly decreased number of EBs, particularly hematopoietic EBs, following primary differentiation (Fig. 1). Furthermore, when day 9 EBs were induced to secondary (hematopoietic) differentation, there was a significant decrease in the ratio of erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation in mutant YHC074 cells. In order to confirm these results in an isogenic background, we stably transfected S17-10H1 cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones were found to overexpress Rps19 and were further studied in secondary differentiation experiments. There was a significant decrease in erythroid and myeloid colony formation and in BFU-E size from mutant S17-10H1 cells when compared to the Rps19-overexpressing clone, suggesting a direct relationship between the levels of Rps19 protein and hematopoietic growth and differentiation. Conclusion Using two ES cell lines with slightly different Rps19 mutations and genetic backgrounds, we have recapitulated the major DBA erythroid growth and differentiation defect, as well as the defect in ribosome assembly and rRNA processing caused by Rps19 haploinsufficiency. Disclosures No relevant conflicts of interest to declare.
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Bi, Changwei, Na Lu, Yiqing Xu, Chunpeng He, and Zuhong Lu. "Characterization and Analysis of the Mitochondrial Genome of Common Bean (Phaseolus vulgaris) by Comparative Genomic Approaches." International Journal of Molecular Sciences 21, no. 11 (May 27, 2020): 3778. http://dx.doi.org/10.3390/ijms21113778.
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The common bean (Phaseolus vulgaris) is a major source of protein and essential nutrients for humans. To explore the genetic diversity and phylogenetic relationships of P. vulgaris, its complete mitochondrial genome (mitogenome) was sequenced and assembled. The mitogenome is 395,516 bp in length, including 31 unique protein-coding genes (PCGs), 15 transfer RNA (tRNA) genes, and 3 ribosomal RNA (rRNA) genes. Among the 31 PCGs, four genes (mttB, nad1, nad4L, and rps10) use ACG as initiation codons, which are altered to standard initiation codons by RNA editing. In addition, the termination codon CGA in the ccmFC gene is converted to UGA. Selective pressure analysis indicates that the ccmB, ccmFC, rps1, rps10, and rps14 genes were under evolutionary positive selection. The proportions of five amino acids (Phe, Leu, Pro, Arg, and Ser) in the whole amino acid profile of the proteins in each mitogenome can be used to distinguish angiosperms from gymnosperms. Phylogenetic analyses show that P. vulgaris is evolutionarily closer to the Glycininae than other leguminous plants. The results of the present study not only provide an important opportunity to conduct further genomic breeding studies in the common bean, they also provide valuable information for future evolutionary and molecular studies of leguminous plants.
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Mirabello, Lisa, Bari J. Ballew, Neelam Giri, Kevin B. Jacobs, Meredith Yeager, Joseph F. Boland, Belynda D. Hicks, et al. "RPS29 is Mutated in a Multi-Case Diamond Blackfan Anemia Family." Blood 120, no. 21 (November 16, 2012): 511. http://dx.doi.org/10.1182/blood.v120.21.511.511.
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Abstract Abstract 511 Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red blood cell aplasia, variable physical anomalies, and increased risk of leukemia, myelodysplastic syndrome, lymphoma, and certain solid tumors, including osteosarcoma. DBA has been considered to be a disorder of ribosomal biogenesis because approximately 50% of cases are due to a mutation or deletion in 1 of 9 ribosomal protein genes (RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, or RPS26). However, mutations in GATA1, a hematopoietic transcription factor, have recently been reported to also cause DBA. DBA is inherited in an autosomal dominant manner, but de novo germline mutations have also been reported. Our IBMFS cohort study conducts detailed clinical evaluations and medical record review of patients with DBA and their family members. We evaluated a large family with DBA in which mutation testing for the 10 known genes was negative. The male proband had steroid-responsive anemia as a child; he was in remission until he was treated with chemotherapy for squamous cell lung cancer at age 55 years. His healthy sister is an obligate carrier because her daughter was diagnosed with DBA as a child; her daughter had steroid-responsive anemia. The proband's maternal 1st cousin had steroid-responsive DBA as a child which relapsed during pregnancy. Her 3 children (the proband's 2nd cousins) had DBA. One died due to complications of transfusion-related iron overload, one had successful hematopoietic stem cell transplantation for steroid-refractory and transfusion-dependent anemia at age 26 years, and one has been off treatment and in remission for over 20 years. Except for the proband with lung cancer and a transfusion-dependent individual, all affected individuals had elevated red blood cell adenosine deaminase consistent with DBA. We performed whole-exome sequencing on the 5 clinically affected individuals (a male proband, his affected first cousin, 2 affected second cousins, and an affected niece), his obligate carrier sister, and the unaffected father of the proband's niece. Genomic DNA was used to create an enriched multiplexed sequencing library (Nimblegen v2); this was followed by paired-end sequencing using an Illumina HiSeq™. We detected a total of 229,024 exonic nucleotide variants across this family, including 2,484 uncommon (minor allele frequency <5%) non-synonymous amino-acid substitutions. After removing variants that were present in publically available databases (1000Genomes, ESP, Kaviar, and dbSNP) and applying quality control filters, 731 candidate variants remained. We then evaluated these variants for autosomal dominant inheritance in this family. Remarkably, there was only 1 nonsynonymous variant present in all 5 affected individuals and in the obligate carrier, but absent from the unaffected non-obligate carrier parent. This nonsynonymous variant was present in exon 2 of the ribosomal protein S29 (RPS29) gene (chromosome 14q). This region of the RPS29 protein is very highly evolutionarily conserved. Bioinformatic analyses suggested that this variant is highly likely to affect protein function. SIFT, Polyphen 2, and Condel algorithms all predict this variant to be deleterious. RPS29 gene encodes the 40S ribosomal protein S29, which is a component of the small 40S ribosomal subunit and is important for ribosomal RNA processing and ribosome biogenesis. In addition, Zebrafish models of RPS29 mutations have significant defects in hematopoietic stem cell and red blood cell development. We are currently sequencing RPS29 in additional patients with DBA. In summary, whole-exome sequencing has allowed us to uncover another human DBA gene, RPS29, which will likely lead to improved understanding of this complex disorder. Disclosures: No relevant conflicts of interest to declare.
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Bhar, Saleh, Lisa Mirabello, Nimrat Chatterjee, Christopher Williams, Blanche P. Alter, Neelam Giri, Richard Person, et al. "De Novo RPS20 Mutations in Diamond Blackfan Anemia." Blood 124, no. 21 (December 6, 2014): 2667. http://dx.doi.org/10.1182/blood.v124.21.2667.2667.
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Abstract Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by pure red blood cell aplasia, variable congenital anomalies and increased risk of malignancy. Approximately 50-60% of cases are due to germline, heterozygous mutations in 1 of 13 genes that encode components of either the small (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26 and RPS29) or large (RPL5, RPL11, RPL15, RPL26, RPL31 and RPL35A) ribosomal subunits. The mutations may be either inherited or de novo. Consistent with DBA being a ribosomopathy, defects in ribosome assembly, altered ribosomal RNA processing and nucleolar stress are observed in cells of patients with DBA and ribosomal protein gene mutations. Mutations in GATA1, a hematopoietic transcription factor, have also been reported in rare X-linked recessive cases. Whole exome sequencing was independently carried out on two white/Hispanic probands with genetically uncharacterized DBA (BMF92 and NCI-62-1). Both presented with transfusion-dependent anemia during the first week of life. Bone marrow evaluations revealed marked erythroid hypoplasia characteristic of DBA. One of the probands (BMF92) experienced a spontaneous remission of his anemia toward the end of the first year of life, possibly related to corticosteroids administered for bronchiolitis. Subsequently, his erythrocyte adenosine deaminase(eADA) level was measured and elevated [10.5 units (mol/min/gm hg); reference range 0.42-3.5 units]. BMF92’s hypoplastic anemia later recurred and was found to be steroid refractory. NCI-62-1 is a male, now 12 years of age and red cell transfusion dependent. BMF92 and NCI-62-1 are of normal stature and have no documented congenital anomalies, although both have experienced chronic colitis. In both cases, family history for DBA was negative and parental eADA levels were within normal limits. Whole exome sequencing of peripheral blood DNA, validated by Sanger or Ion Torrent targeted sequencing, identified heterozygous variants of unknown significance in the ribosomal protein gene RPS20. Parental studies revealed both variants to be de novo. The variants were mutated at the same genomic position, but resulted in different amino acid substitutions in RPS20 [hg19 chr8 56985758, c.251A>T, p.I84N (BMF92) and 56985758, c.251A>C, p.I84S (NCI-62-1)]. Buccal swab analysis performed in BMF92 confirmed the presence of the variant. Both variants were novel, based on inspection of several databases, including dbSNP, 1000 Genomes, NHLBI Exome Sequencing Project, clinical WES data at the Whole Genome Laboratory at Baylor College of Medicine, Kaviar, Human Genome Mutation Database and ClinVar, totally well over 10,000 individuals as well as the COSMIC database, which reports somatic mutations in cancer. In silicoanalyses were consistent with the variants being damaging and disease causing (PolyPhen-2, SIFT and Mutation Taster), affecting highly evolutionarily conserved residues (GERP, PhyloP and Sitewise likelihood-ratio score) and decreasing the stability of the protein structure (MUpro and I-Mutant2.0). The above data strongly suggest that the RPS20 variants in these cases are disease causing. A recent report implicated a germline RPS20 truncating mutation in a four-generation pedigree with familial nonpolyposis colorectal carcinoma; however, the mutation carriers were reported to not manifest features of DBA (Nieminen et al., Gastroenterology 2014). This raises the likelihood of allele-specific effects. Functional assays of our probands’ mutations, including quantification of RPS20 protein and ribosomal RNA precursor steady state levels in patient versus control lymphoblastoid cell lines as well as analysis of the impact of mutant RPS20 protein expression on p53 and downstream targets are underway to determine their contribution to the DBA phenotype. Disclosures No relevant conflicts of interest to declare.
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Arbiv, Omri Avraham, Bozana Zlateska, Robert J. Klaassen, Conrad Fernandez, Rochelle Yanofsky, John K. Wu, Nancy Robitaille, et al. "Molecular Analysis of Diamond Blackfan Anemia and Genotype-Phenotype Correlation: Experience from the Canadian Inherited Marrow Failure Registry." Blood 126, no. 23 (December 3, 2015): 3621. http://dx.doi.org/10.1182/blood.v126.23.3621.3621.
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Abstract Background/Objectives: Diamond Blackfan anemia (DBA) is an inherited disorder characterized by chronic hypoproductive anemia, physical malformations, and an increased risk of malignancies. At least 12 DBA genes have been identified, which include various ribosomal protein genes and the transcription factor GATA1. The aims of our study were (1) to identify the mutation spectrum of DBA patients, utilizing a cohort of patients enrolled on the Canadian Inherited Marrow Failure Registry (CIMFR) and (2) to determine whether specific hematological abnormalities, malformations, and outcomes are associated with specific mutations. Methods: Patients were enrolled on the CIMFR, which is a multicenter cohort study of inherited bone marrow failure syndromes (IBMFS). Genetic testing was performed using one or more of the following tests: Sanger sequencing, next generation sequencing (NGS) DBA gene panel, a comprehensive NGS IBMFS gene panel developed in our laboratory, or comparative genetic hybridization (CGH). Severity of the hematological disease was dichotomized according to a patient's requirement for chronic treatment: those who were maintained on corticosteroids, blood transfusions, or received a hematopoietic stem cell transplantation were considered to have a more severe phenotype than those who did not require hematological treatment. Chi-square tests with a Fisher's exact test correction were used to compare genetic groups with at least 5 patients on observed phenotypes. Results: 71 patients with DBA have been enrolled in our registry. A causal mutation has been identified in 36 of these patients, with the following rates: RPS19 (n=11), RPL11 (n=7), RPL5 (n=6), RPS26 (n=5), RPL35a (n=2), RPS24 (n=2), and one of each RPS7, RPS29, RPS17. Remarkably, a substantial number of patients in our population-based cohort (19.4%) had mild hematological phenotype requiring no therapy. Patients with RPL11 mutations tended to have a less severe DBA phenotype, while patients with RPS19 mutations tended to have a more severe phenotype (p=0.04). In terms of non-hematological malformations, we found no differences in cardiac, stature and craniofacial malformations across the groups compared (all p>0.1). However, patients with RPL5 mutations had significantly more hand malformations (p=0.02), and patients with RPS26 mutations had more genitourinary malformations (p=0.04). To control for the impact of mutation severity on the observed phenotype, we compared the prevalence of mutations that are predicted to result in truncated or lack of protein from the respective allele (large copy-number variation, nonsense, or indel frameshift) to mutations that are predicted to be hypomorphic or affect function (splicing, indel/inframe and, missense) between mutation categories. There were no differences among genetic groups in the severity of their mutations (p=0.58). Conclusions: Mutations in a wide spectrum of ribosomal protein genes underlie DBA cases in Canada, which approximate those observed by other registries in Western countries. Patients with DBA caused by RPL11 mutations tended to have a milder hematological phenotype, while patients with RPS19 mutation tended to have a more severe phenotype. Mutations in RPS26 and RPL5 are associated with genitourinary and hand malformations, respectively. Our findings may help improve counseling of DBA patients and their family. Future studies are needed to replicate our results and determine whether these findings can help personalize care. Disclosures Lipton: Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.
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Goldberg, Tracie A., Sharon Singh, Adrianna Henson, Abdallah Nihrane, Jeffrey Michael Lipton, Steven Ellis, and Johnson M. Liu. "Unique Primitive Erythropoiesis Defect In Rpl5-Deficient Murine Embryonic Stem Cell Model of Diamond Blackfan Anemia." Blood 116, no. 21 (November 19, 2010): 877. http://dx.doi.org/10.1182/blood.v116.21.877.877.
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Abstract Abstract 877 Background: Diamond Blackfan anemia (DBA), a rare inherited bone marrow failure syndrome, is characterized mainly by erythroid hypoplasia but is also associated with congenital anomalies, short stature and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS17, RPS19, RPS24, RPL5, RPL11, RPL35a), which renders erythroid precursors highly sensitive to death by apoptosis. The ontogeny and basis of the hematopoietic defect are unclear. The typical presentation of anemia occurs at 2–3 months of age, although there are rare cases of hydrops fetalis. Marked phenotypic variations exist among members of the same family and also between subsets of patients with different mutations. Methods: We studied in vitro hematopoietic differentiation of two murine embryonic stem (ES) cell lines: YHC074, Rps19 mutant with the pGT0Lxf gene trap vector inserted in intron 3 of Rps19, and D050B12, Rpl5 mutant with the FlipRosaβgeo gene trap vector inserted in intron 3 of Rpl5. Wild-type parental cell lines were used as controls. For primary differentiation and generation of embryoid bodies (EBs), ES cells were cultured in serum-supplemented methylcellulose medium containing stem cell factor (SCF). After 7 days, the cultures were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 12 for hematopoietic percentage. For secondary differentiation into definitive hematopoietic colonies, day 10 EBs were disrupted, and individual cells were suspended in serum-supplemented methylcellulose medium containing SCF, IL-3, Il-6 and epo. Definitive hematopoietic colonies were counted on day 10. Primitive erythropoiesis differentiation assays were performed by disruption of day 4 EBs, followed by suspension of cells in methylcellulose medium containing plasma-derived serum and epo. Primitive erythropoiesis colonies were counted on day 7. Results: We confirmed haploinsufficient expression (∼50% wild type) of Rps19 in YHC074 and Rpl5 protein in D050B12 by Western blot analysis. By polysome analysis, we found a selective reduction in the 40S subunit peak in the Rps19 mutant cell line and in the 60S subunit peak in the Rpl5 mutant cell line. Both types of mutants produced a significantly decreased number of EBs, particularly hematopoietic EBs, compared to parental cell lines. EB size was not compromised in the Rps19 mutant cell line, while Rpl5 mutant ES cells produced significantly smaller EBs, compared to its parental cells. Upon differentiation of cells to definitive hematopoietic colonies, both Rps19 and Rpl5 mutants showed a similar reduction in the erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation ratio. Primitive erythropoiesis was conserved in the Rps19 mutant (Figure 1. 1, top panel). By contrast, the Rpl5 mutant demonstrated a severe primitive erythropoiesis defect (Figure 1. 1, bottom panel). For confirmation of these results in an isogenic background, we stably transfected YHC074 ES cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones expressed Rps19 at the wild-type level. Upon differentiation of a chosen clone, we demonstrated correction of the EB defect and the definitive erythropoiesis defect, suggesting that the hematopoietic differentiation defects seen are directly related to levels of Rps19 protein. We are currently working on correction of the D050B12 ES cells in a similar manner. Conclusion: Murine ES cell lines with Rps19 and Rpl5 mutations exhibit ribosomal protein haploinsufficiency, demonstrate respective ribosome assembly defects, and recapitulate the major DBA hematopoietic differentiation defect. In addition, a unique defect in primitive erythropoiesis in the Rpl5 mutant ES cell line suggests that the Rpl5 mutation in this mouse strain affects early-stage embryogenesis, a finding which may offer insight into the ontogeny of DBA hematopoiesis and may offer an explanation for phenotypic variations seen in patients (such as hydrops fetalis). Disclosures: No relevant conflicts of interest to declare.
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Dal Bo, Michele, Federico Pozzo, Riccardo Bomben, Antonella Zucchetto, Erika Tissino, Dania Benedetti, Massimo Degan, et al. "Nucleophosmin-1 and Ribosome-Associated Components Are Constitutively Overexpressed in NOTCH1 Mutated IGHV Unmutated CLL." Blood 120, no. 21 (November 16, 2012): 3880. http://dx.doi.org/10.1182/blood.v120.21.3880.3880.
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Abstract Abstract 3880 Background: activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012). Aim: to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL. Methods: the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation. Results: in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels. Conclusion: NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset. Disclosures: No relevant conflicts of interest to declare.
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Farrar, Jason E., Adrianna Vlachos, Eva Atsidaftos, Hannah Carlson-Donohoe, Steven R. Ellis, Thomas C. Markello, Robert J. Arceci, Jeffrey M. Lipton, and David M. Bodine. "SNP Array Genotyping Reveals Constitutional and Mosaic Losses of Ribosomal Protein Gene Regions In Patients with Diamond Blackfan Anemia without Ribosomal Protein Gene Mutations." Blood 116, no. 21 (November 19, 2010): 1168. http://dx.doi.org/10.1182/blood.v116.21.1168.1168.
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Abstract Abstract 1168 Background: DBA is a congenital anemia that results from failure of adequate erythrocyte expansion from erythroid precursors, often associated with congenital physical abnormalities. Mutations of eleven ribosomal protein (RP) genes have been confirmed in association with DBA, with several other RP gene changes of uncertain significance reported in isolated cases. However, despite a series of sequencing studies in DBA including all of the RP genes, no genetic abnormality has been identified in ∼40% of patients, suggesting the possibility of additional genetic changes. Aim: We performed SNP array genotyping on DBA blood samples without identified RP gene mutations to determine whether allelic loss of one or more RP genes, which would not be identified by sequencing, might be responsible for DBA in these patients. Method: We performed SNP array genotyping on 23 DBA probands, 5 parents, 1 affected and 1 unaffected sibling. Genomic DNA from peripheral blood mononuclear cells was hybridized to HumanOmni1-Quad BeadChips and copy number variants (CNVs) were identified using a hidden Markov model-based algorithm integrating signal intensity with SNP distribution. Regions of putative copy variation were queried for overlap with RP genes. Fractional mosaicism was estimated by modeling B-allele frequency data against mosaic models using an iterated, non-linear continuous distribution function regression model. Results: We identified regional monosomy of chromosomal regions containing RP genes with established relevance to DBA in 3 probands: 1) a 16 Kb deletion involving RPS26, 2) a 1.6 Mb deletion that includes RPS17, and 3) an 828 Kb deletion involving RPS19. The latter patient had macrocephaly with developmental delay and was not steroid responsive. The former two patients were steroid-responsive and lacked physical abnormalities. In addition to constitutional copy loss at RP gene loci, we identified 3 examples of mosaic regional copy loss involving RP genes. Mosaicism is visualized by SNP genotyping as a symmetric splitting around 0.5 of the B-allele frequency plot into two distinct histogram peaks in regions of reduced signal intensity (Fig 1a & b). One patient, discussed in detail separately, demonstrated two discrete regions of mosaic loss on 5q in a region that includes RPS14 and is associated with 5q- MDS (Fig 1a). The monosomic fraction was estimated at 64% in mixed peripheral blood DNA. Similar analysis of DNA from sorted lymphoid and myeloid peripheral blood populations demonstrated near total monosomy in myeloid and disomy of these regions in lymphoid DNA. This abnormality was not found in either parent, further confirming somatic copy loss. Two siblings evaluated from a family of 3 affected siblings demonstrated variable mosaicism of 3q, including the telomeric region containing RPL35a (Fig 1b). The degree of deletion was greatest at the telomeric end (23% at 187M-qter) and decreased approaching the centromere. DNA from a third sibling and father were not available; however no similar abnormality was detected in the mother. All siblings have modest neutropenia but lack physical abnormalities. None responded to corticosteroid therapy though two achieved spontaneous remission at 12 and 16 years of age. The father had a history of anemia and developed MDS progressing to AML at 44 years of age. A third variably mosaic chromosomal abnormality was identified on the long arm of chromosome 15 in one proband. The involved region spans the entire long arm, and like the 3q abnormality, shows the highest fractional mosaicism at the telomere. This patient is also steroid-unresponsive and lacks physical abnormalities. RPS17, RPL4, RPLP1 and RPS27L all lie in the telomeric half of 15q. Conclusion: We detected deletions of known or suspected DBA-related genes in a cohort of patients in whom no mutations could be found by RP sequencing. The rate of detection (6/23, 26%) suggests that deletions may explain a portion of patients in whom RP gene mutations cannot be identified. These data demonstrate the novel finding of chromosome-specific variable mosaicism in a hematologic disorder, a finding which suggests inheritance (3q-) or potentially acquisition (15q-) of a factor predisposing to regional chromosomal instability. Finally, these findings suggest the possibility of hematopoietic mosaicism as a determinant of remission from anemia seen in ∼20% of patients with DBA. Disclosures: No relevant conflicts of interest to declare.
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Quarello, Paola, Emanuela Garelli, Adriana Carando, Maria Francesca Campagnoli, Alfredo Brusco, Irma Dianzani, and Ugo Ramenghi. "High Frequency of RPL11 Gene Mutation in Italian Patients with Diamond-Blackfan Anemia." Blood 112, no. 11 (November 16, 2008): 3100. http://dx.doi.org/10.1182/blood.v112.11.3100.3100.
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Abstract Diamond Blackfan anemia (DBA, MIM#205900) is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors. About 40% of cases show malformations. Anemia is corrected by steroid treatment in half of the patients; non-responders need chronic transfusions or stem cell transplantation. Defects in the RPS19 gene, encoding the ribosomal protein (RP) S19, are the main known cause of DBA and account for more than 25% of patients. Mutations in other RPs (RPS24, RPS17, RPL35a) have been reported in a minority of patients (Gazda et al., 2006; Cmejla et al., 2007; Farrar et al., 2008) and show that DBA is a disorder of ribosome synthesis. Recently, Gadza et al. (2007) reported the involvement of two new genes (RPL5, RPL11) encoding for RPs of the large subunit in a considerable percentage of DBA patients without mutations in RPS19 or RPS24 (10% and 6,5%, respectively). Here we present the results of RPL11 mutation screening in Italian DBA patients without RPS19 or RPS24 mutations. Preliminary data of RPL5 analysis are also reported. The human RPL11 gene includes 6 exons and spans 4622 bp on chromosome 1. We identified 12 heterozygous mutations in 80 analyzed probands (15%), 10 of which were deletions of 1–47 nucleotides causing frameshift and a premature termination; another deletion (12nt) resulted in a loss of 4 aminoacids. One acceptor splice site defect (intron 5) was also detected. RPL11 mutations were spread in all exons except for exon 1. The human RPL5 gene spans 9888 bp on chromosome 1, and includes 8 exons. We identified one heterozygous mutation in RPL5 in 14 analyzed patients (7%); it is a single nucleotide deletion in exon 5 causing frameshift and a premature termination. None of these sequence changes were found on the NCBI SNP dalabase (http://www.ncbi.nlm.nih.gov/SNP/). Our mutations are expected to alter the genetic information drastically and to cause haploinsufficency. Depletion of RPs compromises ribosome biogenesis and disturbs rRNA processing at different levels (Robledo et al. 2008). Interestingly, all mutated patients (11/13) showed one or more somatic malformations; specifically, thumb anomalies and growth retardation were present in 7/11 (64%) and 6/11 (54%) patients, respectively. The erythrocyte adenosine deaminase activity was performed in 6/13 mutated patients; all of them showed an increased activity. The majority of RPL11 mutated patients were transfusion-dependent at last follow-up (58%,7/12); the RPL5 mutated patient was in clinical remission after steroid treatment. No one of the mutated patients has so far developed neoplasia. Our data show that RPL11 mutations are more frequent than expected (Gazda et al. 2007). They seem to confer a relevant association with a malformation phenotype as compared to RPS19 mutations.
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Pospisilova, Dagmar, Radek Cmejla, Jana Cmejlova, Helena Handrkova, Jan Stary, and Zdenka Cerna. "The Frequency and Characteristics of the Ribosomal Protein L5 (RPL5) and Ribosomal Protein L11 (RPL11) Gene Mutations in Patients with Diamond-Blackfan Anemia in the Czech National Registry." Blood 112, no. 11 (November 16, 2008): 3103. http://dx.doi.org/10.1182/blood.v112.11.3103.3103.
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Abstract Introduction: Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia that is usually diagnosed during early infancy. Apart from defects in red cell maturation, the disorder is also associated with various physical anomalies in 40% of patients. Mutations in the ribosomal protein (RP) S19 were found in 25% of patients, while mutations in other proteins of a small ribosomal subunit (RPS17 and RPS24) were published only in a small fraction of patients. Recently, mutations in RPL5, RPL11 and RPL35a of a large ribosomal subunit were also disclosed in several DBA patients. Results: The Czech DBA registry currently comprises 31 patients. Mutations in RPL5 were identified in 8/31 patients (26%), and mutations in RPL11 in 2/31 patients (6.5%), implying that mutations in RPL5 account for more Czech DBA cases than mutations in RPS19 (22.6%). As for the classification of mutations, all types were identified, including a nonsense mutation (in RPL11), point mutations (in RPL11 and RPL5), a supposed splicing defect and a small insertion and deletions (all in RPL5). Except for point mutations, all other changes were predicted to cause frameshift with premature stop codon. Since identified alterations were found neither in dbSNP nor in 52 healthy controls, and in two families mutations segregate with the disease, we conclude that they represent true DBA-causative mutations. Although the Czech DBA Registry is rather small, we performed a direct comparison of the group of patients with RPS19 mutations (n=7) with the group of patients with RPL5 mutations (n=8). No differences were found in sex ratio, steroid responsiveness, severity or course of the disease or the treatment outcome. However, patients with RPL5 mutations were generally born small for gestational age (SGA) compared with patients from the RPS19-mutated group. Only one patient (12.5%) with an RPL5 mutation was born with normal birth weight compared to four patients (57.1%) with RPS19 mutations. The second difference was even more striking: all patients with RPL5 mutations had flat thenar and some also an additional thumb anomaly, while no thumb anomalies were observed in patients with RPS19 mutations. It is questionable whether normal RPL5 function is in some way more important for proper thumb development than RPS19. Discussion: The identification of mutations in the genes in DBA patients is also interesting from another point of view. Both proteins RPL5 and RPL11 have been reported to be implicated in the activation of p53 through the interaction with the MDM2 protein, suppressing its E3 ubiquitin ligase function that otherwise directs p53 to a rapid degradation. It is noteworthy that yet another RP of a large ribosomal subunit was described, having exactly the same function – RPL23. Because no RPL23 mutations in our DBA patients were found, the primary function of RPL5 and RPL11 in ribosome biogenesis and/or translation underlies DBA phenotype rather than the conjoint role of RPL5, RPL11 and RPL23 in the p53 regulation. Conclusions: We identified 6 and 2 different mutations in the RPL5 and RPL11 genes, respectively, expanding the repertoire of known DBA-associated mutations. No mutations in the RPL23 were identified, suggesting that aberrant p53 activation due to mutations in RPL5 and RPL11 seems unlikely to be the primary cause of DBA. Patients with RPL5 mutations are more commonly born SGA and have higer frequency of thumb anomalies.
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Zhao, Xue-Li, and Zhang-Ming Zhu. "Comparative Genomics and Phylogenetic Analyses of Christia vespertilionis and Urariopsis brevissima in the Tribe Desmodieae (Fabaceae: Papilionoideae) Based on Complete Chloroplast Genomes." Plants 9, no. 9 (August 28, 2020): 1116. http://dx.doi.org/10.3390/plants9091116.
Full textAbstract:
Taxonomic and phylogenetic relationships of Christia, Urariopsis, Uraria and related genera within the tribe Desmodieae (Fabaceae: Papilionoideae) have long been controversial. Here, we report the complete chloroplast (cp) genomes of Christia vespertilionis and Urariopsis brevissima and perform comparative and phylogenetic analyses with Uraria lagopodioides and other relatives in the Desmodieae. The cp genomes of C. vespertilionis and U. brevissima are 149,656 and 149,930 bp long, with 128 unique genes (83 protein-coding genes, 37 tRNA genes and 8 rRNA genes), respectively. Comparative analyses revealed 95-129 simple sequence repeats (SSRs) and eleven highly variable regions (trnK-rbcL, rbcL-atpB, ndhJ-trnF, trnL-trnT, psbD-rpoB, accD-cemA, petA-psbL, psbE-petL, rps11-rps19, ndhF-ccsA, and rps15-ycf1) among six Desmodieae species. Phylogenetic analyses clearly resolved two subtribes (Desmodiinae and Lespedezinae) of Desmodieae as monophyletic, and the newly reported C. vespertilionis and U. brevissima clustered in subtribe Desmodiinae. A sister relationship of C. vespertilionis to U. lagopodioides was supported. Evidence was presented to support the treatment of Urariopsis as a distinct genus rather than in synonymy with Uraria. The results provide valuable information for further studies on species delimitation, phylogenetics, population genetics, and the evolutionary process of speciation in the Desmodieae.