BackgroundDiamond-Blackfan anemia is a rare, clinically heterogeneous, congenital red cell aplasia: 40% of patients have congenital abnormalities. Recent studies have shown that in western countries, the disease is associated with heterozygous mutations in the ribosomal protein (RP) genes in about 50% of patients. There have been no studies to determine the incidence of these mutations in Asian patients with Diamond-Blackfan anemia. Design and MethodsWe screened 49 Japanese patients with Diamond-Blackfan anemia (45 probands) for mutations in the six known genes associated with Diamond-Blackfan anemia: RPS19, RPS24, RPS17, RPL5, RPL11, and RPL35A. RPS14 was also examined due to its implied involvement in 5q-syndrome. ResultsMutations in RPS19, RPL5, RPL11 and RPS17 were identified in five, four, two and one of the probands, respectively. In total, 12 (27%) of the Japanese Diamond-Blackfan anemia patients had mutations in ribosomal protein genes. No mutations were detected in RPS14, RPS24 or RPL35A. All patients with RPS19 and RPL5 mutations had physical abnormalities. Remarkably, cleft palate was seen in two patients with RPL5 mutations, and thumb anomalies were seen in six patients with an RPS19 or RPL5 mutation. In contrast, a small-for-date phenotype was seen in five patients without an RPL5 mutation. ConclusionsWe observed a slightly lower frequency of mutations in the ribosomal protein genes in patients with Diamond-Blackfan anemia compared to the frequency reported in western countries. Genotype-phenotype data suggest an association between anomalies and RPS19 mutations, and a negative association between small-for-date phenotype and RPL5 mutations.Key words: protein genes, Diamond-Blackfan anemia, RPL5 mutation. DiamondBlackfan anemia. Haematologica 2010;95(8):1293-1299. doi:10.3324/haematol.2009 This is an open-access paper. Citation: Konno Y, Toki T, Tandai S, Xu G, Wang RN, Terui K, Ohga S, Hara T, Hama A, Kojima S, Hasegawa D, Kosaka Y, Yanagisawa R, Koike K, Kanai R, Imai T, Hongo T, Park M-J, Sugita K, and Ito E. Mutations in the ribosomal protein genes in Japanese patients with Mutations in the ribosomal protein genes in Japanese patients with Diamond-Blackfan anemia

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Both nuclear factor erythroid 2 45 kDa subunit (p45) and BTB and CNC homolog 1 (Bach) transcription factors can form dimers with one of the small Maf proteins, and these heterodimers bind to the musculoaponeu-rotic fibrosarcoma oncogene (Maf) recognition element (MARE). MARE is known to act as a critical cis-regulatory element of ery-throid and megakaryocytic genes. Although detailed analyses of p45-null mutant mice and small maf compound mutant mice revealed that these factors are both critical for platelet production, the functional contributions of Bach1 and the relationship or redundancy between Bach1 and p45 in megakaryocytes remain to be clarified. To address these issues, we generated trans-genic lines of mice bearing human BACH1 cDNA under the control of the GATA-1 locus hematopoietic regulatory domain. The trans-genic mouse lines showed significant throm-bocytopenia associated with impaired matu-ration of the megakaryocytes, and they developed myelofibrosis. The megakaryo-cytes in the transgenic mice exhibited reduced proplatelet formation, and the modal ploidy class of megakaryocytes was 2N, indicating the impairment of endomitosis. Transcription of the p45 target genes was down-regulated and we indeed found that BACH1 binds to the thromboxane synthase gene, one of the target genes for p45 in megakaryocytes. These findings thus provide evidence that BACH1 acts as a tran-scriptional repressor in the regulation of MARE-dependent genes in megakaryocytes. (Blood. 2005;105:3100-3108)

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Both p45 and BACH transcription factors can form dimers with one of the small Maf proteins, and these heterodimers bind to Maf recognition elements (MARE). MARE is known to act as a critical cis-regulatory element of erythroid and megakaryocytic genes. While detailed analyses of p45 -null mutant mice and small maf compound mutant mice revealed that these factors are both critical for platelet production, the functional contributions of BACH1 and the relationship or redundancy between BACH1 and p45 in megakaryocytes remain to be clarified. To address these issues, we generated transgenic lines of mice bearing human BACH1 cDNA under the control of the GATA-1 locus hematopoietic regulatory domain. The transgenic mouse lines showed significant thrombocytopenia associated with impaired maturation of the megakaryocytes, and they developed myelofibrosis. The megakaryocytes overexpressing the BACH1 transgene exhibited reduced proplatelet formation. Since the phenotype of the BACH1 transgenic mice resembled that of the p45 -deficient mice, we examined the expression of the p45 NF-E2 target genes in the primary megakaryocytes from fetal liver cells of the BACH1 transgenic mice. RT-PCR analyses showed that expression of the hematopoietic-specific ß1-tubulin, thromboxane synthase ( TXAS), and of the 3ß-hydroxy-steroid dehydrogenase genes was significantly downregulated in the megakaryocytes from BACH1 transgenic mice. The TXAS gene is a well-known MARE-dependent gene containing functional MAREs in its promoter and in the second intron. To ask whether BACH1 actually binds to MARE in the megakaryocytic genes, we then performed chromatin immunoprecipitation (ChIP) analysis with a BACH1-specific antibody. A ChIP assay with a human megakaryocytic cell line, UT-7/TPO, demonstrated that BACH1 bound to the promoter and enhancers region in vivo. As expected, co-transfection with BACH1 or Bach1-MafK fusion protein (B1K) expression plasmids repressed the reporter gene activity driven by the TXAS promoter. These findings thus provide evidence that BACH1 acts as a transcriptional repressor in the regulation of MARE-dependent genes in megakaryocytes.

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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.