Mutations in the ribosomal protein genes in Japanese patients with Diamond-Blackfan anemia.

BACKGROUND: Diamond-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 METHODS: We 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. RESULTS: Mutations 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. CONCLUSIONS: We 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.

Transgenic expression of BACH1 transcription factor results in megakaryocytic impairment.

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 musculoaponeurotic fibrosarcoma oncogene (Maf) recognition element (MARE). MARE is known to act as a critical cis-regulatory element of erythroid 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 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 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 transcriptional repressor in the regulation of MARE-dependent genes in megakaryocytes.

B-cell-specific transcription factor BACH2 modifies the cytotoxic effects of anticancer drugs.

The transcription factor Bach2, a member of the CNC family of proteins, binds to the Maf recognition element (MARE) by forming homodimers or dimerizing with small Maf transcription factors. Bach2-expressing cells show reduced proliferation and undergo spontaneous cell death. The inhibition of BCR/ABL tyrosine kinase activity by STI571 in chronic myeloid leukemia (CML) cell lines and CD34+ cells from patients with CML in lymphoid crisis results in induction of BACH2 expression. We show here that BACH2 modifies the in vitro cytotoxicity of anticancer drugs. The cytotoxic effects of commonly used anticancer agents were studied by overexpression of BACH2 in RAJI lymphoid cells, a cell line that does not express endogenous BACH2. Cell growth inhibition was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Clones overexpressing BACH2 were more sensitive to etoposide, doxorubicin, and cytarabine than control RAJI cells, whereas there were no significant differences in the sensitivity of either cells to methotrexate or vincristine. Interestingly, we found that the former drugs were oxidative stressors that induced the nuclear accumulation of BACH2. In contrast, methotrexate or vincristine did not induce production of intracellular reactive oxygen species (ROS) and nuclear accumulation of BACH2. These results, coupled with our previous data showing that BACH2 promotes oxidative stress-induced cell death, suggest that combination chemotherapy involving STI571 and anticancer drugs that produce ROS may be of benefit in the treatment of Philadelphia chromosome 1 (Ph1)-positive leukemia.