Shin’ichiro Yasunaga

A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness

Using a candidate gene approach, we identified a novel human gene, OTOF, underlying an autosomal recessive, nonsyndromic prelingual deafness, DFNB9. The same nonsense mutation was detected in four unrelated affected families of Lebanese origin. OTOF is the second member of a mammalian gene family related to Caenorhabditis elegans fer-1. It encodes a predicted cytosolic protein (of 1,230 aa) with three C2 domains and a single carboxy-terminal transmembrane domain. The sequence homologies and predicted structure of otoferlin, the protein encoded by OTOF, suggest its involvement in vesicle membrane fusion. In the inner ear, the expression of the orthologous mouse gene, mainly in the sensory hair cells, indicates that such a role could apply to synaptic vesicles.

OTOF Encodes Multiple Long and Short Isoforms: Genetic Evidence That the Long Ones Underlie Recessive Deafness DFNB9

We have recently reported that OTOF underlies an autosomal recessive form of prelingual sensorineural deafness, DFNB9. The isolated 5-kb cDNA predicted a 1,230 amino acid (aa) C-terminus membrane-anchored cytosolic protein with three C2 domains. This protein belongs to a family of mammalian proteins sharing homology with the Caenorhabditis elegans fer-1. The two other known members of this family, dysferlin and myoferlin, both have six predicted C2 domains. By northern blot analysis, a 7-kb otoferlin mRNA could be detected in the human brain. We isolated the corresponding cDNA, which is expected to encode a 1,977-aa-long form of otoferlin with six C2 domains. A 7-kb cDNA derived from the murine orthologous gene, Otof, was also identified in the inner ear and the brain. The determination of the exon-intron structure of the human and murine genes showed that they are composed of 48 coding exons and extend approximately 90 kb and approximately 80 kb, respectively. Alternatively spliced transcripts could be detected that predict several long isoforms (six C2 domains) in humans and mice and short isoforms (three C2 domains) only in humans. Primers were designed to explore the first 19 OTOF exons, henceforth permitting exploration of the complete coding sequence of the gene in DFNB9 patients. In a southwestern Indian family affected by DFNB9, a mutation in the acceptor splice site of intron 8 was detected, which demonstrates that the long otoferlin isoforms are required for inner ear function.

Expression of Polycomb-group (PcG) protein BMI-1 predicts prognosis in patients with acute myeloid leukemia.

Expression of Polycomb-group (PcG) protein BMI-1 predicts prognosis in patients with acute myeloid leukemia

IL-4 and IL-13: Their Pathological Roles in Allergic Diseases and their Potential in Developing New Therapies

The incidence of allergic diseases has dramatically increased in recent decades, and it is socially and medically important to establish more useful strategies to overcome allergic disorders. Various kinds of drugs are utilized for allergic patients; however, some cases are unresponsive to these drugs and in others there are undesired adverse effects. On the other hand, a substantial body of evidence has accumulated pointing to the pivotal role of Th2-cytokines, interleukin (IL)-4, and IL-13, in the pathogenesis of bronchial asthma. The evidence is categorized as (1) expression of these cytokines in the bronchial lesions, (2) genetic association of the signaling molecules of these cytokines, (3) analyses of mouse models. In addition, the molecular mechanism of the signal transduction of these cytokines has also been well characterized. Based on such information, IL-4 and IL-13 have emerged as promising means of improving allergic states, and several IL-4/IL-13 antagonists have been developed, among which soluble IL-4 receptor is now in human trials. Identifying the structure of the IL-13 variant and of the IL-4/IL-13-inducing genes would be of great use. It is expected that in the near future, several drugs will emerge based on these strategies, which will give us wider choice in treating patients, depending on the pathogenesis of the diseases.

Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity

Polycomb-group (PcG) genes encode multimeric nuclear protein complexes, PcG complex 1 and 2. PcG complex 2 was proved to induce transcription repression and to further methylate histone H3 at lysine-27 (H3K27). Subsequently PcG complex 1 is recruited through recognition of methylated H3K27 and maintains the transcription silencing by mediating monoubiquitination of histone H2A at lysine-119. Genetic evidence demonstrated a crucial role for PcG complex 1 in stem cells, and Bmi1, a member of PcG complex 1, was shown to sustain adult stem cells through direct repression of the INK4a locus encoding cyclin-dependent kinase inhibitor, p16CKI, and p19ARF. The molecular functions of PcG complex 1, however, remain insufficiently understood. In our study, deficiency of Rae28, a member of PcG complex 1, was found to impair ubiquitin-proteasome-mediated degradation of Geminin, an inhibitor of DNA replication licensing factor Cdt1, and to increase protein stability. The resultant accumulation of Geminin, based on evidence from retroviral transduction experiments, presumably eliminated hematopoietic stem cell activity in Rae28-deficient mice. Rae28 mediates recruiting Scmh1, which provides PcG complex 1 an interaction domain for Geminin. Moreover, PcG complex 1 acts as the E3 ubiquitin ligase for Geminin, as we demonstrated in vivo as well as in vitro by using purified recombinant PcG complex 1 reconstituted in insect cells. Our findings suggest that PcG complex 1 supports the activity of hematopoietic stem cells, in which high-level Geminin expression induces quiescence securing genome stability, by enhancing cycling capability and hematopoietic activity through direct regulation of Geminin.

Neurodevelopmental abnormalities associated with severe congenital neutropenia due to the R86X mutation in the HAX1 gene

Objective: Severe congenital neutropenia (SCN), also known as Kostmann syndrome (SCN3, OMIM 610738), includes a variety of haematological disorders caused by different genetic abnormalities. Mutations in ELA2 are most often the cause in autosomal dominant or sporadic forms. Recently, mutations in HAX1 have been identified as the cause of some autosomal recessive forms of SCN, including those present in the original pedigree first reported by Kostmann. We sought to determine the relationship between HAX1 gene mutations and the clinical characteristics of Japanese cases of SCN. Methods: The genes implicated in SCN (ELA2, HAX1, Gfi-1, WAS, and P14) were analysed in 18 Japanese patients with SCN. The clinical features of these patients were obtained from medical records. Immunoblotting of HAX1 was performed on cell extracts from peripheral blood leucocytes from patients and/or their parents. Results: We found five patients with HAX1 deficiency and 11 patients with mutations in the ELA2 gene. In HAX1 deficiency, a homozygous single base pair substitution (256C>T), which causes the nonsense change R86X, was identified in three affected individuals. Two sibling patients showed a compound heterozygous mutation consisting of a single base pair substitution (256C>T) and a 59 bp deletion at nucleotides 376–434. There was no detectable phenotype in any heterozygous carrier. All patients with HAX1 deficiency had experienced developmental delay. Three patients carrying R86X also suffered from epileptic seizures. In contrast, no SCN patient with heterozygous mutations in the ELA2 gene suffered from any neurodevelopmental abnormality. Conclusions: These findings suggest that the R86X mutation in the HAX1 gene is an abnormality in Japanese SCN patients with HAX1 deficiency and may lead to neurodevelopmental abnormalities and severe myelopoietic defects.

BMI-1 expression is enhanced through transcriptional and posttranscriptional regulation during the progression of chronic myeloid leukemia.

BMI-1 plays a critical role in regulating the activity of hematopoietic stem and progenitor cells. Patients with chronic myeloid leukemia (CML) are at a risk of developing blastic crisis (BC) even after the emergence of imatinib mesylate. In this study, to determine the relevance of BMI-1 to BC, we investigated the expression of BMI-1 in CD34+ cells at each of the chronic phase (CP), the accelerated phase (AP), and BC by flow cytometry. Interestingly, the level of BMI-1 expression was significantly higher in CP than in controls and was further increased during the course of the disease progression (control—5.66%; CP—36.93%; AP and BC—76.41%). Curiously, mRNA levels for BMI-1 were almost consistent during the disease progression from CP to BC (control—2.21; CP—9.77; AP and BC—9.70 (BMI-1/glyceraldehyde-3-phosphate dehydrogenase ratio)). Since we further found that overexpression of BCR–ABL in human embryonic kidney-293 cells enhanced BMI-1 expression and that BMI-1 expression was increased in K562 cells, derived from patients with BC, in the presence of proteasomal inhibitors, BMI-1 was presumed to be positively regulated by BCR–ABL and further by posttranscriptional modification in the course of the disease progression. We suggest the usefulness of BMI-1 expression in CD34+ cells as a molecular marker for monitoring patients with CML.

Extensive polymorphism of a (CA)n microsatellite located in the HLA-DQA1/DQB1 class II region.

A highly polymorphic (CA)n microsatellite marker (DQCAR), located between the DQA1 and the DQB1 genes, was characterized in four ethnic groups. Based on length polymorphism, 12 alleles could be defined. The marker is located 1- to 2-kb telomeric to the DQB1 gene and 10 kb centromeric to the DQA1 gene and was shown to be in tight linkage disequilibrium with HLA-DQ. Analysis of the linkage disequilibrium pattern revealed little additional diversity in DQ1-associated haplotypes. Almost all DQ1 subjects examined were DQCAR 103 or DQCAR 107 (13 and 15 CA repeats, respectively). In contrast, significant haplotypic diversity was observed for most DQ2-, DQ3-, and DQ4-associated haplotypes. These haplotypes often had longer allele sizes (DQCAR > 111, more than 17 CA repeats) and more DQCAR alleles per haplotype. These haplotypes also carried DQCAR alleles of different sizes, even though they bore the same DQA1 and DQB1 alleles, and sometimes the same DRB1 allele as well. These results indicate that DQCAR could be a useful marker to better define disease associations with HLA. Our results are also consistent with the hypothesis that CAR alleles with higher numbers of repeats have higher mutation rates and that recombination within the HLA-DR/DQ region is haplotype dependent.

Poly-ADP Ribosylation of Miki by tankyrase-1 Promotes Centrosome Maturation

During prometaphase, dense microtubule nucleation sites at centrosomes form robust spindles that align chromosomes promptly. Failure of centrosome maturation leaves chromosomes scattered, as seen routinely in cancer cells, including myelodysplastic syndrome (MDS). We previously reported that the Miki (LOC253012) gene is frequently deleted in MDS patients, and that low levels of Miki are associated with abnormal mitosis. Here we demonstrate that Miki localizes to the Golgi apparatus and is poly(ADP-ribosyl)ated by tankyrase-1 during late G2 and prophase. PARsylated Miki then translocates to mitotic centrosomes and anchors CG-NAP, a large scaffold protein of the γ-tubulin ring complex. Due to impairment of microtubule aster formation, cells in which tankyrase-1, Miki, or CG-NAP expression is downregulated all show prometaphase disturbances, including scattered and lagging chromosomes. Our data suggest that PARsylation of Miki by tankyrase-1 is a key initial event promoting prometaphase.

Synergistic and persistent effect of T-cell immunotherapy with anti-CD19 or anti-CD38 chimeric receptor in conjunction with rituximab on B-cell non-Hodgkin lymphoma

Using artificial receptors, it is possible to redirect the specificity of immune cells to tumour‐associated antigens, which is expected to provide a useful strategy for cancer immunotherapy. Given that B‐cell non‐Hodgkin lymphoma (B‐NHL) cells invariably express CD19 and CD38, these antigens may be suitable molecular candidates for such immunotherapy. We transduced human peripheral T cells or a T‐cell line with either anti‐CD19‐chimeric receptor (CAR) or anti‐CD38‐CAR, which contained an anti‐CD19 or anti‐CD38 antibody‐derived single‐chain variable domain respectively. Retroviral transduction led to anti‐CD19‐CAR or anti‐CD38‐CAR expression in T cells with high efficiency (>60%). The T cell line, Hut78, when transduced with anti‐CD19‐CAR or anti‐CD38‐CAR, exerted strong cytotoxicity against the B‐NHL cell lines, HT and RL, and lymphoma cells isolated from patients. Interestingly, use of both CARs had an additive cytotoxic effect on HT cells in vitro. In conjunction with rituximab, human peripheral T cells expressing either anti‐CD19‐CAR or anti‐CD38‐CAR enhanced cytotoxicity against HT‐luciferase cells in xenografted mice. Moreover, the synergistic tumour‐suppressing activity was persistent in vivo for over 2 months. These results provide a powerful rationale for clinical testing of the combination of rituximab with autologous T cells carrying either CAR on aggressive or relapsed B‐NHLs.