Recombinant DNA technology is an outcome of molecular biology and has revolutionized any field in biology and medicine since its emergence in early 70s. I (M. Yamada) have been engaged in the spread and wide application of the techniques to the medical field since I arrived at my position in National Children’s Medical Research Center in 1985, and continue to do after reformation of our research organization to National Research Institute for Child Health and Development.
In our department, we are studying on the structure and function of genes to elucidate the pathogenesis mechanism of genetic disorders and childhood cancers. Our Department is proud to be a pioneer to identify causative mutations and genes for three disorders: the amelogenin gene (AMG) for X-linked amelogenesis imperfecta, trinucleotide repeat expansion for dentatorubral-pallidoluysian atrophy (DRPLA), and a unique misssense mutation of PAX6 for isolated foveal hypoplasia. DRPLA is a hereditary neurodegenerative disorder, and a little more common in Japan but very rare in Western countries. We found CAG repeat expansion in DRPLA and then isolated the responsible gene in 1994. DRPLA is now recognized as a polyglutamine disease, together with Huntington's disease and spinocerebellar ataxia, and we are currently studying on the cell death mechanism in relation with apoptosis, and the normal function of the DRPLA product to aim to elucidate pathogenesis in the specific area of the brain. The PAX6 gene encodes a transcription factor with DNA binding through its paired box domain, and is well known to control the eye morphogenesis since its isolation as a gene responsible for aniridia (lack of the iris) in 1991. We looked for PAX6 mutations in genomic DNA isolated from aniridia patients and extended such screening to a variety type of eye anomalies. A missense mutation occurring in the C-terminal portion of the paired domain was detected in a family with isolated foveal hypoplasia. This was the first mutation detected in the C-terminal portion of the paired domain throughout any member of the paired family of all the species, and this finding objected a previous concept that the C-terminal portion of the paired domain was dispensable. We have also proposed that the N-terminal portion regulates genes involving in morphogenesis of the anterior segments while the C-terminal portion regulates genes for the retina layer. This is an example of success in "candidate disease approach" we proposed.
1) the structure and function of human genes for
elucidation of genetic diseases
1-1) the DRPLA gene, a trinucleotide repeat
disorder
1-2) subtle alternative splicing
1-3) PAX6 and eye malformation
1-4)
DNA polymorphism
1-5) interaction of transcription factors
2) Investigation of
apoptosis regulation
2-1) Identification of glucocorticoid target genes
2-2)
Basic study on caspases 3) Analysis of PTCH, the responsible gene for nevoid
basal cell carcinoma syndrome
Currently, our Department is consisted with the following members:
Masao Yamada, Director, Ph. D.
Toshiyuki Miyashita, Head of Laboratory, M. D., Ph. D.
Keiko Tadokoro, Staff, Ph. D.
Masashi Toyoda, Post-Doctoral fellow, Ph. D.
Kazuaki Nagao, Associate
In addition, we have many persons in our laboratory, who are engaged in medical practice in the NCCHD hospital as well as outside hospitals, graduate students and undergraduates of universities, and visitors from other organizations together with technicians and supporting staffs in our Department.
Dentatorubral pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by selective neuron loss in the cerebellar and pallidal outflow pathways. Patients show a combination of ataxia and extrapyramidal signs (chorea, athetosis) to varying degrees. We detected expansion of CAG repeats on chromosome 12p13 in the patients (Nagafuchi et al. Nature Genetics, 6:14-18, 1994), and then determined the entire cDNA sequence of the gene (Nagafuchi et al. Nature Genetics, 8:177-182, 1994). Several other neurodegenerative disorders including Huntington disease have been shown to be caused by expansion of CAG repeats in the coding region of respective genes. As the CAG unit is situated in the translational frame and encodes glutamine, these disorders are collectively called as polyglutamine diseases. As repeat expansion is a novel type of mutations associated with a subset of disorders and shows unique features, it attracts a broad attention. The molecular mechanism underlying cell death has been demonstrated as induction of apoptosis at the final step, and several processes have been proposed for the route where the cohesive force of the polyglutamine tract is central. In contrast, it is still uncertain how and why a specific subset of neurons are degenerated in respective polyglutamine diseases. To address this issue, we have been studying on the normal functions of the DRPLA gene and its product.
After our sequence report on DRPLA cDNA, other laboratories also reported cDNA sequences for the human DRPLA gene and orthologues. Besides of the number of iteration of the CAG repeats, which is polymorphic and expandable, some other sequence lacked 3 nt occurring in other sequences, which resulted in the absence of the single glutamine residue at 94 in the DRPLA protein. When these sequences appeared in the public database, we originally thought that such tiny discrepancies may caused by sequencing and human errors. However, we gradually considered the possibility of alternative splicing, as the inconsistent 3 nt was situated at the boundary of exon 4 and 5. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. In DRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus.
Based on these facts, we searched databases and revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR, and found interesting features associated with this phenomena. Inclusion of 3 nt resulted in inclusion of a single amino acid residue for most cases despite of the phase of translational frame. The expression ratio of the included to excluded form sometimes drastically altered, indicating that a splice control system operates even in the narrow span rather than reflection of infidelity in splice machinery.
Our cells have a suicide mechanism that instructs cells when it is time to die. Unfortunately, defects in the regulation of this cell suicide program can occur, leading to diseases characterized by either too much cell death (stroke, neurodegenerative disorders, AIDS etc.) or too little cell death (cancer, auto-immunity etc.). In fact, it is estimated that over half of the major medical illnesses for which effective treatments or preventions are currently lacking can be attributed directly or indirectly to defective regulation of programmed cell death mechanisms.
2-1) Identification of glucocorticoid target genes Glucocorticoid receptor (GR) is a member of the nuclear hormone receptor superfamily. Its ligands, glucocorticoids (GCs), induce cell cycle arrest and cell death via apoptosis in normal thymocytes, immature T cells and many leukemic cells. Therefore, GCs are essential therapeutic agents for many types of leukemias, lymphomas and autoimmune and allergic disorders. Despite their importance, the mechanisms underlying the clinical effects of GCs are poorly understood. We identified 93 genes that were induced by GC using oligonucleotide microarrays. Some of them were direct transcriptional targets of GR and functional glucocorticoid response elements were identified in upper intronic regions. These are predicted to be clinically important genes since they potentially mediate GC-induced apoptosis in leukemic cells.
2-2) Basic study on caspases Caspases belong to a family of cysteine proteases that executes apoptotic cell death. NF-kB regulates the expression of various genes involved in cell growth and differentiation, immune response and inhibition of apoptosis. Recently, some death effector domain (DED)-containing proteins, such as FADD and c-FLIP were reported to activate NF-kB. We previously reported that the prodomain-only isoforms of caspase-8 and -10 (PDCasp8/10) containing two DEDs could inhibit Fas-mediated apoptosis. During these two years, we demonstrated these isoforms also activate NF-kB, implying this to be one of the mechanisms by which these polypeptides inhibit apoptosis. Among upstream kinases that activate NF-kB, NIK and RIP, but not RICK or IKKb could directly bind to PDCasp8/10. In addition, both modules of DED in PDCasp8/10 were required for these interactions as well as NF-kB activation. We also found that caspase-8 and -10 have roles in a non- or anti-apoptotic signaling pathway leading to NF-kB activation through RIP, NIK and IKKb by using dominant negative mutants and small interfering RNAs.
(NF-kB = NF- kappa- B, IKKb = IKK-beta, if you see a curious font, )
The Patched (PTCH) gene, which encodes one component of the Shh receptor, is responsible for the inherited cancer predisposition disorder known as Gorlin’s or nevoid basal cell carcinoma syndrome (NBCCS). We have been investigating PTCH mutations in NBCCS patients and have detected mutations in 13 out of 17 cases analyzed so far. This study greatly increased the detection rate of mutations in NBCCS. During these two years, we identified 7 and 5 isoforms of human and mouse PTCH mRNA, respectively, which are generated by the complex alternative use of 5’ exons as the first exon, most of which had not been reported before. We investigated spatial and temporal expression profiles of these isoforms. These mRNA isoforms encode four protein variants that have distinct amino-termini. Among these, the one lacking first transmembrane domain was found to be significantly unstable compared with others. This study may shed light on the mechanism whereby a single PTCH gene plays a role in both tumor cell growth and embryonic development.
Masao Yamada, Ph. D. (Director, Department of Genetics)
From Annual Report published in 2004/5
Department of Genetics, National Research Institute for Child Health and Development
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