Keisuke Hamada

De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy

Early infantile epileptic encephalopathy with suppression-burst (EIEE), also known as Ohtahara syndrome, is one of the most severe and earliest forms of epilepsy. Using array-based comparative genomic hybridization, we found a de novo 2.0-Mb microdeletion at 9q33.3–q34.11 in a girl with EIEE. Mutation analysis of candidate genes mapped to the deletion revealed that four unrelated individuals with EIEE had heterozygous missense mutations in the gene encoding syntaxin binding protein 1 (STXBP1). STXBP1 (also known as MUNC18-1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein that is essential in synaptic vesicle release in several species. Circular dichroism melting experiments revealed that a mutant form of the protein was significantly thermolabile compared to wild type. Furthermore, binding of the mutant protein to syntaxin was impaired. These findings suggest that haploinsufficiency of STXBP1 causes EIEE.

Dominant-Negative Mutations in α-II Spectrin Cause West Syndrome with Severe Cerebral Hypomyelination, Spastic Quadriplegia, and Developmental Delay

A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding alpha-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the alpha/beta spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) alpha-II spectrin could assemble heterodimers with beta-II spectrin, but alpha-II (mut)/beta-II spectrin heterodimers were thermolabile compared with the alpha-II (WT)/beta-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of alpha-II (mut)/beta-II and alpha-II (mut)/beta-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of alpha/beta spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy.

Mutations in POLR3A and POLR3B Encoding RNA Polymerase III Subunits Cause an Autosomal-Recessive Hypomyelinating Leukoencephalopathy

Congenital hypomyelinating disorders are a heterogeneous group of inherited leukoencephalopathies characterized by abnormal myelin formation. We have recently reported a hypomyelinating syndrome characterized by diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC). We performed whole-exome sequencing of three unrelated individuals with HCAHC and identified compound heterozygous mutations in POLR3B in two individuals. The mutations include a nonsense mutation, a splice-site mutation, and two missense mutations at evolutionally conserved amino acids. Using reverse transcription-PCR and sequencing, we demonstrated that the splice-site mutation caused deletion of exon 18 from POLR3B mRNA and that the transcript harboring the nonsense mutation underwent nonsense-mediated mRNA decay. We also identified compound heterozygous missense mutations in POLR3A in the remaining individual. POLR3A and POLR3B encode the largest and second largest subunits of RNA Polymerase III (Pol III), RPC1 and RPC2, respectively. RPC1 and RPC2 together form the active center of the polymerase and contribute to the catalytic activity of the polymerase. Pol III is involved in the transcription of small noncoding RNAs, such as 5S ribosomal RNA and all transfer RNAs (tRNA). We hypothesize that perturbation of Pol III target transcription, especially of tRNAs, could be a common pathological mechanism underlying POLR3A and POLR3B mutations.

Missense mutations in the DNA-binding/dimerization domain of NFIX cause Sotos-like features

Sotos syndrome is characterized by prenatal and postnatal overgrowth, characteristic craniofacial features and mental retardation. Haploinsufficiency of NSD1 causes Sotos syndrome. Recently, two microdeletions encompassing Nuclear Factor I-X (NFIX) and a nonsense mutation in NFIX have been found in three individuals with Sotos-like overgrowth features, suggesting possible involvements of NFIX abnormalities in Sotos-like features. Interestingly, seven frameshift and two splice site mutations in NFIX have also been found in nine individuals with Marshall–Smith syndrome. In this study, 48 individuals who were suspected as Sotos syndrome but showing no NSD1 abnormalities were examined for NFIX mutations by high-resolution melt analysis. We identified two heterozygous missense mutations in the DNA-binding/dimerization domain of the NFIX protein. Both mutations occurred at evolutionally conserved amino acids. The c.179T>C (p.Leu60Pro) mutation occurred de novo and the c.362G>C (p.Arg121Pro) mutation was inherited from possibly affected mother. Both mutations were absent in 250 healthy Japanese controls. Our study revealed that missense mutations in NFIX were able to cause Sotos-like features. Mutations in DNA-binding/dimerization domain of NFIX protein also suggest that the transcriptional regulation is abnormally fluctuated because of NFIX abnormalities. In individuals with Sotos-like features unrelated to NSD1 changes, genetic testing of NFIX should be considered.

The 8th and 9th tandem spectrin-like repeats of utrophin cooperatively form a functional unit to interact with polarity-regulating kinase PAR-1b.

Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor, dystroglycan (DG). The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal actin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polarity-regulating kinase, PAR-1b, interacts with the utrophin-DG complex, and positively regulates the interaction between utrophin and DG. In this study, we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b. Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin-DG complex. Interestingly, PAR-1b also binds and phosphorylates R8-9 of dystrophin, and colocalizes with dystrophin at the skeletal muscle membrane. These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain.

A novel allosteric mechanism on protein-DNA interactions underlying the phosphorylation-dependent regulation of Ets1 target gene expressions.

Cooperative assemblies of transcription factors (TFs) on target gene enhancers coordinate cell proliferation, fate specification, and differentiation through precise and complicated transcriptional mechanisms. Chemical modifications, such as phosphorylation, of TFs induced by cell signaling further modulate the dynamic cooperativity of TFs. In this study, we found that various Ets1-containing TF-DNA complexes respond differently to calcium-induced phosphorylation of Ets1, which is known to inhibit Ets1-DNA binding. Crystallographic analysis of a complex comprising Ets1, Runx1, and CBFβ at the TCRα enhancer revealed that Ets1 acquires robust binding stability in the Runx1 and DNA-complexed state, via allosteric mechanisms. This allows phosphorylated Ets1 to be retained at the TCRα enhancer with Runx1, in contrast to other Ets1 target gene enhancers including mb-1 and stromelysin-1. This study provides a structure-based model for cell-signaling-dependent regulation of target genes, mediated via chemical modification of TFs.

Rational Approach to the Synthesis, Evaluation, and 68Ga Labeling of a Novel 4-Anilinoquinoline Epidermal Growth Factor Receptor Inhibitor as a New Imaging Agent That Selectively Targets the Epidermal Growth Factor Receptor Tyrosine Kinase

Certain small-molecule inhibitors that target epidermal growth factor receptor (EGFR), such as Gefitinib, Erlotinib, and Lapatinib, provide a new approach for cancer treatment. In accordance with the pharmacophore model for inhibitor competition at EGFR-binding site, this study proposes a rationalized design for a novel 4-anilinoquinoline EGFR tyrosine kinase inhibitor, [6,7-dimethoxyethoxy]-quinolin-4-yl]-(3-ethynylphenyl)-amine (YCU07). This is the first study to apply ring-closing metathesis toward synthesis of the quinoline nucleus for this 4-anilinoquinoline EGFR inhibitor. YCU07 expressed significant inhibitory activity for EGFR tyrosine kinase in A431 cells, as confirmed by an ABTS microwell peroxidase substrate system read colorimetrically at 405 nm. Injection of (68)Ga-labeled glutamic acid polypeptide (GAP)-YCU07 conjugate in nude mice implanted with A431 was imaged by animal PET camera (LabPET8; Gamma Medica-Ideas) and computed tomography (eXplore Locus; GE Healthcare), to evaluate its biodistribution. (68)Ga-GAP-YCU accumulated in the receptor-positive tumors, with uptake values of 1.50% +/- 0.09% and 2.36% +/- 0.36% of injected activity per gram tissue at 30 and 90 minutes, respectively.

Pseudohomozygous type II hyperlipoproteinemia.

SummaryA 12-month-old boy with clinical findings suggestive of homozygous familial hypercholesterolemia, who had no secondary causes of hypercholesterolemia, and whose parents had no lipid abnormalities, was reported. No abnormalities were noted in the low density lipoprotein (LDL) receptor activities of the fibroblasts from patient, parents and sibling.These features, combined with extreme responsiveness to available therapy, are strikingly suggestive of a new clinical hypercholesterolemic syndrome with manifestations resembling homozygous familial hypercholesterolemia.

Puretic syndrome-gingival fibromatosis with hyaline fibromas

SummaryA 21-month-old Japanese boy with Puretić syndrome (gingival fibromatosis with hyaline fibromas) was reported. On the basis of the present case and 17 cases in the literature, it was concluded that the disorder was a very rare connective tissue disease with an autosomal recessive mode of inheritance.

Crystallization of the Ets1-Runx1-CBFβ-DNA complex formed on the TCRα gene enhancer.

Gene transcription is regulated in part through the assembly of multiple transcription factors (TFs) on gene enhancers. To enable examination of the mechanism underlying the formation of these complexes and their response to a phosphorylation signal, two kinds of higher-order TF-DNA assemblies were crystallized composed of an unmodified or phosphorylated Ets1 fragment, a Runx1(L94K) fragment and a CBFβ fragment on the T-cell receptor (TCR) α gene enhancer. Within these complexes, the Ets1 and Runx1 fragments contain intrinsically disordered regulatory regions as well as their DNA-binding domains. Crystals of the complex containing unmodified Ets1 belonged to space group P212121, with unit-cell parameters a = 78.7, b = 102.1, c = 195.0 Å, and diffracted X-rays to a resolution of 2.35 Å, and those containing phosphorylated Ets1 belonged to the same space group, with unit-cell parameters a = 78.6, b = 101.7, c = 194.7 Å, and diffracted X-rays to a similar resolution. To facilitate crystallization, a Runx1 residue involved in a hydrophobic patch that was predicted to be engaged in crystal packing based on the previously reported structures of Runx1-containing crystals was mutated.