Shin-ichi Horike

15q11.2–13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain

Copy number variations (CNVs) within human 15q11.2-13.3 show reduced penetrance and variable expressivity in a range of neurologic disorders. Therefore, characterizing 15q11.2-13.3 chromatin structure is important for understanding the regulation of this locus during normal neuronal development. Deletion of the Prader-Willi imprinting center (PWS-IC) within 15q11.2-13.3 disrupts long-range imprinted gene expression resulting in Prader-Willi syndrome. Previous results establish that MeCP2 binds to the PWS-IC and is required for optimal expression of distal GABRB3 and UBE3A. To examine the hypothesis that MeCP2 facilitates 15q11.2-13.3 transcription by linking the PWS-IC with distant elements, chromosome capture conformation on chip (4C) analysis was performed in human SH-SY5Y neuroblastoma cells. SH-SY5Y neurons had 2.84-fold fewer 15q11.2-13.3 PWS-IC chromatin interactions than undifferentiated SH-SY5Y neuroblasts, revealing developmental chromatin de-condensation of the locus. Out of 68 PWS-IC interactions with15q11.2-13.3 identified by 4C analysis and 62 15q11.2-13.3 MeCP2-binding sites identified by previous ChIP-chip studies, only five sites showed overlap. Remarkably, two of these overlapping PWS-IC- and MeCP2-bound sites mapped to sites flanking CHRNA7 (cholinergic receptor nicotinic alpha 7) encoding the cholinergic receptor, nicotinic, alpha 7. PWS-IC interaction with CHRNA7 in neurons was independently confirmed by fluorescent in situ hybridization analysis. Subsequent quantitative transcriptional analyses of frontal cortex from Rett syndrome and autism patients revealed significantly reduced CHRNA7 expression compared with controls. Together, these results suggest that transcription of CHRNA7 is modulated by chromatin interactions with the PWS-IC. Thus, loss of long-range chromatin interactions within 15q11.2-13.3 may contribute to multiple human neurodevelopmental disorders.

An inversion inv(4)(p12–p15.3) in autistic siblings implicates the 4p GABA receptor gene cluster

Introduction: We describe the case of two brothers diagnosed with autism who both carry a paracentic inversion of the short arm of chromosome 4 (46,XY, inv(4)(p12–p15.3)). We have determined that this inversion is inherited from an apparently unaffected mother and unaffected maternal grandfather. Methods/Results: Using fluorescence in situ hybridisation analysis and Southern blot hybridisation we identified the breakpoints. The proximal breakpoint (4p12) maps to a region containing a cluster of gamma-aminobutyric acid A (GABA(A)) receptor genes, and directly interrupts the GABRG1 gene, the distal-most gene of the cluster. We also identified an insertion/deletion polymorphism for a ∼2 kb LINE1 (L1) element that occurs within intron 7 of GABRG1. Our genotype analysis amongst autism families indicated that the L1 deletion allele did not show increased transmission to affected individuals. No linkage disequilibrium was evident between the L1 and single nucleotide polymorphisms in adjacent GABA(A) receptor genes on 4p, where a recent study has identified significant association with autism. Discussion: Despite this, the identification of an inversion breakpoint disrupting GABRG1 provides solid support for the genetic involvement of the short arm of chromosome 4 in the genetic aetiology of autism, and for the hypothesis of disrupted GABA neurotransmission in autism.

Preparation and some properties of perfluoroalkoxy-substituted phthalocyanine complexes of iron(III), nickel(II) and zinc(II)

Abstract Tetrakis(2′, 2′, 3′, 3′, 4′, 4′, 5′, 5′-octafluoropentoxy)phthalocyanine complexes of iron(III), nickel(II) and zinc(II) were prepared and characterized. The complexes were soluble in polar solvents such as ethanol and methanol but insoluble in less polar solvents such as benzene and toluene. The electron-withdrawing fluorine atoms in the substituents strongly affected the electrochemical and spectroscopic properties of the complexes.

Screening of DNA methylation at the H19 promoter or the distal region of its ICR1 ensures efficient detection of chromosome 11p15 epimutations in Russell–Silver syndrome

Over a 10‐year period blood samples were collected from 57 individuals with growth restriction and RSS‐like features. Our goal was to identify epigenetic abnormalities in this cohort, including uniparental disomy of chromosome 7 (UPD7), methylation changes at chromosome11p15, as well as new epigenomic alterations. We evaluated the methylation status of 7 imprinting control regions on chromosomes 7, 11, 14, and 15. UPD7 and chromosome 7 structural abnormalities had been previously identified in five patients. Epigenetic alterations on chromosome 11p15 were identified in 11 patients. Of interest, in 3 of these 11 patients, the epigenetic alterations were limited to the H19 promoter and the distal region of its associated imprinting center, ICR1. In addition, in one patient, we detected methylation changes consistent with maternal UPD at all tested imprinted regions. This patient series suggests that epimutations on chromosome 11p15 can be most efficiently detected in RSS patients by screening for DNA methylation defects at the H19 promoter or the distal region of ICR.

DLX5 expression is monoallelic and Dlx5 is up-regulated in the Mecp2-null frontal cortex

There has been a controversy as to whether DLX5 is imprinted in human brain and lymphoblastoid cell lines (LCLs), and whether expression of Dlx5 and Dlx6 (Dlx5/6 exist as a bigene cluster [1]), is dysregulated in the Mecp2-null mouse frontal cortex. In the September issue of Am J Hum Genet. (Vol 81: 492–506, 2007) [2], Schüle et al. published a paper entitled ‘DLX5 and DLX6 expression is bi-allelic and not modulated by Mecp2 deficiency.’ This study concluded that DLX5 was not imprinted in normal human LCLs nor in human brain, contradicting the conclusions of Okita et al. (Genomics 81:556–559, 2003) [3] and Horike et al. (Nature Genetics 37:31–40, 2005) [4]. Schüle et al. also claimed that expression of Dlx5 and Dlx6 in mouse brain varies greatly among individual mice and is not necessarily up-regulated in the frontal cortex of Mecp2-null mice. These findings directly contradict those of Horike et al. We have now repeated and verified our previous experiments that are relevant to the points raised by Schüle et al. Further, we have also expanded our analyses to determine why Schüle et al., were not able to reproduce our findings. Here are our findings in response to their assertions. (1) Schüle et al. presented quantitative RT-PCR (qRT-PCR) data obtained from frontal cortex tissue samples of male wild-type and Mecp2-null mice from several litters. They reported that the expression levels of Dlx5, Dlx6 and the imprinted gene Peg3 were all highly variable among individual mice, independent of genotype (0.5–3.5-fold difference relative to a single wild-type mouse selected among several litters). Schüle et al. therefore concluded that the findings of Horike et al. – that there is a 2-fold increase in expression of Dlx5 and Dlx6 in Mecp2-null mice compared to wild type littermates – were the result of ‘biological noise.’ Considering that these genes are tightly regulated during development, we were surprised at their observations that their expression was completely random even among wild type animals from the same litter. We repeated these experiments using three sets of littermates of Mecp2-null and wild-type mice (42 or 51 days old) from an independent breeding colony of Mecp2-heterozygous mice (generated by Adrian Bird’s laboratory) [5] kindly provided by Masayuki Itoh (National Institute of Neuroscience, Japan), who carefully bred the Mecp2 heterozygous mice to retain the original phenotype. In all five Mecp2-null mice examined using Gapdh as a control, we were able to reproduce the ~2fold (1.7–2.6-fold) increase in expression of Dlx5 in Mecp2-null mice, compared with wild-type mice (Fig. 1A), just as originally reported by Horike et al. We similarly verified up-regulation of Dlx6 in the same brain subregions of the Mecp2-null mice tested (representative data are shown in Fig. 1B). We further examined expression levels of Peg3, which Schüle et al. also report as having high variability in expression. In contrast to their findings, we observed consistent expression levels with a small standard error of the mean (S.E.M.) between mice of the same genotype and saw similar levels in the frontal cortex between wild-type and Mecp2-null mice (Fig. 1C). Expression levels of genes in brains are best compared among littermates, since mice from different litters often show some variability in gene expression. Nonetheless, in our study, difference in expression levels of the control Gapdh typically do not exceed more than 20%, even among mice from different litters. Therefore, our results consistently show non-random expression of Dlx5 and Dlx6 relative to Gapdh among littermates, accurately reflecting the genotype of individual mice within each litter. In the frontal cortex of Mecp2-null mice, Dlx5 and Dlx6 are moderately up-regulated compared to wild-type mice, while there is no change in expression for Peg3. The differences between our results and those of Schüle et al. likely arose from differences in reproducibility in dissection of the frontal cortex and in the purity/quality of RNA; RNA degradation can dramatically alter qRT-PCR results. Because Dlx5 is expressed DLX5 expression is monoallelic and Dlx5 is up-regulated

Oxytocin for Male Subjects with Autism Spectrum Disorder and Comorbid Intellectual Disabilities: A Randomized Pilot Study.

Approximately half of autism spectrum disorder (ASD) individuals suffer from comorbid intellectual disabilities (IDs). Oxytocin (OXT) receptors are highly expressed in temporal lobe structures and are likely to play a modulatory role in excitatory/inhibitory balance, at least based on animal model findings. Thus, it is feasible that in the highly representative group of Kanner-type ASD subjects, OXT could have a beneficial effect on social communication and social interaction. The aim of this pilot study is to investigate the feasibility and adverse events, such as epilepsy, of the long-term administration of intranasal OXT for adolescent and adult ASD subjects with ID because such patients frequently have seizures. We also addressed the question on how to scale the OXT effects to the core symptoms of social deficits because of the relative difficulty in obtaining objective measurements. Twenty-nine males (aged 15–40 years old) participated in a randomized, double-blind, and placebo-controlled crossover study (each for 8 weeks) with OXT (16 IU/day). Except for seizures experienced by one participant, other serious adverse events did not occur. The primary and secondary outcomes measured using the Childhood Autism Rating Scale and several standard scales, respectively, revealed no difference between the OXT and placebo groups. Instead, in an exploratory analysis, the social interactions observed in the play sessions or in daily life were significantly more frequent in the initial half period in the OXT-first arm of the crossover trial. There were also significant correlations between the plasma OXT concentration and subscale scores for irritability on the Aberrant Behavior Checklist. In conclusion, this pilot study demonstrates that long-term administration of intranasal OXT is tolerable in a representative cohort of ASD individuals with ID and suggests that future multicenter trials of OXT are warranted and should include measurements of reciprocal social interactions based on daily life under closer surveillance for epilepsy. Trial registration: UMIN000007250.

Catalytic oxygenation of olefin with dioxygen and tetra-t-butylphthalocyanine complexes in the presence of sodium borohydride

Abstract Catalytic oxygenation of styrene with dioxygen was performed by tetra-t-butylphthalocyanine complexes of manganese(III), iron(III), and cobalt(II) in the presence of sodium borohydride using ethanol as a solvent. The yield of 1-phenylethanol increased with the passage of time in the case of the manganese(III) and iron(III) complexes, but an induction period appeared in the case of the cobalt(II) complex. The initial reaction rate was 2.8 × 10−3 or 3.4 × 10−2 mmol min−1 for the reaction catalyzed by the manganese(III) or cobalt(II) complex respectively. No oxygenation product was formed in anaerobic ethanol with any of the complexes, but ethylbenzene was formed exclusively in the presence of the cobalt(II) complex. A radical scavenger, 2,2,6,6-tetramethylpiperidine-1-oxyl, inhibited the reaction considerably with the manganese(III) or iron(III) complex, but inhibited the reaction slightly with the cobalt(II) complex. The amount of oxygenation product from α-methylstyrene was larger than that from styrene in the case of the manganese(III) and iron(III) complexes, but the amount of oxygenation product from α-methylstyrene was almost the same as that from styrene in the case of the cobalt(II) complex. A radical mechanism is proposed for the oxygenation reaction of olefin catalyzed by the complexes. In the case of the cobalt(II) complex, a σ-bound intermediate may also contribute to the reaction.

Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation.

Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish.

Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human chromosome 3, via microcell-mediated chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT. To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated chromosome 3 regions produced by a novel chromosomal engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT. We found a telomerase repressor region located within a 7-Mb interval on chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements.

A translocation t(6;7)(p11-p12;q22) associated with autism and mental retardation: localization and identification of candidate genes at the breakpoints.

Objectives Our aim is to use information from cytogenetic anomalies to identify candidate genes for autism. Methods We have identified a male patient with mental retardation and autism who has a balanced translocation involving chromosomes 6 and 7, described as t(6;7)(p11–p12;q22). This translocation was inherited from an apparently normal father. Results Using fluorescence in situ hybridization, we have localized the breakpoints on both the chromosomes; and using bioinformatic genomic analysis, we have identified a number of potential candidate genes at these loci. These include the neural pentraxin 2 gene, NPTX2, and a novel gene encoding a transmembrane protein, TMEM130, which contains a polycystic kidney domain on 7q22. On 6p12 the breakpoint directly interrupts isoform 2 of the human homologue of the mouse dystonin gene. We also performed a 250 K single nucleotide polymorphism microarray analysis and comparative genomic hybridization using a bacterial artificial chromosome microarray to look for minor genomic deletions or duplications in the probands DNA. The single nucleotide polymorphism microarray analysis identified a number of copy number variants, remote from the translocation breakpoints, containing potential candidate genes. Conclusion It is conceivable that one or more of the copy number variant regions or either of the two breakpoint locations and the dystonin gene, in particular, may be a new locus for a form of mental retardation, which may also include autistic features.