Aaron Jeffries

Allelic Skewing of DNA Methylation Is Widespread across the Genome

DNA methylation is assumed to be complementary on both alleles across the genome, although there are exceptions, notably in regions subject to genomic imprinting. We present a genome-wide survey of the degree of allelic skewing of DNA methylation with the aim of identifying previously unreported differentially methylated regions (DMRs) associated primarily with genomic imprinting or DNA sequence variation acting in cis. We used SNP microarrays to quantitatively assess allele-specific DNA methylation (ASM) in amplicons covering 7.6% of the human genome following cleavage with a cocktail of methylation-sensitive restriction enzymes (MSREs). Selected findings were verified using bisulfite-mapping and gene-expression analyses, subsequently tested in a second tissue from the same individuals, and replicated in DNA obtained from 30 parent-child trios. Our approach detected clear examples of ASM in the vicinity of known imprinted loci, highlighting the validity of the method. In total, 2,704 (1.5%) of our 183,605 informative and stringently filtered SNPs demonstrate an average relative allele score (RAS) change > or =0.10 following MSRE digestion. In agreement with previous reports, the majority of ASM ( approximately 90%) appears to be cis in nature, and several examples of tissue-specific ASM were identified. Our data show that ASM is a widespread phenomenon, with >35,000 such sites potentially occurring across the genome, and that a spectrum of ASM is likely, with heterogeneity between individuals and across tissues. These findings impact our understanding about the origin of individual phenotypic differences and have implications for genetic studies of complex disease.

Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits

Autism spectrum disorder (ASD) defines a group of common, complex neurodevelopmental disorders. Although the aetiology of ASD has a strong genetic component, there is considerable monozygotic (MZ) twin discordance indicating a role for non-genetic factors. Because MZ twins share an identical DNA sequence, disease-discordant MZ twin pairs provide an ideal model for examining the contribution of environmentally driven epigenetic factors in disease. We performed a genome-wide analysis of DNA methylation in a sample of 50 MZ twin pairs (100 individuals) sampled from a representative population cohort that included twins discordant and concordant for ASD, ASD-associated traits and no autistic phenotype. Within-twin and between-group analyses identified numerous differentially methylated regions associated with ASD. In addition, we report significant correlations between DNA methylation and quantitatively measured autistic trait scores across our sample cohort. This study represents the first systematic epigenomic analyses of MZ twins discordant for ASD and implicates a role for altered DNA methylation in autism.

Molecular and phenotypic characterization of ring chromosome 22.

We performed a phenotype study of 35 individuals (19 males, 16 females) with ring chromosome 22 or r(22) with a mean age of 10 years. In common with other studies, a phenotype of moderate‐to‐profound learning difficulties and delay or absence of speech affected all individuals with the exception of the case with the smallest deletion. Autistic traits were significantly associated with r(22), as shown by an autism screening questionnaire. Mild and variable dysmorphic features, predominantly craniofacial and distal limb, were observed. Internal organ involvement was uncommon. Even though ring chromosomes are reportedly associated with growth abnormalities, only 2 out of 24 individuals showed evidence of growth failure, while 2 showed accelerated growth. Chromosome 22 long arm deletions, as determined by hemizygosity for informative microsatellite markers, varied from <67 kb to 10.2 Mb in size (or <0.15 to 21% of total chromosome length), with no significant differences in the parental origin of the ring chromosome. Few phenotypic features correlated with deletion size suggesting a critical gene, or genes, of major effect lies close to the telomere. Loss of the SHANK3/PROSAP2 gene has been proposed to be responsible for the main neurological developmental deficits observed in 22q13 monosomies. This study supports this candidate gene by identifying a phenotypically normal r(22) individual whose ring chromosome does not disrupt SHANK3. All other r(22) individuals were hemizygous for SHANK3, and we propose it to be a candidate gene for autism or abnormal brain development.

Knockdown of the psychosis susceptibility gene ZNF804A alters expression of genes involved in cell adhesion

Genome-wide association studies have convincingly implicated several novel genes in susceptibility to schizophrenia and bipolar disorder. The first genome-wide significant association with the broad phenotype of psychosis was with a polymorphism in the ZNF804A gene. However, the biological function(s) of ZNF804A have, to date, been entirely unknown. In this study, we manipulated the expression of ZNF804A in neural progenitor cells derived from human cortical neuroepithelium and assessed its effects on the cellular transcriptome. Gene ontology analysis of differentially expressed genes indicated a significant effect of ZNF804A knockdown on the expression of genes involved in cell adhesion, suggesting a role for ZNF804A in processes such as neural migration, neurite outgrowth and synapse formation. Several highly significant gene expression changes were confirmed in repeat cell culture experiments. Most consistent gene expression changes were seen for C2ORF80, a gene of as-yet-unknown function, and STMN3, a gene involved in neurite outgrowth and axonal and dendritic branching. These data, generated in a hypothesis-free manner, provide a basis for more targeted investigations of ZNF804A function.

RE1 Silencing Transcription Factor Maintains a Repressive Chromatin Environment in Embryonic Hippocampal Neural Stem Cells

The control of gene expression in neural stem cells is key to understanding their developmental and therapeutic potential, yet we know little of the transcriptional mechanisms that underlie their differentiation. Recent evidence has implicated the RE1 silencing transcription factor (REST) in neuronal differentiation. However, the means by which REST regulates transcription in neural stem cells remain unclear. Here, we show that REST recruits distinct corepressor platforms in neural stem cells. REST is able to both silence and repress neuronal genes in embryonic hippocampal neural stem cells by creating a chromatin environment that contains both repressive local epigenetic signature (characterized by low levels of histones H4 and H3K9 acetylation and elevated dimethylation of H3K9) and H3K4 methylation, which are characteristic of gene activation. Furthermore, inhibition of REST function leads to activation of several neuron‐specific genes but does not lead to overt formation of mature neurons, supporting the notion that REST regulates part, but not all, of the neuronal differentiation program.

Purmorphamine increases DARPP-32 differentiation in human striatal neural stem cells through the Hedgehog pathway.

Transplantation of neural stem cells (NSCs) is a promising therapeutic approach for Huntingtons disease (HD). HD is characterized by a progressive loss of medium-sized spiny neurons (MSNs) in the striatum. DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa) is expressed in 98% of these MSNs. To establish an effective cell therapy for HD, the differentiation of human NSCs into MSNs is essential. Enhancing differentiation of NSCs is therefore an important aspect to optimize transplant efficacy. A comparison of 5 differentiation protocols indicated that the Hedgehog agonist purmorphamine (1 μM) most significantly increased the neuronal differentiation of a human striatal NSC line (STROC05). This 3-fold increase in neurons was associated with a dramatic reduction in proliferation as well as a decrease in astrocytic differentiation. A synergistic effect between purmorphamine and cell density even further increased neuronal differentiation from 20% to 30% within 7 days. Upon long-term differentiation (21 days), this combined differentiation protocol tripled the number of DARPP-32 cells (7%) and almost doubled the proportion of calbindin cells. However, there was no effect on calretinin cells. Differential expression of positional specification markers (DLX2, MASH1, MEIS2, GSH2, and NKX2.1) further confirmed the striatal identity of these differentiated cells. Purmorphamine resulted in a significant upregulation of the Hedgehog (Hh) signaling pathway (GLI1 expression). Cyclopamine, an Hh inhibitor, blocked this effect, indicating that purmorphamine specifically acts through this pathway to increase neuronal differentiation. These results demonstrate that small synthetic molecules can play a pivotal role in directing the differentiation of NSCs to optimize their therapeutic potential in HD.

Effects of cis-regulatory variation differ across regions of the adult human brain

Cis-regulatory variation is considered to be an important determinant of human phenotypic variability, including susceptibility to complex disease. Recent studies have shown that the effects of cis-regulatory polymorphism on gene expression can differ widely between tissues. In the present study, we tested whether the effects of cis-regulatory variation can also differ between regions of the adult human brain. We used relative allelic expression to measure cis-effects on the RNA expression of five candidate genes for neuropsychiatric illness (ZNF804A, NOS1, RGS4, AKT1 and TCF4) across multiple discrete brain regions within individual subjects. For all five genes, we observed significant differences in allelic expression between brain regions in several individual subjects, suggesting regional differences in the effects of cis-regulatory polymorphism to be a common phenomenon. As well as highlighting an important caveat for studies of regulatory polymorphism in the brain, our findings indicate that it is possible to delineate brain areas in which cis-regulatory variants are active. This may provide important insights into the fundamental biology of neuropsychiatric phenotypes with which such variants are associated.

Stochastic choice of allelic expression in human neural stem cells.

Monoallelic gene expression, such as genomic imprinting, is well described. Less well‐characterized are genes undergoing stochastic monoallelic expression (MA), where specific clones of cells express just one allele at a given locus. We performed genome‐wide allelic expression assessment of human clonal neural stem cells derived from cerebral cortex, striatum, and spinal cord, each with differing genotypes. We assayed three separate clonal lines from each donor, distinguishing stochastic MA from genotypic effects. Roughly 2% of genes showed evidence for autosomal MA, and in about half of these, allelic expression was stochastic between different clones. Many of these loci were known neurodevelopmental genes, such as OTX2 and OLIG2. Monoallelic genes also showed increased levels of DNA methylation compared to hypomethylated biallelic loci. Identified monoallelic gene loci showed altered chromatin signatures in fetal brain, suggesting an in vivo correlate of this phenomenon. We conclude that stochastic allelic expression is prevalent in neural stem cells, providing clonal diversity to developing tissues such as the human brain. Stem Cells2012;30:1938–1947

β-1,3-Glucuronyltransferase-1 gene implicated as a candidate for a schizophrenia-like psychosis through molecular analysis of a balanced translocation

We have mapped and sequenced both chromosome breakpoints of a balanced t(6;11)(q14.2;q25) chromosome translocation that segregates with a schizophrenia-like psychosis. Bioinformatics analysis of the regions revealed a number of confirmed and predicted transcripts. No confirmed transcripts are disrupted by either breakpoint. The chromosome 6 breakpoint region is gene poor, the closest transcript being the serotonin receptor 1E (HTR1E) at 625 kb telomeric to the breakpoint. The chromosome 11 breakpoint is situated close to the telomere. The closest gene, β-1,3-glucuronyltransferase (B3GAT1 or GlcAT-P), is 299 kb centromeric to the breakpoint. B3GAT1 is the key enzyme during the biosynthesis of the carbohydrate epitope HNK-1, which is present on a number of cell adhesion molecules important in neurodevelopment. Mice deleted for the B3GAT1 gene show defects in hippocampal long-term potentiation and in spatial memory formation. We propose that the translocation causes a positional effect on B3GAT1, affecting expression levels and making it a plausible candidate for the psychosis found in this family. More generally, regions close to telomeres are highly polymorphic in both sequence and length in the general population and several studies have implicated subtelomeric deletions as a common cause of idiopathic mental retardation. This leads us to the hypothesis that polymorphic or other variation of the 11q telomere may affect the activity of B3GAT1 and be a risk factor for schizophrenia and related psychoses in the general population.

Radiation dose rates from adult patients undergoing nuclear medicine investigations

Adult patients undergoing nuclear medicine investigations may subsequently come into close contact with members of the public and hospital staff. In order to expand the available dosimetry and derive appropriate recommendations, dose rates were measured at 0.1, 0.5 and 1.0 m from 80 adult patients just before they left the nuclear medicine department after undergoing one of eight 99Tcm studies, an 123I thyroid, an 111In leucocyte or a 201Tl cardiac scan. The maximum departure dose rates at these distances of 150, 30 and 7.3 microSv h-1 were greater than those found in similar published studies of adult and paediatric patients. To limit the dose to an infant to less than 1 mSv, an 111In leucocyte scan is the only investigation for which it may be necessary to restrict close contact between the infant and a radioactive parent, depending on the dose rate near the surface of the patient, the parents habits and how fretful is the infant. It is unlikely that a ward nurse will receive a dose of 60 microSv in a working day if caring for just one radioactive adult patient, unless the patient is classified as totally helpless and has undergone a 99Tcm marrow, bone or brain scan. The data and revised calculations of effective exposure times based on a total close contact time of 9 h in every 24 h period should allow worst case estimates of radiation dose to be made and recommendations to be formulated for other circumstances, including any future legislative changes in dose limits or derived levels.