Increased copy number for methylated maternal 15q duplications leads to changes in gene and protein expression in human cortical samples

<p>Abstract</p> <p>Background</p> <p>Duplication of chromosome 15q11-q13 (dup15q) accounts for approximately 3% of autism cases. Chromosome 15q11-q13 contains imprinted genes necessary for normal mammalian neurodevelopment controlled by a differentially methylated imprinting center (imprinting center of the Prader-Willi locus, PWS-IC). Maternal dup15q occurs as both interstitial duplications and isodicentric chromosome 15. Overexpression of the maternally expressed gene <it>UBE3A </it>is predicted to be the primary cause of the autistic features associated with dup15q. Previous analysis of two postmortem dup15q frontal cortical samples showed heterogeneity between the two cases, with one showing levels of the GABA_Areceptor genes, <it>UBE3A </it>and <it>SNRPN </it>in a manner not predicted by copy number or parental imprint.</p> <p>Methods</p> <p>Postmortem human brain tissue (Brodmann area 19, extrastriate visual cortex) was obtained from 8 dup15q, 10 idiopathic autism and 21 typical control tissue samples. Quantitative PCR was used to confirm duplication status. Quantitative RT-PCR and Western blot analyses were performed to measure 15q11-q13 transcript and protein levels, respectively. Methylation-sensitive high-resolution melting-curve analysis was performed on brain genomic DNA to identify the maternal:paternal ratio of methylation at PWS-IC.</p> <p>Results</p> <p>Dup15q brain samples showed a higher level of PWS-IC methylation than control or autism samples, indicating that dup15q was maternal in origin. <it>UBE3A </it>transcript and protein levels were significantly higher than control and autism in dup15q, as expected, although levels were variable and lower than expected based on copy number in some samples. In contrast, this increase in copy number did not result in consistently increased <it>GABRB3 </it>transcript or protein levels for dup15q samples. Furthermore, <it>SNRPN </it>was expected to be unchanged in expression in dup15q because it is expressed from the single unmethylated paternal allele, yet <it>SNRPN </it>levels were significantly reduced in dup15q samples compared to controls. PWS-IC methylation positively correlated with <it>UBE3A </it>and <it>GABRB3 </it>levels but negatively correlated with <it>SNRPN </it>levels. Idiopathic autism samples exhibited significantly lower <it>GABRB3 </it>and significantly more variable <it>SNRPN </it>levels compared to controls.</p> <p>Conclusions</p> <p>Although these results show that increased <it>UBE3A</it>/UBE3A is a consistent feature of dup15q syndrome, they also suggest that gene expression within 15q11-q13 is not based entirely on copy number but can be influenced by epigenetic mechanisms in brain.</p

Significant transcriptional changes in 15q duplication but not Angelman syndrome deletion stem cell-derived neurons

Abstract Background The inability to analyze gene expression in living neurons from Angelman (AS) and Duplication 15q (Dup15q) syndrome subjects has limited our understanding of these disorders at the molecular level. Method Here, we use dental pulp stem cells (DPSC) from AS deletion, 15q Duplication, and neurotypical control subjects for whole transcriptome analysis. We identified 20 genes unique to AS neurons, 120 genes unique to 15q duplication, and 3 shared transcripts that were differentially expressed in DPSC neurons vs controls. Results Copy number correlated with gene expression for most genes across the 15q11.2-q13.1 critical region. Two thirds of the genes differentially expressed in 15q duplication neurons were downregulated compared to controls including several transcription factors, while in AS differential expression was restricted primarily to the 15q region. Here, we show significant downregulation of the transcription factors FOXO1 and HAND2 in neurons from 15q duplication, but not AS deletion subjects suggesting that disruptions in transcriptional regulation may be a driving factor in the autism phenotype in Dup15q syndrome. Downstream analysis revealed downregulation of the ASD associated genes EHPB2 and RORA, both genes with FOXO1 binding sites. Genes upregulated in either Dup15q cortex or idiopathic ASD cortex both overlapped significantly with the most upregulated genes in Dup15q DPSC-derived neurons. Conclusions Finding a significant increase in both HERC2 and UBE3A in Dup15q neurons and significant decrease in these two genes in AS deletion neurons may explain differences between AS deletion class and UBE3A specific classes of AS mutation where HERC2 is expressed at normal levels. Also, we identified an enrichment for FOXO1-regulated transcripts in Dup15q neurons including ASD-associated genes EHPB2 and RORA indicating a possible connection between this syndromic form of ASD and idiopathic cases.https://deepblue.lib.umich.edu/bitstream/2027.42/140784/1/13229_2018_Article_191.pd

A Single-Tube Quantitative High-Resolution Melting Curve Method for Parent-of-Origin Determination of 15q Duplications

The most common chromosomal abnormalities associated with autism are 15q11–q13 duplications. Maternally derived or inherited duplications of 15q pose a substantial risk for an autism phenotype, while paternally derived duplications may be incompletely penetrant or result in other neurodevelopmental problems. Therefore, the determination of maternal versus paternal origin of this duplication is important for early intervention therapies and for appropriate genetic counseling to the families. We adapted a previous single-reaction tube assay (high-resolution melting curve analysis) to determine the parent of origin of 15q duplications in 28 interstitial duplication 15q samples, one family and two isodicentric subjects. Our method distinguished parent origin in 92% of the independent samples as well as in the familial inherited duplication and in the two isodicentric samples. This method accurately determines parental origin of the duplicated segment and measures the dosage of these alleles in the sample. In addition, it can be performed on samples where parental DNA is not available for microsatellite analysis. The development of this single-tube assay will make it easier for genetic testing laboratories to provide parent-of-origin information and will provide important information to clinical geneticists about autism risk in these individuals

Dental Pulp Stem Cells Model Early Life and Imprinted DNA Methylation Patterns.

Early embryonic stages of pluripotency are modeled for epigenomic studies primarily with human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSCs). For analysis of DNA methylation however, ESCs and iPSCs do not accurately reflect the DNA methylation levels found in preimplantation embryos. Whole genome bisulfite sequencing (WGBS) approaches have revealed the presence of large partially methylated domains (PMDs) covering 30%-40% of the genome in oocytes, preimplantation embryos, and placenta. In contrast, ESCs and iPSCs show abnormally high levels of DNA methylation compared to inner cell mass (ICM) or placenta. Here we show that dental pulp stem cells (DPSCs), derived from baby teeth and cultured in serum-containing media, have PMDs and mimic the ICM and placental methylome more closely than iPSCs and ESCs. By principal component analysis, DPSC methylation patterns were more similar to two other neural stem cell types of human derivation (EPI-NCSC and LUHMES) and placenta than were iPSCs, ESCs or other human cell lines (SH-SY5Y, B lymphoblast, IMR90). To test the suitability of DPSCs in modeling epigenetic differences associated with disease, we compared methylation patterns of DPSCs derived from children with chromosome 15q11.2-q13.3 maternal duplication (Dup15q) to controls. Differential methylation region (DMR) analyses revealed the expected Dup15q hypermethylation at the imprinting control region, as well as hypomethylation over SNORD116, and novel DMRs over 147 genes, including several autism candidate genes. Together these data suggest that DPSCs are a useful model for epigenomic and functional studies of human neurodevelopmental disorders. Stem Cells 2017;35:981-988

Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders

A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSCs) are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSCs that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSCs. We immortalized control DPSCs using human telomerase reverse transcriptase (hTERT) and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSCs share morphological and electrophysiological properties with non-immortalized DPSCs. We also show that differentiation of DPSCs into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NRSF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSCs can be obtained from teeth stored for ≥72 h, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSCs for the study of disease

Additional file 1: Tables S1–S9. of Significant transcriptional changes in 15q duplication but not Angelman syndrome deletion stem cell-derived neurons

Complete GSEA analysis for all samples and FOXO1 and functional enrichment analysis of ASD brain vs Dup15q DPSC neurons. S9 is a table of primers used for qRT-PCR. (XLSX 68 kb

Identification of novel candidate disease genes from de novo exonic copy number variants

Background: Exon-targeted microarrays can detect small ( Methods: We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association. Results: In this clinical population, among rare CNVs involving any single gene reported in 7200 patients (11%), we identified 145 de novo autosomal CNVs (117 losses and 28 intragenic gains), 257 X-linked deletion CNVs in males, and 1049 inherited autosomal CNVs (878 losses and 171 intragenic gains); 111 known disease genes were potentially disrupted by de novo autosomal or X-linked (in males) single-gene CNVs. Ninety-one genes, either recently proposed as candidate disease genes or not yet associated with diseases, were disrupted by 147 singlegene CNVs, including 37 de novo deletions and ten de novo intragenic duplications on autosomes and 100 X-linked CNVs in males. Clinical features in individuals with de novo or X-linked CNVs encompassing at most five genes (224 bp to 1.6 Mb in size) were compared to those in individuals with larger-sized deletions (up to 5 Mb in size) in the internal CMA database or loss-of-function single nucleotide variants (SNVs) detected by clinical or research whole-exome sequencing (WES). This enabled the identification of recently published genes (BPTF, NONO, PSMD12, TANGO2, and TRIP12), novel candidate disease genes (ARGLU1 and STK3), and further confirmation of disease association for two recently proposed disease genes (MEIS2 and PTCHD1). Notably, exon-targeted CMA detected several pathogenic single-exon CNVs missed by clinical WES analyses. Conclusions: Together, these data document the efficacy of exon-targeted CMA for detection of genic and exonic CNVs, complementing and extending WES in clinical diagnostics, and the potential for discovery of novel disease genes by genome-wide assay.Peer reviewe

La gestión emocional de los docentes de educación física

Treball Final de Grau en Mestre o Mestra de Educació Primària. Codi: MP1040. Curs acadèmic: 2016/2017El tema elegido para este TFG trata sobre la inteligencia emocional con el objetivo de analizar la gestión emocional del docente y cómo influye en la actitud de las niñas y los niños. Concretamente, ser capaces de afrontar ciertas situaciones y gestionar sus emociones de manera que las pueda afrontar de una forma ecológica para las partes implicadas, de forma que la clase pueda continuar con la dinámica adecuada, la cual comentaremos durante el trabajo. La razón por la cual se ha elegido este tema es porque hemos visto y vivenciado durante la estancia en prácticas en el CEIP Pio XII que, durante la clase de educación física, aparecen situaciones que pueden afectarnos emocionalmente a los docentes como personas que somos y puede repercutirnos en nuestra labor docente, concretamente, en el área de la educación física. Por ello, también proponemos una serie de herramientas y recomendaciones para ayudar a los docentes de educación física, para que como dice Elia López (2011, p.32) se fomente en las clases un clima afectivo, distendido y desinhibido. Para analizar la propuesta, se partirá de una puesta en práctica, que explicaremos, acerca del tema y en el análisis de nuestra conducta a la hora de impartir las clases para ver y analizar la actitud de los/as niños/as, durante las clases de expresión corporal en 6º curso, ya que, en la expresión corporal, como comentaremos es importante la parte emocional tanto del profesor como de los alumnos