Increased copy number for methylated maternal 15q duplications leads to changes in gene and protein expression in human cortical samples
© Scoles et al; licensee BioMed Central Ltd. 2011
Received: 17 September 2011
Accepted: 12 December 2011
Published: 12 December 2011
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 UBE3A 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 GABAA receptor genes, UBE3A and SNRPN in a manner not predicted by copy number or parental imprint.
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.
Dup15q brain samples showed a higher level of PWS-IC methylation than control or autism samples, indicating that dup15q was maternal in origin. UBE3A 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 GABRB3 transcript or protein levels for dup15q samples. Furthermore, SNRPN was expected to be unchanged in expression in dup15q because it is expressed from the single unmethylated paternal allele, yet SNRPN levels were significantly reduced in dup15q samples compared to controls. PWS-IC methylation positively correlated with UBE3A and GABRB3 levels but negatively correlated with SNRPN levels. Idiopathic autism samples exhibited significantly lower GABRB3 and significantly more variable SNRPN levels compared to controls.
Although these results show that increased UBE3A/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.
Keywordsautism imprinting copy number variation 15q duplication methylation epigenetic
Autism is a common neurodevelopmental disorder affecting 1 in 110 children , but its genetic etiology is complex and heterogeneous [2, 3] Among the leading genetic causes of autism are abnormalities in proximal chromosome 15q, collectively referred to as "duplication 15 syndrome" (dup15q), which occur in ~1-3% of autism cases [4–6]. Dup15q syndrome is a clinically heterogeneous neurodevelopmental disorder characterized by varying degrees of cognitive impairment, gross motor delays, seizures, dysmorphic features and autism in 85% of cases [5, 7, 8].
Chromosome 15q11-q13 is a genetic hotbed for neurodevelopmental disorders because of a high density of low copy repeats (LCRs) that increase susceptibility to errors in meiotic recombination, resulting in deletions as well as duplications [9, 10]. The parental origin of deletions and duplications influences the expression of 15q11-q13 through genomic imprinting. Imprinting of 15q11-q13 is regulated by the bipartite imprinting center (IC), which contains a differentially methylated region at the 5' end of SNRPN that controls expression throughout 15q11-q13 . Loss of 15q11-q13 paternally expressed genes through deletions or maternal uniparental disomy (UPD) results in Prader-Willi syndrome (PWS), whereas the maternal deficiency at this locus results in a phenotypically distinct neurodevelopmental disorder, Angelman syndrome (AS).
Maternally derived duplications of chromosome 15, specifically 15q11-q13, are associated with an autistic phenotype, whereas paternally derived duplications primarily show normal phenotypes but may manifest neurological features other than autism [7, 12]. In addition, individuals with PWS with maternal UPD show a higher occurrence of autism compared to those with PWS 15q11-q13 deletions [13, 14], suggesting that overexpression at maternally expressed genes confers risk for autism [13, 15]. The E3 ubiquitin ligase gene (UBE3A) is the only known paternally imprinted gene in the locus, and its maternal allele-specific transcription is limited to postnatal neurons [16, 17]. While ATP10A was previously described as maternally expressed [18, 19], recent studies have shown variable imprinting in humans and lack of imprinting in mouse [20, 21]. Paternally expressed genes within 15q11-q13 include the splicing factor encoding SNRPN, necdin (NDN), MAGEL2 and several large clusters of small nucleolar RNAs (snoRNAs). A cluster of three receptor subunit genes for the neurotransmitter GABAA (GABRB3, GABRA5, GABRG3) are biallelically expressed in control brain tissue samples, but show epigenetic alterations that result in monoallelic expression in a subset of autism cortical samples .
Although increased copy number is generally assumed to increase transcript levels, the epigenetic and neurodevelopmental complexities associated with 15q11-q13 confound this simple explanation of UBE3A overexpression as the sole molecular cause of the dup15q phenotype. In addition to imprinting, the 15q11-q13 locus is subject to the interchromosomal higher organization of homologous pairing between maternal and paternal alleles [23–25]. Chromosome 15 duplications result in disrupted homologous pairing in dup15q brain tissue samples  and a neuronal cell line model of dup15q . In addition, disruption of 15q11-q13 pairing by dup15q in neurons has been shown to result in reduced transcript levels of NDN, SNRPN, GABRB3 and CHRNA7. In a prior analysis of two dup15q cortical tissue samples, one showed reduced levels of the paternally derived transcripts SNRPN, snoRNAs, NDN and the biallelically expressed GABRB3, GABRA5 and GABRG3 transcripts that corresponded to PWS-like behaviors .
To further understand the genetic and epigenetic effects on transcript levels that lead to the pathogenesis of dup15q syndrome, we performed an extensive analysis of a panel of eight dup15q cortical tissue samples as compared to control and idiopathic autism samples. The dup15q samples showed changes in both methylation and transcription levels, with UBE3A showing significantly higher, SNRPN showing significantly lower and GABRB3 showing variable transcript levels as compared to controls. Interestingly, UBE3A transcript positively correlated with maternal allele-specific methylation of the Prader-Willi imprinting control locus (PWS-IC) within the dup15q samples. These results support the hypothesis that elevated UBE3A levels in the brain are a major contributor to the dup15q phenotype but also are consistent with observations that dup15q syndrome results in transcriptional and epigenetic changes that are variable and not based solely on copy number.
RNA extraction and cDNA synthesis
Frozen cerebral cortex samples from Brodmann area 19 (BA19) were obtained through the Autism Tissue Program from the University of Maryland Brain and Tissue Bank for Neurodevelopmental Disorders, the Harvard Brain and Tissue Resource Center and the University of Miami Brain and Tissue Bank for Neurodevelopmental Disorders. Brain tissues were stored at -80°C until processing. While the tissues were kept frozen on dry ice, 0.1 to 0.15 g was sliced and homogenized using TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA, USA). All RNA work was done on a bench using pipettes wiped down with Ambion RNaseZap wipes (Invitrogen/Life Technologies, Austin, TX, USA) prior to beginning work to prevent RNase contamination of the sample. To eliminate DNA contamination, the RNA was treated with DNase I (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions and precipitated using sodium acetate and ethanol. Single-stranded cDNA was synthesized using QuantiTect Reverse Transcription Kit (QIAGEN, Valencia, CA, USA) and incubated for 15 minutes at 42°C followed by 15 minutes at 50°C with a 3-minute deactivation step at 95°C. For each reaction, a tube without RT was used as a control for genomic DNA contamination. After completion, each reaction was diluted 4.75-fold with DNase- and RNase-free water.
Primers were designed using mRNA sequences extracted from the UCSC Genome Browser  with the February 2009 human reference sequence (GRCh37). Primers were designed to cross an intron or span intron-exon boundaries to limit genomic DNA contamination using Primer3 software  or Biosearch Technologies RealTimeDesign software (Biosearch Technologies Inc, Novato, CA, USA; http://www.biosearchtech.com/realtimedesign). These primer sequences are shown in Additional file 1. Each reaction was carried out in triplicate, and outliers were removed according to the method described by Bookout et al.. PCR amplification of cDNA was performed using 200 nM primers and EXPRESS SYBR GreenER Universal Master Mix (Invitrogen/Life Technologies). Cycling conditions were 20 seconds at 95°C followed by 40 cycles of 2 seconds at 95°C and 30 seconds at 60°C. The reaction was performed using the Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany), and crossing points were analyzed using Realplex software. For each reaction, we ran a well of no RT and no cDNA control to evaluate genomic DNA contamination, nonspecific product formation or other contamination. Reaction conditions were as follows: 1× EXPRESS SYBR GreenER Universal Master Mix, 200 nM primers and 3.5 μl of cDNA. Cycling conditions were as follows: All samples were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative cycle threshold (CT) method (Applied Biosystems, Foster City, CA, USA) to measure fold changes relative to the calibrator, following normalization to GAPDH. Melting curve analysis was also performed to determine homogeneous product formation with no primer-dimers and no nonspecific products.
Quantitative PCR for copy number analysis
Genomic DNA was isolated from brain tissue samples by using the Gentra Puregene Tissue Kit (QIAGEN). PCR conditions were the same as described above using primers shown in Additional file 1.
Determination of methylation percentage at the imprinting center
Methylation-sensitive high-resolution melting-curve analysis (MS-HRM) was performed as described by Urraca et al.. Briefly, 500 ng of DNA was treated with bisulfite. The treated sample (50 ng) was then used for MS-HRM on the LightCycler 480 Real-Time PCR System (Roche Applied Science, Indianapolis, IN, USA) using primers and conditions previously described . Since the paternal SNRPN IC allele is completely unmethylated and the maternal allele is methylated, the normalized fluorescence intensity reveals the percentage of methylated (maternal) to unmethylated (paternal) allele present in a given sample. Additional copies of a maternal duplication of this region results in a higher percentage of maternal allele-specific (methylated) fluorescent signal.
Protein level analyses
Protein extracts were isolated from the same frozen brain tissue samples using TRIzol reagent and analyzed for UBE3A and GABRB3 levels as described previously .
Quantitative determination of genomic copy number and PWS-IC methylation in human brain samples
Quantitation of UBE3A, SNRPN and GABRB3 transcript levels
To determine the effect dup15q and PWS-IC methylation may have on the transcript levels of the genes within this duplicated region, qRT-PCR was performed on RNA samples isolated from each brain tissue sample. Three chromosome 15 transcripts were amplified, including maternally expressed UBE3A, paternally expressed SNRPN and biallelically expressed GABRB3 (shown in Figure 2). The results were normalized to the chromosome 12 housekeeping control gene GAPDH by using the comparative CT method. The transcript levels, described as fold changes from average control, are shown for each brain tissue sample in Figure 1, columns 8 to 10. To visualize the direction and level of change from the expected transcript levels, heat map colors in Figure 1 were utilized, in which red is higher, purple is lower and white is < 20% change from expected.
GABRB3 is biallelically expressed, so the idic15, int dup(15) and tricentric derivative chromosome M:P ratios were expected to be 2:1, 1.5:1 and 3:1, respectively, compared to control samples (Figure 2). GABRB3 levels in the dup15q cortex samples instead were highly variable, as three samples showed 0.3× lower expression and five samples showed 1.5× higher GABRB3 levels compared with typically developing controls (Figures 1, 4b and 4e). Figure 4b shows that the biallelically expressed GABRB3 exhibited no significant changes in the quantity of transcript in dup15q samples as a group; however, the variance in GABRB3 was significantly different from both the control group (P < 0.001) and the autism group (P < 0.001; t-test). Figure 4e of the distribution of GABRB3 levels shows two groups of dup15q samples clustered as either higher or lower than control samples. The idiopathic autism group showed a significant reduction in GABRB3 transcript compared with controls as reported previously .
SNRPN is paternally expressed and in all cases has one expressed copy, because all of the chromosome 15 duplications used were maternally derived. Surprisingly similar to an idic15 brain sample we previously reported from an individual with PWS-like features . SNRPN was lower than expected by at least 0.77× in six of eight dup15q samples (Figures 1 and 4f) and significantly lower in dup15q samples compared to control or autism samples (Figure 4c). SNRPN levels were reduced from control in sample 6856 and increased from control in sample 7436, similar to findings of the previous study of BA9 prefrontal cortex . Idiopathic autism showed no significant change in SNRPN level, but did show an increase in variability of the ten samples compared to control (P = 0.006; Levene's test for equality of variances) and to dup15q (P = 0.045; Levene's test for equality of variances).
Correlation analyses of 15q transcript and protein levels with copy number and maternal PWS-IC methylation
This paper reports the largest study of dup15q brain samples to date. Our results demonstrate that duplication of the 15q11-q13 region alters the expression not only of UBE3A, as expected, but also the expression of SNRPN and GABRB3 in ways not always predicted by copy number, confirming our prior small-scale study . Previously, UBE3A overexpression from the duplicated maternal allele had been hypothesized to be the sole explanation for autism comorbidity in dup15q syndrome as well as the increase in autism spectrum disorder (ASD) phenotypes in PWS maternal UPD compared to deletion cases [13, 33]. It is important to keep in mind that the PWS-IC is methylated on all maternal alleles, regardless of allele copy number . Even in studies of various nonneuronal cell lines, however, where UBE3A is expressed biallelically, increases in UBE3A transcript in the dup15q cells were observed [34–36]. Our study replicates the prior findings of increased UBE3A levels in human cortex, showing a twofold increase in dup15q samples. In contrast, GABRB3 expression was not analyzed in any of the prior studies in cell lines, because GABRB3 is a neuronally expressed gene. SNRPN is expressed in nonneuronal cell lines, but researchers in prior studies did not find SNRPN levels to be different from those of controls in nonneuronal cells [34–36]. In our investigation of dup15q human cortex samples, however, SNRPN levels were significantly lower than in controls, a result that we did not expect, since all of the samples (control, autism and dup15) should express one copy of the SNRPN gene from the single paternal allele present. Our results therefore demonstrate the tissue-specific epigenetic complexities associated with dup15q syndrome in humans which simple copy number changes are inadequate to explain.
Epigenetic patterns and mechanisms are often tissue-specific, and the brain shows high levels of DNA methylation despite being primarily nonmitotic in postnatal life . Our recent genomic analysis of DNA methylation showed large genomic regions that are highly methylated in neurons compared to fibroblasts that span large regions of 15q11-q13 . Interestingly, in this study, we observed tissue-specific differences in PWS-IC methylation between brain tissues as compared to blood samples analyzed previously  by MS-HRM, with brain tissue showing a higher percentage of baseline maternal allele-specific methylation in controls. The MS-HRM analysis of the PWS-IC upstream of SNRPN showed that, when normalized to brain, a M:P methylation ratio of 2.9:1 was observed, indicating that the duplications are maternal in origin. The increased methylation observed in dup15q samples is consistent with findings of previous studies in blood from int dup(15) samples showing that the duplication is maternal, not paternal, in origin. However, it is possible that the paternal allele may be methylated at one or more individual bases in the dup15q samples only. The recent discovery of 5-hydroxymethylcytosine (5-hmC) [39, 40] may be of significance in this regard, because more 5-hmC has been found in brain than in other tissues  and 5-hmC is thought to affect gene regulation through DNA demethylation  or by converting 5-methylcytosine (5-mC) to 5-hmC [43–45]. Further investigation of the methylation status of the PWS-IC in brain samples is needed to determine whether the bisulfite-converted sites are protected by 5-hmC or 5-mC.
UBE3A transcript and protein levels were increased twofold on average in dup15q samples compared to controls in our study, consistent with the hypothesis that there is increased maternal allele-specific expression of UBE3A in dup15q autism brain. These levels were slightly lower than expected from maternally expressed genes with an average of three maternal alleles, but this may reflect the complex transcriptional and posttranslational regulation of UBE3A. The function of UBE3A as a transcriptional coactivator has been largely unexplored in the context of human genetic disease, but, in a Drosophila model of 15q duplication syndrome, elevated levels of an enzymatically defective version of Dube3a were able to induce transcription of the dopamine regulator GTP cyclohydrolase I and elevate dopamine levels in the fly brain . UBE3A can trans-ubiquitinate itself in vivo, leading to self-degradation, supporting the idea that there is an upper limit for UBE3A protein induction that may be reached in as few as two active copies of the duplicated region.
Dup15q sample 6,856 showed a 2.5-fold increase in UBE3A compared to no significant change as seen previously for a different brain region from this individual . Brain region differences in transcript levels within the same individual may explain some of the clinical heterogeneity seen within the dup15q syndrome. They may also potentially be explained by the stochastic nature of the epigenetic dysregulation. Interestingly, the epigenetic measure that best correlated with UBE3A levels in the dup15q brain samples was the level of PWS-IC methylation. Since the correlation was positive rather than negative, we hypothesize that maternal PWS-IC methylation acts as a long-range enhancer of UBE3A expression. The methyl-binding protein MeCP2 binds to the methylated PWS-IC allele [25, 31, 47, 48], and MECP2 mutation has been shown to correspond with reduced UBE3A and GABRB3 levels in human brain . Therefore, increased binding of MeCP2 to highly methylated PWS-IC in brain may act as a positive transcriptional regulator of UBE3A and, to a lesser extent, GABRB3 in human cortex.
In contrast to UBE3A, GABRB3 exhibited no significant change in the mean expression in the dup15q cortical samples compared to controls. Instead, significant variability in GABRB3 levels, as well as an interesting bimodal separation in GABRB3 levels of the dup15q samples, was observed in dup15q samples. This result is similar to our findings in a prior study of two samples with discordant GABRB3 levels , as well as the finding of reduced GABRB3 levels in 56% of autism cortex samples . SNRPN levels were decreased overall in all dup15q samples in this study, which deviates from our previous study result from sample 7014 at a different brain region, BA9, examined previously . This unexpected result of reduced SNRPN in dup15q postmortem cortex samples suggests that an increase in maternal dosage of the region epigenetically affects transcription of a paternally expressed gene, possibly in a tissue- or region-specific manner. In contrast to UBE3A and GABRB3, which positively correlated with PWS-IC methylation, SNRPN levels showed a negative correlation with PWS-IC methylation. These results suggest that although maternal methylation of the PWS-IC is repressive to SNRPN expression, as expected, there appears to be a long-range enhancing effect of PWS-IC methylation on UBE3A and GABRB3. Homologous chromosome pairing of maternal and paternal 15q11-q13 alleles occurs in human lymphocytes, neuronal cells and brain [23–25]. Both dup15q brain samples and a neuronal cell culture model of dup15q in SH-SY5Y neuronal cells showed significant disruption of homologous pairing that corresponded to reduced SNRPN and lower than expected GABRB3 levels [24, 26].
This study, together with previous studies of dup15q syndrome, shows that dup15q brain samples are epigenetically complex and that 15q11-q13 transcripts in brain do not behave solely as predicted by copy number. These findings should be important for understanding ASD cases with other de novo copy number variations on other chromosomes, in particular large duplications . The bimodal pattern of GABRB3 deficiencies seen in these 8 dup15q samples may provide some insight into the relationship between dup15q and seizures. Maternal UPD PWS individuals have a higher incidence of seizures than individuals with deletions , suggesting that these people may also have epigenetically induced GABRB3 deficiency. GABRB3 and UBE3A are well-characterized candidate genes for ASD because they are associated with normal brain development and have been shown to be reduced in idiopathic autism, Angelman syndrome and Rett syndrome . Although these results provide support for the hypothesis that overexpression of the maternally expressed UBE3A gene in the brain is the primary underlying cause of the ASD phenotype in dup15q, the changes in GABRB3 and SNRPN expression not predicted by copy number may also influence the phenotypic variability observed in ASD.
duplication of 15q11-q13
imprinting control locus
- int dup(15):
interstitial duplication 15q
GABAA receptor β3
glyceraldehyde 3-phosphate dehydrogenase
low copy repeat
methylation-sensitive high-resolution melting-curve analysis
imprinting center of the Prader-Willi locus
reverse transcriptase polymerase chain reaction
small nucleolar RNA
small nucleoriboprotein N
ubiquitin ligase 3A
We are grateful to all of the families who donated the tissues that made this study possible. This work was supported by National Institutes of Health awards R01 HD048799, 2R01HD041462, and R01 NS076263 (to JML), a Shainberg Neuroscience Fund award (to LTR) and a University of Tennessee Health Science Center Neuroscience Institute Fellowship (to NA).
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