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].