Relationships between UBE3A and SNORD116 expression and features of autism in chromosome 15 imprinting disorders
Chromosome 15 (C15) imprinting disorders including Prader–Willi (PWS), Angelman (AS) and chromosome 15 duplication (Dup15q) syndromes are severe neurodevelopmental disorders caused by abnormal expression of genes from the 15q11–q13 region, associated with abnormal DNA methylation and/or copy number changes. This study compared changes in mRNA levels of UBE3A and SNORD116 located within the 15q11–q13 region between these disorders and their subtypes and related these to the clinical phenotypes. The study cohort included 58 participants affected with a C15 imprinting disorder (PWS = 27, AS = 21, Dup15q = 10) and 20 typically developing controls. Semi-quantitative analysis of mRNA from peripheral blood mononuclear cells (PBMCs) was performed using reverse transcription droplet digital polymerase chain reaction (PCR) for UBE3A and SNORD116 normalised to a panel of internal control genes determined using the geNorm approach. Participants completed an intellectual/developmental functioning assessment and the Autism Diagnostic Observation Schedule-2nd Edition. The Dup15q group was the only condition with significantly increased UBE3A mRNA levels when compared to the control group (p < 0.001). Both the AS and Dup15q groups also had significantly elevated SNORD116 mRNA levels compared to controls (AS: p < 0.0001; Dup15q: p = 0.002). Both UBE3A and SNORD116 mRNA levels were positively correlated with all developmental functioning scores in the deletion AS group (p < 0.001), and autism features (p < 0.001) in the non-deletion PWS group. The findings suggest presence of novel interactions between expression of UBE3A and SNORD116 in PBMCs and brain specific processes underlying motor and language impairments and autism features in these disorders.
Angelman syndrome (AS), Prader–Willi syndrome (PWS) and chromosome 15 duplication syndrome (Dup15q) are neurodevelopmental disorders that are associated with varying degrees of intellectual disability (ID) and social communication deficits1,2, and arise from different deletions or duplications at the 15q11–q13 imprinted region3.
PWS was the first example of genomic imprinting identified in humans4. Cardinal features include a poor suck with failure to thrive, infantile hypotonia and hypogonadism. Food seeking and hyperphagia emerges at approximately 6 years of age, leading to morbidity if not externally controlled. Mild ID (mean full scale IQ [FSIQ] between 55 and 69) is typical, frequently accompanied by compulsions, tantrums and skin picking5. AS is characterised by microcephaly, gait ataxia, seizures, ID, and absence of speech6. Dup15q is associated with variable cognitive impairment and motor delays. An overlapping feature between AS and Dup15q is the presence of seizures3.
DNA methylation and/or copy number changes on chromosome 15 are thought to cause PWS and AS specific phenotypes3,7. Loss of paternal gene expression from the chromosome 15q11–q13 region is the primary cause of PWS7, while the absence of the maternal gene expression in the same region is the primary cause of AS3. For PWS the lack of expression of key genes result from: (i) two deletion subtypes (typical—type I and type II deletions; and atypical smaller or larger 15q deletions) in ~60% of cases; (ii) three maternal disomy subtypes in ~35% of cases; and (ii) two imprinting centre defects (ICD; epimutation and microdeletion) in ~5% of cases5,8. Similarly, deletions from the maternally contributed chromosome 15 are the most common cause of AS (~70% of cases). Paternal uniparental disomy (patUPD) occurs in approximately 8% of AS cases and ICD in approximately 7% of cases3. Approximately, 10% of AS cases result from a mutation in the ubiquitin-protein ligase E3A gene (UBE3A). Both PWS and AS have a frequency of approximately 1 in 15,000 births9.
Dup15q syndrome results from duplications or triplications of the PWS/AS imprinted 15q11–q13 region. Triplication typically arises through the presence of a supernumerary chromosome (isodicentric 15 [idic15]), while the duplication is caused by interstitial tandem duplication (int dup). Hereafter, we use Dup15q to encompass these subtypes, unless otherwise stated. In maternal Dup15q, autism features are more common and severe, as compared to AS and PWS, with severity directly proportional to the number of maternal copies present9. In contrast, paternal Dup15q has a less severe phenotype than maternal Dup15q10. Despite Dup15q being a cause of autism spectrum disorder (ASD), reported in 1–3% of ASD cases9, prevalence in the general population has not been well established, with one study reporting 1 in 14,000 in the general population11.
While some genotype–phenotype correlations have emerged in each of the syndromes, primarily around the different molecular classes, there is a need for peripheral tissue biomarkers in humans as the phenotypes are highly variable in each disorder, and their specific subtypes do not fully explain this variability12. For PWS, those with the typical 15q11–q13 deletions have been reported to have lower Verbal IQ (VIQ) scores than those with matUPD13. In addition, PWS individuals with the larger typical 15q11–q13 type I deletion involving chromosome 15 breakpoints BP1 and BP3 have been reported to have more behavioural problems, specifically self-injury and compulsions compared to those having the smaller typical 15q11–q13 type II deletion involving breakpoints BP2 and BP314. The larger type I deletion encompasses four extra genes (i.e., NIPA2, NIPA1, GCP5, and CYFIP1), which may account for the additional clinical findings.
For AS, mouse models have been used to implicate loss of UBE3A expression in the brain as the primary cause of specific deficits in AS15. However, few studies have been performed in human peripheral tissues16,17. UBE3A is a key gene in neurodevelopment and is thought to be imprinted only in neurons, where it is expressed from the maternal allele in humans and mice15. Interestingly in other cell types UBE3A has bi-allelic expression18 and is thought to be consistently expressed from both alleles in different peripheral tissues15. The silencing of UBE3A on the paternal allele in neurons is thought to be regulated by a paternally expressed antisense transcript of UBE3A. This antisense transcript is part of the 3′end of SNRPN–SNURF transcript that comprises multiple small nuclear RNAs (snoRNAs)19. SnoRNA C/D box cluster 116 (SNORD116) is one of the snoRNAs that serves as a precursor to the antisense UBE3A, and through this process may regulate UBE3A silencing on the paternal allele16. Importantly, in PWS SNORD116 has been reported to be completely silenced, in neurons as well as other cell types in peripheral tissues, and this may contribute to phenotype severity17.
This study, for the first time, examines UBE3A and SNORD116 mRNA levels (controlled for the allele copy number and subtype) in peripheral blood mononuclear cells (PBMCs) using a highly sensitive and quantitative droplet digital PCR (ddPCR) method developed for analysis of gene expression. Expression changes for these genes in PBMCs are investigated between the chromosome 15 imprinting disorders, the different subtypes and typically developing controls. For AS and PWS subtypes genotype–phenotype studies are also described, with the focus on relationships between the UBE3A and SNORD116 mRNA levels in PBMCs and brain specific phenotypes including formal assessments targeting behavioural features and intellectual functioning. It was hypothesised that expression of these genes in PBMCs reflects immune processes in the brain related to gene expression in microglial cells (of the same cellular lineage) that support neuronal processes in the brain related to intellectual/developmental functioning and autism features in these disorders20.