Gender influences monoallelic expression of ATP10A in human brain

Abstract

Human chromosome 15q11-13 and the syntenic region of mouse chromosome 7 contain multiple imprinted genes necessary for proper neurodevelopment. Due to imprinting, paternal 15q11-13 deficiencies lead to Prader-Willi syndrome (PWS) while maternal 15q11-13 deficiencies cause Angelman syndrome (AS). The mechanisms involved in parental imprinting of this locus are conserved between human and mouse, yet inconsistencies exist in reports of imprinting of the maternally expressed gene Atp10a/ATP10A. Excess maternal 15q11-13 dosage often leads to autism-spectrum disorder therefore further investigation to characterize the true imprinting status of ATP10A in humans was warranted. In this study, we examined allelic expression of ATP10A transcript in 16 control brain samples, and found that 10/16 exhibited biallelic expression while only 6/16 showed monoallelic expression. Contrary to the expectation for a maternally expressed imprinted gene, quantitative RT-PCR revealed significantly reduced ATP10A transcript in Prader-Willi syndrome brains with two maternal chromosomes due to uniparental disomy (PWS UPD). Furthermore, a PWS UPD brain sample with monoallelic ATP10A expression demonstrated that monoallelic expression can be independent of imprinting. Investigation of factors that may influence allelic ATP10A expression status revealed that gender has a major affect, as females were significantly more likely to have monoallelic ATP10A expression than males. Regulatory sequences were also examined, and a promoter polymorphism that disrupts binding of the transcription factor Sp1 also potentially contributes to allelic expression differences in females. Our results show that monoallelic expression of human ATP10A is variable in the population and is influenced by both gender and common genetic variation.

Figure 1

Analysis of ATP10A allelic expression in cerebral cortex. (a) Genomic DNA (upper panel) and cDNA (lower panel) from heterozygous control brain samples was digested with AclI to examine allelic expression. Upper band corresponds to uncut C allele and lower band corresponds to digested G allele. cDNA band quantification shown below the image reflects the percent that each allele contributes to the total densitometry. (b) Sequencing chromatograms of ATP10A cDNA confirm the allelic expression shown in (a). Arrows indicate the SNP differentially cleaved by AclI. (c) qRT-PCR analysis of ATP10A transcript in controls (n = 8), Angelman syndrome deletions (n = 2), Prader-Willi syndrome deletions (n = 2), Prader-Willi syndrome maternal uniparental disomy (n = 2), and Rett syndrome (n = 5). Error bars represent the S.E.M. for each category with significance values, * p < 0.02 and ** p < 0.001.

Figure 2

Allelic expression analysis of ATP10A in PWS UPD brain. Sequencing chromatograms of ATP10A brain gDNA (upper panels) show that both PWS UPD individuals are heterozygous for polymorphisms within ATP10A. Sequencing of ATP10A cDNA (lower panels) reveals that PWS 1447 showed biallelic expression while PWS 1290 exhibited monoallelic expression of ATP10A.

Figure 3

DNA methylation analysis of ATP10A regulatory sequences. (a) Schematic of ATP10A gene organization with vertical lines representing coding exons. Gray boxes indicate the relative locations of the 5′ CpG island and the intronic MeCP2 binding site. (b) Bisulfite sequencing of the 5′ CpG island in PWS 1290 and PWS 1447. Each horizontal line represents an individual clone, with filled circles representing methyled CpG sites and unfilled circles representing unmethylated CpG sites. (c) Bisulfite sequencing results from the MeCP2 binding site with polymorphic CpG sites indicated with stars (site 3 and site 7). A control sample heterozygous for polymorphisms in site 3 and site 7 shows that both sites are normally methylated. PWS 1290 lacks both polymorphic CpG sites while PWS 1447 lacks only site 3.

Figure 4

ATP10A CpG island methyation in control brain. Bisulfite sequencing was used to assess methylation of the ATP10A 5′ CpG island in control brain samples with monoallelic (light grey) and biallelic (dark grey) ATP10A expression. Percent methylation represents the number of methylated CpG sites divided by the total number of possible CpG sites for all clones analyzed (10-12 clones per individual).