The E6-Ap ubiquitin-protein ligase (UBE3A) gene is localized within a narrowed Angelman syndrome critical region

Abstract

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct clinical phenotypes resulting from maternal and paternal deficiencies, respectively, in human chromosome 15qll-q13. Although several imprinted, paternally expressed transcripts have been identified within the PWS candidate region, no maternally expressed gene has yet been identified within the AS candidate region. We have developed an integrated physical map spanning the PWS and AS candidate regions and localized two breakpoints, including a cryptic t(14;15) translocation associated with AS and a non-AS 15q deletion, which substantially narrow the AS candidate region to approximately 250 kb. Mapping data indicate that the entire transcriptional unit of the E6-AP ubiquitin-protein ligase (UBE3A) gene lies within the AS region. The UBE3A locus expresses a transcript of approximately 5 kb at low to moderate levels in all tissues tested. The mouse homolog of UBE3A was cloned and sequenced revealing a high degree of conservation at nucleotide and protein levels. Northern and RT-PCR analysis of Ube3a expression in mouse tissues from animals with segmental, paternal uniparental disomy failed to detect substantially reduced or absent expression compared to control animals, failing to provide any evidence for maternal-specific expression from this locus. Recent identification of de novo truncating mutations in UBE3A taken with these observations indicates that mutations in UBE3A can lead to AS and suggests that this locus may encode both imprinted and biallelically expressed products.

Figure 1

(A) A schematic physical map of the 15q11–q13 PWS/AS common deletion interval is shown with the centromere toward the left and the telomere toward the right. Genes and genomic markers are shown in boxes. Sites of differential methylation are indicated by asterisks (*) over PW71 and SNRPN. Wavy vertical lines represent chromosomal breakpoints. The common PWS and AS deletion breakpoints are near either end of the map, and the Se family centromeric deletion breakpoint maps between SRRPN and UBE3A, defining the PWS and AS candidate regions, indicated over the map. Breakpoints in the 15q non-AS deletion case and the t(14;15) translocation case, together with the Se breakpoint, define the narrowed AS critical region indicated above UBE3A. (B) YAC, PAC, cosmid, STS, and gene map of the ∼1-Mb region surrounding the Se breakpoint. Genomic clones are indicated by horizontal lines; gene and STS markers are indicated by broken vertical lines.

Figure 2

Detection of the t(14;15) cryptic translocation breakpoints. An 8-kb EcoRI fragment from cosmid 24 detects a single translocation junction fragment in EcoRI-digested DNA from the mother but not from the child, who is unbalanced. Both der(14) and der(15) breakpoints are seen in the HindIII panel, but the unbalanced, affected child has only the der(14) and not the der(15) chromosome, therefore displaying only one of the two breakpoint fragments seen in the mother.

Figure 3

Northern analysis of UBE3A gene expression. Hybridization of UBE3A and control GAP3D cDNA probes to a Northern filter (Clontech) containing 2 μg poly(A)⁺ RNA per lane is shown.

Figure 4

Comparison of peptide sequences for human and mouse UBE3A. The deduced peptide sequence of the mouse cDNA was compared to the published human peptide sequence, using the Wisconsin package; this analysis revealed 94% amino acid identity between human and mouse for UBE3A.

Figure 5

Northern analysis of Ube3a gene expression in mouse tissues from control and paternal UPD mice using probes corresponding to the coding region (A) and the 3′ UTR region (B). Comparison to the ethidium bromide-stained gel (not shown) reveals no significant difference between the control and UPD animals.