Imprinted expression of ATP10C in brain. a, RT-PCR of samples from normal brain, PWS brain, and AS brain, demonstrating minimal expression of ATP10C in samples from AS brain but normal levels of expression in samples from PWS brain, suggesting preferential maternal expression of ATP10C. ATP10C primers, as indicated in figure 1 as follows: A, 5′-TGGTGCACAGAACCCAGAGC; B, 5′-AATCGAAACCCAGTGTGTGC (755 bp); C, 5′-ATTCTTCACGGGCATTGGTGC; and D, 5′-TTCCTGGTCAACTGACGTGC (715 bp). Other primers are as follows: APBA2, 5′F-TGGACAAACCACCAATAGGC and 5′R-ATCTTCTTCCTGGTCATGGGC (696 bp); SNRPN, 5′F-GCTCCATCTACTCTTTGAAGC and 5′R-CTTTTCTTCACGCTCTGGTTGC (338 bp); UBE3A, 5′F-CTCTTCTTGCAGTTTACAACG and 5′R-CTTGAGTATTCCGGAAGTAAAAGC (152 bp). Δ = 15q11-13 deletion (in fig. 1a, the deletion including BP2-BP3); UPD = chromosome 15 uniparental disomy; OC = occipital cortex; TC = temporal cortex; Br = total brain. b, Sequence analyses of RT-PCR products for ATP10C exon 21 (left-side traces) and exon 9 (right-side traces) polymorphisms, demonstrating preferential expression of a single allele in all brain regions tested. As shown in the top row, both nucleotides are detected in genomic DNA of heterozygous, normal individuals, with the trace for the 4582 polymorphism typically overrepresenting the “G” allele and with the trace for the 1728 polymorphism detecting both nucleotides at roughly equal levels (“N”). Sequence traces from RT-PCR products from brain samples of normal individuals 3253 and 1858 show preferential expression of a single allele in all regions tested. The normal fibroblast line GM00242 demonstrates slight but consistent preferential expression of the maternal “A” allele; parental origin of alleles for individuals 3253 and 1858 could not be determined. Duplicate RNA isolates for each individual/brain region were reverse-transcribed, PCR was performed with several primer combinations, and the various PCR products were sequenced (not shown); preferential expression of the same allele (4582 “A” in 3253 and 1728 “T” in 1858) was observed in almost all products sequenced. FC = frontal cortex; H = hippocampus; SPC = superior parietal cortex; CH = cerebellar hemisphere; SV = superior vermis; FB = fibroblast.

Genomic and physical map of ATP10C. a, Diagram showing that ATP10C maps immediately distal to UBE3A and is transcribed in the same orientation. Imprinted genes are represented by shaded (in the case of maternal expression) and blackened boxes (in the case of paternal expression). Genomic-sequence contigs overlapping ATP10C are shown below the map (where they are denoted by the prefix “NT_”). b, Diagram showing that ATP10C comprises 21 exons spanning >160 kb. Exon sizes (in bp) are shown above the gene, and intron sizes (in kb) are shown below the gene. Hatched exons represent sequence present in the KIAA0566 EST AB011138; shaded exons represent sequence present in Image clone 784559. Alternate splicing of exons 19–21 is indicated above the gene; the translation start site in exon 1 is indicated by an arrow. Contig sequence/RPCI-11 BACs spanning ATP10C are shown below the gene. Genomic sequences from BAC clones are indicated by solid black lines, which are broken by a slash at sites of sequence gaps. Sequence contigs (denoted by the prefix “NT_”) and sequence accession numbers (AC) also are shown; an asterisk (*) indicates sequence not corresponding to its assigned BAC. A–D are primers used in RT-PCR of an imprinted-expression panel (fig. 2a). The solid-black star indicates exon 9 polymorphism 1728 C→T (T→M); the star containing a white center indicates exon 21 polymorphism 4582 A→G.