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The Human Aminophospholipid-Transporting ATPase Gene ATP10C Maps Adjacent to UBE3A and Exhibits Similar Imprinted Expression

2001; Elsevier BV; Volume: 68; Issue: 6 Linguagem: Inglês

10.1086/320616

1537-6605

Laura B. K. Herzing, Soo‐Jeong Kim, Edwin H. Cook, David H. Ledbetter,

Genomic variations and chromosomal abnormalities

Maternal duplications of the imprinted 15q11-13 domain result in an estimated 1%–2% of autism-spectrum disorders, and linkage to autism has been identified within 15q12-13. UBE3A, the Angelman syndrome gene, has, to date, been the only maternally expressed, imprinted gene identified within this region, but mutations have not been found in autistic patients. Here we describe the characterization of ATP10C, a new human imprinted gene, which encodes a putative protein homologous to the mouse aminophospholipid-transporting ATPase Atp10c. ATP10C maps within 200 kb distal to UBE3A and, like UBE3A, also demonstrates imprinted, preferential maternal expression in human brain. The location and imprinted expression of ATP10C thus make it a candidate for chromosome 15–associated autism and suggest that it may contribute to the Angelman syndrome phenotype. Maternal duplications of the imprinted 15q11-13 domain result in an estimated 1%–2% of autism-spectrum disorders, and linkage to autism has been identified within 15q12-13. UBE3A, the Angelman syndrome gene, has, to date, been the only maternally expressed, imprinted gene identified within this region, but mutations have not been found in autistic patients. Here we describe the characterization of ATP10C, a new human imprinted gene, which encodes a putative protein homologous to the mouse aminophospholipid-transporting ATPase Atp10c. ATP10C maps within 200 kb distal to UBE3A and, like UBE3A, also demonstrates imprinted, preferential maternal expression in human brain. The location and imprinted expression of ATP10C thus make it a candidate for chromosome 15–associated autism and suggest that it may contribute to the Angelman syndrome phenotype. Most of the imprinted transcripts that, to date, have been identified within 15q11-13 (fig. 1a) exhibit exclusive paternal expression, loss of which results in the contiguous gene–deletion syndrome Prader-Willi syndrome (PWS). Mutation of UBE3A, the only known maternally expressed gene in this region, is sufficient to cause the classic Angelman syndrome (AS) phenotype (MIM 105830) (Kishino et al. Kishino et al., 1997Kishino T Lalande M Wagstaff J UBE3A/E6-AP mutations cause Angelman syndrome.Nat Genet. 1997; 15: 70-73Crossref PubMed Scopus (939) Google Scholar; Matsuura et al. Matsuura et al., 1997Matsuura T Sutcliffe JS Fang P Galjaard RJ Jiang YH Benton CS Rommens JM Beaudet AL De novo truncating mutations in E6-AP ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome.Nat Genet. 1997; 15: 74-77Crossref PubMed Scopus (635) Google Scholar), although the phenotype of patients with AS and a 15q11-13 deletion is more severe (Cassidy et al. Cassidy et al., 2000Cassidy S Dykens E Williams C Prader-Willi and Angelman syndromes: sister imprinted disorders.Am J Med Genet. 2000; 97: 136-146Crossref PubMed Scopus (213) Google Scholar). Paternal duplication of 15q11-13 has no obvious phenotype, but maternal duplication results in a spectrum of phenotypes, ranging from language delay to autism (MIM 209850) (Browne et al. Browne et al., 1997Browne CE Dennis NR Maher E Long FL Nicholson JC Sillibourne J Barber JCK Inherited interstitial duplications of proximal 15q: genotype-phenotype correlations.Am J Hum Genet. 1997; 61: 1342-1352Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar; Cook et al. Cook et al., 1997Cook Jr, EH Lindgren V Leventhal BL Courchesne R Lincoln A Shulman C Lord C Courchesne E Autism or atypical autism in maternally but not paternally derived proximal 15q duplication.Am J Hum Genet. 1997; 60: 928-934PubMed Google Scholar). The phenotype is more severe in proportion to the number of intrachromosomal maternal copies of this segment, and patients carrying a marker chromosome containing two additional copies of maternal 15q11-13 have AS-like features (Wolpert et al. Wolpert et al., 2000Wolpert CM Menold MM Bass MP Qumsiyeh MB Donnelly SL Ravan SA Vance JM Gilbert JR Abramson RK Wright HH Cuccaro ML Pericak-Vance MA Three probands with autistic disorder and isodicentric chromosome 15.Am J Med Genet. 2000; 96: 365-372Crossref PubMed Scopus (79) Google Scholar). Autism may also be a component of AS (Steffenburg et al. Steffenburg et al., 1996Steffenburg S Gillberg CL Steffenburg U Kyllerman M Autism in Angelman syndrome: a population-based study.Pediatr Neurol. 1996; 14: 131-136Abstract Full Text PDF PubMed Scopus (182) Google Scholar). The association of these autism-spectrum phenotypes with UBE3A is unclear, since mutations in UBE3A have not been found in karyotypically normal autistic individuals (Veenstra-VanderWeele et al. Veenstra-VanderWeele et al., 1999Veenstra-VanderWeele J Gonen D Leventhal BL Cook EH Mutation screening of the UBE3A/E6-AP gene in autistic disorder.Mol Psychiatry. 1999; 4: 64-67Crossref PubMed Scopus (46) Google Scholar), and linkage analyses suggest the existence of candidate loci between UBE3A and D15S156 (Cook et al. Cook et al., 1998Cook Jr, EH Courchesne RY Cox NJ Lord C Gonen D Guter SJ Lincoln A Nix K Haas R Leventhal BL Courchesne E Linkage-disequilibrium mapping of autistic disorder, with 15q11-13 markers.Am J Hum Genet. 1998; 62: 1077-1083Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar; Bass et al. Bass et al., 2000Bass MP Menold MM Wolpert CM Donnelly SL Ravan SA Hauser ER Maddox LO Vance JM Abramson RK Wright HH Gilbert JR Cuccaro ML DeLong GR Pericak-Vance MA Genetic studies in autistic disorder and chromosome 15.Neurogenetics. 2000; 2: 219-226Crossref PubMed Scopus (89) Google Scholar). Together, these results support a hypothesis that another gene in this region may play a role in some forms of autism and in modification of the AS phenotype. In the course of mapping the 15q11-13 region (Christian et al. Christian et al., 1998Christian SL Bhatt NK Martin SA Sutcliffe JS Kubota T Huang B Mutirangura A Chinault AC Beaudet AL Ledbetter DH Integrated YAC contig map of the Prader-Willi/Angelman region on chromosome 15q11-q13 with average STS spacing of 35 kb.Genome Res. 1998; 2: 146-157Google Scholar), our laboratory had localized STS-N35112, contained within the expressed-sequence tag (EST) KIAA0566 (AB011138) (Ishikawa et al. Ishikawa et al., 1998Ishikawa K Nagase T Suyama M Miyajima N Tanaka A Kotani H Nomura N Ohara O Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro.DNA Res. 1998; 5: 169-176Crossref PubMed Scopus (168) Google Scholar), to the ∼1-Mb interval between SNRPN and D15S986, by minimal-tile YAC mapping. The sequence of KIAA0566 is contained within AC023449.3 and AC020639.16, which were used to identify 3′ exon-intron boundaries of the KIAA0566 transcript. Sequence-homology database searches have identified a further six bacterial artificial chromosomes (BACs), which both hybridize, by FISH, to chromosome 15 and are positive, by PCR, for KIAA0566 exons (not shown). Comparisons between Unigene Resources ESTs (accession number Hs.44697) identified a single 3′ splice variant (fig. 1b). KIAA0566 corresponds to the putative 3′ end of the human ATP10C gene (LocusLink accession number 10080; also, numerous GenBank accession numbers, which are available at the GenBank Overview web site and are listed below in the “Electronic Database Information” section). The 5′ portion of the complete ATP10C transcript was reconstructed by the sequencing of Image clone 784559, which partially overlaps the 5′ end of KIAA0566, revealing a further 615 bp of upstream open reading frame (ORF). This sequence in turn overlaps RPCI-11 BAC 2C7 (AC016266.5), and analysis of the 2C7 flanking sequence suggested a further extension of the ORF, including a methionine, resulting in a gene of 21 exons encoding a putative protein of 1,499 amino acids. Expression of RNA corresponding to the complete ORF from fibroblasts and various brain regions has been confirmed by reverse-transcriptase PCR (RT-PCR). Comparison of the human ATP10C sequence with that of the mouse P-ATPase class V homologue (AF156549) (Halleck et al. Halleck et al., 1999Halleck MS Lawler Jr, JF Blackshaw S Gao L Nagarajan P Hacker C Pyle S Newman JT Nakanishi Y Ando H Weinstock D Williamson P Schlegel RA Differential expression of putative transbilayer amphipath transporters.Physiol Genomics. 1999; 1: 139-150Crossref PubMed Scopus (68) Google Scholar), recently mapped to the syntenic mouse chromosome 7 (Dhar et al. Dhar et al., 2000Dhar M Webb LS Smith L Hauser L Johnson D West DB A novel ATPase on mouse chromosome 7 is a candidate gene for increased body fat.Physiol Genomics. 2000; 4: 93-100Crossref PubMed Google Scholar), yields a total similarity, between the full-length mouse and human genes, of 84% at the nucleotide level and 80% at the protein level. Genomic sequence (AC023449.3) overlapping exons 5–21 of ATP10C is contained within the chromosome 15 contig NT_010364, which contains UBE3A and thus indicates that ATP10C transcription is in the same orientation as that of UBE3A (fig. 1a). The sequence of 5′ATP10C, including exon 2, is contained within the chromosome 15 contig NT_010345. To complete the contig across ATP10C, we identified BACs RPCI-11 259N18 and CIT-D 2060C24, by BAC end-sequence homology. These BACs both hybridize, by FISH, to chromosome 15 and are positive, by PCR, for exons 2–3 and exons 2–-21, respectively, and complete the chromosome 15 genomic contig from PIX1 through GABRB3 (fig. 1b). To determine the imprint status of ATP10C, we tested intron-spanning primer pairs (e.g., C and D or A and B; see fig. 1b) on a chromosome 15–expression panel, comprising cDNA from samples of normal brain, PWS brain, and AS brain, which express, respectively, both the maternal and paternal 15q11-13 gene alleles, the maternal allele only, or the paternal allele only. The expression panel demonstrated that there was a paucity of expression from AS brain samples (fig. 2a), suggesting preferential maternal expression and highlighting ATP10C as a second imprinted, maternally expressed gene in 15q11-13. No difference in ATP10C transcription level is apparent in a fibroblast expression panel (not shown). Similarly, by this assay, UBE3A also exhibits only exclusively maternal expression in adult brain (Rougeulle et al. Rougeulle et al., 1997Rougeulle C Glatt H Lalande M The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain.Nat Genet. 1997; 17: 14-15Crossref PubMed Scopus (309) Google Scholar).Figure 2Imprinted 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ongoing sequence analysis of ATP10C in a cohort of autistic and normal patients has identified two polymorphisms, one a silent change in exon 21 and the other resulting in a threonine-to-methionine amino acid change in exon 9 (figs. 1b and 2b). Sequencing of the RT-PCR products from five heterozygous, normal individuals demonstrated consistent preferential expression of one allele in each case. Discrepancy in peak heights for each allele was most marked in the samples from various brain regions from the two individuals from whom such tissue was available, although it was not possible to determine the parental origin of the preferentially expressed allele. The allele-signal differences between genomic and cDNA samples from fibroblasts were less marked but also consistent and, in the line (GM00242; Coriell Cell Repository) where the allele origin was known, it was the maternal allele that was preferentially expressed (fig. 2b). This suggests that regulation of the degree of differential ATP10C imprinted expression is tissue specific, whereas the imprint itself may be more ubiquitous. Imprinting of ATP10C expression is further supported by the observation of an obesity phenotype in mouse, occurring only with maternal p-locus deletions that span the region containing the mouse homologue, pfatp (Dhar et al. Dhar et al., 2000Dhar M Webb LS Smith L Hauser L Johnson D West DB A novel ATPase on mouse chromosome 7 is a candidate gene for increased body fat.Physiol Genomics. 2000; 4: 93-100Crossref PubMed Google Scholar). Taken together with the minimal expression in AS brain, these results indicate that ATP10C represents the second in a cluster of maternally expressed imprinted genes in 15q11-13. Work is currently ongoing to determine whether imprinted ATP10C expression is regulated by the SNRPN Imprinting Center (IC) in a manner similar to the regulation of UBE3A expression. ATP10C is an interesting candidate for the autism-spectrum phenotypes associated with chromosome 15 and maternal rearrangements thereof, as well as for contributing to the severity of deletion-associated AS. In addition to its map location and imprint status, the putative function of ATP10C as an aminophospholipid-transporting ATPase would involve it in maintenance of cell membrane integrity (Halleck et al. Halleck et al., 1998Halleck MS Pradhan D Blackman C Berkes C Williamson P Schlegel RA Multiple members of a third subfamily of P-type ATPases identified by genomic sequences and ESTs.Genome Res. 1998; 8: 354-361Crossref PubMed Scopus (52) Google Scholar), and it may therefore be critical for cell signaling in the CNS. Indeed, loss of members of another subfamily of P-type ATPases is responsible for Menkes disease and Wilson disease (DiDonato and Sarkar DiDonato and Sarkar, 1997DiDonato M Sarkar B Copper transport and its alterations in Menkes and Wilson diseases.Biochim Biophys Acta. 1997; 1360: 3-16Crossref PubMed Scopus (161) Google Scholar). Specific localization of Atp10c transcript in mouse brain to cerebellar granule cells, the hippocampus, and cells surrounding the corpus callosum (Halleck et al. Halleck et al., 1999Halleck MS Lawler Jr, JF Blackshaw S Gao L Nagarajan P Hacker C Pyle S Newman JT Nakanishi Y Ando H Weinstock D Williamson P Schlegel RA Differential expression of putative transbilayer amphipath transporters.Physiol Genomics. 1999; 1: 139-150Crossref PubMed Scopus (68) Google Scholar) is especially intriguing, since all these areas (a) have been suggested as being preferentially involved in autism (Lord et al. Lord et al., 2000Lord C Cook EH Leventhal BL Amaral DG Autism spectrum disorders.Neuron. 2000; 28: 355-363Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar) and (b) overlap regions of imprinted Ube3a expression in mouse (Albrecht et al. Albrecht et al., 1997Albrecht U Sutcliffe JS Cattanach BM Beechey CV Armstrong D Eichele G Beaudet AL Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.Nat Genet. 1997; 17: 75-78Crossref PubMed Scopus (378) Google Scholar). Identification of imprinted ATP10C expression therefore paves the way both for more-extensive analysis of chromosome 15 imprinting mechanisms and for further understanding of the etiology of neurologic defects in AS and autism. Note added in proof.— As this report was in press, Meguro et al. (Meguro et al., 2001Meguro M Kashiwagi A Mitsuya K Nakao M Kondo I Saitoh S Oshimura M A novel maternally expressed gene, ATP10C, encodes a putative aminophospholipid translocase associated with Angelman syndrome.Nat Genet. 2001; 28: 19-20Crossref PubMed Google Scholar) reported imprinted expression of ATP10C in lymphoblasts from patients with AS and deletion of either 15q11-13 or IC. We thank S. L. Christian for initial STS (sequence-tagged site) mapping and for sharing her depth of chromosome 15 knowledge. We thank S. Zhang and J. Chung for excellent technical assistance. Samples from AS brain were a generous gift from D. Driscoll. Samples from normal brain and PWS brain were obtained, in part, via Miami Brain and Tissue Bank for Developmental Disorders contract NOI-HD-8-3284. Support for L.B.K.H. was provided by a Cure Autism Now (CAN) Foundation Young Investigator Award. Research funds were provided in part by the International Rett Syndrome Association and by National Institutes of Health grants R01 HD36111 (to D.H.L.) and R01 MH52223 and K02 MH01389 (both to E.H.C.).