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. 1999 Jul 6;96(14):8064-9.
doi: 10.1073/pnas.96.14.8064.

A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome

N J Smilinich  1 , C D Day, G V Fitzpatrick, G M Caldwell, A C Lossie, P R Cooper, A C Smallwood, J A Joyce, P N Schofield, W Reik, R D Nicholls, R Weksberg, D J Driscoll, E R Maher, T B Shows, M J Higgins
Affiliations

A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome

N J Smilinich et al. Proc Natl Acad Sci U S A. .

Abstract

Loss of imprinting at IGF2, generally through an H19-independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith-Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island (KvDMR1) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2. Reverse transcription-PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1-associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and/or its associated antisense RNA (KvLQT1-AS) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains.

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Figures

Figure 1
Figure 1
Map of the 1,000-kb 11p15.5 imprinted domain. The relative location and sizes of genes are drawn approximately to scale, with genes shown to be regulated by genomic imprinting as solid black boxes. Imprinted expression of ORCTL2S, TAPA1(CD81), TH, and INS (gray boxes) has not yet been established, although Tapa1 and Ins have restricted allele-specific expression in the mouse (22). Presented below the map is an enlargement of the KvLQT1 locus showing its exon–intron structure (nomenclature from Lee et al.; ref. 38) as determined by comparison of the mRNA (GenBank accession no. U89364) and genomic sequences (GenBank accession nos. AC001228, AC003675, U90095, AC002403, AC000377, and AC003693), and the position of the intronic, differentially methylated CpG island KvDMR1 and NotI (N) sites. The direction of transcription of the maternally expressed KvLQT1 gene and the paternally expressed antisense transcript (KvLQT1-AS) are indicated. The approximate locations of the Beckwith–Wiedemann syndrome (BWS) and rhabdoid tumor (Rhd) 11p15.5 rearrangement breakpoints are shown. cen, centromere; tel, telomere.
Figure 2
Figure 2
The human and mouse KvDMR1 loci. (a) Physical map of the human locus showing restriction sites with respect to hybridization probes DMRP and BX2 (solid black boxes), the KvDMR1 CpG island, the position of two direct repeat structures, and several ESTs transcribed in the opposite (antisense) direction compared with KvLQT1. The dashed line connecting these ESTs indicates that amplification products joining these cDNA fragments could be obtained by RT-PCR by using EST-specific primers. The bidirectional arrows (primer names indicated above) correspond to positive RT-PCR assays used to “sample” the genomic DNA sequence for expressed sequences. (b) Physical map of the syntenic mouse locus (same symbols as in a). A region of homology (83% identity over 437 bp) between the mouse and human loci is indicated by the striped box (the open end indicates that the downstream extent of homology is unknown, as the mouse sequence is not complete).
Figure 3
Figure 3
Differential methylation of KvDMR1. (a) DNA isolated from normal lymphoblastoid (GM00131, GM07048, and GM06991), peripheral blood lymphocyte (802, 804, and 808), or fibroblast (GM08333) cells was digested with EcoRI and NotI and hybridized with DMRP (see Fig. 2a). (b) Southern blot of BamHI/NotI-digested DNA from Wilms’ tumors (WT) with loss of heterozygosity (LOH) or without LOH (no LOH) in 11p15 and hybridized with the BX2 probe (Fig. 2a). (c) Hybridization of sc34 to HindIII (H) and HindIII/EagI (H/E) double digests of adult kidney DNA from reciprocal [C57BL/6J (B6) × PWK]F1 animals (for F1 hybrids, the maternal parent is specified first). (d) The BX2 probe was hybridized to EcoRI/NotI digests of human DNA from somatic tissues (peripheral blood lymphocytes or brain), testes (ts), sperm (sp), and fetal ovaries (ov). The ratio of the intensity of the upper and lower bands is shown in the accompanying histogram. The faint band at 3–3.5 kb present in all lanes is a cross-hybridizing locus observed when experiments are done at reduced stringency. The additional band seen in the sperm lane is likely due to a heterogeneous methylation at this cross-reacting locus.
Figure 4
Figure 4
Expression of an antisense transcript associated with KvDMR1. (a) RNA was isolated from human fetal tissues and analyzed by RT-PCR with the indicated primer pairs (see Fig. 2a for location of primers). Primers 686.3 and 592.20 were designed from EST 68627 and EST 592241, respectively, and were used to show the connection of these two ESTs. The + and − indicate that the PCR templates were from cDNA (+RT) or mock cDNA (−RT), respectively. (b, Upper) Southern blot of BamHI/NotI-digested DNA from a panel of eight single human chromosome 11 somatic cell hybrids (47) hybridized with DMRP. (b, Lower) RT-PCR analysis of same hybrids with primers specific for EST 592241. (c) Paternal-specific expression at the mouse KvDMR1 locus. Arrows indicate the presence of both alleles in DNA from F1 animals. In F1 fetal liver RNA from a C57BL/6J × PWK mating, as well as in the reciprocal cross, only the paternal allele was detected. The weak bands in the −RT lane result from contaminating DNA in the RNA samples, because the PCR primers do not amplify across an intron.
Figure 5
Figure 5
(a) Southern blot of EcoRI/NotI digests of DNA from patients with nonrearrangement BWS and normal controls hybridized with DMRP. Densitometric analysis showed an increase in the cleaved unmethylated (paternal) band with respect to the uncleaved methylated (maternal) band in patients with BWS known to have paternal UPD (lanes 1 and 2) compared with normal individuals (lanes 12 and 13). Methylation at KvDMR1 was absent in the patients with BWS shown in lanes 3, 5, and 8. (b) Loss of imprinting at IGF2 and KvDMR1 in a BWS-associated inv(11). (Left) PCR and RT-PCR analysis at the AvaII/ApaI polymorphism in IGF2 (48). The lane labeled DNA AvaII illustrates that the BWS inv(11) cells and normal skin fibroblast control cells are heterozygous for the AvaII site. The presence of both alleles in the RNA +RT AvaII lane indicates that IGF2 is biallelically expressed. +RT and −RT indicate that the PCR templates were from cDNA (+RT) or mock cDNA (−RT). m, monoallelic; b, biallelic. (Right) Southern hybridization of the DMRP probe to EcoRI/NotI digests of DNA from individuals with BWS with the inv(11), a t(11;22), and t(11;16), as well as a rhabdoid tumor with a t(11;22) (40), all of which disrupt KvLQT1 (39). DNA from the inv(11) fetus with BWS showed an absence of the methylated allele.
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