Human Molecular Genetics

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Human Molecular Genetics

Pages 1209-1217

©1999 Oxford University Press

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LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids
Introduction
Results
   Screening for differentially expressed transcripts using monochromosomal hybrids
   Paternal expression of LIT1 RNA in the KvLQT1 locus
   Maternal methylation at the intronic CpG island in the KvLQT1 locus
   Loss of maternal methylation at the intronic CpG island in BWS patients
   Normal imprinting of LIT1 in Wilms' tumors
Discussion
Materials And Methods
   Cell lines
   EST screen
   Sequence analysis
   Expression analysis
   Assessment of allelic expression
   Methylation analysis
Acknowledgements
References

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LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids

Kohzoh Mitsuya¹, Makiko Meguro¹, Maxwell P. Lee², Motonobu Katoh¹, Thomas C. Schulz¹, Hiroyuki Kugoh¹, Mitsuaki A. Yoshida², Norio Niikawa³, Andrew P. Feinberg², Mitsuo Oshimura^{1, 5, *}

¹Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Nishimachi 86, Yonago, Tottori 683-8503, Japan, ²Departments of Medicine, Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 1064 Ross, 720 Rutland Avenue, Baltimore, MD 21205, USA, ³Department of Cytogenetics, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan, ⁴Department of Human Genetics, Nagasaki University School of Medicine, Nagasaki 852-8523, Japan and ⁵CREST, Japan Science and Technology Corp. (JST), Tokyo 170-0013, Japan.
Erratum

Received February 2, 1999; Revised and Accepted April 23, 1999

Mammalian imprinted genes are frequently arranged in clusters on particular chromosomes. The imprinting cluster on human chromosome 11p15 is associated with Beckwith-Wiedemann syndrome (BWS) and a variety of human cancers. To clarify the genomic organization of the imprinted cluster, an extensive screen for differentially expressed transcripts in the 11p15 region was performed using monochromosomal hybrids with a paternal or maternal human chromosome 11. Here we describe an imprinted antisense transcript identified within the KvLQT1 locus, which is associated with multiple balanced chromosomal rearrangements in BWS and an additional breakpoint in embryonal rhabdoid tumors. The transcript, called LIT1 (long QT intronic transcript 1), was expressed preferentially from the paternal allele and produced in most human tissues. Methylation analysis revealed that an intronic CpG island was specifically methylated on the silent maternal allele and that four of 13 BWS patients showed complete loss of maternal methylation at the CpG island, suggesting that antisense regulation is involved in the development of human disease. In addition, we found that eight of eight Wilms' tumors exhibited normal imprinting of LIT1 and five of five tumors displayed normal differential methylation at the intronic CpG island. This contrasts with five of six tumors showing loss of imprinting of IGF2. We conclude that the imprinted gene domain at the KvLQT1 locus is discordantly regulated in cancer from the imprinted domain at the IGF2 locus. Thus, this positional approach using human monochromosomal hybrids could contribute to the efficient identification of imprinted loci in humans.

INTRODUCTION

Genomic imprinting is an epigenetic modification leading to the functional inequality of paternal and maternal genomes in somatic cells (1,2). This phenomenon plays an important role in early development and disrupted imprinting manifests as disorders involving developmental abnormalities, congenital diseases, malignant tumors and abnormal behavior (3). More than 20 imprinted genes have been identified in mammals to date (4), nevertheless genetic studies of uniparental disomies in mice and humans have demonstrated evidence for imprinting effects on many other chromosomal regions (5,6). Although it is obvious that DNA methylation has a crucial role in genomic imprinting (7), DNA replication timing, chromosome nuclear organization and mitotic recombination frequencies are also associated with this process (8-11).

Intriguingly, it has become apparent that several imprinted genes are closely linked with oppositely imprinted transcripts (12-14). An intronic CpG island in the mouse Igf2r gene is indispensable for repression of the paternally inherited allele and an unmethylated paternal CpG island is associated with the production of an oppositely imprinted antisense RNA (15). Recently, the differentially methylated region of Igf2r was shown to consist of two cis-acting elements that bind specific proteins (16). Multiple imprinted sense and antisense transcripts have also been found in a region upstream of the Igf2 gene (17). These transcripts are associated with a direct repeat cluster which flanks differentially methylated regions of potential regulatory elements. In addition, previous studies have suggested that non-coding RNAs could induce silencing or activation of a chromosomal domain (18). These observations imply a general role for antisense transcripts in the imprinting process.

Human chromosome 11p15 harbors at least eight imprinted genes in a chromosomal domain (4,19). This imprinted domain is genetically linked with BWS, a congenital overgrowth disorder with a predisposition to several embryonal tumors (20-22). Frequent loss of heterozygosity (LOH) in the same region of 11p15 has been reported in a variety of adult tumors as well as embryonal tumors, including Wilms' tumor and rhabdomyosarcoma (23). Functional assays using microcell-mediated chromosome transfer identified a region harboring at least one tumor suppressor gene in 11p15 (24). Recently, a potassium channel gene, KvLQT1, which causes the long QT syndrome, was demonstrated to encompass a cluster of BWS chromosomal rearrangement breakpoints (BWSCR1) and one rhabdoid tumor breakpoint (25). It is suggested that the BWS-associated translocations within the KvLQT1 gene disrupt an important imprinting element in the chromosomal domain. The molecular etiology of BWS, however, is not yet established.

Previously, mouse/human somatic cell hybrids containing an inactive human X chromosome have been demonstrated to maintain the inactive state of the X chromosome. Somatic cell hybrids have therefore been used to study the inactivation process and to identify genes that escape from X inactivation (26-28). To develop an in vitro assay system for the investigation of imprinted loci in humans, we have established human monochromosomal hybrids with a human chromosome of defined parental origin, via microcell-mediated chromosome transfer (29,30). Expression and methylation studies demonstrated that the appropriate imprinting status of human loci was maintained in mouse A9 hybrids. In the present study, we screened expressed sequence tags (ESTs) which have been localized to the 11p15 region (31) using monochromosomal hybrids to identify transcripts that were expressed differentially between parental alleles. Here we describe the identification of LIT1, a novel imprinted antisense RNA within the KvLQT1 locus. In addition, we demonstrated that disrupted imprinting of LIT1 might contribute to the development of BWS but not Wilms' tumor.

RESULTS

Screening for differentially expressed transcripts using monochromosomal hybrids

To determine the allelic expression profiles of 100 ESTs in the 11p15 region, we performed RT-PCR analysis using mouse A9 hybrids containing a paternal or maternal human chromosome 11. Figure 1A showed that a cDNA clone corresponding to the human H19 gene was expressed only from the maternal chromosome. In contrast, H88273 was expressed only from the paternal chromosome, while equal allelic expression was detected for a cDNA clone corresponding to the gene for the dopamine D4 receptor (DRD4), which has previously been reported to be biallelically expressed in the human brain (32). To verify the allelic expression of H88273 in humans, we sought to identify DNA polymorphisms within the transcribed region. Direct sequencing of PCR products from genomic DNAs of 36 individuals identified three single nucleotide polymorphisms (SNPs) in the H88273 cDNA sequence (Fig. 2A). Subsequently, we performed RT-PCR on RNA from five informative individuals with primers flanking the polymorphisms. Since two of the three SNPs introduced an RsaI or MnlI restriction site, restriction analysis was performed on the PCR products. Restriction digestion of the amplified products revealed only the paternally inherited allele (Fig. 2B and C). Because a diagnostic restriction enzyme site was not available, paternal expression was confirmed by direct sequencing of the PCR products in one individual heterozygous for the remaining C/G polymorphism (Fig. 2D).

Figure 1. Identification of differentially expressed transcripts using monochromosomal hybrid cells. (A) Allele-specific expression profiles of human ESTs on chromosome 11p15. cDNAs from human chromosome donor fibroblasts (Fi), A9 hybrids containing a paternal (P) or maternal (M) copy of chromosome 11 and mouse A9 recipient cells (A9) were amplified using primers for 100 ESTs localized to the 11p15 region. (B) Physical map of the imprinted cluster on human chromosome region 11p15. BWSCR1 is the BWS breakpoint cluster region 1. 1632, CV581, B901, B23.1 and 1217 are cytogenetic translocation breakpoints of BWS germline rearrangements and TM87-16 is a rhabdoid tumor translocation breakpoint (22). The arrows indicate the deduced transcriptional orientation. Two novel non-imprinted genes, TSSC4 and TSSC6, have recently been reported by Lee et al. (62) to locate telomeric to the KvLQT1 gene. CpG islands were identified using the GRAIL program. The extent of the overlapping genomic contigs spanning the KvLQT1 gene is shown by the horizontal bars, with accession numbers listed below. The lower panel shows the location of KvLQT1 exons (closed boxes) and paternally expressed ESTs (dots) in relative spacing. Exon numbers are indicated below the boxes (25). The maternally methylated intronic CpG island is represented by the open box at the bottom of the figure.

Figure 2. Exclusive paternal expression of LIT1 in normal human fibroblasts and lymphoblasts. (A) Transcribed sequence polymorphisms in LIT1. The SNPs are indicated by asterisks. PCR was performed using primers (arrowheads) flanking the polymorphisms. (B and C) Assessment of allelic expression by restriction analysis. PCR products from normal human fibroblasts and lymphoblasts were digested with RsaI (B) and MnlI (C). The undigested and digested alleles are designated allele a and b, respectively. (D) Assessment of allelic expression by direct sequence analysis. The C/G polymorphism is indicated by the arrowhead.

Paternal expression of LIT1 RNA in the KvLQT1 locus

Database searches revealed that the transcript represented by H88273 was located within intron 10 of the KvLQT1 gene and was transcribed in the opposite orientation from KvLQT1, indicating that it is an antisense transcript of KvLQT1. As previous studies have suggested that isoform 1 of KvLQT1 is maternally repressed in human fetal tissues (25), we examined the expression profiles of different isoforms of KvLQT1 using isoform-specific PCR primers. As shown in Figure 3A, isoform 1 was expressed in various fetal tissues and was maternal specific in A9 hybrids. In contrast, expression of isoform 2 was restricted to heart and kidney, while no expression was detected in A9 hybrids. Tissue distribution of the antisense transcript was found to be very similar to the distribution of KvLQT1 isoform 1. In addition, further BLAST analysis of the GenBank dbEST identified nine additional ESTs located within a 60 kb region spanning exon 10 of KvLQT1 (Fig. 1B). All of these transcripts were also orientated in the divergent direction to KvLQT1 and were found to exhibit exclusive paternal expression in A9 hybrids (Fig. 3B, and data not shown). RT-PCR analysis using primers for the distinct ESTs suggested that the paternally expressed ESTs were derived from the same transcript (data not shown). Furthermore, sequences from cDNAs corresponding to these paternally expressed transcripts did not exhibit extended open reading frames and introns, implying that the transcript is not translated, as is the case for the antisense RNA in the mouse Igf2 locus (17). These observations suggest that the KvLQT1 antisense RNA could extend over 60 kb, which is termed LIT1.

Figure 3. (A) Allelic expression and tissue distribution of KvLQT1 isoforms and LIT1. To assess the expression profiles of KvLQT1 isoforms, RT-PCR analysis was performed using isoform-specific primers. (B) Paternal expression of human ESTs identified within the 60 kb region encompassing KvLQT1 exon 10. RT-PCR was performed using primers corresponding to these ESTs in the presence of GC Melt, to resolve the GC-rich secondary structure of the DNA fragments. (C) Imprinting studies of IGF2 in BWS patients. Allelic expression of IGF2 was assessed using primers flanking an ApaI RFLP in the 3[prime] untranslated region. The undigested and digested alleles are designated allele a and b, respectively. Patient 2 shows biallelic expression of IGF2. Patients 9 and 13 exhibit loss of methylation at the intronic CpG island but retain normal IGF2 imprinting.

Maternal methylation at the intronic CpG island in the KvLQT1 locus

Analysis of the GC density of 300 kb from the KvLQT1 genomic region identified a region containing the 5[prime] end of the AA359588 cDNA which has an apparently higher GC density than flanking sequences. Figure 4A indicates a GC-rich region that resembles mammalian CpG islands and two direct repeat clusters located within intron 10 of the KvLQT1 gene. It has previously been reported that differentially methylated regions often overlap or are adjacent to direct repeat clusters in the vicinity of imprinted genes (33). To assess parent-of-origin-specific methylation of LIT1, we performed methylation analysis of a 40 kb region containing the intronic CpG island using A9 hybrids. A methylation-sensitive PCR assay revealed that the intronic CpG island was specifically methylated on the silent maternal allele, while no parent-of-origin-specific methylation was detected in the flanking regions (data not shown). Subsequently, the methylation status of the CpG island was confirmed by Southern analysis using the methylation-sensitive restriction endonuclease NotI. DNA was digested either with BamHI alone or by double digestion with BamHI plus NotI (Fig. 4B). Partial digestion of the 6.0 kb BamHI fragment in normal human fibroblast DNA showed that the CpG island was methylated monoallelically. Consistent with this, the 6.0 kb methylated fragment was detected specifically in the hybrid with a maternal chromosome 11, demonstrating maternal methylation at the intronic CpG island.

Figure 4. Loss of maternal methylation at the intronic CpG island in BWS patients. (A) The intronic CpG island and direct repeat clusters within KvLQT1 intron 10. The location of cDNA clones corresponding to ESTs AA359588 and AA155639 are indicated at the top. EST AA155639 was used as a probe for methylation analysis of the intronic CpG island. Recognition sites of restriction enzymes are depicted as vertical lines: B, BamHI; N, NotI; S, SmaI. The upper plot shows the GC content and CpG observed/expected ratios calculated using CpG view v.1.5 (Division of Genetic Resources, National Institute of Health, Tokyo, Japan). As shown in the lower plot, aligning the sequence to itself indicates two clusters of short direct repeats (DR1 and DR2). (B) Southern blot analysis of the intronic CpG island in the KvLQT1 locus. The methylation status was analyzed after double digestion with BamHI plus NotI endonucleases. The 6.0 kb BamHI fragment encompassing the intronic CpG island is digested with NotI, resulting in a 4.2 kb fragment. FB indicates human chromosome donor fibroblast DNA digested with BamHI alone. The common signal present in all the rodent cell lines is probably due to weak cross-hybridization with the mouse counterpart (arrowhead). (C) Southern blot analysis of the CpG island upstream of KvLQT1. The same blot used in (B) was stripped and rehybridized with the indicated probe. The upstream CpG island is represented by the open box. Exon 1a and the transcriptional start site of the KvLQT1 gene are shown by a closed box with an arrow above the restriction map. The 8.6 kb BamHI fragment was completely digested by the methylation-sensitive endonuclease NotI in all cases, indicating absence of methylation on both parental chromosomes. Methylation analysis of both the upstream and intronic CpG islands using the methylation-sensitive endonuclease SmaI gave the same result (data not shown).

Loss of maternal methylation at the intronic CpG island in BWS patients

Since the differentially methylated CpG island is located within BWSCR1, we investigated allelic methylation in 13 Japanese BWS patients. The analysis showed that the methylated fragment was absent in four of 13 patients (31%), suggesting no maternal methylation at the CpG island (Fig. 4B). The remaining nine patients exhibited normal differential methylation. Interestingly, two of these were translocation cases (Table 1), clearly demonstrating that the translocation at 11p15.5 did not affect LIT1 imprinting. Although we could not determine the allelic expression of LIT1 because no polymorphisms were available in the patients, it is plausible that loss of maternal methylation at the intronic CpG island results in biallelic expression of LIT1 and has direct or indirect effects on subsequent BWS phenotypes. In contrast to the intronic CpG island, an upstream CpG island located in the 5[prime] portion of KvLQT1 was completely unmethylated on both parental alleles (Fig. 4C). Digestion with the methylation-sensitive endonuclease SmaI gave the same results in all cases examined (data not shown), implying that maternal expression of KvLQT1 did not correlate with allele-specific methylation at the upstream CpG island. To address the question of whether loss of maternal methylation at the intronic CpG island relates to loss of imprinting (LOI) of IGF2, we examined the allelic expression of IGF2 in these patients. As illustrated in Figure 3C, two of two informative cases with abnormal methylation at the intronic CpG island showed normal IGF2 imprinting (patients 9 and 13). The results clearly demonstrate that LOI of LIT1 is not accompanied by LOI of IGF2 in BWS patients. In contrast to previous reports showing the frequent (67-82%) LOI of IGF2 in BWS patients (34,35), only one of six informative cases (17%) exhibited biallelic expression of IGF2 in this study (Fig. 3C and Table 1). In addition, we also examined the methylation of H19 and found that the promoter region was hypermethylated in a patient with LOI of IGF2 (data not shown). This observation of LOI of IGF2 accompanied by abnormal methylation of H19 confirmed that of Reik et al. (36), further supporting a crucial role of LIT1 in the development of BWS.

Table 1. Analysis of imprinting of LIT1 and IGF2 in BWS patients

Patient	LIT1		IGF2
	Allele-specific expression	CpG island methylation	Allele-specific expression
1a	I	I
2	I	I	LOI
3		I
4		I
5a		I
6		I
7	I	I	I
8	I	I	I
9		LOI	I
10b		LOI
11		I	I
12b		LOI
13		LOI	I

LOI, loss of imprinting; I, normal imprinting.
^aTranslocation cases. Patients 1 and 6 carried a t(11;12)(p15.5;q24.11) and t(11;X)(p15.5;q22.1), respectively.
^aThese non-UPD cases were determined by polymorphic analysis of multiple 11p15.5 markers (data not shown).

Normal imprinting of LIT1 in Wilms' tumors

LIT1 is the second paternally expressed transcript from 11p15, which provides the opportunity to investigate whether altered imprinting of LIT1 could play a role in cancers, similar to that of IGF2, and whether imprinting of IGF2 and LIT1 is coordinately regulated. We used two transcribed polymorphisms for this purpose, including that within H88273. The second, within AA331124, is reported separately (37). We typed genomic DNA from 30 Wilms' tumors and identified eight heterozygous (informative) samples. RT-PCR was then carried out on RNA isolated from both normal and tumor tissues. Eight of eight cases showed monoallelic expression of LIT1 in both normal and tumor tissues (Fig. 5A and data not shown). Typing of parental DNA confirmed that it was the paternal allele that was expressed from both normal kidney and tumor samples (Table 2). We also examined methylation modification at the intronic CpG island. Methylation-sensitive NotI digestion was used in genomic Southern blot experiments. DNA was digested with either BamHI alone (Fig. 5B, lane 1) or by double digestion with BamHI plus NotI (Fig. 5B, lanes 2-4). BamHI digestion generated a 6.0 kb fragment (Fig. 5B, lanes 1-4), while BamHI and NotI double digestion generated a 4.2 kb fragment in the absence of methylation, using the cDNA clone 592241 as a probe, which corresponds to EST AA155639. The presence of both the 6.0 and 4.2 kb bands, with equal intensity, demonstrated differential methylation of DNA in these tumors (Fig. 5B) and matched normal tissue (Table 2), which was observed in all three tumors and matched normal kidney tissues examined. We also determined the imprinting status of IGF2 in these tumors and matched normal tissues. Consistent with our previous report (12), five of six tumors showed biallelic expression and four normal tissues showed normal imprinting. In three cases of tumors displaying LOI of IGF2 (Table 2, patients 2, 3 and 12), LIT1 was imprinted normally. Therefore, we conclude that imprinting of LIT1 is not altered in Wilms' tumor. This result contrasts with IGF2, which shows frequent LOI in Wilms' tumor (12).

Figure 5. (A) Monoallelic expression of LIT1 in Wilms' tumors. Tumor 5 gDNA indicates the genotyping of genomic DNA from the tumor from patient 5 using Lit102/Lit202. The heterozygosity is clearly demonstrated as double peaks of C and T at nucleotide 222 (arrow). Tumor 5 cDNA shows monoallelic expression of LIT1. In this case the expressed allele is T. Tumor 12 gDNA indicates the genotyping of genomic DNA from the tumor of patient 12 using Lit105/Lit205. Tumor 12 is heterozygous, as demonstrated by the two peaks of A and G at nucleotide 18 (arrow). cDNA of tumor 12 also shows monoallelic expression of LIT1. In this case the expressed allele is G. (B) Differential methylation at the intronic CpG island in Wilms' tumors. B, single digest with BamHI; BN, double digest with BamHI and NotI. Lanes 1 and 2, tumor DNA from patient 8; lanes 3 and 4, tumor DNA from patients 7 and 15, respectively.

Table 2. Analysis of imprinting of LIT1 and IGF2 in Wilms' tumors

Patient	LIT1				IGF2
	Allele-specific expression		CpG island methylation		Allele-specific expression
	Normal	Tumor	Normal	Tumor	Normal	Tumor
1	I	I
2	I	I			I	LOI
3	I	I				LOI
4	I	I
5	I	I
6						LOI
7			I	I
8	I	I	I	I	I
9					I
10						LOI
11	I	I			I	I
12		I				LOI
13				I
14				I
15			I	I

LOI, loss of imprinting; I, normal imprinting.

DISCUSSION

One of the main results of this study is that imprinting of LIT1 is not altered in Wilms' tumor, in contrast to IGF2, which shows frequent LOI. These results are most consistent with a two-domain model of imprinting of 11p15, which we elaborate in detail elsewhere (37). We propose that there are two imprinting domains at 11p15, a centromeric domain containing TSSC3, TSSC5, CDKN1C and KvLQT1 and a telomeric domain spanning ASCL2, IGF2 and H19. We hypothesize that these two domains are regulated separately and that abnormal imprinting of the more telomeric domain, including IGF2, is primarily involved in human cancer. According to our model, the two domains would have overlapping but non-identical functions, consistent with Haig's parental conflict model (38).

Recently, a differentially methylated intronic CpG island, which is the promoter for a paternally expressed non-coding RNA, was demonstrated to be required for imprinted maternal expression of the mouse Igf2r gene (15). Reciprocal expression of closely linked imprinted genes was also shown for the IGF2/H19, UBE3A/AS-RNA regions (12,13), suggesting that mechanistic interactions, such as transcription competition between the reciprocally expressed gene pairs, might play a role in imprinting mechanisms. The major imprinting clusters on human chromosomes 11 and 15 contain a number of imprinted genes, which implies long-range regulation through common regulatory elements. Indeed, microdeletions in the 5[prime] end of the SNRPN gene disrupt imprinting of the entire cluster lying within a 4 Mb region on chromosome 15q11-q13 (39,40). The recent observation that deletion of H19 in mice affects the imprinting of Igf2 and Ins2, but not that of Mash2, Cdkn1c and Kvlqt1, indicates that the telomeric genes could be regulated by a separate mechanism (41). In addition, biallelic expression of IGF2 in a BWS family with normal H19 imprinting suggests an H19-independent pathway to the disruption of IGF2 imprinting (42). By analogy with the imprinting cluster on human chromosome 15, it has been postulated that the BWS-associated translocation locus could disrupt an important imprinting element in the vicinity of KvLQT1 (43). In this report, we have demonstrated that the paternally expressed antisense transcript LIT1 within the KvLQT1 locus was a direct target of methylation, as previously predicted by others (41). Consequently, this imprinting cluster may be regulated by a somewhat complex mechanism involving antisense regulation.

The observations presented in this study also have important implications for understanding the molecular etiology of BWS. Previously, a considerable number of BWS patients have been reported to exhibit biallelic expression of IGF2, which is frequently linked to hypermethylation of H19 (34-36). Germline mutations in CDKN1C have also been found in a minority of patients (44-46). The present finding that loss of maternal methylation of LIT1 is not associated with LOI of IGF2 in BWS patients suggests a BWS pathway that is completely independent of IGF2, as discussed in detail elsewhere (37). On the basis of transcription competition in the imprinted domain, we suggest that loss of LIT1 imprinting could cause silencing of the normally expressed maternal copy of a closely linked imprinted gene within the same centromeric domain. Alternatively, chromosomal translocations could separate the target gene from transcriptional regulatory elements which might be shared with LIT1 and locate telomeric to BWSCR1, resulting in an identical consequence. Thus, disrupted imprinting of LIT1 could contribute to BWS pathophysiology in a substantial number of patients not associated with loss of IGF2 imprinting. Although differences in the imprinting status of KvLQT1 have been observed in mice and humans (41,43,47,48), targeted disruption in mice could be used to test this possibility. To analyze the human 11p15.5 region, a shuttle system using DT40 cells that exhibit a high frequency of homologous recombination is also available (49-51).

So far, four systematic screening systems have been developed for the identification of imprinted loci in mice (52-56). In this report, we demonstrated that human monochromosomal hybrids provide a valuable resource for the efficient identification of imprinted loci in humans. We have identified at least one additional imprinted transcript on human chromosome 15 using this system of monochromosomal hybrids (M. Meguro, K. Mitsuya, M. Kohda, A. Kashiwagi, T.C. Schulz, H. Kugoh, M. Nakao and M. Oshimura, manuscript in preparation). A similar approach could also be applied to additional matched maternal and paternal pairs of human monochromosomal hybrids. In particular, early embryonic lethality has been demonstrated for both paternal and maternal uniparental disomies for mouse chromosome 12, suggesting the presence of multiple imprinted loci (5). Imprinting effects have also been proposed for human chromosome 14q, which has significant homology to mouse chromosome 12 (6,57). More recent findings provided evidence for X-linked imprinted loci that affect the social cognitive abilities or the survival of male embryos (58,59). No imprinted transcript has been described in these chromosomal regions and the positional approach as described here could be useful for the identification of imprinted loci in the yet-to-be-characterized regions.

MATERIALS AND METHODS

Cell lines

Mouse A9 hybrids containing a paternal or maternal human chromosome 11 tagged with pSV2bsr were constructed by microcell-mediated chromosome transfer (29,30). Hybrid cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 3 µg/ml Blasticidin S (Calbiochem, La Jolla, CA). Fibroblast and EBV-transformed lymphoblast cultures were obtained using standard procedures.

EST screen

Total RNA was extracted from frozen cells by guanidinium isothiocyanate extraction. First strand cDNA synthesis was carried out with an oligo(dT)₁₅ primer and MMLV reverse transcriptase (Life Technologies, Grand Island, NY). RT-PCR was performed on the cDNA with Taq polymerase (Perkin Elmer, Foster City, CA) using a step-down protocol. In general, the reaction parameters were as follows: 95°C initial denaturation for 10 min, 95°C for 30 s, 60°C annealing for 30 s, 72°C for 30 s. Nine rounds of PCR were carried out in this manner, with the annealing temperature being reduced by 2°C every three cycles to a final annealing temperature of 56°C. Twenty-four subsequent rounds of PCR were carried out as follows: 95°C for 30 s, 54°C for 30 s, 72°C for 30 s, followed by a 2 min final extension at 72°C. Primers for 100 ESTs that have been mapped previously to 11p15 (31) were obtained from Research Genetics (Huntsville, AL). Primer sequences corresponding to the ESTs are available at http://ncbi.nml.nih.gov/genemap/ . Amplified fragments were resolved on 2% agarose gels followed by staining with SYBR Green I (Molecular Probes, Eugene, OR).

Sequence analysis

DNA and EST database searches were performed using the BLAST programs on the NCBI server (http://www.ncbi.nlm.nih.gov/ ). Genomic sequences from the KvLQT1 locus were filtered for human repetitive elements using RepeatMasker programs (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker ). cDNA clones corresponding to human ESTs were assembled with the ESTblast programs (http://www.hgmp.mrc.ac.uk/ESTBlast/ ) and were obtained from Genome Systems (St Louis, MO) and the American Type Culture Collection (Rockville, MD). After correcting sequence ambiguities that were present in the human ESTs, PCR primers were synthesized and used to amplify cDNA fragments. Before sequencing, PCR-amplified DNA fragments were treated to inactivate the unincorporated primers and deoxynucleotide triphosphates in the samples. PCR products were mixed with exonuclease I and shrimp alkaline phosphatase (Amersham Pharmacia, Piscataway, NJ), followed by heat inactivation according to the manufacturer's recommendations. Pretreated PCR products were directly sequenced on both strands using ThermoSequenase (Amersham Pharmacia) on a LI-COR DNA sequencer 4200.

Expression analysis

Multiple human fetal tissue cDNAs (Clontech, Palo Alto, CA) were used as templates in a PCR reaction for analysis of KvLQT1 isoforms and LIT1. PCR products were separated on 2% agarose gels. Southern blot analysis was performed by the use of radioactively end-labeled internal primers as probes and analyzed with a BAS-2500 bioimaging analyzer (Fuji Film, Tokyo, Japan). For PCR amplification and internal probing, the following oligonucleotides were used: isoform 1, forward (F), 5[prime]-CGCGTCTACAACTTCCTCG-3[prime], reverse (R), 5[prime]-ACCGAGTCCCCGT-3[prime], internal (I), 5[prime]-TGTCCACCATCGAGCAGTAT-3[prime]; isoform 2, F, 5[prime]-TTTCTGGCTCTCGGGAATTT-3[prime], R, 5[prime]-ATCCAGAAGAGAGTCCCCGT-3[prime], I, 5[prime]-TGTCCACCATCGAGCAGTAT-3[prime]; H88273, F, 5[prime]-CAGCACAAAGAGGTTTTTGACAG-3[prime], R, 5[prime]-GAGTTTAAAACACGTGTGTGCATT-3[prime], I, 5[prime]-TCCTCAGATAGGGTGGAGA-3[prime].

Assessment of allelic expression

cDNAs from normal human fibroblasts and lymphoblasts were amplified using the primers and amplification conditions described above. Endonucleases RsaI and MnlI (New England Biolabs, Beverly, MA) were used for restriction analysis of the PCR-produced DNA fragments. The C/G polymorphism in H88273 was analyzed by direct sequencing using the same primers. Controls for DNA contamination in the total RNA preparation were performed for all sets of primers with the same procedure as for RT-PCR, but without reverse transcriptase. The primers for imprinting analysis of Wilms' tumor patients using a polymorphism in EST AA331124 (37) were Lit105 (5[prime]-GATCCTNTCCAGGCAGCTTCTTCCACA-3[prime]) and Lit205 (5[prime]-CATAAGGTAGGTAAGTTTGTGTCCCTG-3[prime]). The primers for polymorphisms in EST H88273 were Lit102 (5[prime]-TCTGGTTCATGTCACTCTGTGGAGCAG-3[prime]) and Lit202 (5[prime]-CTCCCAAAAGCAGAGTTTTGGCAATAT-3[prime]). RT-PCR and sequencing of PCR products was used to analyze allele-specific gene expression. RNA was treated with RNase-free DNase (Boehringer Mannheim, Indianapolis, IN) before the reverse transcription reaction. Reverse transcription was carried out using AMV reverse transcriptase (Boehringer Mannheim). Reverse transcription reactions were always carried out in the presence or absence of reverse transcriptase. PCR reactions contained 0.5 µM primers, 0.2 µM dNTP, 50 ng DNA, 1× PCR buffer (Life Technologies) and 0.5 U Taq DNA polymerase (Life Technologies) in 25 µl and were performed with a Robocycler (Stratagene, La Jolla, CA) as follows: 40 cycles of 95°C for 45 s, 60°C for 30 s and 72°C for 30 s, followed by extension at 72°C for 10 min. PCR products were purified using QIAquick (Qiagen, Chatsworth, CA) and directly sequenced on an ABI377 automated sequencer. Allelic expression of IGF2 was assessed using an ApaI polymorphism, as described previously (60).

Methylation analysis

The methylation status of the KvLQT1 locus was assessed by a methylation-sensitive PCR assay. Genomic DNA was extracted by standard phenol-chloroform extraction methods and digested with the methylation-sensitive endonuclease HpaII prior to PCR amplification. Primer sequences and reaction conditions are available on request. Southern hybridization was carried out to verify the methylation status of the CpG island, as described previously (29). The probe used for Southern analysis of the CpG island upstream of KvLQT1 was generated from human genomic DNA by PCR (primers F, 5[prime]-CCTCTACCCAGACATCCCATGGCTGAT-3[prime] and R, 5[prime]-CACAGGAGAGTTCGCCCTAGCCCTGTA-3[prime]) in the presence of GC Melt (Clontech). Genomic DNA was digested with BamHI plus NotI and electrophoresed on 0.8% agarose gels. The DNA was then transferred to Hybond-N⁺ filters and hybridized with oligolabeled probes. For methylation analysis of Wilms' tumors, genomic DNA was also digested with BamHI or BamHI plus NotI. Digested DNA was extracted with phenol-chloroform, and ethanol precipitated. DNA was resolved on 0.8% agarose gels, transferred to Hybond-N⁺ filters and fixed by UV crosslinking. Filters were hybridized with the cDNA clone 592241 probe prepared by the random priming method (61). Hybridizations were carried out at 65°C overnight in Church-Gilbert buffer (0.5 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA). The filters were washed with 0.1× SSC and 0.1% SDS at 65°C.

ACKNOWLEDGEMENTS

The authors thank A. Kashiwagi and M. Kohda for technical assistance and Drs M. Nakao, T. Tada, R. Katoh and H. Sasaki for valuable suggestions. K.M. is a Research Fellow of the Japan Society for the Promotion of Science. The Feinberg laboratory contributed the analysis of Wilms' tumor specimens to this work. This work was supported by the Japan Science and Technology Corp., Japan (M.O.), and NIH grant CA65145 (A.P.F.).

REFERENCES

1. Surani, M.A., Barton, S.C. and Norris, M.L. (1984) Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature, 308, 548-550. MEDLINE Abstract

2. McGrath, J. and Solter, D. (1984) Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell, 37, 179-183. MEDLINE Abstract

3. Hall, J.G. (1997) Genomic imprinting: nature and clinical relevance. Annu. Rev. Med., 48, 35-44. MEDLINE Abstract

4. Morison, I.M. and Reeve, A.E. (1998) A catalogue of imprinted genes and parent-of-origin effects in humans and animals. Hum. Mol. Genet., 7, 1599-1609. MEDLINE Abstract

5. Cattanach, B.M. and Beechey, C.V. (1997) Genomic imprinting in the mouse: possible final analysis. In Reik, W. and Surani, A. (eds), Genomic Imprinting. Oxford University Press, New York, NY, pp. 118-145.

6. Ledbetter, D.H. and Engel, E. (1995) Uniparental disomy in humans: development of an imprinting map and its implications for prenatal diagnosis. Hum. Mol. Genet., 4, 1757-1764. MEDLINE Abstract

7. Constância, M., Pickard, B., Kelsey, G. and Reik, W. (1998) Imprinting mechanisms. Genome Res., 8, 881-900. MEDLINE Abstract

8. Lalande, M. (1997) Parental imprinting and human disease. Annu. Rev. Genet., 30, 173-195.

9. Feil, R. and Kelsey, G. (1997) Genomic imprinting: a chromatin connection. Am. J. Hum. Genet., 61, 1213-1219. MEDLINE Abstract

10. Pàldi, A., Gyapay, G. and Jami, J. (1995) Imprinted chromosomal regions of the human genome display sex-specific meiotic recombination frequencies. Curr. Biol., 5, 1030-1035. MEDLINE Abstract

11. Robinson, W.P. and Lalande, M. (1995) Sex-specific meiotic recombination in the Prader-Willi/Angelman syndrome imprinted region. Hum. Mol. Genet., 4, 801-806. MEDLINE Abstract

12. Rainier, S., Johnson, L.A., Dobry, C.J., Ping, A.J., Grundy, P.E. and Feinberg, A.P. (1993) Relaxation of imprinted genes in human cancer. Nature, 362, 747-749. MEDLINE Abstract

13. Rougeulle, C., Cardoso, C., Fontés, M., Colleaux, L. and Lalande, M. (1998) An imprinted antisense RNA overlaps UBE3Aand a second maternally expressed transcript. Nature Genet., 19, 15-16. MEDLINE Abstract

14. Hayward, B.E., Moran, V., Strain, L. and Bonthron, D.T. (1998) Bidirectional imprinting of a single gene: GNAS1encodes maternally, paternally and biallelically derived proteins. Proc. Natl Acad. Sci. USA, 95, 15475-15480. MEDLINE Abstract

15. Wutz, A., Smrzka, O.W., Schweifer, N., Schellander, K., Wagner, E.F. and Barlow, D.P. (1997) Imprinted expression of the Igf2rgene depends on an intronic CpG island. Nature, 389, 745-749. MEDLINE Abstract

16. Birger, Y., Shemer, R., Perk, J. and Razin, A. (1999) The imprinting box of the mouse Igf2rgene. Nature, 397, 84-88. MEDLINE Abstract

17. Moore, T., Constancia, M., Zubair, M., Bailleul, B., Feil, R., Sasaki, H. and Reik, W. (1997) Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2.Proc. Natl Acad. Sci. USA, 94, 12509-12514. MEDLINE Abstract

18. Willard, H.F. and Salz, H.K. (1997) Remodelling chromatin with RNA. Nature, 386, 228-229. MEDLINE Abstract

19. Reik, W. and Maher, E.R. (1997) Imprinting in clusters: lessons from Beckwith-Wiedemann syndrome. Trends Genet., 13, 330-334. MEDLINE Abstract

20. Ping, A.J., Reeve, A.E., Law, D.J., Young, M.R., Boehnke, M. and Feinberg, A.P. (1989) Genetic linkage of Beckwith-Wiedemann syndrome to 11p15. Am. J. Hum. Genet., 44, 720-723. MEDLINE Abstract

21. Sait, S.N., Nowak, N.J., Singh-Kahlon, P., Weksberg, R., Squire, J., Shows, T.B. and Higgins, M.J. (1994) Localization of Beckwith-Wiedemann and rhabdoid tumor chromosome rearrangements to a defined interval in chromosome band 11p15.5. Genes Chromosomes Cancer, 11, 97-105. MEDLINE Abstract

22. Hoovers, J.M., Kalikin, L.M., Johnson, L.A., Alders, M., Redeker, B., Law, D.J., Bliek, J., Steenman, M., Benedict, M., Wiegant, J., Lengauer, C., Taillon-Miller, P., Schlessinger, D., Edwards, M.C., Elledge, S.J., Ivens, A., Westerveld, A., Little, P., Mannens, M. and Feinberg, A.P. (1995) Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments. Proc. Natl Acad. Sci. USA, 92, 12456-12460. MEDLINE Abstract

23. Seizinger, B.R., Klinger, H.P., Junien, C., Nakamura, Y., Beau, M.L., Cavenee, W., Emanuel, B., Ponder, B., Naylor, S., Mitelman, F., Louis, D., Menon, A., Newsham, I., Decker, J., Kaelbling, M., Henry, I. and Deimling, A.V. (1991) Report of the committee on chromosome and gene loss in human neoplasia. Cytogenet. Cell Genet., 58, 1080-1096.

24. Koi, M., Johnson, L.A., Kalikin, L.M., Little, P.F., Nakamura, Y. and Feinberg, A.P. (1993) Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from chromosome 11. Science, 260, 361-364. MEDLINE Abstract

25. Lee, M.P., Hu, R.J., Johnson, L.A. and Feinberg, A.P. (1997) Human KVLQT1gene shows tissue-specific imprinting and encompasses Beckwith-Wiedemann syndrome chromosomal rearrangements. Nature Genet., 15, 181-185. MEDLINE Abstract

26. Brown, C.J. and Willard, H.F. (1994) The human X-inactivation centre is not required for maintenance of X-chromosome inactivation. Nature, 368, 154-156. MEDLINE Abstract

27. Brown, C.J., Carrel, L. and Willard, H.F. (1997) Expression of genes from the human active and inactive X chromosomes. Am. J. Hum. Genet., 60, 1333-1343. MEDLINE Abstract

28. Miller, A.P. and Willard, H.F. (1998) Chromosomal basis of X chromosome inactivation: identification of a multigene domain in Xp11.21-p11.22 that escapes X inactivation. Proc. Natl Acad. Sci. USA, 95, 8709-8714. MEDLINE Abstract

29. Meguro, M., Mitsuya, K., Sui, H., Shigenami, K., Kugoh, H., Nakao, M. and Oshimura, M. (1997) Evidence for uniparental, paternal expression of the human GABA_A receptor subunit genes, using microcell-mediated chromosome transfer. Hum. Mol. Genet., 6, 2127-2133. MEDLINE Abstract

30. Mitsuya, K., Meguro, M., Sui, H., Schulz, T.C., Kugoh, H., Hamada, H. and Oshimura, M. (1998) Epigenetic reprogramming of the human H19 gene in mouse embryonic cells does not erase the primary parental imprint. Genes Cells, 3, 245-255. MEDLINE Abstract

31. Deloukas, P. et al.(1998) A physical map of 30,000 human genes. Science, 282, 744-746. MEDLINE Abstract

32. Cichon, S., Nöthen, M.M., Wolf, H.K. and Propping, P. (1996) Lack of imprinting of the human dopamine D4 receptor (DRD4)gene. Am. J. Med. Genet., 67, 229-231. MEDLINE Abstract

33. Reik, W. and Walter, J. (1998) Imprinting mechanisms in mammals. Curr. Opin. Genet. Dev., 8, 154-164. MEDLINE Abstract

34. Weksberg, R., Shen, D.R., Fei, Y.L., Song, Q.L. and Squire, J. (1993) Disruption of insulin-like growth factor 2 imprinting in Beckwith-Wiedemann syndrome. Nature Genet., 5, 143-150. MEDLINE Abstract

35. Joyce, J.A., Lam, W.K., Catchpoole, D.J., Jenks, P., Reik, W., Maher, E.R. and Schofield, P.N. (1997) Imprinting of IGF2and H19:lack of reciprocity in sporadic Beckwith-Wiedemann syndrome. Hum. Mol. Genet., 6, 1543-1548. MEDLINE Abstract

36. Reik, W., Brown, K.W., Schneid, H., Le Bouc, Y., Bickmore, W. and Maher, E.R. (1995) Imprinting mutations in the Beckwith-Wiedemann syndrome suggested by altered imprinting pattern in the IGF2-H19domain. Hum. Mol. Genet., 4, 2379-2385. MEDLINE Abstract

37. Lee, M.P., DeBaun, M.R., Mitsuya, K., Galonek, H.L., Brandenburg, S., Oshimura, M. and Feinberg, A.P. (1999) Loss of imprinting of a paternally expressed transcript, with antisense orientation to KvLQT1, occurs frequently in Beckwith-Wiedemann syndrome and is independent of insulin-like growth factor IIimprinting. Proc. Natl Acad. Sci. USA, 96, 5203-5208. MEDLINE Abstract

38. Moore, T. and Haig, D. (1991) Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet., 7, 45-49. MEDLINE Abstract

39. Dittrich, B., Buiting, K., Korn, B., Rickard, S., Buxton, J., Saitoh, S., Nicholls, R.D., Poustka, A., Winterpacht, A., Zabel, B. and Horsthemke, B. (1996) Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPNgene. Nature Genet., 14, 163-170. MEDLINE Abstract

40. Yang, T., Adamson, T.E., Resnick, J.L., Leff, S., Wevrick, R., Francke, U., Jenkins, N.A., Copeland, N.G. and Brannan, C.I. (1998) A mouse model for Prader-Willi syndrome imprinting-centre mutations. Nature Genet., 19, 25-31. MEDLINE Abstract

41. Caspary, T., Cleary, M.A., Baker, C.C., Guan, X.J. and Tilghman, S.M. (1998) Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster. Mol. Cell. Biol., 18, 3466-3474. MEDLINE Abstract

42. Brown, K.W., Villar, A.J., Bickmore, W., Clayton-Smith, J., Catchpoole, D., Maher, E.R. and Reik, W. (1996) Imprinting mutation in the Beckwith-Wiedemann syndrome leads to biallelic IGF2expression through an H19-independentpathway. Hum. Mol. Genet., 5, 2027-2032. MEDLINE Abstract

43. Paulsen, M., Davies, K.R., Bowden, L.M., Villar, A.J., Franck, O., Fuermann, M., Dean, W.L., Moore, T.F., Rodrigues, N., Davies, K.E., Hu, R.J., Feinberg, A.P., Maher, E.R., Reik, W. and Walter, J. (1998) Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5. Hum. Mol. Genet., 7, 1149-1159. MEDLINE Abstract

44. Hatada, I., Ohashi, H., Fukushima, Y., Kaneko, Y., Inoue, M., Komoto, Y., Okada, A., Ohishi, S., Nabetani, A., Morisaki, H., Nakayama, M., Niikawa, N. and Mukai, T. (1996) An imprinted gene p57^KIP2is mutated in Beckwith-Wiedemann syndrome. Nature Genet., 14, 171-173. MEDLINE Abstract

45. O'Keefe, D., Dao, D., Zhao, L., Sanderson, R., Warburton, D., Weiss, L., Anyane-Yeboa, K. and Tycko, B. (1997) Coding mutations in p57^KIP2 are present in some cases of Beckwith-Wiedemann syndrome but are rare or absent in Wilms' tumors. Am. J. Hum. Genet., 61, 295-303. MEDLINE Abstract

46. Lee, M.P., DeBaun, M., Randhawa, G., Reichard, B.A., Elledge, S.J. and Feinberg, A.P. (1997) Low frequency of p57^KIP2 mutation in Beckwith-Wiedemann syndrome. Am. J. Hum. Genet., 61, 304-309. MEDLINE Abstract

47. Gould, T.D. and Pfeifer, K. (1998) Imprinting of mouse Kvlqt1is developmentally regulated. Hum. Mol. Genet., 7, 483-487. MEDLINE Abstract

48. Jiang, S., Hemann, M.A., Lee, M.P. and Feinberg, A.P. (1998) Strain-dependent developmental relaxation of imprinting of an endogenous mouse gene, Kvlqt1. Genomics, 53, 395-399. MEDLINE Abstract

49. Dieken, E.S., Epner, E.M., Fiering, S., Fournier, R.E. and Groudine, M. (1996) Efficient modification of human chromosomal alleles using recombination-proficient chicken/human microcell hybrids. Nature Genet., 12, 174-182. MEDLINE Abstract

50. Koi, M., Lamb, P.W., Filatov, L., Feinberg, A.P. and Barrett, J.C. (1997) Construction of chicken × human microcell hybrids for human gene targeting. Cytogenet. Cell Genet., 76, 72-76. MEDLINE Abstract

51. Kuroiwa, Y., Shinohara, T., Notsu, T., Tomizuka, K., Yoshida, H., Takeda, S., Oshimura, M. and Ishida, I. (1998) Efficient modification of a human chromosome by telomere-directed truncation in high homologous recombination-proficient chicken DT40 cells. Nucleic Acids Res., 26, 3447-3448. MEDLINE Abstract

52. Hayashizaki, Y., Shibata, H., Hirotsune, S., Sugino, H., Okazaki, Y., Sasaki, N., Hirose, K., Imoto, H., Okuizumi, H., Muramatsu, M., Komastubara, H., Shiroishi, T., Moriwaki, K., Katsuki, M., Hatano, N., Sasaki, H., Ueda, T., Mise, N., Takagi, N., Plass, C. and Chapman, V.M. (1994) Identification of an imprinted U2af binding protein related sequence on mouse chromosome 11 using the RLGS method. Nature Genet., 6, 33-40. MEDLINE Abstract

53. Hatada, I., Sugama, T. and Mukai, T. (1993) A new imprinted gene cloned by a methylation-sensitive genome scanning method. Nucleic Acids Res., 21, 5577-5582. MEDLINE Abstract

54. Kaneko-Ishino, T., Kuroiwa, Y., Miyoshi, N., Kohda, T., Suzuki, R., Yokoyama, M., Viville, S., Barton, S.C., Ishino, F. and Surani, M.A. (1995) Peg1/Mestimprinted gene on chromosome 6 identified by cDNA subtraction hybridization. Nature Genet., 11, 52-59. MEDLINE Abstract

55. Hagiwara, Y., Hirai, M., Nishiyama, K., Kanazawa, I., Ueda, T., Sakaki, Y. and Ito, T. (1997) Screening for imprinted genes by allelic message display: identification of a paternally expressed gene Impacton mouse chromosome 18. Proc. Natl Acad. Sci. USA, 94, 9249-9254. MEDLINE Abstract

56. Kikyo, N., Williamson, C.M., John, R.M., Barton, S.C., Beechey, C.V., Ball, S.T., Cattanach, B.M., Surani, M.A. and Peters, J. (1997) Genetic and functional analysis of neuronatinin mice with maternal or paternal duplication of distal Chr 2. Dev. Biol., 190, 66-77. MEDLINE Abstract

57. Robin, N.H., Harari-Shacham, A., Schwartz, S. and Wolff, D.J. (1997) Duplication 14 (q24.3q31) in a father and daughter: delineation of a possible imprinted region. Am. J. Med. Genet., 71, 361-365. MEDLINE Abstract

58. Skuse, D.H., James, R.S., Bishop, D.V., Coppin, B., Dalton, P., Aamodt-Leeper, G., Bacarese-Hamilton, M., Creswell, C., McGurk, R. and Jacobs, P.A. (1997) Evidence from Turner's syndrome of an imprinted X-linked locus affecting cognitive function. Nature, 387, 705-708. MEDLINE Abstract

59. Naumova, A.K., Leppert, M., Barker, D.F., Morgan, K. and Sapienza, C. (1998) Parental origin-dependent, male offspring-specific transmission-ratio distortion at loci on the human X chromosome. Am. J. Hum. Genet., 62, 1493-1499. MEDLINE Abstract

60. Mitsuya, K., Sui, H., Meguro, M., Kugoh, H., Jinno, Y., Niikawa, N. and Oshimura, M. (1997) Paternal expression of WT1in human fibroblasts and lymphocytes. Hum. Mol. Genet., 6, 2243-2246. MEDLINE Abstract

61. Feinberg, A.P. and Vogelstein, B.A. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132, 6-13. MEDLINE Abstract

62. Lee, M.P., Brandenburg, S., Landes, G.M., Adams, M., Miller, G. and Feinberg, A.P. (1999) Two novel genes in the center of the 11p15 imprinted domain escape genomic imprinting. Hum. Mol. Genet., 8, 683-690. MEDLINE Abstract

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*To whom correspondence should be addressed at: Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Nishimachi 86, Yonago, Tottori 683-8503, Japan. Tel: +81 859 34 8260; Fax: +81 859 34 8134; Email: oshimura{at}grape.med.tottori-u.ac.jp

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				Y. Li, H. Nagai, T. Ohno, M. Yuge, S. Hatano, E. Ito, N. Mori, H. Saito, and T. Kinoshita Aberrant DNA methylation of p57KIP2 gene in the promoter region in lymphoid malignancies of B-cell phenotype Blood, September 18, 2002; 100(7): 2572 - 2577. [Abstract] [Full Text] [PDF]

				K. Nakabayashi, L. Bentley, M. P. Hitchins, K. Mitsuya, M. Meguro, S. Minagawa, J. S. Bamforth, P. Stanier, M. Preece, R. Weksberg, et al. Identification and characterization of an imprinted antisense RNA (MESTIT1) in the human MEST locus on chromosome 7q32 Hum. Mol. Genet., July 15, 2002; 11(15): 1743 - 1756. [Abstract] [Full Text] [PDF]

				R. Weksberg, C. Shuman, O. Caluseriu, A. C. Smith, Y.-L. Fei, J. Nishikawa, T. L. Stockley, L. Best, D. Chitayat, A. Olney, et al. Discordant KCNQ1OT1 imprinting in sets of monozygotic twins discordant for Beckwith-Wiedemann syndrome Hum. Mol. Genet., May 16, 2002; 11(11): 1317 - 1325. [Abstract] [Full Text] [PDF]

				E. Balint, A. C. Phillips, S. Kozlov, C. L. Stewart, and K. H. Vousden Induction of p57KIP2 expression by p73beta PNAS, March 19, 2002; 99(6): 3529 - 3534. [Abstract] [Full Text] [PDF]

				R. Weksberg, J. Nishikawa, O. Caluseriu, Y.-L. Fei, C. Shuman, C. Wei, L. Steele, J. Cameron, A. Smith, I. Ambus, et al. Tumor development in the Beckwith-Wiedemann syndrome is associated with a variety of constitutional molecular 11p15 alterations including imprinting defects of KCNQ1OT1 Hum. Mol. Genet., December 1, 2001; 10(26): 2989 - 3000. [Abstract] [Full Text] [PDF]

				R. M. John, J. F. -X. Ainscough, S. C. Barton, and M. A. Surani Distant cis-elements regulate imprinted expression of the mouse p57 Kip2 (Cdkn1c) gene: implications for the human disorder, Beckwith-Wiedemann syndrome Hum. Mol. Genet., July 1, 2001; 10(15): 1601 - 1609. [Abstract] [Full Text] [PDF]

				J. Bliek, S. M. Maas, J. M. Ruijter, R. C.M. Hennekam, M. Alders, A. Westerveld, and M. M.A.M. Mannens Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS Hum. Mol. Genet., March 1, 2001; 10(5): 467 - 476. [Abstract] [Full Text] [PDF]

				M. Meguro, K. Mitsuya, N. Nomura, M. Kohda, A. Kashiwagi, R. Nishigaki, H. Yoshioka, M. Nakao, M. Oishi, and M. Oshimura Large-scale evaluation of imprinting status in the Prader-Willi syndrome region: an imprinted direct repeat cluster resembling small nucleolar RNA genes Hum. Mol. Genet., February 1, 2001; 10(4): 383 - 394. [Abstract] [Full Text] [PDF]

				V. A. Erdmann, M. Z. Barciszewska, M. Szymanski, A. Hochberg, N. d. Groot, and J. Barciszewski The non-coding RNAs as riboregulators Nucleic Acids Res., January 1, 2001; 29(1): 189 - 193. [Abstract] [Full Text] [PDF]

				J. R Engel, A. Smallwood, A. Harper, M. J Higgins, M. Oshimura, W. Reik, P. N Schofield, and E. R Maher Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome J. Med. Genet., December 1, 2000; 37(12): 921 - 926. [Abstract] [Full Text]

				S. Engemann, M. Strodicke, M. Paulsen, O. Franck, R. Reinhardt, N. Lane, W. Reik, and J. Walter Sequence and functional comparison in the Beckwith-Wiedemann region: implications for a novel imprinting centre and extended imprinting Hum. Mol. Genet., November 1, 2000; 9(18): 2691 - 2706. [Abstract] [Full Text] [PDF]

				S.-i. Horike, K. Mitsuya, M. Meguro, N. Kotobuki, A. Kashiwagi, T. Notsu, T. C. Schulz, Y. Shirayoshi, and M. Oshimura Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome Hum. Mol. Genet., September 1, 2000; 9(14): 2075 - 2083. [Abstract] [Full Text] [PDF]

				V. M. Barbour, C. Tufarelli, J. A. Sharpe, Z. E. Smith, H. Ayyub, C. A. Heinlein, J. Sloane-Stanley, K. Indrak, W. G. Wood, and D. R. Higgs alpha -Thalassemia resulting from a negative chromosomal position effect Blood, August 1, 2000; 96(3): 800 - 807. [Abstract] [Full Text] [PDF]

				M. Paulsen, O. El-Maarri, S. Engemann, M. Strodicke, O. Franck, K. Davies, R. Reinhardt, W. Reik, and J. Walter Sequence conservation and variability of imprinting in the Beckwith-Wiedemann syndrome gene cluster in human and mouse Hum. Mol. Genet., July 22, 2000; 9(12): 1829 - 1841. [Abstract] [Full Text] [PDF]

				B. E. Hayward and D. T. Bonthron An imprinted antisense transcript at the human GNAS1 locus Hum. Mol. Genet., March 22, 2000; 9(5): 835 - 841. [Abstract] [Full Text] [PDF]

				D. Prawitt, T. Enklaar, G. Klemm, B. Gartner, C. Spangenberg, A. Winterpacht, M. Higgins, J. Pelletier, and B. Zabel Identification and characterization of MTR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression Hum. Mol. Genet., January 22, 2000; 9(2): 203 - 216. [Abstract] [Full Text] [PDF]

				T. Caspary, M. A. Cleary, E. J. Perlman, P. Zhang, S. J. Elledge, and S. M. Tilghman Oppositely imprinted genes p57Kip2 and Igf2 interact in a mouse model for Beckwith-Wiedemann syndrome Genes & Dev., December 1, 1999; 13(23): 3115 - 3124. [Abstract] [Full Text]

				S. F. Wroe, G. Kelsey, J. A. Skinner, D. Bodle, S. T. Ball, C. V. Beechey, J. Peters, and C. M. Williamson An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus PNAS, March 28, 2000; 97(7): 3342 - 3346. [Abstract] [Full Text] [PDF]

				C. Schwienbacher, L. Gramantieri, R. Scelfo, A. Veronese, G. A. Calin, L. Bolondi, C. M. Croce, G. Barbanti-Brodano, and M. Negrini Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas PNAS, May 9, 2000; 97(10): 5445 - 5449. [Abstract] [Full Text] [PDF]

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