Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia
Steven J. Kuerbitz1,2,3,
Joshua Pahys1,
Alison Wilson4,
Nicole Compitello1 and
Todd A. Gray4,5
1 Department of Pediatrics and
2 Department of Genetics, Case Western Reserve University and Ireland Cancer Center, Cleveland, OH, 44109, USA
3 Present addresses: Department of Pediatrics, Children's Hospital Medical Center of Akron, Akron, OH 44308-106, USA and
4 Wadsworth Center and Genomics Institute, Albany, NY 12208, USA
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Abstract
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Recent studies have demonstrated imprinting of the human neuronatin (NNAT) gene. NNAT maps to 20q11.2-q12, a region exhibiting loss of heterozygosity in acute myeloid leukemia and myelodysplastic/myeloproliferative disease. To investigate possible epigenetic dysregulation of genes in this region relevant to leukemogenesis, we analyzed methylation of the NNAT gene in normal tissues and in leukemias. We found a differential methylation pattern, typical of imprinted genes, at sites in the CpG island containing NNAT exon 1 in normal pituitary, peripheral blood cells and bone marrow-derived CD34-positive hematopoietic progenitor cells. Substantial or complete loss of the unmethylated NNAT allele was observed in leukemia cell lines and in 20 of 29 (69%) acute myeloid or lymphoid leukemia samples. While most highly expressed in brain, NNAT mRNA was also detected in normal hematopoietic progenitor cells and in leukemia cells exhibiting the normal methylation pattern, although not in hypermethylated leukemia cells. Demethylation by treatment of hypermethylated leukemia cells with 5-aza-2'-deoxycytidine resulted in reactivation of NNAT expression, concomitant with a reversion to the normal methylation pattern. The data demonstrate that hypermethylation of the NNAT locus is a frequent event in both myeloid and lymphoid acute leukemias of childhood. Aberrant hypermethylation of the NNAT locus suggests that the dysregulation of genes at 20q11.2-q12 in leukemia may be the result of epigenetic as well as genetic events.
Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; 5-azaCdR, 5-aza-2'-deoxycytidine; G6PD, glucose-6-phosphate dehydrogenase; MDS, myelodysplastic syndrome; MPD, myeloproliferative disease; PBMN, peripheral blood mononuclear cell; P. vera, polycythemia vera; SNRPD, small nuclear ribonucleoprotein D
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Introduction
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Deletion of a segment of human chromosome 20q observed in acute myeloid leukemia (AML), polycythemia vera (P. vera) and other myeloproliferative diseases (MPD) or myelodysplastic syndromes (MDS) suggests that a gene or group of genes in this region regulates hematopoiesis (16). Cytogenetic and molecular mapping studies have narrowed the interval containing this candidate tumor suppressor locus to the chromosome 20q12 region (711).
One gene that maps to this interval has recently been shown to be imprinted in humans; the neuronatin (NNAT) gene is imprinted and actively transcribed exclusively from the paternally inherited allele (12). Genomic imprinting is a process by which homologous alleles of a gene are differentially expressed in the same cell dependent upon the parental origin of the alleles (1316). The murine neuronatin gene (Nnat) on subdistal chromosome 2 is similarly imprinted (17,18). Imprinting was initially demonstrated in this region by the generation of mice with both allelic copies inherited from the mother (maternal uniparental disomy) or from the father (paternal uniparental disomy). These mice exhibit variant somatic and neuropathologic phenotypes and neonatal death (19), suggesting that biparental genetic contribution of imprinted genes in this region is required for normal development. The Nnat gene encodes a putative protein with structural similarity to proteolipid proteins that function as regulatory subunits of membrane-associated cation-translocation ATPases (18,20,21).
While deletion represents a mechanism for physical removal of a gene, epigenetic silencing offers an alternative method of gene inactivation. In fact, some tumor suppressor genes are more frequently epigenetically silenced than physically deleted in many cancers. The normally imprinted H19/IGF2 locus has been shown to be epigenetically dysregulated in some Wilms' tumors and chronic myelogenous leukemias (CML) (22,23). Therefore, we hypothesized that NNAT, an imprinted gene in a region frequently deleted in leukemia, is epigenetically silenced in leukemogenesis. We examined peripheral blood leukocytes (PBL) from normal individuals, and from pediatric patients with acute lymphoblastic leukemia (ALL) and AML, for DNA methylation at NNAT. Our results show that, while normal PBL show the differential methylation pattern expected of an imprinted gene, aberrant hypermethylation at the NNAT locus is a frequent event in acute leukemia in children. Furthermore, we find that the aberrant hypermethylation pattern is replaced by a normal differential methylation pattern upon hematologic remission. We also show that, although NNAT is transcribed in normal hematopoietic tissues, it is transcriptionally repressed, concordant with hypermethylation. Additionally, treatment of a leukemic cell line with 5-aza-2'-deoxycytidine (5-azaCdR) reinstates normal methylation and expression. The data suggest that epigenetic dysregulation of the imprinted NNAT gene and/or other genes in this region may play a role in leukemogenesis.
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Materials and methods
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Tissue samples and cell culture
Leukemia cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and were grown at 37°C in 5% CO2. Acute leukemia specimens were collected at the time of diagnostic evaluation from the bone marrow or peripheral blood of patients presenting to the Pediatric Hematology/Oncology service at Rainbow Babies and Children's Hospital (Cleveland, OH). Morphologic and immunophenotypic characterization of the leukemia cells was performed as part of the diagnostic evaluation. Informed consent was obtained from patients and/or parents. Normal bone marrow-derived hematopoietic progenitor cells expressing the CD34 antigen were obtained from the Hematopoietic Stem Cell core facility at the Ireland Cancer Center of Case Western Reserve University. Typically,
75% of cells yielded by the selection process express CD34.
Southern blot analysis
Human genomic DNA was prepared by standard methods. Ten micrograms of genomic DNA were digested to completion with 100 U BamHI with or without 100 U of the methylation-sensitive restriction enzymes FspI, NruI or BssHII. Digested DNA samples were separated in 1% agarose gels and transferred to nylon membranes. Membranes were hybridized overnight with the probe HNN1, which was labeled with [32P]dCTP by random hexamer priming. After washing, filters were subjected to autoradiography.
The 1.3 kb NNAT genomic probe HNN1 was generated by PCR using primers: forward (F), 5'-GATCCTGAGGCAGCTACAGCCTCG-3' and reverse (R), 5'-GCCCGCCACCTAAGTGCGCATGC-3' with total genomic or cloned genomic templates.
RTPCR analysis of NNAT expression
Total cellular RNA was extracted from cell pellets or frozen, pulverized tissue (24). Reverse transcription (RT) was carried out as described previously using 5 µg of total RNA as template for cDNA synthesis (25). A 3 µl aliquot of the RT reaction was used as a template in PCR reactions with primers corresponding to NNAT exon 1 and exon 3 sequences: E1F, 5'-CCAACAGCGGACTCCGAGACCAG and E3R, 5'-GTGTATGCCAGCTTCTGCAGGGAG-3'. RTPCR for GAPDH was performed as described previously (26). Immune tissue NNAT expression survey was performed with 0.5 µl aliquots of a Human Immune System multiple cDNA panel (Clontech, Palo Alto, CA), amplified with primers for NNAT (NNAT1 5'-TTTCTCGACCACCCACCTAC-3' and NNAT2 5'-TGTCCCGACTTTGTCCAGAT-3') or the SNRPD core mRNA splicing factor (SNRPDs 5'-CGGACCGAAGAGAAGAAAAG-3' and SNRPDas 5'-AAGCACCCACTCCAATGAAC-3') as a positive control.
Demethylation studies
KG1a myeloid leukemia cells were cultured in the presence of 1 µM 5-azaCdR for 72 h. Genomic DNA and RNA were prepared from cells as described above.
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Results
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Methylation of the NNAT locus in normal pituitary and blood cells
To assess methylation of the NNAT CpG island in normal cells, we generated a genomic probe, HNN1, extending 1 kb 5' from the transcriptional start site (21). Methylation-insensitive BamHI, and methylation-sensitive FspI, NruI and BssHII restriction sites were identified (Figure 1A
). Analysis of genomic DNA derived from human pituitary, which robustly expresses NNAT (25,26), reveals a differential methylation pattern at multiple sites within the CpG island, as detected by the methylation-sensitive restriction enzymes FspI, NruI and BssHII. Double digestion of DNA with BamHI and each of these enzymes creates a prominent 6 kb band representing the full-length BamHI fragment, indicating methylation at all methylation-sensitive restriction sites in
50% of alleles (Figure 1B
). In addition, a prominent band of 1.3 kb is detected in BamHIFspI digests, indicating an absence of methylation at the upstream FspI site in
50% of alleles. A minor band of 1.9 kb indicates that <10% of alleles are methylated at the upstream FspI site yet lack methylation at the downstream site (Figure 1A and B
, lane 4). A similar pattern of methylation is observed at the two BssHII sites (Figure 1A and B
, lane 3). Analysis of the single NruI site in intron 1 revealed methylation and unmethylated alleles in about equal proportions (Figure 1A and B
, lane 2).

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Fig. 1. Structure and methylation of the human neuronatin gene. (A) Filled boxes represent NNAT exons as determined previously (20,21). Restriction enzyme sites are BamHI (B), BssHII (Bs), FspI (F) and NruI (N). The 1.3 kb NNAT probe HNN1 is depicted below the map as a hatched line. Solid lines below the map indicate products resulting from digestion of genomic DNA with the indicated methylation-sensitive restriction enzyme plus BamHI. Open and closed circles indicate unmethylated and methylated restriction sites, respectively. (B) Southern blot of genomic DNA derived from normal human pituitary probed with HNN1. Restriction digests for each sample are shown above the lane as abbreviated in (A), and the 6 kb BamHIBamHI and 1.6 kb BamHINruI fragments are indicated by arrows in the left margin. (C) Southern blot of two normal PBMN samples. Annotations as in (B).
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Analysis of genomic DNA from human peripheral blood mononuclear (PBMN) cells from two normal individuals reveals a differential methylation pattern similar to that of pituitary DNA (Figure 1C
). Approximately equal proportions of alleles with full methylation of BssHII sites and alleles lacking methylation at the upstream site are evident in both DNA samples (Figure 1C
, lanes 3 and 5). As observed in pituitary DNA, a small proportion of alleles are methylated at the upstream BssHII site yet lack methylation at the downstream site. Incomplete methylation in this region has been noted previously (12). DNA from both individuals shows alleles with methylated or unmethylated NruI sites in about equal proportions (Figure 1C
, lanes 2 and 4). This BamHINruI digestion pattern is observed in PBMN-derived DNA from three additional individuals, as well as in normal human liver, kidney and cultured human diploid fibroblasts (data not shown). This differential methylation pattern is similar to that observed at the imprinted murine NNAT locus, which is methylated at restriction sites in the exon 1 CpG island on the transcriptionally silent maternally derived allele, but unmethylated at sites on the transcribed paternally derived allele (17).
Aberrant NNAT hypermethylation in acute leukemia
Because aberrant DNA methylation is associated with dysregulation of imprinted genes in 11p15.5 in Wilms' tumor (27), we analyzed the NNAT locus to identify possible aberrant methylation in DNA from human leukemia cell lines and primary childhood leukemias. A representative Southern blot is shown in Figure 2A
. BamHINruI-digested DNA from normal peripheral blood cells again shows the expected bands of 6 and 1.6 kb, representing methylated and unmethylated NNAT alleles, respectively, in about equal proportions (Figure 2A
, lane 1). In contrast, analysis of DNA from each of five myeloid or lymphoid leukemia cell lines (four shown, Figure 2A
, lanes 25), reveals a complete loss of the normal 1.6 kb band, indicating uniform methylation at the NruI site. In addition, 20 of 29 primary leukemia samples (69%) reveal hypermethylation, indicating loss or de novo methylation of unmethylated NNAT alleles (five shown, Figure 2A
, lanes 610). Traces of the 1.6 kb BamHINruI fragment in two samples (Figure 2A
, lanes 8 and 9) are likely to result from the presence of circulating normal cells in the primary sample. Samples exhibiting hypermethylation include eight of 13 early B-lineage ALL, two of four T-lineage ALL and 10 of 12 AML. Hypermethylation of BssHII and FspI sites in the NNAT CpG island is also detected in leukemia cells (data not shown). Analysis of several samples informative for a closely linked polymorphic marker indicates the presence of both alleles, suggesting that LOH is not responsible for absence of the unmethylated fragment (see Discussion; data not shown).

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Fig. 2. The NNAT CpG island is hypermethylated in leukemias. (A) Hypermethylation of the NNAT CpG island in leukemia cell lines and samples. A BamHI plus NruI double digest Southern blot of DNA derived from normal human PBMN cells (PB, lane 1); the Burkitt lymphoma cell line RAJI (lane 2); and AML cell lines K562, ML-1 and HL-60 (lanes 35); primary leukemia samples including AML (lanes 6 and 9), T-lineage ALL (lane 7) and B-lineage ALL (lanes 8 and 10). PBMN DNA was under-loaded relative to leukemia samples. The blot was probed with HNN1. (B) Differential methylation of the NNAT CpG island in CD34+ hematopoietic progenitor cells and remission-derived blood cells. Shown is a Southern blot of BamHINruI digested DNA from normal PBMN cells (lane 1), normal CD34+ hematopoietic progenitor cells (34+, lane 2), leukemia samples (L, lanes 3, 5, 7 and 9) and peripheral blood cells obtained from patients (Pats. AD) during hematologic remission corresponding to the leukemia samples in the preceding lanes (R, lanes 4, 6, 8 and 10). The blot was probed with HNN1.
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To rule out the possibility that the predominance of methylated alleles of the NNAT CpG island observed in leukemias reflects the normal methylation state in hematopoietic progenitor cells comprising a very small proportion of cells in peripheral blood, we analyzed DNA prepared from human bone marrow cells selected for expression of the human hematopoietic progenitor cell antigen CD34 (CD34+). Expression of this glycoprotein identifies pluripotent and early lineage-committed hematopoietic progenitor cells (28). As shown in Figure 2B
, BamHINruI-digestion of DNA from CD34+ cells (lane 2) reveals the same differential methylation pattern seen in PBMNs (lane 1). Four leukemia patients with a hypermethylated NNAT profile were re-evaluated upon hematologic remission. In each case, DNA from remission PBMNs exhibits a prominent 1.6 kb band (Figure 2B
, lanes 4, 6, 8 and 10) compared with DNA from the corresponding leukemia samples (Figure 2B
, lanes 3, 5, 7 and 9). Together, the data strongly suggest that hypermethylation of the NNAT CpG island is an aberrant event frequently and closely associated with leukemic transformation in human leukocytes.
NNAT expression in normal hematopoietic cells and leukemia cells
Expression of human and rodent neuronatin is subject to developmental and tissue-specific regulation. Murine Nnat expression is highest in the fetal brain, with the distribution and intensity of expression declining with maturation (17,20,26). Although Nnat expression was not detected in most non-neural tissues, hematopoietic cells were not examined in those studies. We therefore used RTPCR to assess NNAT expression in normal human hematopoietic cells and in leukemia cells. As a control for integrity of the mRNA, ubiquitous GAPDH or SNRPD expression was also assayed and was detected in all samples (Figure 3AC
, bottom panels). Two NNAT mRNA isoforms were detected by RTPCR in RNA from human pituitary (Figure 3A
, top panel, lane 1). These bands represent alternatively spliced products containing (NNAT
) or lacking (NNATß) exon 2, as has been shown previously in human cells (20). Surprisingly, NNAT expression was also detected in RNA from a population of normal bone marrow cells enriched for hematopoietic progenitor cells expressing CD34 (Figure 3A
, top panel, lane 5). NNAT transcripts were not readily detectable in RNA from normal, mature CD34-negative PBMNs (Figure 3A
, top panel, lane 3) or from human THP1 monocytic leukemia cells (Figure 3A
, top panel, lane 7), which exhibit full methylation at NNAT (data not shown).

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Fig. 3. Expression of human NNAT determined by RTPCR. (A) NNAT expression in normal pituitary (Pit, lanes 1 and 2), PBMN (PB, lanes 3 and 4), CD34+ cells (34+, lanes 5 and 6) and a NNAT hypermethylated AML cell line, THP1 (lanes 7 and 8). Paired amplification reactions were performed either with (+) or without () prior reverse transcription. (B) NNAT transcription in CD34+ leukemias correlates with methylation status. A B-lineage ALL sample exhibiting normal differential NNAT methylation (lanes 1 and 2); B-lineage ALL samples (lanes 36) and an AML sample (lanes 7 and 8), all exhibiting NNAT hypermethylation, and normal human pituitary (lane 9). (C) Hematopoietic/immune system tissue expression of NNAT. cDNA aliquots (Clontech) were amplified from pooled samples representing a wide range of tissue types (C, lane 1), bone marrow (BM, lane 2), fetal liver (Li, lane 3), lymph node (LN, lane 4), spleen (S, lane 5) and thymus (Th, lane 6). The positions of NNAT and NNATß splice forms are indicated in the right margins, as are positive controls GAPDH and SNRPD.
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To determine whether NNAT expression is correlated with the methylation pattern in leukemia cells, we assayed NNAT expression in RNA derived from five CD34+ leukemia samples. Two CD34+ ALL samples (one B-lineage, one T-lineage) that display a normal differential methylation pattern at the NNAT CpG island expressed NNAT by RTPCR (one shown, Figure 3B
, top panel, lane 1). Two other B-lineage ALL samples, and one AML sample, each of which expressed CD34 but exhibited aberrant hypermethylation of the NNAT CpG island, lacked NNAT RNA expression (Fig. 3B
, top panel, lanes 3, 5 and 7, respectively). Blasts comprised >90% of nucleated cells in each leukemic marrow specimen. Aberrant hypermethylation is concordant, therefore, with the absence of NNAT transcription in these leukemia samples. NNAT is variably expressed in a broad range of hematopoietic/immune tissues (Figure 3C
, top panel). Interestingly, different NNAT
and NNATß splice form ratios appear in the control (pooled tissues, Figure 3C
, lane 1), liver (lane 3) and thymus (lane 6); the significance of this is at present unclear.
Demethylation-associated reactivation of NNAT expression in leukemia cells
As a hallmark of epigenetic transcriptional repression is reversibility, we treated hypermethylated leukemia cells with 5-azaCdR to attempt demethylation of the NNAT locus over a 72 h exposure period. As shown in Figure 4A
, KG1a myeloid leukemia cells were fully methylated at the NNAT CpG island NruI site (lane 1), while partial demethylation of the NNAT CpG island was evident after 72 h incubation in 1 µM 5-azaCdR. This shift toward the normal differential methylation pattern was also seen with BssHII and FspI restriction enzymes (data not shown). As expected, untreated KG1a cells did not express NNAT mRNA (Figure 4B
, lane 1). Expression of NNAT was detectable by RTPCR, however, in 5-azaCdR-treated KG1a cells by 24 h and was robust by 72 h (Figure 4B
, lanes 3 and 5, respectively). This result is consistent with the silencing of NNAT expression in leukemia cells as a result of epigenetic transcriptional repression associated with hypermethylation of the CpG island.

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Fig. 4. NNAT demethylation and expression in 5-azaCdR-treated leukemia cells. KG1a myeloid leukemia cells were incubated for up to 72 h in 1 µM 5-azaCdR. (A). Southern blot of genomic DNA prepared from cells harvested after 0, 24 or 72 h of culture in the presence of 5-azaCdR. DNAs were double digested with BamHINruI and hybridized with the probe HNN1. The 6.0 and 1.6 kb fragments are indicated by arrows in the left margin. (B) Total RNA was prepared from cells treated for 0 (lanes 1 and 2), 24 (lanes 3 and 4) and 72 (lanes 5 and 6) h. The positive control (C, lane 7) utilized RNA from the cell line HOS. RNA was used in RTPCR reactions with (+) or without () reverse transcriptase for NNAT (top panel) and GAPDH (bottom panel).
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Discussion
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The imprinted NNAT gene maps to human chromosome 20q11.2-q12 (12), a region frequently deleted in myeloid disorders. Because aberrant regulation of genes in this region could be relevant to leukemogenesis, we performed a methylation analysis of the imprinted NNAT locus in normal human tissues and in pediatric acute leukemias. Methylation analysis was focused on the CpG island encompassing exon 1 of the NNAT gene and revealed methylation of
50% of alleles in normal tissues, consistent with the differential methylation pattern characteristic of imprinted genes. In mice and in humans, the methylated and transcriptionally silent allele is maternal in origin, whereas the unmethylated and expressed allele is paternally derived (12,17,18,29,30). While normal tissues, including CD34+ hematopoietic progenitor cells, exhibit differential NNAT CpG island methylation, hypermethylation was evident in 69% of the leukemia samples analyzed. Furthermore, methylation status reflects transcriptional competence, as we show that CD34+ leukemia samples with differential methylation transcribe NNAT, while biallelically methylated samples are transcriptionally silent. This aberrant hypermethylation correlates with the leukemic phenotype, as patients with NNAT hypermethylation revert to the normal differential methylation pattern in remission. Therefore, epigenetic dysregulation of the imprinted NNAT locus is frequently associated with pediatric leukemogenesis.
Aberrant methylation of imprinted genes has been implicated in the pathogenesis of childhood cancer. Biallelic CpG island methylation of the imprinted H19 gene, associated with transcriptional repression of both H19 alleles, has been observed in Wilms' tumor (27), along with aberrant biallelic expression of the adjacent, paternally expressed IGF2 gene (27,3133); this pattern was also seen in AML (34) and CML (22). This methylation-assayable loss of imprinting (27) may also alter expression of other imprinted genes in the 11p15.5 region (35). A `loss of imprinting' status at the NNAT locus must await studies that show neighboring genes or CpG islands are coordinately normally imprinted but epigenetically dysregulated in cancer. The NNAT gene may therefore represent a sentinel locus whose methylation status effectively marks a new region of aberrant imprinting in childhood cancer.
As an alternative to hypermethylation, a uniform, fully methylated pattern at the NNAT CpG island in leukemia cells could reflect deletion of the normally unmethylated paternally inherited allele. However, KG1a cells, which show only a methylated NNAT pattern and do not transcribe NNAT, nevertheless retain two identifiable alleles of a closely linked microsatellite marker, D20S478 (7) (data not shown). Moreover, three of six primary leukemia specimens showing a single methylated NNAT Southern blot fragment were also informative for this marker (data not shown). While normal tissues corresponding to the three non-informative hypermethylated leukemic samples were not similarly genotyped, our data suggest that hypermethylation, rather than allelic loss, is the primary mechanism for the absence of the unmethylated NNAT Southern blot fragment. Furthermore, if NNAT LOH did occur in the non-informative samples, there is an apparent selection bias for loss of the unmethylated allele as none of the leukemia samples analyzed in this study showed loss of the methylated BamHINruI fragment. Should a putative tumor suppressor gene be normally imprinted, the actively transcribed allele would be expected to be specifically lost, as removal of a normally silent allele would have no phenotypic impact. We therefore hypothesize deletions in chromosome 20q11.2-q12 associated with cancer will show parental origin bias, specifically, loss of the paternal allele harboring the unmethylated NNAT gene. Whether allelic deletion at 20q11.2-q12 in leukemia exhibits parent-of-origin bias has not yet been addressed.
Expression of the NNAT gene has been identified previously in brain tissues (17,18,20,26) and in the SV-40 transformed pancreatic cell lines betaTC3 (36). Interestingly, rat Nnat was identified in a screen for genes that are down regulated in the progression from immortalization to transformation; however, neuronatin's significance in the transition to transformation is unknown (37). We detect NNAT expression in bone marrow-derived CD34+ hematopoietic progenitor cells, and variable expression in other tissues. CD34+ leukemia cells that exhibit a normal pattern of methylation express NNAT mRNA, whereas aberrant hypermethylation of the NNAT CpG island in CD34+ cells is accompanied by transcriptional silencing. As a large proportion of childhood leukemias are CD34+ (38,39), epigenetic dysregulation of NNAT in 69% of samples in this study suggests that loss of neuronatin expression may be an important factor in leukemic transformation.
Whether loss of NNAT expression is directly relevant to leukemogenesis, or whether aberrant methylation of this locus potentially marks the inactivation of other genes at 20q11.2-q12, is unclear. Findings that localize the common interval of chromosome 20q allelic loss to a region adjacent to NNAT suggest that deletion of flanking genes or control elements contributes to myeloid disease in adults (9,10,34). The data must now be re-evaluated, given that imprinted domains can be mega bases in size and regulated by distant elements. Interestingly, the compact three-exon NNAT gene lies within the only intron of the bladder cancer-associated protein (BLCAP) gene, which is not imprinted in human brain, in contrast to NNAT (12). Tissue-specific imprinting has not yet been carefully examined and other genes in the vicinity have not been evaluated to determine whether NNAT is the only imprinted gene in an otherwise non-imprinted region or, alternatively, whether BLCAP is protected from local imprinting effects. The GenBank record for BLCAP (GenBank accession AF053470) states that it was originally identified as being down regulated in bladder cancer, raising the possibility that its down regulation is epigenetically based, as is NNAT.
Chromosome 20q allelic loss in up to 9% of leukemias reported in previous studies (8,9,10,40) indicates the presence of a tumor suppressor gene in this region and is supported by our finding of NNAT hypermethylation in a majority of pediatric leukemias. Combined, LOH and now hypermethylation in chromosome 20q in leukemia suggest that this region may be one of the most frequently mutated genomic regions in leukemia. The data presented here not only strongly support a role for dysregulation of chromosome 20q11.2-q12 genes in leukemia but they also provide a new highly associated biomarker in cancer.
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Notes
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5 To whom correspondence should be addressed Email: gray{at}wadsworth.org 
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Acknowledgments
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We thank Dr George Dubyak for providing the THP1 leukemia cell lines and Dr Leona Cuttler for providing human pituitary tissue for analysis. We also thank Drs Huntington F.Willard, Rob Nicholls and Stanton Gerson for reviewing the manuscript. This work was supported in part by a grant to S.J.K from the Rainbow Babies and Children's Hospital Board of Trustees and a Joesph S.Silber Student Fellowship awarded to J.P. by the American Cancer Society, Ohio Division.
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Received December 27, 2001;
accepted January 2, 2002.