00019606-200106000-00005ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2001 Lippincott Williams & Wilkins, Inc.10June 2001
p 100-104Rearrangement in the Coding Region of the MYCN Gene in a Subset of Amplicons in a Case of Neuroblastoma With MYCN AmplificationOriginal ArticlesChen, Beiyun M.D., Ph.D.; Jhanwar, Suresh C. Ph.D.; Ladanyi, Marc M.D.From the Departments of Pathology and Medicine, Memorial Sloan-Kettering Cancer Center, New York, New YorkAddress correspondence and reprint requests to Beiyun Chen, MD, PhD, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021 (e-mail:
[email protected] mskcc.org).AbstractThe MYCN gene is often amplified but rarely rearranged in neuroblastoma. We report, for the first time, a
rearrangement within the MYCN coding region in a metastatic neuroblastoma in a 3-year-old boy with MYCN amplification in his primary tumor. The
rearrangement occurred 46 nucleotides downstream from the ATG codon in exon 2 of MYCN. The amplification level of the rearranged copies of the MYCN gene was lower than that of the unrearranged copies of MYCN. These results indicate that the
rearrangement occurred after initial MYCN gene amplification. Monochromosomal somatic cell hybrid mapping of the novel region fused to exon 2 of MYCN localized it to chromosome 2, suggesting that this
rearrangement resulted from an interstitial deletion, presumably within the MYCN amplicon itself.The MYCN gene is often amplified up to several hundred-fold in neuroblastoma cell lines and tumors (3,6,9). The amplification is associated with increased expression of both mRNA and protein and is significantly correlated with poor prognosis (2,10). The amplified DNA region encompasses much more than the MYCN transcription unit and can range from 100 kilobases to several megabases (1,8). The individual amplicons, each containing many copies of MYCN, are often arranged in a regular pattern, where variable numbers of MYCN amplicons are repeated in a 5´→3´ direction (1,8,10).Structural analysis of the amplicons has shown that rearrangements often occur, but at some distance from the amplified MYCN genes (5,11). The rearrangements occur less frequently in primary tumors than in cell lines (8). These rearrangements are typically too far (i.e., >30 kb) from the MYCN gene to be detectable by conventional Southern blot analysis using an MYCN probe, with a single reported exception (4). In one patient's tumor, the
rearrangement occurred 723 bp downstream from the 3´ end of exon 3 of MYCN and therefore did not directly alter the coding region of the MYCN gene (4). Both the rearranged MYCN gene and the normal MYCN gene were amplified to the same degree in that case.In the course of routine clinical analysis of MYCN gene amplification in neuroblastoma samples, the authors encountered a case with
rearrangement and amplification of MYCN. The rearranged MYCN gene was moderately amplified, whereas the germline MYCN gene was highly amplified. The
rearrangement, resulting from an interstitial deletion on chromosome 2, occurred within exon 2 of the MYCN gene and is described in detail here.MATERIALS AND METHODSDNA extraction and Southern blot analysisA bone marrow aspirate from a 3-year-old boy with neuroblastoma was submitted for MYCN gene amplification analysis. The sample was received on ice and processed immediately without further storage. DNA was isolated by SDS-proteinase K lysis and phenol-chloroform extraction. Ten micrograms of DNA was digested with restriction enzymes (Eco RI, Hin dIII, Bgl II, Sac I) (Boehringer Mannheim, Indianapolis, IN), size-fractionated in 1% agarose gels, and transferred to SureBlot nylon membranes (Intergen, Purchase, NY). The membranes were hybridized successively to three MYCN probes, one each for exon 1 (polymerase chain reaction [PCR]-generated probe of 401 bp using a forward primer 5´-TCC TGG GAA CTG TGT TGG-3´ and a reverse primer 5´-TTC GCA GAA GCC ATT ACT C-3´), exon 2 (Nb-1 probe) (1,3), and exon 3 (PCR-generated probe of 714 bp using a forward primer 5´-CCG CTC AAG AGT GTC GTC C-3´ and a reverse primer 5´-CCC AGG GGT ATC AAA TGG C-3´) of the MYCN gene (12). The membranes were sequentially or simultaneously hybridized with a probe to the immunoglobulin heavy chain gene on chromosome 14 as an internal control for normalization of DNA loading. The probes were labeled with [α-32P]dCTP by the conventional random primer method. The membranes were washed. The signals were collected and analyzed on a phosphorimager (Bio-Rad Molecular Imager, Hercules, CA). Genomic DNA from human placenta was used as negative control.Fluorescence in situ hybridizationFluorescence in situ hybridization analysis was performed using LSI N-myc probe (Vysis, Downers Grove, IL). The cytogenetic pellet was stored at 4°C in fixative solution (ethanol–acetic acid 3:1) until analysis. The air-dried slides were prepared and hybridized according to methods routinely used in the laboratory. The denaturation was performed in 70% formamide–2× SSC at 70°C to 73°C for 5 minutes. The slides were immediately processed through an ice-cold ethanol series (70%, 80%, 95%, and 100%) for 3 minutes each. After drying, 10 μL of a probe mixture was applied to the area of interest on the slide, and a coverslip was applied. Hybridization was performed overnight at 37°C in a humidified chamber, followed by three washes at 45°C in 50% formamide–2× SSC for 10 minutes each and then at 37°C in 2× SSC for 10 minutes. After posthybridization washes, the slides were counterstained with DAPI and coverslipped. The number of MYCN gene copies varied from 20 to 60 signals per cell, indicating amplification of approximately 30 times.Inverse PCR and sequencingGenomic DNA from the patient's bone marrow aspirate was digested with Bgl II for 20 hours. DNA fragments were purified by phenol–chloroform extraction followed by ethanol precipitation. Circularization of the fragments with T4 ligase was carried out at a final DNA concentration of 2 μg/mL and T4 ligase concentration of 0.02 Weiss units/μL for 16 hours at 15°C. After ethanol precipitation the circularized DNA was linearized with Eco RI, which cuts outside the unknown sequence of interest. The linearized DNA fragments were purified as mentioned above and 0.1 μg DNA was subjected to long-range PCR amplification (XL PCR kit, Perkin Elmer, Branchburg, NJ) with MYCN primers flanking the unknown sequence (Fig. 1). The PCR conditions were as follows: 35 cycles of 94°C for 45 seconds, 58°C for 1 minute, and 72°C for 5 minutes, followed by a final 10-minute extension at 72°C in the presence of 1 mmol/L magnesium acetate and 30 pmol of each primer. The PCR product was directly sequenced on an automated sequencer using the same PCR primers for the first round. Internal primers were chosen according to the new sequence. Long-range PCR was performed again on the genomic DNA and the PCR product was sequenced using the same PCR primers. This stepwise procedure was repeated until the complete sequence was obtained.JOURNAL/dimp/04.03/00019606-200106000-00005/figure1-5/v/2021-02-17T195814Z/r/image-tiff
Schematic diagram of inverse long-range polymerase chain reaction (PCR). Open area:MYCN gene region. Shaded area: unknown sequence. P1–P6: PCR primers.Somatic cell hybrid mapping by PCRTo identify the chromosomal origin of the sequence downstream of the MYCN breakpoint, primers were chosen from the downstream sequence for PCR amplification of a somatic cell hybrid DNA panel. The hybrid DNA panel consisted of DNA isolated from 24 human/rodent somatic cell hybrids, each containing one intact human chromosome (Coriell Institute for Medical Research, Camden, NJ).RESULTSClinical summaryThe patient was diagnosed with an unresectable stage 3 neuroblastoma at age 3. The tumor arose from the left adrenal gland and involved the retroperitoneal lymph nodes. Adverse biologic prognostic factors present at diagnosis included MYCN amplification, diploid chromosomal content, chromosome 1p deletion, and unfavorable Shimada histopathology. Treatment included four cycles of high-dose cyclophosphamide (140 mg/kg) with doxorubicin (75 mg/m2) and vincristine (0.067 mg/kg) (HD-CAV), three cycles of cisplatin (200 mg/m2)–etoposide (600 mg/m2) (P/VP), immunotherapy using 3F8 monoclonal antibody, and targeted radioimmunotherapy using 20 mCi/kg 131I-3F8. Residual disease was entirely resected after three cycles of HD-CAV and one cycle of P/VP, and the tumor bed was treated with external-beam radiation therapy (21 Gy). Relapse in bone marrow was documented 13 months from diagnosis and death occurred 1 month later.Southern blot analysisUsing an MYCN exon 2 probe, nongermline bands were detected with all four enzymes. A 3.4-kb nongermline band was detected with Bgl II digestion, a 6.6-kb nongermline band with Sac I, a 14-kb nongermline band with Hin dIII digestion, and a 7.6-kb nongermline band with Eco RI digestion (Fig. 2). Interestingly, the rearranged MYCN gene was only moderately amplified (3-fold), whereas the germline MYCN gene was highly amplified (33-fold). Using an MYCN exon 1 probe (Fig. 3), a nongermline band was detected with Sac I and Hin dIII digestion, but not with Bgl II or Eco RI digestion (data not shown). An MYCN exon 3 probe did not detect any nongermline band with any of the enzymes used (data not shown). These results indicated that the MYCN gene in the bone marrow sample was interrupted between the Eco RI site and the Sac I site in the intron 1 and exon 2 region, and that the 3´ downstream portion of the MYCN gene was lost from the rearranged gene copies.JOURNAL/dimp/04.03/00019606-200106000-00005/figure2-5/v/2021-02-17T195814Z/r/image-png
Southern blot analysis using MYCN exon 2 probe. Lanes 1, 3, 5, and 7: human placental DNA as germline control. Lanes 2, 4, 6, and 8: patient bone marrow DNA with neuroblastoma. Lanes 1 and 2:Bgl II digestion. Lanes 3 and 4:Sac I digestion. Lanes 5 and 6:Hin dIII digestion. Lanes 7 and 8:Eco RI digestion. Lane 9: DNA size standard. Normalization of DNA loading by hybridization with a control probe confirmed 3-fold amplification of the rearranged bands and 33-fold amplification of the germline bands (not shown).JOURNAL/dimp/04.03/00019606-200106000-00005/figure3-5/v/2021-02-17T195814Z/r/image-tiff
Restriction maps of germline MYCN gene (A) and rearranged MYCN gene (B). H, Hin dIII; S, Sac I; Bg, Bgl II; E, Eco RI; B, Bam HI. Horizontal arrows indicate position and size of MYCN probes used in Southern blot analysis for exon 1 (probe 1), exon 2 (probe 2), and exon 3 (probe 3). Positions of noncoding and coding regions of MYCN exons are indicated by open and shaded rectangles, respectively. Dashed line indicates the sequence fused to the MYCN gene (B). Vertical arrow (↓) indicates breakpoint.Fluorescence in situ hybridizationFluorescence in situ hybridization analysis showed that 7% of cells had >20 copies of the MYCN gene per cell, whereas the remaining 93% of cells had 2 copies per cell. This indicates that there was only one population of cells with a high-level amplification of MYCN.Sequence of the breakpoint regionBecause the 3.4-kb nongermline band created by Bgl II digestion was well within the limit of long-range PCR, the entire sequence of this fragment was obtained using an inverse long-range PCR strategy. The breakpoint was at nucleotide 188 of exon 2, which is 46 nucleotides downstream of the ATG codon of the MYCN gene (Fig. 4). The sequence downstream from the breakpoint showed no homology to any known gene sequence in GenBank or the Human Genome Database it mapped to chromosome 2, as indicated by PCR amplification in a human monochromosomal somatic cell hybrid DNA panel (data not shown).JOURNAL/dimp/04.03/00019606-200106000-00005/figure4-5/v/2021-02-17T195814Z/r/image-tiff
Sequence of the breakpoint region. Underlined bold letters indicate the MYCN sequence. The triple asterisk indicates the start of coding region 5´ of the breakpoint. Vertical arrow (↓) indicates breakpoint. The predicted amino acid sequence of the fusion protein is shown under the DNA sequence. (GenBank accession AF317388)DISCUSSIONRearrangement of the amplified DNA containing the MYCN gene is known to occur in the regions surrounding the MYCN gene but has not been reported to involve the MYCN gene itself directly (4,5,8,11). However, there has been one report by Hiyama et al. (4) documenting a
rearrangement in the 3´ flanking region of the MYCN gene in a neuroblastoma. The
rearrangement was detected in the primary tumor and in the lymph node metastasis but not in the hepatic metastasis. Both the rearranged gene and the normal gene were amplified to the same degree. Based on these results, it was suggested that two clones (one with the
rearrangement and another without the
rearrangement) coexisted in the primary tumor. The clone with the
rearrangement then gave rise to the lymph node metastasis, and the
rearrangement occurred before, not during, amplification.The current authors report, for the first time, a
rearrangement occurring within the coding region of the MYCN gene in a neuroblastoma sample. An interesting finding was that the rearranged MYCN gene was only moderately amplified, whereas the germline MYCN gene was highly amplified. The
rearrangement in this case occurred at the beginning of the coding region, 46 nucleotides from the ATG codon, and would lead to the synthesis of a markedly truncated fusion protein of 48 amino acids. The authors hypothesize that this interstitial deletion occurred within an amplicon, either early or late after initial MYCN gene amplification. If the
rearrangement occurred soon after MYCN amplification, the interruption of the MYCN coding region presumably reduced the selective advantage provided by this subset of amplicons, leading to a gradual decrease in its representation. In an initial period, these deleted amplicons may have expanded simply as “passengers” alongside functional MYCN amplicons. Alternatively, the deleted MYCN genes may coexist on the same amplicons with functional MYCN genes, and their lower amplification level may be due to the later occurrence of the interstitial deletion involving MYCN in the natural history of this neuroblastoma. Hiyama et al. (4) had shown that a rearranged MYCN gene was highly amplified in a case where the
rearrangement occurred at 723 bp downstream of exon 3. This finding and the current data suggest that a functional MYCN gene is required for the gene amplification. Previous studies have shown that the MYCN gene is located at the 5´ end of the 130-kb core region within the MYCN amplicon and that the only necessary oncogenic element of the amplicon is the MYCN gene (8). The current findings provide further anecdotal evidence for this pathobiology.Amplification of the MYCN gene is associated with aggressive clinical behavior. MYCN is a classic protooncogene. Amplification leads to overproduction of the MYCN protein, a nuclear transcription factor, which presumably contributes to the aggressive behavior of these tumors by deregulating certain critical target genes (7). The tumor in this case did not respond to multimodal therapy, and the patient died of the disease. The rearranged MYCN gene described here would, if expressed, have produced only a short polypeptide of 48 amino acids. Therefore, it is unlikely that the amplicons containing this rearranged MYCN gene played a significant role in the aggressive behavior of this particular neuroblastoma.Acknowledgements:The authors thank Dr. Brian Kushner for providing clinical data and Dr. Nai-Kong Cheung for helpful discussion.REFERENCES1. Akiyama K, Kanda N, Uamada M, et al. Megabase-scale analysis of the origin of MYCN amplicons in human neuroblastomas. Nucl Acids Res 1994; 22:187–93.[Context Link][CrossRef][Medline Link]2. Brodeur GM. Molecular basis for heterogeneity in human neuroblastoma. Eur J Cancer 1995; 31A:505–10.[Context Link][CrossRef][Medline Link]3. Brodeur G, Seeger RC, Schwab M, et al. Amplification of MYCN in untreated human neuroblastomas correlates with advanced disease stage. Science 1984; 224:1121–4.[Context Link][CrossRef][Medline Link]4. Hiyama E, Hiyama K, Yokoyama T. Neuroblastoma with DNA amplification and
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Schematic diagram of inverse long-range polymerase chain reaction (PCR). Open area:MYCN gene region. Shaded area: unknown sequence. P1–P6: PCR primers.
Southern blot analysis using MYCN exon 2 probe. Lanes 1, 3, 5, and 7: human placental DNA as germline control. Lanes 2, 4, 6, and 8: patient bone marrow DNA with neuroblastoma. Lanes 1 and 2:Bgl II digestion. Lanes 3 and 4:Sac I digestion. Lanes 5 and 6:Hin dIII digestion. Lanes 7 and 8:Eco RI digestion. Lane 9: DNA size standard. Normalization of DNA loading by hybridization with a control probe confirmed 3-fold amplification of the rearranged bands and 33-fold amplification of the germline bands (not shown).
Restriction maps of germline MYCN gene (A) and rearranged MYCN gene (B). H, Hin dIII; S, Sac I; Bg, Bgl II; E, Eco RI; B, Bam HI. Horizontal arrows indicate position and size of MYCN probes used in Southern blot analysis for exon 1 (probe 1), exon 2 (probe 2), and exon 3 (probe 3). Positions of noncoding and coding regions of MYCN exons are indicated by open and shaded rectangles, respectively. Dashed line indicates the sequence fused to the MYCN gene (B). Vertical arrow (↓) indicates breakpoint.
Sequence of the breakpoint region. Underlined bold letters indicate the MYCN sequence. The triple asterisk indicates the start of coding region 5´ of the breakpoint. Vertical arrow (↓) indicates breakpoint. The predicted amino acid sequence of the fusion protein is shown under the DNA sequence. (GenBank accession AF317388)
Rearrangement in the Coding Region of the <em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">MYCN</em> Gene in a Subset of Amplicons in a Case of Neuroblastoma With <em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">MYCN</em> AmplificationChen Beiyun M.D. Ph.D.; Jhanwar, Suresh C. Ph.D.; Ladanyi, Marc M.D.Original ArticlesOriginal Articles210p 100-104
