A physical, transcript, and deletion map of chromosome region 12p12.3 flanked by ETV6 and CDKN1B: hypermethylation of the LRP6 CpG island in two leukemia patients with hemizygous del(12p).
FISH analyses and loss of heterozygosity studies have delineated a commonly deleted region in hematological malignancies flanked by ETV6 and CDKN1B on chromosome 12p12.3. The same chromosomal region is also a target for deletions in certain solid tumors. As an initial step toward the cloning of a potential tumor suppressor gene at 12p12.3, we mapped the ETV6-CDKN1B region physically using bacterial artificial chromosome (BAC) and P1-derived clone (PAC) contigs. The 1.2-Mb high-resolution, contiguous map extends from D12S1095 to D12S929 and consists of 19 PACs and 20 BACs. Pulsed-field gel electrophoresis experiments confirmed the integrity of the clone-based map and identified six CpG islands in the region. A transcript map was generated by performing hybridization selection experiments with the genomic clones, by evaluating known 12p ESTs for their presence in the contig, and by sequence analysis of CpG islands in the region. Altogether evidence was gathered for the presence of the recently published LRP6 gene and at least seven other new genes in this chromosomal region. The CLAPS3 gene, mapped between D12S391 and D12S358, was reassigned to chromosome 5 since genomic sequencing demonstrated the chromosome 12p sequence to be a pseudogene. Polymorphic CA repeats were identified approximately every 100 kb, which will support future analysis of loss of heterozygosity in tumors. Fluorescence in situ hybridization analysis of leukemia patients with del(12p) further refined the commonly deleted segment to 600 kb between ETV6 and D12S358, which apparently excludes CDKN1B. Methylation changes of the CpG islands in the ETV6-CDKN1B interval were assessed by Southern analysis for leukemia patients with hemizygous 12p deletions. A "de novo" methylation was detected only at the LRP6 CpG island in 2 of 22 leukemia patients tested and was confirmed by methylation-sensitive PCR and sequencing. The genomic structure of LRP6 was elucidated to allow screening for inactivating mutations, but only intragenic polymorphisms were identified. Hypermethylation of CpG islands associated with gene promoters is reported as a common mechanism for gene silencing and tumor suppressor inactivation. Therefore the consequences of the LRP6 CpG island methylation and its role in the observed phenotype need further investigation.
Human Genome Laboratory, Center for Human Genetics-Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium.
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M
Baens
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Wlodarska
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Corveleyn
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Hoornaert
A
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Hagemeijer
P
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Marynen
D018797:Cell Cycle Proteins; D002871:Chromosome Banding; D002872:Chromosome Deletion; D002874:Chromosome Mapping; D002881:Chromosomes, Human, Pair 12 / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics*; D005091:Exons; D020224:Expressed Sequence Tags; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007438:Introns; D007938:Leukemia / Q000235:genetics*; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D008297:Male; D008745:Methylation; D018895:Microsatellite Repeats; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D020161:Physical Chromosome Mapping; D011110:Polymorphism, Genetic; D050783:Proto-Oncogene Proteins c-ets; D011973:Receptors, LDL / Q000235:genetics*; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics*; D025521:Tumor Suppressor Proteins; D000097764:ETS Translocation Variant 6 Protein
D018797:Cell Cycle Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D008869:Microtubule-Associated Proteins; D050783:Proto-Oncogene Proteins c-ets; D011973:Receptors, LDL; D012097:Repressor Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D000097764:ETS Translocation Variant 6 Protein; C555475:LRP6 protein, human