| Research Abstract |
Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s)might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have found a homozygously deleted (HD)region within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines. The 800-kb PAC contig covering the entire region of homozygous deletion was made and sequenced (about 85%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes (DFF45, PGD, CORT, HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) within the region. They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma.
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RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor. To assess this possibility, we investigated these genes' transcriptional expression before and after the treatment with demethylation reagent to eight neuroblatoma cell lines. All cell lines, when treated or not treated with the reagent, showed certain level of expression, suggesting that these genes were not suppressed by genomic methylation in these cell lines. However, one of them exhibited slight increase of expression after the de-methylation, we therefore started to analyze its promoter region. To further identify novel genes in this region and also detect a promoter region of the gene, we isolated 4 mouse BAC clones covering a synthenic portion of the HD region. Approximately 660-kb of genomic sequencing has been accomplished. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Less
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