Genomic analysis of a homozygously deleted region in neuroblastoma cell lines on human chromosome 1p36

染色体1 p远端区域的杂合性丢失是许多人类癌症的常见现象，包括MYCN扩增和预后不良的神经母细胞瘤（NBL）。我们在两个NBL细胞系的1p36.2-p36.3的最小重叠区域内发现了一个同源性缺失（HD）区域。制备覆盖整个纯合缺失区域的800-kb PAC重叠群并测序（约85%）。缺失区域的估计长度为500 kb。到目前为止，我们已经在该区域内确定了六个基因（DFF 45，PGD，CORT，HDNB 1/UFD 2，KIAA 0591 F/KIF 1B-beta和PEX 14）。这些基因包括与细胞凋亡、糖代谢、泛素-蛋白酶体途径、神经元微管相关运动分子和过氧化物酶体生物发生有关的基因。至少有三个基因（HDNB 1/UFD 2，KIAA 0591 F/KIF 1B-beta和PEX 14）在原发性神经母细胞瘤的有利亚群中以高水平差异表达，而在不利亚群中以低水平差异表达。 ...更多信息 RT-PCR-SSCP分析表明，目前所鉴定的基因突变很少。由于1 p远端区域被报道为印记基因，这些差异表达的基因可能是候选NBL抑制基因的新成员。为了评估这种可能性，我们研究了这些基因在去甲基化试剂处理8个神经母细胞瘤细胞系前后的转录表达。所有细胞系，当用试剂处理或未处理时，均显示一定水平的表达，表明这些基因在这些细胞系中未被基因组甲基化抑制。然而，其中一个在去甲基化后表现出轻微的表达增加，因此我们开始分析其启动子区域。为了进一步鉴定该区域中的新基因并检测该基因的启动子区域，我们分离了4个小鼠BAC克隆，其覆盖了HD区域的非特异性部分。已经完成了大约660 kb的基因组测序。纯合缺失区的全序列测定和基因预测将阐明该区域更详细的结构，并可能导致发现额外的候选基因。少

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. … More 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