Mutation and LOH analysis of ACO2 in colorectal cancer:: no evidence of biallelic genetic inactivation -: art. no. e73
Mutation and LOH analysis of ACO2 in colorectal cancer:: no evidence of biallelic genetic inactivation -: art. no. e73
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DOI：
10.1136/jmg.40.5.e73
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发表时间：
2003-05-01
影响因子：
4
通讯作者：
Aaltonen, LA
中科院分区：
文献类型：
--
作者：
Laiho, P;Hienonen, T;Aaltonen, LA
材料与方法收集1994年5月至1998年6月在赫尔辛基大学医学遗传学系的1000例新鲜冰冻结直肠腺癌标本。用BAT 26单核苷酸标记物测定了样品的微卫星不稳定性（MSI）状态。23，24我们选择了在我们之前的104个MSI阴性CRC的全基因组分析中在染色体22 q13中显示洛缺失的所有23个样本，其中372个微卫星标记，22并通过肿瘤和正常DNA中ACO 2的基因组测序对其进行分析。因为目标基因座的洛缺失是一个纳入标准，所以除了肿瘤DNA外，还对正常DNA进行测序，以排除肿瘤进展期间ACO 2变异体的丢失。这种方法允许检测所有推定的体细胞和种系序列变化。从新鲜冷冻标本中提取肿瘤DNA。病理学家在DNA提取之前对肿瘤组织的比例进行了组织学评估。所有标本均显示60%以上的癌组织。正常DNA从血液或正常结肠粘膜获得。代表正常粘膜的标本总是来自一个单独的网站，而不是从肿瘤边缘。其中15个样本是散发性的，8个样本有一个一级亲属患有CRC。由于MSI阴性和轻微的癌症家族史，这八个家族样本尚未筛选出已知的CRC易感基因。然而，家族性腺瘤性息肉病、Peutz-Jeghers综合征和幼年性息肉病在临床上被排除。患者的年龄从30岁到89岁不等。
MATERIALS AND METHODSOver 1000 fresh frozen colorectal adenocarcinoma specimens were collected in the Department of Medical Genetics, University of Helsinki between May 1994 and June 1998. The microsatellite instability (MSI) status of the samples was previously determined using BAT26 mononucleotide marker. 23, 24 We chose all 23 samples which had displayed LOH in chromosome 22q13 in our previous genome wide analysis of 104 MSI negative CRCs with 372 microsatellite markers, 22 and analysed them by genomic sequencing of ACO2 in tumour and normal DNA. Because LOH at the locus of interest was an inclusion criterion, normal DNA was sequenced in addition to tumour DNA to exclude loss of ACO2 variants during tumour progression. This approach allowed detection of all putative somatic and germline sequence changes. Tumour DNA was extracted from fresh frozen specimens. A pathologist evaluated the proportion of tumour tissue histologically before DNA extraction. All samples displayed over 60% carcinoma tissue. Normal DNA was obtained either from blood or normal colon mucosa. The specimens representing normal mucosa were always derived from a separate site rather than from tumour margins. Fifteen of the samples were sporadic and eight had one first degree relative affected with CRC. Because of the MSI negativity and mild family history of cancer, these eight familial samples have not been screened for known genes predisposing to CRC. Familial adenomatous polyposis, Peutz-Jeghers syndrome, and juvenile polyposis were, however, clinically excluded. The age of the patients varied from 30 to 89.