Georgia Health Sciences University Cancer Center, Georgia Health Sciences University, Augusta, Georgia, United States of America.
PLoS One. 2013;8(3):e58731. doi: 10.1371/journal.pone.0058731. Epub 2013 Mar 7.
BACKGROUND: The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients. METHODS: We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A. RESULTS: Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15:1, a ratio significantly higher than the expected 2:1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations. CONCLUSIONS: Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.
背景：位于 8 号染色体短臂上的 Cub 和 Sushi 多结构域 1（CSMD1）基因编码一种 I 型跨膜蛋白，其功能目前尚不清楚。CSMD1 表达在许多上皮癌中经常丢失。我们的目标是描述 CSMD1 体细胞突变、等位基因失衡、DNA 甲基化与结直肠癌患者临床特征之间的关系。
方法：我们使用 454FLX 焦磷酸测序平台对 54 例结直肠肿瘤中的 CSMD1 编码区进行测序，共检测了 72 个涵盖整个编码序列的扩增子。我们使用多个 CSMD1 位点的杂合性 SNP 等位基因比值来确定等位基因平衡并推断杂合性丢失。最后，我们对 76 例结直肠肿瘤进行甲基化特异性 PCR，以确定 CSMD1 和已知甲基化靶点 ALX4、RUNX3、NEUROG1 和 CDKN2A 的 DNA 甲基化状态。
结果：使用 454FLX 测序并经 Sanger 测序验证，在 54 例结直肠肿瘤中的 6 例（11%）中发现了 16 个 CSMD1 体细胞突变。16 个体细胞突变中的非同义与同义突变比为 15:1，明显高于预期的 2:1 比值（p=0.014）。该比值表明 CSMD1 蛋白序列中的突变存在正选择。在 37 例有信息的病例中，19 例存在 CSMD1 等位基因失衡（56%）。存在等位基因失衡和 CSMD1 突变的患者明显比没有体细胞突变的患者年轻（平均年龄为 41 岁）（平均年龄为 68 岁）。大多数肿瘤在 CSMD1 编码序列内的一个或多个 CpG 位点发生甲基化，CSMD1 甲基化与两个已知的甲基化靶点 ALX4 和 RUNX3 显著相关。C:G>T:A 取代明显过表达（47%），表明广泛的胞嘧啶甲基化易导致体细胞突变。
结论：深度扩增子测序和甲基化特异性 PCR 显示，CSMD1 改变与结直肠肿瘤中较早的临床表现相关，因此进一步提示 CSMD1 是一种肿瘤抑制基因。
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