Identification of Novel SNPs in Glioblastoma Using Targeted Resequencing

High-throughput sequencing opens avenues to find genetic variations that may be indicative of an increased risk for certain diseases. Linking these genomic data to other “omics” approaches bears the potential to deepen our understanding of pathogenic processes at the molecular level. To detect novel single nucleotide polymorphisms (SNPs) for glioblastoma multiforme (GBM), we used a combination of specific target selection and next generation sequencing (NGS). We generated a microarray covering the exonic regions of 132 GBM associated genes to enrich target sequences in two GBM tissues and corresponding leukocytes of the patients. Enriched target genes were sequenced with Illumina and the resulting reads were mapped to the human genome. With this approach we identified over 6000 SNPs, including over 1300 SNPs located in the targeted genes. Integrating the genome-wide association study (GWAS) catalog and known disease associated SNPs, we found that several of the detected SNPs were previously associated with smoking behavior, body mass index, breast cancer and high-grade glioma. Particularly, the breast cancer associated allele of rs660118 SNP in the gene SART1 showed a near doubled frequency in glioblastoma patients, as verified in an independent control cohort by Sanger sequencing. In addition, we identified SNPs in 20 of 21 GBM associated antigens providing further evidence that genetic variations are significantly associated with the immunogenicity of antigens.

高通量测序（High-throughput sequencing）为挖掘可提示特定疾病风险升高的遗传变异开辟了新路径。将此类基因组数据与其他"组学"研究手段相结合，有望深化我们对分子层面致病过程的认知。为筛选多形性胶质母细胞瘤（glioblastoma multiforme, GBM）相关的新型单核苷酸多态性（single nucleotide polymorphisms, SNPs），本研究采用特异性靶点富集与下一代测序（next generation sequencing, NGS）联用的实验策略。我们构建了覆盖132个GBM相关基因外显子区域的微阵列，用于富集两例GBM患者的肿瘤组织及对应外周血白细胞中的靶序列。富集得到的靶基因通过Illumina平台完成测序，所得序列读段被比对至人类参考基因组。通过该实验策略，我们共鉴定出6000余个单核苷酸多态性，其中1300余个位于本次研究靶向的基因区域内。整合全基因组关联分析（genome-wide association study, GWAS）数据库与已知疾病相关单核苷酸多态性数据集，我们发现部分鉴定得到的单核苷酸多态性此前已被报道与吸烟行为、体重指数、乳腺癌及高级别胶质瘤存在关联。尤为关键的是，SART1基因中rs660118位点的乳腺癌相关等位基因在GBM患者群体中的出现频率近乎翻倍，该结果通过独立对照队列的桑格测序（Sanger sequencing）得到了验证。此外，我们在21个GBM相关抗原中的20个内鉴定出单核苷酸多态性，为遗传变异与抗原免疫原性存在显著关联提供了进一步实验证据。