Application of next generation sequencing in the whole exome sequencing for pediatric T lymphoblastic lymphoma
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摘要:
目的 建立从石蜡包埋的儿童T淋巴母细胞淋巴瘤（T-LBL）病理组织中提取基因组DNA，采用二代测序技术行全外显子测序检测的方法并分析其可行性。 方法 从10例儿童T-LBL石蜡包埋组织中提取基因组DNA，PCR反应扩增后采用二代测序技术行全外显子测序检测并确定致病位点，所获得的致病突变采用Sanger测序法测序确认，比较两种方法的一致性。 结果 10例石蜡包埋T-LBL组织标本中均成功提取到基因组DNA，采用二代测序技术开展全外显子测序测序均检测到致病突变；二代测序检测结果经Sanger测序法验证，证实致病突变确实存在，且与Sanger测序结果一致。 结论 基于二代测序技术的全外显子测序测序可用于检测石蜡包埋的儿童T-LBL病理标本的致病突变。 Abstract:Objective To establish the methods of extract DNA and detect whole exome sequencing (WES) by using next generation sequencing (NGS) technique from paraffin embedded samples for children with T lymphoblastic lymphoma (T-LBL) and to explore its feasibility. Methods Genomic DNA was extracted from 10 cases of paraffin embedded samples with pediatric T-LBL. Genomic DNA samples were amplified by polymerase chain reactions, NGS technique was used for WES detection and pathogenic mutations were founded. The pathogenic mutations were confirmed by Sanger sequencing, comparison of consistency between NGS technique and Sanger sequencing was evaluated. Results Genomic DNA samples were successfully extracted from the 10 T-LBL specimens of paraffin embedded tissues. The pathogenic mutations were detected by NGS technique and the result was consistent with the Sanger sequencing method. Conclusion NGS technology can be used for WES and detection of pathogenic mutation for paraffin embedded pediatric T-LBL specimens. -
表 1 10例T-LBL患者疾病相关基因突变
患者序号 疾病相关基因突变 1 JAK3: NM_000215:exon11:c.G1533C:p.M511I NF1: NM_001042492:exon19:c.G2299T:p.E767X 2 TSC1: NM_001162426:exon10:c.C1006T:p.R336W 3 FBXW7: NM_033632:exon9:c.C1393T:p.R465C TSC1: NM_001162426:exon10:c.C1006T:p.R336W 4 CDKN1B: NM_004064:exon1:c.A247T:p.S83C CDKN1B: NM_004064:exon1:c.G245C:p.G82A CDKN1B: NM_004064:exon1:c.A241G:
p.K81E CDKN1B: NM_004064:exon1:c.242_243insCCTCC:p.K81fs SMARCA4: NM_001128844:exon20:c.C2738A:p.P913Q5 NOTCH1: NM_017617:exon34:c.C7375T:p.Q2459X FLG: NM_002016:exon3:c.G8438A:p.G2813E 6 RASA2: NM_006506:exon23:c.2334_2335insTG:p.A778fs CDH1: NM_004360:exon10:c.C1409T:p.T470I 7 NOTCH1: NM_017617:exon34:c.C7507T:p.Q2503X BCOR: NM_017745:exon4:c.T2633C:p.V878A 8 FBXW7: NM_033632:exon9:c.C1393T:p.R465C DMBT1: NM_007329:exon45:c.C5578T:p.R1860X 9 PHF6: NM_032458:exon4:c.C346T:p.R116X DMBT1: NM_007329:exon45:c.C5578T:p.R1860X ETV6: NM_001987:exon7:c.T1171C:p.Y391H 10 USH2A: NM_206933:exon22:c.A4634C:p.K1545T KMT2D: NM_003482:exon38:c.G10661A:p.R3554H 
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