中国健康人血清游离表皮生长因子受体外显子18-22序列分析

Gene Sequence Analysis of EGFR Exon 18-22 in Free DNA in Serum of Chinese Healthy Individual

目的分析中国健康人血清游离DNA中表皮生长因子受体（EGFR）外显子18～22基因序列是否存在突变，为监测EGFR酪氨酸激酶选择性抑制剂（EGFR TKIs）的临床疗效提供正常参考数据。方法采用高灵敏度DNA提取方法结合高保真巢式PCR和基因测序技术对42例中国健康人外周血血清游离DNA中EGFR TKIs作用靶点所在结构域即EGFR外显子18-22的基因序列和氨基酸序列进行分析。结果从外周血血清游离DNA中均扩增出EGFR外显子18～22的基因片段，片段大小（分别为400，374，404，408和419bp）与预期设计片段完全相符。健康人外周血游离DNA中存在EGFR基因突变，突变频率出现最多的是EGFR外显子20，41名健康人样本中有7例样本出现1处相同位点的沉默点突变（4838499G→A），其氨基酸序列（Glu）没有发生改变；EGFR外显子18也出现点突变，34名健康人样本中有1名健康者样本出现3处点突变，其中2处点突变（4830989 G→C；4831025 G→C）导致氨基酸序列发生改变（Glu→Gln；Ala-Pro），另外1处点突变（4831000 A→C）为沉默突变，其氨基酸序列（Thr）没有发生变化；EGFR外显子19、外显子21～22没有出现基因突变。结论通过巢式PCR结合基因测序可以分析外周血游离DNA中EGFR基因序列，健康人外周血游离EGFR外显予18和20存在基因点突变，其中外显子18的点突变导致了氨基酸序列改变。

Objective To analyze the mutations of epidermal growth factor receptor （EGFR） exon 18-22 in free DNA in serum of Chinese healthy individuals, and to provide normal reference for evaluating clinical therapeutic effects of EGFR tyrosine kinase inhihitors （TKIs）. Methods High sensitivity DNA extraction methods,high fidelity nested PCR and sequencing were used to detect DNA sequence and amino acid sequence of EGFR exon 18-22 in serum DNA of 42 healthy individuals. Results The gene fragment of EGFR exon 18-22 were identified at 400,374,404,408 and 419 bp,respectively. DNA mutation were detected in EGFR exon 18 and exon 20. Silent mutations（4838499 G→A）in exon 20 were found in 7 cases of 41 healthy individuals ,and their amino acid sequences （Glu） were not changed. Three point mutations （4830989 G→C; 4831025 G→C; 4831000 A→C）in exon 18 were found in 1 of 34 healthy individuals. 2 of 3 point mutations（4830989 G→C and 4831025 G→C）changed their amino acid sequences （Glu and Ala,respectively） to Gin and Pro,respeetively.and the point mutation 4831000 A→C was silent mutation,its amino acid sequence（Thr） was not changed. The gene mutation was not found in exon 19,exon 21 and exon 22. Conclusion EGFR sequence in serum DNA can be detected by high fidelity nested PCR and gene sequencing. The point mutation was found in EGFR exon 18 and 20,and amino acid sequences of exon 18 were changed.