鼻咽癌组织中PIK3CA基因热点突变区的突变筛查

Screening for Mutations in the Hotspot Mutation Regions of PIK3CA Gene in Nasopharyngeal Carcinoma

背景与目的:近期研究发现磷脂酰肌醇激酶-3催化亚单位α基因(phosphatidylinositol3-kinasecatalyticalphapolypeptidegene,PIK3CA)在多种肿瘤中存在高频的体细胞突变,并且突变常发生在PIK3CA的第9外显子和20外显子两个热点突变区。对这两个热点突变的研究表明,这些突变能增强该酶的活性、有助于肿瘤细胞的侵袭、对抗凋亡等。因此提示PIK3CA基因是与多种肿瘤发生、发展相关的癌基因。本研究旨在检测鼻咽癌组织中PIK3CA两个热点突变区的突变,以及该基因的变异与鼻咽癌发病的关系。方法:在鼻咽癌组织及患者外周血中筛查PIK3CA的突变热点区第9外显子和第20外显子。对46例散发性鼻咽癌组织标本采用PCR产物克隆测序,对与之匹配的46例鼻咽癌患者外周血标本和3个鼻咽癌细胞系(CNE1,CNE2,SUNE1),则将PCR产物直接测序。结果:在46例鼻咽癌组织标本中在PIK3CA热点突变区第9号外显子检出2例突变(4.3%):1例为T1563G(521Asn→Lys);另1例为A1646G(549Asp→Gly)。在46例鼻咽癌标本第9外显子中18例检出A1634C-G1658C-del1659T“复合突变”,进一步研究表明该“复合突变”可能为22号染色体上同源区域的序列。在对PIK3CA另外一个突变热点区第20外显子的检测中,46例鼻咽癌组织标本中都没检测到突变。另外,在3个鼻咽癌细胞系和46例鼻咽癌匹配外周血DNA标本用PCR-直接测序法均未检测到第9外显子和第20外显子突变。结论:PIK3CA基因第9外显子与第20外显子鼻咽癌中较少发生突变;克隆测序检测体细胞突变具有更高的敏感性,通过该方法在第9外显子发现的“复合突变”可能是22q11.2的CatEyeSyndromeregion高度同源区域的序列而非PIK3CA基因座的变异。

BACKGROUND ＆ OBJECTIVE： Recent studies showed high frequency of phosphatidylinositol 3-kinase catalytic alpha polypeptide （PIK3CA） mutations in various human cancers; notably, these mutations frequently locate in the hotspot mutation regions of PIK3CA exon 9 and exon 20 with functional significance in tumorigenesis, invasion, and anti-apoptosis. This study was to screen for mutations in the hotspot mutation regions of PIK3CA in nasopharyngeal carcinoma （NPC）, and explore the correlation of PIK3CA mutations to tumorigenesis of NPC. METHODS： PIK3CA exon 9 and exon 20 in 46 specimens of sporadic primary NPC tissues were screened by polymerase chain reaction （PCR）-clone sequencing; those in 46 samples of matched NPC peripheral blood and 3 NPC cell lines CNE1, CNE2, and SUNE1 were directly sequenced. RESULTS： Among the 46 specimens of NPC, 2 （4.3%） had point mutation in PIK3CA exon 9 [T1563G （521Asn→Lys） and A1646G （549Asp→Gly）], 18 had multiple mutations in PIK3CA exon 9 （A1634C-G1658C-del 1659T）, which might be the homologous sequence of Cat Eye Syndrome region on 22q11.2; none had mutation in PIK3CA exon 20. Moreover, no mutation was detected in PIK3CA exon 9 and exon 20 in the 46 matched NPC peripheral blood samples and CNE1, CNE2, and SUNE1 cells. CONCLUSIONS：PIK3CA exon 9 and exon 20 rarely mutate in NPC. Clone sequencing is more sensitive than direct sequencing in screening for somatic mutation. A1634C-G1658C-del 1659T mutations in PIK3CA exon 9, detected by clone sequencing, are supposed to be the homologous sequence of Cat Eye Syndrome region on 22q11.2 instead of mutations in PIK3CA.