中国人群中STK15基因内两种非同义SNP的连锁不平衡和单体型分析

STK15基因编码一种丝氨酸苏氨酸蛋白激酶，哺乳动物细胞中其过量表达将导致中心体扩增、染色体不稳定和细胞癌变。STK15基因外显子3中有3种非同义单核苷酸多态(SNP)，即：91A→T(131F)、169G→A(V571)和311C→T(S104L)。新近研究发现，91A→T与人类肿瘤遗传易感性相关。应用PCR-RFLP技术确定了91A→T(131F)和169G→A(V571)两种SNP在中国人群中的基因型和单体型。采用巢式PCR方法扩增了193例正常个体的DNA样品，通过错配正向巢式内引物引入EcoRⅠ酶切位点。巢式PCR扩增产物用限制性内切酶EcoRⅠ和ACCⅡ双酶切消化，其中EcoRⅠ能酶切91A，AccⅡ能切开169G，用聚丙烯酰胺凝胶电泳银染法鉴定双酶切结果，发现了4种可能的单体型中的3种，其单体型频率分别为：p(91A-169G)=68．65％，p(91T-169A)=10．88％，p(91T-169G)=20．47％，p(91A-169A)=0％；它们组成的6种基因型及频率分别为：91A-169G／91A-169G(46．11％)，91A-169G／91T-169A(14．51％)，91A-169G／91T-169G(30．57％)．91T-169G／91T-169G(3．11％)，91T-169G／91T-169A(4．15％)，91T-169A／91T-169A(1．55％)。等位基因及单体型数据分析结果表明，91A→T(131F)和169G→A(V571)之间存在连锁不平衡。

Linkage Disequilibrium and Haplotype Analysis of Two Single Nucleotide Polymorphisms in STK15 in Chinese

STK15 (Serine/Threonine protein kinase 15) is a serine/threonine kinase encoding gene,whose overexpression in mammalian cells leads to centrosome amplification,chromosomal instability,and oncogenic transformation.91A→T,a single nucleotide polymorphism (SNP) in exon 3 of STK15,has recently been shown to be associated with human cancer susceptibility.Within exon 3 of STK15,there are three nonsynonymous SNPs: 91A→T,169G→A and 311C→T.We have determined STK15 genotypes and haplotypes composed of 91A→T and 169G→A by PCR-RFLP in a randomly sampled cohort of 193 normal individuals from Northeast China.DNA samples from all individuals were subjected to first round of PCR using a pair of specific primers.For the subsequent nested PCR,a mismatch forward primer,which could introduce an EcoRⅠ restriction site to the 91A allele,was included.The nested PCR products were digested with the restriction endonucleases EcoRⅠ and AccⅡ.The double restriction digests were separated by polyacrylamide gel electrophoresis.Three haplotypes,91A-169G,91T-169A and 91T-169G,were detected and their frequencies were 68.65%,10.88% and 20.47%,respectively.Six genotypes composed of the above three haplotypes were found,and their frequencies were 91A-169G/91A-169G (46.11%),91A-169G/91T-169A (14.51%),91A-169G/91T-169G (30.57%),91T-169G/91T-169G (3.11%),91T-169G/91T-169A (4.15%), 91T-169A/91T-169A (1.55%).Whereas no 91A-169A haplotype was detected in all individuals examined in the current study,linkage disequilibrium (LD) between the two SNPs was found.