| ObjectivesSingle nucleotide polymorphisms (SNPs) are thought to be the genetic basis of most human diseases, or at least positional markers for our genetic heritage. Recent studies show that low - penetrance disease susceptibility genes, which are relatively common in the population, may confer a much higher attributable risk in the general population than rare mutations in high - penetrance disease susceptibility genes. In the last several years, attention in the human genetics community has increasingly turned from the study of single - gene, Men-delian disorders to the search for DNA sequence variants associated with increased risks for the development of common, or complex diseases caused by the interaction of hereditary and environmental influences. The great majority of SNPs are not associated with changes in gene structure or function, but a small proportion are believed to be directly responsible for genetic disease. One of the central challenges of modern human genetics is to identify these deleterious SNPs. Studies to investigate the role of SNPs in genetic susceptibility to human diseases will offer the possibility of designing novel targeted therapies in the future.Worldwide, gastric cancer is the second largest cause of cancer - related death. The disease is most common in China; the death rate for gastric cancer is 20 deaths per 100 000. Studies on the development of gastric cancer suggest that genetic predisposition, infection, and diet are part of a complex interaction.The human STK15 gene maps to the chromosome 20q13.2 - q13.3 regionand encodes Aurora kinase A (also called STK15 and BTAK) , a member of the serine/threonine kinase family. The open reading frame of the full - length STK15 cDNA sequence encodes a 403 - amino acid protein with a molecular mass of approximately 46 kD. Aurora kinase A has been implicated in regulating centrosome function, spindle assembly, spindle maintenance and mitotic commitment in cells. Overexpression of this kinase was reported to induce a centrosome amplification, chromosome instability and tumorigenic transformation of cells, showed that Aurora kinase A phosphorylates p53 at ser315 and that over-expression of aurora kinase A leads to increased degradation of p53 causing downregulation of checkpoint - response pathways and facilitating oncogenic transformation of cells. 91 A—>T, a nonsynonymous single nucleotide polymorphism in exon 3 of STK15 resulting in an ile31 -to - phe (D1F) substitution, has recently been shown to be associated with human cancer susceptibility. Ewart - Toland et al found that the ile31 variant of STK15 was preferentially amplified and associated with a degree of aneuploidy in human tumors. Nonsynonymous SNPs result in single amino acid substitutions, therefore, they could affect conformation and function of Aurora kinase A.This study aimed at investigating genetic polymorphisms of STK15 gene and the contribution of STK15 polymorphisms to the risk of gastric cancer in Chinese.Thoracic aortic dissection (TAD) and thoracic aortic aneurysms (TAA) recently made an appearance among the 15 leading causes of death in the United States; however, the mechanisms underlying these disease processes remain poorly understood. The medial layer of the aorta is composed of vascular smooth muscle cells (VSMCs) and the extracellular matrix (ECM) proteins, primarily elastin and collagen. Maintaining a balanced composition appears to be critical for preserving the important functional properties of the thoracic aorta, especially its mechanical compliance to pulsatile blood flow. Disturbances in the metabolic balance that result in excessive ECM degradation may lead to progressive aortic wall deterioration, expansion and rupture.Matrix metalloproteinases (MMPs) are a large family of > 20 zinc - dependent proteolytic enzymes. These enzymes play vital roles in diseases relatedto ECM metabolism and aortic wall remodeling, which may be relevant to the development of aneurysms or dissection. Recent studies have shown that excessive MMP9 activation occurs in abdominal aortic aneurysms (AAA) and may contribute to the rapid growth and rupture. Increased MMP9 expression has also been identified in patients with thoracic aortic disease.Genetic analysis of TAA or TAD remains relatively unexplored, with the exception of patients with connective tissue disorders, such as Marfan or Ehler -Danlos syndromes, which are caused by rare genetic mutations, and familial cases. Given the extensive literature surrounding the role of MMP9 in abdominal aortic aneurysms, this gene emerges as a potential target for investigation into thoracic aortic disease. The purpose of this case - control study was to examine the contribution of three SNPs in the MMP9 gene to the risk of TAA and TAD in a Caucasian population. The 3 SNPs are - 8202A—*G (nucleotide position numbered relative to the start of transcription) in the 5' untranscribed region, IVS4 +3G—?T at the fourth intron, and 2003 A-*G (Q668R) at the twelfth ex-on.Materials and Methods1 Detection of Single Nucleotide Polymorphisms in the STK15 Gene by Denaturing High Performance Liquid ChromatographyThis study consisted of 118 normal individuals from Northeast China, and all subjects were Han Chinese, the majority ethnic group of China. Peripheral blood samples of 118 subjects were collected in EDTA vacutainer tubes, immediately iced, and processed within a few hours. DNA was extracted from peripheral blood by the use of Proteinase K and Phenol/Chloroform.All STK15 coding regions and their flanking intronic sequences were amplified from genomic DNA samples by PCR. Based on the sequence of the STK15 gene, 8 pairs of PCR primers were designed and synthesized commercially. The PCR reactions were carried out in final volume of 20 jxL, using 2 jxL lOx PCR buffer, 0.5 U Taq DNA polymerase, 200 jxmol/L dNTPs , 1 p,mol/L of each primer, and 20 ng of sample DNA. The thermocycler was programmed with aninitial step of denaturation at 94X for 3 min. 35 cycles were run under the following conditions; denaturation at 94 X for 30 s, annealing at 51 -54t! for 30 s, elongation at 72X1 for 1 min. The last round of elongation was for 7 min at 72*C. The PCR products were detected by Agarose gel electrophorisis.DHPLC was performed on a WAVE DNA fragment analysis system. To enhance heteroduplex formation, the untreated PCR product was denatured at 95 X for 5 min followed by gradual reannealing to 25 X. Products were automatically loaded on a DNAsep column and eluted with a linear acetonitrile ( ACN) gradient in a 0.1 M triethylamine acetate buffer (pH 7) , at a constant flow rate of 0.9 ml/min. The start and end points of the gradient were adjusted according to the size of the PCR product. Analysis per amplified sample took 7.2 -8.5 min, including column regeneration and equilibration. Samples were analysed at the melt temperature (Tm) determined by using the WAVE - Maker software. Each fragment was analysed at one, two, or three different temperatures, depending on the melting pattern of the fragment. Eluted DNA fragments were detected by an UV detector. PCR products displaying variant DHPLC melt profiles were directly sequenced.2 Linkage Disequilibrium and Haplotype Analysis of Two Single Nucleotide Polymorphisms in STK15 in ChineseThis study consisted of 193 normal individuals from Northeast China, and all subjects were Han Chinese, the majority ethnic group of China.Peripheral blood samples of 193 subjects were collected in EDTA vacutain-er tubes, immediately iced, and processed within a few hours. DNA was extracted from peripheral blood by the use of Proteinase K and Phenol/Chloroform.First, a standard PCR reaction was run with the patient sample using the outer primers. Then, a subsequent nested PCR reaction was run with the inner nested primers using the product of the first reaction as the amplification target. 2 pairs of PCR primers were designed and synthesized commercially. For the subsequent nested PCR, a mismatch forward inner nested primer, which could introduce an EcoRI restriction site to the 91A allele, was included.The PCR reactions were carried out in final volume of 20 L, using 2 jxL lOx PCR buffer, 0.5 U Taq DNA polymerase, 200 |xmol/L dNTPs , 1 jxmol/Lof each primer, and 20 ng of sample DNA. The thermocycler was programmed with an initial step of denaturation at 94 X for 3 min. 35 cycles were run under the following conditions: denaturation at 94 X for 30 s, annealing at 53 X, for 30 s, elongation at 72 X for 1 min. The last round of elongation was for 7 min at 72 X. The PCR products were detected by Agarose gel electrophorisis. After purification, PCR products were sequenced commercially.The nested PCR products were digested with the restriction endonucleases EcoRI and AccII. The double restriction digestion reactions were carried out in final volume of 10 jxL at 37 X overnight, using 1 uX lOx M buffer, 5 U EcoRI, 5 U AccII, and 500 ng of the nested PCR products.2 julL of the double restriction products were separated by 8% polyacrylam-ide gel electrophoresis. After electrophoresis, the gel was separated from the plates and treated for 10 min in fixation solution with gentle shaking. After incubating for 15 min in staining solution, the gel was washed two times with distilled water for 2 min, and then the gel was transferred to developing solution to visualize the silver — staining DNA bands.The formulas used to measure linkage disequilibrium (LD) were:~~ ij ij.maxIfDii<0, Dijtmax=: min(piPj, [1 -pj [1 -p.]) , Dijtmax=min([l- pj Pj,Pi[l- Pj]3. STK15 polymorphisms and susceptibility to gastric cancer This study consisted of 68 patients with gastric cancer and 75 controls, and all subjects were Han Chinese. STK15 genotypes and haplotypes composed of 91 A—+T and 169G—kA were determined by PCR - RFLP (polymerase chain reaction - restriction fragment length polymorphism) . 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 prod-ucts were digested with the restriction endonucleases EcoRI and AccII. The double restriction digests were separated by polyacrylamide gel electrophoresis.The associations between STK15 polymorphisms and gastric cancer were estimated by odds ratios (ORs) and their 95% confidence interval (CIs) , which were calculated by unconditional logistic regression models. All analysis was carried out with Statistical Product and Service Solutions software ( Version 12. 0; SPSS Inc. , Chicago, IL).4. MMP9 polymorphisms and susceptibility to thoracic aortic aneurysm and thoracic aortic dissectionThis study consisted of 85 patients and 111 controls, and all subjects were Caucasians. Sixty patients had thoracic aortic dissection (TAD )and 28 patients had degenerative thoracic aortic aneurysm (TAA) that were not caused by aortic dissection. Three patients with both a TAD and degenerative TAA were included in both groups for this analysis. Blood samples were obtained from 161 subjects (24 TAA, 26 TAD, and 111 controls) and tissue samples were obtained from 32 subjects (1 TAA and 31 TAD). Tissue was collected from the 3 patients included in both the TAA and TAD groups. Blood was collected in 4 mL EDTA tubes. Within 15 minutes of collection, blood was stored at -80 X..Using DNAzol DNA extraction kit (Invitrogen, CA) , genomic DNA was i-solated from either peripheral blood or aortic tissue. The Assays - on - Demand TaqMan SNP Genotyping Assays of three SNPs { consisting of a 20X mix of unla-beled PCR primers and TaqMan MGB probe, Applied Biosystems, CA) were used to determine MMP9 genotypes. Genotyping was accomplished by ICycler iQ Real - Time Detection System ( Bio - Rad, C A ). Allelic discrimination real -time PCR was accomplished with a 25 - jxL reaction mixture containing 20 ng of DNA, 12.5 jxL TaqMan Universal PCR Master Mix (Applied Biosystems) , and 1.25 uX 20X TaqMan SNP Genotyping Assay Mix (Applied Biosystems). The PCR profile consisted of an initial melting step of 10 min at 95 T , followed by 40 cycles of 15 s at 92 X. , and 1 min at 60 X.The associations between MMP9 polymorphisms and TAA and TAD were estimated by odds ratios (ORs) and their 95% confidence intervals (CIs) , which were calculated by unconditional logistic regression models. All analysiswas carried out with Statistical Product and Service Solutions software ( Version 12.0; SPSS Inc. , Chicago, IL).Results1 Detection of Single Nucleotide Polymorphisms in the STK15 Gene by Denaturing High Performance Liquid ChromatographyTwo nonsynonymous SNPs, 91A-*T(I31F)ftl 169G-+A (V57I), were i-dentified in the coding region. A SNP, IVS4 +30C—?G, was identified in the adjacent intronic sequence. There was no 31G—kA(GIIR) in Chinese.A stable tandem repeat (twofold tandem repeat) was found at the second intron in all subjects, and the repeat unit length was 61 bases. The repeat unit was TAAATTGAAT AATCTGTAAT CTCATTCACA TTTATAAACC CACATG-GAGG TTGGTCTTGT C.2 Linkage Disequilibrium and Haplotype Analysis of Two Single Nucleotide Polymorphisms in STK15 in ChineseThere could be 4 haplotypes theoretically, but only 3 haplotype, 91A -169G, 91T - 169A and 91T - 169G, were detected and their frequencies were 68. 65% , 10. 88% and 20.47% , respectively. Whereas no 91A - 169A haplotype was detected in all individuals examined in the current study. 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% ).D = 0.075,x = 0.27( >0. 1),D' = 1.0. X2 = 103. 18,P<0.001. Since D was greater than zero, 91A was associated with 169G and 91T was associated with 169A. A value of r2 >0.1 is suggested as a criterion for meaningful LD. The case of D' = 1 is known as complete LD. D' value of 1.0 result is due to the presence of only three of four possible haplotypes. By definition, the presence of only three of four possible haplotypes results in D' being equal to unity. According to D,r2 and D' , 91 A—+T and 169G—>A are in Linkage Disequilibri-urn (LD).3. STK15 polymorphisms and susceptibility to gastric cancerThe frequency of 91T - 169G haplotype in cases was significantly lower than that in control group (X2 = 4.903,OR 0.501,P = 0.03 ). The frequency of 91A - 169G / 91T - 169A genotype in female cases was significantly higher than that in female controls (OR 6.563, 95% CI 1.192 -36.143).Carriers of the 91A - 169G but not the 91T - 169G (91A - 169G / 91A -169G + 91A - 169G / 91T - 169A) had a 2. 5 - fold elevated risk for gastric cancer (95% CI 1.220 -5.074). It was found that the OR for gastric cancer patients carrying the 91T - 169G allele (91A - 169G / 91T - 169G + 91T -169G/91T-169G + 91T-169G/91T-169A) was 0.473 (95% CIO.231 -0.968 ). There were 3 carriers of the 91T -169 A / 91T - 169 A in controls, whereas no 91T — 169A / 91T - 169A genotype was found in cases.4. MMP9 polymorphisms and susceptibility to thoracic aortic aneurysm and thoracic aortic dissectionThe rare — 8202 G allele frequency was significantly higher in TAA and TAD cases than controls ( P < 0.001). There were 96.4% of TAA cases carrying the - 8202G allele, which was significantly higher than that of controls. Subjects carrying the -8202G allele had a 13 -fold elevated risk for TAA compared with subjects with the AA genotype. The -8202G allele was found in 88. 3% of the TAD cases, which was significantly higher than controls {58.6% , P <0.001). It was found that the adjusted OR for TAD patients carrying the -8202G allele compared with the AA genotype was 4.3 (95% CI 1.7 -10.7). If all TAA and TAD cases were combined into a single group, the -8202G allele frequency was 0.535, which was significantly higher than controls (0.360; P <0.001) , the adjustedOR for the -8202G allele carriers compared with the AA genotype was 4.9 (95% CI 2.0 -11.6).There was no significant association between TAA or TAD and FVS4 + 3G —>-T (P >0.05). It was also found that the distribution of the allele and genotype frequencies for 2003 A—*G (Q668R) were not statistically different between TAA or TAD cases and controls ( P > 0.05 ). |
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