A common variant of the MACC1 gene is significantly associated with overall survival in colorectal cancer patients.

BACKGROUND: The newly discovered metastasis-associated in colon cancer-1 (MACC1) gene is a key regulator of the HGF/MET pathway. Deregulation of HGF/MET signaling is reported as a prognostic marker for tumorigenesis, early stage invasion, and metastasis. High expression levels of MACC1 have been associated with colon cancer metastasis and reduced survival. Potential links between the genetic diversity of the MACC1 locus and overall survival are unknown. We therefore investigated the association between MACC1 tagging single nucleotide polymorphisms (SNPs) and overall survival in a large cohort of colorectal cancer patients. METHODS: The study included 318 subjects with histopathologically proven colorectal cancer at the Academic Teaching Hospital Feldkirch, Austria. Survival data were provided by the federal agency for statistics in Austria. Genomic DNA was isolated from formalin-fixed paraffin-embedded specimens; six tagging SNPs (rs1990172, rs3114446, rs10275612, rs3095007, rs3095009, and rs7780032), capturing most of the common variants of the MACC1 locus, were genotyped by SNaPshot assays. RESULTS: Over a mean follow up period of 5.3 (± 1.0) years, 94 deaths were recorded. Carriers of the G-allele of SNP rs1990172 showed a significantly decreased overall survival (additive HR = 1.38 [1.05-1.82]; p = 0.023). Multivariate analysis adjusted for age and UICC tumor stage confirmed this result (HR = 1.49 [1.12-1.98]; p = 0.007). Other investigated genetic variants of the MACC1 gene were not significantly associated with overall survival (p-values > 0.05). CONCLUSIONS: For the first time, our study investigated the influence of MACC1 tagging polymorphisms on overall survival suggesting SNP rs1990172 as a predictor for reduced overall survival in colorectal cancer patients. Further studies will be required to validate our findings.

Different types of K-Ras mutations are conversely associated with overall survival in patients with colorectal cancer.

A glycine to valine substitution at codon 12 (G12V) in Kirsten-Ras (K-Ras) gene has been associated with reduced overall survival in colorectal cancer patients; however, the effect of other K-Ras mutations than G12V still remains unclear. Therefore, we investigated the role of different K-Ras mutations on overall survival in a homogeneous, large patient cohort with standardized therapy and uniform analysis of K-Ras mutation status. The study included 342 patients with histopathologically proven colorectal cancer. Survival data were provided by the federal agency for statistics in Austria. Occurrence of K-Ras mutations at codons 12, 13 and 61 were determined by capillary sequencing. The overall K-Ras mutation frequency in carcinoma tissue was 28%. Carriers of the G12V mutation at the K-Ras gene showed a significantly decreased overall survival compared to carriers of the wild-type [HR=2.56 (1.15-5.69)]. Other mutations than G12V were associated with better overall survival compared to wild-type [HR=0.44 (0.2-0.99)]. In conclusion, for the first time, our study showed clearly that different types of K-Ras mutations are conversely associated with overall survival in patients with colorectal cancer.

Reconstitution in vitro of the GDP-fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster.

The deoxyhexose sugar fucose has an important fine-tuning role in regulating the functions of glycoconjugates in disease and development in mammals. The two genetic model organisms Caenorhabditis elegans and Drosophila melanogaster also express a range of fucosylated glycans, and the nematode particularly has a number of novel forms. For the synthesis of such glycans, the formation of GDP-fucose, which is generated from GDP-mannose in three steps catalysed by two enzymes, is required. By homology we have identified and cloned cDNAs encoding these two proteins, GDP-mannose dehydratase (GMD; EC 4.2.1.47) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GER or FX protein; EC 1.1.1.271), from both Caenorhabditis and Drosophila. Whereas the nematode has two genes encoding forms of GMD (gmd-1 and gmd-2) and one GER-encoding gene (ger-1), the insect has, like mammalian species, only one homologue of each (gmd and gmer). This compares to the presence of two forms of both enzymes in Arabidopsis thaliana. All corresponding cDNAs from Caenorhabditis and Drosophila, as well as the previously uncharacterized Arabidopsis GER2, were separately expressed, and the encoded proteins found to have the predicted activity. The biochemical characterization of these enzymes is complementary to strategies aimed at manipulating the expression of fucosylated glycans in these organisms.

Significant survival impact of MACC1 polymorphisms in HER2 positive breast cancer patients.

BACKGROUND: Deregulation of hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (MET) signalling has been associated with poor clinical outcome in breast cancer and other cancers. The recently discovered metastasis-associated in colon cancer-1 (MACC1) gene is a key regulator of the HGF/MET pathway. Potential links between genetic variants of the MACC1 gene and survival in breast cancer patients are unknown. In the present study, we therefore aimed to investigate the influence of MACC1 polymorphisms on event-free and overall survival in patients with human epidermal growth factor 2 (HER2)-positive breast cancer. METHODS: The present study included 164 consecutive white patients with HER2-positive breast cancer. Three MACC1 polymorphisms, rs1990172, rs975263 and rs3735615, already associated with cancer prognosis or with potential functional effects, were genotyped by the 5' nuclease assay. RESULTS: Multivariate Cox regression analysis adjusted for age and tumour stage showed increased risk for progression or death for carriers of the rare allele (G-allele) of single nucleotide polymorphism (SNP) rs1990172 (hazard ratios (HR) = 2.26; p = 0.004 and HR = 3.13; p = 0.001 for event-free survival and overall survival, respectively). In addition, we were able to demonstrate an adverse effect on cancer prognosis for carriers of the rare allele (T-allele) of SNP rs975263 (HR = 2.17; p = 0.007 and HR = 2.80; p = 0.003 for event-free survival and overall survival, respectively). The rare allele (C-allele) of SNP rs3735615 showed a significant protective impact on event-free survival as well as overall survival (HR = 0.25; p = 0.001, and HR = 0.16; p = 0.001, respectively). CONCLUSIONS: This study provides first evidence that MACC1 polymorphisms are associated with clinical outcome for HER2-positive breast cancer patients. Further studies are warranted to validate these findings.

Association of a common genetic variant of the IGF-1 gene with event-free survival in patients with HER2-positive breast cancer.

PURPOSE: Insulin-like growth factor 1 (IGF-1) stimulates mitosis and inhibits apoptosis. High circulating IGF-1 levels are linked with an increased risk of colorectal and breast cancer. Recently, IGF-1 single nucleotide polymorphisms (SNPs), especially variant rs2946834, have been associated with poor clinical outcome in patients with colorectal cancer. In the present study, we aimed to investigate the influence of IGF1 polymorphisms associated with IGF-1 plasma levels on event-free survival in patients with HER2-positive breast cancer. METHODS: The present study included 161 consecutive white patients with HER2-positive breast cancer. Event-free survival was calculated as the time from cancer diagnosis to either relapse or death from any cause. Genomic DNA was extracted from archived formalin-fixed paraffin-embedded tumor tissue samples; five IGF-1 polymorphisms (rs2946834, rs6220, rs1520220, rs5742694, and rs5742678), all associated with IGF-1 levels, were genotyped by SNaPshot assays. RESULTS: Kaplan-Meier analysis showed a poorer clinical outcome for carriers of the rare allele of SNP rs2946834 (log-rank test, p = 0.020). Concordantly, in univariate Cox regression analyses, the rare allele of SNP rs2946834 was significantly associated with a decreased event-free survival (HR = 3.06 [1.14-8.22]; p = 0.027). Multivariate analysis adjusted for age and tumor stage confirmed this result (HR = 4.02 [1.36-11.90]; p = 0.012). Other investigated polymorphisms of the IGF1 gene were not significantly associated with event-free survival (all p values >0.05). CONCLUSIONS: This study provides first evidence that IGF1 rs2946834 polymorphism is associated with clinical outcome of HER2-positive breast cancer patients. Further studies are warranted to validate these findings.

Are AHSG polymorphisms directly associated with coronary atherosclerosis?

BACKGROUND: Fetuin-A (AHSG) has been proposed as a new cardiovascular risk factor. Fetuin-A levels as well as AHSG single nucleotide polymorphisms (SNPs) have been associated with intima media thickness and incident vascular events, respectively. However, the association between AHSG variants and angiographically determined coronary artery disease (CAD) has not been reported yet. Therefore, we aimed at investigating the association of AHSG SNPs with angiographically characterized coronary atherosclerosis. METHODS: We genotyped AHSG variants rs4917, rs2248690, rs2518136, and rs2077119 in a cross-sectional study including 1,649 patients undergoing coronary angiography. Significant CAD was diagnosed in the presence of coronary stenoses ≥50%. RESULTS: Variant rs2077119 deviates significantly from Hardy-Weinberg equilibrium and was excluded from further analysis. Neither under an additive, nor under a recessive or dominant model of inheritance, the association between investigated AHSG variants and angiographically determined CAD reached statistical significance. Haplotypes derived from these AHSG variants also were not significantly associated with coronary lesions. Further, no significant associations between investigated SNPs and the extent or severity of CAD could be observed (all p-values>0.05). CONCLUSION: Our data do not support a significant direct association between AHSG variants rs4917, rs2248690, and rs2518136 and clinical atherosclerosis as exemplified by angiographically characterized coronary atherosclerosis.

Identification of hypoxia-induced genes in human SGBS adipocytes by microarray analysis.

Hypoxia in adipose tissue is suggested to be involved in the development of a chronic mild inflammation, which in obesity can further lead to insulin resistance. The effect of hypoxia on gene expression in adipocytes appears to play a central role in this inflammatory response observed in obesity. However, the global impact of hypoxia on transcriptional changes in human adipocytes is unclear. Therefore, we compared gene expression profiles of human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes under normoxic or hypoxic conditions to detect hypoxia-responsive genes in adipocytes by using whole human genome microarrays. Microarray analysis showed more than 500 significantly differentially regulated mRNAs after incubation of the cells under low oxygen levels. To gain further insight into the biological processes, hypoxia-regulated genes after 16 hours of hypoxia were classified according to their function. We identified an enrichment of genes involved in important biological processes such as glycolysis, response to hypoxia, regulation of cellular component movement, response to nutrient levels, regulation of cell migration, and transcription regulator activity. Real-time PCR confirmed eight genes to be consistently upregulated in response to 3, 6 and 16 hours of hypoxia. For adipocytes the hypoxia-induced regulation of these genes is shown here for the first time. Moreover in six of these eight genes we identified HIF response elements in the proximal promoters, specific for the HIF transcription factor family members HIF1A and HIF2A. In the present study, we demonstrated that hypoxia has an extensive effect on gene expression of SGBS adipocytes. In addition, the identified hypoxia-regulated genes are likely involved in the regulation of obesity, the incidence of type 2 diabetes, and the metabolic syndrome.

Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF.

Mutations in the oncogenes KRAS and BRAF have been identified as prognostic factors in patients with colorectal diseases and as predictors of negative outcome in epidermal growth factor receptor-targeted therapies. Therefore, accurate mutation detection in both genes, KRAS and BRAF, is of increasing clinical relevance. We aimed at optimizing allele-specific real-time PCR assays for the detection of common mutations in KRAS and the BRAF Val600Glu mutation using allele-specific PCR primers for allelic discrimination and probes (TaqMan) for quantification. Each reaction mix contains a co-amplified internal control to exclude false-negative results. Allele-specific real-time PCR assays were evaluated on plasmid model systems providing a mutation detection limit of 10 copies of mutant DNA in proportions as low as 1% of the total DNA. Furthermore, we analyzed 125 DNA samples prepared from archived, formalin-fixed, paraffin-embedded colorectal carcinomas and compared results with those obtained from direct-sequence analysis. All mutations determined by sequence analysis could be recovered by allele-specific PCR assays. In addition, allele-specific PCR assays clearly identified three additional samples affected by a mutation. We propose these allele-specific real-time PCR assays as a low-cost and fast diagnostic tool for accurate detection of KRAS and BRAF mutations that can be applied to clinical samples.

Single nucleotide polymorphisms of TCF7L2 are linked to diabetic coronary atherosclerosis.

BACKGROUND: Coronary artery disease (CAD) shares common risk factors with type 2 diabetes (T2DM). Variations in the transcription factor 7-like 2 (TCF7L2) gene, particularly rs7903146, increase T2DM risk. Potential links between genetic variants of the TCF7L2 locus and coronary atherosclerosis are uncertain. We therefore investigated the association between TCF7L2 polymorphisms and angiographically determined CAD in diabetic and non-diabetic patients. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped TCF7L2 variants rs7903146, rs12255372, and rs11196205 in a cross-sectional study including 1,650 consecutive patients undergoing coronary angiography for the evaluation of established or suspected stable CAD. Significant CAD was diagnosed in the presence of coronary stenoses ≥50%. Variant rs7903146 in the total study cohort was significantly associated with significant CAD (adjusted additive OR = 1.29 [1.09-1.53]; p = 0.003). This association was strong and significant in T2DM patients (n = 393; OR = 1.91 [1.32-2.75]; p = 0.001) but not in non-diabetic subjects (OR = 1.09 [0.90-1.33]; p = 0.370). The interaction risk allele by T2DM was significant (p(interaction) = 0.002), indicating a significantly stronger impact of the polymorphism on CAD in T2DM patients than in non-diabetic subjects. TCF7L2 polymorphisms rs12255372 and rs11196205 were also significantly associated with CAD in diabetic patients (adjusted additive OR = 1.90 [1.31-2.74]; p = 0.001 and OR = 1.75 [1.22-2.50]; p = 0.002, respectively). Further, haplotype analysis demonstrated that haplotypes including the rare alleles of all investigated variants were significantly associated with CAD in the whole cohort as well as in diabetic subjects (OR = 1.22 [1.04-1.43]; p = 0.013 and OR = 1.67 [1.19-2.22]; p = 0.003, respectively). CONCLUSIONS/SIGNIFICANCE: These results suggest that TCF7L2 variants rs7903146 rs12255372, and rs11196205 are significantly associated with angiographically diagnosed CAD, specifically in patients with T2DM. TCF7L2 therefore appears as a genetic link between diabetes and atherosclerosis.

Evaluation of the association of genetic variants on the chromosomal loci 9p21.3, 6q25.1, and 2q36.3 with angiographically characterized coronary artery disease.

OBJECTIVES: The chromosomal loci 9p21.3, 6q25.1, and 2q36.3, represented by their respective leading variants rs1333049, rs6922269 and rs2943634, have been linked with a history of coronary artery disease (CAD) by genome-wide association studies. Whereas the association of variant rs1333049 with CAD was analysed in several subsequent studies, replication studies of variants rs6922269 and rs2943634 are missing. Furthermore, no direct association with coronary atherosclerosis has been established. We therefore aimed at investigating the association of the above variants with coronary atherosclerosis. METHODS: We performed genotyping in two large cohorts of consecutive Caucasian patients undergoing coronary angiography for the evaluation of suspected or established stable CAD, comprising 671 and 940 patients, respectively, with a total of 1611 subjects. RESULTS: In models of dominant inheritance, variant rs1333049 conferred a significantly increased risk of significant coronary stenoses with lumen narrowing >or=50% in both study cohorts, with adjusted odd ratios (OR) of 1.71 (1.15-2.52); p=0.007 and 1.55 (1.10-2.18); p=0.012, respectively. Variant rs6922269 in neither cohort was significantly associated with CAD. Although carriers of the A allele of variant rs2943634 were at an increased risk of significant coronary stenoses in the second cohort (OR=1.41 (1.06-1.88); p=0.018), no such association was found for the first cohort nor for both cohorts combined. CONCLUSION: Our data from two populations show that variant rs1333049 is significantly associated with angiographically characterized CAD. In contrast, variant rs6922269 did not show any impact on coronary atherosclerosis. The association between variant rs2943634 and CAD warrants further investigation.

Significant impact of chromosomal locus 1p13.3 on serum LDL cholesterol and on angiographically characterized coronary atherosclerosis.

OBJECTIVES: Recently, a significant impact of a new locus on chromosome 1p13.3 on serum LDL (low-density lipoprotein) cholesterol and clinical events of coronary artery disease (CAD) was described. Potential associations between variants on this locus and angiographically characterized coronary atherosclerosis are unknown. We therefore aimed at investigating the association of variants of locus 1p13.3 with coronary atherosclerosis. METHODS: We performed genotyping of variants rs599839, rs646776, and rs4970834 on chromosome 1p13.3 in a large cohort of 1610 consecutive Caucasian patients undergoing coronary angiography, where lesions of 50% or more were classified as significant. RESULTS: Compared to the homozygous common allele the rare alleles of variants rs599839, rs646776, and rs4970834 were significantly associated with decreased serum LDL cholesterol (132+/-40mg/dl vs. 125+/-36mg/dl, P=0.003, 132+/-40mg/dl vs. 124+/-36mg/dl, P<0.001, and 131+/-40mg/dl vs. 125+/-37mg/dl, P=0.005, respectively). Further, carriers of the rare alleles of variants rs599839 and rs646776 were at a significantly lower risk of significant coronary stenoses than subjects who were homozygous for the frequent alleles, with odds ratios (ORs) of 0.78 [0.63-0.96]; P=0.019 and 0.74 [0.60-0.91]; P=0.004, respectively. After multivariate adjustment including LDL cholesterol, the protective effect of the rare allele of variant rs646776, but not of variant rs599839, on CAD risk remained significant (OR=0.77 [0.61-0.98], P=0.034). CONCLUSIONS: We conclude that chromosomal locus 1p13.3 is significantly associated with both, serum LDL cholesterol and coronary atherosclerotic lesions.