MassARRAY Genotyping Research Articles

Association between genetic variants in the promoter region of a novel antisense long noncoding RNA RP11-392P7.6 and colorectal cancer risk.

There is a widespread occurrence of antisense transcripts' regulation on cancer-related genes in cancer biology. RP11-392P7.6 is antisense to the coding region of cancer-related gene GPRC5D, which has been found recently. The aim of this study was to investigate the associations of tagSNPs in the promoter region of RP11-392P7.6 with the risk of colorectal cancer. We conducted a two-stage case-control study, with a discovery set (320 cases and 319 controls) and a validation set (501 cases and 538 controls). Four tagSNPs (rs1531970, rs1642199, rs4763903, and rs10845671) were selected based on 1000 Genomes Project data and genotyped by using the Sequenom MassARRAY genotyping platform. In the discovery set, three tagSNPs (rs1642199, rs4763903, and rs10845671) were revealed promising associations with the risk of colorectal cancer, among which the rs10845671 variants were further replicated in the validation set (OR = 1.47, 95% CI = 1.10-1.20 in heterozygote codominant model; OR = 1.38, 95% CI = 1.04-1.83 in dominant model). When combined the two sets, the above positive associations remained unchanged. Rs10845671 was found to be associated with an increased risk of colorectal cancer (OR = 1.43, 95% CI = 1.14-1.81 in heterozygote codominant model; OR = 1.35, 95% CI = 1.08-1.69 in dominant model). These findings indicate that rs10845671 may contribute to the susceptibility to colorectal cancer and be a candidate biomarker for colorectal cancer risk prediction. Environ. Mol. Mutagen. 58:434-442, 2017. © 2017 Wiley Periodicals, Inc.

Correlation between clinical response to sorafenib in hepatocellular carcinoma treatment and polymorphisms of P-glycoprotein (ABCB1) and of breast cancer resistance protein (ABCG2): monocentric study.

We studied the relation between the polymorphism of P-glycoprotein (P-gp) and of breast cancer resistance protein (BCRP), encoded by ABCB1 and ABCG2 genes, respectively, and the pharmacokinetic variability and clinical response during the treatment with sorafenib of hepatocellular carcinoma. At the Paul Brousse Hospital in Villejuif, France, 47 consecutive patients with advanced HCC treated with a single agent sorafenib, were enrolled. Sorafenib exposure was measured by its plasma concentration 3h after oral administration of 400mg (bid) by liquid chromatography. All enrolled patients were genotyped for ABCB1 (rs2032582; rs1045642) and ABCG2 (rs2231137; rs2231142; rs2622604) by blood genomic DNA extraction and Mass ARRAY genotyping. The clinical response was evaluated after 3months of treatment according to the RECIST criteria. Significant associations between sorafenib exposure and the studied polymorphisms were observed for ABCB1 3435C>T, ABCG2 34G>A, ABCG2 1143C>T and ABCG2 421C>A, but not for ABCB1 2677G>TA SNP. In heterozygous patients for ABCB1 3435 C>T, ABCG2 34 G>A and ABCG2 1143 C>T polymorphisms were significantly associated with the lowest sorafenib plasma levels. Those patients presented a tendency to have the best clinical evolution. Heterozygous forms of the studied polymorphisms could be associated with a better therapeutic response.

Characterization of BRCA1 and BRCA2 variants in multi-ethnic Asian cohort from a Malaysian case-control study

BackgroundGenetic testing for BRCA1 and BRCA2 has led to the accurate identification of individuals at higher risk of cancer and the development of new therapies. Approximately 10-20% of the genetic testing for BRCA1 and BRCA2 leads to the identification of variants of uncertain significance (VUS), with higher proportions in Asians. We investigated the functional significance of 7 BRCA1 and 25 BRCA2 variants in a multi-ethnic Asian cohort using a case-control approach.MethodsThe MassARRAY genotyping was conducted in 1,394 Chinese, 406 Malay and 310 Indian breast cancer cases and 1,071 Chinese, 167 Malay and 255 Indian healthy controls. The association of individual variant with breast cancer risk was analyzed using logistic regression model adjusted for ethnicity, age and family history.ResultsOur study confirmed BRCA2 p.Ile3412Val is presented in >2% of unaffected women and is likely benign, and BRCA2 p.Ala1996Thr which is predicted to be likely pathogenic by in-silico models is presented in 2% of healthy Indian women suggesting that it may not be associated with breast cancer risk. Single-variant analysis suggests that BRCA1 p.Arg762Ser may be associated with breast cancer risk (OR = 7.4; 95% CI, 0.9–62.3; p = 0.06).ConclusionsOur study shows that BRCA2 p.Ile3412Val and p.Ala1996Thr are likely benign and highlights the need for population-specific studies to determine the likely functional significance of population-specific variants. Our study also suggests that BRCA1 p.Arg762Ser may be associated with increased risk of breast cancer but other methods or larger studies are required to determine a more precise estimate of breast cancer risk.

Characterization of Three CYP2C19 Gene Variants by MassARRAY and Point of Care Techniques: Experience from a Czech Centre.

Distribution of cytochrome P450 2C19 enzyme gene (CYP2C19) variants affecting metabolism of clopidogrel was determined in 526 Czech patients after percutaneous coronary intervention using MassARRAY genotyping and compared to distribution in other populations of European descent. Fifty-three (10%) patients underwent parallel determination of CYP2C19 genotypes from buccal swabs by a point of care technique with 100% concordance to the main genotyping platform. Observed CYP2C19 genotypes were related to clopidogrel metabolism phenotypes and discussed in population context. Hereby, presented methodologies provide accurate CYP2C19 genotyping results in a relatively short time of one up to 12h and may, therefore, find the relevant place in the field of genotype-guided antiplatelet therapy.

Association between miR-199a rs74723057 and MET rs1621 polymorphisms and the risk of hepatocellular carcinoma.

MicroRNAs (miRNAs) can regulate gene expression at post-transcriptional levels, thereby influence cancer risk. The aim of the current study is to investigate association between miR-199a rs74723057 and MET rs1621 and HCC risk in 1032 HCC patients and 1060 cancer-free controls. These two SNPs were genotyped by using the Agena MassARRAY genotyping system. Odds ratio (OR) and 95% confidence interval (95%CI) were calculated to assess the strength of the associations. We found that compared with the wild-type AA genotype of MET rs1621, the variant GG genotype was associated with a decreased risk for HCC (OR = 0.24, 95% CI = 0.06–0.96, P = 0.043). No association between miR-199a rs74723057 and HCC risk was observed. In addition, an interaction effect on HCC risk between the selected two SNPs was found. Among those who carried the CG/GG genotypes of miR-199a rs74723057, those who carried the GG genotype of MET rs1621 had a reduced risk of HCC, when compared with those who carried the AG/AA genotypes of MET rs1621 (OR = 0.15, 95% CI = 0.03~0.73, P for interaction = 0.018). Our results suggest that MET rs1621 polymorphism, alone and combined with miR-199a rs74723057, may influence susceptibility to HCC. Further large-scale association studies and functional studies are needed to validate our findings.

Whole-Genome Sequencing and iPLEX MassARRAY Genotyping Map an EMS-Induced Mutation Affecting Cell Competition in Drosophila melanogaster.

Cell competition, the conditional loss of viable genotypes only when surrounded by other cells, is a phenomenon observed in certain genetic mosaic conditions. We conducted a chemical mutagenesis and screen to recover new mutations that affect cell competition between wild-type and RpS3 heterozygous cells. Mutations were identified by whole-genome sequencing, making use of software tools that greatly facilitate the distinction between newly induced mutations and other sources of apparent sequence polymorphism, thereby reducing false-positive and false-negative identification rates. In addition, we utilized iPLEX MassARRAY for genotyping recombinant chromosomes. These approaches permitted the mapping of a new mutation affecting cell competition when only a single allele existed, with a phenotype assessed only in genetic mosaics, without the benefit of complementation with existing mutations, deletions, or duplications. These techniques expand the utility of chemical mutagenesis and whole-genome sequencing for mutant identification. We discuss mutations in the Atm and Xrp1 genes identified in this screen.

Influence of genetic polymorphisms in cytochrome P450 oxidoreductase on the variability in stable warfarin maintenance dose in Han Chinese

The aim of this study was to investigate whether any of the single-nucleotide polymorphisms (SNPs) in the POR gene were significantly associated with CYP activity and expression, and could contribute to the total variability in stable warfarin maintenance doses in Han Chinese. A total of 408 patients treated at the First Affiliated Hospital of Sun Yat-Sen University were eligible for the study and had attained a stable warfarin maintenance dose at the start of the investigation. Demographics, warfarin maintenance doses, and concomitant medications were documented. Genomic DNA was extracted from peripheral blood samples and genotyped for ten SNPs (CYP 2C9*2 and *3, CYP4F2 rs2108622, VKORC1 -1639C>T, and potential POR genes of rs10239977, rs3815455, rs41301394, rs56256515, rs1057868, and rs2286823) using the Sequenom MassARRAY genotyping system. A predictive model of warfarin maintenance dose was established and indicated that age, gender, body surface area, aspirin use, CYP2C9*3, CYP4F2 rs2108622, VKORC1 -1639C>T, and POR*37 831-35C>T accounted for 42.4 % of dose variance in patients undergoing anticoagulant treatment. The contribution of POR*37 831-35C>T to warfarin dose variation was only 3.9 %. For the first time, the SNP POR*37 831-35C>T was confirmed as a minor but statistically significant factor associated with interindividual variation in warfarin maintenance dose in Han Chinese. The POR*37 gene polymorphism should be considered in future algorithms for faster and more reliable achievement of stable warfarin maintenance doses.

PADI2 gene confers susceptibility to breast cancer and plays tumorigenic role via ACSL4, BINC3 and CA9 signaling.

BackgroundPeptidylarginine deiminase (PAD) post-translationally converts arginine residues to citrulline residues. Recent studies have suggested that PADI2 (PAD isoform 2), a member of the PAD family, is involved in the tumorigenic process of some tumors, especially breast cancer. However, little is known about the mechanisms of PADI2 in tumorigenesis. This study aimed to elucidate the tumorigenic role and regulatory pathway of PADI2 in breast tumors.MethodsThe Sequenom MassARRAY and TaqMan genotyping methods were used to investigate the correlation between PADI2 gene SNPs and various tumor risks. PCR array analyses, including cancer pathway finder and signal transduction PCR arrays, were performed to investigate the tumorigenic pathway of PADI2 in the MCF-7 breast cancer cell line following treatment with anti-PADI2 siRNA. Cell proliferation, apoptosis and transwell migration assays were performed to observe the effect of PADI2 in MCF-7 cells treated with anti-PADI2 siRNA.ResultsBoth Sequenom MassARRAY and TaqMan genotyping assays demonstrated that SNP rs10788656 in the PADI2 gene was significantly associated with breast cancer. PCR arrays indicated that inhibiting PADI2 expression significantly increased expression of CA9 and decreased expression of ACSL4 and BIRC3 in MCF-7 cells, which was verified using real-time PCR. Inhibiting PADI2 expression also significantly decreased the migration ability of MCF-7 cells but did not affect cell proliferation or apoptosis.ConclusionsThe PADI2 gene confers susceptibility to breast cancer. PADI2 expression contributes to abnormal migration of breast tumor cells. PADI2 affects tumorigenesis in breast tumor cells by regulating the expression of ACSL4, BINC3 and CA9, which are known to promote abnormal lipid metabolism and cell invasion of tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0335-0) contains supplementary material, which is available to authorized users.

The first genetic linkage map of Primulina eburnea (Gesneriaceae) based on EST-derived SNP markers.

Primulina eburnea is a promising candidate for domestication and floriculture, since it is easy to culture and has beautiful flowers. An F₂ population of 189 individuals was established for the construction of first-generation linkage maps based on expressed sequence tags-derived single-nucleotide polymorphism markers using the massARRAY genotyping platform. Of the 232 screened markers, 215 were assigned to 18 LG according to the haploid number of chromosomes in the species. The linkage map spanned a total of 3774.7 cM with an average distance of 17.6 cM between adjacent markers. This linkage map provides a framework for identification of important genes in breeding programmes.

MiRSNPs of miR1274 and miR3202 Genes that Target MeCP2 and DNMT3b Are Associated with Lung Cancer Risk: A Study Conducted on MassARRAY Genotyping.

Genetic variants of miRNAs that target DNMTs and MBDs involved in DNA methylation were scanned with current databases, and 35 miRSNPs in 22 miRNA genes were identified. The aim of the study was to determine the association between these variants of miRNA genes and lung cancer (LC). DNA samples were isolated from blood samples and genotyped using a Sequenom MassARRAY System. An association between the rs188912830 gene variant of miR3202 that targets the MeCP2 protein and LC was indicated in both subtypes. The presence of the C-allele in patients with LC and its subtypes was significantly lower, and the absence of the C-allele was determined to increase the risk of LC by 7,429-times compared to the presence (p=0,010). The rs318039 gene variant of miR1274 that targets DNMT3b was found to be associated with LC subtypes. When allele distributions were compared, the numbers of individuals with the C-allele were significantly lower in the NSCLC and SCLC groups. No significant associations were found for the rs72563729 variant of the miR200b gene that targets DNMT3a or for the rs145416750 variant of the miR513c gene that targets TRDMT1. The other 33 variants were found to be ancestral genotypes. Consequently, rs188912830 and rs318039 variations were associated with LC subtypes. Importantly, this study is the first to indicate the functional characterisation of miRSNPs of genes that target DNA methylation.

Association of IL33/ST2 signal pathway gene polymorphisms with myocardial infarction in a Chinese Han population.

This study investigated the relationship between IL-33/ST2 signal pathway gene polymorphisms and myocardial infarction (MI) in Han Chinese. A case-control association analysis was performed on a total of 490 MI patients (MI group) and 929 normal subjects (NC group). Sequenom Mass Array and Taqman genotyping technique were used to analyze the tag single nucleotide polymorphisms (SNPs) in the genes encoding IL-33, ST2, and IL-1RaP (rs11792633, rs1041973 and rs4624606). The results showed that the frequencies of rs4624606 genotypes AA, TT, AT were 0.031, 0.647, 0.322 in MI group and 0.026, 0.712, 0.263 in NC group, and the allele frequencies of A and T were 0.192, 0.808 in MI group and 0.157, 0.843 in NC group. There were significant differences in rs4624606 genotypes and allele frequencies between MI group and NC group (P<0.05). For rs11792633, the allele frequencies of C and T were 0.45, 0.55 in MI group and 0.454, 0.546 in NC group with no significant differences found between the two groups. Compared with genotype CC+TC, rs11792633 genotype TT had an increased risk of hypertension (P<0.05). However, there were no significant differences in the frequencies of rs11792633 genotypes between the two groups. No significant differences were noted in the frequencies of rs1041973 genotype and allele between the two groups. Logistic regression analysis showed that rs4624606 genotypes AT and AA+AT were both significantly associated with MI (AT: OR=1.325, P=0.029, 95% CI=1.03-1.705; AA+AT: OR=1.316, P=0.028, 95% CI=1.03-1.681) after factors such as age, gender, smoking, drinking, body mass index (BMI), triglyceride (TG) and cholesterol were adjusted. Those carrying rs4624606 genotype AT or AA+AT had an increased risk of MI. No associations were found between the polymorphisms of the other two loci with MI. It was concluded that, in the IL33/ST2 signal pathway, the A allele of rs4624606 polymorphism of IL-1RaP gene is a potential independent risk factor for MI, and the genotypes AA+AT and AT are associated with the incidence of MI.

Transcriptional expression of Bcl-2 as predictive of response to neoadjuvant chemotherapy with trastuzumab in HER2-positive ER-positive breast cancer patients.

602 Background: Several trials have confirmed that pathologic complete response (pCR) rates after neoadjuvant chemotherapy (NAC) are significantly lower in HER2+/ER+ patients (pts) than in HER2+/ER− pts. To elucidate this phenomenon, we investigated the association of anti-apoptotic Bcl-2, which is frequently overexpressed in ER+ tumors, with to NAC resistance. Methods: Pretreatment formalin-fixed tumor tissues were collected from 75 HER2+ pts receiving NAC comprising anthracyclines, taxanes, and trastuzumab. mRNA expression was detected by PCR amplificationand quantitative primer extension using MassARRAY (Sequenom, CA). The panel of 15 genes comprised EGFR, HER2, HER3, IGF1R, FGFR, PTEN, INPP4B, SRC-1, DUSP4, ESR1, PgR, FOXA1, PDL1, CTLA-4, and Bcl-2, whose expressions were dichotomized according to median values. PIK3CA mutations were detected using MassARRAY genotyping. ER, PTEN, and Bcl-2 protein expressions were scored from 0 to 3 according to the frequency and intensity by immunohistochemisty (IHC)...

Association of C(-106)T Polymorphism in Aldose Reductase Gene with Diabetic Retinopathy in Chinese Patients with Type 2 Diabetes Mellitus

To identify the possible association between C(-106)T polymorphism of the aldose reductase (ALR) gene and diabetic retinopathy (DR) in a cohort of Chinese patients with type 2 diabetes mellitus (T2DM). From November 2009 to September 2010, patients with T2DM were recruited and assigned to DR group or diabetic without retinopathy (DWR) group according to the duration of diabetes and the grading of 7-field fundus color photographs of both eyes. Genotypes of the C(-106)T polymorphism (rs759853) in ALR gene were analyzed using the MassARRAY genotyping system and an association study was performed. A total of 268 T2DM patients (129 in the DR group and 139 in the DWR group) were included in this study. No statistically significant differences were observed between the 2 groups in the age of diabetes onset (P=0.10) and gender (P=0.78). The success rate of genotyping for the study subjects was 99.6% (267/268), with one case of failure in the DR group. The frequencies of the T allele in the C(-106)T polymorphism were 16.0% (41/256) in the DR group and 19.4% (54/278) in the DWR group (P=0.36). There was no significant difference in the C(-106)T genotypes between the 2 groups (P=0.40). Compared with the wild-type genotype, odds ratio (OR) for the risk of DR was 0.7 (95% CI, 0.38-1.3) for the heterozygous CT genotype and 0.76 (95% CI, 0.18-3.25) for the homozygous TT genotype. The risk of DR was positively associated with microalbuminuria (OR=4.61; 95% CI, 2.34-9.05) and insulin therapy (OR=3.43; 95% CI, 1.94-6.09). Microalbuminuria and insulin therapy are associated with the risk of DR in Chinese patients with T2DM. C(-106)T polymorphism of the ALR gene may not be significantly associated with DR in Chinese patients with T2DM.

Association of Single Nucleotide Polymorphisms in the Prostaglandin-endoperoxide Synthase 2 (PTGS2) and Phospholipase A2Group IIA (PLA2G2A) Genes with Susceptibility to Esophageal Squamous Cell Carcinoma

The prostaglandin-endoperoxide synthase 2 (PTGS2) and phospholipase A2 group IIA (PLA2G2A) genes encode enzymes that are involved in arachidonic acid and prostaglandin biosynthesis. Dysregulation of both genes is associated with inflammation and carcinogenesis, including esophageal squamous cell carcinoma (ESCC). We therefore hypothesized that there is an association between single nucleotide polymorphisms (SNPs) in these genes and susceptibility to ESCC. We performed a gene-wide tag SNP-based association study to examine the association of SNPs in PTGS2 and PLA2G2A with ESCC in 269 patients and 269 healthy controls from Taihangshan Mountain, Henan and Hebei Provinces, the rural area of China which has the highest incidence of esophageal cancer in the world. Thirteen tag SNPs in PLA2G2A and 4 functional SNPs in PTGS2 were selected and genotyped using a high-throughput Mass Array genotyping platform. We found a modest increased risk of ESCC in subjects with the PTGS2 rs12042763 AA genotype (OR=1.23; 95% CI, 1.00- 3.04) compared with genotype GG. For PLA2G2A, a decreased risk of ESCC was observed in subjects with the rs11677 CT (OR=0.51, 95%CI, 0.29-0.85) or TT genotype (OR=0.51, 95%CI, 0.17-0.96) or the T carriers (CT+TT) (OR=0.52, 95%CI, 0.31-0.85) when compared with the CC genotype. Also for PLA2G2A, rs2236771 C allele carriers were more frequent in the control group (P=0.02). Subjects with the GC (OR=0.55, 95%CI, 0.33-0.93) or CC genotype (OR=0.38, 95% CI, 0.16-0.94) or the C carriers (GC+CC) (OR=0.52, 95%CI, 0.32- 0.85) showed a negative association with ESCC susceptibility. Our results suggest that PTGS2 and PLA2G2A gene polymorphisms may modify the risk of ESCC development.

Abstract C03: A comprehensive study of genomic alterations reveals deregulation of the cell cycle in most esophageal squamous cell carcinomas

Abstract Objective: Esophageal squamous cell carcinoma (ESCC) is a major cause of cancer related mortality. To determine the molecular basis of ESCC development, this investigation sought to comprehensively identify the characteristic genome-wide changes in ESCC, including exonic mutations and structural alterations. The clinical implications of these genetic changes are also analyzed. Methods: Exome sequencing was applied to detect somatic mutations in nine pairs of ESCC and matched blood samples, followed by validation with MassARRAY genotyping and Sanger sequencing. Whole-genome SNP arrays were employed to study copy number changes and the loss of heterozygosity in 55 ESCCs. Results: 108 non-synonymous mutations in 102 genes were identified and validated in nine patients, suggesting a low somatic mutation rate (average 12 per tumor) in our ESCC cases. The chromatin modification process was found to be enriched in gene ontology analysis. Mutated TP53 was seen in five of the nine tumors (55.6%) undergone exome sequencing and in 23/46 tumors in the validation set. Integrating with the SNP array data, we found that tumor genomes with TP53 mutations were much more unstable than those without these mutations. In the landscape of genomic alterations, deletion of 9p21.3 covering CDKN2A (30.9%), amplification of 11q13.3 covering CCND1 (30.9%), and point mutation of TP53 (50.9%) occurred in two-thirds of ESCCs, suggesting deregulation of G1 in cell cycle is the key event in ESCC. Furthermore, six minimal common regions were found to be significantly altered in ESCC and three (9p21.3, 7p11.2, 3p12.1) were associated with lymph node metastasis. Conclusions: Frequent TP53 mutation and genomic instability are highly correlated in ESCC. The amplification of CCND1, deletion of CDKN2A/B, and somatic mutation of TP53 play pivotal roles via G1 deregulation and therefore classify this cancer into major groups. These findings provide clinical significance for future molecular diagnosis and therapeutic targeting. Citation Format: Jian Bai, Qiyan Wang, Kenan Gong, Hong Cai, Yang Ke, Changqing Zeng. A comprehensive study of genomic alterations reveals deregulation of the cell cycle in most esophageal squamous cell carcinomas. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C03.

Association between six single nucleotide polymorphisms of EGLN1 gene and adaptation to high-altitude hypoxia

To investigate the association between SNPs located in 5'UTR and intron of prolyl hydroxylase 2 (EGLN1 or PHD2) and adaptation to high-altitude hypoxia, the SNPs (rs2066140, rs2808584, rs2491405, rs2486741, rs2486734 and rs21533646) of EGLN1 gene were genotyped using Sequenom MassArray genotyping system in 152 unrelated healthy Tibetan individuals (3 650 m altitude) and 192 Han (5 00 m altitude), and the haplotypes of these SNPs were constructed and analyzed. Our results showed all the homozygous genotypes of six SNPs loci were significantly different between the two groups (P<0.05). The frequencies of haplotypes G-G (rs2066140 and rs2808584) and G-C (rs2486741 and rs2486734) of high-altitude group were significantly different from low-altitude group (P<0.05). In addition, the frequencies of haplotypes C-A (rs2066140 and rs2808584) and C-T (rs2486741 and rs2486734) of high-altitude group were significantly lower than those in low-altitude group (P<0.05). Our results indicate that the polymorphism of homozygous genotype in six SNPs and their haplotypes were associated with adaptation to high-altitude hypoxia.

Replication of Type 2 Diabetes Candidate Genes Variations in Three Geographically Unrelated Indian Population Groups

Type 2 diabetes (T2D) is a syndrome of multiple metabolic disorders and is genetically heterogeneous. India comprises one of the largest global populations with highest number of reported type 2 diabetes cases. However, limited information about T2D associated loci is available for Indian populations. It is, therefore, pertinent to evaluate the previously associated candidates as well as identify novel genetic variations in Indian populations to understand the extent of genetic heterogeneity. We chose to do a cost effective high-throughput mass-array genotyping and studied the candidate gene variations associated with T2D in literature. In this case-control candidate genes association study, 91 SNPs from 55 candidate genes have been analyzed in three geographically independent population groups from India. We report the genetic variants in five candidate genes: TCF7L2, HHEX, ENPP1, IDE and FTO, are significantly associated (after Bonferroni correction, p<5.5E−04) with T2D susceptibility in combined population. Interestingly, SNP rs7903146 of the TCF7L2 gene passed the genome wide significance threshold (combined P value = 2.05E−08) in the studied populations. We also observed the association of rs7903146 with blood glucose (fasting and postprandial) levels, supporting the role of TCF7L2 gene in blood glucose homeostasis. Further, we noted that the moderate risk provided by the independently associated loci in combined population with Odds Ratio (OR)<1.38 increased to OR = 2.44, (95%CI = 1.67–3.59) when the risk providing genotypes of TCF7L2, HHEX, ENPP1 and FTO genes were combined, suggesting the importance of gene-gene interactions evaluation in complex disorders like T2D.

A Regulatory Promoter Polymorphism and Hypermethylation of Intron 1 Are Associated with Reduced Expression of KLF5 and Inferior Survival in AML

AbstractAbstract 3508 Background:Krüppel-like factor 5 (KLF5) is a transcription factor with regulatory roles in cell growth, survival, and differentiation. Our previous studies have indicated that KLF5 is required for myeloid blood lineage differentiation and is a target of genomic methylation in Acute Myeloid Leukaemia (AML) which is associated with reduced expression. Further, we have shown that treatment of leukaemia cell lines, displaying KLF5 hypermethylation, with demethylating agents is associated with induction of KLF5 expression, supporting an inhibitory role of methylation on expression (Diakiw et al, Leuk Res 36:110). Other recent studies have shown that KLF5 expression is reduced by the presence of a heterozygous genotype at a promoter SNP (rs3812852) and plays a role in pathogenic processes in heart and brain. We investigated both methylation and SNP genotype as mechanisms of KLF5 gene regulation in AML and their relationship to patient outcome. Methods:We analysed 232 diagnostic bone marrow mononuclear cell samples from a retrospective cohort of patients with AML. The genotype of the rs3812852 SNP was determined by Sequenom MassARRAY genotyping. DNA methylation of the KLF5 gene locus (intron 1) was assessed using Sequenom MassARRAY epityper. KLF5 gene expression was determined for 85 of these samples using quantitative real-time PCR. Of the 232 patients, 161 received treatment with induction chemotherapy and were included in survival analysis. Results:We found that 28 (12%) patients were heterozygous (G/A) for the SNP (rs3812852) in the KLF5 promoter, with one patient having the rare homozygous genotype (G/G) and 204 (88%) patients the common homozygous genotype (A/A). KLF5 gene expression correlated with the SNP genotype, with the heterozygous patients displaying significantly reduced KLF5 expression compared to normal controls (CD34 cells from normal donors, p=0.044). Consistent with our previous study on a smaller test cohort we observed that hypermethylation of this region of KLF5 is common and is present in 50% of samples in our larger cohort. AML patients with high methylation of KLF5 had significantly reduced KLF5 expression compared to normal controls and AML patients with low KLF5 methylation (p=0.032 and p=0.048 respectively). The correlation of gene expression with the SNP genotype and KLF5 methylation groups was also true when considered together, as patients with the heterozygous genotype (A/G) and high methylation had lower KLF5 expression than patients with the common homozygous genotype (A/A) and low KLF5 methylation (Figure 1A).Cox multi-variate analysis (variables of age, WCC and mutational status of FLT3-ITD, IDH1/2, NPM1) of survival of patients treated with induction chemotherapy showed that the presence of a G/A genotype at the KLF5 promoter SNP was not an independent predictor of overall survival (p= 0.502). In contrast hypermethylation of KLF5 intron 1 was a highly significant independent predictor of poorer overall survival (median survival 10.4 months versus 20.7 months, HR 1.5, p=0.001, Figure 1B). Importantly, when the heterozygous SNP genotype (A/G) and high methylation status were combined, this group of patients had even poorer OS (median survival 5.2 months, HR 3.6, Figure 1C) and this was significant compared to the group with homozygous SNP genotype (A/A) and low methylation (p=0.002). Conclusions:We have shown that both methylation of KLF5 intron 1 and a heterozygous genotype at a promoter polymorphism are associated with reduced KLF5 expression in AML. KLF5 hypermethylation is an independent predictor of poor prognosis and combined with the heterozygous SNP genotype defines a group with particularly poor survival indicating a connection with KLF5 gene expression and response to treatment. Importantly, as the majority of the effect is associated with hypermethylation of KLF5, these patients (50%) may benefit from treatment with demethylating agents. [Display omitted] Disclosures:Wei:Celgene: Research Funding.

Effect of specific ADRB1/ADRB2/AGT genotype combinations on the association between survival and carvedilol treatment in chronic heart failure

The aim of the present study was to determine whether carvedilol-treated chronic heart failure patients have a different prognosis when stratified for a specific combination of a gain-of-function genotype of the adrenergic β-1 receptor gene (ADRB1) (Arg389-homozygous), two gain-of-function genotypes of the angiotensinogen gene (AGT) (Thr174-homozygous and Thr235-homozygous), and a downregulated genotype of the adrenergic β-2 receptor gene (ADRB2) (Gln27-carrier). Genotyping of 618 patients was carried out using the Sequenoms MassARRAY genotyping system. Outcome was all-cause mortality and statistics were calculated using a multivariable Cox proportional hazards model. Internal validation was performed using the bootstrap procedure. Eighty-seven of the 618 patients included in the study were treated with carvedilol. There was a significant interaction between the outcome of carvedilol treatment and the combination of the gain-of-function ADRB1 genotype (Arg389-homozygous) and the gain-of-function AGT genotype (Thr174-homozygous) (P(interaction)=0.003; hazard ratio 2.19, 95% confidence interval 1.26-3.78, P=0.005). There was also a significant interaction when the downregulated ADRB2 genotype (Gln27-carrier) was added to the ADRB1/AGT combination (P(interaction)=0.0005; hazard ratio 2.67, 95% confidence interval 1.51-4.72, P=0.0007). Two hundred and four patients were treated with metoprolol. There was no interaction between metoprolol treatment and the specific genotype combinations as there was no difference in the overall survival. The validity of the results was supported by the bootstrap procedure. We found a doubling of the hazard of mortality in carvedilol-treated patients with the combination of the gain-of-function ADRB1 genotype (Arg389-homozygous), the gain-of-function AGT genotype (Thr174-homozygous), and the downregulated ADRB2 genotype (Gln27-carrier). This might be valuable when stratifying chronic heart failure patients to the right β-blocker therapy.

Association of ELMO1 gene polymorphisms with diabetic nephropathy in Chinese population.

Genetic variations in the engulfment and cell motility 1 (ELMO1) gene have recently been identified to be associated with nephropathy attributed to Type 2 diabetes mellitus (T2DM). Since T2DM-associated complications are proved to be more common among Asians than Western individuals, and Chinese people have a high incidence of diabetic nephropathy (DN), this study sought to analyze the association of ELMO1 gene polymorphisms with DN to probe into the effects of ELMO1 gene on susceptibility of DN in Chinese population. We genotyped 6 polymorphism sites of ELMO1 gene in 200 unrelated Chinese subjects (123 T2DM with DN case subjects and 77 T2DM without DN control subjects). Genotyping was detected by a Sequenom MassARRAY genotyping system. The strongest associations in ELMO1 gene with DN occurred at rs741301 [odds ratio (OR) 1.89; p=0.004] and rs10951509 (OR 1.76; p=0.02). Unconditional logistic regression analysis identified that the rs741301 polymorphism (presence of A allele, adjusted OR 3.27; p=0.03) and duration of T2DM (adjusted OR 1.15; p=0.04) were independent predictors for DN. The marker rs741301 located in intron 18 of ELMO1 gene was in strong linkage disequilibrium (LD) with rs11769038 (D'=0.91). Furthermore, haplotype analysis identified that haplotype 1 [CAAAGA] (OR 1.95; p=0.01), haplotype 2 [CAAAGG] (OR 0.50; p=0.01), and haplotype 9 [TGCGGG] (OR 0.17; p=0.007) of ELMO1 were significantly associated with DN. This study first investigated the association of ELMO1 gene polymorphisms with DN in a Chinese population, supporting its key role as a candidate gene in the susceptibility of DN.