High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology

Olivier, Michael; Chuang, Lee‐Ming; Chang, Mau‐Song; Chen, Ying‐Tsung; Pei, Dee; Ranade, Koustubh; Witte, Anniek De; Allen, Jennifer; Tran, Nguyet; Curb, David; Pratt, R. T. C.; Neefs, Henk; Indig, Monika de Arruda; Law, Scott M.; Neri, Bruce; Wang, Lu; Cox, David

doi:10.1093/nar/gnf052

Cited by 77 publications

(66 citation statements)

References 14 publications

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“…As a quality standard, we randomly included six positive (two homozygous wild-type allele carriers, two heterozygous and two homozygous risk allele carriers) and two negative (all components excluding DNA) sequenced controls in each TaqMan reader plate. Because all controls were correctly identified, we assumed that the genotyping error rate of this method did not exceed 0.3% [23].…”

Section: Methodsmentioning

confidence: 99%

Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms

Schäfer¹,

Tschritter²,

Machicao³

et al. 2009

Diabetologia

119

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Aims/hypothesis Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene are associated with type 2 diabetes and reduced insulin secretion. The transcription factor TCF7L2 is an essential factor for glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells. We studied whether a defect in the enteroinsular axis contributes to impaired insulin secretion in carriers of TCF7L2 polymorphisms. Methods We genotyped 1,110 non-diabetic German participants for five single nucleotide polymorphisms in TCF7L2. All participants underwent an OGTT; GLP-1 secretion was measured in 155 participants. In 210 participants, an IVGTT combined with a hyperinsulinaemic-euglycaemic clamp was performed. In another 160 participants from the Netherlands and 73 from Germany, a hyperglycaemic clamp (10 mmol/l) was performed. In 73 German participants this clamp was combined with a GLP-1 infusion and an arginine bolus. Results The OGTT data confirmed that variants in TCF7L2 are associated with reduced insulin secretion. In contrast, insulin secretion induced by an i.v. glucose challenge in the IVGTT and hyperglycaemic clamp was not different between the genotypes. GLP-1 concentrations during the OGTT were not influenced by the TCF7L2 variants. However, GLP-1-infusion combined with a hyperglycaemic clamp showed a significant reduction in GLP-1-induced insulin secretion in carriers of the risk allele in two variants (rs7903146, rs12255372, p<0.02). Conclusions/interpretation Variants of TCF7L2 specifically impair GLP-1-induced insulin secretion. This seems to be rather the result of a functional defect in the GLP-1 signalling in beta cells than a reduction in GLP-1 secretion. This defect might explain the impaired insulin secretion in carriers of the risk alleles and confers the increased risk of type 2 diabetes.

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Section: Methodsmentioning

confidence: 99%

Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms

Schäfer¹,

Tschritter²,

Machicao³

et al. 2009

Diabetologia

119

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“…All SNPs were genotyped using the biplex Invader assay, as previously described [38]. SNPs were genotyped on a set of 80 samples representing 10 independent three-generation CEPH families from Utah.…”

Section: Genotyping Of Single Nucleotide Polymorphismsmentioning

confidence: 99%

“…In addition, we included SNPs in and around the other apolipoprotein genes that were reported previously in association studies: SNPs 34, 35, and 36 are nonsynonymous changes in APOA4; SNPs 38 (APOC3 −482), 39 (APOC3 −455), and 41 (SstI) are located in APOC3; and SNP 43 (XmnI polymorphism) is located in APOA1 (see Table 1). All SNPs were genotyped using the biplex Invader assay, as previously described [38]. SNPs were genotyped on a set of 80 samples representing 10 independent three-generation CEPH families from Utah.…”

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confidence: 99%

Haplotype analysis of the apolipoprotein gene cluster on human chromosome 11

et al. 2004

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Members of the apolipoprotein gene cluster (APOA1/C3/A4/A5) on human chromosome 11q23 play an important role in lipid metabolism. Polymorphisms in both APOA5 and APOC3 are strongly associated with plasma triglyceride concentrations. The close genomic locations of these two genes as well as their functional similarity have hindered efforts to define whether each gene independently influences human triglyceride concentrations. In this study, we examined the linkage disequilibrium and haplotype structure of 49 SNPs in a 150-kb region spanning the gene cluster. We identified a total of five common APOA5 haplotypes with a frequency of greater than 8% in samples of northern European origin. The APOA5 haplotype block did not extend past the 7 SNPs in the gene and was separated from the other apolipoprotein gene in the cluster by a region of significantly increased recombination. Furthermore, one previously identified triglyceride risk haplotype of APOA5 (APOA5*3) showed no association with three APOC3 SNPs previously associated with triglyceride concentrations, in contrast to the other risk haplotype (APOA5*2), which was associated with all three minor APOC3 SNP alleles. These results highlight the complex genetic relationship between APOA5 and APOC3 and support the notion that APOA5 represents an independent risk gene affecting plasma triglyceride concentrations in humans. KeywordsSingle nucleotide polymorphism; Apolipoprotein A5; Haplotype; Linkage disequilibrium; Recombination; Four-gamete testThe apolipoprotein gene cluster on human chromosome 11q23 contains four apolipoprotein genes (APOA1/C3/A4/A5) in a genomic interval of approximately 60 kb [1]. Three of these genes (APOA1/C3/A4) have been well described, and each plays an important role in lipid metabolism in humans and mice. For example, mice lacking apoA1 have significantly reduced plasma high-density lipoprotein (HDL) cholesterol levels [2]. In contrast, mice lacking apoC3 show lower concentration of plasma triglycerides compared to control littermates [3].In humans, analyses of genetic sequence variants around these three genes have revealed polymorphisms associated with plasma lipid levels (for a review, see [4] in APOA1 primarily affect HDL-cholesterol cocentrations, while variation in APOC3 is primarily associated with altered plasma triglyceride concentrations. In APOC3, two rare alleles in the promoter region (−482C →T and −455T →C) and a minor allele in the 3′UTR (SstI polymorphism, 3238G →C) have repeatedly been associated with elevated plasma triglyceride concentrations in several human populations [5][6][7][8][9][10][11][12][13][14][15]. However, the lack of strong functional data at least for the SstI polymorphism raises questions whether the association seen in humans is due to these sequence variants in APOC3 directly or due to other functional variants in APOC3 or possibly in one of the neighboring apolipoprotein genes.Recently, we identified a fourth member (APOA5) of the chromosome 11 apolipoprotein gene cluster, located approximatel...

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“…As a quality standard, we randomly included six positive (2×A/A, 2×A/G, 2×G/G) and two negative (all components excluding DNA) sequenced controls in each TaqMan reader plate. Because all controls were correctly identified, we assumed that the genotyping error rate of this method did not exceed 0.3%, as demonstrated previously in a study designed to validate this method [17].…”

Section: Methodsmentioning

confidence: 99%

A novel functional polymorphism (?336A/G) in the promoter of the partitioning-defective protein-6? gene is associated with increased glucose tolerance and lower concentrations of serum non-esterified fatty acids

et al. 2005

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Aims/hypothesis: Partitioning-defective protein-6α (Par6α) has recently been demonstrated to negatively regulate insulin signalling in murine myoblasts. To address whether Par6α plays a role in human physiology, the present study investigated whether mutations exist in the Par6α gene and whether these mutations, if present, are associated with pre-diabetic phenotypes in non-diabetic subjects. Methods: The complete gene (part of the promoter [2.1 kb], all exons/introns and the 3′ untranslated region) encoding Par6α was analysed in 664 non-diabetic subjects. We investigated possible associations between single nucleotide polymorphisms and percentage of body fat, glucose tolerance (as determined by OGTT), serum NEFA concentrations and whole-body insulin sensitivity (estimated during the OGTT, and for a subgroup of 242 subjects determined by the euglycaemic-hyperinsulinaemic clamp). Results: A rare A/G polymorphism was found 336-bp upstream of the translational start codon (allele frequency 0.03). The data for subjects homozygous and heterozygous for −336G (R/G, n=43) were combined and compared with those for subjects homozygous for −336A (A/A, n=621). Subjects with the R/G genotype had lower fasting (4.84± 0.09 mmol/l, means±SEM, p=0.049) and 2-h (5.50±0.02 mmol/l, p=0.050) plasma glucose concentrations than subjects with the A/A genotype (5.02±0.02 and 5.94±0.06 mmol/l, respectively). Subjects with the R/G genotype also had lower fasting (448±31 μmol/l, p=0.018) and 2-h serum NEFA concentrations (61±7 μmol/l, p= 0.015) than subjects with the A/A genotype (529±9 and 75± 2 μmol/l, respectively), adjusted for age, sex and percentage of body fat. There were no differences in adiposity or whole-body insulin sensitivity between the two genotype groups (all p> 0.36). A luciferase reporter gene assay revealed that the −336G promoter variant had a significantly lower (−22.8%, p=0.006) transcriptional activity in transfected C2C12 murine myoblasts than the −336A promoter variant. Conclusions/interpretation: A novel functional variant in the promoter of the Par6α gene is associated with reduced fasting glycaemia, increased glucose tolerance and reduced serum NEFA concentrations.

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High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology

Cited by 77 publications

References 14 publications

Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms

Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms

Haplotype analysis of the apolipoprotein gene cluster on human chromosome 11

A novel functional polymorphism (?336A/G) in the promoter of the partitioning-defective protein-6? gene is associated with increased glucose tolerance and lower concentrations of serum non-esterified fatty acids

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