Mutagenically Separated Polymerase Chain Reaction Research Articles

Prevalence of Cholesteryl Ester Storage Disease

Cholesteryl ester storage disease (CESD) is an autosomal recessive chronic liver disease caused by lysosomal acid lipase (LAL) deficiency. The gene is located on chromosome 10q23.2-q23.3, and the enzyme is essential for triglycerides and cholesteryl ester hydrolysis in lysosomes. CESD is characterized by hypercholesterolemia, hypertriglyceridemia, HDL deficiency, and abnormal lipid deposition in many organs. In the liver this results in hepatomegaly caused by hepatic steatosis and fibrosis that can lead to micronodular cirrhosis.1 Disease onset takes place during childhood or adolescence. Males and females are affected in about equal numbers. Patients rarely reach the age of 30. Biochemically, the disorder is recognized by largely reduced lysosomal acid lipase activity.2,3 Complete absence of LAL activity causes Wolman Disease, which is normally fatal within the first 6 months of life.1,4 Several groups have identified mutations in the LAL gene underlying CESD and Wolman disease.5–9 Mutations causing Wolman disease produce an enzyme with no residual activity or no enzyme at all, whereas CESD-causing mutations encode for LAL which retains some enzyme activity.4,10 A G-to-A transition at position −1 of the exon 8 splice donor (E8SJM, E xon 8 S plice J unction M utation) leads to an in-frame deletion of exon 8. The resulting protein is 24 amino acids shorter and has no residual LAL activity, however E8SJM does not cause Wolman Disease because 2% to 4% of normally spliced LAL is present in homozygote carriers.11,12 The vast majority of CESD …

The association of PC-1 (ENPP1) K121Q polymorphism with metabolic syndrome in patients with coronary heart disease

BackgroundMetabolic syndrome (MS) is a clinical feature, closely associated with insulin resistance, one of the prime underlying causes of overall cardiovascular morbidity, including coronary heart disease (CHD). Considering the association between PC-1 121Q genotype and insulin resistance phenotype, the aim of the present study was to investigate the contribution of PC-1 K121Q polymorphism to the development of MS and its concomitant disorders in CHD patients. MethodsA total of 130 Caucasians from Serbia, including 80 CHD patients (aged 59.4±8.6 years, of a mean BMI 28.9±3.9 kg/m2) and 50 control subjects (aged 48.0±6.4 years, of a mean BMI 29.6±2.1 kg/m2), were genotyped for PC-1 K121Q using a mutagenic separated PCR assay, in order to determine the prevalence of the PC 121Q variant in individuals suffering from CHD and its association with MS. ResultsThe frequency of PC-1 121Q allele found in CHD patients was 28.5%, with significantly (P<0.01) higher prevalence in those with MS (40% vs. 10%). Both MS (P<0.01) and its components [central obesity (P<0.01), low HDL-cholesterol (P<0.01) and high triglycerides (P<0.05)] were significantly more prevalent in CHD 121Q carriers compared to CHD patients who exhibited the wild-type genotype. A binary logistic regression model has revealed that PC-1 121Q allele carriers had a 5.5 fold increased odds (95%CI: 1.4–20.9, P=0.01) for the MS compared to wild-type carriers. The PC-1 121Q allele contributed to MS components as well, although these associations did not reach statistical significance. ConclusionThe findings of the present study support the hypothesis that the PC-1 (ENPP1) 121Q allele is associated with the genetic susceptibility for MS in patients with CHD. Further studies and more extensive research in this area are needed, not only to confirm this association, but to elucidate it in more details.

The C-Reactive Protein +1444C/T Alteration Modulates the Inflammation and Coagulation Response in Human Endotoxemia

C-reactive protein (CRP) plays a major role in the immune system and is an independent risk marker of cardiovascular disease. However, CRP's role in atherogenesis as innocent bystander, causative, or even protective agent, remains unresolved. The (+)1444C/T alteration in the CRP gene has been reported to determine basal CRP concentrations. We hypothesized that this alteration may also be associated with the degree of inflammatory response and coagulation activation in a well-standardized model of systemic inflammation. We administered 2 ng/kg endotoxin [Escherichia coli bacterial lipopolysaccharide (LPS)] intravenously to stimulate inflammation in 91 healthy young Caucasian male paid volunteers (age range, 19-40 years). Participants were confined to bed rest and fasted for 8.5 h after LPS infusion. We collected blood samples before LPS infusion and at 0, 2, 6, and 24 h after LPS infusion to measure inflammation markers [interleukin 6 (IL6), tumor necrosis factor-alpha (TNFalpha)], temperature, and coagulation markers (prothrombin fragment F(1+2), D-dimer). We analyzed the CRP 3' untranslated variant with a mutagenic separated PCR assay. Basal concentrations of high-sensitivity CRP were approximately 40% lower in (+)1444CC alteration carriers than in T homozygous (TT) allele carriers (P = 0.04). In contrast, basal IL6 concentrations were 2-fold higher in wild-type C homozygous (CC) than in TT individuals (P = 0.01). In response to the LPS challenge, CC individuals had 4-fold higher peak TNFalpha concentrations (P <0.01), >2.5-fold higher peak IL6 concentrations (P <0.01), and increased temperature (P <0.01). Twenty-four hours after LPS challenge, prothrombin fragment F(1+2) concentrations were 75% higher and D-dimer concentrations 50% higher in CC than in TT individuals (P <0.05). Genetic factors regulating CRP concentrations also modulate the individual response to endotoxin-stimulated inflammation.

Polymorphisms in the Interleukin-1 Gene Cluster in Children and Young Adults with Systemic Meningococcemia

An association has been described between mortality in children with meningococcal disease and functional polymorphisms in the interleukin-1 (IL1) cluster. We undertook a multicenter study to evaluate associations of these polymorphisms in a Central European population. The study involved 95 Middle European pediatric hospitals. We collected blood samples from, and clinical information about, 285 previously healthy children with meningococcal infection. We used a newly developed multiplexed mutagenic separated PCR assay to analyze 6 polymorphisms within the IL1 cluster: IL1A (-889)C/T, IL1A (+4845)G/T, IL1B (-511)C/T, IL1B (-31)C/T, IL1B (+3954), and IL1RA (+2018)C/T. We studied the same polymorphisms in a comparison group of 481 healthy newborns. Genotype frequencies between patients and the comparison group differed significantly only for the IL1RA (+2018)C/T variant: The CC genotype was more frequent in patients (11%) than in healthy controls (5%; P = 0.008). In the patient group, the C allele was significantly more prevalent (67%) in nonsurvivors than in survivors (42%; P = 0.02). The IL1RA (+2018)C/T polymorphism is associated with the risk of meningococcal disease and with its outcome.

Association of the T+294C polymorphism in PPAR δ with low HDL cholesterol and coronary heart disease risk in women

The +T294C polymorphism in PPARdelta represents a functional SNP affecting transcriptional activity of the PPARdelta gene. To address whether this polymorphism is associated with the risk for coronary heart disease and/or plasma lipid levels in women, we studied a group of 967 female patients with hyperlipidaemia in the presence (n=453) or absence (n=514) of coronary heart disease. 967 female patients with or without coronary heart disease were genotyped using mutagenically separated polymerase chain reaction (MS-PCR). Statistical analysis was performed according to genotype with parameters of lipid metabolism as dependant variables. A highly significant association between the rare C allele and lower plasma HDL concentrations was found in female subjects. The effect remained significant after correcting for multiparametric testing according to Bonferoni and was seen only in subjects with a BMI below the median. Moreover, a significant association of the C-allele with coronary heart disease and BMI was obtained. Regarding the entire group, trends towards higher VLDL and LDL levels were observed. Our data show for the first time that the PPARdelta +T294C polymorphism is associated with lipid levels and coronary heart disease in women. However, the molecular mechanism of action remains to be elucidated.

Simultaneous analysis of MDR1 C3435T, G2677T/A, and C1236T genotypes by multiplexed mutagenically separated PCR

P-glycoprotein (PGP) encoded by the multi-drug-resistance 1 (MDR1) gene is a member of the ATP-binding cassette (ABC) transporter family, drug-transporting proteins involved in the bioavailability and pharmacokinetics of various drugs. Several single nucleotide polymorphisms (SNPs) in the MDR1 gene have been identified so far that may influence PGP expression levels and function. Thus, genotyping for MDR1 polymorphisms and determining specific haplotypes may become an important tool in predicting individual susceptibility to developing drug resistance. We developed a new multiplexed allele-specific PCR method based on the principle of mutagenically separated PCR (MS-PCR) for rapid and reliable simultaneous genotyping of the C3435T polymorphism in exon 26 of the MDR1 gene and two additional SNPs (G2677T/A in exon 21 and C1236T in exon 12), which are in linkage disequilibrium with MDR1 C3435T. The accuracy and reliability of this method was confirmed by sequencing the respective regions in the MDR1 gene. This newly developed MDR1 MS-PCR will facilitate fast, accurate and economic analysis of MDR1 genotypes and will provide important information in optimizing individual therapeutic approaches.

Study on the application of parent-of-origin specific DNA methylation markers to forensic genetics

In paternity test, especially in motherless cases, the allele inherited from father (obligatory gene, OG) often cannot be determined. The paternity exclusion probability (PE) of a genetic marker is reduced considerably. Therefore, it is necessary to develop a new technique, by which the parental origin of alleles can be determined without genealogical analysis. In this paper, we explored the possibility of using parent-of-origin specific DNA methylation markers to determine the parental origin of alleles, choosing the imprinted single nucleotide polymorphism (SNP) locus rs220028 (A/G) as a model system. We typed the SNP by mutagenically separated PCR (MS-PCR). The frequencies of alleles were A = 0.5085, G = 0.4915; the unbiased heterozygosity was 0.5020. In order to discriminate between the maternal allele and paternal allele, post-digestion MS-PCR, a novel PCR based methylation analysis and SNP typing technique was developed and performed on 18 heterozygous children, and the methylated maternal allele was detected specifically. As a pilot study on the use of epigenetic markers in forensic genetics, our results demonstrated the feasibility of using parent-of-origin specific DNA methylation markers to determine the parental origin of alleles.

Study of Antiretroviral Drug???Resistant HIV-1 Genotypes in Northern Thailand: Role of Mutagenically Separated Polymerase Chain Reaction as a Tool for Monitoring Zidovudine-Resistant HIV-1 in Resource-Limited Settings

As the number of HIV-1-infected individuals receiving antiretroviral drugs has been rapidly increasing in developing countries, there is an urgent need for drug resistance genotype information of non-B subtype HIV-1 and for the establishment of a practical system of monitoring drug-resistant viruses. This study first sequenced the reverse transcriptase region of HIV-1 in 112 infected individuals who had been treated with zidovudine (AZT)/didanosine or AZT/zalcitabine as dual therapy at a government hospital in northern Thailand and then compared the above sequence method with mutagenically separated polymerase chain reaction (MS-PCR) for detecting M41L and K70R mutations. Concordant rates of detecting M41L and K70R mutations by the 2 methods were 96.9% (93/96) and 92.7% (89/96), respectively. The M41L and K70R MS-PCR could detect 86.4% of AZT-resistant strains with any resistance mutation, which was determined by the sequencing method. Then 292 drug-naive individuals were screened for the presence of drug-resistant HIV-1 by the MS-PCR assay and it was found that 2 individuals (0.7%) carried viruses with either the M41L or K70R mutation. It is feasible to test a large number of samples with MS-PCR, which is sensitive, cheap, and easy to perform and does not require sophisticated equipment. The M41L and K70R MS-PCR is potentially a useful tool to monitor the spread of AZT-resistant HIV-1 in resource-limited countries.

Simple and rapid detection of uncoupling protein-2 $minus;866G/A polymorphism by mutagenically separated polymerase chain reaction

Background: Uncoupling protein-2 (UCP2), a recently identified member of the mitochondrial transporter superfamily, is a candidate gene for obesity. A common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo. Methods: We developed a rapid and simple method, mutagenically separated polymerase chain reaction (MS-PCR) for genotyping UCP2 −866G/A polymorphism. Two reverse mutagenic allele-specific primers of different lengths for the UCP2 −866G/A polymorphic site were paired with the same forward primer in the same PCR reaction. Results: Agarose gel electrophoresis (3.5%) showed at least one of the two allelic products and provided a within-assay quality control to exclude false-negative results. The 203-bp fragment of the PCR products was A allele-specific and the 183-bp fragment was G allele-specific. The frequencies of the UCP2 −866G/A genotypes in 72 Japanese subjects were AA: 21 (29.2%), AG: 32 (44.4%), and GG: 19 (26.4%). The results were confirmed by the PCR-RFLP genotyping method, in which a 360-bp fragment of PCR products was cut into 290- and 70-bp fragments by the restriction enzyme MluI when the G allele was present. This Japanese group showed a higher frequency of the AA genotype, which is associated with a low prevalence of obesity, than Caucasian populations. Conclusions: The MS-PCR technique is a simple, rapid, and reliable method for genotyping UCP2 −866G/A polymorphism.

Simple and rapid detection of uncoupling protein-2 −866G/A polymorphism by mutagenically separated polymerase chain reaction

Background: Uncoupling protein-2 (UCP2), a recently identified member of the mitochondrial transporter superfamily, is a candidate gene for obesity. A common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo. Methods: We developed a rapid and simple method, mutagenically separated polymerase chain reaction (MS-PCR) for genotyping UCP2 −866G/A polymorphism. Two reverse mutagenic allele-specific primers of different lengths for the UCP2 −866G/A polymorphic site were paired with the same forward primer in the same PCR reaction. Results: Agarose gel electrophoresis (3.5%) showed at least one of the two allelic products and provided a within-assay quality control to exclude false-negative results. The 203-bp fragment of the PCR products was A allele-specific and the 183-bp fragment was G allele-specific. The frequencies of the UCP2 −866G/A genotypes in 72 Japanese subjects were AA: 21 (29.2%), AG: 32 (44.4%), and GG: 19 (26.4%). The results were confirmed by the PCR-RFLP genotyping method, in which a 360-bp fragment of PCR products was cut into 290- and 70-bp fragments by the restriction enzyme MluI when the G allele was present. This Japanese group showed a higher frequency of the AA genotype, which is associated with a low prevalence of obesity, than Caucasian populations. Conclusions: The MS-PCR technique is a simple, rapid, and reliable method for genotyping UCP2 −866G/A polymorphism.

The interleukin-6 G(-174)C promoter polymorphism does not determine plasma interleukin-6 concentrations in experimental endotoxemia in humans.

Interleukin 6 (IL-6) is a pleiotropic cytokine that plays an essential role in the pathogenesis of acute and chronic infections. As the role of the IL-6 G(-174)C polymorphism in determining serum concentrations of IL-6 is controversial, we studied the genotype-specific IL-6 response in a well-standardized model of systemic inflammation. A total of 76 healthy young males (age range, 19-35 years) received a single bolus of 2 ng/kg endotoxin [lipopolysaccharide (LPS)] intravenously. Plasma IL-6 was measured by enzyme immunoassay at 0, 2, 6, and 24 h after LPS infusion, and the IL-6 promoter genotype was analyzed by a mutagenic separated PCR assay. IL-6 increased 300-fold 2 h after LPS challenge and returned almost to normal within 24 h. Neither basal IL-6 nor the IL-6 response to LPS was significantly affected by the IL-6 promoter genotype. The IL-6 G(-174)C promoter polymorphism does not significantly influence basal concentrations of IL-6 or peak IL-6 in human endotoxemia.

2P-0415 Rapid detection of uncoupling protein 2-866G/A mutation by mutagenically separated polymerase chain reaction (MS-PCR)

2P-0415 Rapid detection of uncoupling protein 2-866G/A mutation by mutagenically separated polymerase chain reaction (MS-PCR)

The E-selectin S128R polymorphism is not a risk factor for coronary artery disease in patients with diabetes mellitus type 2

Aim/hypothesis: E-selectin is thought to play a key role in the early stages of vascular disease by facilitating the attachment of leukocytes to the endothelium. Recently, a polymorphism in the E-selectin gene (S128R) has been associated with higher E-selectin levels in patients with diabetes mellitus and with premature coronary artery disease. The impact of the S128R polymorphism on the development of diabetic coronary artery disease has not been investigated yet. Patients and methods: A total of 254 patients recruited from the Division of Cardiology, University of Vienna with diabetes mellitus type 2 was assessed for the E-selectin S128R polymorphism by a novel mutagenic separated PCR, allowing fast and reliable genotyping without restriction enzyme digest. Ninety-five patients had a history of myocardial infarction, 90 were admitted with stable coronary artery disease whereas in 69 the presence of CAD could be excluded. Results: Of all 254 individuals tested, 197 (77.6%) exhibited wildtype E-selectin 128S genotype, 54 (21.3%) were heterozygous S128R and 3 (1.1%) were homozygous for the 128R allele. In all groups the genotype frequencies did not differ significantly. No associations were found between E-selectin genotype and coronary artery disease or myocardial infarction. Conclusion/interpretation: In subjects suffering from diabetes mellitus type 2 the E-selectin S128R polymorphism is not associated with coronary artery disease nor with an increased risk for myocardial infarction. Thus, screening for this polymorphism is not indicated for risk assessment of CAD in patients with diabetes mellitus.

Mutagenically separated PCR assay for rapid detection of M41L and K70R zidovudine resistance mutations in CRF01_AE (subtype E) human immunodeficiency virus type 1.

A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01_AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries.

The K121Q polymorphism in the plasma cell membrane glycoprotein 1 gene predisposes to early myocardial infarction.

Elevated plasma cell membrane glycoprotein 1 (PC-1) expression and the frequent PC-1 K121Q polymorphism have recently been associated with insulin resistance. Since insulin resistance represents an important risk factor for atherosclerotic vascular disease and myocardial infarction, we investigated the involvement of the PC-1 K121Q polymorphism in the development of myocardial infarction. We analyzed two independent series of cardiovascular patients at a defined end-point of atherosclerotic vascular disease (those having suffered myocardial infarction) for the PC-1 K121Q mutation by a newly developed mutagenic separated PCR assay. In both patient groups the presence of the Q allele was significantly associated with younger age at the time of first myocardial infarction, suggesting a more rapid progression of endothelial dysfunction. In a multivariate analysis carriers of the 121Q allele from Vienna and from Central Germany exhibited a 2.6- and a 4.2-increased odds, respectively, for suffering myocardial infarction within the first tertile of age (<51 and <48 years, respectively). Our data indicate that the PC-1 121Q allele might predispose independently of other well established risk factors for early myocardial infarction. Testing for the PC-1 K121Q polymorphism might be valuable in patients with a family history of atherosclerotic vascular disease and myocardial infarction.

Single strand conformation polymorphism (SSCP) as a quick and reliable method to genotype M235T polymorphism of angiotensinogen gene

Objectives: Conflicting results on the relationship between M235T polymorphism of angiotensinogen (AGT) gene and diabetic nephropathy are reported in the literature, probably due to the small number of subjects, to different inclusion criteria and the different genotype analysis methods used. The aim of the present study was to set up a fast, cheap and reliable method to allow the genotyping of M235T polymorphism in a large number of subjects. Design and methods: We developed in our laboratory a new specifically designed PCR-SSCP method for M235T genotyping whose specificity was compared with that of Allele Specific PCR (ASPCR) and Mutagenically Separated PCR (MS-PCR). The exact M235T genotype was estabilished by direct sequencing. The new PCR-SSCP method was then used to genotype a population of 1171 hypertensive, normoalbuminuric type II diabetes mellitus patients. The patients were also genotyped for ACE I/D polymorphism. For comparison a group of hypertensive non diabetic patients ( n = 88) were also screened. Results: The PCR-SSCP method identified the M235T polymorphism with no misinterpretation at variance with ASPCR and MS-PCR methods that showed a preferential amplification of the T allele. The rare Y248C polymorphism of the AGT gene was also detected by PCR-SSCP. In diabetic hypertensive patients the prevalence of TT genotype was higher than in normotensive healthy controls and equivalent to that found in hypertensive non diabetic patients. Conclusions: The PCR-SSCP method for detection of M235T polymorphism is a powerful and sensitive tool for rapid, cheap and efficient screening of a large number of samples. The results obtained with this method demonstrate an association of the TT genotype of AGT gene with hypertension, both in diabetic and non diabetic patients.

The mutagenically separated polymerase chain reaction is a rapid and reliable method for genotyping of the tumour necrosis factor-α promoter polymorphism (−308 G/A)

Background: The tumour necrosis factor-α (TNFα) promoter polymorphism (−308 G/A) has been shown to be associated with the susceptibility to and/or the severity of diverse diseases such as infections, autoimmunity, and malignancies. We developed a genotyping technique based on the mutagenically separated polymerase chain reaction (MS-PCR) which may be useful in the clinical risk assessment. Methods: Different length allele-specific primers and an unspecific complementary strand primer were used in a one-tube assay. At least one PCR product was generated in a single reaction obviating the need for an internal control amplification. Introduction of additional base substitutions into the allele-specific primers led to a clear-cut separation between the alleles through the reduction of cross-reactions during amplification. The only post-PCR step required was the separation of allelic PCR products by size upon agarose gel electrophoresis. Results: The allele frequencies in 300 German healthy Caucasians were 0.84 for TNF1 (−308 G) and 0.16 for TNF2 (−308 A) in accordance with published data obtained with the conventional RFLP method. No significant deviation from Hardy–Weinberg equilibrium was observed. The specificity of MS-PCR was confirmed by sequence-based typing. Conclusions: MS-PCR is a rapid, reliable, and cost-effective technique for genotyping of the TNFα promoter polymorphism (−308 G/A).

Simple detection of point mutations associated with HIV-1 drug resistance

A novel assay is described for the detection of HIV-1 drug resistance that is simple, cheap and sensitive. HIV-1 drug resistance in B and non-B HIV-1 subtypes was investigated using Mutagenically-Separated PCR (MS–PCR) — a competitive semi-nested PCR which uses mutagenic primers. The assay was assessed for sensitivity, specificity and its ability to detect mutant virus within a mixed mutant–wild-type population. Gene sequencing was carried out simultaneously for comparison. MS–PCR detected five copies of HIV-1 RNA from laboratory isolates and 50 copies from patient samples. We demonstrate 100% specificity of detection for wild type or mutant virus for clades A, B, C, D and E. For mixed populations of virus, MS–PCR can detect at least a 10% mix of wild type:mutant, or vice-versa. When applied to African patient samples MS–PCR detected 91.6% of the codons tested. Concordance with sequencing data was 88.8% for protease and 97.2% for RT. MS-PCR is sensitive and specific for the detection of mutations in HIV-1, and can be adapted easily to test for resistance at any codon of interest.

Effects of the angiotensinogen gene M235T and A(-6)G variants on blood pressure and other vascular risk factors in a Spanish population.

Angiotensinogen (AGT) gene polymorphism has shown significant differences in the allelic frequencies between hypertensive and normotensive subjects. This allele frequency varies among ethnic groups. There are still some controversies related to the 235T-variant as a marker for essential hypertension. As part of an extensive case-control study carried out in a Spanish population, we selected the 237 subjects with a diagnosis of essential hypertension according to the established criteria. A group of 242 normotensives matched for age and gender was used as control. Smoking habits, a previous diabetes and hypertension medical history, body mass index (BMI) and blood pressure (BP) values were recorded. Glucose, plasma creatinine, lipid profile with Lp(a), homocysteine and microalbuminuria were measured. Angiotensinogen M235T-gene polymorphism was determined by polymerase chain reaction (PCR) from genomic DNA. A(-6)G polymorphism was determined by mutagenically separated PCR (MS-PCR). BP values, BMI and microalbuminuria were significantly higher in hypertensive subjects; 31.6% of hypertensives and 40.1% normotensives were active smokers. M235T-genotype frequencies were not different in the hypertensive and normotensive population. Similarly, homocigotic AA predominate in the hypertensives but without statistical significance. The association of 235T-genotype or the changes in the promoter activity due to A(-6) substitution with essential hypertension was not confirmed in the multivariate regression analyses. Only a previous family history of hypertension and BMI were significantly associated with hypertension. Journal of Human Hypertension (2000) 14, 789-793

A high-throughput MS-PCR method on MADGE gels for ANG II type-1 receptor A1166C polymorphism

We have developed a highly accurate, low-cost, single-step, mutagenically separated polymerase chain reaction (MS-PCR) method for the determination of angiotensin II type-1 receptor (AT(1)) A1166C gene polymorphism. The genotypes are determined using the microtiter array diagonal gel electrophoresis (MADGE) system. We have compared the MS-PCR method with allele-specific oligonucleotide hybridization and Dde I digestion techniques for determining the AT(1) A1166C genotype. The combination of MS-PCR and MADGE serves as a model for high-throughput single-nucleotide polymorphism genotyping in large population studies.