Science, medicine, and the future: Genetics and cardiovascular risk.
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Cardiovascular Diseases↗
Biomedical subjects
I N Day
Publications and source records attributed to I N Day.
An efficient procedure for genotyping single nucleotide polymorphisms.
Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.
DNA↗
Apolipoprotein E4 and coronary heart disease in middle-aged men who smoke: a prospective study.
BACKGROUND: The common isoforms of apolipoprotein E (apoE), E2, E3, and E4, are important determinants of plasma lipid concentrations, and the epsilon4 allele is associated with raised risk of coronary heart disease. We investigated whether the effect of smoking on coronary heart disease risk is affected by APOE genotype. METHODS: We enrolled 3052 middle-aged men who were free of coronary heart disease for prospective cardiovascular surveillance in the second Northwick Park Heart Study (NPHSII). Smoking habit was ascertained at baseline and yearly by questionnaire. APOE genotype was identified by PCR and restriction enzyme digestion. Endpoints were fatal coronary heart disease, non-fatal myocardial infarction, and coronary artery surgery and silent myocardial infarction at follow-up. FINDINGS: During 18836 person years of surveillance, 96 men had an acute myocardial infarction, 26 needed coronary artery surgery, and 14 had silent myocardial infarctions. Compared with never-smokers, risk of coronary heart disease in ex-smokers was 1.34 (95% CI 0.86-2.08) and in smokers it was 1.94 (1.25-3.01). This risk was independent of other classic risk factors. In never-smokers, risk was closely similar in men with different genotypes. Risk in men homozygous for the epsilon3 allele was 1.74 (1.10-2.77) in ex-smokers and 1.68 (1.01-2.83) in smokers, whereas in men carrying the epsilon4 allele risk was 0.84 (0.40-1.75) and 3.17 (1.82-5.50), respectively, with no significant differences in risk in the epsilon2 carriers. For the epsilon3 group, the genotype effect on risk was no longer significant after adjustment for classic risk factors (including plasma lipids). However, even after adjustment, smokers who were carriers of the epsilon4 allele, showed significantly raised risk of coronary heart disease compared with the non-smoking group (2.79, 1.59-4.91, epsilon4-smoking interaction p=0.007). INTERPRETATION: Smoking increases the risk of coronary heart disease in men of all genotypes but particularly in men carrying the epsilon4 allele.
Apolipoprotein E4↗
Positive associations between single nucleotide polymorphisms in the IGF2 gene region and body mass index in adult males.
We previously demonstrated an association between the insulin-like growth factor 2 (IGF2) gene 3'-untranslated region (3'-UTR) ApaI polymorphism and body mass index (BMI) in over 2500 middle-aged Caucasoid males. In the same cohort, we have now tested association with 11 more markers, including seven novel single nucleotide polymorphisms (SNPs), spanning >30 kb across the IGF2 gene. Three SNPs showed significant positive associations with BMI: 6815 A/T in the IGF2 P1 promoter (P = 0.00012, n = 2394) and the newly identified SNPs 1156 C/T in intron 2 (P = 0.017, n = 1567) and 1926 C/G in the 3'-UTR (P = 0.0062, n = 1872). There was strong pairwise linkage disequilibrium (LD) between the ApaI and 1926 C/G sites, whereas LD between ApaI and 6815 A/T, and between ApaI and 1156 T/C, was minimal. Univariately 6815 A/T, 1156 T/C and ApaI explained 1.03, 1.02 and 0.67% of the variation in BMI. Multi-way analysis of variance (ANOVA) models showed that 6815 A/T and 1156 T/C explained a further 0.4 and 0.8% of the variation beyond that accounted for by ApaI and the association of 1926 C/G with BMI disappeared after adjustment. The 6815 A/T, 1156 T/C and ApaI markers in effect constitute independent affirmations of our original hypothesized candidate gene region. In a stepwise multi-way ANOVA model, all three terms were significantly independently associated with BMI. The total proportion of BMI variance explained by this model was 2.25%, strongly suggesting that IGF2 genetic variation is a significant determinant of body weight in middle-aged males.
3' Untranslated Regions↗
Microarray analysis of nicotine-induced changes in gene expression in endothelial cells.
Cigarette smoking causes vascular endothelial dysfunction and is a major risk factor for cardiovascular diseases. Nicotine, a major constituent of cigarette smoke, has been shown to alter gene expression in endothelial cells; however, the regulatory pathways involved remain to be defined. We hypothesized that there might be distinct pathways that could be identified by systematic transcriptome analysis. Using the cDNA microarray approach, we ascertained the expression of over 4,000 genes in human coronary artery endothelial cells and identified a number of nicotine-modulated genes encoding a protein involving in signal transduction or transcriptional regulation. Among these were phosphatidylinositol phosphate kinase and diacylglycerol kinase, which are regulators of the inositol phospholipid pathway. Changes were also detected for transcription factors cAMP response element binding protein and nuclear factor-kappaB, of which the activities of both have been previously shown to be altered in nicotine-stimulated cells. The data from this study are relevant to understanding the mechanisms underlying the pathophysiological effect of nicotine and smoking, particularly on endothelial function and pathogenesis of atherosclerosis.
Cells, Cultured↗
Birthweight, vitamin D receptor genotype and the programming of osteoporosis.
Studies of the association between polymorphisms of the gene for the vitamin D receptor (VDR) and adult bone mass have been inconsistent, pointing to the possibility that gene--environment interactions may vary in different populations. We have demonstrated previously an association between weight in infancy (a marker of the intrauterine and early post-natal environment) and each of adult bone mass and VDR genotype. We therefore sought to extend these observations in an elderly UK cohort and to investigate the possibility of an interaction between these genetic and early environmental markers of later osteoporosis risk. One hundred and sixty-five men and 126 women aged 61--73 years for whom birth records were available underwent bone mass measurements at baseline and follow-up 4 years later. Whole-blood samples were obtained, DNA extracted using standard techniques and polymorphisms in the VDR and collagen type I alpha 1 (Col IA1) genes identified. In the cohort as a whole, there were no significant associations between either birthweight or VDR genotype and bone mineral density (BMD) or bone loss rate at either site. However, the relationship between lumbar spine BMD and VDR genotype varied according to birthweight. Among individuals in the lowest third of birthweight, spine BMD was higher (P = 0.01) in individuals of genotype 'BB' after adjustment for age, sex and weight at baseline. In contrast, spine BMD was reduced (P = 0.04) in individuals of the same genotype who were in the highest third of the birthweight distribution. A significant (P = 0.02) statistical interaction was also found between VDR genotype and birthweight as determinants of BMD. Similar but slightly weaker associations were seen between lumbar spine bone mineral content (BMC) and VDR genotype in the lowest birthweight tertile. When examining the relationship between Col1A1 genotype and bone mass, lumbar spine BMC was higher in individuals of genotype 'Ss' or 'ss' in the lowest birthweight tertile (P = 0.02) after adjustment for age, sex and weight at baseline. These results suggest that genetic influences on adult bone size and mineral density may be modified by undernutrition in utero.
Aged↗
Epidemiology and the genetic basis of disease.
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Genetic Predisposition to Disease↗
Polymorphisms in matrix metalloproteinase-1, -3, -9, and -12 genes in relation to subarachnoid hemorrhage.
BACKGROUND AND PURPOSE: Intracranial aneurysm, which underlies the vast majority of subarachnoid hemorrhage incidences, has a multifactorial etiology, and the importance of genetic factors is increasingly recognized. Development and rupture of intracranial aneurysms involve degradation and remodeling of the vascular wall matrix in which the matrix metalloproteinases (MMPs) play an important role. The possible impact of MMP gene polymorphisms on susceptibility to intracranial aneurysms is still controversial, with conflicting data from different reported studies. METHODS: In this study we analyzed 5 different functional promoter polymorphisms in the MMP-1, MMP-3, MMP-9, and MMP-12 genes in a sample of 92 patients with aneurysmal subarachnoid hemorrhage and 158 healthy control subjects, all from southern England. RESULTS: No significant difference was detected between the patient and control groups in genotype distribution of any of the polymorphisms studied. CONCLUSIONS: The data do not support the hypothesis that MMP gene variations influence the development of intracranial aneurysms in the population studied.
Adult↗
Higher resolution microplate array diagonal gel electrophoresis: application to a multiallelic minisatellite.
The 5' polymorphic region of the insulin (INS, MIM# 176730) gene contains a variable tandem repetition of 14-15 bp (a variable number of tandem repeats (VNTR) locus). After PCR amplification, we achieved precise sizing of class I alleles (range 641 to 843 bp) on 96-well open-face polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels, obtaining resolution of the 2% mobility difference which represents one tandem repeat. PCR products were run double-stranded, but no additional bands were generated except in the case of differences of three, two, and one repeat between alleles; none compromised allele identification, and in the latter case the heteroduplex was a useful confirmation signal. No end labelling of primers was required, as the sensitive Vistra Green intercalating dye for double strands was used for visualization of bands from diluted samples. Duracryl, a high mechanical-strength polyacrylamide derivative, proved to have good resolution properties for electrophoresis. A co-run ladder ensured precise binning without inter-lane variability. Simultaneous electrophoresis of gels in a thermostatically controlled tank allowed up to 1,000 samples to be run in 90 min. Gels were analyzed using a FluorImager 595 fluorescent scanning system, and alleles identified using a combination of Phoretix software for band migration measurement and Microsoft Excel to compute allele sizes. Unlike other systems for minisatellite allele sizing, throughput was not limited (in time or cost) by electrophoresis.
Alleles↗
The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit.
Long PCR followed by nested PCR has previously been used to determine CYP2A6 160H alleles, but the method proved unreliable. We have optimized this approach in a DNA bank of 1032 subjects (age range 59-74 years) to give reliable results, yielding indirect molecular evidence and very strong statistical evidence of hitherto unrecognized common alleles (designated O) recalcitrant to the long PCR. Coding three alleles (160L, 160H and O) and an approach to association analysis originally developed to deal with null alleles implicit in ABO blood group phenotyping, the contribution of 160H (functionally null) to reduced smoking habit has been clearly measured for the first time, unconfounded by alleles null to the long PCR. The most significant findings (p < 0.01) are that the possession of a 160H allele, compared with not possessing a 160H allele, is associated with a mean age of starting regular smoking 3 years later (95% CI +/- 1.93 years, average start age 20-21 years rather than 17-18 years); and that the average likelihood of quitting smoking at any time is 1.75 fold (95% CI, 1.17-2.61) for those possessing an 160H allele compared with those who have no 160H allele. This suggests that a smoking subject with a genotype predicted to confer 50% of the ability to eliminate nicotine via the CYP2A6 pathway has almost twice the likelihood of quitting smoking.
Aged↗
Tools for molecular genetic epidemiology: a comparison of MADGE methodology with other systems.
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Electrophoresis↗
Universal primer quantitative fluorescent multiplex (UPQFM) PCR: a method to detect major and minor rearrangements of the low density lipoprotein receptor gene.
A method based on quantitative fluorescent multiplex PCR has been developed to detect major rearrangements of the low density lipoprotein receptor gene (LDLR) which account for approximately 5% of mutations. The method involves two PCR reactions; the first (P1) amplifies the selected exons using unique primer sequences tagged with newly designed universal primers, while the second (P2) amplifies the P1 amplicons using the universal primers. One of the P2 universal primers is labelled with a fluorescent dye which is incorporated into the PCR products which are then electrophoresed on an ABI DNA sequencer. The relative amounts of the amplified peak areas are determined and compared to ratios obtained for DNA from four normal controls and known major rearrangements. The multiplex set developed is based on LDLR exons 3, 5, 8, 14, and 17 and 86% of reported major rearrangements would be detectable by this assay as well as any deletions and insertions of greater than 1 bp. The method was evaluated using DNA from 15 reported deletions and duplications which were all correctly identified. Two groups of UK patients with a clinical diagnosis of familial hypercholesterolaemia (FH) and where no mutation had been identified in LDLR or APOB (14 children and 42 adults) were screened for the presence of major LDLR rearrangements by this assay. Three major rearrangements were detected and a 4 bp duplication was identified in a fourth patient. Since it avoids the problems associated with Southern blotting, this method will be useful for detecting gene rearrangements.
Adolescent↗
Elimination of dumbbell bands and enhancement of resolution in MADGE using delayed start electrophoresis.
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DNA Mutational Analysis↗
SNP genotyping by combination of 192-well MADGE, ARMS and computerized gel image analysis.
A new modification of the microplate array diagonal gel electrophoresis (MADGE) system accommodates the dual amplification refractory mutation system (ARMS) products of 96 samples on one 192-well gel. Simultaneous electrophoresis of a number of horizontal ARMS-MADGE gels achieves high throughput. Gels are imaged digitally, here using the FluorImager 595 fluorescent scanning system. Customized software by Phoretix enables rapid computerized calling of band patterns in ARMS-MADGE arrays, in which the two wells receiving a pair of allele-specific assays for a single template are juxtaposed to form one virtual track, with genotype data exported directly into Microsoft Excel for statistical analysis. An ARMS assay of the A/T base change at the -23/HphI RFLP in the insulin gene promoter, which initiates from 2.5 ng template DNA, was used here to demonstrate this improved general approach for population SNP analyses.
Electrophoresis↗
Comparison of three methods for single nucleotide polymorphism typing for DNA bank studies: sequence-specific oligonucleotide probe hybridisation, TaqMan liquid phase hybridisation, and microplate array diagonal gel electrophoresis (MADGE).
In the near future the number of SNPs identified and mapped will increase and the need for high throughput SNP typing will be paramount for comprehensive examination by association of the role of genomic regions in disease traits. A range of higher throughput methods for typing SNPs is now in routine use in many laboratories worldwide. In this report, we analyse the relative advantages and disadvantages of three such methods, TaqMan, PCR-SSOP, and ARMS-MADGE, currently in use in our laboratories. Throughputs achievable are similar, but there are major differences in cost and time for set-up, equipment, consumables, and staff time, which may determine the choice for individual laboratories.
Costs and Cost Analysis↗
Microplate array diagonal gel electrophoresis for mutation research in DNA banks.
Molecular genetic epidemiology, association and linkage studies in populations, human or other species, is now yielding powerful new insights into disease and susceptibility genes. Inter alia, the subject requires laboratory analytical methodologies focused on achieving high throughput. Here we review one suite of methodology suitable for such laboratories. Microplate array diagonal gel electrophoresis (MADGE) was invented for molecular genetic epidemiological studies. It combines direct compatibility with microplates, convenient polyacrylamide gel electrophoresis and economy of time and reagents, at minimal capital cost, and enables one user to run up to several thousand gel lanes per day for direct assay of single-base variations. Melt-MADGE combines temporal thermal ramp apparatus to achieve similar throughput for de novo mutation scanning.
Animals↗
Microplate array diagonal gel electrophoresis (MADGE), CpG-PCR and temporal thermal ramp-MADGE (Melt-MADGE) for single nucleotide analyses in populations.
Important requirements for molecular genetic epidemiological studies are economy, sample parallelism, convenience of setup and accessibility, goals inadequately met by existent approaches. We invented microplate array diagonal gel electrophoresis (MADGE) to gain simultaneously the advantages of simple setup, 96-well microplate compatibility, horizontal electrophoresis, and the resolution of polyacrylamide. At essentially no equipment cost (one simple plastic gel former), 10-100-fold savings on time for sample coding, liquid transfers, and data documentation, in addition to volume reductions and gel re-use, can be achieved. MADGE is compatible with ARMS, restriction analysis and other pattern analyses. CpG-PCR is a general PCR approach to CpG sites (10-20% of all human single base variation): both primers have 3' T, and are abutted to the CpG, forcing a TaqI restriction site if the CpG is intact. Typically, a 52 bp PCR product is then cut in half. CpG-PCR also illustrates that PAGE-MADGE readily permits analysis of 'ultrashort' PCRs. Melt-MADGE employs real-time-variable-temperature electrophoresis to examine duplex mobility during melting, achieving DGGE-like de novo, mutation scanning, but with the conveniences of arbitrary programmability, MADGE compatibility and short run time. This suite of methods enhances our capability to type or scan thousands of samples simultaneously, by 10-100-fold.
CpG Islands↗
Evidence for a third genetic locus causing familial hypercholesterolemia. A non-LDLR, non-APOB kindred.
Monogenically inherited hypercholesterolemia is most commonly caused by mutations at the low density lipoprotein receptor (LDLR) locus causing familial hypercholesterolemia (FH) or at the apolipoprotein B (APOB) locus causing the disorder familial defective apoB (FDB). Probands from 47 kindreds with a strict clinical diagnosis of FH were selected from the Cardiovascular Genetics Research Lipid Clinic, Utah, for molecular genetic analysis. Using a combination of single-strand conformation polymorphism (SSCP) and direct sequencing, 12 different LDLR gene mutations were found in 16 of the probands. Three of the probands were carriers of the APOB R3500Q mutation. In five of the remaining 28 pedigrees where no mutation had been detected, samples from enough relatives were available to examine co-segregation with the LDLR region using the microsatellite marker D19S221, which is within 1 Mb centromeric of the LDLR locus, and D19S394, sited within 150 kb telomeric of the LDLR locus. In four of the families there was strong evidence for co-segregation between the LDLR locus and the phenotype of hypercholesterolemia, but in one large family with 18 living affected members and clear-cut bimodal hypercholesterolemia, there were numerous exclusions of co-segregation. Using length polymorphic markers within and outside the APOB gene, linkage of phenotype in this family to the APOB region was similarly excluded. In this large family, the degree of hypercholesterolemia, prevalence of tendon xanthomata, and occurrence of early coronary disease were indistinguishable from the other families studied. In summary, the data provide unequivocal evidence that a third locus can be etiological for monogenic familial hypercholesterolemia and should be reinvigorating to research in this field.
Adolescent↗