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Biomedical subjects

S D O'Dell

Publications and source records attributed to S D O'Dell.

At least 19 recordsLinked to original sources
PubMed2007

SHP-2 and PI3-kinase genes PTPN11 and PIK3R1 may influence serum apoB and LDL cholesterol levels in normal women.

Insulin regulates apoB metabolism via activation of PI3K or regulation of MTP via MAPK/ERK signalling. SHP-2 enhances both pathways through increased IRS-1 phosphorylation. We hypothesized that variants in the SHP-2 gene PTPN11 and PI3K p85alpha subunit gene PIK3R1 may influence fasting levels of plasma apoB and/or LDL cholesterol. We tested association of tagging SNPs (tSNPs) in each gene with serum lipids in a large sample of unselected population-based Caucasian female twins (n=2771, mean age 47.4+/-12.5 years) and then tested interaction between tSNPs in determining apoB and LDL levels. PTPN11 tSNP rs11066322 was associated with apoB (P=0.007) and rs11066320 was associated with LDL cholesterol (P=0.016). PIK3R1 tSNP rs251406 was associated with apoB (P=0.0003) and rs706713 was associated with LDL cholesterol (P=0.009). PTPN11 tSNP rs11066322 interacted with PIK3R1 tSNP rs251406 in determining serum apoB levels (P=0.012) and with PIK3R1 tSNP rs40318 in determining LDL cholesterol levels (P=0.009). Association of single tSNPs with both apoB and LDL cholesterol as well as interactions between the two genes suggest that variants influencing SHP-2 activity may modulate the acute pathway by which insulin regulates these lipids.

Adult↗
PubMed2006

Phosphatidylinositol 3-kinase p85alpha regulatory subunit gene PIK3R1 haplotype is associated with body fat and serum leptin in a female twin population.

AIMS/HYPOTHESIS: Phosphatidylinositol 3-kinase (PI3K) couples the leptin and insulin signalling pathways via the insulin receptor substrates IRS1 and IRS2. Hence, defective activation of PI3K could be a novel mechanism of peripheral leptin or insulin resistance. We investigated associations of tagging single-nucleotide polymorphisms (tSNPs) in the PI3K p85alpha regulatory subunit gene PIK3R1 with anthropometry, leptin, body fat and insulin sensitivity in a female twin population of European extraction. MATERIALS AND METHODS: Eight tSNPs were genotyped in 2,778 women (mean age 47.4+/-12.5 years) from the St Thomas' UK Adult Twin Registry (Twins UK). RESULTS: SNP rs1550805 was associated with serum leptin (p=0.028), BMI (p=0.025), weight (p=0.019), total fat (p=0.004), total fat percentage (p=0.002), waist circumference (p=0.025), central fat (p=0.005) and central fat percentage (p=0.005). SNPs rs7713645 and rs7709243 were associated with BMI (p=0.020 and p=0.029, respectively), rs7709243 with weight, total and central fat (p=0.026, p=0.031 and p=0.023, respectively) and both SNPs with fasting glucose (p=0.003 and p=0.001, respectively) and glucose 2-h post OGTT (p=0.023 and p=0.007, respectively). Subjects with haplotype 222 (frequency 7.2%) showed higher serum leptin concentration (p=0.007) and body fat measures (p< or =0.001 for all), and those with haplotype 221 (frequency 38.7%) showed higher fasting and 2-h glucose (p=0.035 and p=0.021, respectively) compared with subjects with the most common haplotype, 111 (frequency 45.5%). CONCLUSIONS/INTERPRETATION: Association of the PIK3R1 SNP rs1550805 with serum leptin and body fat may reflect a diminished ability of PI3K to signal via IRS1 or IRS2 in response to leptin.

Adipose Tissue↗
PubMed2006

AMP-kinase alpha2 subunit gene PRKAA2 variants are associated with total cholesterol, low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol in normal women.

BACKGROUND: 5'-AMP-activated protein kinase (AMPK) inactivates critial ensymes in fatty acid and cholesterol synthesis. We hypothesised that the serum lipid profile may be influenced by genetic variation in the AMPK catalytic alpha2 subunit. METHOD: We examined association of 5 tagging SNPs (tSNPs) in the PRKAA2 gene with serum lipids in 2777 normal Caucasian females (mean age 47.4+/-12.5 years). RESULTS: All tSNPs were associated with total- and LDL-cholesterol, (p<0.001 to 0.034), explaining variances of 0.13-0.59% and 0.11-0.55% respectively. One haplotype (frequency 34.7%) showed lower total- and LDL-cholesterol compared with the most common haplotype (frequency 45.7%) (p< or =0.001), explaining 0.78% of total- and 0.75% of LDL-cholesterol. Another haplotype (frequency 10.5%) was significantly associated with lower HDL-cholesterol (p = 0.005), explaining 0.59% of variance. CONCLUSIONS: PRKAA2 gene variants are significantly associated with serum lipoproteins in a large sample of normal female Caucasians.

AMP-Activated Protein Kinases↗
PubMed2006

Common polymorphisms in SOCS3 are not associated with body weight, insulin sensitivity or lipid profile in normal female twins.

AIMS/HYPOTHESIS: Inhibition of signal transduction by suppressor of cytokine signalling-3 (SOCS-3) potentially influences resistance to insulin and leptin. The aim of this study was to test the association between three single-nucleotide polymorphisms (SNPs) representative of common linkage disequilibrium clusters in SOCS3 (rs4969169, rs12953258 and rs8064821) and obesity measures, insulin sensitivity measures and serum lipids in the general population. METHODS: The three SNPs, which had rare allele frequencies >0.06, were genotyped in 2,777 female twins of European extraction (mean age 47.4+/-12.5 years) from the St Thomas' UK Adult Twin Registry (Twins UK). RESULTS: Minor allele frequencies were as follows: rs4969169=0.067, rs12953258=0.097 and rs8064821=0.101. Individual SOCS3 SNPs were not associated with general or central obesity, or with two indices of insulin sensitivity (homeostasis model assessment and insulin sensitivity measure). CONCLUSIONS/INTERPRETATION: The results do not indicate that any of the three SNPs studied are associated with obesity, insulin measures or lipid measures.

Adult↗
PubMed2001

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↗
PubMed2000

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↗
PubMed2000

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↗
PubMed1999

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↗
PubMed1999

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↗
PubMed1998

Microplate-array diagonal-gel electrophoresis (MADGE) and melt-MADGE: tools for molecular-genetic epidemiology.

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 the direct assay of single-base variations. Melt-MADGE adds temporal-thermal-ramp apparatus to achieve similar throughput for de novo mutation scanning.

Electrophoresis↗
PubMed1998

Insulin-like growth factor II (IGF-II).

Insulin-like growth factor II (IGF-II) plays a key role in mammalian growth, influencing foetal cell division and differentiation and possibly metabolic regulation. The mature 67 amino acid peptide shares sequence homology with both insulin and IGF-I. The liver is the main endocrine source of IGFs, but autocrine/paracrine activity is found in most tissues. The type 1 receptor mediates most of the biological effects of IGF-I and IGF-II; the type 2 receptor is involved with IGF-II degradation. Binding proteins may both localise IGFs to the receptors and regulate their activities. The IGF2 gene is maternally imprinted in mouse and human. Relaxation of IGF2 imprinting occurs in the Beckwith-Wiedemann syndrome of somatic overgrowth, sporadic Wilms' tumour and a number of other cancers. In the general adult population, the IGF2-INS gene cluster may also influence body weight, in which case IGF-II function could become a target for therapeutic intervention in obesity.

Adult↗
PubMed1998

W64R mutation in beta-3-adrenergic receptor gene and weight in a large population sample.

We have investigated the W64R (Trp64Arg) mutation in the beta-3-adrenergic receptor in 2270 healthy British males aged 50-61 y. The frequency of the rare R allele was 0.07 (95% confidence interval (CI) 0.06-0.08). The men showed an absence of association between W64R genotype and weight or height, both in the whole sample and in each quintile of the body mass index (BMI), and there was no association with tendency to gain weight. The W64R heterozygous state appears not to be a major contributing factor to obesity in the general population.

Alleles↗
PubMed1997

Spectrum of LDL receptor gene mutations in heterozygous familial hypercholesterolemia.

Familial hypercholesterolemia by usual definition reflects mutations of the LDL-receptor gene. Extensive molecular characterization of mutations ascertained mainly through homozygotes (the Dallas collection) has been presented by Hobbs et al. (Hum Mutat 1:445-446, 1992). This paper catalogues a spectrum of 134 mutations (27 novel mutations in 45 patients, 24 previously described mutations in 89 patients) ascertained through heterozygotes from the analysis of 791 patients with definite, probable, or possible FH, mainly from the UK, using high-throughput modifications of the single-strand conformation polymorphism technique. From a composite database of LDL receptor gene mutations complied from these two sets and from the literature, deductions are made about ascertainment bias, mutation rates, and molecular heterogeneity. Calculations suggest that there may be a large number of rare amino acid variants in the general population not causing classic FH. Approaches to, and feasibility of, molecular diagnostics are considered.

Genetic Variation↗
PubMed1997

Apal polymorphism in insulin-like growth factor II (IGF2) gene and weight in middle-aged males.

In 1474 healthy Caucasoid men aged 45-65 y, insulin-like growth factor II (IGF2) Apal AA homozygotes showed a mean body weight 4 kg lower than Apal GG homozygotes (77.6 +/- 10.9 kg vs 81.6 +/- 11.5 kg, P = 0.003) with heterozygotes (GA) intermediate (80.1 +/- 11.9 kg). The mean serum IGF-II concentration in 44 Apal AA individuals was significantly higher than in 48 Apal GG individuals (683.3 +/- 146.9 ng/ml vs 614.0 +/- 124.0 ng/ml, P = 0.01). An INS Pstl polymorphism showed no association with weight and it was also found to be in minimal linkage disequilibrium with the IGF2 Apal site (coefficient 0.016). The IGF2 Apal AA genotype is therefore associated with lower mean body weight but higher serum IGF-II concentrations than the GG genotype. Apal GG homozygotes incur a 1.67-fold risk of pathological Body Mass index (BMI) (> 30 kg/m2) compared with AA homozygotes.

Base Sequence↗
PubMed1997

Identification of a common low density lipoprotein receptor mutation (R329X) in the south of England: complete linkage disequilibrium with an allele of microsatellite D19S394.

Familial hypercholesterolaemia is commonly caused by mutations in the low density lipoprotein receptor (LDLR) gene and more than 300 different mutations have been described worldwide. Some mutations occur at relatively higher frequency in certain populations, reflecting both chance and demography, most evident in founder populations. As part of a study of kindreds of 78 probands from Southampton and south west Hampshire, we identified the same mutation (R329X) in 9/78 (11.5%) probands. In all (100%) of these probands, length allele 259nt of the 17 allele microsatellite D19S394, sited approximately 250 kilobases telomeric and 5' to the LDLR gene, was observed, although in the general population this allele has a prevalence of only 16.1%. A simple diagnostic assay for R329X was constructed in conjunction with more detailed family studies. Both the R329X and linked D19S394 allele also cosegregated with the FH phenotype within each kindred. Although R329X involves a CpG site, it is highly likely that the families are identical by descent for R329X, we surmise with a common ancestor within 500 to 1000 years, although the mutation is not restricted to this geographical area. This relationship illustrates that the linkage disequilibrium of gene LDLR with marker D19S394 will enable rapid recognition using D19S394 genotype of possible common FH mutation(s) within a cohort of FH patients from a particular locality or ethnic group.

Alleles↗
PubMed1996

PCR induction of a TaqI restriction site at any CpG dinucleotide using two mismatched primers (CpG-PCR).

A final common pathway has been devised for analysis of de novo mutation at any CpG site. Artificial restriction sites can be introduced in known DNA sequences by using either or both sense and antisense mismatched PCR primers. Sitting of the primers directly adjacent 5' and 3' to the CpG site yields a 52-bp PCR product, resulting from the sum of the two 25-mer oligonucleotides plus the two intervening bases (C and G), and also yields consistent digestion fragments. Three out of four possible four-base palindromes (TaqI, HHaI, and MspI) were investigated for mutations R329X and E80K in the human LDL receptor gene, and for mutations R395W and R612C, and TaqI site was forced using PCR in which both primers had 3' mismatched T. Both empirically and on theoretical grounds, Taq1 is the forced restriction site of choice. The approach has been adapted to the high-throughput microplate diagonal gel electrophoresis (MADGE) system for selective analysis of mutations at CpG sites, which may account for 20% of all single base variation in the human genome.

Base Composition↗
PubMed1995

Electrophoresis for genotyping: temporal thermal gradient gel electrophoresis for profiling of oligonucleotide dissociation.

Traditional use of an oligonucleotide probe to determine genotype depends on perfect base pairing to a single-stranded target which is stable to a higher temperature than when imperfect binding occurs due to a mismatch in the target sequence. Bound oligonucleotide is detected at a predetermined single temperature 'snapshot' of the melting profile, allowing the distinction of perfect from imperfect base pairing. In heterozygotes, the presence of the alternative sequence must be verified with a second oligonucleotide complementary to the variant. Here we describe a system of real-time variable temperature electrophoresis during which the oligonucleotide dissociates from its target. In 20% polyacrylamide the target strand has minimal mobility and released oligonucleotide migrates extremely quickly so that the 'freed' rather than the 'bound' is displayed. The full profile of oligonucleotide dissociation during gel electrophoresis is represented along the gel track, and a single oligonucleotide is sufficient to confirm heterozygosity, since the profile displays two separate peaks. Resolution is great, with use of short track lengths enabling analysis of dense arrays of samples. Each gel track can contain a different target or oligonucleotide and the temperature gradient can accommodate oligonucleotides of different melting temperatures. This provides a convenient system to examine the interaction of many different oligonucleotides and target sequences simultaneously and requires no prior knowledge of the mutant sequence(s) nor of oligonucleotide melting temperatures. The application of the technique is described for screening of a hotspot for mutations in the LDL receptor gene in patients with familial hypercholesterolaemia.

Autoradiography↗