Nutrient regulation of ß-cell function: what do islet cell/animal studies tell us?

Diabetes mellitus is widely recognised as one of the most serious metabolic diseases worldwide, and its incidence in Asian countries is growing at an alarming rate. Type 2 diabetes (T2DM) is closely associated with age, sedentary lifestyle and poor diet. In T2DM, ß-cell dysfunction will occur before hyperglycaemia develops. Excessive levels of glucose, lipid and various inflammatory factors interact at the level of the pancreatic islet to promote ß-cell dysfunction. Pancreatic ß-cell lines have been widely utilised since the early 1980s and have contributed a large volume of important information regarding molecular, metabolic and genetic mechanisms that regulate insulin secretion. The purpose of this review is to describe the origin and characteristics of the most commonly used ß-cell lines and their contribution to discovery of fundamental regulatory processes that control insulin production and release. Pancreatic islets obtained from rodents as well as other animals have additionally provided information on the architecture and three-dimensional design of this endocrine tissue that allows precise regulation of hormone release. Understanding the nature of failure of physiologic and metabolic processes leading to insufficient insulin release and subsequent diabetes has allowed development of novel anti-diabetic therapeutics, now in common use, worldwide.

A map of the class III region of the sheep major histocompatibilty complex.

BACKGROUND: The central, or class III, region of the major histocompatibility complex (MHC) is an important gene rich sub-region of the MHC of mammals and contains many loci implicated in disease processes and potential productivity traits. As a prelude to identifying MHC loci associated with productivity traits in sheep, we have used BAC and cosmid libraries of genomic DNA to generate a physical map of the sheep MHC class III region. This map will facilitate association studies and provide insights into the distribution of recombination events in this chromosomal segment. RESULTS: Twenty eight sheep genes were identified in 10 BAC clones which spanned approximately 700 kbp of a chromosomal region adjacent to the class I region of the sheep MHC and which therefore covers most, if not all, of the class III of the sheep MHC. The relative positions of 17 of these genes was established as well as two additional groups of genes for which the intragroup order was not known. Cosmid mapping permitted a more detailed mapping of the complement genes present in the class III and showed a local inversion (relative to humans) of one pair of the duplicated complement C4 and CYP21 loci. A panel of 26 single nucleotide polymorphisms (SNPs) was identified in 10 loci, covering approximately 600 kbp of the mapped region. CONCLUSION: This report provides a physical map covering approximately 700 kbp of the class III of the sheep MHC together with a SNP panel which will facilitate disease and productivity association studies. The presence of a local inversion (relative to humans) of one pair of the duplicated C4 and CYP21 loci and a previously described dinucleotide tandem repeat locus (BfMs) has been located within an intron of the SK12VL gene.

HLA-B*5701 typing: evaluation of an allele-specific polymerase chain reaction melting assay.

Inheritance of HLA-B*5701 is a strong predictor of a hypersensitivity reaction to the anti-HIV drug abacavir. The identification of susceptible individuals prior to the institution of abacavir therapy is therefore of clinical importance and has generated demand for a simple and rapid diagnostic test for carriage of HLA-B*5701. In this study, we describe the development of such a method based on allele-specific polymerase chain reaction (AS-PCR) and melting curve analysis. Ninety-six patient samples including 36 HLA-B*5701-positive samples and 60 HLA-B*5701-negative samples were analysed. Compared with sequence-based typing, this method had 100% sensitivity and specificity for the HLA-B*5701 allele. In conclusion, the AS-PCR/melting curve approach minimises post-polymerase chain reaction handling processing and provides an attractive alternative to currently described AS-PCR methods.

Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to HIV-1 reverse-transcriptase inhibitor abacavir.

BACKGROUND: The use of abacavir--a potent HIV-1 nucleoside-analogue reverse-transcriptase inhibitor--is complicated by a potentially life-threatening hypersensitivity syndrome in about 5% of cases. Genetic factors influencing the immune response to abacavir might confer susceptibility. We aimed to find associations between MHC alleles and abacavir hypersensitivity in HIV-1-positive individuals treated with abacavir. METHODS: MHC region typing was done in the first 200 Western Australian HIV Cohort Study participants exposed to abacavir. Definite abacavir hypersensitivity was identified in 18 cases, and was excluded in 167 individuals with more than 6 weeks' exposure to the drug (abacavir tolerant). 15 individuals experienced some symptoms but did not meet criteria for abacavir hypersensitivity. p values were corrected for comparisons of multiple HLA alleles (p(c)) by multiplication of the raw p value by the estimated number of HLA alleles present within the loci examined. FINDINGS: HLA-B*5701 was present in 14 (78%) of the 18 patients with abacavir hypersensitivity, and in four (2%) of the 167 abacavir tolerant patients (odds ratio 117 [95% CI 29-481], p(c)<0.0001), and the HLA-DR7 and HLA-DQ3 combination was found in 13 (72%) of hypersensitive and five (3%) of tolerant patients (73 [20-268], p(c)<0.0001 ). HLA-B*5701, HLA-DR7, and HLA-DQ3 were present in combination in 13 (72%) hypersensitive patients and none of the tolerant patients (822 [43-15 675], p(c)<0.0001). Other MHC markers also present on the 57.1 ancestral haplotype to which the three markers above belong confirmed the presence of haplotype-specific linkage disequilibrium, and mapped potential susceptibility loci to a region bounded by C4A6 and HLA-C. Within the entire abacavir-exposed cohort (n=200), presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 had a positive predictive value for hypersensitivity of 100%, and a negative predictive value of 97%. INTERPRETATION: Genetic susceptibility to abacavir hypersensitivity is carried on the 57.1 ancestral haplotype. In our population, withholding abacavir in those with HLA-B*5701, HLA-DR7, and HLA-DQ3 should reduce the prevalence of hypersensitivity from 9% to 2.5% without inappropriately denying abacavir to any patient.

Lack of evidence for association between endothelial nitric oxide synthase gene polymorphisms and coronary artery disease in the Australian Caucasian population.

BACKGROUND: Genetic polymorphism in the gene for endothelial nitric oxide synthase (eNOS) has been identified as a potential risk factor for the development of premature coronary artery disease (CAD). We determined whether the eNOS 4ab, G894T, and T-786C polymorphisms are associated with premature coronary artery disease. DESIGN: A case-control study. METHODS: PCR-based assays were used to compare the frequency of eNOS gene polymorphisms in 573 Caucasian subjects aged under 50 years presenting with symptomatic CAD and documented by coronary angiography, with or without myocardial infarction, to that of 624 similarly aged community controls without a history of symptomatic CAD. RESULTS: We found no difference in the frequency of 4ab genotypes between cases and controls: in the CAD subjects, the 4aa, 4ab, and 4bb genotype frequencies were 1.9%, 24.3% and 73.8% respectively, compared to 2.2%, 25.5% and 72.3% respectively for the controls. There was also no significant difference between cases and controls in the frequency of any allele (4a/4b, 894G/894T, -786C/-786T), or genotype for any of the polymorphisms. Similarly, logistic regression analysis showed no evidence for an association of the polymorphisms with premature CAD or myocardial infarction or any indication of an interaction between the polymorphisms and other CAD risk factors, including smoking. CONCLUSIONS: In a large case-control study, and in contrast to some earlier positive findings by others, we have found no evidence for an association between several eNOS gene polymorphisms and premature CAD in an Australian Caucasian population.

Lipoprotein lipase D9N, N291S and S447X polymorphisms: their influence on premature coronary heart disease and plasma lipids.

Lipoprotein lipase (LPL) plays a pivotal role in lipoprotein metabolism. Three recently described exonic polymorphisms of the gene, D9N, N291S and S447X, have been variably found to influence plasma lipids while effects on coronary heart disease (CHD) are less well documented. Two predominantly Caucasian groups were studied: CHD patients <50 years of age, with angiographically documented CHD; and a randomly recruited community control group without a history of heart disease. The 9N allele of the D9N polymorphism was present in 25 of 428 (5.8%) of Caucasian males with CHD and in seven of 291 (2.4%) of corresponding community subjects (odds ratio, 2.5; 95% confidence interval (CI), 1.1-5.9; P=0.03) and was also significantly over-represented in the Caucasian males with myocardial infarction (MI) (21 of 308 or 6.8%; odds ratio, 2.6; 95% CI, 1.1-5.9; P=0.01). The distributions of the other two polymorphisms were similar in the CHD and community groups. In multivariate models adjusted for age, sex, diabetes, body mass index, smoking, lipid levels and race, the D9N polymorphism remained significantly related to both CHD and MI, with an odds ratio >2. There were, generally, trends to more adverse fasting plasma high-density lipoprotein (HDL) cholesterol and triglycerides in carriers of the 291S and 9N alleles, and the opposite trends for triglycerides in 447X carriers. In the community group, male carriers of 291S (n=13) had significantly (20%) lower HDL cholesterol than corresponding non-carriers (n=323), 0.98+/-0.07 mmol/l (mean+/-S.E.) versus 1.22+/-0.02 mmol/l (P<0.005), while HDL cholesterol was not different in male carriers (n=8) and non-carriers (n=296) of 9N (1.23+/-0.13 mmol/l versus 1.22+/-0.02 mmol/l). Multivariate analysis confirmed that the 291S allele carrier status conferred a significantly lower HDL cholesterol (P=0.001) and the 447X allele lower triglyceride (P<0.01) in the community group. In conclusion, LPL 9N carrier status was unequivocally related to premature CHD and to MI in males, strongly supporting recent results in older aged males. The somewhat different effects of the D9N and N291S polymorphisms on plasma lipids, and the absence of a clear effect of the N291S on CHD, raise the possibility that the effect of 9N carrier status might be mediated through effects on LPL function in addition to those influencing fasting plasma lipids.

Angiotensin-converting enzyme gene polymorphism and premature coronary heart disease.

Since the initial report of the association of the deletion/insertion (D/I) polymorphism in the gene for angiotensin-converting enzyme (ACE) with myocardial infarction (MI), there has been considerable controversy. Some have found the D allele to be associated with MI, coronary heart disease (CHD) or other cardiac pathology, while others have not. In the present study 713 consecutive patients, < 50 years of age, documented prospectively with angiographic CHD (> 50% diameter stenosis of at least one coronary artery), with or without MI, were studied, along with 688 community control subjects, also < 50 years of age, selected randomly from the electoral rolls and without a history of CHD or MI. Genotyping was done by standard methods. Most of the subjects in both groups were Anglo-Celtic Caucasians (547 in the CHD group and 642 in the community group), and the report concerns primarily these subjects. ACE genotype distributions were not different between the Caucasian community control group and the CHD or the MI subgroups; the odds ratios and 95% confidence limits for the CHD group were 0.96 (0.73-1.27) for the D allele and 1.02 (0.80-1.31) for D homozygotes; for the MI group these values were 1.00 (0.83-1.20) and 0.99 (0.74-1.32) respectively. This negative result was supported in multivariate analysis accounting for conventional risk factors. There was a significant racial difference in ACE genotypes between Caucasians, Asians and Australian Aborigines in the CHD group (P < 0.001); for example, in this group, 158 of 540 (29%) Caucasians had the DD genotype compared with eight of 84 (10%) Aboriginals (P < 0.001) and six of 59 (10%) Asians (P = 0.002). Failure to account for such racial differences would have led to erroneous conclusions. In conclusion, we found no evidence that the D/I ACE gene polymorphism plays a role in the development of CHD or MI at an early age in a Western Australian Caucasian population. While this result refers uniquely to premature CHD and MI, and could be population specific, it is in general agreement with recent meta-analysis of the larger previous studies.

Elite athletes and the gene for angiotensin-converting enzyme.

The deletion (D) allele of the gene for angiotensin-converting enzyme (ACE) is associated with higher plasma and tissue levels of the enzyme and has also been related to a variety of cardiovascular complications, particularly myocardial infarction. On the basis of indirect evidence, we hypothesized that inheritance of the D allele would contribute to elite athletic ability. Over a period of 4 yr, 120 Caucasian athletes who were national (Australian) representatives in sports demanding a high level of aerobic fitness were recruited. Their ACE genotypes were compared with those of a community control group recruited randomly from the electoral roll. There was no difference in ACE genotype frequencies between the two groups. The DD genotype frequency was 30% in athletes and 29% in the control group, and the II genotype frequency was 22.5 and 22%, respectively. The results do not exclude the possibility that ACE genotype could be related to some attribute relating to a specific type of elite athletic ability or that there may be a difference between genders. Larger studies are desirable.

Butyrycholinesterase K variant and Alzheimer's disease.

This study attempted to corroborate findings on the association between butyrylcholinesterase K variant and Alzheimer's disease. This was performed on an autopsy-confirmed series of patients with Alzheimer's disease and controls. The butyrylcholinesterase K variant was found to be of increased allele frequency in patients with sporadic Alzheimer's disease. When related to APOE epsilon4 typing the association was specific but not sensitive for the diagnosis of Alzheimer's disease.

Comparison of the LDL-receptor binding of VLDL and LDL from apoE4 and apoE3 homozygotes.

Compared with apolipoprotein E3 (apoE3), apoE2 is less effective in mediating the binding of lipoproteins to the low-density lipoprotein (LDL) receptor. The influence of the E4 isoform, which is associated with adverse effects on plasma lipids and coronary heart disease, is less clear. We compared the ability of very low density lipoprotein (VLDL) and LDL from paired E4/4 and E3/3 subjects to compete against 125I-labeled LDL for binding with the LDL receptor on cultured fibroblasts and Hep G2 cells. The concentrations of VLDL or LDL required to inhibit binding of 125I-LDL by 50% (IC50, microgram apoB/ml) were determined, and results were assessed in terms of an IC50 ratio, E4/4 IC50 relative to E3/3 IC50, to reduce the influence of interassay variability. In Hep G2 cells, E4/4 VLDL was more effective than E3/3 VLDL in competing for the LDL receptor, the IC50 ratio being lower than unity (0.73 +/- 0.31, P < 0.05, two-tailed t-test). IC50 values themselves were marginally lower in E4/4 than E3/3 subjects (3.7 +/- 1.3 vs. 6.1 +/- 3.7, P < 0.08). However, there was no difference between E4/4 and E3/3 VLDL in competing for the LDL receptor on fibroblasts or between E4/4 and E3/3 LDL in competing for the LDL receptor on either cell type. These results suggest that inheritance of apoE4 is associated with an increased affinity of VLDL particles for LDL receptors on hepatocytes and may partly explain the influence of the E4 isoform on lipid metabolism.

PIa1/a2 polymorphism of glycoprotein IIIa and risk of coronary artery disease and restenosis following coronary angioplasty.

Glycoprotein IIIa (GpIIIa), a platelet protein that mediates platelet aggregation and thrombus formation, has attained recent widespread interest following a report that a common genetic variant of GpIIIa, known as Pla2, is an inherited risk factor for the development of premature coronary artery disease (CAD). We determined the frequency of the Pla2 allele in 589 prospectively recruited subjects aged <50 years presenting with symptomatic CAD with or without myocardial infarction and documented by coronary angiography (group I), 207 subjects investigated prospectively for restenosis 6 months after coronary balloon angioplasty (group II), and 570 control subjects without a history of angina or myocardial infarction, randomly selected from the community. Detection of the Pla2 allele was based on MspI digestion of polymerase chain reaction (PCR) amplified deoxyribonucleic acid (DNA) spanning the Pla1/a2 locus. The accuracy of genotyping was verified with a second independent method based on BstXI digestion of DNA amplified with mutagenic PCR primers. The frequency of the Pla2 allele was similar (p >0.1) in group I (170 of 1,178, 14%), group II (49 of 414, 12%), and control subjects (160 of 1,140, 14%). Among group I subjects, there was no relation (p >0.1) between the Pla2 allele frequency and the number of coronary vessels with >50% diameter obstruction, and current or previous myocardial infarction; among group II subjects, there was no difference between those with and without restenosis after angioplasty (26 of 242 and 23 of 172, respectively, p >0.1). We conclude that the Pla2 allele is not associated with a significantly elevated risk of premature CAD, myocardial infarction, or restenosis after coronary angioplasty.

The effect of homocysteine on DNA synthesis in cultured human vascular smooth muscle.

Elevated plasma homocysteine is an established risk factor for vascular disease although the mechanisms are unclear. Homocysteine has been reported to stimulate DNA synthesis and proliferation in rat aortic smooth muscle cells. Human vascular smooth muscle cells (HVSMC) from saphenous veins (n = 8), internal mammary arteries (n = 6) and umbilical arteries (n = 2) were studied. To reflect DNA synthesis, 3H-thymidine incorporation, during 24 h exposure to homocysteine in concentrations from 0.0625 to 10 mM, was studied. Incorporation was significantly increased up to 0.5 or 1 mM and thence was progressively depressed, the maximum stimulation being 24 +/- 5(S.E.)% in vein (P < 0.005) and 34 +/- 4% in mammary artery (P < 0.001) while incorporation fell to approximately 25% of the control values at 10 mM (P < 0.001). Qualitatively similar results were obtained in umbilical arteries. Homocysteine had a biphasic effect on DNA synthesis in cultured HVSMC but the higher inhibitory concentrations are well above those commonly found in vivo. While the conditions of exposure to homocysteine render close analogy to the clinical situation impossible, homocysteine can stimulate HVSMC, offering one possible mechanism for the involvement of homocysteine in the pathogenesis of atherosclerosis.

Methylenetetrahydrofolate reductase gene and coronary artery disease.

BACKGROUND: Hypermocysteinemia has been substantiated as a risk factor for occlusive vascular disease. A common mutation (nucleotide 677 C-->T) has been described recently in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, which results in a valine for alanine substitution, a thermolabile enzyme, and a tendency to elevate plasma homocysteine levels and which has been proposed to contribute importantly to coronary artery disease. METHODS AND RESULTS: To study the potential influence of the mutation on ischemic heart disease, we screened 555 whites with angiographically documented coronary artery disease and 143 unrelated control subjects without a history of angina or myocardial infarction randomly selected from the community. The patients were in two groups: group 1 comprised 358 prospectively recruited individuals younger than 50 years, and group 2, 197 patients investigated prospectively for restenosis 6 months after coronary angioplasty. The frequency of homozygosity for the mutation was 10.5% in control subjects, 10.6% in group 1, and 9.1% in group 2 patients. There was no relationship between MTHFR genotype and number of coronary vessels with > 50% diameter obstruction, prior myocardial infarction, or restenosis after coronary angioplasty. Plasma folate concentrations in control subjects (n = 90) and patients (n = 208) showed closely similar distributions. CONCLUSIONS: Although it is accepted that moderate hyperhomocysteinemia significantly increases the risk for coronary, cerebrovascular, and peripheral vascular diseases, our data suggest that a mutation of the MTHFR gene, which has been associated with a thermolabile form of the enzyme and with hyperhomocysteinemia in subjects with plasma folate below the median, does not appear to be significantly associated with risk for premature coronary artery disease or for restenosis after coronary angioplasty.

Familial defective apolipoprotein B-100 (FDB): effect of simvastatin therapy on LDL-receptor binding.

Heterozygotes for familial defective apolipoprotein B-100 (FDB) have two populations of low density lipoprotein (LDL), one bearing normal apolipoprotein B-100 (apo B) and the other bearing defective apo B which exhibits a much lower affinity for the LDL-receptor. If HMGCoA reductase inhibitors such as simvastatin lowered LDL mainly by up-regulating LDL-receptor mediated clearance, they should decrease the overall binding affinity of LDL from an FDB heterozygote by selectively decreasing LDL bearing normal apo B. We compared how LDL from FDB heterozygotes competed with normal 125I-labelled LDL for binding to LDL-receptors while on and off therapy with simvastatin. The LDL of FDB heterozygotes had 40% (n = 10) the affinity of normal LDL (n = 12) for the LDL receptor on cultured fibroblasts, and 55% (n = 6) of normal LDL (n = 6) for that on HepG2 cells. Treatment of FDB subjects with simvastatin (n = 10) decreased serum LDL by 22% but had no effect on its binding affinity for LDL receptors, indicative of lowering of LDL containing both normal and defective apo B. This is consistent with the major LDL lowering effect being associated with decreased synthesis of LDL, rather than enhanced LDL-receptor clearance.

Angiotensin-converting enzyme and apolipoprotein E genotypes and restenosis after coronary angioplasty.

BACKGROUND: An insertion/deletion (I/D) polymorphism in the gene for angiotensin-converting enzyme (ACE) has been associated with myocardial infarction and other cardiac pathology. There is evidence for a role of the renin-angiotensin system in cell growth and in the repair of damaged arterial walls, so the ACE gene was postulated to be a candidate gene affecting the important clinical problem of restenosis after percutaneous transluminal balloon coronary angioplasty (PTCA). Because restenosis is influenced by the apolipoprotein E (apoE) genotype, the possibility of a relation between ACE and apoE genotypes and restenosis was also sought. METHODS AND RESULTS: Subjects (< 70 years of age) were prospectively followed and had coronary angiography 6 months after PTCA to determine the presence or absence of restenosis. Those who had angiography earlier and did not have restenosis (> or = 50% loss of gain at PTCA plus > or = 50% luminal diameter stenosis) also had angiography at 6 months. The whole group (n = 207) had a higher DD genotype frequency than did 136 population control subjects (38% versus 26%, P < .02); in PTCA patients, the frequency was the same in those with and without prior myocardial infarction. The distribution of ACE genotypes was not different in the 88 patients with and 119 patients without restenosis, while the epsilon 4/4 genotype was more frequent in those with restenosis (8 of 88 versus 3 of 118, P < .05). There was no effect of the ACE genotype in noncarriers of the epsilon 4 allele, but there was a significant effect in epsilon 4 carriers (P < .005). The combined D and epsilon 4 carrier state showed a 16-fold increase in the odds ratio for restenosis (P < .02). Multiple linear regression examining the loss of lumen as a continuous variable showed significant independent effects of the ACE and apoE genotypes. CONCLUSIONS: Overall, the ACE genotype had no clear influence on restenosis, but there was an interaction between ACE and apoE genotypes. The combined carrier state for the D and apoE epsilon 4 alleles substantially increased restenosis. For loss of lumen as a continuous variable, there were significant effects of both ACE and apoE genotypes. While the observations may not affect current management, they no doubt have implications in pathophysiology.

Apolipoprotein epsilon 4 homozygosity--a determinant of restenosis after coronary angioplasty.

Conventional coronary risk factors have not consistently been found to be related to restenosis after coronary angioplasty. Apolipoprotein E (apo E) gene polymorphism and/or plasma apo(a) levels were determined in 195 subjects undergoing prospective follow up and angiographic study 6 months after elective balloon angioplasty of a previously untreated coronary obstruction. Restenosis (stenosis > or = 50% plus loss of > or = 50% of initial gain) had occurred in 59 of 150 subjects for whom E genotypes were available. The apo epsilon 4 allele frequency in those with restenosis was higher than those without (0.20 vs. 0.10, P < 0.01), attributable to an excess of epsilon 4 homozygotes in the restenosis group (5 of 59 vs. 1 of 91, P < 0.04). Restenosis was not related to plasma apo(a) and the apo epsilon 4 allele was not associated with elevated levels of apo(a) as has been reported elsewhere. No relationship was found between E genotype and serum lipid and lipoprotein levels; paradoxically, LDL cholesterol was significantly lower and HDL cholesterol higher in those with restenosis. In conclusion, homozygosity for apolipoprotein epsilon 4 appears to be an important determinant of restenosis after coronary angioplasty.

Urine albumin excretion in healthy subjects.

The relationship between cardiovascular risk factors and urine albumin excretion were studied in 474 healthy office workers. Albumin concentration was measured fresh in first morning midstream urines. Lifestyle details, oscillometric BP and lipids were assessed. Subjects with urine albumin concentration above the median (5.30 mg/l) were compared with those with albumin concentration below the median. Subjects with above median urinary albumin concentration had higher systolic blood pressure (mean 115.2 vs. 113.1 mm Hg for above median, respectively, P = 0.06), were more likely to be male (56.8 vs. 45.0%, respectively, P = 0.01) and to have lower levels of high density lipoprotein (HDL)-cholesterol (mean 1.34 vs. 1.41 mmol/l, P = 0.006). Multivariate analysis following adjustment for urine creatinine concentration to allow for urine volume confirmed the relationship with systolic blood pressure (P = 0.01) and sex (P = 0.02), and in addition revealed a relationship with alcohol intake approaching significance (mean intake 70.8 and 76.0 g/week, respectively, P = 0.06). The univariate finding of increased albuminuria with lower HDL-cholesterol appeared to be attributable to the associated relationships with male sex and lower alcohol intake. The relationships between albumin excretion and BP, male sex and alcohol intake may reflect the effects of asymptomatic developing arterial disease. The relationship with BP may also be a consequence of effects on glomerular hydrostatic or interstitial renal pressure on albumin filtration or resorption. Very low level urine albumin excretion in healthy subjects is associated with factors which predict arterial disease. Urine albumin excretion may prove to be a useful early marker of cardiovascular disease in population studies.

A robust strategy for screening and confirmation of familial defective apolipoprotein B-100.

The diagnosis of familial defective apolipoprotein B-100 (FDB) has been facilitated by the use of mutagenic polymerase chain reaction (PCR) primers to introduce restriction sites at the FDB gene locus. We describe a two-test strategy for diagnosing FDB that overcomes the potential for error in single-test methods based on such techniques. We introduce an Sau96I restriction site for PCR products of the normal apolipoprotein B allele. Incomplete digestion of the PCR product with Sau96I suggests an FDB heterozygote, although a false-positive result due to nonideal digestion conditions remains a possibility. In such cases we use a second test that introduces an ScaI restriction site in PCR products of the FDB allele. The diagnosis is confirmed if half of this product can be digested with ScaI. Both tests use 0.25 units of Taq polymerase and are robust with respect to annealing temperature (31-58 degrees C) and to Mg2+ concentration (1.0-3.2 mmol/L).

Apolipoprotein epsilon 4 homozygosity in young men with coronary heart disease.

We have compared apolipoprotein E gene polymorphism in 91 Australian men aged 30-50 who had been referred for coronary angioplasty and in 172 healthy younger men. 5 of the 19 patients who were less than 40 years of age were homozygous for the epsilon 4 allele, representing a 16-fold increase in prevalence compared with controls. In patients aged 40-50 the epsilon 4 allele frequency was 60% higher than it was in controls. Inheritance of epsilon 4 seems to confer risk of premature ischaemic heart disease in males, homozygotes being especially at risk at a younger age.