Familial aggregation of albuminuria and arterial hypertension in an Aboriginal Australian community and the contribution of variants in ACE and TP53.

BACKGROUND: Aboriginal Australians are at high risk of cardiovascular, metabolic and renal diseases, resulting in a marked reduction in life expectancy when compared to the rest of the Australian population. This is partly due to recognized environmental and lifestyle risk factors, but a contribution of genetic susceptibility is also likely. METHODS: Using results from a comprehensive survey of one community (N = 1350 examined individuals), we have tested for familial aggregation of plasma glucose, arterial blood pressure, albuminuria (measured as urinary albumin to creatinine ratio, UACR) and estimated glomerular filtration rate (eGFR), and quantified the contribution of variation at four candidate genes (ACE; TP53; ENOS3; MTHFR). RESULTS: In the subsample of 357 individuals with complete genotype and phenotype data we showed that both UACR (h2 = 64%) and blood pressure (sBP h2 = 29%, dBP, h2 = 11%) were significantly heritable. The ACE insertion-deletion (P = 0.0009) and TP53 codon72 polymorphisms (P = 0.003) together contributed approximately 15% of the total heritability of UACR, with an effect of ACE genotype on BP also clearly evident. CONCLUSIONS: While the effects of the ACE insertion-deletion on risk of renal disease (especially in the setting of diabetes) are well recognized, this is only the second study to implicate p53 genotype as a risk factor for albuminuria - the other being an earlier study we performed in a different Aboriginal community (McDonald et al., J Am Soc Nephrol 13: 677-83, 2002). We conclude that there are significant genetic contributions to the high prevalence of chronic diseases observed in this population.

Relations between markers of renal function, coronary risk factors and the occurrence and severity of coronary artery disease.

OBJECTIVE: While renal failure greatly increases coronary risk, mild renal impairment is not usually considered a major risk factor. To explore this we assessed relations between measures of mild impairment of renal function and coronary artery disease (CAD) together with other risk factors. METHODS AND RESULTS: In 408 consecutive patients aged 75 years or less with angiographically defined normal or obstructed coronary arteries and an estimated glomerular filtration rate (eGFR) >45 mL/min per 1.73 m(2), we assessed relations between severity of CAD and levels of plasma cystatin C, creatinine, eGFR, lipid profile, C-reactive protein (CRP), homocysteine, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). With univariate ANOVA, the severity of CAD was significantly associated with all indices of renal function: increased plasma cystatin C (p=0.003) and creatinine (p=0.004) and decreased eGFR (p=0.008). An elevated plasma cystatin C was associated with increases in ADMA, SDMA, CRP, homocysteine and age. ADMA, SDMA, CRP and homocysteine levels were not associated with CAD severity. eGFR was negatively associated only with SDMA and homocysteine. In multivariate analysis, increased plasma cystatin C predicted both the occurrence and the severity of CAD more strongly than other measures of renal function. CONCLUSIONS: We conclude that mild renal impairment detected by elevated cystatin C is associated with both the occurrence and the severity of CAD, independent of the other risk factors we measured and that mild renal impairment results in increased plasma levels of homocysteine, ADMA and SDMA. Our findings suggest a possible mechanistic link between CAD and mild renal impairment in which cystatin C may serve as an early marker for CAD and may also participate directly in atherogenesis.

Asymmetric dimethylarginine (ADMA) in vascular, renal and hepatic disease and the regulatory role of L-arginine on its metabolism.

Asymmetric dimethylarginine (ADMA), an inhibitor of nitric oxide synthase (NOS), has been identified as a new and emerging contributor to, or marker for, cardiovascular risk. The ADMA-mediated regulation of nitric oxide (NO) production is determined by the quantitative bioavailability of intracellular and extracellular ADMA. Dimethylarginine dimethylaminohydrolase (DDAH), which is ubiquitously expressed in tissues, especially liver and kidney, converts the majority of the ADMA to citrulline. In this review, we discuss a new regulatory mechanism for the metabolism of ADMA in which L-arginine acts as a competitive inhibitor of DDAH activity. This novel regulatory pathway is consistent with ADMA contributing to cardiovascular risk when levels are increased but not when levels are within the normal range. The pathway then has a physiological role in the regulation of NO production by preventing overproduction of NO. The regulatory role of L-arginine on ADMA may explain the unexpected outcomes in some L-arginine supplementation studies. This paper also reviews associations between the metabolism of ADMA and insulin resistance, smoking and homocysteine which are all associated with an increased risk of vascular disease.

Diversity of cystathionine beta-synthase haplotypes bearing the most common homocystinuria mutation c.833T>C: a possible role for gene conversion.

Homozygosity or compound heterozygosity for the c.833T>C transition (p.I278 T) in the cystathionine beta-synthase (CBS) gene represents the most common cause of pyridoxine-responsive homocystinuria in Western Eurasians. However, the frequency of the pathogenic c.833C allele, as observed in healthy newborns from several European countries (q(c.833C) approximately equals 3.3 x 10(-3)), is approximately 20-fold higher than expected on the basis of the observed number of symptomatic homocystinuria patients carrying this mutation (q(c.833C) approximately equals 0.18 x 10(-3)), implying clinical underascertainment. Intriguingly, the c.833C mutation is also present in combination with a 68-bp insertion, c.[833C; 844_845ins68], in a substantial proportion of chromosomes from nonhomocystinuric individuals worldwide. We have sought to study the relationship between the pathogenic and nonpathogenic c.833C-bearing chromosomes and to determine whether the pathogenic c.[833C; -] chromosomes are identical-by-descent or instead arose by recurrent mutation. Initial haplotype analysis of 780 randomly selected Czech and sub-Saharan African wild-type chromosomes, employing 12 intragenic markers, revealed 29 distinct CBS haplotypes, of which 10 carried the c.[833C; 844_845ins68] combination; none carried an isolated c.833C or c.844_845ins68 mutation. Subsequent examination of 69 pathogenic c.[833C; -] chromosomes, derived from homocystinuria patients of predominantly European origin, disclosed three unrelated haplotypes that differed from their wild-type counterparts by virtue of the presence of c.833C, thereby indicating that c.833T>C transition has occurred repeatedly and independently in the past. Since c.833T does not reside within an obvious mutational hotspot, we surmise that the three pathogenic and comparatively prevalent c.[833C; -] chromosomes may have originated by recurrent gene conversion employing the common nonpathogenic c.[833C; 844_845ins68] chromosomes as templates.

Asymmetric dimethylarginine in homocystinuria due to cystathionine beta-synthase deficiency: relevance of renal function.

OBJECTIVE: Vascular disease is associated with increased plasma asymmetric dimethylarginine (ADMA) and homocysteine, and both are increased in renal failure. In cystathionine beta-synthase deficiency (CBS) there is severe hyperhomocysteinaemia, precocious vascular disease, and endothelial dysfunction. We investigated whether ADMA levels are elevated in CBS patients with and without renal impairment, and whether lowering plasma homocysteine also lowers ADMA. METHODS: We measured plasma homocysteine, arginine, asymmetric and symmetric dimethylarginines, nitrate + nitrite, creatinine and cystatin C in 23 CBS-deficient patients and 24 age-matched controls. RESULTS: In the patients, nitrate + nitrite and the ratio L: -arginine/ADMA were markedly reduced (21.6 +/- 6.1 vs 57.7 +/- 7.5 micromol/L and 132.9 +/- 24.7 vs 181.9 +/- 56.1, respectively, p < 0.001 for both), reflecting endothelial dysfunction. Plasma ADMA for the group was moderately increased (0.55 +/- 0.08 vs 0.49 +/- 0.07 micromol/L, p = 0.018), but this was due to significantly higher levels than controls in only those 7 of the 23 patients who had elevated cystatin C levels (0.59 +/- 0.08 vs 0.49 +/- 0.07 mg/L, p = 0.007). Posttreatment total homocysteine in patients varied widely (15-285, median 92 micromol/L), but was not correlated with ADMA or other measured variables. In three newly-diagnosed patients, marked reduction of total homocysteine during treatment produced minimal changes in ADMA. CONCLUSIONS: ADMA levels were significantly increased only in the CBS-deficient patients with elevated cystatin C levels, and not in those with normal renal function. The reported relationship between hyperhomocysteinaemia and ADMA may not be direct, but could be secondary to reduced renal function.

Relations between plasma asymmetric dimethylarginine (ADMA) and risk factors for coronary disease.

BACKGROUND: Elevated plasma levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide production, are reported to be associated with coronary artery disease (CAD). METHODS: We measured plasma levels of ADMA and related compounds, nitrate+nitrite (NO(x)), total homocysteine (tHCY) and assessed renal function and lipid profiles in 145 patients--75 with triple vessel coronary disease and 70 with no detectable coronary disease. RESULTS: Levels of ADMA, l-arginine, l-arginine/ADMA and plasma NO(x) were not different in the two groups but smokers with triple vessel disease had higher ADMA and lower NO(x) levels than the non-smokers, relationships also present for all smokers and non-smokers in the two groups combined. In all 145 patients ADMA, symmetric dimethylarginine (SDMA) and tHCY levels were significantly higher in patients with glomerular filtration rate (GFR) <81 mL/min/1.73 m(2) than in patients with GFR> or =81 mL/min/1.73 m(2). There was a modest positive correlation between tHCY and ADMA and both were strongly correlated with SDMA which is excreted by the kidney. ADMA, SDMA and tHCY were negatively correlated with GFR. CONCLUSIONS: We suggest that the reported ADMA increases in CAD patients are due to an associated reduction in renal function and to smoking habit.

Obesity, Type II diabetes and the Ala54Thr polymorphism of fatty acid binding protein 2 in the Tongan population.

Genetic variation of fatty acid binding protein 2 (FABP2) may contribute to the high prevalence of obesity and Type II diabetes in Tonga. To explore this we assessed the frequency of the FABP2 Ala54Thr polymorphism, obesity, and Type II diabetes in Tongans and possible inter-relationships. We investigated 1022 Tongan subjects, 433 men and 589 women aged 15-85 years, to identify possible associations between the FABP2 Ala54Thr polymorphism, obesity, Type II diabetes, BMI, glucose tolerance and standard lipid variables. The prevalence of the polymorphism was compared with that reported for other ethnic populations (studies from: Japanese, Finnish, African American, Native Canadian and Inuit, Swedish, Guadeloupe Indians, European males, and Caucasian populations). We found that 84% of the Tongan men and 93% of the Tongan women were overweight or obese (BMI> or =25kg/m2). The mean BMI+/-SD was not significantly different among those who were and were not carrying the Thr allele (males: Ala/Ala 30.4+/-5.4 and Thr carriers 29.8+/-5.1; females: Ala/Ala 33.8+/-6.4 and Thr carriers 33.6+/-5.1). The genotype frequencies were 76.2% Ala/Ala, 22.8% Ala/Thr, and 1.0% Thr/Thr. The Alal/Ala frequency is higher than the prevalences reported for all populations studied. The Thr allele was significantly associated with lower total cholesterol and LDL cholesterol in both sexes and in women also with lower HDL cholesterol. We conclude that there is a high prevalence of the FABP2 Ala54Thr polymorphism in Tongans. The polymorphism may be involved in lipid metabolism as the Thr allele is associated with low total and LDL cholesterol levels in this population.

A 45-bp insertion/deletion polymorphism of uncoupling protein 2 in relation to obesity in Tongans.

We compared the current prevalence of increased BMI and type 2 diabetes in a representative group of Tongan subjects with measurements made in 1973, and we determined the distribution and possible interrelations with the UCP2 insertion/deletion (ins/del) polymorphism of these variables. We documented the BMI, glucose tolerance, and standard lipid variables in 1012 Tongan subjects (429 men and 583 women, ages 15 to 85 years) during 1998 and 2000 and compared the BMI findings with those of the 1973 survey. We also genotyped for the UCP2 ins/del polymorphism, assessed its association with obesity and type 2 diabetes, and compared its prevalence with those reported for other ethnic populations. The mean BMI +/- SD was greatly increased in both men (30.2 +/- 5.4 kg/m(2)) and women (33.8 +/- 6.2 kg/m(2)), representing increases since 1973 of 11.9% and 19.4%, respectively. The genotype frequencies were 97% for the del/del genotype and 3% for the ins/del genotype; we found no ins/ins homozygotes. This distribution is strikingly different from those reported for white, South Indian, Pima Native-American, and Asian populations (49 to 77% for del/del genotype). We conclude that there is a marked prevalence of obesity in Tonga, a prevalence that has increased since 1973. We also conclude that there is a unique, near-uniform distribution of the UCP2 45-bp ins/del polymorphism in Tongans. This may be the result of a founder effect and may be relevant to the prevalence of obesity and type 2 diabetes in Tonga.

Obesity, Type II diabetes and the beta 2 adrenoceptor gene Gln27Glu polymorphism in the Tongan population.

As there is a high prevalence of obesity in Tonga, we aimed to determine the distribution of the beta2 adrenoceptor gene Gln(27)Glu polymorphism and to assess its relevance to obesity and to Type II diabetes, known to be prevalent in that population. A random sample of 1022 individuals from Tonga were genotyped for the Gln(27)Glu polymorphism in the beta 2 adrenoceptor gene. To assess the prevalence of obesity we measured body-mass index (BMI), fat-free mass, percentage fat and waist-to-hip ratio (WHR). To assess glucose metabolism we measured HbA(1c), fasting blood glucose, fasting serum insulin, and 1- and 2-h glucose; we also measured serum lipid and creatinine levels. We found that 84% of the Tongan men and 93% of the women were overweight or obese (BMI > or = 25 kg/m(2)) and 15.1% had Type II diabetes. Genotype frequencies among the 1022 Tongans were: Gln/Gln 90.3% and Gln/Glu 9.6%; we found one Glu/Glu homozygote. The mean BMI (+/-S.D.) for men was not significantly different for those who were homozygous (30.2+/-5.4 kg/m(2)) or heterozygous (30.1+/-5.5 kg/m(2)) for the Gln allele; this was also true for women (33.7+/-6.2 kg/m(2) for homozygous and 34.0+/-5.6 kg/m(2) for heterozygous). The Glu allele was not associated with other measures of obesity or abnormal glucose metabolism in this generally overweight population. There is a unique frequency of the Gln/Glu beta 2 adrenoceptor polymorphism among Tongans. We found no association of the polymorphism with obesity measures or Type II diabetes-related variables in the Tongan population among whom we documented a high prevalence of obesity and Type II diabetes and a low frequency of the Glu allele.

Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.

Atherosclerosis is a state of heightened oxidative stress. Oxidized LDL is present in atherosclerotic lesions and used as marker for coronary artery disease, although in human lesions lipids associated with HDL are as oxidized as those of LDL. Here we investigated specific changes occurring to apolipoprotein A-I (apoA-I) and apoA-II, as isolated HDL and human plasma undergo mild, chemically induced oxidation, or autoxidation. During such oxidation, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. Placing plasma at -20 degrees C prevents autoxidation, whereas metal chelators and butylated hydroxytoluene offer partial protection. Independent of the oxidation conditions, apoA-I and apoA-II (dimer) with two MetO residues accumulate as relatively stable oxidation products. Compared to controls, serum samples from subjects with the endothelial cell nitric oxide synthase a/b genotype that is associated with increased coronary artery disease contain increased concentrations of apoA-I with two MetO residues. Our results show that during the early stages, oxidation of HDL gives rise to specifically oxidized forms of apoA-I and apoA-II, some of which may be useful markers of in vivo HDL oxidation, and hence potentially atherosclerosis.

Homocysteine as a risk factor for cognitive impairment in stroke patients.

BACKGROUND: Elevated total homocysteine (tHcy) levels are associated with an increased risk of cerebrovascular disease. It is uncertain whether tHcy is also an independent risk factor for cognitive impairment. METHODS: We examined 95 stroke subjects 3 months after their strokes, and 55 healthy comparison subjects, with a detailed neuropsychological assessment, and MRI brain scans in a proportion (n = 97). Baseline measurements of tHcy, serum folate and B(12), creatinine and plasma fibrinogen levels were obtained. RESULTS: tHcy levels were higher in the stroke subjects by a mean 34%. These levels were significantly correlated with the first factor of a principal component analysis of the neuropsychological data, after controlling for age, folate, B(12) and creatinine levels. The correlation of Hcy levels was particularly significant with frontal-executive functioning and attention. tHcy levels were significantly correlated with number of infarcts and total stroke volume in the stroke group, but not with T(2)-weighted deep white matter hyperintensity scores, after correction for age. In the control group, tHcy levels were significantly correlated with ventricle-to-brain ratios as measures of brain atrophy. CONCLUSION: This study provides evidence that high tHcy levels are associated with cognitive impairment, in particular that of frontal-executive function. The major component of this association is accounted for by small and large strokes, but non-vascular neurotoxic effects of tHcy also appear to play a role. tHcy must receive greater attention as a risk factor for cognitive impairment.

The molecular basis of cystathionine beta-synthase deficiency in Australian patients: genotype-phenotype correlations and response to treatment.

Cystathionine beta-synthase (CBS) deficiency is the most common cause of homocystinuria. It is inherited as an autosomal recessive trait and common clinical features are: dislocation of the optic lens, osteoporosis, mental retardation, and thromboembolism. We determined the molecular basis of CBS deficiency in 36 Australian patients from 28 unrelated families, using direct sequencing of the entire coding region of the CBS gene. The G307S and I278T mutations were the most common mutations. They were present in 19% and 18% of independent alleles, respectively. In total, seven novel and 20 known mutations were detected. Of those, the two novel missense mutations (C109R and G347S), as well as two known missense mutations (L101P and N228K), were expressed in E. Coli. All mutant proteins completely lacked catalytic activity. Furthermore, we studied the correlation between genotype and the biochemical response to pyridoxine treatment in the patients of whom 13 were pyridoxine responsive, 21 were non-responsive, and two were partially responsive. The G307S mutation always resulted in a severe non-responsive phenotype, whereas I278T resulted in a milder B6 responsive phenotype. From our results, we were also able to establish three other mild mutations: P49L, R369C, and V371M.

Effect of CYP1A1 MspI polymorphism on cigarette smoking related coronary artery disease and diabetes.

CYP1A1, is one of the key detoxifying enzymes catabolizing cigarette smoking derived toxins and may be relevant to smoking-induced atherogenesis. Recently a CYP1A1 MspI polymorphism at the 3'-flanking region of the gene has been found to be associated with smoking related cancer risk and may, therefore, also be associated with vascular disease. To explore this, we investigated interactive effects between smoking and the CYP1A1 MspI polymorphism on coronary artery disease (CAD), diabetes and hypertension in 701 patients (aged < or =65 years) consecutively referred to Eastern Heart Clinic for angiographic investigation. The frequencies of the TT (80.2%), TC (17.7%) and CC (2.1%) genotypes were in Hardy-Weinberg equilibrium with the rare C allele frequency 0.11. The C allele carriers had an increased risk for triple vessel disease (three major epicardial coronary arteries with > or =50% luminal obstruction, OR, 3.44; 95%CI, 1.46-8.09; P=0.0046) in light smokers (< or =20 packyears). We further identified an interactive effect between smoking, the CYP1A1 MspI polymorphism and type 2 diabetes (chi(2)=9.508, P=0.002). The C allele carriers who were smokers had an increased risk of diabetes (OR, 2.44; 95%CI, 1.32-4.49; P=0.0059). Our study suggests that CYP1A1 may participate in the pathogenesis of atherosclerosis and in the development of diabetes and its vascular complications. The presence of the rare C allele of the CYP1A1 gene in smokers may enhance predisposition to severe CAD and type 2 diabetes. These findings contribute to the understanding of cardiovascular risk and to smoking related vascular disease.

Glutathione S-transferase mu1 deficiency, cigarette smoking and coronary artery disease.

BACKGROUND: While genetic variation accounts for a large proportion of interindividual differences in coronary artery disease (CAD) development, environmental factors such as cigarette smoking may genotype-dependently initiate or accelerate the risk. Glutathione S-transferase mu1 (GSTM1) is one of the GST isoenzymes and contributes to the detoxification process of organic compounds produced by cigarette smoking. In the present study we explored the hypothesis that GSTM1 deficiency, caused by GSTM1 null allele, may predispose subjects to cigarette smoking related CAD risk. DESIGN: Cross-sectional. METHODS: We genotyped the GSTM1 null allele in 868 angiographically characterized CAD patients who were consecutively recruited in the present study. RESULTS: The frequency of the null genotype in this high-risk patient population was 57.1% (55.4% for males and 61.0% for females). While 75.7% male and 50.7% female null GSTM1 patients had significant CAD as defined by one or more significantly stenosed coronary arteries, 79.3% male and 48.3% female patients with positive GSTM1 also had the significant CAD (P > 0.05). However, although 54.3% male and 55.2% female GSTM1 null patients had triple vessel disease, only 45.7% male and 44.5% female GSTM1 positive patients had the severe disease. Controlling for cigarette smoking did not change the relationship. The occurrences of MI were 37.9% in male and 31.4% in female with the null genotype whereas they were 42.8% in male 37.6% in female with positive GSTM1 (P > 0.05). Using logistic regression analyses, we found no interactions between GSTM1 genotype and cigarette smoking in relation to CAD or MI. CONCLUSIONS: While our data may be consistent with that the GSTM1 null genotype predisposes subjects to cigarette smoking related severe CAD, interactive effect on CAD risk is minor and insignificant. GSTM1 deficiency alone is not sufficient to cause CAD.

The p53Pro72Arg polymorphism is associated with albuminuria among aboriginal Australians.

Albuminuria is a widely recognized marker of renal disease and cardiovascular risk. This is especially true in Aboriginal Australians living in remote communities who suffer high rates of end-stage renal disease and cardiovascular mortality. During a survey of risk factors for renal and cardiovascular disease in one such community, an association between a common polymorphism at codon 72 (Arg/Pro) of the p53 gene and markers of renal disease was sought. A cross-sectional community survey including 217 people was performed. Genotypes of the polymorphism were distributed in Hardy-Weinberg equilibrium, with p53Arg allele frequency of 0.45 (range, 0.41 to 0.50). Overall prevalence of albuminuria was high (31% microalbuminuria; 14% overt albuminuria). Urine albumin/creatinine ratio (ACR) was significantly associated with the number of p53Pro alleles (P = 0.01), and there was an interaction with tobacco smoking (P = 0.04). The p53 genotype was also associated with increasing HbA1c, but the relationship between p53 and ACR was independent of this. This is a previously unreported association. This study does not address the mechanism, but this finding, if confirmed, expands the described effects of p53 in cellular proliferation and apoptosis to include a role in the course of renal and possibly cardiovascular disease in this population.