Klaus Lindpaintner

Genetic mapping of a gene causing hypertension in the stroke-prone spontaneously hypertensive rat.

The stroke-prone spontaneously hypertensive rat (SHRSP) is a well-characterized model for primary hypertension in humans. High blood pressure in SHRSP shows polygenic inheritance, but none of the loci responsible have previously been identified. To locate genes controlling this quantitative trait, we mapped a large collection of DNA polymorphisms in a cross between SHRSP and the normotensive WKY strain. Here we report strong genetic evidence that a gene, Bp1, having a major effect on blood pressure maps to rat chromosome 10 with a LOD score of 5.10 and is closely linked to the rat gene encoding angiotensin-converting enzyme (ACE), an enzyme that plays a major role in blood pressure homeostasis and is an important target of anti-hypertensive drugs. We also find significant, albeit weaker, linkage to a locus, Bp2, on chromosome 18. We discuss the implications of genetic dissection of quantitative disease-related phenotypes in mammals.

Evidence for Association and Genetic Linkage of the Angiotensin-Converting Enzyme Locus With Hypertension and Blood Pressure in Men but Not Women in the Framingham Heart Study

BACKGROUND There is controversy regarding the association of the angiotensin-converting enzyme deletion-insertion (ACE D/I) polymorphism with systemic hypertension and with blood pressure. We investigated these relations in a large population-based sample of men and women by using association and linkage analyses. METHODS AND RESULTS The study sample consisted of 3095 participants in the Framingham Heart Study. Blood pressure measurements were obtained at regular examinations. The ACE D/I polymorphism was identified by using a polymerase chain reaction assay. In logistic regression analysis, the adjusted odds ratios for hypertension among men for the DD and DI genotypes were 1.59 (95% confidence interval [CI], 1.13 to 2.23) and 1.18 (95% CI, 0.87 to 1.62), respectively, versus II (chi2 P=.02). In women, adjusted odds ratios for the DD and DI genotypes were 1.00 (95% CI, 0.70 to 1.44) and 0.78 (95% CI, 0.56 to 1.09), respectively (P=.14). In linear regression analysis, there was an association of the ACE DD genotype with increased diastolic blood pressure in men (age-adjusted P=.03, multivariate-adjusted P=.14) but not women. Quantitative trait linkage analyses in 1044 pairs of siblings, by using both ACE D/I and a nearby microsatellite polymorphism of the human growth hormone gene, supported a role of the ACE locus in influencing blood pressure in men but not in women. CONCLUSIONS In our large, population-based sample, there is evidence for association and genetic linkage of the ACE locus with hypertension and with diastolic blood pressure in men but not women. Our data support the hypothesis that ACE, or a nearby gene, is a sex-specific candidate gene for hypertension. Confirmatory studies in other large population-based samples are warranted.

Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease

Van Buchem disease is an autosomal recessive skeletal dysplasia characterised by generalised bone overgrowth, predominantly in the skull and mandible. Clinical complications including facial nerve palsy, optic atrophy, and impaired hearing occur in most patients. These features are very similar to those of sclerosteosis and the two conditions are only differentiated by the hand malformations and the tall stature appearing in sclerosteosis. Using an extended Dutch inbred van Buchem family and two inbred sclerosteosis families, we mapped both disease genes to the same region on chromosome 17q12-q21, supporting the hypothesis that van Buchem disease and sclerosteosis are caused by mutations in the same gene. In a previous study, we positionally cloned a novel gene, called SOST, from the linkage interval and identified three different, homozygous mutations in the SOST gene in sclerosteosis patients leading to loss of function of the underlying protein. The present study focuses on the identification of a 52 kb deletion in all patients from the van Buchem family. The deletion, which results from a homologous recombination between Alu sequences, starts approximately 35 kb downstream of the SOST gene. Since no evidence was found for the presence of a gene within the deleted region, we hypothesise that the presence of the deletion leads to a down regulation of the transcription of the SOST gene by a cis regulatory action or a position effect.

Absence of Association or Genetic Linkage between the Angiotensin-Converting–Enzyme Gene and Left Ventricular Mass

BACKGROUND Homozygous carries of the D allele of the angiotensin-converting-enzyme (ACE) gene have been reported to be at increased risk for various cardiovascular disorders, including left ventricular hypertrophy. We investigated the potential role of the ACE gene in influencing left ventricular mass. METHODS Quantitative echocardiographic data and DNA samples were available for 2439 subjects from the Framingham Heart Study. ACE genotypes were determined by an assay based on the polymerase chain reaction. (The D allele of the ACE gene contains a deletion, whereas the I [insertion] allele does not.) Left ventricular mass and the prevalence of left ventricular hypertrophy, adjusted for clinical covariates, were analyzed according to genotype. Genetic linkage between the ACE locus and left ventricular mass was evaluated by quantitative analysis of pairs of siblings. RESULTS The ACE genotype was associated neither with left ventricular mass nor with the prevalence of left ventricular hypertrophy. Mean (+/-SE) left ventricular mass (adjusted for sex) among subjects carrying the DD, DI, and II genotypes was 165+/-1.6, 165+/-1.3, and 166+/-2.0 g, respectively (P=0.90). The prevalence of left ventricular hypertrophy among the three genotype groups was 15.6 percent, 13.6 percent, and 15.6 percent, respectively (P=0.36), and the adjusted relative risk of left ventricular hypertrophy associated with the DD genotype was 1.10 (95 percent confidence interval, 0.86 to 1.19). Linkage analysis in 759 pairs of siblings using both the ACE D/I marker and a microsatellite polymorphism at the neighboring locus for the human growth hormone gene failed to support any role of ACE in influencing left ventricular mass. CONCLUSIONS The ACE genotype showed no association with echocardiographically determined left ventricular mass, nor did it confer an increased risk of left ventricular hypertrophy. We found no appreciable role of the ACE gene in influencing left ventricular mass.

Increased Platelet Aggregability Associated With Platelet GPIIIa PlA2 Polymorphism

The platelet glycoprotein IIb/IIIa (GP IIb/IIIa) plays a pivotal role in platelet aggregation. Recent data suggest that the PlA2 polymorphism of GPIIIa may be associated with an increased risk for cardiovascular disease. However, it is unknown if there is any association between this polymorphism and platelet reactivity. We determined GP IIIa genotype and platelet reactivity phenotype data in 1422 subjects from the Framingham Offspring Study. Genotyping was performed using PCR-based restriction fragment length polymorphism analysis. Platelet aggregability was evaluated by the Born method. The threshold concentrations of epinephrine and ADP were determined. Allele frequencies of PlA1 and PlA2 were 0.84 and 0.16, respectively. The presence of 1 or 2 PlA2 alleles was associated with increased platelet aggregability as indicated by incrementally lower threshold concentrations for epinephrine and ADP. For epinephrine, the mean concentrations were 0.9 micromol/L (0.9 to 1.0) for homozygous PlA1, 0.7 mmol/L (0.7 to 0.9) for the heterozygous PlA1/PlA2, and 0.6 micromol/L (0.4 to 1.0) for homozygous PlA2 individuals, P=0.009. The increase in aggregability induced by epinephrine remained highly significant (P=0.007) after adjustment for covariates. For ADP-induced aggregation, the respective mean concentrations were 3.1 micromol/L (3.0 to 3.2), 3.0 micromol/L (2.9 to 3.2), and 2.8 micromol/L (2.4 to 3.3); P=0.19 after adjustment for covariates. Our findings indicate that molecular variants of the gene encoding GP IIIa play a role in platelet reactivity in vitro. Our observations are compatible with and provide an explanation for the reported association of the PlA2 allotype with increased risk for cardiovascular disease.

Risk of Coronary Artery Disease Associated With Polymorphism of the Cytochrome P450 Epoxygenase CYP2J2

Background—Cytochrome P450 (CYP) 2J2 is expressed in the vascular endothelium and metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs). The EETs are potent endogenous vasodilators and inhibitors of vascular inflammation. However, it is not known whether genetic polymorphisms of CYP2J2 are associated with increased cardiovascular risks. Methods and Results—All 9 exons of the CYP2J2 gene and its proximal promoter were sequenced in 132 patients to identify potential variants. Functional consequence of a single nucleotide polymorphism (SNP) in the promoter of CYP2J2 was further evaluated by use of transcription factor-binding and reporter assays. A total of 17 polymorphisms were identified. One of the most relevant polymorphisms in terms of frequency and functional importance is located at −50 (G-50T) in the proximal promoter of CYP2J2. Screening of 289 patients with coronary artery disease and 255 control subjects revealed 77 individuals with the G-50T SNP (17.3% of coronary artery disease patients, 10.6% of control subjects; P=0.026). The association of the G-50T polymorphism remained significant after adjustment for age, gender, and conventional cardiovascular risk factors (OR, 2.23; 95% CI, 1.04 to 4.79). The G-50T mutation resulted in the loss of binding of the Sp1 transcription factor to the CYP2J2 promoter and resulted in a 48.1±2.4% decrease in CYP2J2 promoter activity (P<0.01). Plasma concentrations of stable EET metabolites were significantly lower in individuals with the G-50T SNP. Conclusions—A functionally relevant polymorphism of the CYP2J2 gene is independently associated with an increased risk of coronary artery disease.

Genetic and Environmental Contributions to Platelet Aggregation The Framingham Heart Study

Background—Platelet aggregation plays an important role in arterial thrombosis in coronary heart disease, stroke, and peripheral arterial disease. However, the contribution of genetic versus environmental influences on interindividual variation in platelet aggregability is poorly characterized. Methods and Results—We studied the heritability of platelet aggregation responses in 2413 participants in the Framingham Heart Study. The threshold concentrations of epinephrine and ADP required to produce biphasic platelet aggregation and collagen lag time were determined. Mixed-model linear regression was used to calculate correlation coefficients within sibships and within spouse pairs. Variance and covariance component methods were used to estimate the proportion of platelet aggregation attributable to measured covariates versus additive genetic effects. After accounting for environmental covariates, the adjusted sibling correlations for epinephrine, ADP, and collagen lag time were 0.24, 0.22, and 0.31, respectively (P =0.0001 for each). In contrast, adjusted correlations for spouse-pairs were −0.01, 0.05, and −0.02, respectively (all P >0.30). The estimated heritabilities were 0.48, 0.44, and 0.62, respectively. Measured covariates accounted for only 4% to 7% of the overall variance in platelet aggregation, and heritable factors accounted for 20% to 30%. The platelet glycoprotein IIIa PlA2 polymorphism and the fibrinogen Hind III &bgr;-148 polymorphism contributed <1% to the overall variance. Conclusions—In our large, population-based sample, heritable factors play a major role in determining platelet aggregation, and measured covariates play a lesser role. Future studies are warranted to identify the key genetic variants that regulate platelet function and to lay the groundwork for rational pharmacogenetic approaches.

Cardiac angiotensinogen and its local activation in the isolated perfused beating heart.

Increasing evidence suggests that the renin-angiotensin system modulates cardiovascular homeostasis both via its circulating, plasma-borne components and through locally present, tissue-resident systems with site-specific activity. The existence of such a system in the heart has been proposed, based on biochemical studies as well as on the demonstration of renin and angiotensinogen messenger RNA in cardiac tissue. We conducted the present study to determine whether biologically active angiotensin peptides may be cleaved within the heart from locally present angiotensinogen. Isolated, perfused rat hearts were exposed to infusions of purified hog renin; the coronary sinus effluent was collected and subsequently assayed for angiotensin I (Ang I) and angiotensin II (Ang II) by high-pressure liquid chromatography and specific radioimmunoassay. Both Ang I and II were undetectable under control conditions but appeared promptly after the addition of renin. Dose-dependent peak values for Ang I release ranged from 2.42 +/- 0.65 fmol/min to 1.38 +/- 0.18 pmol/min during renin infusions at concentrations between 10 microunits/ml and 5 milliunits/ml. Ang II levels measured in the perfusate reflected a mean fractional intracardiac conversion of Ang I to Ang II of 7.18 +/- 1.09%. Generation of Ang I and Ang II was inhibited in the presence of specific inhibitors of renin and converting enzyme, respectively. To investigate the source of angiotensinogen, we measured spontaneous angiotensinogen release from isolated perfused hearts. In the absence of renin in the perfusate, angiotensinogen was initially released in high, but rapidly declining, concentrations and subsequently at a low, but stable, rate. Prior perfusion with angiotensinogen-rich plasma resulted in enhanced early angiotensinogen release but did not alter the second, delayed phase, suggesting that, in addition to plasma-derived substrate, locally produced angiotensinogen may also participate in the intracardiac formation of angiotensin. Supporting this interpretation, hearts from animals pretreated with dexamethasone showed increased angiotensinogen messenger RNA concentrations as well as increased rates of angiotensinogen release not only during the early but also during the late phase. Our study newly demonstrates that Ang I and II may be formed within the isolated heart from locally present substrate, which appears to be derived in part from the circulating pool and in part from endogenous synthesis. These findings add support to the concept of a functionally active and locally integrated cardiac renin-angiotensin system and emphasize its potential physiological and pathological relevance.

Genetic Polymorphism of Methylenetetrahydrofolate Reductase and Myocardial Infarction A Case-Control Study

BACKGROUND Elevated total plasma homocyst(e)ine (tHcy; the composite of homocysteine-derived moieties in their oxidized and reduced forms) levels are a risk factor for coronary heart disease, stroke, and venous thrombosis. tHcy plasma levels are influenced by folate, vitamins B6 and B12, as well as by hereditary factors. A point mutation (C677T) in the gene encoding methylenetetrahydrofolate reductase, an enzyme involved in homocysteine remethylation, has been reported to render the enzyme thermolabile and less active and has been associated with elevated tHcy in homozygous carriers (+/+ genotype) as well as with increased risk of premature cardiovascular disease. METHODS AND RESULTS We investigated whether this mutation influences risk for myocardial infarction (MI) and plasma levels of tHcy and whether this effect may be modified by dietary folate intake in 190 MI cases and 188 control subjects from the Boston Area Health Study. Genotype frequencies were 37.8% for -/-, 47.8% for +/-, and 14.4% for +/+ in the control group and 50.0% for -/-, 34.7% for +/-, and 15.3% for +/+ in the case group. The relative risk for MI associated with the +/+ genotype (compared with +/- and -/-) was 1.1 (95% CI, 0.6 to 1.9; P = .8). Stratification by folate intake values above and below the median did not significantly alter these results. Plasma tHcy levels were 9.9 +/- 2.7 mumol/L in -/- individuals, 10.6 +/- 3.8 mumol/L in +/- individuals, and 9.1 +/- 2.3 mumol/L in +/+ individuals (Ptrend = NS; determined in 68 cases and 59 control subjects). CONCLUSIONS Our data show that homozygosity for the C677T mutation in this largely white, middle-class US population is not associated with increased risk for MI, irrespective of folate intake. This suggests that this mutation does not represent a useful marker for increased cardiovascular risk in this and in similar populations.

Angiotensin-Converting Enzyme I/D Polymorphism and Arterial Wall Thickness in a General Population The Vobarno Study

BACKGROUND It has been reported that the D allele of an insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene is associated with conditions of increased cardiovascular risk, including left ventricular hypertrophy. METHODS AND RESULTS Considering that a genetically determined overactivity of the renin-angiotensin system may influence cardiac as well as vascular growth, we investigated possible relations between ACE I/D genotype and carotid artery wall thickness (B-mode ultrasound) in 199 subjects, 50 to 64 years old, sampled from the general population of Vobarno, a small town in northern Italy. ACE DD genotype was associated with significantly higher common carotid artery intima-media thickness (P = .003). The occurrence of carotid atherosclerotic plaques was similar in the different genotypes. There was no association of the ACE I/D genotype with blood pressure values (either casual of 24-hour ambulatory monitored). CONCLUSIONS ACE DD genotype may be considered a risk factor for the development of common carotid intima-media thickening in our study population.