Genetics in drug discovery and development: challenge and promise of individualizing treatment in common complex diseases.
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Drug Design↗
Biomedical subjects
K Lindpaintner
Publications and source records attributed to K Lindpaintner.
Interstrain differences in angiotensin I-converting enzyme mRNA and activity levels. Comparison between stroke-prone spontaneously hypertensive rats and Wistar-Kyoto rats.
Plasma angiotensin I-converting enzyme (ACE) levels are different between the stroke-prone spontaneously hypertensive rat (SHRSPHD) and the normotensive Wistar-Kyoto (WKYHD) rat. This interstrain variability in plasma ACE levels is independent of blood pressure and is genetically linked to the ACE gene. The present study explored the hypothesis of an interstrain variability of tissue ACE activity and ACE gene expression levels. Tissue ACE levels were studied by enzymic activity measurement in the membrane fraction, and ACE mRNA levels were quantified by solution hybridization-ribonuclease protection assay. In lung, heart, kidney, and duodenum, membrane-bound ACE activity and ACE mRNA amount were significantly higher in WKYHD rats compared with SHRSPHD rats. No difference was observed in the testis where a specific isoform of the enzyme is produced. Our results suggest that in addition to determine differential plasma ACE levels between the WKYHD and SHRSPHD strains, the interstrain genetic variability also determines differential ACE mRNA and membrane-bound enzyme levels in somatic tissues. This likely reflects a difference in the ACE gene expression due to genetically determined regulatory mechanisms operative in all somatic tissues.
Animals↗
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.
Adult↗
Angiotensinogen gene and hypertension in Chinese.
The renin-angiotensin system plays a major role in regulating blood pressure and maintaining electrolyte and volume homeostasis. Previously, the angiotensinogen gene, which encodes the key substrate for renin within this system, has been reported linked to and associated with essential hypertension in White Europeans, African-Caribbeans, and Japanese. Therefore, we investigated whether the angiotensinogen gene might be similarly implicated in the pathogenesis of essential hypertension in Chinese by carrying out linkage analysis in 310 hypertensive sibling pairs. Genotypes for two diallelic DNA polymorphisms observed at amino acid residues 174 (T174M) and 235 (M235T) within the coding sequence and for two highly informative dinucleotide (GT)-repeat sequences (one in the 3' flanking region, and one at a distance of 6.1 cM from the gene) were determined. Affected sibpair analysis conducted according to three different algorithms (S.A.G.E./SIBPAL, MAPMAKER/ SIBS, and APM methods) revealed no evidence for linkage of the angiotensinogen gene to hypertension. Our data indicate that molecular variants of this gene do not appear to contribute materially to the pathogenesis of primary hypertension among Chinese (a notion supported by concomitant, direct estimates of power), and that the disease relevance of this gene may vary therefore depending on ethnicity.
Alleles↗
Salt susceptibility maps to chromosomes 1 and 17 with sex specificity in the Sabra rat model of hypertension.
Random genome screening was initiated in the Sabra rat model of hypertension in search of genes that account for salt sensitivity or salt resistance in terms of the development of hypertension. Female salt-sensitive Sabra hypertension-prone (SBH/y) rats were crossed with male salt-resistant Sabra hypertension-resistant (SBN/y) rats, resulting in an F2 cohort consisting of 100 males and 132 females. Systolic blood pressure (BP) was measured in rats at 6 weeks of age under basal conditions and after 4 weeks of salt loading. Genotypes for 24 polymorphic microsatellite markers localized to chromosome 1 and for 8 markers localized to chromosome 17 were determined in F2 and cosegregation with BP was evaluated by ANOVA and multipoint linkage analysis. Basal BP did not cosegregate with any locus on chromosomes 1 or 17. In contrast, BP after salt loading showed significant cosegregation with three QTLs, two on chromosome 1 and one on chromosome 17, designated SS1a, SS1b, and SS17, respectively; the maximal logarithm of the odds (LOD) scores were 4.71, 4.91, and 3.43, respectively. Further analysis revealed sexual dimorphism. In male F2, BP response to salt loading cosegregated with one QTL (LOD score 4.52) and a second QTL (LOD score 2.98), both on chromosome 1 and coinciding with SS1a and SS1b, respectively. In female rats, BP response cosegregated with one QTL on chromosome 1 (LOD score 3.08) coinciding with SS1b, and with a second QTL on chromosome 17 (LOD score 3.66) coinciding with SS17. In males, the additive effects of the two QTLs on chromosome 1 accounted for most of the BP variance to salt loading, whereas in females the additive effects of the QTLs on chromosomes 1 and 17 accounted for over two thirds of the variance. These results identify three putative gene loci on chromosomes 1 and 17 that contribute importantly to salt sensitivity and/or resistance and uncover sex specificity in the role that salt susceptibility genes fulfill in the development of hypertension.
Analysis of Variance↗
Three candidate genes and angiotensin-converting enzyme inhibitor-related cough: a pharmacogenetic analysis.
Unexplained, persistent cough limits the use of angiotensin-converting enzyme (ACE) inhibitors in a significant number of patients. It has been speculated that occurrence of this adverse effect is genetically predetermined; in particular, variants of the genes encoding ACE, chymase, and B2-bradykinin receptor have been implicated. To investigate this question, we determined genotypes for common polymorphisms for these three genes in subjects with a history of ACE inhibitor-related cough. Specificity of the adverse effect was confirmed by a blinded, double-crossover design protocol in which subjects were rechallenged with either lisinopril or placebo. In 99 case subjects and 70 control subjects (who failed to develop cough on rechallenge with ACE inhibitor) thus selected, frequencies for the ACE D and I alleles were 0.56 and 0.44 (cases) and 0.56 and 0.44 (controls), respectively; frequencies for chymase A and B alleles (absence/presence of BstXI site) were 0.56 and 0.44 (cases) and 0.46 and 0.54 (controls), respectively; frequencies for B2-bradykinin receptor + and - alleles (presence/absence of a 21 to 29 nonanucleotide sequence) were 0.52 and 0.48 (cases) and 0.53 and 0.47 (controls), respectively. All observed genotype frequencies were in Hardy-Weinberg equilibrium. There was no evidence for association between genotype at either gene examined and cough (adjusted for gender and age). Our data indicate that common genetic variants of ACE, chymase, and B2-bradykinin receptor do not explain the occurrence of ACE inhibitor-related cough.
Angiotensin-Converting Enzyme Inhibitors↗
Tissue- and development-specific expression of multiple alternatively spliced transcripts of rat neuronal nitric oxide synthase.
Nitric oxide (NO) functions as an intercellular messenger and mediates numerous biological functions. Among the three isoforms of NO synthase that produce NO, the ubiquitously expressed neuronal NO synthase (nNOS) is responsible for a large part of NO production, yet its regulation is poorly understood. Recent reports of two alternative spliceforms of nNOS in the mouse and in man have raised the possibility of spatial and temporal modulation of expression. This study demonstrates the existence of at least three transcripts of the rat nNOS gene designated nNOSa, nNOSb, and nNOSc, respectively, with distinct 5' untranslated first exons that arise from alternative splicing to a common second exon. Expression of the alternative transcripts occurs with a high degree of tissue and developmental specificity, as demonstrated by RNase protection assays on multiple tissues from both fetal and adult rats. Furthermore, terminal differentiation of rat pheochromocytoma-derived PC12 cells into neurons is associated with induction of nNOSa, suggesting, likewise, development- and tissue-specific transcriptional control of nNOS isoform expression. Physical mapping using a rat yeast artificial chromosome clone shows that the alternatively spliced first exons 1a, 1b, and 1c are separated by at least 15-60 kb from the downstream coding sequence, with exons 1b and 1c being positioned within 200 bp of each other. These findings provide evidence that the biological activity of nNOS is tightly and specifically regulated by a complex pattern of alternative splicing, indicating that the notion of constitutive expression of this isoform needs to be revised.
Alternative Splicing↗
Molecular cloning and expression of rat kallistatin gene.
We have previously purified and cloned human kallistatin and rat kallikrein-binding protein (RKBP), which are tissue kallikrein inhibitors belonging to the serine proteinase inhibitor superfamily. In this study, we have cloned and sequenced the gene encoding rat kallistatin with Phe-Phe-Ser-Ala-Gln at positions P2-P3', which is identical to the reactive center of human kallistatin. Rat kallistatin is highly similar to human kallistatin, sharing 68% and 57% sequence identity at the cDNA and the amino acid levels. The rat kallistatin gene exists in a single copy and is located on chromosome 6. An SphI RFLP is found between SHR and WKY rats at or near the rat kallistatin gene locus. Two amino acid polymorphisms of the rat kallistatin gene between these two strains were found by sequence analysis. A candidate promoter in the 5'-flanking region (109 bp) of the rat kallistatin gene has been identified by reporter assays. The expression of rat kallistatin in the liver is growth-dependent and down-regulated during acute phase inflammation. Recombinant rat kallistatin produced in E. coli is able to bind to tissue kallikrein, and the interaction is inhibited by heparin. These characteristics define rat kallistatin as the counterpart of human kallistatin.
Amino Acid Sequence↗
Evidence for primary genetic determination of heart rate regulation: chromosomal mapping of a genetic locus in the rat.
BACKGROUND: We investigated whether an accelerated heart rate (HR), observed in the stroke-prone spontaneously hypertensive rat (SHRSP(HD)), is a primary, genetically determined trait and whether it contributes to blood pressure (BP) regulation in this model of polygenic hypertension. METHODS AND RESULTS: We measured BP and HR in SHRSP(HD) and normotensive Wistar-Kyoto rats (WKY), as well as in F2 hybrids bred from crossing the two strains, at baseline and after 12 days of dietary NaCl loading. Random marker genome screening and cosegregation analysis were performed on F2 hybrids derived from SHRSP(HD)/WKY-0(HD) (n=115) and SHRSP(HD)/WKY-1(HD) (n=139) crosses (WKY-0(HD) and WKY-1(HD) are two congenic WKY strains). HR in SHRSP(HD) was significantly higher than in WKY-0(HD) both at baseline (404+/-30 versus 375+/-46 bpm; P=.0034) and after NaCl (437+/-23 versus 364+/-40 bpm; P=10(-9)). BP in F2 hybrids showed no significant correlation with HR either at baseline or after NaCl loading. HR after NaCl loading but not at baseline was significantly linked in a recessive fashion to a locus on chromosome 3: in animals homozygous for the SHRSP(HD) allele, HR was 414+/-49 compared with 383+/-44 bpm in heterozygotes and WKY homozygotes (F(210,1)=19.7, P=1.4x10(-5), lod score=5.9). The putative BP-relevant gene at this locus, termed HR-SP1, showed no evidence of linkage to any of the BP parameters measured. CONCLUSIONS: Our results demonstrate that a genetic locus on rat chromosome 3, HR-SP1, contributes directly to the regulation of HR in SHRSP(HD) but exhibits no effect on BP. Thus, in addition to its modulation by reflex-mediated neurohumoral mechanisms, HR is also under the direct influence of primary genetic factors.
Analysis of Variance↗
Genetics of interventional cardiology. Old principles, new frontiers.
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Cardiovascular Diseases↗
Angiotensin AT1 receptor inhibition. Effects on hypertrophic remodeling and ACE expression in rats with pressure-overload hypertrophy due to ascending aortic stenosis.
BACKGROUND: We tested the hypothesis that long-term administration of the specific angiotensin II subtype 1 (AT1)-receptor blocker BMS-186295 will regress hypertrophy and modify left ventricular angiotensin converting enzyme (ACE) expression in rats with ascending aortic stenosis. METHODS AND RESULTS: Six weeks after surgery, rats with ascending aortic stenosis were randomized to receive either the AT1-receptor blocker BMS-186295 50 mg.kg-1.d-1 (n = 49), amlodipine 2.5 mg.kg-1.d-1 (n = 48) as a positive control for systemic vasodilation, or no drug (n = 48) and compared with sham-operated rats (n = 39). Drug treatment was continued for 15 weeks. Left ventricular ACE mRNA levels were measured by ribonuclease protection assay. The left ventricular/body weight ratio was increased 43% in hearts from rats with untreated left ventricular hypertrophy (LVH) versus control hearts (P < .05). However, there was no difference in either the left ventricular/body weight ratio (2.78 +/- 0.08 versus 2.81 +/- 0.20 mg/g; P = NS) or myocyte cross-sectional area in the AT1-blocker-treated versus untreated LVH hearts. Amlodipine also showed no effect on regression of hypertrophy. In vivo left ventricular systolic pressure was significantly higher in untreated LVH versus sham-operated rats (193 +/- 8 versus 118 +/- 4 mm Hg; P < .05), and there was a similar severe elevation of left ventricular systolic pressure in the AT1-blocker- and amlodipine-treated LVH groups (189 +/- 9 and 188 +/- 16 mm Hg; P = NS versus untreated LVH). In vivo left ventricular end-diastolic pressure was higher in the untreated LVH than in the sham-operated rats (14.8 +/- 2.3 versus 7.0 +/- 0.5 mm Hg; P < .05). Left ventricular end-diastolic pressure was lower in the AT1-blocker-treated (11.0 +/- 1.7 mm Hg) and amlodipine-treated rats (11.5 +/- 1.8 mm Hg) and was similar to left ventricular end-diastolic pressure in the sham-operated rats (P = NS). Left ventricular ACE mRNA levels were elevated in untreated LVH rats but were normalized in both the AT1-blocker-treated rats and amlodipine-treated rats. CONCLUSIONS: Long-term AT1-receptor blockade did not regress LVH in rats with persistent systolic pressure overload due to ascending aortic stenosis. However, both AT1-receptor blockade and amlodipine improved in vivo left ventricular end-diastolic pressure in association with the normalization of left ventricular ACE mRNA levels.
Angiotensin Receptor Antagonists↗
PIA1/A2 polymorphism of platelet glycoprotein IIIa and risks of myocardial infarction, stroke, and venous thrombosis.
BACKGROUND: The gene encoding the platelet glycoprotein IIIa receptor (GPIIIa), shows polymorphism (PIA1/A2). In a previous study, men with acute coronary ischaemia were more likely than controls to carry the PIA2 allele. This receptor has an important role in acute thrombus formation; these findings therefore raise the possibility of inherited platelet risk factors for both arterial and venous thrombosis. We investigated whether the PIA2 allele is associated with myocardial infarction, stroke, and venous thrombosis in a large prospective cohort of men in the USA. METHODS: 14916 initially healthy men participating in the Physicians Health Study provided baseline blood specimens for DNA analysis and were followed prospectively for a mean of 8.6 years. 374 men had a first myocardial infarction, 209 stroke, and 121 venous thrombosis during follow-up (704 cases). Distribution of the PIA1/A2 polymorphism was investigated by a PCR based on restriction-fragment length polymorphism in these men and in a sample of 704 matched study participants who remained free of thrombosis during follow-up (controls). FINDINGS: The frequency of the PIA2 allele was similar to the control frequency (14.8%) among men who had myocardial infarction (13.5%, p = 0.4), stroke (13.4%, p = 0.5), or venous thrombosis (14.5%, p = 0.9). The relative risk of any vascular event among men homozygous or heterozygous for PIA2 compared with men homozygous for PIA1 was 0.96 (95% CI 0.8-1.2). We found no evidence of association between the PIA2 allele and myocardial infarction (relative risk 0.93 [95 0.7-1.2]), stroke (0.93 [0.7-1.3]), or venous thrombosis (1.07 [0.7-1.6]). There was no evidence of association in subgroup analyses by age, smoking status, and presence of family history of premature coronary disease, hypercholesterolaemia, hypertension, or diabetes. Aspirin use had no effect on these findings. INTERPRETATION: In a large cohort of apparently healthy men, carriage of the GPIIIa PIA2 allele was not associated with any increase in subsequent risk of myocardial infarction, stroke, or venous thrombosis.
Adult↗
Construction and characterization of a 10-genome equivalent yeast artificial chromosome library for the laboratory rat, Rattus norvegicus.
Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20-30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species.
Animals↗
Arterial and venous thrombosis is not associated with the 4G/5G polymorphism in the promoter of the plasminogen activator inhibitor gene in a large cohort of US men.
BACKGROUND: The 4G allele of the 4G/5G polymorphism in the promoter of the plasminogen activator inhibitor (PAI-1) gene is associated with increased PAI-1 activity. In a small group of young Swedish men, this allele has been reported to predict risk of myocardial infarction. Whether this polymorphism increases risk of arterial and venous thrombosis among middle-aged men is unknown. METHODS AND RESULTS: Among 14916 men 40 to 84 years old participating in the Physicians' Health Study who provided baseline blood samples for DNA analysis, 374 suffered first myocardial infarction and 121 had venous thromboembolism during 8.6 years of follow-up. Distributions of the 4G/5G polymorphism in the PAI-1 gene promoter were assessed in these men as well as in a sample of study participants matched on age and smoking who did not develop vascular occlusion during the prospective follow-up period. The distributions of the 4G/4G, 4G/5G, and 5G/5G genotypes among men who developed myocardial infarction (0.27, 0.51, 0.22; P = .7) or venous thromboembolism (0.30, 0.49, 0.21; P = .5) were virtually identical to those of men who remained free of vascular disease (0.27, 0.50, 0.23). Thus, the relative risk of future thrombosis among those with the 4G/4G genotype compared with those without the 4G/4G genotype was 1.02 (95% CI, 0.8 to 1.3). There was no effect modification by age, smoking status, family history of premature thrombosis, history of hypertension, hypercholesterolemia, or aspirin use. CONCLUSIONS: These data indicate that the 4G/5G polymorphism in the promoter of the PAI-1 gene is not a major pathogenetic risk factor for arterial or venous thrombosis among middle-aged men.
Adult↗
Two specific and simple methods for genotyping of the paraoxonase/arylesterase A/B polymorphism.
We developed an amplification refractory mutation system (ARMS) and mutagenically separated PCR (MS-PCR) assays for genotyping of the paraoxonase/arylesterase (PONA) A/B polymorphism as practical, reliable, and more economic alternatives to the conventional restriction fragment length polymorphism (RFLP) technique for detection of this mutation.
Aryldialkylphosphatase↗
Urinary albumin excretion and atherosclerosis in essential hypertension.
1. Increased urinary albumin excretion is common in patients with essential hypertension and is at least to some extent correlated with prevailing blood pressure levels. However, the generalized vascular dysfunction present in advanced atherosclerotic disease may independently influence this parameter. 2. To evaluate this possibility, we assessed blood pressure, ultrasonographic carotid thickness, cardiac mass, minimum forearm vascular resistances, metabolic parameters and the angiotensin-converting enzyme genotype in patients with untreated essential hypertension and atherosclerotic peripheral vascular disease (n = 11). The results were compared with similar data obtained in matched groups of patients with uncomplicated hypertension and with normotensive control subjects (n = 11 per group). 3. Urinary albumin excretion was higher in hypertensive patients with atherosclerosis than in those without complications; carotid thickness was higher in atherosclerotic patients and a positive, statistically significant correlation existed between this parameter and urinary albumin excretion. In the same patient group, systolic blood pressure, fasting insulin and triacylglycerol levels were elevated and correlated with urinary albumin levels. However, differences in urinary albumin excretion persisted after taking into account the influence of those parameters by analysis of covariance. The distribution of angiotensin-converting enzyme genotype patterns and values of cardiac mass and minimum forearm vascular resistances did not differ significantly among the experimental groups. 4. The data suggest that vascular status may influence urinary albumin excretion in patients with essential hypertension, while confirming the importance of systolic blood pressure levels as a determinant of the raised urinary albumin excretion.
Adult↗
Mapping of both autosomal recessive and dominant variants of pseudoxanthoma elasticum to chromosome 16p13.1.
Pseudoxanthoma elasticum (PXE) is a classic inherited disorder of the elastic tissue characterized by progressive calcification of elastic fibers with a pathognomonic histological appearance. The clinical manifestations of PXE typically involve the skin, the eye and the cardiovascular system, resulting in skin lesions, decreased vision and vascular disease. Clinically, a more common autosomal recessive and a less common autosomal dominant pattern of inheritance, with high penetrance, have been described; the estimated prevalence of the disease is 1 in 70,000-100,000. Previous failure to link the disease to any of several candidate genes prompted us to conduct a genome-wide screen on a collection of 38 families with two or more affected siblings, using allele sharing algorithms. Excess allele sharing was found on the short arm of chromosome 16 and confirmed by conventional linkage analysis, localizing the disease gene under a recessive model with a maximum two point lod score of 21.27 on chromosome 16p13.1, an area so far devoid of any obvious candidate genes. Under a dominant transmission pattern linkage with a maximum two point lod score of 14.53 was observed to the same region. Linkage heterogeneity analysis predicted the presence of allelic heterogeneity with different variants of a single gene that resides in this chromosomal region accounting for recessive and dominant forms of PXE.
Adult↗