Risk factor-gene interaction in carotid atherosclerosis: effect of gene polymorphisms of renin-angiotensin system.

The risk factor-gene interaction in carotid atherosclerosis was investigated in 205 community-dwelling healthy subjects aged 50 years or more in Japan. The intima-media thickness (IMT) of the common carotid artery was evaluated by ultrasonography with a 7.5-MHz probe. Gene polymorphisms were determined for each subject with angiotensin-converting enzyme (ACE) insertion/deletion (I/D), angiotensinogen (AGT) M235T, angiotensin II type 1 receptor (AT1R) A1166C, and apolipoprotein E (apoE) genotypes. There was no genotype-specific difference in carotid IMT among any genes examined. Combinations of genotypes did not increase carotid IMT compared with subjects without these genotypes. In the total population, multiple regression analysis showed that age, systolic blood pressure (SBP), sex, and body mass index (BMI) were significantly associated with carotid IMT. However, the association between risk factors and IMT was genotype-specific. Age was significantly associated with IMT in ACE D carriers, but not in subjects with the ACE II genotype. Analysis of covariance adjusted with other risk factors showed that the age-dependent change in IMT was significantly different between subjects with the ACE II genotype and the ACE D carriers (F[1.196] = 4.97; P = 0.027). Similarly, the regression of IMT on SBP was significantly different between AGT TT and AGT MT + MM (F[1.196] = 7.20; P = 0.0079). The regression of IMT on BMI was also significantly different between apo E4 carriers and noncarriers (F[1.196] = 6.78; P = 0.0099). Furthermore, general linear model analysis with risk factors, genotype, and risk factor-genotype interactions revealed that the age*ACE genotype interaction, the SBP*AGT genotype interaction, and the BMI*apoE genotype interaction were significantly associated with IMT. These findings further support the role of risk factor-gene interaction in carotid atherosclerosis.

Association of ACE I/D polymorphism with cardiovascular risk factors.

Since the identification of an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene, the D allele has been recognized to be associated with cardiovascular disease. Moreover, significant associations of this polymorphism with multiple cardiovascular risk factors have been reported, although some studies failed to detect such associations. In the present study, we investigated the association of the ACE gene polymorphism with the parameters of multiple risk factors in 300 Japanese men who participated in a medical check-up. This investigation detected a significant association of the polymorphism with systolic blood pressure (P=0.007) and diastolic blood pressure (P=0.026), with their highest values in DD subjects and lowest values in II subjects. This significant association is consistent with the proposition that the polymorphism influences blood-pressure variability in men. Furthermore, we investigated the association of the polymorphism with four major disorders (obesity, hyperlipidemia, hypertension, diabetes mellitus) correlated with the risk for cardiovascular disease in the same 300 subjects. This investigation failed to detect any significant association of the polymorphism with each disorder. However, there was a trend that all four disorders were more frequent in ID and DD subjects than in II subjects. We therefore analyzed the association between the ACE gene polymorphism and having at least one of the four disorders in the same population. This analysis detected a significant difference: that ID and DD subjects had at least one of the four disorders more frequently than II subjects (P=0.008; odds ratio=1.89, 95% confidence interval= 1.19-2.99). Taken together, the results of this study are compatible with the proposition that the ACE polymorphism is associated with cardiovascular disease partially mediated through the four disorders in our population.

[A very elderly autopsy case of cecal cancer with pulmonary lymphangitis carcinomatosa].

A 91-year-old man was admitted with colliquative diarrhea, anorexia and weight loss. He had a history of healed tuberculosis, hypertension and atherosclerotic abdominal aortic aneurysms. On admission, shortness of breath without cough, exertional dyspnea, and ascites were also noticed. His chest X-ray and CT showed almost normal findings in the lung fields except for calcified old pleurisy. Since laboratory tests revealed thrombocytopenia, low fibrinogen, and increased CA19-9. DIC induced by an unknown cancer was diagnosed. He died on the eighth day due to progressive respiratory failure which did not respond to oxygen therapy. Autopsy revealed that he had a poorly differentiated adenocarcinoma in the cecum complicated with pulmonary lymphangitis carcinomatosa. Lymphangitis should be considered in the case of unexplained progressive respiratory failure developing in patient with cancer, even in the absence of X-ray findings.

Helicases and aging.

Studying monogenic hereditary disorders that manifest age-related phenotypes in cells, tissues, and the total organism would be helpful for clarifying the mechanisms of aging. In this context, seven human disorders that manifest age-related phenotypes have been found to be caused by aberrations of five proteins with seven helicase motifs conserved in most of the helicases. These disorders are xeroderma pigmentosum, Cockayne syndrome, trichothiodystrophy, Bloom syndrome, Werner syndrome, X-linked alpha-thalassemia/mental retardation syndrome, and Juberg-Marsidi syndrome. A decline of probably pleiotropic and fundamental function of helicases in these disorders is, therefore, implied to underlie not only the various age-related phenotypes of the disorders but also the pleiotropic and universal nature of ordinary aging. Consistent with this implication, studies of these seven disorders suggest that their various age-related phenotypes are caused by aberrations in multiple processes, especially transcription. Furthermore, a few studies imply some association between aberration of the helicases and phenotypes in ordinary aging.

Deletion polymorphism of ACE gene is associated with higher blood pressure after hospitalization in normotensive subjects.

Blood pressure has been shown to decrease in response to hospital admission. Several parameters including the decline of sympathetic nervous activity and negative sodium balance have been shown to be involved in this phenomenon. We investigated genetic influence on office BP and BP after hospitalization. One hundred and sixty-three men from the general population, free from antihypertensive medication, were enrolled in the present study. They stayed at the hospital for general medical check-up. BP was measured on the day of admission, and again the following day. Mean systolic blood pressure was significantly decreased after hospitalization from 117.3 +/- 9.9 mmHg to 115.3 +/- 12.8 mmHg (p=0.042). Subjects with DD+ID genotype showed a significantly higher systolic blood pressure after hospitalization than that of subjects with genotype II. There were no genotype specific differences in diastolic blood pressure or changes in blood pressure by the administration. In summary, systolic blood pressure after hospitalization was significantly higher in normotensive male subjects who possessed the D allele of ACE I/D polymorphism.

Transcriptional activation by the Werner syndrome gene product in yeast.

Werner syndrome (WS) is characterized by the premature occurrence of many age-related features. Before the cloning of the gene for WS (WRN), several reports suggested that transcriptional defects of genes may relate to the mechanisms of the occurrence of WS and natural aging. Because WRN, which encodes a helicase (WRN-H), has been cloned, we are attempting to clarify the mechanism of the transcriptional abnormalities found in WS cells, using WRN and WRN-H. In this article, we studied transcriptional activation of a promoter by WRN-H in a yeast assay system as a first step. The results showed that WRN-H functions as a transcriptional activator in the system. Furthermore, we performed additional transcriptional assays using various parts of WRN to define the critical region of WRN-H for transcriptional activation in yeast. The results revealed the critical region for the activation most likely mapped to the region of 315 to 403 aa. The region of 404 to 1309 aa may also effect activation in the presence of the critical region. The two regions contain an acidic domain, and the region of 404 to 1309 aa also contains a helicase domain. If this transcriptional activation by WRN-H occurs also in human cells in vivo, direct activation of the promoters by WRN-H could explain the results of somatic cell hybrid studies as well as the overexpressed genes detected in WS cells. However, our results should be interpreted with caution, because thus far, the transcriptional activation by WRN-H were only demonstrated using one promoter in a yeast system.

Molecular and epidemiological studies of Werner syndrome in the Japanese population.

Werner syndrome (WS) is an autosomal recessive genetic disease characterized by many age-related features. The gene responsible for WS (WRN) has been isolated and contains a helicase domain, but its function is unknown. Six different mutations throughout the WRN gene have been reported in the Japanese population. We have studied whether patients with a specific mutation exhibit distinct phenotypes from others. Fourteen patients with different mutations showed almost the same signs and symptoms and, therefore, the C terminal part of the product appears to be crucial for its functions, although other parts may be important as well. Haplotype analyses using 13 microsatellites covering the 2.8-3.0 cM WRN region showed that two out of six different mutations had founder chromosomes. These two founder chromosomes may be evenly distributed throughout the western part of Japan, suggesting that these mutations go back to a time earlier than 1400 years ago.

A high-density STS map based on a single contig of YAC and P1 clones in the chromosome 8p12-p21 region.

We have constructed a yeast artificial chromosome (YAC) and P1 contig in the 8p12-p21 region. The contig comprises 16 overlapping YAC clones and 44 overlapping P1 clones. Twelve dinucleotide-repeat polymorphic sequence-tagged site (STS)-markers that were previously isolated mainly from these YAC and P1 clones were genetically mapped. A total of 46 nonpolymorphic STS markers were newly established mainly from the YAC and P1 clone end fragments, and 28 of the 46 nonpolymorphic STSs, as well as the 12 polymorphic STSs, were also mapped physically onto the contig based on STS content analysis of YAC pools and of the P1 and YAC clones. As a result, the YAC and P1 clones were assembled into a single contig covering a minimum of 1.5 Mb physically and 2.8 cM genetically with 12 polymorphic and 28 nonpolymorphic STSs within the 8p12-p21 region. Average STS spacing in the contig was estimated to be 40 kb/STS. In addition, further characterization of the contig suggested that this contig includes a region where genetic recombination occurs frequently. Thus, the resulting cloned region, together with densely mapped STS markers on the contig, should help to promote our understanding of this region.

Association of a polymorphic variant of the Werner helicase gene with myocardial infarction in a Japanese population.

The Werner syndrome (WS) is a rare autosomal recessive progeroid syndrome characterized by the premature onset of multiple age-related disorders, including atherosclerosis, cancer, non-insulin-dependent diabetes mellitus (NIDDM), ocular cataracts and osteoporosis [Epstein et al., 1966]. The major cause of death (at a median age of 47) is myocardial infarction (MI) [Epstein et al., 1966]. The WS mutation involves a member (WRN) of the RecQ family of helicases and may perturb DNA replication, repair, recombination, transcription, or chromosomal segregation [Yu et al., 1996]. We now report data on 149 MI cases and age-matched controls suggesting that a polymorphic WRN variant is associated with increased risk for MI. Based on our data, homozygosity for a cysteine at amino acid 1367 (the most prevalent genotype) predicts a 2.78 times greater risk of MI (95% confidence intervals: 1.23 to 6.86). The variant was not significantly associated with NIDDM. The two alleles (cysteine vs. arginine) could influence helicase activity, turnover, macromolecular interactions or, alternatively, could be markers for haplotypes influencing WRN regulation or reflecting gene action at linked loci. However, given the caveats implicit in genetic association studies, it is imperative that the present results be replicated in independent populations.

Mutations in the consensus helicase domains of the Werner syndrome gene. Werner's Syndrome Collaborative Group.

Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid protein with homology to DNA and RNA helicases. Previous work identified four WS mutations in the 3' end of the gene, which resulted in predicted truncated protein products of 1,060-1,247 amino acids but did not disrupt the helicase domain region (amino acids 569-859). Here, additional WS subjects were screened for mutations, and the intron-exon structure of the gene was determined. A total of 35 exons were defined, with the coding sequences beginning in the second exon. Five new WS mutations were identified: two nonsense mutations at codons 369 and 889; a mutation at a splice-junction site, resulting in a predicted truncated protein of 760 amino acids; a 1-bp deletion causing a frameshift; and a predicted truncated protein of 391 amino acids. Another deletion is >15 kb of genomic DNA, including exons 19-23; the predicted protein is 1,186 amino acids long. Four of these new mutations either partially disrupt the helicase domain region or result in predicted protein products completely missing the helicase region. These results confirm that mutations in the WRN gene are responsible for WS. Also, the location of the mutations indicates that the presence or absence of the helicase domain does not influence the WS phenotype and suggests that WS is the result of complete loss of function of the WRN gene product.

Homozygous and compound heterozygous mutations at the Werner syndrome locus.

The Werner syndrome (WS) is a rare autosomal recessive progeroid disorder. The Werner syndrome gene (WRN) has recently been identified as a member of the helicase family. Four distinct mutations were previously reported in three Japanese and one Syrian WS pedigrees. The latter mutation was originally described as a 4 bp deletion spanning a spliced junction. It is now shown that this mutation results in a 4 bp deletion at the beginning of an exon. Nine new WRN mutations in 10 additional WS patients, both Japanese and Caucasian, are described. These include three compound heterozygotes (one Japanese and two Caucasian). The new mutations are located all across the coding region.

Narrowing the position of the Werner syndrome locus by homozygosity analysis-extension of homozygosity analysis.

Werner syndrome (WS) is an autosomal recessive disorder characterized by the premature occurrence of many age-related features. Previously, the WS gene (WRN) was mapped between D8S131 and D8S87, in an 8.3-cM interval. In this study, regions of homozygosity in 36 WS patients from inbred families were searched for by genotyping for 35 dinucleotide repeat polymorphic markers to narrow down the WRN critical region. The region most consistently homozygous in these patients was between the D8S1219/D8S1220 cluster and D8S278, within a 4.4-cM interval. For 16 markers mapped in this interval, 24 WS patients (22 Japanese patients and 2 Caucasian patients) in whom consanguinity failed to be proved were also genotyped, under the assumption that some of these patients might still be from consanguineous marriages. The data were analyzed by Fisher's exact test with a 2 x 2 contingency table for the 22 Japanese patients, excluding the 2 Caucasian patients. The frequencies of homozygosity in the 22 patients at 10 of 16 markers tested were significantly higher than those detected in the general population. Analysis of homozygosity patterns indicated that the region most consistently homozygous was between D8S1445 and D8S278. Thus the WRN locus is most likely between the two markers D8S1445 and D8S278, in a 1.6-cM interval.