Association of novel promoter single nucleotide polymorphisms in vasopressin V1a receptor gene with essential hypertension in nonobese Japanese.

We studied the association between four novel single nucleotide polymorphisms (SNPs) in the promoter region of V1aR gene and essential hypertension in 620 Japanese subjects (365 hypertensives and 255 healthy). A significant association was found between one of the genotypes and alleles at SNP -6951 and hypertension in a subsample of nonobese individuals. This association demonstrated an independent risk for nonobese hypertension.

A case of thyrotropin-producing pituitary adenoma, accompanied by an increase in anti-thyrotropin receptor antibody after tumor resection.

We describe a rare, but interesting, case of TSH-producing adenoma (TSHoma), accompanied by increases in both anti-TSH receptor antibody (TRAb) and thyroid-stimulating antibody (TSAb) after tumor resection. A 21-yr-old woman was referred to our department for further evaluation of pituitary tumor. In a nearby hospital, she had been diagnosed as having pituitary tumor. Her serum free T4, free T3, and TSH levels were all elevated concomitantly. On the basis of a diagnosis of pituitary adenoma with TSH production, transsphenoidal resection of the pituitary adenoma was performed. Two weeks after the operation, the blood concentrations of TSH were undetectable, whereas both TRAb and TSAb levels were elevated. TSAb levels gradually increased further from 2 weeks to 3 months after the operation, accompanied by an increase in TSH and free T4 levels. TSH is an important hormone in maintaining physiology and regulating immunomodulators in thyrocytes, as it can influence a variety of immune-regulating cytokine-like activities and inhibit expressions of Fas antigen, intracellular adhesion molecule-1, and class II trans-activator. Changes in TSH would modulate the immune circumstances in the thyroid, and then induce TRAb and TSAb. Autoimmune parameters with thyroid function should be observed carefully when managing patients with TSHoma.

A novel mutation of the thiazide-sensitive sodium chloride cotransporter gene in a Japanese family with Gitelman syndrome.

Gitelman syndrome is an inherited renal disorder characterized by impaired NaCl reabsorption in the distal convoluted tubule leading to hypokalemia, hypomagnesemia and normocalcemic hypocalciuria. It has been shown that this syndrome results from mutations in the gene encoding the thiazide-sensitive sodium chloride cotransporter (TSC). We performed the mutational analysis in the TSC gene of a 30-year-old Japanese woman with Gitelman syndrome and found two mutations at adjacent spots in both alleles. One was a frame shift mutation which generated stop codon at position 671, the other was a single nucleotide mutation, which resulted in an aminoacid substitution at position 672, Met to Ile. Her 52-year-old mother and two daughters had neither hypokalemia nor hypomagnesemia. However, her mother and her 8-year-old daughter had the Met672Ile mutation as heterozygotes. Her 4-year-old daughter had the same frame shift mutation as her mother, a heterozygotic mutation. These results suggest that Gitelman syndrome requires 2 compound heterozygotic mutations and the coexistence of the large deletion in the C-terminal domain with Met672Ile substitution of the TSC could impair the transporter activity underling the hypokalemia and hypomagnesemia in this patient.

Gene analysis of the calcium channel 1 subunit and clinical studies for two patients with hypokalemic periodic paralysis.

Hypokalemic periodic paralysis (HypoPP) is a skeletal muscle disorder in which episodic attacks of muscle weakness occur; they are associated with decreased serum potassium (K+) levels. Recent molecular approaches have clarified that the condition is caused by mutations in the skeletal muscle voltage-gated calcium channel 1 subunit (CACNA1S). We describe two unrelated patients with HypoPP, followed by their relevant clinical studies and gene analysis. Clinical studies included an oral glucose tolerance test (OGTT), food-loading and insulin tolerance tests (ITT). For Case 1, serum K+ levels were extremely decreased following insulin tolerance testing compared with levels for controls. These results support the hypothesis that no efflux of K+ ion occurs in patients because of low activity of adenosine triphosphate (ATP)-sensitive K+ channel (KATP) channels. Mutational analysis of the CACNA1S gene showed a duplicate insertion of 14 base pairs (bp) from 52 to 65 in intron 26, present in the heterozygous state in both patients. No other mutations were detected in the CACNA1S gene, the muscle sodium channel gene (SCN4A) or the voltage-gated K+ channel gene (KCN3) of either patient. Further analysis showed that this duplicate insertion of 14 bp in intron 26 of the CACNA1S gene was found in 23.7% of healthy subjects. K+ dynamics studies are useful for confirming this syndrome, while further gene analysis for various ion channels using amplification and direct sequencing are required to evaluate the molecular basis of the disorder in the individual patient.

Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan.

Vascular endothelial cells produce nitric oxide (NO), which contributes to the regulation of blood pressure and regional blood flow. Although Endothelial NO synthase (eNOS) gene polymorphisms have been shown to have a positive association with coronary artery disease, the linkage between eNOS gene polymorphisms and hypertension has been controversial. In the present study, therefore, we identified genotypes for Glu298Asp and variable number tandem repeats in intron 4 (4b/a) in 183 hypertensive and 193 normotensive populations. The Glu298Asp variant had a significant association with hypertension (odds ratio, 1.8; 95% confidence interval, 1.1-3.0). The allele frequencies of 298Asp for Glu298 in hypertensive patients were significantly higher than those in normotensive subjects (0.128 vs 0.080, p<0.05). Diastolic and mean arterial blood pressures were significantly higher in hypertensive subjects with the 298Asp allele than those without the variant allele (p<0.05). However, disequilibrium of 4b/a polymorphism was absent between these two groups. These results suggest that the Glu298Asp variant may be a genetic susceptibility factor for hypertension.

[Endothelial nitric oxide synthase gene polymorphism and hypertension].

Nitric oxide (NO) is produced by endothelial cells and serves as a potent vasodilator. Several lines of evidence have shown that NO plays an important role in the regulation of blood pressure and regional blood flow. Endothelial NO synthase (eNOS) gene polymorphisms exhibit a positive association in myocardial infarction and smoking-dependent risk of coronary artery disease. However, the relationship between eNOS gene polymorphisms and hypertension is controversial. To examine the possible involvement of the eNOS gene in the genetic basis for hypertension, we identified genotypes for 2 eNOS gene polymorphisms in 166 hypertensive and 174 normotensive populations in Aomori prefecture, in northern Japan. The specific genotypes for Glu298Asp missense variant and variable numbers of tandem repeats in intron 4 (eNOS 4b/4a) were isolated using allele specific gene amplification and restriction fragment length polymorphism. The 298Asp variant was significantly correlated to hypertension in these groups (odds ratio, 1.8; 95% confidence interval, 1.1-3.2). The allelic frequencies of 298Asp for Glu298 in hypertensive patients were significantly higher than those in normotensive subjects (0.136 vs 0.083, p < 0.05). However, disequilibrium of eNOS4b/4a was absent between these 2 groups. These results suggest that Glu298Asp is a genetic susceptibility factor for hypertension.

[Hypertension and gene polymorphisms].

For our understanding of the genetic factors of human essential hypertension, gene polymorphisms have played a significant role as DNA markers in association and linkage studies. We found positive linkages between hypertension and 4 gene polymorphisms including angiotensinogen Met235Thr, angiotensin converting enzyme I/D, aldosterone synthase CYP11B2 T-344C, and endothelial nitric oxide synthase Glu298Asp in the Aomori population. These results suggest that the 4 gene polymorphisms might be genetic risk factors for hypertension in this district. However, there has been a frustration with the inconsistencies of accumulated evidence. Because, the genetic associations tend to vary across race, ethnicity, and ecological states. Thus, the rates of racial inter-mixture can explain regional differences in disease susceptibility. We emphasize that human lineage based analysis across populations may lead to the better understanding of the variability.

Diagnosis of a case of Gitelman's syndrome based on renal clearance studies and gene analysis of a novel mutation of the thiazide-sensitive Na-Cl cotransporter.

Gitelman's syndrome is a recessively inherited renal tubular disorder characterized by low plasma potassium and magnesium levels, reduced calcium excretion, metabolic alkalosis, and increased plasma renin activity and plasma aldosterone concentration with normal blood pressure levels. A 23-yr-old man was referred to our department for further evaluation of hypokalemia. The patient also had hypomagnesemia and markedly reduced urinary calcium excretion. Renal clearance studies and gene analysis of the thiazide-sensitive Na-Cl cotransporter (TSC) were performed in the patient. In response to an iv injection of furosemide, chloride clearance (CCl) increased markedly, while distal fractional chloride reabsorption CH2O/(CH2O+CCl) was considerably reduced. In contrast, thiazide ingestion had no significant effects on these parameters. The patient had compound heterozygous mutations in the alleles encoding the TSC gene, one of which has not been formerly reported. Renal clearance studies and TSC gene analysis by amplification and direct sequencing are useful diagnostic tools for confirming a diagnosis of Gitelman's syndrome.

Angiotensin converting enzyme gene polymorphism and its enzyme activity in serum in young Japanese females.

To assess the potential association between the angiotensin converting enzyme (ACE) gene polymorphism and the activity of the renin-angiotensin system in a Japanese population, we determined the ACE genotype and its enzyme activity in serum in 108 young Japanese females. Genomic DNA was extracted from blood samples and amplified by polymerase chain reaction (PCR). PCR primers flanked the polymorphic region in intron 16 of the ACE gene. The distribution of the DD, ID and II ACE genotypes was 10, 55 and 35%, respectively. The estimated allele frequencies of the deletion and the insertion were 0.375 and 0.625, respectively. The mean serum ACE activity in DD subjects was about 1.4 times that of II subjects (p < 0.01), with ID subjects having intermediate levels (p < 0.05), whereas the renin profile were not statistically different among the three groups. These results indicate a significant association between ACE gene polymorphism and serum ACE activity levels, suggesting a mechanism by which genotype might have a hearing on the physiology of the renin-angiotensin system axis.

Analysis of aldehyde dehydrogenase 2 gene polymorphism and ethanol patch test as a screening method for alcohol sensitivity.

To assess clinical availability of the aldehyde dehydrogenase (ALDH) 2 gene polymorphism to detect alcohol sensitivity among a Japanese population, we determined the ALDH 2 genotypes and also compared to an ethanol patch test in 119 young Japanese. Their alcohol sensitivity was evaluated by a questionnaire on the frequency of alcohol-associated symptoms when they drink. Genomic DNA was extracted from blood samples and amplified by polymerase chain reaction (PCR). PCR primers were flanking the polymorphic region in exon 12 of the ALDH 2 gene. The distribution of the typical homozygote, the heterozygote and the atypical homozygote was 63.9, 31.9 and 4.2%, respectively. Gene frequencies of the typical and atypical alleles calculated from the genotype frequencies were 0.80 and 0.20. The atypical genotypic homozygotes were positively associated with facial flushing symptom, but not with positive response for ethanol patch test. These results indicate that ALDH 2 genotypes determination is essential to detect alcohol sensitivity whereas the ethanol patch test has some limitations.