Kotoko Kosuge

Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension

Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. The prostacyclin synthase (PGIS) gene is a candidate gene for cardiovascular disease. The purpose of this study was to locate possible mutations in the PGIS gene related to hypertension and cerebral infarction. Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we discovered a T to C transition at the +2 position of the splicing donor site of intron 9 in patients with essential hypertension (EH). In vitro expression analysis of an allelic minigene consisting of exons 8-10 revealed that the nucleotide transition causes skipping of exon 9. This in turn alters the translational reading frame of exon 10 and introduces a premature stop codon (TGA). A three-dimensional model shows that the splice site mutation produces a truncated protein with a deletion in the heme-binding region. This splice site mutation was found in only one subject in 200 EH patients and 200 healthy controls. Analysis of the patients family members revealed the mutation in two of the three siblings. The urinary excretion of prostacyclin metabolites in subjects with the mutation was significantly decreased. All subjects displaying the splice site mutation in the PGIS gene were hypertensive. In this study, we report a novel splicing mutation in the PGIS gene, which is associated with hypertension in a family. It is thought that this mechanism may involve in the pathophysiology of their hypertension.

Two medium-chain acyl-coenzyme A synthetase genes, SAH and MACS1, are associated with plasma high-density lipoprotein cholesterol levels, but they are not associated with essential hypertension.

Background and objectives SAH has been proposed as a candidate gene for essential hypertension (EH) because elevated expression of SAH was observed in the kidneys of spontaneously hypertensive rats. Recently, a homology search of SAH in the human genome revealed the presence of the SAH gene family, which includes SAH, MACS1, MACS2, and MACS3. SAH and MACS1 are located within a 150-kb region on human chromosome 16p13.11. SAH and MACS1 are thought to function as acyl-coenzyme A synthetases, which are involved in fatty acid metabolism. In the present study, we analyzed six single nucleotide polymorphisms (SNPs) in the SAH and MACS1 genes in a Japanese population, and examined whether these SNPs contribute to EH and multiple risk factors. Methods and results We performed association studies of six SNPs in 287 EH patients and 259 normotensive subjects. Multiple logistic linear regression analysis revealed that the allele frequencies of these six SNPs in SAH and MACS1 genes were not significantly different between EH patients and normotensives. SNP in exon 8 of the A/G polymorphism of the MACS1 gene and the G/T SNP in intron 3 of the SAH gene were associated with plasma levels of plasma high-density lipoprotein cholesterol. Conclusions SNPs in the MACS1 and SAH genes contribute to plasma levels of high-density lipoprotein cholesterol.

Structure and polymorphisms of the human natriuretic peptide receptor C gene

Natriuretic peptides (NPs) regulate cardiovascular homeostasis, including natriuresis, diuresis, vasodilation, regulation of endocrine secretion, and inhibition of cellular growth. Atrial natriuretic peptide receptor C (NPRC) has a short cytoplasmic domain that lacks guanylyl cyclase activity. We used information available for the cDNA of human NPRC to amplify products covering all genomic regions of the gene by long polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR. PCR products were sequenced directly after extraction and purification. The human NPRC gene spans >65 kb and contains eight exons and seven introns. All of the exon-intron junction sequences contain the GT/AG consensus junction sequence. We then used the PCR-single-strand conformation polymorphism (PCR-SSCP) to identify polymorphisms of the human NPRC gene. All eight exons and neighboring introns were analyzed by PCR-SSCP for 96 subjects, and migration variants were observed for intron 1, exon 2, and exon 5. Direct sequencing of these variants revealed the following sequence differences: a C to T transition in intron 1, an A to C transition in exon 2, and a C to T transition in exon 5. PCR-restriction fragment length polymorphism analysis (PCR-RFLP) was used to evaluate all three variations. We have determined the structural organization and identified polymorphic sites in the human NPRC gene. The results of this study will facilitate further genetic analyses of the human NPRC gene function.

A novel mutation in Ca2+-sensing receptor gene in familial hypocalciuric hypercalcemia

Missense mutations in the calcium-sensing receptor (CaSR) gene have previously been identified in patients with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism. We identified a newborn with hypercalcemia in our hospital by mass screening. The family members were studied, and we found a novel CaSR missense mutation with polymerase chain reaction single-strand conformational polymorphism analysis. The mother, grandmother, and aunt of the baby all had FHH. A heterozygous missense mutation in exon 6 that substitutes a glutamic acid for the glycine at codon 557 (Gly557 Glu), which corresponds to the extracellular domain of CaSR, was identified and shown to cosegregate with the disease. Identification of the mutation responsible for the FHH phenotype in this family may facilitate rapid testing of individuals at risk for FHH.

Association of the insulin-like growth factor1 gene with myocardial infarction in Japanese subjects

During adult life, the insulin/insulin-like growth factor1 (IGF1) signaling pathway plays an important role in cardiovascular function. Several reports have suggested that low baseline levels of IGF1 increase the risk of fatal ischemic heart disease. Thus, IGF1 may be involved in cardiovascular disease. The aim of the present study was to investigate the relationship between the human IGF1 gene and myocardial infarction (MI) in the Japanese population via the use of single nucleotide polymorphisms (SNPs). After selecting six SNPs in the human IGF1 gene (rs2162679, rs7956547, rs2288378, rs2072592, rs978458 and rs6218), we performed a case-control study using each of the SNPs and haplotypes in 320 MI patients and 307 non-MI controls. Multiple logistic regression analysis demonstrated that the GG+GA variant of rs2162679 (p=0.009) and the AA+GA variant of rs2072592 (p=0.026) exhibited a resistant effect for MI. The haplotype-based case-control study revealed that the frequency of the A-T-G-G haplotype for rs2162679-rs7956547-rs2072592-rs978458 was significantly higher in the MI group (47.3%) as compared to the non-MI group (41.4%) (p=0.037, odds ratio=1.270). The frequency of the A-T-G-T haplotype for rs2162679-rs7956547-rs978458-rs6218 was also significantly higher in the MI group (47.3%) as compared to the non-MI group (41.3%) (p=0.033, odds ratio=1.276). The current results suggest that specific SNPs and haplotypes can be utilized as genetic markers for MI risk or MI resistance. In addition, IGF1 or a neighboring gene might be associated with increased or decreased susceptibility to MI.

Association between SIRT2 gene polymorphism and height in healthy, elderly Japanese subjects

T he silent information regulator factor 2 (SIR2; a nicotinamide adenine dinucleotide–dependent histone deacetylase) protein is related to life span extension in a diverse range of species. In mammals there are 7 sirtuins, all possessing a highly conserved central nicotinamide adenine dinucleotide–binding site and common catalytic domain. Sirtuin 2 (SIRT2) is the mammalian ortholog of the yeast HST2, one of the four SIR2 paralogs. Similar to SIR2, HST2 is upregulated by caloric restriction and oxidative stress and extends life span. SITR2 acts as an important regulator of cell differentiation through modulation of forkhead transcription factor 1 acetylation/phosphorylation. Forkhead transcription factor 1 is a homolog of daf-16, a key regulator of the insulin-like growth factor 1 signaling pathway, and is necessary for increasing life span. An association between chronic obstructive pulmonary disease and SIRT2 single nucleotide polymorphisms (SNPs) has been reported, but an association of the SIRT2 gene with longevity and other phenotypes has not been reported. We focused on the biological pleiotropic roles of SIRT2 and investigated their association with various laboratory and anthropometric data in elderly Japanese subjects.

The insulin-like growth factor-1 gene is associated with cerebral infarction in Japanese subjects

Atherosclerosis leads to cerebral infarction (CI) and the insulin/insulin-like growth factor-1 (IGF1) signaling pathway plays an important role in this process during adult life. The purpose of this study was to investigate the relationship between the human IGF1 gene and CI in the Japanese population via a case-control study that also included a separate analysis of the two gender groups. A total of 155 CI patients and 316 controls were genotyped for six single nucleotide polymorphisms (SNPs) of the human IGF1 gene (rs2162679, rs7956547, rs2288378, rs2072592, rs978458 and rs6218). All data were analyzed for three separate groups: the total subjects, men and women. The logistic regression analysis revealed that the GG + AG variant of rs2162679 (P = 0.047), the AA + GA variant of rs2072592 (P = 0.005) and the CC + TC variant of rs6218 (P = 0.015) exhibited a protective effect for CI in the total subject group. For the women and the total subjects groups, the overall distribution of the haplotype established by rs7956547-rs978458 was significantly different between the CI patients and the non-CI subjects. For the total subjects, the frequency of the T-G haplotype (rs7956547-rs978458) was also significantly higher (P = 0.034), whereas the frequency of the T-A haplotype (rs7956547-rs978458) was significantly lower (P = 0.008) in the CI patients versus the non-CI subjects. For women, the frequency of the T-A haplotype (rs7956547-rs978458) was significantly lower (P = 0.021) in the CI patients as compared with the non-CI subjects. The specific SNPs and haplotypes can be utilized as genetic markers for CI resistance or CI risk.