Dolkun Rahmutula

Nonsense mutation of prostacyclin synthase gene in a family

We found a nonsense mutation in exon 2 of the human prostacyclin-synthase gene in a family with essential hypertension and cerebral infarction. Prostacyclin (PGI2) is an inhibitor of platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. Prostacyclin synthase (PGIS), which catalyses the formation of PGI2 from prostaglandin H2, is widely distributed, predominantly in vascular endothelial and smooth muscle cells. We have reported the organisation of this gene. We searched for possible point mutations in the exons using peripheral blood from 100 patients with essential hypertension by PCR and single strand conformation polymorphism (PCR-SSCP) analysis. One patient had an abnormally migrating band on exon 2. Sequencing of this exon showed a nonsense mutation in codon 26 (CGA/TGA). This nucleotide change makes Bst EII the restriction site. 300 people (150 with essential hypertension and 150 healthy controls) were screened by PCR and Bst EII digestion. The mutation was found in one patient with essential hypertension and in none of the controls. The patient was shown to be heterozygous for this mutation.This mutation of the stop codon is 76 bp downstream from ATG, the start codon in cDNA, thus a large part of mRNA,

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.

Systematic screening of type B human natriuretic peptide receptor gene polymorphisms and association with essential hypertension

C-type natriuretic peptide (CNP) dilates arteries, lowers blood pressure and inhibits proliferation of vascular smooth muscle cells via the type B natriuretic peptide receptor (NPRB). The CNP-NPRB system may play a crucial role in the development of cardiovascular disease. We recently determined the structure of the human NPRB gene. In the present study, our objectives are to identify the polymorphisms of the NPRB gene and investigate the association of this gene with essential hypertension (EH). We used the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to study the NPRB gene polymorphism, and conducted an association study using a novel polymorphic marker. PCR-SSCP analysis of all 22 exons was done in 90 subjects, and abnormally-migrating bands were observed in the analyses of exon 11 and intron 18. Direct sequencing of these DNA fragments revealed the following sequence alterations: a C to T transition at nucleotide (nt) 2077 in exon 11 and a 9-bp insertion/deletion (I/D) in intron 18. PCR-restriction fragment length polymorphism analysis (PCR-RFLP) was developed to detect the C2077T transition. PCR-RFLP analyses of healthy subjects revealed that the C2077T polymorphism had complete linkage to GT repeats in intron 2 reported previously. The I/D polymorphism was identified by polyacrylamide gel electrophoresis, and it was not linked to any known polymorphic alleles of this gene. Therefore, the possible association between the I/D polymorphism and EH was investigated. A total of 123 individuals with EH and 123 age-matched normotensive control subjects were studied. Overall distributions of allele frequencies in the two groups were not significantly different. Although the I/D polymorphism in intron 18 of the NPRB gene was not associated with EH, the results of this study, which identified two novel polymorphisms in the human NPRB gene, will facilitate further genetic analysis of this gene and cardiovascular disease.

A T1083C Polymorphism in the Human Adenosine A2a Receptor Gene is not Associated With Essential Hypertension

The adenosine A2a receptor (A2aAR) gene is thought to be involved in essential hypertension because adenosine elicits vasodilation and decreases arterial blood pressure via this receptor, and because disruption of the A2aAR gene increases blood pressure in mice. Therefore, using a restriction fragment length polymorphism (RFLP) of the A2aAR gene, we performed an association study in patients with essential hypertension. One hundred forty-two patients with essential hypertension and 142 age-matched subjects with normal blood pressure were studied. Polymerase chain reaction (PCR) was applied to amplify the T1083C polymorphic site in the A2aAR gene, and restriction analysis of the PCR product was employed to score the T and C alleles. Overall distributions of allele frequencies in the two groups were not significantly different. Thus, the alleles detected by this RFLP polymorphism in the A2aAR gene are not associated with essential hypertension.