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Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension
BBRC Biochemical and Biophysical Research Communications 297 (2002) 1135–1139 www.academicpress.com
Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension Tomohiro Nakayama,a,* Masayoshi Soma,b Yoshiyasu Watanabe,b Buaijiaer Hasimu,b Mikano Sato,b Noriko Aoi,b Kotoko Kosuge,b Katsuo Kanmatsuse,b Shinichiro Kokubun,a Jason D. Marrow,c and John A. Oatesc a
c
Division of Receptor Biology, Advanced Medical Research Center, Nihon University School of Medicine, Ooyaguchi-kamimachi 30-1, Itabashi-ku, Tokyo 173-8610, Japan b Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan Division of Clinical Pharmacology, Vanderbilt University School of Medicine, 21st South at Garland Avenue, Nashville, TN 37232, USA Received 6 September 2002
Abstract 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 patient’s 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. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Genetics; Hypertension; Prostaglandins; Prostacyclin; Splicing mutation
Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction [1–3]. Prostacyclin synthase (CYP 8A1, EC 5.3.99.4), a catalyst of prostacyclin formation from prostaglandin H2, is widely distributed and predominantly found in vascular endothelial and smooth muscle cells [4,5]. The prostacyclin synthase (PGIS) gene is localized to 20q13.11-13 [6] and thought to be a candidate gene for cardiovascular disease. We recently reported the organization of this gene [7]. Furthermore, we identified a family with a nonsense mutation in exon 2 of the prostacyclin
synthase gene and this family displays a history of hypertension and cerebral infarction [8]. These findings suggested that abnormality of the prostacyclin synthase gene may lead to altered vasodilation and platelet aggregation. In the present study, we searched for new mutations and polymorphisms in the PGIS gene and assessed the relationship with essential hypertension (EH) and atherothrombotic disease.
Materials and methods *
Corresponding author. Fax: +81-3-5375-8076. E-mail address: [email protected] (T. Nakayama).
Subjects. The study group consisted of 200 patients (mean age 53:6 11:1 years) with EH diagnosed according to ISH criteria. These
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criteria included a sitting systolic blood pressure (SBP) of more than 160 mmHg and/or diastolic blood pressure (DBP) of more than 100 mmHg (Grade II). All patients had family history of hypertension and were not being treated with anti-hypertensive drugs. Subjects diagnosed with secondary hypertension were excluded. Two hundred normotensive (NT) healthy subjects (mean age 53:2 10:9 years) were also studied as controls. Normotensive subjects had no family history of hypertension, and in all cases, their SBP was less than 140 mmHg and their DBP less than 85 mmHg. A positive family history was defined as hypertension diagnosed in at least one immediate blood relative: grandparents, parents, or siblings. Informed consent was obtained from each individual according to a protocol approved by the Ethics Committee of Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital. Biochemical analysis. The plasma concentration of total cholesterol and the serum concentrations of creatinine and uric acid were measured by standard methods in the Clinical Laboratory Department of Nihon University Hospital. A prostacyclin metabolite, 2,3-dinor-6-keto-PGF1a, was measured utilizing gas chromatography/mass spectrometry in the negative-ion chemical ionization mode, as described previously [9]. As there had been no reason previously to focus on the lower limit of the normal range of 2,3-dinor-6-keto-PGF1a excretion, we elected to analyze the prostacyclin metabolite in the urine of 15 normal individuals utilizing exactly the same method employed for analysis. The Mann–Whitney U test was used to compare these values with 15 normal controls. Morrow and Minton [10] describe the method employed in the analysis of the 11-dehydro-TXB2 in the paper. Single strand conformation polymorphism. For polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of each exon and both acceptor and donor sites of intron, oligonucleotide primers were designed from the genomic sequence of the prostacyclin synthase gene [7,11] (GenBank/EMBL/DDBJ Data Bank Accession Nos. D84115, D84116, D84117, D84118, D84119, D84120, D84121, D84122, D84123, and D84124). DNA was extracted from whole blood according to standard procedures [12]. PCR-SSCP was used as described [13] on DNA from 90 patients with EH.
Sequencing analysis. PCR products showing an abnormally migrating band and normal controls were gel purified and subjected to automated DNA sequencing analyses with fluorescence-labeled dideoxyterminators (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin–Elmer) according to manufacturer’s instructions (ABI PRISM 310 Genetic Analyzer, PE Applied Biosystems, Foster City, CA) [14]. Sequencing was performed for both strands. Genotyping. Genotyping was confirmed by PCR-restriction fragment length polymorphism (RFLP) analysis. We used a sense primer set in exon 8 and an antisense primer in intron 9 (sense primer including artificial mutation shown underlined: 50 -GCG GTC AAC AGC ATC AAA CCG-30 ; antisense primer, 50 - texas red labeled TGC CAA CCA GGG GAT CAG GAG-30 ). PCR conditions were initial denaturation at 94 °C for 3 min followed by 30 cycles at 96 °C for 30 s, 63 °C for 1 min, and 72 °C for 1 min and a final extension at 72 °C for 10 min. PCR products were digested with an AccII and separated in polyacrylamide-sequencing gels using a LASER automated sequencer (SQ5500E, HITACHI, Tokyo). The wild-type allele lacks the AccII site and yields a 73-base pair (bp) band, whereas the mutation-type allele is digested by AccII into 52- and 21-bp fragments. In vitro expression study. Exons 8–10 of the patient’s mutant PGIS gene and those of the normal PGIS gene from a control subject were amplified using the forward primer (adding EcoRI adapter at the 50 -end) and the reverse primer (adding NotI adapter at 50 -end) by the Long and Accurate PCR System (TaKaRa Shyuzo, Shiga, Japan). The respective PCR products, consisting of 5 kbp, were purified utilizing a Microcon 100 column (US Amicon, Beverly, MA). The resulting products were ligated to pCR2.1 vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA). Ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer). The inserts were cleaved from the plasmid DNA with the restriction enzymes EcoRI and NotI (Toyobo Biochemicals, Osaka, Japan) and then ligated to the expression vector pcDNA1.1/Amp (Invitrogen, Fig. 1). The plasmids were cloned and then purified with Qiagen resin (QIAprep Spin Miniprep Kit, Hilden, Germany). One lg mutant or normal plasmids was transfected into human umbilical vein endothelial cells (HUVEC) by the lipofectamine method [14]. After
Fig. 1. (A) Allelic minigene construct consisting of exon 8–10. Splicing site mutation is shown by asterisk. The +2 position in the splicing down site of intron 9 contained a T to C transition. The fragment between exon 8 and exon 10 was ligated to the EcoRI and the NotI sites of pcDNA1.1 vector. Bold bars display primers for RT-PCR. The RT-PCR wild-type product included exon 9, while that of mutant-type displays a 255 bp fragment. (B) RT-PCR products electrophoresed in agarose gel. Lane 1 is a molecular DNA marker of / X174/HincII digest. Lane 2 is the RT-PCR mutanttype product showing the 255 bp fragment. Lane 3 is the RT-PCR wild-type product showing the 407 bp fragment.
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Results and discussion
sure of 130 mmHg. He was found to have been hypertensive by the age of 22. An electrocardiogram and echocardiogram revealed left ventricular hypertrophy, probably due to high blood pressure. Urinary protein excretion was 2+ and serum creatinine level was slightly increased at 1.3 mg/dL. The patient’s serum total cholesterol and HDL-cholesterol levels were 180 and 58 mg/ dL, respectively. He was admitted to our hospital due to cardiac failure at the age of 54. After discharge from our hospital, he was admitted to another hospital and died of cardiac and renal failure at the age of 57. As the patient (subject 4) was already deceased by the time we discovered the mutation, we analyzed the mutation in a number of other family members (Fig. 2). Two (subjects 1 and 2) of the three siblings (subjects 1, 2, and 3) tested displayed the mutation. All subjects displaying this mutation had hypertension. Interestingly, serum creatinine levels in subjects with mutation were elevated (subjects 1, 2, and 4 were 1.4, 1.6, and 1.3, respectively) compared with those in subjects without mutation (subjects 3 and 5 were 1.0 and 0.7, respectively). These suggested that the mutation of PGIS causes hypertension complicated with renal dysfunction. Samples could only be taken from subjects 1, 2, and 5 (a daughter of subject 4) because subject 3 did not agree to sample collection. The data are listed in Fig. 2. All results were corrected with urinary creatinine excretions. Prostacyclin metabolite 2,3-dinor-6-keto-PGF1a in both samples 1 and 2 was reduced below the lower limit of the normal range (mean SD, 0:179 0:015). Sample 1 was reduced below normal with a P value of 0.027 and sample 2 was reduced below the normal range with a P value of 0.037. By contrast, urinary 2,3-dinor-6-ketoPGF1a excretion in sample 5 was slightly elevated (P ¼ 0.005). For the assay of 11-dehydro-thromboxane B2 (11-dehydro-TXB2), the upper limit of normal is 0.644 ng/mg creatinine. 11-Dehydro-TXB2 in sample 1 was elevated above the upper limit of the normal range,
Using SSCP analysis, we discovered a T to C transition at +2 the position in the splicing donor site of intron 9 in patients with EH (Fig. 1A). In vitro expression analysis of an allelic minigene consisting of exons 8–10 showed that this nucleotide transition causes skipping of exon 9 (Figs. 1A and B). This exon skipping alters the translational reading frame of exon 10 and creates a premature stop codon (TGA) 64 bp downstream from the boundary of exons 8–10 (Fig. 1A). Therefore, this transition is a splicing site mutation, which yields a truncated protein read with mismatch-codons from the codon 403. This mutation was found in only one subject following the screening of 200 EH patients and 200 NT controls, and was heterozygous. The patient was a Japanese man with EH who presented with a systolic blood pressure of 220 mmHg and diastolic blood pres-
Fig. 2. Genealogy of the patient with splicing site mutation of PGIS.
48 h, the transfected cells were washed once with phosphate-buffered saline, then digested with trypsin, and collected. mRNA was extracted from the cells with an RNAzol B RNA Isolation solvent (TEL-TEST, Friendswood, Texas). First-strand complementary DNA (cDNA) was synthesized with the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, England, UK). The resulting cDNA was subjected to PCR amplification with 2 lmol/L primers (sense: 50 -CGGGAGAG AATTCAACCTGCGACGTGG-30 , 1113–1139 nucleotides count from the A of start codon; antisense: 50 -TCCCTGTGTCATGGGCGGAT GCGGTA-30 , 1486–1511, Fig. 1A) and 2.5 U Taq DNA polymerase. PCR conditions were 35 cycles of denaturation at 98 °C for 25 s, annealing at 65 °C for 30 s, and extension at 72 °C for 1 min. PCR products were excised from the 1.5% agarose gel (Sawady, Tokyo, Japan) and purified with Microcon 100. Portions of the purified PCR products were sequenced. Molecular modeling. The strategy used for constructing the PGIS three-dimensional model followed that previously established method [15] developed for TXAS [16] and other mammalian P450s [17]. A sequence similarity alignment was made for PGIS and the hemoprotein domain of the P450BM-3 , and the main-chain conformation of PGIS was built with the Quanta-Charmm protein modeling package by transferring the crystal coordinates of P450BM-3 to the aligned components of PGIS. Thus, the conserved helices and strand framework in P450BM-3 provided the backbone section coordinates of PGIS. The backbone segments were linked to each other using a fragment searching approach [18–20] and a database containing 58-protein three-dimensional structures, developed by Ruan et al. [16]. Several three-dimensional structural candidates were obtained from the database and one was chosen based on the best similarity of primary structure and the best fit of Cadistance, with a cutoff value of root mean square deviation [16,17,21]. The three-dimensional 0.5 A structural coordinates of the heme in PGIS were adopted directly from the X-ray structure of P450BM-3 and then fixed into the backbone structure of PGIS. The substrate, PGH2, structure was constructed, energy-minimized, and subjected to conformation search. One of the 200 conformations of the PGH2 three-dimensional structure with the best score was docked into the proposed PGIS substrate-binding pocket of the constructed three-dimensional model of PGIS, which corresponded to the P450BM-3 substrate-binding cavity. An energy minimization with 500 steps of steepest descent was performed for the PGIS three-dimensional structural model containing heme and PGH2 structures.
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while that in samples 2 and 5 was within normal limits. No history of aspirin or nonsteroidal anti-inflammatory drug use was reported by any members of the family. The data on the thromboxane B2 metabolite provided confirmatory evidence that the reduction in 2,3-dinor6-keto-PGF1a could not be ascribed to the administration of aspirin or nonsteroidal anti-inflammatory drugs, which would have suppressed the levels of 11-dehydroTXB2. The fact that 11-dehydro-TXB2 was slightly elevated in sample 1 would be consistent with excessive in vivo platelet activation in a patient with reduced prostacyclin biosynthesis. The hypothesis that an inherent defect in prostacyclin biosynthesis is present in affected individuals was therefore supported. PGIS belongs to a new family (CYP8) in the P450 superfamily [22]. Cytochrome P450BM-3 from Bacillus megaterium catalyzes the monooxygenation of fatty acids, including arachidonic acid (AA). Its catalytic function and primary structure resemble mammalian microsomal cytochrome P450’s. The heme domain of P450BM-3 shows about 25–30% sequence identity to several microsomal P450 enzymes [23]. Since AA is a functional substrate of P450BM-3 and the substrate is closely related to the substrate of PGIS, we reasoned that the crystal structure of the hemoprotein domain could serve as a useful template for constructing a threedimensional model of PGIS (Fig. 3). Comparison of the amino acid sequence of human PGIS with P450s shows that the PGIS has a significant sequence similarity to other P450s in the C-terminal region including the Cyspocket near the helix L [24]. The cysteine residue in the pocket has been shown to constitute the fifth ligand of the heme iron [25–29]. Thus, it is very much possible
Fig. 3. PGIS residues predicted from the three-dimensional model to be important in heme site and changed region by the splice site mutation. Arrows indicate heme and Cys 441. Black denotes the changed or truncated region.
that this invariant cysteine residue with a polar side chain at position 441 of the PGIS plays an important role in the catalytic activity of the enzyme. The method of site-directed mutagenesis revealed that the mutants of alanine and serine for Cys441 resulted in a diminished enzyme activity (13%), without alteration in the expressed protein level [24]. The splice site mutation discovered in our investigation produces a truncated protein, which lacks the heme-binding region. Therefore, the allele with the mutation cannot produce the mature protein. Thus, the PGIS activity of subjects having the mutation is decreased. It is thought that this mechanism may involve in the pathophysiology of their hypertension. Previously we identified a nonsense mutation in exon 2 of the human prostacyclin synthase gene in a family with a history of hypertension and cerebral infarction [13]. This mutation was found in only 1 woman in a group comprised of 150 EH and 150 normotensive individuals. This result suggested that PGIS gene abnormalities might be associated with EH. We hypothesized that a genetic variant with a functional difference could be linked to EH, even if its frequency in the population was very low. The presence of genetic heterogeneity, however, makes prediction of the degrees of overlap between many genes in a population difficult. Our study suggests that the differences in prostacyclin activities are allele-dependent and these differences may influence the risk of EH. Recently, we isolated a variable number of tandem repeat (VNTR) polymorphisms upstream in the 50 flanking region of this gene and showed that the VNTR was associated with a risk of cerebral infarction [8], but not with EH [30]. Furthermore, each allele of the prostacyclin synthase gene has a different transcriptional activity. Alleles with smaller numbers of tandem repeats have lower transcriptional activity. The region between )150 and the start codon is GC-rich, and it has been reported that this position contains the basal promoter region [8]. These experiments suggested that the VNTR may influence the basic transcriptional activity and that this influence by PGIS may be stronger for cerebral infarction than for EH. In the present study, a novel splicing mutation was discovered in intron 9 of the PGIS gene in only 1 man in a group comprised of 200 EH and 200 NT individuals. In vitro expression studies revealed that this mutation causes exon skipping at exon 9, resulting in a premature stop codon. This mutation may therefore influence the enzymatic activities of PGIS. As the PGIS mRNA is not detected in peripheral white blood cells and all subjects in this family did not agree to collection of other tissue, we could not confirm the exon skipping in the body of patients. The splicing mutation shows no linkage with the nonsense mutation in exon 2 [13] or the VNTR in the promoter region associated with cerebral infarction [8].
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The splicing mutation must therefore affect PGIS activity independently the nonsense mutation and VNTR. Further studies are needed to clarify whether this mutation in the PGIS gene can cause cardiovascular diseases such as EH, cerebral infarction, and myocardial infarction.
Acknowledgments This work was supported financially by grants from the Ministry of Education Science and Culture of Japan (High-Tech Research Center Nihon University) the alumni association of Nihon University School of Medicine the Tanabe Biomedical Conference Toray-Yamanouchi Pharmacology Company Japan, and by NIH Grants GM15431, DK48831, and CA77839. Jason Morrow is the recipient of a Burrough’s Welcome Fund Clinical Scientist Award in Translation Research.
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Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension Tomohiro Nakayama,a,* Masayoshi Soma,b Yoshiyasu Watanabe,b Buaijiaer Hasimu,b Mikano Sato,b Noriko Aoi,b Kotoko Kosuge,b Katsuo Kanmatsuse,b Shinichiro Kokubun,a Jason D. Marrow,c and John A. Oatesc a
c
Division of Receptor Biology, Advanced Medical Research Center, Nihon University School of Medicine, Ooyaguchi-kamimachi 30-1, Itabashi-ku, Tokyo 173-8610, Japan b Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan Division of Clinical Pharmacology, Vanderbilt University School of Medicine, 21st South at Garland Avenue, Nashville, TN 37232, USA Received 6 September 2002
Abstract 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 patient’s 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. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Genetics; Hypertension; Prostaglandins; Prostacyclin; Splicing mutation
Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction [1–3]. Prostacyclin synthase (CYP 8A1, EC 5.3.99.4), a catalyst of prostacyclin formation from prostaglandin H2, is widely distributed and predominantly found in vascular endothelial and smooth muscle cells [4,5]. The prostacyclin synthase (PGIS) gene is localized to 20q13.11-13 [6] and thought to be a candidate gene for cardiovascular disease. We recently reported the organization of this gene [7]. Furthermore, we identified a family with a nonsense mutation in exon 2 of the prostacyclin
synthase gene and this family displays a history of hypertension and cerebral infarction [8]. These findings suggested that abnormality of the prostacyclin synthase gene may lead to altered vasodilation and platelet aggregation. In the present study, we searched for new mutations and polymorphisms in the PGIS gene and assessed the relationship with essential hypertension (EH) and atherothrombotic disease.
Materials and methods *
Corresponding author. Fax: +81-3-5375-8076. E-mail address: [email protected] (T. Nakayama).
Subjects. The study group consisted of 200 patients (mean age 53:6 11:1 years) with EH diagnosed according to ISH criteria. These
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 3 4 1 - 0
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criteria included a sitting systolic blood pressure (SBP) of more than 160 mmHg and/or diastolic blood pressure (DBP) of more than 100 mmHg (Grade II). All patients had family history of hypertension and were not being treated with anti-hypertensive drugs. Subjects diagnosed with secondary hypertension were excluded. Two hundred normotensive (NT) healthy subjects (mean age 53:2 10:9 years) were also studied as controls. Normotensive subjects had no family history of hypertension, and in all cases, their SBP was less than 140 mmHg and their DBP less than 85 mmHg. A positive family history was defined as hypertension diagnosed in at least one immediate blood relative: grandparents, parents, or siblings. Informed consent was obtained from each individual according to a protocol approved by the Ethics Committee of Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital. Biochemical analysis. The plasma concentration of total cholesterol and the serum concentrations of creatinine and uric acid were measured by standard methods in the Clinical Laboratory Department of Nihon University Hospital. A prostacyclin metabolite, 2,3-dinor-6-keto-PGF1a, was measured utilizing gas chromatography/mass spectrometry in the negative-ion chemical ionization mode, as described previously [9]. As there had been no reason previously to focus on the lower limit of the normal range of 2,3-dinor-6-keto-PGF1a excretion, we elected to analyze the prostacyclin metabolite in the urine of 15 normal individuals utilizing exactly the same method employed for analysis. The Mann–Whitney U test was used to compare these values with 15 normal controls. Morrow and Minton [10] describe the method employed in the analysis of the 11-dehydro-TXB2 in the paper. Single strand conformation polymorphism. For polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of each exon and both acceptor and donor sites of intron, oligonucleotide primers were designed from the genomic sequence of the prostacyclin synthase gene [7,11] (GenBank/EMBL/DDBJ Data Bank Accession Nos. D84115, D84116, D84117, D84118, D84119, D84120, D84121, D84122, D84123, and D84124). DNA was extracted from whole blood according to standard procedures [12]. PCR-SSCP was used as described [13] on DNA from 90 patients with EH.
Sequencing analysis. PCR products showing an abnormally migrating band and normal controls were gel purified and subjected to automated DNA sequencing analyses with fluorescence-labeled dideoxyterminators (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin–Elmer) according to manufacturer’s instructions (ABI PRISM 310 Genetic Analyzer, PE Applied Biosystems, Foster City, CA) [14]. Sequencing was performed for both strands. Genotyping. Genotyping was confirmed by PCR-restriction fragment length polymorphism (RFLP) analysis. We used a sense primer set in exon 8 and an antisense primer in intron 9 (sense primer including artificial mutation shown underlined: 50 -GCG GTC AAC AGC ATC AAA CCG-30 ; antisense primer, 50 - texas red labeled TGC CAA CCA GGG GAT CAG GAG-30 ). PCR conditions were initial denaturation at 94 °C for 3 min followed by 30 cycles at 96 °C for 30 s, 63 °C for 1 min, and 72 °C for 1 min and a final extension at 72 °C for 10 min. PCR products were digested with an AccII and separated in polyacrylamide-sequencing gels using a LASER automated sequencer (SQ5500E, HITACHI, Tokyo). The wild-type allele lacks the AccII site and yields a 73-base pair (bp) band, whereas the mutation-type allele is digested by AccII into 52- and 21-bp fragments. In vitro expression study. Exons 8–10 of the patient’s mutant PGIS gene and those of the normal PGIS gene from a control subject were amplified using the forward primer (adding EcoRI adapter at the 50 -end) and the reverse primer (adding NotI adapter at 50 -end) by the Long and Accurate PCR System (TaKaRa Shyuzo, Shiga, Japan). The respective PCR products, consisting of 5 kbp, were purified utilizing a Microcon 100 column (US Amicon, Beverly, MA). The resulting products were ligated to pCR2.1 vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA). Ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer). The inserts were cleaved from the plasmid DNA with the restriction enzymes EcoRI and NotI (Toyobo Biochemicals, Osaka, Japan) and then ligated to the expression vector pcDNA1.1/Amp (Invitrogen, Fig. 1). The plasmids were cloned and then purified with Qiagen resin (QIAprep Spin Miniprep Kit, Hilden, Germany). One lg mutant or normal plasmids was transfected into human umbilical vein endothelial cells (HUVEC) by the lipofectamine method [14]. After
Fig. 1. (A) Allelic minigene construct consisting of exon 8–10. Splicing site mutation is shown by asterisk. The +2 position in the splicing down site of intron 9 contained a T to C transition. The fragment between exon 8 and exon 10 was ligated to the EcoRI and the NotI sites of pcDNA1.1 vector. Bold bars display primers for RT-PCR. The RT-PCR wild-type product included exon 9, while that of mutant-type displays a 255 bp fragment. (B) RT-PCR products electrophoresed in agarose gel. Lane 1 is a molecular DNA marker of / X174/HincII digest. Lane 2 is the RT-PCR mutanttype product showing the 255 bp fragment. Lane 3 is the RT-PCR wild-type product showing the 407 bp fragment.
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sure of 130 mmHg. He was found to have been hypertensive by the age of 22. An electrocardiogram and echocardiogram revealed left ventricular hypertrophy, probably due to high blood pressure. Urinary protein excretion was 2+ and serum creatinine level was slightly increased at 1.3 mg/dL. The patient’s serum total cholesterol and HDL-cholesterol levels were 180 and 58 mg/ dL, respectively. He was admitted to our hospital due to cardiac failure at the age of 54. After discharge from our hospital, he was admitted to another hospital and died of cardiac and renal failure at the age of 57. As the patient (subject 4) was already deceased by the time we discovered the mutation, we analyzed the mutation in a number of other family members (Fig. 2). Two (subjects 1 and 2) of the three siblings (subjects 1, 2, and 3) tested displayed the mutation. All subjects displaying this mutation had hypertension. Interestingly, serum creatinine levels in subjects with mutation were elevated (subjects 1, 2, and 4 were 1.4, 1.6, and 1.3, respectively) compared with those in subjects without mutation (subjects 3 and 5 were 1.0 and 0.7, respectively). These suggested that the mutation of PGIS causes hypertension complicated with renal dysfunction. Samples could only be taken from subjects 1, 2, and 5 (a daughter of subject 4) because subject 3 did not agree to sample collection. The data are listed in Fig. 2. All results were corrected with urinary creatinine excretions. Prostacyclin metabolite 2,3-dinor-6-keto-PGF1a in both samples 1 and 2 was reduced below the lower limit of the normal range (mean SD, 0:179 0:015). Sample 1 was reduced below normal with a P value of 0.027 and sample 2 was reduced below the normal range with a P value of 0.037. By contrast, urinary 2,3-dinor-6-ketoPGF1a excretion in sample 5 was slightly elevated (P ¼ 0.005). For the assay of 11-dehydro-thromboxane B2 (11-dehydro-TXB2), the upper limit of normal is 0.644 ng/mg creatinine. 11-Dehydro-TXB2 in sample 1 was elevated above the upper limit of the normal range,
Using SSCP analysis, we discovered a T to C transition at +2 the position in the splicing donor site of intron 9 in patients with EH (Fig. 1A). In vitro expression analysis of an allelic minigene consisting of exons 8–10 showed that this nucleotide transition causes skipping of exon 9 (Figs. 1A and B). This exon skipping alters the translational reading frame of exon 10 and creates a premature stop codon (TGA) 64 bp downstream from the boundary of exons 8–10 (Fig. 1A). Therefore, this transition is a splicing site mutation, which yields a truncated protein read with mismatch-codons from the codon 403. This mutation was found in only one subject following the screening of 200 EH patients and 200 NT controls, and was heterozygous. The patient was a Japanese man with EH who presented with a systolic blood pressure of 220 mmHg and diastolic blood pres-
Fig. 2. Genealogy of the patient with splicing site mutation of PGIS.
48 h, the transfected cells were washed once with phosphate-buffered saline, then digested with trypsin, and collected. mRNA was extracted from the cells with an RNAzol B RNA Isolation solvent (TEL-TEST, Friendswood, Texas). First-strand complementary DNA (cDNA) was synthesized with the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, England, UK). The resulting cDNA was subjected to PCR amplification with 2 lmol/L primers (sense: 50 -CGGGAGAG AATTCAACCTGCGACGTGG-30 , 1113–1139 nucleotides count from the A of start codon; antisense: 50 -TCCCTGTGTCATGGGCGGAT GCGGTA-30 , 1486–1511, Fig. 1A) and 2.5 U Taq DNA polymerase. PCR conditions were 35 cycles of denaturation at 98 °C for 25 s, annealing at 65 °C for 30 s, and extension at 72 °C for 1 min. PCR products were excised from the 1.5% agarose gel (Sawady, Tokyo, Japan) and purified with Microcon 100. Portions of the purified PCR products were sequenced. Molecular modeling. The strategy used for constructing the PGIS three-dimensional model followed that previously established method [15] developed for TXAS [16] and other mammalian P450s [17]. A sequence similarity alignment was made for PGIS and the hemoprotein domain of the P450BM-3 , and the main-chain conformation of PGIS was built with the Quanta-Charmm protein modeling package by transferring the crystal coordinates of P450BM-3 to the aligned components of PGIS. Thus, the conserved helices and strand framework in P450BM-3 provided the backbone section coordinates of PGIS. The backbone segments were linked to each other using a fragment searching approach [18–20] and a database containing 58-protein three-dimensional structures, developed by Ruan et al. [16]. Several three-dimensional structural candidates were obtained from the database and one was chosen based on the best similarity of primary structure and the best fit of Cadistance, with a cutoff value of root mean square deviation [16,17,21]. The three-dimensional 0.5 A structural coordinates of the heme in PGIS were adopted directly from the X-ray structure of P450BM-3 and then fixed into the backbone structure of PGIS. The substrate, PGH2, structure was constructed, energy-minimized, and subjected to conformation search. One of the 200 conformations of the PGH2 three-dimensional structure with the best score was docked into the proposed PGIS substrate-binding pocket of the constructed three-dimensional model of PGIS, which corresponded to the P450BM-3 substrate-binding cavity. An energy minimization with 500 steps of steepest descent was performed for the PGIS three-dimensional structural model containing heme and PGH2 structures.
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while that in samples 2 and 5 was within normal limits. No history of aspirin or nonsteroidal anti-inflammatory drug use was reported by any members of the family. The data on the thromboxane B2 metabolite provided confirmatory evidence that the reduction in 2,3-dinor6-keto-PGF1a could not be ascribed to the administration of aspirin or nonsteroidal anti-inflammatory drugs, which would have suppressed the levels of 11-dehydroTXB2. The fact that 11-dehydro-TXB2 was slightly elevated in sample 1 would be consistent with excessive in vivo platelet activation in a patient with reduced prostacyclin biosynthesis. The hypothesis that an inherent defect in prostacyclin biosynthesis is present in affected individuals was therefore supported. PGIS belongs to a new family (CYP8) in the P450 superfamily [22]. Cytochrome P450BM-3 from Bacillus megaterium catalyzes the monooxygenation of fatty acids, including arachidonic acid (AA). Its catalytic function and primary structure resemble mammalian microsomal cytochrome P450’s. The heme domain of P450BM-3 shows about 25–30% sequence identity to several microsomal P450 enzymes [23]. Since AA is a functional substrate of P450BM-3 and the substrate is closely related to the substrate of PGIS, we reasoned that the crystal structure of the hemoprotein domain could serve as a useful template for constructing a threedimensional model of PGIS (Fig. 3). Comparison of the amino acid sequence of human PGIS with P450s shows that the PGIS has a significant sequence similarity to other P450s in the C-terminal region including the Cyspocket near the helix L [24]. The cysteine residue in the pocket has been shown to constitute the fifth ligand of the heme iron [25–29]. Thus, it is very much possible
Fig. 3. PGIS residues predicted from the three-dimensional model to be important in heme site and changed region by the splice site mutation. Arrows indicate heme and Cys 441. Black denotes the changed or truncated region.
that this invariant cysteine residue with a polar side chain at position 441 of the PGIS plays an important role in the catalytic activity of the enzyme. The method of site-directed mutagenesis revealed that the mutants of alanine and serine for Cys441 resulted in a diminished enzyme activity (13%), without alteration in the expressed protein level [24]. The splice site mutation discovered in our investigation produces a truncated protein, which lacks the heme-binding region. Therefore, the allele with the mutation cannot produce the mature protein. Thus, the PGIS activity of subjects having the mutation is decreased. It is thought that this mechanism may involve in the pathophysiology of their hypertension. Previously we identified a nonsense mutation in exon 2 of the human prostacyclin synthase gene in a family with a history of hypertension and cerebral infarction [13]. This mutation was found in only 1 woman in a group comprised of 150 EH and 150 normotensive individuals. This result suggested that PGIS gene abnormalities might be associated with EH. We hypothesized that a genetic variant with a functional difference could be linked to EH, even if its frequency in the population was very low. The presence of genetic heterogeneity, however, makes prediction of the degrees of overlap between many genes in a population difficult. Our study suggests that the differences in prostacyclin activities are allele-dependent and these differences may influence the risk of EH. Recently, we isolated a variable number of tandem repeat (VNTR) polymorphisms upstream in the 50 flanking region of this gene and showed that the VNTR was associated with a risk of cerebral infarction [8], but not with EH [30]. Furthermore, each allele of the prostacyclin synthase gene has a different transcriptional activity. Alleles with smaller numbers of tandem repeats have lower transcriptional activity. The region between )150 and the start codon is GC-rich, and it has been reported that this position contains the basal promoter region [8]. These experiments suggested that the VNTR may influence the basic transcriptional activity and that this influence by PGIS may be stronger for cerebral infarction than for EH. In the present study, a novel splicing mutation was discovered in intron 9 of the PGIS gene in only 1 man in a group comprised of 200 EH and 200 NT individuals. In vitro expression studies revealed that this mutation causes exon skipping at exon 9, resulting in a premature stop codon. This mutation may therefore influence the enzymatic activities of PGIS. As the PGIS mRNA is not detected in peripheral white blood cells and all subjects in this family did not agree to collection of other tissue, we could not confirm the exon skipping in the body of patients. The splicing mutation shows no linkage with the nonsense mutation in exon 2 [13] or the VNTR in the promoter region associated with cerebral infarction [8].
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The splicing mutation must therefore affect PGIS activity independently the nonsense mutation and VNTR. Further studies are needed to clarify whether this mutation in the PGIS gene can cause cardiovascular diseases such as EH, cerebral infarction, and myocardial infarction.
Acknowledgments This work was supported financially by grants from the Ministry of Education Science and Culture of Japan (High-Tech Research Center Nihon University) the alumni association of Nihon University School of Medicine the Tanabe Biomedical Conference Toray-Yamanouchi Pharmacology Company Japan, and by NIH Grants GM15431, DK48831, and CA77839. Jason Morrow is the recipient of a Burrough’s Welcome Fund Clinical Scientist Award in Translation Research.
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