Introduction

Polymorphic sites within genes encoding G protein-coupled receptor (GPCRs) may yield possible correlations between genotypes and disease-susceptibility phenotypes or responsiveness to drug therapy (Rana et al. 2001). A number of investigators have already reported numerous polymorphisms in all domains of many GPCR genes (Rana et al. 2001; Small et al. 2003). Eight GPCR genes selected for the work reported here are described in the following paragraphs:

  • Angiotensin II receptors types 1 and 2 (AGTR1 and AGTR2 respectively), mediated by the renin-angiotensin system, play important roles in cardiovascular pathophysiology (Wagenaar et al. 2002) and are considered the most important receptors in that context (Whitebread et al. 1989). Stimulation of AGTR1 leads to physiological effects such as vasoconstriction, inflammation, and proliferation—all of those processes being involved in cardiovascular disease (Griendling et al. 1996; Wagenaar et al. 2002). A polymorphism in the 3' untranslated region (UTR) of the AGTR1 gene has been associated with increased risk of essential hypertension (Bonnardeaux et al. 1994; Hingorani et al. 1995; Wang et al. 1997; Kainulainen et al. 1999). For its part, AGTR2 not only plays a role in cardiovascular functions (Hein et al. 1995) but also mediates programmed cell death (Yamada et al. 1996). Mutations in AGTR2 cause X-linked mental retardation (Vervoort et al. 2002).

  • Angiotensin receptor-like 1 (AGTRL1), also known as APJ, was originally isolated from human genomic DNA by polymerase chain reactions (O'Dowd et al. 1993).

  • Arginine vasopressin receptor 1A (AVPR1A) mediates contraction and proliferation of cells, aggregation of platelets, and lysis of glycogen (Jard 1983). A number of investigators have examined potential associations of AVPR1A genotypes with altered susceptibility to essential hypertension (Thibonnier et al. 2000) and autism (Kim et al. 2002).

  • Arginine vasopressin receptor 2 (AVPR2) mediates antidiuretic effects (Jard 1983). Mutations in the AVPR2 gene are responsible for X-linked congenital nephrogenic diabetes insipidus (Bichet et al. 1997; Oksche and Rosenthal 1998; Morello and Bichet 2001; Rana et al. 2001).

  • Dopamine receptor D1 (DRD1) regulates growth and differentiation of neurons (Lankford et al. 1988), and mediates several behavioral responses (Clark and White 1987). A number of investigators have examined potential associations of DRD1 genotypes with altered susceptibilities to psychiatric diseases (Liu et al. 1995; Cichon et al. 1996; Kojima et al. 1999), drug addiction (Comings et al. 1997), alcoholism (Heinz et al. 1996; Hietala et al. 1997), and essential hypertension (Sato et al. 2000).

  • Integrin alpha 2b (ITGA2B), also called platelet glycoprotein IIb, plays a key role in platelet aggregation (French and Seligsohn 2000). Mutations in the ITGA2B gene cause Glanzmann's thrombasthenia, an autosomal recessive disorder of platelets [Glanzmann Thrombasthenia Database (http://med.mssm.edu/glanzmanndb/)].

  • Prostaglandin I2 receptor (PTGIR), also known as prostacyclin receptor, plays important roles in relaxation of vascular smooth muscle and in preventing blood coagulation (Smyth and FitzGerald 2002). A polymorphism (Arg212His) associated with impaired binding and activation of this receptor has been reported (Stitham et al. 2002).

To investigate in detail the nature of apparent genotype/phenotype correlations involving the eight genes described above, we began by searching for additional SNPs in their promoter regions, exons, and introns (except for repetitive elements), and report here a total of 178 genetic variations among 96 Japanese chromosomes, of which 97 had not been reported before.

Subjects and methods

Total genomic DNAs were isolated from peripheral leukocytes of 48 unrelated Japanese individuals by the standard phenol/chloroform extraction method after informed consent was obtained from each participant. On the basis of sequence information from GenBank, we designed protein-coupled receptor (PCR) primers to amplify DNA fragments from all eight GPCR genes, excluding repetitive elements, by invoking the Repeat Masker computer program (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker). PCR experiments and DNA sequencing were performed according to methods described previously (Iida et al. 2001; Saito et al. 2001; Sekine et al. 2001). All SNPs detected by the PolyPhred computer program (Nickerson et al. 1997) were confirmed by sequencing both strands of each PCR product.

Results and discussion

Exon-intron boundaries in eight selected GPCR genes were defined by comparison of genomic sequences with cDNA sequences; all accession numbers are listed in Table 1. We screened 96 Japanese chromosomes for SNPs in these genes by means of direct DNA sequencing.

Table 1. Accession numbers for the genomic and cDNA sequences used in this study
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Subsequent re-sequencing of about 86 kilobases (kb) of genomic DNA (34.4 kb for the AGTR1 gene, 7.9 kb for AGTR2, 5.7 kb for AGTRL1, 9.8 kb for AVPR1A, 6.0 kb for AVPR2, 6.8 kb for DRD1, 11.0 kb for ITGA2B, and 4.2 kb for PTGIR) identified 147 SNPs and 31 insertion/deletion polymorphisms (Table 2). Fig. 1a–h documents the location of each variation we found; detailed information about nucleotide positions and substitutions is summarized in Table 3, 4, 5, 6, 7, 8, 9, and 10. On average, we identified one SNP in every 584 nucleotides. Of the 178 genetic variations identified by our screening procedures, including insertion/deletion polymorphisms, 97 (54%) had not been reported previously.

Table 2. Summary of genetic variations in eight G protein-coupled receptor (GPCR) genes. SNP single-nucleotide polymorphism
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Fig. 1.
figure 1

Locations of single-nucleotide polymorphisms (SNPs) in the AGTR1 (a), AGTR2 (b), AGTRL1 (c), AVPR1A (d), AVPR2 (e), DRD1 (f), ITGA2B (g), and PTGIR (h) genes (vertical lines). Open boxes, exons; hatching, unsequenced regions of repetitive elements; ATG, initiation codons; TGA, TAG, and TAA, stop codons

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Table 3. Summary of genetic variations detected in the AGTR1 gene. AGTR angiotensin receptor 1, NCBI National Center for Biotechnology Information, SNP single nucleotide polymorphism, UTR untranslated region, del deletion, ins insertion
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Table 4. Summary of genetic variations detected in the AGTR2 gene. AGTR2 angiotensin receptor 2, NCBI National Center for Biotechnology Information, SNP single nucleotide polymorphism
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Table 5. Summary of genetic variations detected in the AGTRL1 gene. AGTRL1 angiotensin receptor-like 1, NCBI National Center for Biotechnology Information, SNP single nucleotide polymorphism
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Table 6. Summary of genetic variations detected in the AVPR1A gene. AVPR1A arginine vasopressin receptor 1A, NCBI National Center for Biotechnology Information, SNP single nucleotide polymorphism
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Table 7. Summary of genetic variations detected in the AVPR2 gene. AVPR2 arginine vasopressin receptor 2, NCBI National Center for Biotechnology Information, SNP single nucleotide polymorphism
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Table 8. Summary of genetic variations detected in the DRD1 gene. DRD1 dopamine receptor D1, NCBI National Center for Biotechnology Information, SNP single nucleotide polymorphism
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Table 9. Summary of genetic variations detected in the ITGA2B gene. ITGA2B integrin, alpha 2b, NCBI National Center for Biotechnology Information, SNP single nucleotide polymorphism
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Table 10. Summary of genetic variations detected in the PTGIR gene. PTGIR prostaglandin I2 receptor, NCBI National Center for Biotechnology Information, SNP single nucleotide polymorphism
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Among the 147 SNPs mapped in our experiments, 21 were located in 5' flanking regions, 76 in introns, 32 in exons, and 18 in 3' flanking regions (Table 11). Among the 32 SNPs detected in exons, six were located in 5'UTRs, 13 in coding regions, and 13 in 3'UTRs. Four of the 13 SNPs detected in coding regions would cause substitution of amino acids, and two of those had not been reported before (Gly250Glu in AVPR2, and Ala128Thr in PTGIR) (Table 12).

Table 11. Number and regions of single nucleotide polymorphisms (SNPs) detected in eight GPCR genes. GPCR G protein-coupled receptor, UTR untranslated region
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Table 12. Novel single nucleotide polymorphisms (SNPs) detected in Exon s in eight GPCR genes. GPCR G protein-coupled receptor, UTR untranslated region
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Because AGTR1 is expressed in vascular smooth-muscle cells and regulates vasoconstriction in response to angiotensin II, a number of investigators have examined potential associations of AGTR1 genotypes with altered susceptibility to essential hypertension (Duncan et al. 2001). A polymorphism in the 3'UTR of this gene has been associated with increased risk of essential hypertension (Bonnardeaux et al. 1994; Hingorani et al. 1995; Wang et al. 1997; Kainulainen et al. 1999). However, other investigators have been unable to confirm the association between this variant and hypertension (Schmidt et al. 1997; Takami et al. 1998; Kikuya et al. 2003; Ono et al. 2003). Our results should contribute to a better understanding of ethnic differences that might influence correlations between genotypes and phenotypes with respect to susceptibility to hypertension.

DRD1 mediates several behavioral responses (Clark and White 1987). Although potential correlations between DRD1 genotypes and disease susceptibilities have been investigated extensively (Liu et al. 1995; Cichon et al. 1994; Heinz et al. 1996; Comings et al. 1997; Hietala et al. 1997; Kojima et al. 1999; Sato et al. 2000), no conclusive results have emerged as yet.

Our screening experiments detected two novel nonsynonymous polymorphisms (Gly250Glu in AVPR2, and Ala128Thr in PTGIR). As both were located in cytoplasmic domains, these polymorphisms could have phenotypic consequences (Birnbaumer et al. 1992; Boie et al. 1994; Stitham et al. 2003). These and other polymorphisms published herein may have significant value for researchers who wish to examine relationships between GPCR genotypes and susceptibilities to certain diseases.