Introduction

One of major goals in our laboratory is to establish “personalized medicine” on the basis of single nucleotide polymorphisms (SNPs) regarding responsiveness or adverse effects to drugs and susceptibility to common diseases. To achieve this aim, we have been conducting extensive screening programs to isolate SNPs in the Japanese population. Particularly, we have focused on SNP mapping in 209 genomic regions that correspond to genes involved in the metabolism of drugs and have constructed high-density SNP maps containing more than 6,500 genetic variations (Iida et al. 2001a,b,c,d,e, 2002a,b,c,d, 2003; Saito et al. 2001a,b, 2002a,b,c,d, 2003a,b; Sekine et al. 2001). Moreover, these efforts have allowed us to identify a total of five mutations that may affect the gene products, including two nonsense mutations in CYP genes and three frame-shift mutations (two in CYP genes and one in the MGST1 gene) among Japanese healthy donors (see a review by Iida and Nakamura 2003).

G-protein coupled receptors are cell-surface proteins and are responsible for the signal transduction for a variety of extracellular chemical signals such as photons, Ca2+, nucleotides, amino acids, peptides, amines, fatty derivatives, and various polypeptide ligands (see a review by Howard et al. 2001). On recognition of such ligands, these proteins mediate a variety of intracellular signals via activation of heterotrimeric G-proteins that in turn, activate various effector proteins, consequently resulting in a physiologic response for the cell (Marinissen and Gutkind 2001). G-protein coupled receptors are members of a large multigene family characterized by similar structure, which consists of seven alpha-helical transmembrane segments connected by three intracellular and three extracellular loops, an extracellular amino-terminal domain, and an intracellular carboxyl-terminal domain. So far, a large number of studies have reported genetic variations of G-protein coupled receptor gene loci and their role in etiology or predisposition of human diseases (Rana et al. 2001). G-protein coupled receptors are also considered to be good potential targets for drug development (Howard et al. 2001).

To expand the genetic basis for drug development as well as eventually to enable clinicians to predict efficacy and adverse drug reactions, we explored SNPs among 23 genes encoding G-protein coupled receptors. In this report, we provide high-resolution maps of these gene loci containing a total of 300 SNPs and 33 genetic variations of other types that we detected in DNAs from 48 Japanese volunteers.

Subjects and methods

Samples of peripheral blood were obtained with written informed consent form 48 healthy Japanese volunteers for this study. The SNP screening method described in an earlier report by Haga et al. (2002) was the principal technique applied in this study. In brief, on the basis of genomic sequences corresponding to each of the receptor genes from the GenBank database in the US National Center for Biotechnology Information (NCBI), we designed primers to amplify all selected genes in their entirety, excluding only regions that corresponded to repetitive sequences predicted by the RepeatMasker program (http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker). All gene symbols mentioned through this report are according to the nomenclature in LocusLink of NCBI.

Each polymerase chain reaction (PCR) was performed using 20 ng of a mixture of genomic DNAs from three individuals. All 16 mixed samples were amplified in the GeneAmp PCR system 9700 (PE Applied Biosystems, Foster City, Calif., USA) under the following conditions: initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturaion at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, and post-extension at 72°C for 7 min. Products obtained from the PCR experiments served as templates for direct sequencing and detection of SNPs, using the fluorescent dye-terminator cycle-sequencing method. 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

By direct sequencing of DNA from 96 chromosomes, we screened SNPs in a total of approximately 174.4-kb genomic sequences that accounted for 60% of the 289.4-kb genomic region encompassing the loci of 23 selected genes, excluding the regions corresponding to human repetitive sequences. We identified a total of 333 variations, including 300 SNPs and 33 genetic variations of other types, and constructed a fine-scale SNP map of each locus (Fig. 1, Table 1). Detailed information for each variation identified in this study is summarized in Table 2. The overall genomic distribution of SNP was 1 in every 581 nucleotides in the 174.4-kb region that we sequenced. The overall frequencies of nucleotide substitutions in our test population were counted as 31% for A/G, 36.3% for C/T, 10% for C/G, 9.3% for T/G, 7.3% for A/C, and 6% for A/T; transitions occurred 2.1 times more frequently than transversions. By comparing SNPs detected in this study with previous reports from elsewhere (Sturm et al. 2001; Lubrano-Berthelier et al. 2003; Schalin-Jantti et al. 2003) or with the dbSNP database at NCBI, we were able to consider 132 of the 300 SNPs (44%) to be novel as of the end of December 2003.

Fig. 1
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Fine-scale single nucleotide polymorphism (SNP) maps of 23 gene loci encoding G-protein coupled receptors. Exons and introns are represented by rectangles and horizontal lines, respectively. SNPs are indicated above the lines (designations correspond to the left-most column of Table 1). Genetic variations of other types, where present, are indicated below the maps. However, the complete 5′ untranslated sequences and/or 3′ untranslated sequences of ADRA2B, EDG5, MC3R, MC4R, SSTR3, GPR3, and GPR10 were yet unidentified in database we used

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Table 1 Summary of genetic variations identified in 23 gene-encoding G-protein coupled receptors. SNP Single nucleotide polymorphism
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Table 2 Characterization of 333 genetic variations in 23 gene loci corresponding to G-protein coupled receptors. dbSNP Database of single-nucleotide polymorphisms, ins insertion polymorphism, del deletion polymorphism
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We also identified two novel nonsense mutations in the Japanese healthy donors. One is a 322C→T (Arg108Stop) in ADORA3 belonging to adenosine receptor gene family. The other is a 706C→T (Arg236Stop) in an anonymous G protein-coupled receptor 1 gene. Both SNPs, each of which was identified in a single individual among the 48 volunteers tested, would be predicted to lack nearly half of the C-terminal part of each gene product. However, since these mutations were found in the healthy donors, we have no information regarding its potential effects on the carrier’s susceptibility to any diseases or drug responses. In all cases, more detailed genetic and functional studies will be necessary to clarify a relationship between these mutations and functional properties of these receptors.

Altogether, we have collected a total of 333 genetic variations, including 300 SNPs and 33 genetic variations of other types, among the 23 genes encoding G-protein coupled receptors by screening 96 Japanese chromosomes. We hope our SNP catalog can contribute to further investigations for identifying genes associated with drug efficacy and/or adverse drug reactions and for designing personalized medical care.