Introduction

The G-protein coupled receptors (GPCRs) represent the largest class of cell-surface receptors that recognize extracellular messengers. All members of the GPCR family, which consists of approximately 950 members in humans (Takeda et al. 2002), are predicted to share seven predicted α-helical transmembrane domains, extracellular N-termini and intracellular C-termini, and several conserved structure motifs (Howard et al. 2001). Since these receptors mediate important cellular signals, their mutations and polymorphisms are shown to be responsible for or associated with a large number of diseases. They are also known to be the targets of therapeutic agents; 50% of all modern drugs are considered to target GPCRs (see reviews by George et al. 2002; Spiegel 1995).

To establish “personalized medicine” on the basis of individual genetic variations, we have systematically explored single nucleotide polymorphisms (SNPs) in the genomic regions corresponding to drug-related genes (Iida et al. 2001a–e, 2002a–d, 2004b; Saito et al. 2001a, 2001b, 2002a–d, 2003a; Sekine et al. 2001). As a part of this program, we previously reported SNPs in genomic regions corresponding to genes encoding GPCRs and other known drug targets and constructed fine-scale SNP maps containing more than 1,100 SNPs in 63 genes (Iida et al. 2003, 2004a, 2004b; Saito et al. 2003b). We further extended our SNP discoveries of the GPCRs in 96 chromosomes from healthy Japanese donors and here report a total of 156 novel SNPs and 32 genetic variations of other types for 29 additional members of the GPCR gene family GPR5-9, GPR11-18, GPR20, GPR21-27, GPR29-31, GPR34, GPR35, and GPR37, GPR39, and GPR40.

Subjects and methods

Samples of peripheral blood were obtained with written informed consent form 48 healthy Japanese volunteers. Polymerase chain reaction (PCR) experiments and DNA sequencing were performed according to methods described previously (Iida et al. 2003). In brief, on the basis of genomic sequences corresponding to each of GPCR 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 as well as intron 1 of GPR39 because it is very long, at 227 kb. Each 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, CA, USA) under the following conditions: initial denaturation at 94°C for 2 min followed by 35 cycles of denaturation 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. All gene names and gene symbols mentioned in this report are according to the nomenclature in LocusLink of the NCBI.

Results and discussion

Sequencing of an approximately 183.3-kb genomic region corresponding to the 29 GPCR loci in 96 Japanese chromosomes identified a total of 390 genetic variations, 358 SNPs and 32 genetic variations of other types (Table 1). The overall distribution of SNP was one in every 512 nucleotides on average. By comparing our data with the SNPs deposited in the dbSNP database in the NCBI (as of the end of December 2004), we judged 156 SNPs to be novel (Tables 1, 2). The exon–intron organization of each gene and locations of SNPs identified within each locus are shown schematically in Fig.1; detailed information is given in Table 2. Sub-regional distributions of novel SNPs were as follows: 44 in 5′ flanking regions, five in 5′ untranslated regions (UTRs), 11 in coding regions, 47 in introns, 12 in 3′ UTRs, and 37 in 3′ flanking regions. The overall frequencies of nucleotide substitutions were counted as 28% for A/G, 28% for C/T, 17% for A/C, 14% for C/G, 9% for G/T, and 4% for A/T. The transitions occurred 1.3 times more frequently than transversions. In addition, of the 19 SNPs in coding regions, we found a total of eight novel nonsynonymous substitutions: 404A>T (Tyr135Phe) in exon 1 of GPR7, 217C>G (Leu73Val) in exon 2 of GPR11, 149C>T (Ala50Val) in exon 1 and 371G>A (Arg124His) in exon 1 of GPR14, 235G>T (Ala79Ser) in exon 2 of GPR16, 343G>A (Val115Ile) in exon 1 of GPR21, 1201G>A (Gly401Arg) in exon 2 of GPR24, and 743G>A (Arg248His) in exon 2 of GPR30. These SNPs might effect on the function of the corresponding GPCRs.

Table 1 Summary of genetic variations identified in 29 genes encoding GPCRs. SNP Single nucleotide polymorphism
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Table 2 Identification of 156 novel single nucleotde polymorphisms and 32 genetic variations of other types in 29 GPCR loci found among 96 Japanese chromosomes
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Fig. 1
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Fine-scale single nucleotide polymorphism (SNP) maps of 29 gene loci encoding G-protein coupled receptors (GPCRs). Exons and introns are represented by rectangles and horizontal lines, respectively. The SNPs are indicated above the lines (designations correspond to the left-most column of Table 2). Genetic variations of other types, where present, are indicated below the maps. However, the complete 5′ untranslated sequences and/or 3′ untranslated sequences of GPR5, GPR6, GPR7, GPR8, GPR14, GPR20, GPR21, GPR22, GPR25, GPR27, GPR31, GPR35, GPR39 and GPR40 were yet unidentified in database we used

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Altogether, we have collected a total of 156 novel SNPs and 32 genetic variations of other types by screening of 29 genes encoding GPCRs. 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.