Introduction

Heterotrimeric guanine nucleotide binding proteins (G proteins), which are composed of α, β, γ subunits, act as a molecular switch that mediate a wide variety of extracellular signals from G-protein coupled receptors to effector molecules within cells (see a review by Dhanasekaran et al. 1998). On the basis of sequence similarities of the α subunits of G protein, they are divided into four groups: Gs, Gi, Gq, and G12 (Hurowitz et al. 2000). It is also reported that members of the G12 subfamily, consisting of Gα12 and Gα13, are ubiquitously expressed and share more than 67% amino-acid sequence identity (Strathmann and Simon 1991). The gene encoding human Gα12, GNA12, was originally identified as a transforming gene by means of an expression cloning method to search the putative oncogene for soft-tissue sarcoma (Chan et al. 1993). Overexpression of wild-type Gα12 leads to the oncogenic transformation of NIH3T3 cells in a serum-dependent manner. Subsequent functional analyses revealed that Gα12 as well as Gα13 are involved in the regulation of various signaling pathways, such as cell growth, differentiation, cytoskeletal changes, and apoptosis (see reviews by Radhika and Dhanasekaran 2001; Kurose 2003).

Single nucleotide polymorphisms (SNPs) at some gene loci are indicated to be useful as DNA markers of individual risk for adverse drug reactions or susceptibility to complex diseases. To establish the bases of SNP information for genetic studies of complex diseases and responsiveness to drug therapy, we have been focusing on isolating SNPs in gene loci encoding proteins involved in the metabolism, transport, and signaling of drugs. So far, high-density SNP maps containing approximately 6,800 genetic variations have been constructed (Iida et al. 2001a,b,c,d,e; 2002a,b,c,d; 2003; 2004; Saito et al. 2001a,b; 2002a,b,c,d; 2003a,b; Sekine et al. 2001). Furthermore, we reported several distinct mutations in the genes encoding drug metabolizing enzymes and potential drug receptors among Japanese healthy donors (a review by Iida and Nakamura 2003; Iida et al. 2004). As an addition to SNP information reported earlier, we provide here 20 novel SNPs and 12 genetic variations of other types in the GNA12 locus.

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. 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, 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 postextension 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

We performed direct sequencing of DNAs from 48 healthy individuals in a total of 36-kb regions (excluding the parts corresponding to human repetitive sequences) that corresponded to 29.3% of the 123-kb genomic region containing GNA12. We identified 86 SNPs in this region (SNPs were distributed every 419 nucleotides on average). By comparing our data with the SNPs deposited in the dbSNP database in the National Center for Biotechnology Information, USA, we considered 20 of these SNPs to be novel as of the beginning of April 2004. The exon-intron organization of GNA12 and locations and detailed information of the 20 novel SNPs are illustrated schematically in Fig. 1 and Table 1, respectively. Subregional distributions of novel SNPs were as follows: 16 in introns, two in the coding region, and two in the 3′ flanking region. The overall frequencies of nucleotide substitutions were counted as 30% for A/G, 35% for C/T, 10% for A/C, and 25% for C/G. The transitions occurred 1.9 times more frequently than transversions. Both of the two substitutions found in the coding region were synonymous substitutions: one was 534G>A (Ser178Ser) in exon 3, and the other was 1062C>T (Ile354Ile) in exon 4. We also identified 12 genetic variations of other types in the region.

Fig. 1
figure 1

Genomic organization and locations of 32 genetic variations in GNA12 locus. Exons and introns are represented by rectangles and horizontal lines, respectively. Single nucleotide polymorphisms (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 3′ untranslated sequences of GNA12 was as yet unidentified in the database we used

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Table 1 Characterization of 32 genetic variations in the GNA12 locus. ins insertion polymorphism, del deletion polymorphism. An accession number of the genomic sequence obtained from Genbank is AC006028.3
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Altogether, we have collected 32 genetic variations, including 20 SNPs and 12 genetic variations of other types, in GNA12 locus by screening 96 Japanese healthy donors. We hope that genetic variations can contribute to further investigations for designing personalized medicine.