Introduction

Peptidylarginine deiminases (PADIs) are post-translational modification enzymes that catalyze the conversion of protein-bound arginine residues into citrulline residues in the presence of calcium ions. To date, four human PADI genes, type 1 to type 4, have been isolated and characterized by cDNA sequences, expression patterns, and biochemical analyses (Nakashima et al. 1999; Kanno et al. 2000; Ishigami et al. 2002; Guerrin et al. 2003). Three of four genes, PADI1, PADI3, and PADI4, lie within an approximately 160-kb region on chromosome 1p36.13. PADI2 is located at 86-kb distal to the PADI1,3,4 gene cluster.

We previously reported one of four PADI genes, PADI4, to be a rheumatoid arthritis-susceptibility gene by a case-control association study using single nucleotide polymorphisms (SNPs) and subsequent functional assays (Suzuki et al. 2003). In the course of this association study, we constructed fine-scale SNP maps of the PADI gene cluster region by a direct sequencing method and the SNP information derived from the JSNP database (Haga et al. 2002). In this report, we describe more detailed information of genetic variations in the 83-kb region corresponding to the PADI1/PADI3 loci, including 45 novel SNPs and two insertion–deletion polymorphisms in the Japanese population.

Subjects and methods

Samples of peripheral blood were obtained with written informed consent form 48 healthy Japanese volunteers for this study. Polymerase chain reaction (PCR) experiments and DNA sequencing were performed according to the methods described previously (Iida et al. 2003). In brief, on the basis of the genomic sequence from the Genbank database (accession number AL590644.13), we designed primer sites to amplify each gene locus in its entirely, excluding most of the regions that corresponded to human repetitive sequences predicted by the RepeatMasker program (http://www.repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker). 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

By direct sequencing of DNA from 48 Japanese individuals, we explored SNPs in an 83-kb genomic region including the PADI1/PADI3 loci except most of the regions containing human repetitive sequences. The extent of each screened genomic sequence was 16.8-kb for the PADI1 locus and 13.1-kb for the PADI3 locus. Consequently, we identified a total of 87 SNPs from the regions (SNPs were distributed every 344 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 45 of these SNPs to be novel as of beginning of March 2004; 33 were identified in the PADI1 locus and 12 in the PADI3 locus (Fig. 1). Detailed information for these genetic variations is given in Table 1. Subregional distributions of novel SNPs were as follows: one in 5′ flanking regions, four in coding regions, 35 in introns, two in 3′ untranslated regions, and three in 3′ flanking regions. Of four SNPs identified in the coding regions, three were nonsynonymous substitutions: 1289T>A (Ile430Asn) in exon 11 and 1652G>A (Arg551His) in exon 15 of the PADI1, and 380G>A (Gly127Glu) in exon 4 of the PADI3. All three could affect structures and/or biological functions of the respective gene products. In addition, the overall frequencies of nucleotide substitutions were counted as 31% for A/G, 33% for C/T, 7% for A/C, 11% for C/G, 11% for G/T, and 7% for T/A. The transitions occurred 1.8 times more frequently than transversions. We also found two insertion–deletion polymorphisms in the introns of the PADI1.

Fig. 1
figure 1

A fine-scale single nucleotide polymorphism (SNP) map in the 83-kb region including the PADI1 and PADI3. 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). Two insertion–deletion polymorphisms are indicated below the map

Table 1 Characterization of 45 novel SNPs and two insertion–deletion polymorphisms in the PADI1 and PADI3 gene loci. dbSNP database of single nucleotide polymorphisms, del deletion polymorphsim

The SNP map that we constructed in this study will serve as a useful resource for analyzing gene scans of complex diseases mapped to this local segment on chromosomal band 1p36.13. We hope that the genetic variations can contribute to further investigations for designing personalized medical care.