118 S. Nomura et al. With respect to the genetic aspects of thrombotic diseases such as type 2 diabetes mellitus, the HLA haplotype of patients can possibly be considered a potentially important factor in the aetiology, although its role remains unclear. This study examined the genetic status of HLA, HPA and in patients with type 2 diabetes mellitus and dia- betic ASO. To our knowledge, this is the ﬁrst report detail- ing relationships among these three genetic factors in type 2 diabetes mellitus and ASO patients. Materials and methods Subjects We studied 104 Japanese patients (67 men and 37 women, aged 39–85 years) with type 2 diabetes mellitus, as deﬁned by the American Diabetes Association Criteria (The Expert Committee on the Diagnosis and Classiﬁca- tion of Diabetes Mellitus, 1997). Seventeen non-diabetic ASO patients were used for patient control subjects. A brief clinical proﬁle of the 104 diabetic patients and 17 non-diabetic ASO patients is presented in Table 1. One hundred and twenty-nine controls were randomly selected from among healthy unrelated Japanese individ- uals. Approval was obtained from the Institutional Review Board for these studies, and informed consent was obtained in accord with the Declaration of Helsinki. Diagnosis of ASO All patients were evaluated by the ankle-pressure index (API). Patients with an API > 1.0 were considered not to have ASO. Thirty-three patients with API < 1.0 under- went angiography and plethysmography, and 28 were diagnosed as having ASO (Fontain’s class I–III). Of these, 21 patients with ASO underwent bypass procedures for femoral and trifurcation occlusive disease. Seventeen non- diabetic ASO patients were also diagnosed with Fontain’s class I–III. Of these, 10 patients with ASO underwent bypass procedures. HLA DNA typing by PCR–RFLP Genomic DNAs from 104 diabetic patients, 17 non- diabetic patients and 126 healthy control subjects were Table 1. Clinical characteristics of the diabetic and non-diabetic patients No. of patients Sex ratio (male : female) Age (year) Range Median BMI (kg m−2) Mean ± S.E. Type 2 DM Non-diabetic ASO (–) 76 51 : 25 39–81 54 ASO (+) 28 22 : 6 52–85 66 ASO 17 13 : 4 47–66 59 28.1 ± 1.6 28.3 ± 2.1 24.2 ± 1.7 isolated by phenol extraction of sodium dodecyl sulphate- lysed and proteinase K-treated cells. DNA was ampliﬁed by PCR with Taq DNA polymerase, and HLA typing was performed by the PCR–restriction fragment length poly- morphism (RFLP) method (Ota et al., 1992). The reaction mixture was subjected to 30 cycles of denaturation at 96 – 97 °C for 1 min, annealing at 55 – 62 °C for 1 min, and extension at 72 °C for 2 min in an automated PCR thermal sequencer (Iwaki Glass, Inc., Tokyo, Japan). After ampliﬁcation, aliquots of the products were digested with allele-speciﬁc restriction endonucleases for 3 h after addition of the appropriate reaction buffer. Samples of the ampliﬁed and cleaved DNAs were subjected to electrophoresis on 12% polyacrylamide gel in a minigel apparatus (Mupid-2, Cosmo Bio Co., Ltd, Tokyo, Japan). Cleavage of ampliﬁed fragments was detected by staining with ethidium bromide, and genotypes were determined on the basis of the RFLP band patterns thus generated. genotyping by PCR–preferential homoduplex formation assay (PHFA) We applied PCR–preferential homoduplex formation assay (PHFA) developed by Oka et al. (1994) to DNA typing of neutrophil-speciﬁc antigens (NA1, NA2 and SH) located on the neutrophil receptor, FcγRIIIb. Gene fragments containing the polymorphic sequences corre- sponding to NA1, NA2 and SH of FcγRIIIb, as well as the homologous fragment of FcγRIIIa, were ampliﬁed by PCR using speciﬁc primers (Fujiwara et al., 1996, 1999). Assay procedure was performed according to the manu- facturer’s instructions (Wakunaga Pharm. Co., Ltd, Hiro- shima, Japan). HPA DNA typing by enzyme-linked mini-sequence assay (ELMA) Gene fragments that contained polymorphic sequences corresponding to HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and HPA-6 were speciﬁcally ampliﬁed by the PCR method. Enzyme-linked mini-sequence assay (ELMA) typing was performed according to the manufacturer’s instructions (Smitest HPA Genotype ELMA, Sumitomo Metal Industries, Ltd, Ibaraki, Japan). Statistical analysis The χ2 method with continuity correction and Fisher’s exact test were used for HLA data analysis. Relative risk was calculated according to Wolf’s method with Hold- ane’s correction. Brieﬂy, it was calculated as (a × d)/(b × c), where a, b, c and d are the number of marker-positive patients, marker-negative patients, marker-positive con- trols and marker-negative controls, respectively (Thom- son, 1981). The frequencies of the FcγRIIIb and HPA alleles were estimated by a gene-counting method and tested for deviation from the Hardy–Weinberg equilib- rium using the χ2 test. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, International Journal of Immunogenetics 33, 117–122