Int. J. Environ. Res. Public Health 2009, 6 1002 well as two versions of SNuPe primers (5’ - CCACACAGGTATGGTGTGAA -3’ and 5’ -CCACACAGGTACGGTGTGAA -3’) were designed with DNASIS Pro Ver.2.0 (Hitachi Software Engineering Co. Ltd., Tokyo, Japan), since there is another SNP site 9-bp upstream of the ALAD1-2 polymorphism. Then, mixed SNuPe primer (5’ -CCACACAGGTAYGGTGTGAA -3’) was made. The PCR reactions began with preheating at 80 °C for 15 minutes, followed by denaturing at 94 °C for 4.5 minutes and 40 cycles of denaturing at 94 °C for 30 seconds, annealing at 60 °C for 1 minute, extension at 72 °C for 1 minute, with a final extension at 72 °C for 7 minutes. We then carried out the SNuPe method. The reactions included 25 cycles of denaturing at 94 °C for 10 seconds, annealing at 53 °C for 5 seconds, and extension at 60 °C for 10 seconds. We analyzed the products by using ABI 7700 (Amersham Biosciences Corp.). As for genotyping accuracy, we compared the genotyping results by SNuPe method as well as conventional RFLP, and the agreement rate was 100% except one, which was not determined due to the lack of residual sample. 2.4. Statistical Analysis All values were shown by mean and Standard deviation (SD) except the value mentioned with the Geometric mean (GSD). We compared the mean value of all basic characteristic, clinical, biochemical variables including blood lead level between GG and GC genotypes by Student's t-test or 2 test. The mean value of biomarkers for lead exposure between two genotypes of ALAD gene was compared by using Student’s t-test. All P value <0.05 was considered as statistical significant. 3. Results and Discussion Table 1 shows the basic characteristics of occupationally non lead exposed subjects. The mean (SD) age of all study participants is 43.4 (11.9) yr. The mean blood lead level of all subjects was 3.38 μg/dL and the mean blood lead level of GC genotype was significantly higher than that of GG homozygous genotype. Many previous studies have shown that the ALAD genotype was associated with blood lead levels among environmentally exposed children and occupationally exposed lead workers [16,17]. Subjects with ALAD heterozygous GC, and homozygous CC genotypes were more susceptible to increased blood lead levels than those with the homozygous GG genotype. ALAD2 carriers had significantly higher blood lead level that ALAD1 homozygous [16-19]. The hypothesis of mechanism for lead binding affinity with ALAD2 remained controversial [15,20]. However, some studies found no significant association between ALAD2 carriers and blood lead level [21-23]. Although the association result is inconsistent, our study found the blood lead level in the subjects with heterozygous GC genotype was significantly higher than that in those with homozygous GG genotype, even in the environmentally same exposed lead condition (i.e. very low lead exposure naturally). Age and smoking