Assessment of DNA degradation of buccal cells under humid conditions and DNA repair by DOP-PCR using locked nucleic acids.

We analyzed the degradation level of DNA from buccal cells under humid conditions using quantitative PCR analysis. Gauze samples with buccal cells were incubated for up to 12 months under three different conditions (25 °C/dry, 25 °C/humid, or 40 °C/humid). The degradation was evaluated based on two degradation ratios (129:41 and 305:41 bp). DNA degraded slowly under the 25 °C/humid condition, and significant differences in the two degradation ratios were detected between 25 °C/dry and 25 °C/humid conditions after 12 months. Moreover, the degradation rapidly progressed under the 40 °C/humid condition, and the two degradation ratios in this condition were much lower than those from 25 °C/dry and 25 °C/humid conditions after a short incubation period (3 months). To evaluate the effect of DNA repair on low-copy degraded DNA, degenerate oligonucleotide-primed PCR (DOP-PCR) was performed before short tandem repeats (STR) genotyping. As a standard DOP-PCR, we used a 22-base primer with 10 degenerate sequences (5'-CTCGAGNNNNNNNNNNATGTGG-3'), and additionally designed DOP-PCR primers with 2, 4, 6, or 8 locked nucleic acids (LNAs). When slightly degraded DNA (305:41-bp ratio = 0.60) was used, DOP-PCR significantly increased the fluorescent intensity and success rate of genotyping using Identifiler and Globalfiler kits. In particular, the reaction with four LNAs produced the highest value. However, such benefits were not observed in the analysis of moderately degraded DNA (305:41-bp ratio = 0.13). Although the recovery rates of STR profiles by DOP-PCR were dependent on the degradation level of low-copy DNA, the effectiveness of DOP-PCR highlights the potential of LNA for degenerate sequences.

Estimation of Interindividual Variability of Pharmacokinetics of CYP2C9 Substrates in Humans.

The activity of metabolic enzymes varies across individuals and populations. Activity varies even among individuals sharing the same genotype. Genetic polymorphisms in CYP2C9 cause significant interindividual variability in the metabolism of its substrates. However, the variability of CYP2C9 intrinsic hepatic clearance (CLint,h,CYP2C9) among subjects of the same genotype has not been reported. In this study, we estimated the coefficient of variation (CV) for the intrinsic hepatic clearance of tolbutamide by CYP2C9 for each CYP2C9 genotype using previously reported area under the blood concentration curve (AUC) and oral clearance (CLoral) values in a Monte Carlo simulation with a dispersion model. The CVs for tolbutamide CLint,h,CYP2C9 were estimated to be 18.1%, 23.9%, 25.4%, 22.3%, 13.0%, and 19.8% for CYP2C9*1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. These values are smaller than those previously reported for CYP2D6*1/*1 (43%), CYP2C19*1/*1 (66%), and CYP3A4 (33%). These CV values were used to predict AUC and CLoral variability of other CYP2C9 substrates, which are also substrates of other CYP isoforms. Then, the estimated CVs were consistent with those reported in previous studies of genotyped and ungenotyped subjects. Our estimates of CLint,h,CYP2C9 variability together with the variabilities of other isoforms are useful for predicting the AUC variability of CYP2C9 substrates.

Prediction of inter-individual variability in the pharmacokinetics of CYP2C19 substrates in humans.

Significant inter-individual variability of exposure for CYP2C19 substrates may be only partly due to genetic polymorphism. Therefore, the in vivo inter-individual variability in hepatic intrinsic clearance (CL(int,h)) of CYP2C19 substrates was estimated from reported AUC values using Monte Carlo simulations. The coefficient of variation (CV) for CL(int,h) in poor metabolizers (PM) expected from genotypes CYP2C19*2/*2, CYP2C19*3/*3 or CYP2C19*2/*3 was estimated as 25.8% from the CV for AUC of omeprazole in PMs. With this, CVs of CL(int,h) in extensive metabolizers (EM: CYP2C19*1/*1), intermediate metabolizers (IM: CYP2C19*1/*2 or *3) and ultra-rapid metabolizers (UM), CYP2C19*17/*17 and *1/*17, were estimated as 66.0%, 55.8%, 6.8% and 48.0%, respectively. To validate these CVs, variability in the AUC of CYP2C19 substrates lansoprazole and rabeprazole, partially metabolized by CYP3A4 in EMs and IMs, were simulated using the CV in CL(int,h) for CYP2C19 EMs and IMs and 33% of the CV previously reported for CYP3A4. Published values were within 2.5-97.5 percentile range of simulated CVs for the AUC. Furthermore, simulated CVs for the AUC of omeprazole and lansoprazole in ungenotyped populations were comparable with published values. Thus, estimated CL(int,h) variability can predict variability in the AUC of drugs metabolized not only by CYP2C19 but also by multiple enzymes.

Genotyping of 38 insertion/deletion polymorphisms for human identification using universal fluorescent PCR.

Short insertion/deletion (Indel) polymorphisms of approximately 2-6 bp are useful as biallelic markers for forensic analysis, and the application of Indel genotyping as a supplementary tool would improve human identification accuracy. We examined the allele frequencies of 37 autosomal Indels in the Japanese population and developed a novel dual-color genotyping method for human identification on the basis of universal fluorescent PCR, including the sex-typing amelogenin locus. Target genomic fragment sizes for 38 Indels were 49-143 bp. We analyzed these Indels in 100 Japanese individuals using the M13(-47) sequence as a universal primer. For dual-color genotyping, we designed a novel universal primer with high amplification efficiency and specificity. Using FAM-labeled M13(-47) and HEX-labeled modified M13(-47) primers, fluorescent signals at all loci were clearly distinguished in two independent multiplex PCRs. Average minor allele frequency was 0.39, and accumulated matching probability was 2.12 × 10(-15). Complete profiles were successfully amplified with as little as 0.25 ng of DNA. This method provides robust, sensitive, and cost-effective genotyping for human identification.