Establishment of molecular biological estimation for age from forensic specimens using changes in human genome with aging

我们选择了与胶原代谢相关的8个基因，即脯氨酰4-羟化酶（PH）、赖氨酰羟化酶（LH）、前胶原N-蛋白酶（PNP）、前胶原C-蛋白酶（PCP）、脯氨酰4-羟化酶α多肽2（P4 HA 2）、赖氨酰氧化酶（LOX）、胶原α 1型（COL 1A 1）和基质金属蛋白酶1（胶原酶1; MMP 1），以及推测在皮肤组织中发生变化的7个基因，例如构成弹性纤维的弹性蛋白（ELN）、与神经酰胺代谢相关的β-葡糖脑苷脂酶（GBA）、鞘磷脂酶（SMPD 1）和酸性神经酰胺酶1（ASAH 1），以及酪氨酸酶（TYR），5，6-二羟基吲哚-2-羧酸氧化酶（TYRP 1）和多巴色素互变异构酶（DCT）。针对各基因的启动子和/或增强子区域设计了几种PCR引物组和焦磷酸测序引物，使用这些引物组与从尸检时收集的4名男性和女性个体（年龄范围为0至80岁）的皮肤组织中提取的亚硫酸氢盐模板DNA进行PCR反应，然后进行焦磷酸测序反应。因此，当使用S2引物时，仅在TYR-F1区域的第二个CpG位点处，年龄与甲基化之间显示出强负的正比例。在其他几乎所有地区，所有样品之间的甲基化没有变化，即使样品之间观察到一些显着的变化，这些变化与年龄无关。在本研究中，虽然我们不能最终发现CpG位点甲基化与年龄的明确关系，但由于在正常组织中观察到不同程度的甲基化，因此提示可能存在一些与年龄相关的甲基化CpG位点。

We selected eight genes related to collagen metabolism such as prolyl 4-hydroxylase (PH), lysyl hydroxylase (LH), procollagen N-proteinase (PNP), procollagen C-proteinase (PCP), prolyl 4-hydroxylase, alpha polypeptide, type 2 (P4HA2), lysyl oxidase (LOX), collagen, type I, alpha-1 (COL1A1) and matrix metalloproteinase 1 (collagenase 1; MMP1) in the first fiscal year, and seven genes of which changes are speculated in skin tissues such as elastin (ELN) constituent to elastic fibers, β-glucocerebrosidase (GBA), sphingomyelinase (SMPD1) and acid ceramidase 1 (ASAH1) related to ceramid metabolism, and tyrosinase (TYR), 5,6-dihydroxy-indole-2-carboxylic acid oxidase (TYRP1), and dopachrome tautomerase (DCT) related to melanin biosynthesis in the second year. A few kinds of PCR primer sets and pyrosequencing primers for the promoter and/or enhancer regions at each gene were designed, PCR reactions were performed using those primer sets with bisulfited template DNA extracted from abdomimal skin tissues collected at autopsy from 4 male and female individuals whose ages ranged from 0 to 80, and then the pyrosequencing reactions were performed. Consequently, only at the second CpG site in TYR-F1 region when using the S2 primer, a strong negative direct proportion between age and methylation were shown. In other almost all regions, no changes of methylation among all samples, or even if some significant changes between samples were observed, those were not concerned to the ages. In the present study, although we could not finally find the clear relationship between methylation at CpG sites and age with skin tissues, it was suggested that some methylated CpG sites related to age might exist because some different degrees of methylation were obsereved in normal tissues.