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

我们选择了8个与胶原代谢相关的基因，分别是：脯氨酰4-羟基酶(PH)、赖氨酰羟基酶(LH)、前胶原N-蛋白酶(PNP)、前胶原C-蛋白酶(PCP)、Pro-4-羟基酶、α-多肽、2型(P4HA2)、赖氨酸氧化酶(LOX)、胶原蛋白、I型、α-1(COL1A1)和基质金属蛋白酶1(COL1A1)；其中7个基因的变化被推测在皮肤组织中，如构成弹性纤维的弹性蛋白(Eln)、与神经酰胺代谢有关的β-葡萄糖脑苷酸酶(Gba)、鞘磷脂酶(Smpd1)和酸性神经酰胺酶1(Asah1)，以及第二年与黑色素生物合成有关的酪氨酸酶(Tyr)、5，6-二羟基-吲哚-2-羧酸氧化酶(Tyrosinase)、5，6-二羟基-吲哚-2-羧酸氧化酶(Tyrp1)和多巴铬互变擦除酶(Dct)。针对每个基因的启动子和(或)增强子区域设计了几组聚合酶链式反应(PCR)和焦磷酸测序引物，用从尸检时采集的4例男性和女性腹部皮肤组织提取的亚硫酸盐模板DNA进行聚合酶链式反应(PCR)，然后进行焦磷酸测序反应。因此，仅在TYR-F1区的第二个CpG位点，当使用S2引物时，年龄与甲基化呈强烈的负正相关。在其他几乎所有的地区，所有样本之间的甲基化没有变化，或者即使在样本之间观察到一些显著的变化，这些都与年龄无关。在本研究中，虽然我们最终未能在皮肤组织中找到CpG位点甲基化与年龄之间的明确关系，但由于在正常组织中观察到了不同程度的甲基化，提示可能存在一些与AGE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.