GENETIC HETEROGENEITY AMONG HUMAN SOMATIC CELLS AND ITS ACCUMULATION WITH AGE

摘要/项目总结 从人类健康的角度来看，由于正常人中突变的积累而导致的遗传异质性， 组织增加癌症风险，并且可能是许多退行性疾病的重要因素，例如 成人黄斑变性，充血性心力衰竭，炎症和自身免疫性疾病， 肌肉减少症和肌无力以及骨骼和关节退化。体细胞突变可能是由于 DNA损伤或复制错误。然而，很难检测到体细胞突变， 因为不可能可靠地比较个体患者的基因组序列， 单细胞人结肠隐窝可以用作结肠干细胞在结肠上皮细胞中的克隆代表。 每个地窖的底部使用SNP微阵列，我们已经表明，大规模的人类体细胞对细胞， 差异（缺失、基因转换、重复）存在于结肠隐窝中，并且随着年龄的增长而增加。我们 优化了全基因组测序的方法，而无需将全基因组扩增至> 30 X 使用1000至2000个细胞的单个结肠隐窝进行深度研究，以研究在两个细胞核中积累的突变。 和线粒体基因组。细胞核和线粒体中单碱基突变的增加 在全基因组测序分析中观察到基因组随年龄的变化。基于功率 根据这些初步研究结果，我们建议将研究范围扩大到21个 分布在三个年龄组的个体，以统计学方式验证我们的发现， 与年龄相关的DNA损伤的潜在机制。在目标1中，整个测序文库将是 由五个单个结肠隐窝和大部分组织构建，用于> 30 X的基因组覆盖， 从儿童到老年人的年龄范围内的每个个体人类受试者。目标2： 基因组测序数据将检查点突变和小插入缺失，以及隐窝， 同一个体的生殖系和隐窝间的变化。在目标3中，将鉴定大缺失 以了解DNA断裂和修复的机制。在目标4中，DNA序列基序在 将检查遗传变化的位点，以确定核以及 线粒体基因组这些年龄的潜在生化基础和分子修复途径- 相关的DNA畸变也将被探索。这些努力对于理解DNA 序列是最高风险的类型的DNA损伤或错误倾向的DNA修复，导致 基因变化的积累。本文的研究将为今后研究如何开展工作打下基础 环境和饮食可能会影响人类一生中体细胞遗传变化的进展 以及多态性如何使某些个体更易患上快速变性疾病 或癌症，从而允许考虑预防措施。此外，由于诊断分析依赖于 当体细胞逐渐减少时，这种体细胞遗传异质性的知识将 对精准医疗越来越重要。

ABSTRACT/PROJECT SUMMARY From a human health perspective, genetic heterogeneity due to accumulation of mutations in normal tissues increases cancer risk, and is likely an important factor in many degenerative diseases, such as adult macular degeneration, congestive heart failure, inflammatory & autoimmune disorders, sarcopenia & muscle weakness, and skeletal & joint degeneration. Somatic mutations can occur due to DNA damage or replication errors. However, it has been difficult to detect somatic mutations in individual patients because it has not been possible to reliably compare sequences of the genomes of single cells. Human colon crypts can be used as a clonal representation of the colon stem cell at the base of each crypt. Using SNP microarray, we have shown that large-scale human somatic cell-to-cell differences (deletions, gene conversions, duplications) exist in colon crypts and increase with age. We have optimized methods for whole genome sequencing without whole genome amplification to >30X depth using a single colon crypt of 1000 to 2000 cells to study mutations accumulated in both nuclear and mitochondrial genomes. An increase in single base mutations in both the nuclear and mitochondrial genomes with age was observed in the whole genome sequencing analysis. Based on power calculations using these preliminary findings, we propose to expand the study to include a total of 21 individuals distributed among three age groups to statistically validate our findings and examine potential mechanisms for the age-related DNA damage. In Aim 1, whole sequencing libraries will be constructed from five single colon crypts and the bulk tissue for >30X coverage of the genome from each individual human subject over a range of ages from children to elderly. In Aim 2, the whole genome sequencing data will be examined for point mutations and small indels, and for crypt to germline and crypt-to-crypt changes in the same individual. In Aim 3, large deletions will be identified to discern the mechanism of both the DNA breakage and the repair. In Aim 4, DNA sequence motifs at sites of genetic change will be examined to identify preferred damage sites in the nuclear as well as the mitochondrial genome. The potential biochemical basis and the molecular repair pathway of these age- related DNA aberrations will also be explored. These efforts are important for understanding what DNA sequences are at highest risk for the types of DNA damage or error-prone DNA repair that result in accumulation of genetic change. This study will lay the foundation for future work to study how environment and diet may affect the progression of somatic genetic changes over the human lifespan as well as how polymorphisms may predispose some individuals to more rapid degenerative diseases or cancer and thus permit consideration of preventive measures. Also, as diagnostic analysis relies upon progressively fewer somatic cells, this knowledge of somatic cell genetic heterogeneity will become increasingly important for precision medicine.