RNA Sequence Analysis Reveals Macroscopic Somatic Clonal Expansion Across Normal Tissues

Introduction: Cancer genome studies have significantly contributed to the analysis and discovery of somatic mutations that drive cancer growth. However, studying the genetic makeup of a tumor when it is already fully developed limits our ability to uncover how and which somatic mutations accumulate in normal tissues in the stages preceding cancer initiation. Although efforts have begun to collect and analyze DNA from normal tissues, we still lack a comprehensive catalog of genetic events and clonal properties across a large number of tissues and individuals. By analyzing the information-rich content in RNA now available from recent advances in RNA-sequencing methods, we may be able to significantly expand the scope and scale of these studies.

Materials and Methods: Some mutations found in the DNA can be detected in the corresponding RNA, depending on the mutation allele fraction and sequence coverage. We therefore hypothesized that a careful analysis of RNA sequences from normal bulk tissues could uncover somatic mutations reflecting macroscopic clones within the samples. To this end, we developed a new method called RNA-MuTect, to identify somatic mutations using a tissue-derived RNA sample and its matched-normal DNA. RNA-MuTect is designed to remove RNA-specific false positive variants that are result of sequencing and alignment noise.

Results: We validated RNA-MuTect on both tumor-adjacent and cancer samples from The Cancer Genome Atlas, wherein DNA and RNA were co-extracted from the same samples, and show high sensitivity and precision levels. When applied to the GTEx dataset of normal tissues, including over 6,500 samples from ~500 individuals, and spanning across 30 different normal tissues, multiple somatic mutations were detected in almost all individuals and tissues studied here, including in known cancer genes. The three tissues with the largest number of somatic mutations were sun-exposed skin, esophagus mucosa, and lung, suggesting that environmental exposure can promote somatic mosaicism. Both the individuals’ age and tissue-specific proliferation rate were found to be associated with the number of detected mutations.

Conclusions: Genetic clones carrying somatic mutations are detected to differing extents across normal tissues, and these differences depend on factors such as the tissue’s exposure to environmental mutagens, natural architecture, proliferation rate, and the microenvironment. Some of these clones may be the result of genetic drift. Others, however, may develop due to positive selection driven by certain somatic events, thus potentially representing the earliest stages of tumorigenesis. Higher-resolution studies of normal tissues and pre-cancerous lesions are required for advancing our understanding of both aging and early cancer development.

RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues. Science (80- ). 2019;364: eaaw0726. doi:10.1126/science.aaw0726