One thousand somatic SNVs per skin fibroblast cell set baseline of mosaic mutational load with patterns that suggest proliferative origin

Few studies have been conducted to understand post-zygotic accumulation of mutations in cells of the healthy human body. We reprogrammed 32 skin fibroblast cells from families of donors into human induced pluripotent stem cell (hiPSC) lines. The clonal nature of hiPSC lines allows a high-resolution analysis of the genomes of the founder fibroblast cells without being confounded by the artifacts of single cell whole genome amplification. We estimate that on average a fibroblast cell in children has 1,035 mostly benign mosaic SNVs. On average, 235 SNVs could be directly confirmed in the original fibroblast population by ultra-deep sequencing, down to an allele frequency (AF) of 0.1%. More sensitive droplet digital PCR experiments confirmed more SNVs as mosaic with AF as low as 0.01%, suggesting that 1,035 mosaic SNVs per fibroblast cell is the true average. Similar analyses in adults revealed no significant increase in the number of SNVs per cell, suggesting that a major fraction of mosaic SNVs in fibroblasts arises during development. Mosaic SNVs were distributed uniformly across the genome and were enriched in a mutational signature previously observed in cancers and in de novo variants and which, we hypothesize, is a hallmark of normal cell proliferation. Finally, AF distribution of mosaic SNVs had distinct narrow peaks, which could be a characteristic of clonal cell selection, clonal expansion, or both. These findings reveal a large degree of somatic mosaicism in healthy human tissues, link de novo and cancer mutations to somatic mosaicism and couple somatic mosaicism with cell proliferation.

目前针对健康人体细胞内合子后突变积累的相关研究尚少。我们将32份来自不同供体家族的皮肤成纤维细胞重编程为人类诱导多能干细胞（human induced pluripotent stem cell, hiPSC）系。hiPSC系的克隆特性使得我们可以高分辨率解析原始成纤维细胞的基因组，且不会被单细胞全基因组扩增产生的实验假象所干扰。我们估算得出，儿童体内的成纤维细胞平均携带1035个大多为良性的嵌合单核苷酸变异（single nucleotide variant, SNV）。通过超深度测序，我们可在原始成纤维细胞群体中直接验证其中平均235个SNV，其检测下限可达等位基因频率（allele frequency, AF）0.1%。采用灵敏度更高的液滴数字PCR（droplet digital PCR, ddPCR）实验则验证了更多嵌合SNV，其AF可低至0.01%，这表明每个成纤维细胞平均携带1035个嵌合SNV这一数值为真实平均值。对成人样本的类似分析未发现每个细胞的SNV数量存在显著增加，这提示成纤维细胞中的大部分嵌合SNV产生于发育阶段。嵌合SNV在全基因组范围内均匀分布，且富集于此前在癌症及新生变异中观察到的突变特征；我们推测，该特征是正常细胞增殖的标志性特征。最后，嵌合SNV的AF分布呈现出明显的窄峰特征，这可能是克隆细胞选择、克隆扩增或二者共同作用的结果。本研究的这些发现揭示了健康人体组织中广泛存在的体细胞嵌合现象，将新生突变与癌症突变关联至体细胞嵌合，并阐明了体细胞嵌合与细胞增殖之间的联系。