Maher Geoffrey J, McGowan Simon J, Giannoulatou Eleni, Verrill Clare, Goriely Anne, Wilkie Andrew O M
Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, United Kingdom;
Computational Biology Research Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, United Kingdom;
Proc Natl Acad Sci U S A. 2016 Mar 1;113(9):2454-9. doi: 10.1073/pnas.1521325113. Epub 2016 Feb 8.
De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.
新生点突变主要发生在男性生殖细胞系中，且频率随年龄增长而增加，但此前无法在人类睾丸的生精小管内直接定位特定的、可识别的突变。通过对表现出精原细胞标志物MAGEA4、FGFR3和磷酸化AKT表达改变的小管进行显微切割、全基因组扩增和DNA测序，我们建立了一种原位策略，用于发现和分析致病性新生突变。在14名年龄在39 - 90岁男性的睾丸中，我们从分析的22个小管中的16个中，在五个基因（成纤维细胞生长因子受体FGFR2和FGFR3、酪氨酸磷酸酶PTPN11以及RAS癌基因同源物HRAS和KRAS）中鉴定出11个不同的功能获得性突变；所有突变均与严重疾病相关，范围从先天性或围产期致死性疾病到体细胞获得性癌症。这些结果支持了关于影响生长因子受体 - RAS信号传导的精原细胞突变的自私选择假说，突出了其在老年男性中的普遍性，并能够直接观察到伸长的突变克隆的微观解剖结构。
