Targeted resequencing identifies mutant selfish clones within the testis and unifies the concepts of somatic and germline mutation. G. J. Maher, E. Giannoulatou, S. J. McGowan, A. Goriely, A. O. M. Wilkie Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

   Introduction: The selection and clonal expansion of pathogenic somatic mutations is associated with diseases such as cancer and tissue overgrowth; however such mutations are typically not inherited. In the special context of the testis, we and others have presented evidence that a similar process leads to elevated levels of specific mutations in sperm (up to 104-fold higher than the background rate), a process that we term selfish spermatogonial selection. This mechanism is responsible for the high apparent birth rate of several spontaneous congenital disorders, and may have a wider role in cancer predisposition and neuropsychiatric conditions. Until now, however, it has not been possible to identify directly these mutant clonal expansion events in the cellular architecture of normal human testes. Methods and Results: In sections of formalin-fixed paraffin embedded (FFPE) normal testes from elderly men (aged 62-79 yr), we identified a subset of seminiferous tubules with a morphology and antigenic profile suggestive of mutant clonal expansion. Laser capture microdissection, whole genome amplification (WGA) and targeted resequencing (100 candidate genes using Haloplex technology) of tubules from 8 testes identified previously defined selfish mutations encoding activating substitutions, including FGFR2 Y340C (corresponding to Pfeiffer syndrome when occurring as a germline mutation) and C342S (Crouzon syndrome), FGFR3 Y373C (thanatophoric dysplasia (TD) type I) and HRAS G13R (likely germline lethal). A subsequent targeted screen of 10 mutation hotspots in 6 genes using Ion Torrent PGM sequencing identified further tubules with mutations in FGFR3 (K650E (TD type II)) and HRAS (G13R). The mutations were specifically localised to the tubules with abnormal antigenic properties; surrounding normal tubules were mutation-negative. On histological examination, the mutant tubules had variably reduced spermatogenesis. To exclude confounding technical artefacts, all mutations were verified by Sanger sequencing of non-WGA material. Conclusions: For three genes (FGFR2, FGFR3 and HRAS) our results demonstrate for the first time the occurrence of selfish spermatogonial selection at a cellular level in the testis. This opens up a new approach to analysing the germline profile of mutations in the testis and the signalling pathways affected, which will have implications for understanding the paternal age effect and the many diseases fuelled by a high rate of new mutations.

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