Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes

Geoffrey J. Maher; Simon J. McGowan; Eleni Giannoulatou; Clare Verrill; Anne Goriely; Andrew O. M. Wilkie

doi:10.1073/pnas.1521325113

New Research In

Edited by Aravinda Chakravarti, Johns Hopkins University School of Medicine, Baltimore, MD, and approved January 14, 2016 (received for review October 29, 2015)

Significance

A major goal in genetics is to understand the processes that shape the frequency of new mutations, particularly those causing human disease. Here, we focus on specific mutations in the male germline that, although initially rare, confer a growth or survival advantage to the stem cell, leading to clonal expansion over time: a process similar to early tumor growth and currently described only in humans. Previous studies supporting this “selfish” selection quantified mutations in sperm or testis pieces using methods that destroyed their cellular origins. Here, we pinpoint and identify pathogenic mutations directly within individual seminiferous tubules, the structures that generate spermatozoa. This methodology provides unprecedented precision in documenting the spectrum and prevalence of selfish mutations in men’s testes.

Abstract

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.

Footnotes

↵¹Present address: The Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.
↵²To whom correspondence may be addressed. Email: andrew.wilkie{at}imm.ox.ac.uk or anne.goriely{at}imm.ox.ac.uk.

Author contributions: G.J.M., A.G., and A.O.M.W. designed research; G.J.M., C.V., and A.G. performed research; G.J.M., S.J.M., E.G., A.G., and A.O.M.W. analyzed data; and G.J.M., A.G., and A.O.M.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1521325113/-/DCSupplemental.

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