Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human 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.iscerning the source of spontaneous germline mutations is fundamental to understanding the causes of many diseases, including monogenic developmental disorders (1) and complex conditions such as autism (2, 3) and schizophrenia (4). Recent whole genome sequencing studies of parent-child trios show that most mutations (such as nucleotide substitutions) originate from the paternal germline and increase in frequency with the father's age (5, 6), an issue of particular significance given the demographic shift to delayed reproduction in many populations (7). The deduction that the testes of older men harbor a greater burden of mutations, compared with younger men, is consistent with indirect measures of genetic decline, ranging from high indices of arrested germ cell divisions to complete involution of the seminiferous tubules (7-9). Surprisingly, however, it has not previously been possible to trace specific mutations back to their origins within individual germ cells (spermatogonia) of human testes.One mechanism proposed to contribute to the age-related increase in male mutations is selfish spermatogonial selection, a process equivalent to neoplasia but occurring in the unique context of the germ cell (10). In this process, specific point mutations that confer gain-of-function to components of the growth factor receptor-RAS signaling pathway occur rarely in spermatogonial stem cells of the adult testis but show a steep increase in prevalence with age, attributed to clonal expansion of mutant spermatogonia over time (11)(12)(13)(14)(15)(16). Fertilization of the egg by a mutant sperm leads to serious congenital disorders in the next generation, characterized by multiple malformations and, in some cases, a predisposition to malignancy. These disorders include Apert, Crouzon, and (16, 26). Based on the unexpectedly high birth prevalence of several of the associated congenital d...

Cellular correlates of selfish spermatogonial selection

“…Genetic analysis of this material was experimentally challenging, because the DNA extracted from FFPE tissue is degraded; nevertheless, provided that FFPE material was less than 6 years old, we were able to implement a robust pipeline involving laser capture microdissection, whole genome amplification, and targeted HaloPlex‐based sequencing of >100 genes. An example of the results from one of the testes analysed in our study (Maher et al ., 2016) is shown in Fig. 1A.…”

“…Whilst we contend that these data (Maher et al ., 2016) answer the request by Pohl et al . (2016) for ‘a more careful interpretation of such spermatogonial clusters…to unequivocally determine “selfish spermatogonial selection”‐manifestations’, we do hope that further research will now be undertaken to explore the cellular characteristics of immunopositive tubules, and their relationship to age, in much greater detail.…”

“…In other words, this work directly and unambiguously links the distinct MAGEA4‐/FGFR3‐/phospho‐AKT immunopositive appearance of tubules with the presence of acquired selfish mutations in these elongated cellular clones. Of note, most such tubules showed variably impaired spermatogenesis when compared to their normal neighbours, hinting that some might be incompatible with the formation of mature spermatozoa (Maher et al ., 2016). …”

“…1A and by Maher et al . (2016). For example, by applying our protocol to adjacent sections, we have now established that the tubule labelled (C3) in Fig.…”

“…First, given the estimate that the overall length of seminiferous tubules in a pair of human testes is around 800 m (Glass, 2005), a clone extending along 5 cm of tubule (approximately the maximal length we have been able to document to date) would yield a mutation level of only 1 in 32,000. Second, we have shown a general trend for selfish clones to be associated with impaired spermatogenesis (Maher et al ., 2016), which is expected to reduce transmission to the next generation. Third, most fathers in the population are young, and will have fewer and smaller clones, because elongation of clones is age‐dependent.…”