The Molecular Anatomy of Spontaneous Germline Mutations in Human Testes

Abstract

The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene (FGFR2) is 100–1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 103 to >104 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10−6) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an offspring, thereby unfavorably altering the mutational load in humans. Studying the anatomical distribution of germline mutations can provide new insights into genetic disease and evolutionary change.

Missing heritability: paternal age effect mutations and selfish spermatogonia

“…Recent evidence, based on quantification of mutation levels in sperm 4,5 and testes 6 , indicates that the spermatogonial (pre-meiotic) cells in which PAE mutations originally arose are positively selected and expand clonally (FIG. 1).…”

“…Direct measurements [4][5][6] in normal sperm and testes are laborious, and segregation of normal and mutant alleles in sperm of heterozygotes for PAE mutations is not expected to deviate from 50:50. Recently, the first direct study of new mutations in the human germ line, obtained by whole-genome sequencing Figure 1 | Illustrative depiction of how enrichment of functionally significant paternal age effect mutations could occur.…”

“…This pro cess provides a constant supply of new, functionally significant mutations in the next generation. In practice, fewer than 2% of cell divisions would need to occur symmetrically to account for up to 1,000 fold enrichment in sperm associated with observed paternal age effect (PAE) mutation 6,15 .…”

Evolutionary genetics of affective disorders

“…Another example of pleiotropic effects in 'neuronal' genes is testicular functions in males, given strong patterns of gene co-expression in brain and testis (Guo et al 2005) that are apparently underlain by shared receptor functions such as exocytosis (Meizel 2004). Genes differentially expressed in testis exhibit notably-enhanced signatures of positive selection in humans (Nielsen et al 2005), selection that may generate negative pleiotropic effects in the context of neuronal activities (Qin et al 2007). …”

Cellular correlates of selfish spermatogonial selection

“…1C). Our conclusions on this point are corroborated by work from the Arnheim group, which has documented, using quantitative DNA analysis, the independent spatial distributions of four different mutations across four of the testes (59089, 374‐1, 374‐2 and 854‐2) they analysed (Qin et al ., 2007; Choi et al ., 2008, 2012; Yoon et al ., 2013). Nevertheless, we expect the absolute risk of transmitting any individual mutation to be low, for three reasons.…”