The Molecular Anatomy of Spontaneous Germline Mutations in Human Testes

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 10(3) to >10(4) 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.