Transmission of more Mutations by older Fathers

There are certain disease-causing de novo mutations in the germline with rates per generation orders of magnitude higher than the genome average. Moreover, these mutations occur exclusively in the male germ line, and older men have a higher probability of having an affected child than younger ones, known as the paternal age-effect (PAE). One of the best known PAE mutations is achondroplasia, caused by a single nucleotide substitution in FGFR3. The mechanisms propagating these mutations are not well understood but in the last decade it was shown that for other spontaneous congenital disorders following a PAE, such as Apert syndrome and multiple endocrine neoplasia type 2b (MEN2B), mutations confer a selective advantage to spermatogonial stem cells. It also has been suggested that the selective advantage is the result of changes in the growth factor receptor-RAS signaling pathways caused by the mutant protein. Yet, there are still many open questions on how and to what extent mutations change germline stem-cell behavior, if all PAE mutations are driven by similar mechanisms, and whether other mechanisms such as apoptosis or cell-death counterbalance oncogenic expansions of mutant germline cells. The aim of this proposal is to get a deeper understanding of the mutation mechanisms behind the paternal age effect in FGFR3. Specifically, we will focus on the two achondroplasia mutations (c.1138G>A, c.1138G>C) and the thanatophoric displasia II mutation (c.1948A>G ). These mutations have different mutation rates and show differences in signaling dysregulation of mutant FGFR3 presenting a unique opportunity to assess these properties as effectors in the biology of the paternal age effect. The frequency of these three mutations will be analyzed at different stages of spermatogenesis, specifically in spermatogonia, sperm from ejaculates and sperm with the highest motility in donors of different ages. We have developed a highly sensitive technique based on bead- emulsion amplification that can for the first time measure these mutations. With this project we can elucidate for the first time the very important relationship between mutation, selection, and cell-death at different developmental stages of spermatogenesis and how these factors contribute to the paternal age effect.

Our genome, encoded by DNA, is under constant change. These changes, also known as de novo mutations, can occur in the parents' germline resulting in new traits or disease in our offspring. Recent advances in next generation sequencing have shown that the majority of new mutations originate in the male germline. To date, however, we lack information on individual mutagenic events necessary to study a unique type of mutagenesis the selfish expansion of mutations in the male germline. These mutations have unique characteristics: they are associated with congenital disorders, occur at a thousand-fold higher frequency than other mutations, and drastically increase with paternal age. To date, the mechanisms propagating these paternal-age effect mutations are not completely understood. The aim of this proposal was to examine the origin and expansion of paternal-age effect mutations at different developmental stages of the male germline. For this purpose, we adapted a new ultra-sensitive technology based on the amplification of millions of single DNA fragments in an emulsion that can accurately determine the frequency rare de novo mutations. We focused on four mutations of the fibroblast growth factor receptor 3, FGFR3 that are linked to congenital disorders (achondroplasia, thanatophoric displasia II, and hypochondroplasia), have different mutation rates and affect the signaling of FGFR3 at different degrees. We analyzed the mutation frequency of these four mutations in a dissected testis of an old donor and in sperm collected from different donors ranging from 25 to 60 years old. Two out of four mutations showed that mutant DNA concentrated within different clusters of the old donors testis, resulting from the growth advantage of stem cells in the male germline likely caused by the activation of FGFR3. We did not observe a significant difference in the size or concentration of the clusters. However, mutations with a stronger effect on FGFR3 signaling dysregulation showed a reduced transmission into sperm. With this work, we show for the first time that mutations changing FGFR3 signaling that are expanding in the testes with age, are not necessarily transmitted into sperm at a high frequency. Thus, there are important regulatory mechanisms at different developmental stages of spermatogenesis that affect the downstream transmission of paternal-age mutations. Our analysis forms a basis for understanding this type of mutagenesis and the associated risks of delayed parenthood in our society.