In theory, heritable nucleotide substitutions can arise in any germline cell during scheduled DNA replication or in nondividing cells by means of error-prone DNA repair (see [32
]). Two observations support the importance of the cell division–dependent mutation process. First, neutral germline mutations seem to arise 3–6-fold more frequently in males than females in many organisms, including humans (male-driven evolution, see [33
]). Second, a number of genetic conditions including Apert syndrome appear to increase in the offspring of men as they age (paternal age effect [1
]). Both male-driven evolution and the paternal age effect can be explained by the cell division–dependent mutation process, because cell divisions of self-renewing spermatogonia occur throughout a man's life, whereas the cellular precursors of eggs (oogonia) cease replication during the fetal life of a female [1
We examined the molecular anatomy and frequency of the Apert syndrome C755G mutation in normal human testes to test whether the high mutation frequency was due to an exceptionally high C-to-G transversion mutation rate per cell division. The results show that the observed C755G mutation frequency and distribution within the testes cannot be explained by this hot spot model (p < 10−6).
An alternate hypothesis to explain the high C755G mutation frequency argues that diploid premeiotic cells carrying the C755G mutation have a selective advantage over wild-type cells [4
]. In one case [5
], the authors cited the puzzling observation that the magnitude of the sex bias for the C755G Apert syndrome is at least 99-fold greater in the male germline than in the female germline [34
], whereas estimates of male bias (male-driven evolution) using data on neutral mutations at many different sites would indicate only an ~5-fold male preference [33
]. Rare patients with multiple mutations in the FGFR2
gene that leads to Apert syndrome were also cited as support for a germline selection model [13
]. Finally, in another study [4
], the authors exploited a nearby single nucleotide polymorphism (SNP) to argue that selection acted on the C755G mutation (however, see Text S1
and Figure S1
In our experiments, we were not only able to clearly reject the hot spot model, but also we could show that modifying our model of germline development by incorporating a simple selection scheme led to predictions on mutation frequency and testis distribution consistent with our data. This selection takes place on SrAp cells carrying the C755G mutations that arise at approximately the frequency expected from the existing data on neutral mutations [10
]. The selection model proposes that mutant adult SrAp occasionally divide symmetrically (inferred rate 1 out of 100 divisions on average, or approximately once every 4 y), whereas wild-type SrAp always undergo asymmetric self-renewal divisions. Considering all of the published work [4
] as well as our present results, it now seems very likely that positive selection can be a driving force acting to increase the germline mutation frequency in humans above the frequency at which spontaneous nucleotide substitutions arise.
We would like to emphasize that the type of selection we are discussing is on diploid premeiotic cells. Previous proposals have suggested that C755G mutation bearing sperm may have a selective advantage over wild-type sperm [3,38
]. Selection taking the form of competition among sperm is well known in plants and animals [39
], and is even documented in primates [40
]. However, for this particular mutation, the testis and epididymal sperm data we collected as well as data on ejaculated sperm ([3
] and unpublished data) and Apert syndrome birth data [6
] show similar mutation frequencies. Therefore, while further selection on sperm is possible, we believe that the vast majority of the increase in mutation frequency is due to selection on the diploid premeiotic cells.
Why should a mutation that has a distinct selective disadvantage when present in all the cells of an organism have a selective advantage when present only in a small fraction of the germline cells? It is worth noting here that achondroplasia, the most common cause of dwarfism, has many similarities to that of Apert syndrome (see [5
]). Virtually all of the new achondroplasia mutations arise in the male germline at one nucleotide site (G1138A) in the fibroblast growth factor receptor 3 (FGFR3)
gene and with a mutation frequency even higher than the C755G mutation in FGFR2
. These common characteristics suggest that the G1138A mutation may also increase to such a high frequency by a selective mechanism [4
]. It is interesting to note that both FGFR2 and FGFR3 are receptor tyrosine kinases and can influence downstream members of the signal transduction pathway (for a more detailed discussion see [41
]). Especially relevant may be the fact that some mutations in FGFR2
(although usually not the specific mutations that cause Apert syndrome or achondroplasia) have been associated with certain cancers [43
] and cancer susceptibility [45
Germline selection in diploid germ cells of animals was considered by the population geneticist Ian Hastings [46
]. He examined how mitotic gene conversion and somatic crossing over events in diploid germline cells of animals could lead to loss of heterozygosity of recessive alleles and the possibility of positive or negative selection on such alleles. He calculated that selection against rare germline cells made homozygous for a recessive allele can effectively lower the transmission of the deleterious allele to offspring thereby reducing disadvantageous alleles entering the population and burdening it with reduced viability or fertility. Using plausible models, this reduction in the mutational load could be as large as 100-fold. Similarly, loss of heterozygosity could allow recessive alleles that conferred a germline advantage to be spread more quickly in the population. Experimental literature on germline selection in premeiotic diploid cells in animals is very sparse (see [48
] and references therein) but in one case, wild-type Drosophila
cells were produced by a genetic trick in the germline of females heterozygous for a phenotypically recessive mutation and were found to have a proliferative selective advantage compared to the background heterozygous cells.
Hastings' analysis was primarily concerned with recessive alleles that were already polymorphic in the population. But gain-of-function mutations that arise sporadically in the testis would behave in the same way, because a second event leading to loss of heterozygosity is not required for positive and negative selection to be effective. A new gain-of-function mutation with a germline selective advantage will more likely be transmitted to the next generation, because the effective mutation frequency is elevated beyond the level that can be achieved by the mutation process alone. A disadvantageous gain of function mutation would be less likely to be tested in the population if it were selected against in the germline. Finally, Hastings realized that alleles conferring a selective advantage in the germline may be disadvantageous in the adult and might lead to “mitotic drive” systems that could increase the mutational load of a population. Both Apert syndrome and achondroplasia may be examples of such a system, and additional examples of mutations of medical interest may also exist (see [15
]). The method we have developed can be used to test this hypothesis at any locus in many different species if a sufficiently sensitive mutation assay can be made available.