Saturday, June 09, 2007

The Observed Human Sperm Mutation Frequency Cannot Explain the Achondroplasia Paternal Age Effect

Published online before print October 23, 2002, 10.1073/pnas.232568699
PNAS | November 12, 2002 | vol. 99 | no. 23 | 14952-14957

The observed human sperm mutation frequency cannot explain the achondroplasia paternal age effect
Irene Tiemann-Boege *, William Navidi , Raji Grewal , Dan Cohn ¶, Brenda Eskenazi ||, Andrew J. Wyrobek **, and Norman Arnheim *

*Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089-1340; Department of Mathematical and Computer Sciences, Colorado School of Mines, Golden, CO 80401; New Jersey Neuroscience Institute, 65 James Street, Edison, NJ 08820;¶ Burns and Allen Cedars-Sinai Research Institute/Ahmanson Department of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, CA 90048; ||School of Public Health, University of California, Berkeley, CA 94720; and **Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94550

Communicated by Michael S. Waterman, University of Southern California, Los Angeles, CA and approved September 19, 2002 (received for review August 10, 2002)

The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay.

Abbreviations: FGFR, fibroblast growth factor receptor; USC, University of Southern California; LLNL, Lawrence Livermore National Laboratory; kt-PCR, kinetic PCR; C.I., confidence interval

Geneticists and evolutionary biologists debate the extent to which the lifelong spermatogonial stem cell divisions unique to male gametogenesis contribute to a higher mutation frequency in males (1–8). One source of support for the hypothesis that mutations increase with spermatogonial stem cell divisions comes from the observation of certain human genetic conditions where the incidence of new mutations increases with the age of the father. Epidemiological studies on a number of dominantly inherited conditions indicate that the average age of the fathers is older among unaffected couples having a child with the condition (a sporadic case caused by a new mutation), than the average paternal age in the population (1, 2, 4, 7). Achondroplasia, the most common form of dwarfism, is one of these conditions (9–11). Studies on sporadic achondroplasia cases have reported an exponential increase with paternal age (1, 2, 4, 7, 10).
In mice, a significant increase in the overall male germ cell mutation frequency as measured by the lacI assay was observed between 15 and 28 months of age (12). In humans, no direct molecular measurement of how germ-line nucleotide substitution frequencies change with age exists. Achondroplasia provides a unique opportunity to directly test the relationship between paternal age and sperm mutation frequency at the molecular level. First, 97–99% of the de novo mutations leading to this condition result from a G-to-A transition mutation at base pair 1138 (G1138A) in exon 10 of fibroblast growth factor receptor 3 (FGFR3) (13–15). The cytosine at base pair 1138 is part of a CpG dinucleotide and, if methylated, is highly susceptible to mutation caused by spontaneous deamination (16). Second, all sporadic achondroplasia cases have been found to inherit the G1138A mutation from their father (17). Third, recent data on the population incidence of sporadic achondroplasia (10, 18–20) predict the average frequency of sperm carrying the mutation in normal individuals will be in a range detectable by modern molecular methods (1/15,000 to 1/70,000). Our studies on sperm DNA from men of different ages suggest that the observed increase in G1138A mutation frequency cannot satisfactorily explain the exponential increase in sporadic achondroplasia cases with paternal age.......................

The lifelong spermatogonial stem cell replications have been suggested as an explanation for an increase in the frequency of mutant sperm that results in the increased incidence of sporadic achondroplasia with paternal age (1, 2, 4). A mathematical discrepancy between the cell replication model based on a linear equation to calculate the number of spermatogonial stem cell divisions (4, 26) and the exponential rise in sporadic achondroplasia with paternal age (1, 2, 4, 7, 10) has been pointed out (1, 2, 4, 7). Of course, other age-related mutation mechanisms that do not depend solely on premeiotic cell replications may be responsible for the birth data. However, no matter what the mutation mechanism, our direct measurement of the G1138A mutation frequency in sperm appears to rule out the idea that an age-dependent increase in sperm containing the GC-to-AT transition mutation at position 1138 explains the rapid rise in incidence of sporadic achondroplasia with paternal age (Fig. 3). Below, we discuss six possibilities that might account for this inconsistency.
Fathers of children with sporadic achondroplasia could form a subgroup with distinct mutation properties (because of genetic or environmental factors) compared with our sperm donors. To address this possibility, we studied sperm DNA from four men who fathered a child with achondroplasia. When matched for age to the appropriate sperm donor age group, two (ages 31 and 32) fell within the observed 95% C.I., a third (age 35) had counts that exceeded, by 2-fold, the upper bound of the 95% C.I., whereas the oldest father (age 51) had counts below the lower bound of the 95% C.I. Although the data suggest that fathers of sporadic cases are representative of our sperm donors, the sample size is small. Additional studies will be needed before we can exclude the possibility that population heterogeneity is the explanation for the discrepancy between our mutation data and the achondroplasia paternal age effect.

As yet unappreciated ascertainment biases in the population studies may have overestimated the magnitude of the age effect for the fathers of sporadic achondroplasia cases.

An age-related sperm donor sampling bias could underestimate an age trend in the G1138A frequency data although we can already exclude racial background as a source of bias between young and old donors in both study groups.

Our PCR assay is clearly biased toward overestimating the number of mutants in individuals with <15 counts (Fig. 1). Among the 118 sperm donors, 24 had <15 counts. To assess the impact of this bias, the age distribution of sperm donors expected to father children with sporadic achondroplasia was again compared with the actual age distribution after making a correction to the counts of all individuals with <15 G1138A mutants (Table 1). This correction favored the null hypothesis because it lowered the counts in the youngest age groups and increased the counts in the older age groups. Thus, for ages <40 we set counts <15 equal to 0, while for ages >40 we set counts <15 equal to 15. Despite this severe correction the null hypothesis was still rejected (log likelihood ratio = 20.7268 = 0.0033). The data from the USC and LLNL cohorts were also examined individually by using the same correction (see Table 3). Despite the lower sample size, the USC cohort gave a log likelihood ratio = 25.1466 (= 3.21 x 10–4). The LLNL cohort gave a log likelihood ratio = 10.6348 (= 0.100). Our rather extreme correction is exacerbated in the LLNL cohort because the 18- to 24-year-old category contains only six individuals and all but one of them have <15 counts.

Our results would be formally consistent with the null hypothesis if there was an age-dependent exponential increase in the formation of germ-line premutagenic lesions (16) at the G1138A site that are neither converted to a full mutation or repaired before fertilization (27). One obvious candidate for such a premutagenic lesion is an unrepaired G/T mismatch resulting from deamination of 5-methyl cytosine (16). The cytosine at base pair 1138 is highly methylated in sperm (data not shown and ref. 28). A single sperm with a G/T mismatch would produce PCR product in our assay. However, the observed counts from a population of such sperm would be half of that produced by the same number of sperm carrying A/T transition mutations. A second possible premutagenic lesion contains an apyrimidinic (AP) site on one strand caused by removal of a thymine at a GT mismatch by a glycosylase (16). Taq polymerase is known to pause significantly opposite an abasic site (29) and primers with an internal abasic site can be extended but the extension products are poorly copied during PCR (30). Also, exposure to high temperatures during PCR may lead to strand breaks at abasic sites. It is likely therefore that after the first two steps of our assay, the products from sperm containing a G/AP lesion would be far less than that from an equal number of mutant sperm carrying the AT bp leading to a significant underestimate of the frequency of this premutagenic lesion. Is there any evidence that premutagenic lesions in sperm can lead to achondroplasia? If unrepaired immediately after zygote formation, sperm carrying a G/T lesion would produce a mosaic embryo (+/+ and +/G1138A) after the first cell division. If a G/AP lesion is converted to a G/T or G/G mismatch after zygote formation the embryo can become mosaic for an G1138A or G1138C mutation, respectively, after the first cell division. The G1138C mutation is found in 2% of all sporadic achondroplasia cases (14). Because individuals with the achondroplasia phenotype that have been reported to be mosaic for the G1138A mutation are exceedingly rare (reviewed in ref. 31), cases of sporadic achondroplasia caused by the above repair patterns of premutagenic lesions are likely to be infrequent. On the other hand, sperm carrying a G/T premutagenic lesion could lead to achondroplasia if the G was replaced by an A immediately after zygote formation. The other immediate repair alternative would lead to a WT embryo. Virtually nothing is known about the relative likelihood of the repair alternatives before the first zygotic cell division in early mammalian embryos.

Finally, the discrepancy between the observed G1138A mutation frequency and the achondroplasia paternal age effect might be explained by selection. The G1138A mutation leads to an increased tyrosine kinase activity of FGFR3 protein and influences downstream signal transduction mediated by the Ras-mitogen-activated protein kinase-dependent and/or STAT1 signaling pathways, resulting in a variety of possible biological consequences (32). FGFR3 protein is found in all adult human male germ cells except elongating spermatids (33). The germ cells of the fetal, immature, and adult rat testis exhibit cell- and stage-specific localization of FGFs and FGFRs (including FGFR3 IIIc), which has been taken to imply that signaling via FGF ligands and receptors is spatially and temporally regulated in this organ (34). Although highly speculative, mature sperm derived from cells carrying the FGFR3 achondroplasia mutation may have a selective advantage for sperm motility or capacitation in utero. The molecular mechanisms involved in capacitation are not well known, but protein phosphorylation on tyrosine residues appears to be important (35) and relevant given FGFR3 function. To explain the paternal age effect, any selective advantage would have to increase with age perhaps in association with known changes that occur in the male reproductive system during normal aging (36).

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