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

The observed human sperm mutation frequency cannot explain the achondroplasia paternal age effect Irene Tiemann-Boege*, William Navidi†‡, Raji Grewal‡§, Dan Cohn¶, Brenda Eskenaziʈ, 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 Institute͞Ahmanson Department of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, CA 90048; ʈSchool 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, September 19, 2002 (received for review August 10, 2002) The lifelong spermatogonial stem cell divisions unique to male recent data on the population incidence of sporadic achondro- germ cell production are thought to contribute to a higher muta- plasia (10, 18–20) predict the average frequency of sperm tion frequency in males. The fact that certain de novo human carrying the mutation in normal individuals will be in a range genetic conditions (e.g., achondroplasia) increase in incidence with detectable by modern molecular methods (1͞15,000 to the age of the father is consistent with this idea. Although it is 1͞70,000). Our studies on sperm DNA from men of different assumed that the paternal age effect is the result of an increasing ages suggest that the observed increase in G1138A mutation frequency of mutant sperm as a man grows older, no direct frequency cannot satisfactorily explain the exponential increase molecular measurement of the germ-line mutation frequency has in sporadic achondroplasia cases with paternal age. been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the Methods nucleotide substitution in the fibroblast growth factor receptor 3 Sources of Semen Samples and DNA. Semen samples were collected (FGFR3) gene that causes achondroplasia. Surprisingly, the mag- according to protocols approved by the institutional review nitude of the increase in mutation frequency with age appears boards of the University of Southern California (USC) (60 insufficient to explain why older fathers have a greater chance of donors; mean age 40.65 years, range 18–72) and the University having a child with this condition. A number of alternatives may of California (Berkeley) and Lawrence Livermore National explain this discrepancy, including selection for sperm that carry Laboratory (LNLL) (58 donors; mean age 41.66 years, range the mutation or an age-dependent increase in premutagenic le- 22–80). At least 90% of the donors were of European ancestry. sions that remain unrepaired in sperm and are inefficiently de- Pooled human blood DNA (catalog no. 6550-1) used in the tected by the PCR assay. standards was purchased from CLONTECH. The DNA cell line (NA08859; see below) homozygous for the G1138A mutation eneticists and evolutionary biologists debate the extent to was obtained from Coriell Cell Repositories, Camden, NJ. Gwhich the lifelong spermatogonial stem cell divisions unique to male gametogenesis contribute to a higher mutation frequency in Sperm DNA Quantitation. We quantitated the number of genomes males (1–8). One source of support for the hypothesis that muta- containing the single-copy FGFR3 gene by using real-time or tions increase with spermatogonial stem cell divisions comes from kinetic PCR (kt-PCR; ref. 21). An initial denaturation step at 94°C for 10 min was followed by cycling conditions of 94°C for 40 s and the observation of certain human genetic conditions where the ␮ ϫ incidence of new mutations increases with the age of the father. 66°C for 1 min. Reactions were carried out in 50 l containing 1 Taq Gold Buffer (15 mM Tris⅐HCl͞50 mM KCl at pH 8.0), 1.25 Epidemiological studies on a number of dominantly inherited ␮ ␮ units of Taq Gold, 2 mM MgCl2,80 M of each dNTP, and 0.2 M conditions indicate that the average age of the fathers is older Ј Ј Ј among unaffected couples having a child with the condition (a forward (5 -aggagctggtggagctga-3 ) and reverse primer (5 - tgcgcaggcggcagagcgtc-3Ј), 0.2ϫ SYBR Green I (Molecular Probes), sporadic case caused by a new mutation), than the average paternal ␮ age in the population (1, 2, 4, 7). Achondroplasia, the most common and 2 M 5,6 ROX dye (Sigma). kt-PCR was performed on a form of dwarfism, is one of these conditions (9–11). Studies on GeneAmp 5700 Sequence Detection System (Applied Biosystems). sporadic achondroplasia cases have reported an exponential in- Quantitation of G1138A Mutations in Sperm DNA. crease with paternal age (1, 2, 4, 7, 10). Sperm DNA was extracted from all semen samples by using Puregene DNA In mice, a significant increase in the overall male germ cell Isolation Kits (Gentra Systems). The concentration and purity of mutation frequency as measured by the lacI assay was observed the sperm DNA samples was determined spectrophotometrically between 15 and 28 months of age (12). In humans, no direct after HaeIII or BseDI digestion to reduce the DNA viscosity. molecular measurement of how germ-line nucleotide substitu- Neither enzyme has a recognition sequence in the amplification tion frequencies change with age exists. Achondroplasia pro- target. Sperm DNA from 1͞3 of the samples was also quantitated vides a unique opportunity to directly test the relationship by using kt-PCR (see above). This process confirmed the accu- between paternal age and sperm mutation frequency at the racy of the OD measurements. molecular level. First, 97–99% of the de novo mutations leading In the first step of the assay, FGFR3 fragments containing base 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 Abbreviations: FGFR, fibroblast growth factor receptor; USC, University of Southern Cali- part of a CpG dinucleotide and, if methylated, is highly suscep- fornia; LLNL, Lawrence Livermore National Laboratory; kt-PCR, kinetic PCR; C.I., confidence tible to mutation caused by spontaneous deamination (16). interval. Second, all sporadic achondroplasia cases have been found to ‡W.N. and R.G. contributed equally to this work. inherit the G1138A mutation from their father (17). Third, ††To whom correspondence should be addressed. E-mail: [email protected]. 14952–14957 ͉ PNAS ͉ November 12, 2002 ͉ vol. 99 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.232568699 Downloaded by guest on September 28, 2021 pair 1138 were amplified from 1 ␮g of sperm DNA (duplicate Sensitivity of Allele-Specific PCR. Reconstruction experiments were aliquots) with eight PCR cycles (94°C for 40 s and 66°Cfor1min) used to determine the sensitivity of the allele-specific kt-PCR preceded by a 94°C 10-min Taq Gold activation step. Each 50-␮l step. PCR product obtained by amplifying the homozygous reaction contained 1ϫ Taq Gold Buffer (15 mM Tris⅐HCl͞50 mM achondroplasia cell line DNA was mixed with blood DNA PCR KCl at pH 8.0), 1.25 units of Taq Gold (Applied Biosystems), 2 mM product. Both PCRs used the same conditions as in the first two ␮ ␮ Ј MgCl2,80 M of each dNTP, and 0.2 M forward primer (5 - steps of the assay except there was no MspI digestion. We found tgtgtatgcaggcatcctcagctcc-3Ј) and reverse primer (5Ј-tgcgcaggcg- that the ratio of one G1138A template per 600 WT templates can gcagagcgtc-3Ј). In the second step, 20 units of MspI was added to be distinguished from WT templates alone by a difference of two the amplification reaction and incubated for 30 min at 37°C PCR cycles (data not shown). This is equivalent to detecting one followed immediately by 16 additional amplification cycles. The G1138A mutant in 20,000 genomes because the MspI digestion amount of PCR product produced in these 24 cycles was confirmed in assay step two increases the ratio of mutant to WT by by kt-PCR. In the third step, two 5-␮l aliquots of a 40-fold dilution Ϸ35-fold. of each of the above duplicates were analyzed by using allele- ␮ specific kt-PCR (22). The reactions contained 0.1 ␮M of mutant- Data Collection and Analysis. In each experiment, two 1- g aliquots specific primer (5Ј-atgcaggcatcctcagctaca-3Ј) and reverse nested of sperm DNA per individual from 6–10 different donors of varying primer (5Ј-gccgccaccaccaggatgaac-3Ј), 0.8 units of Taq Gold, 1ϫ ages were analyzed. The allele-specific step for each aliquot was ␮ ϫ Taq Gold buffer, 3.5 mM MgCl2,50 M of each dNTP, 0.2 SYBR done in duplicate. The mean of the four measurements was taken Green I, and 2 ␮M 5,6 ROX dye in a total volume of 50 ␮l. An initial as the count for that sperm donor in that experiment. The final Taq Gold activation step was followed by 35 cycles of 40 s at 94°C count for any donor was calculated by taking the median of the for denaturation and 60 s at 68°C for annealing and extension. To count values obtained in different experiments. In our first statis- confirm that the PCR product was the one desired and not an tical analysis we assume that the assay produced an unbiased amplification artifact (e.g., primer-dimer), we used the GeneAmp estimate of the true counts in any sperm sample. 5700 denaturation profile function to examine every allele-specific reaction. Relationship Between Sporadic Achondroplasia Births and Mutation To avoid carryover contamination by PCR product, each step of Frequency.

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