A germ-line-selective advantage rather than an increased mutation rate can explain some unexpectedly common human disease mutations Soo-Kyung Choi, Song-Ro Yoon, Peter Calabrese*†, and Norman Arnheim*† Molecular and Computational Biology Program, University of Southern California, 1050 Childs Way, Los Angeles, CA 90089-2910 Edited by James F. Crow, University of Wisconsin, Madison, WI, and approved April 16, 2008 (received for review February 7, 2008) Two nucleotide substitutions in the human FGFR2 gene (C755G or mutation hot-spot model (the nucleotide has a higher-than- C758G) are responsible for virtually all sporadic cases of Apert average chance of undergoing a base substitution) at one of these syndrome. This condition is 100–1,000 times more common than sites (C755G). Positive selection for spermatogonial cells car- genomic mutation frequency data predict. Here, we report on the rying a newly arisen mutation is an alternative explanation (4–6, C758G de novo Apert syndrome mutation. Using data on older 11, 12) for an apparently high nucleotide substitution frequency. donors, we show that spontaneous mutations are not uniformly In our earlier study (6), we were unable to reject the selection distributed throughout normal testes. Instead, we find foci where hypothesis as an explanation for the C755G data. C758G mutation frequencies are 3–4 orders of magnitude greater In this work, we studied the other common de novo Apert than the remaining tissue. We conclude this nucleotide site is not nucleotide substitution (C758G). We examined four individual a mutation hot spot even after accounting for possible Luria– testes from three older individuals and two testes from younger Delbruck ‘‘mutation jackpots.’’ An alternative explanation for such donors. By comparing the data on both the C755G and C758G foci involving positive selection acting on adult self-renewing Ap mutations, we were able to ask whether the two mutations were spermatogonia experiencing the rare mutation could not be re- dependent or independent with respect to their anatomical jected. Further, the two youngest individuals studied (19 and 23 distribution throughout each testis. Overall, our results support years old) had lower mutation frequencies and smaller foci at both the selection hypothesis as an explanation for the high frequency mutation sites compared with the older individuals. This implies of both Apert syndrome mutations for being responsible for the that the mutation frequency of foci increases as adults age, and increased chance of older fathers having an affected child thus selection could explain the paternal age effect for Apert (paternal age effect: refs. 1, 11, 13–15). syndrome and other genetic conditions. Our results, now including the analysis of two mutations in the same set of testes, suggest Results that positive selection can increase the relative frequency of C758G Mutation Distribution in Individual Testes. We studied the premeiotic germ cells carrying such mutations, although individu- spatial distribution of de novo C758G mutations within six testes als who inherit them have reduced fitness. In addition, we com- from donors who do not suffer from Apert syndrome. Four of the pared the anatomical distribution of C758G mutation foci with testes were from older donors. Two of these testes were from one both new and old data on the C755G mutation in the same testis donor (374, age 62) and one testis from each of two additional and found their positions were not correlated with one another. donors (854, 54 years; 59089, 45 years). We also examined two testes from younger individuals: one testis each from donors Apert syndrome ͉ paternal age ͉ positive selection ͉ spermatogonia ͉ testis 60832, 23 years, and 59056, 19 years. Each testis was dissected into 192 pieces (six slices each with he frequency at which human germ-line nucleotide substi- 32 pieces), the DNA extracted and a highly specific single- Ttutions arise in each generation can be measured by analysis molecule PCR assay was used to estimate the number of mutant of sporadic cases of human autosomal dominant or sex-linked molecules in each DNA aliquot (see Methods). A sperm sample diseases (1, 2) or by DNA analysis of sperm (3–5). In a recent taken from the epididymis of each testis was also analyzed for article (6), we described an alternative method to study germ-line C758G mutations. The summary data are shown in Table 1 and mutations by dissecting the whole human testis and measuring Fig. 1 [the total DNA content and mutation frequency of each the mutation frequency at a specific nucleotide site in individual piece for all testes are presented in supporting information (SI) pieces. This procedure allowed us to study the spatial distribution Table S1]. The false-positive frequency of the C758G assay was of mutations in this organ and to compare different models of 3.8 ϫ 10Ϫ7 (based on experiments using 31.6 million control the human mutation process. genomes). Individuals born with Apert syndrome (Online Mendelian Among the three older individuals (374, 854, and 59089), the Inheritance in Man no. 101200) exhibit prematurely fused average mutation frequency per testis ranged from 7.3 ϫ 10Ϫ4 to cranial sutures and fused fingers and toes. Most Apert syndrome 1.0 ϫ 10Ϫ4 (Table 1). Each of the four testes is characterized by cases result from a spontaneous fibroblast growth factor receptor 2 gene (FGFR2) mutation in a normal father’s germ line that is transmitted to his offspring. The birth frequency for sporadic Author contributions: P.C. and N.A. designed research; S.-K.C., S.-R.Y., and P.C. performed cases is between 10Ϫ5 and 10Ϫ6 (7, 8). Surprisingly, Ͼ98% of research; S.-K.C., S.-R.Y., P.C., and N.A. analyzed data; and P.C. and N.A. wrote the paper. Apert syndrome cases arise by transversion mutations at only two The authors declare no conflict of interest. sites (C755G and C758G), and a single copy of either mutation This article is a PNAS Direct Submission. is sufficient to cause the disease. The birth frequency of indi- Freely available online through the PNAS open access option. viduals with new mutations at either of these two nucleotide sites *P.C. and N.A. contributed equally to this work. suggests that the mutation frequency at either site is 100- to †To whom correspondence may be addressed. E-mail: [email protected] or petercal@ 1,000-fold greater than expected based on what is known about usc.edu. transversion mutations since humans and chimpanzees last had This article contains supporting information online at www.pnas.org/cgi/content/full/ a common ancestor (9) and mutation data at many human 0801267105/DCSupplemental. MEDICAL SCIENCES disease loci (10). Our previous study (6) allowed us to reject the © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801267105 PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10143–10148 Downloaded by guest on September 29, 2021 Table 1. Summary data on six testes Epididymal sperm Testis Frequency range Percentage of all mutation mutation among testis No. pieces containing testis genomes Testis Age frequency frequency pieces 95% of mutants* in those pieces Mutation C758G 374–1 62y 5.6 ϫ 10Ϫ4 7.3 ϫ 10Ϫ4 Ͻ4 ϫ 10Ϫ6–0.043 12 8.2 374–2 62y 3.1 ϫ 10Ϫ6 1.0 ϫ 10Ϫ4 Ͻ4 ϫ 10Ϫ6–0.039 1 0.27 854–2 54y 2.7 ϫ 10Ϫ4 1.2 ϫ 10Ϫ4 Ͻ4 ϫ 10Ϫ6–0.011 7 3.6 59089–1 45y 8.2 ϫ 10Ϫ5 1.1 ϫ 10Ϫ4 Ͻ4 ϫ 10Ϫ6–0.005 6 4.5 60832–1 23y ‡ 6.5 ϫ 10Ϫ7 Ͻ4 ϫ 10Ϫ6–9.0 ϫ 10Ϫ6 27 15.3 59056–1 19y 2.0 ϫ 10Ϫ6 5.8 ϫ 10Ϫ7 Ͻ4 ϫ 10Ϫ6–3.1 ϫ 10Ϫ5 14 9.4 Mutation C755G 374–1† 62y 4.5 ϫ 10Ϫ4 3.8 ϫ 10Ϫ4 Ͻ10Ϫ6–0.027 12 5.7 374–2 † 62y 3.9 ϫ 10Ϫ5 6.7 ϫ 10Ϫ5 Ͻ10Ϫ6–0.007 5 2.6 854–2 † 54y 1.1 ϫ 10Ϫ4 6.8 ϫ 10Ϫ4 Ͻ2 ϫ 10Ϫ6–0.047 7 5.0 59089–1 45y 9.1 ϫ 10Ϫ5 1.6 ϫ 10Ϫ4 Ͻ4 ϫ 10Ϫ6–0.008 10 4.9 60832–1 23y ‡ 9.4 ϫ 10Ϫ7 Ͻ4 ϫ 10Ϫ6–9 ϫ 10Ϫ6 35 20.1 59056–1 19y 3.0 ϫ 10Ϫ6 1.6 ϫ 10Ϫ5 Ͻ4 ϫ 10Ϫ6–0.004 20 17 *Based on a resolution of 192 pieces/testis. †Previously published data (6). ‡Not enough sperm could be collected. a very small number of ‘‘hot’’ pieces with mutation frequencies sperm (found in that testis’s epididymis) is near one, ranging 3–4 orders of magnitude greater than most of the remaining from 0.29 to 1.76 (see Table 1). For two more pairs, this ratio is pieces (Fig. 1). In every case, 95% of the mutant molecules were higher at 5.33 and 6.18. In ref. 6, we reported that the relatively found in no more than 12 (average 6.5) of the 192 pieces high ratio of 6.18 for the C755G mutation in 854-2 could result examined, whereas, according to a random spatial distribution, from a highly viscous material found only in the one epididymis, many more pieces, which together contain 95% of the genomes, possibly contaminating that sperm sample with nongerm-line would have been expected. In many cases, several pieces with DNA; this explanation is now unlikely, because for the same high mutation frequencies appear to form foci adjacent to one testis and epididymal sperm sample, the ratio for mutation another in the same slice or even between slices (Fig.