De Novo Mutations Reflect Development and Aging of the Human Germline
Authors: J.M. Goldmann, J.A. Veltman, C. Gilissen
Journal: Trends in Genetics (2019)

Both the age of the father and the age of the mother are positively correlated with the number of de novo mutations (DNMs) in offspring, with the effect size of paternal age being larger.DNMs associated with paternal and maternal aging each have unique mutational signatures based on their nucleotide substitution spectrum and genomic locations.DNM clusters have characteristics distinct from nonclustered DNMs, suggesting different underlying mutational causes.Post-zygotic mutations arising during early embryonic development are a frequent phenomenon and differ from nonmosaic germline DNMs in their mutational spectrum.A subset of presumed DNMs can be traced back as low-level mosaic mutations in the somatic tissue of a parent. These mutations have a distinct mutation spectrum and can recur in future offspring. Human germline de novo mutations (DNMs) are both a driver of evolution and an important cause of genetic diseases. In the past few years, whole-genome sequencing (WGS) of parent–offspring trios has facilitated the large-scale detection and study of human DNMs, which has led to exciting discoveries. The overarching theme of all of these studies is that the DNMs of an individual are a complex mixture of mutations that arise through different biological processes acting at different times during human development and life. Human germline de novo mutations (DNMs) are both a driver of evolution and an important cause of genetic diseases. In the past few years, whole-genome sequencing (WGS) of parent–offspring trios has facilitated the large-scale detection and study of human DNMs, which has led to exciting discoveries. The overarching theme of all of these studies is that the DNMs of an individual are a complex mixture of mutations that arise through different biological processes acting at different times during human development and life. Human de novo mutations (DNMs, see Glossary) are germline mutations that newly occurred within one generation. While the vast majority of the genome has been inherited from earlier generations, DNMs provide new genetic variation. The consequences of the new genetic mutation can vary widely. While neutral or advantageous mutations might become established in the genome of our species and thereby contribute to human evolution, changes to crucial genetic sequences can also result in misfunctioning of biological systems, resulting in severe disease. One of the earliest known examples of this was Down syndrome, which is caused by a de novo trisomy of chromosome 21 [1Davenport C.B. Mendelism in man.in: Proceedings of the Sixth International Congress of Genetics. 1932http://www.esp.org/books/6th-congress/facsimile/contents/6th-cong-p135-davenport.pdfGoogle Scholar, 2Allen G. Aetiology of Down's syndrome inferred by Waardenburg in 1932.Nature. 1974; 250: 436-437Crossref PubMed Scopus (8) Google Scholar, 3Lejeune J. et al.[Study of somatic chromosomes from 9 mongoloid children].C. R. Hebd. Seances Acad. Sci. 1959; 248 (in French): 1721-1722PubMed Google Scholar]. In recent years DNMs have been found to be a prominent cause of neurodevelopmental diseases, including intellectual disability, autism, and schizophrenia [4Veltman J.A. Brunner H.G. De novo mutations in human genetic disease.Nat. Rev. Genet. 2012; 13: 565-575Crossref PubMed Scopus (478) Google Scholar, 5Acuna-Hidalgo R. et al.New insights into the generation and role of de novo mutations in health and disease.Genome Biol. 2016; 17: 241Crossref PubMed Scopus (162) Google Scholar]. The unbiased study of de novo point mutations in humans was for many years hampered by the lack of techniques to scan the entire genome in a cost-effective way. The introduction of next-generation sequencing (NGS) technologies has spurred investigations of DNMs in humans [6Koboldt D.C. et al.The next-generation sequencing revolution and its impact on genomics.Cell. 2013; 155: 27-38Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar]. DNMs can refer to a variety of different mutation types, such as single-nucleotide substitutions, insertions, deletions, and copy-number variants (CNVs). In this review we focus on single-nucleotide mutations and review the progress made in this field since the introduction of WGS, exploring their biology and possible underlying mechanisms (Figure 1, Key Figure), but not the potential pathological consequences. One of the most prominent findings concerning germline DNMs is the fact that their number increases steadily with the age of the father at conception. The concept of mutations being associated with age was first described by the human geneticist Wilhelm Weinberg in 1912, who observed that sporadic achondroplasia is more common in last-born children than in first-born children [8Weinberg W. Zur Vererbung des Zwergwuchses.Arch. Rassen Gesell. Biol. 1912; 9 (in German): 710-718Google Scholar]. For several sporadic genetic diseases, the association with paternal age is stronger than the association with maternal age [9Penrose L.S. Parental age and mutation.Lancet. 1955; 266: 312-313Abstract Scopus (198) Google Scholar]. It has been hypothesized that the underlying cause of these associations could be unprecise genome copying during the large number of cell divisions required for continuous sperm cell production in males [8Weinberg W. Zur Vererbung des Zwergwuchses.Arch. Rassen Gesell. Biol. 1912; 9 (in German): 710-718Google Scholar, 9Penrose L.S. Parental age and mutation.Lancet. 1955; 266: 312-313Abstract Scopus (198) Google Scholar, 10Crow J.F. The origins, patterns and implications of human spontaneous mutation.Nat. Rev. Genet. 2000; 1: 40-47Crossref PubMed Scopus (615) Google Scholar]. The first direct observation of DNM rates being higher for offspring of fathers of advanced age was made by WGS of 78 Icelandic parent–offspring trios [11Kong A. et al.Rate of de novo mutations and the importance of father's age to disease risk.Nature. 2012; 488: 471-475Crossref PubMed Scopus (1191) Google Scholar]. This study described that the number of DNMs in the offspring is dependent on the age of the father, with a slope of two additional DNMs per year of life of the father at the time of conception. This association of increased paternal age with increased numbers of DNMs in offspring is generally called the paternal age effect. The main hypothesis of the underlying cause for the paternal age effect is that mutations arise from incidental copying errors during genome replications [8Weinberg W. Zur Vererbung des Zwergwuchses.Arch. Rassen Gesell. Biol. 1912; 9 (in German): 710-718Google Scholar, 9Penrose L.S. Parental age and mutation.Lancet. 1955; 266: 312-313Abstract Scopus (198) Google Scholar, 10Crow J.F. The origins, patterns and implications of human spontaneous mutation.Nat. Rev. Genet. 2000; 1: 40-47Crossref PubMed Scopus (615) Google Scholar, 11Kong A. et al.Rate of de novo mutations and the importance of father's age to disease risk.Nature. 2012; 488: 471-475Crossref PubMed Scopus (1191) Google Scholar]. Genome replications are frequent in the male germline to ensure continuous generation of sperm cells. The stem cells for sperm production are spermatogonia, located in the seminiferous epithelium. Spermatogonia divide to renew themselves and give rise to spermatocytes, which will eventually differentiate into sperm cells [12Sharma S. et al.Spermatogonial stem cells: updates from specification to clinical relevance.Hum. Reprod. Update. 2019; 25: 275-297Crossref PubMed Scopus (29) Google Scholar]. During aging, the mean number of divisions per spermatogonium continues to increase, with each replication potentially introducing new mutations due to copying errors. Due to this, spermatogonia are believed to accumulate mutations during aging, thereby explaining the paternal age effect. Next to this genome-wide paternal age effect, an additional paternal age effect has been observed for highly specific mutations. Due to particular activating mutations in specific genes, affected spermatogonia lineages can generate more sperm cells than other lineages. As a consequence, the effective germline mutation rate in these genes is up to four orders of magnitude higher than the genome-wide rate. This effect has been termed selfish spermatogonial selection (Box 1). The association of these mutations with paternal age is stronger than the linear genome-wide paternal age effect [13Goriely A. Wilkie A.O.M. Paternal age effect mutations and selfish spermatogonial selection: causes and consequences for human disease.Am. J. Hum. Genet. 2012; 90: 175-200Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar].Box 1Selfish Spermatogonial SelectionThe mutations that accumulate in spermatogonia during paternal aging can also have an effect on the fitness of the spermatogonia themselves. While mutations in essential genes might lead to the eradication of the spermatogonial lineage, other mutations can result in selective advantages and lead to clonal expansion; that is, the outgrowth of the specific cell lineage, a process often linked to precancerous lesions. Clonal expansions have been observed in human tissues like bone marrow [14Genovese G. et al.Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.N. Engl. J. Med. 2014; 371: 2477-2487Crossref PubMed Scopus (1541) Google Scholar], skin [15Martincorena I. et al.High burden and pervasive positive selection of somatic mutations in normal human skin.Science. 2015; 348: 880-886Crossref PubMed Scopus (798) Google Scholar], and esophageal epithelium [16Martincorena I. et al.Somatic mutant clones colonize the human esophagus with age.Science. 2018; 362: 911-917Crossref PubMed Scopus (338) Google Scholar], as well as many others [17Yizhak K. et al.RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues.Science. 2019; 364: eaaw0726Crossref PubMed Scopus (180) Google Scholar]. In the human testis, clonal expansion has been observed to be driven by oncogenic mutations of growth factor receptors and components of the RAS–MAPK pathway [13Goriely A. Wilkie A.O.M. Paternal age effect mutations and selfish spermatogonial selection: causes and consequences for human disease.Am. J. Hum. Genet. 2012; 90: 175-200Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 18Maher G.J. et al.Selfish mutations dysregulating RAS–MAPK signaling are pervasive in aged human testes.Genome Res. 2018; 28: 1779-1790Crossref PubMed Scopus (28) Google Scholar]. As a consequence of the expansion of mutated spermatogonia that occurs in the testes of healthy men during aging [19Goriely A. et al.Regulation of cell fate decision of undifferentiated spermatogonia by GDNF.Science. 2003; 287: 1489-1493Google Scholar, 20Goriely A. et al.Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors.Nat. Genet. 2009; 41: 1247-1252Crossref PubMed Scopus (152) Google Scholar, 21Giannoulatou E. et al.Whole-genome sequencing of spermatocytic tumors provides insights into the mutational processes operating in the male germline.PLoS One. 2017; 12: e0178169Crossref PubMed Scopus (18) Google Scholar], the number of sperm carrying these mutations grows continuously at rates much higher than the general, genome-wide paternal age effect. If these mutations are passed on to offspring, they will cause developmental disorders. One prominent example is achondroplasia, a condition characterized by short stature that is caused by mutation of the fibroblast growth factor receptor 3 (FGFR3) gene. Achondroplasia occurs more frequently than expected based on average mutation rates and its occurrence is strongly associated with paternal age [10Crow J.F. The origins, patterns and implications of human spontaneous mutation.Nat. Rev. Genet. 2000; 1: 40-47Crossref PubMed Scopus (615) Google Scholar, 22Dakouane Giudicelli M. et al.Increased achondroplasia mutation frequency with advanced age and evidence for G1138A mosaicism in human testis biopsies.Fertil. Steril. 2008; 89: 1651-1656Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. It was shown that these pathogenic mutations are often present as high-level mosaicisms in the testes of the fathers of achondroplasia patients [20Goriely A. et al.Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors.Nat. Genet. 2009; 41: 1247-1252Crossref PubMed Scopus (152) Google Scholar, 22Dakouane Giudicelli M. et al.Increased achondroplasia mutation frequency with advanced age and evidence for G1138A mosaicism in human testis biopsies.Fertil. Steril. 2008; 89: 1651-1656Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. The mutations that accumulate in spermatogonia during paternal aging can also have an effect on the fitness of the spermatogonia themselves. While mutations in essential genes might lead to the eradication of the spermatogonial lineage, other mutations can result in selective advantages and lead to clonal expansion; that is, the outgrowth of the specific cell lineage, a process often linked to precancerous lesions. Clonal expansions have been observed in human tissues like bone marrow [14Genovese G. et al.Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.N. Engl. J. Med. 2014; 371: 2477-2487Crossref PubMed Scopus (1541) Google Scholar], skin [15Martincorena I. et al.High burden and pervasive positive selection of somatic mutations in normal human skin.Science. 2015; 348: 880-886Crossref PubMed Scopus (798) Google Scholar], and esophageal epithelium [16Martincorena I. et al.Somatic mutant clones colonize the human esophagus with age.Science. 2018; 362: 911-917Crossref PubMed Scopus (338) Google Scholar], as well as many others [17Yizhak K. et al.RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues.Science. 2019; 364: eaaw0726Crossref PubMed Scopus (180) Google Scholar]. In the human testis, clonal expansion has been observed to be driven by oncogenic mutations of growth factor receptors and components of the RAS–MAPK pathway [13Goriely A. Wilkie A.O.M. Paternal age effect mutations and selfish spermatogonial selection: causes and consequences for human disease.Am. J. Hum. Genet. 2012; 90: 175-200Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 18Maher G.J. et al.Selfish mutations dysregulating RAS–MAPK signaling are pervasive in aged human testes.Genome Res. 2018; 28: 1779-1790Crossref PubMed Scopus (28) Google Scholar]. As a consequence of the expansion of mutated spermatogonia that occurs in the testes of healthy men during aging [19Goriely A. et al.Regulation of cell fate decision of undifferentiated spermatogonia by GDNF.Science. 2003; 287: 1489-1493Google Scholar, 20Goriely A. et al.Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors.Nat. Genet. 2009; 41: 1247-1252Crossref PubMed Scopus (152) Google Scholar, 21Giannoulatou E. et al.Whole-genome sequencing of spermatocytic tumors provides insights into the mutational processes operating in the male germline.PLoS One. 2017; 12: e0178169Crossref PubMed Scopus (18) Google Scholar], the number of sperm carrying these mutations grows continuously at rates much higher than the general, genome-wide paternal age effect. If these mutations are passed on to offspring, they will cause developmental disorders. One prominent example is achondroplasia, a condition characterized by short stature that is caused by mutation of the fibroblast growth factor receptor 3 (FGFR3) gene. Achondroplasia occurs more frequently than expected based on average mutation rates and its occurrence is strongly associated with paternal age [10Crow J.F. The origins, patterns and implications of human spontaneous mutation.Nat. Rev. Genet. 2000; 1: 40-47Crossref PubMed Scopus (615) Google Scholar, 22Dakouane Giudicelli M. et al.Increased achondroplasia mutation frequency with advanced age and evidence for G1138A mosaicism in human testis biopsies.Fertil. Steril. 2008; 89: 1651-1656Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. It was shown that these pathogenic mutations are often present as high-level mosaicisms in the testes of the fathers of achondroplasia patients [20Goriely A. et al.Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors.Nat. Genet. 2009; 41: 1247-1252Crossref PubMed Scopus (152) Google Scholar, 22Dakouane Giudicelli M. et al.Increased achondroplasia mutation frequency with advanced age and evidence for G1138A mosaicism in human testis biopsies.Fertil. Steril. 2008; 89: 1651-1656Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. Recent studies have confirmed the observation of the general paternal age effect [23Francioli L.C. et al.Genome-wide patterns and properties of de novo mutations in humans.Nat. Genet. 2015; 47: 822-826Crossref PubMed Scopus (194) Google Scholar, 24Rahbari R. et al.Timing, rates and spectra of human germline mutation.Nat. Genet. 2015; 48: 126-133Crossref PubMed Scopus (247) Google Scholar, 25Wong W.S.W. et al.New observations on maternal age effect on germline de novo mutations.Nat. Commun. 2016; 7: 10486Crossref PubMed Scopus (85) Google Scholar, 26Goldmann J.M. et al.Parent-of-origin-specific signatures of de novo mutations.Nat. Genet. 2016; 48: 935-939Crossref PubMed Scopus (127) Google Scholar, 27Jónsson H. et al.Parental influence on human germline de novo mutations in 1,548 trios from Iceland.Nature. 2017; 549: 519-522Crossref PubMed Scopus (138) Google Scholar, 28Goldmann J.M. et al.Germline de novo mutation clusters arise during oocyte aging in genomic regions with high double-strand-break incidence.Nat. Genet. 2018; 50: 487-492Crossref PubMed Scopus (27) Google Scholar]. The generation of large-scale datasets allowed deeper characterization of the DNMs, including the analysis of mutational spectra and mutational signatures (Box 2). The mutational spectrum of human DNMs has been found to mainly comprise two mutational signatures that are known from somatic mutations in cancer [24Rahbari R. et al.Timing, rates and spectra of human germline mutation.Nat. Genet. 2015; 48: 126-133Crossref PubMed Scopus (247) Google Scholar, 29Alexandrov L.B. et al.Clock-like mutational processes in human somatic cells.Nat. Genet. 2015; 47: 1402-1407Crossref PubMed Scopus (410) Google Scholar]. These two signatures occur in a wide range of somatic tissues and accumulate mutations in a clock-like manner (i.e., at a constant rate). Therefore, in somatic tissues these signatures are likely to represent the impact of aging on DNA. In line with the replication-error hypothesis, one of these signatures is associated with genome replication [29Alexandrov L.B. et al.Clock-like mutational processes in human somatic cells.Nat. Genet. 2015; 47: 1402-1407Crossref PubMed Scopus (410) Google Scholar]. Surprisingly, however, the mutational spectrum of DNMs is not constant and changes with the age of the father at conception. With advancing paternal age, more T>G substitutions and fewer non-CpG C>T substitutions are found in the offspring [27Jónsson H. et al.Parental influence on human germline de novo mutations in 1,548 trios from Iceland.Nature. 2017; 549: 519-522Crossref PubMed Scopus (138) Google Scholar, 28Goldmann J.M. et al.Germline de novo mutation clusters arise during oocyte aging in genomic regions with high double-strand-break incidence.Nat. Genet. 2018; 50: 487-492Crossref PubMed Scopus (27) Google Scholar]. The existence of such differences suggests that, besides the paternal aging-associated mutations, there is another type of mutation that is independent of paternal age. With increasing age of the father, the balance of these types of mutation shifts and the mutational spectrum becomes biased towards more paternal aging-associated mutations.Box 2Mutational SignaturesFrom the analysis of cancer genomes it is known that exposure to mutagens or failure of DNA repair pathways can result in mutations with specific patterns. An example is the light-associated skin cancers, in which C>T and CC>TT nucleotide substitutions are very common [30Pfeifer G.P. et al.Mutations induced by ultraviolet light.Mutat. Res. Mol. Mech. Mutagen. 2005; 571: 19-31Crossref PubMed Scopus (527) Google Scholar]. The advent of NGS technologies allowed the systematic study of mutational patterns in large collections of well-annotated cancer genomes [31Nik-Zainal S. et al.Mutational processes molding the genomes of 21 breast cancers.Cell. 2012; 149: 979-993Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar, 32Alexandrov L.B. et al.Signatures of mutational processes in human cancer.Nature. 2013; 500: 415-421Crossref PubMed Scopus (5077) Google Scholar, 33Alexandrov L. et al.The repertoire of mutational signatures in human cancer.bioRxiv. 2019; (Published online July 3, 2019)https://doi.org/10.1101/322859Google Scholar]. In these analyses, the mutational signatures were analyzed by differentiating the six nucleotide substitution types and stratifying by the nucleotides surrounding the affected base pair, resulting in spectra of 96 mutation types. By mathematically deconvoluting archetypical patterns in the spectra of cancer mutations, these efforts identified 49 single-nucleotide-substitution mutational signatures. These signatures are well annotated and a subset could be linked to underlying mutational mechanisms. The mutational signatures are available online (https://cancer.sanger.ac.uk/cosmic/signatures). Analysis of a mutational spectrum and comparison with the known signatures can hint at the underlying mutational mechanisms. From the analysis of cancer genomes it is known that exposure to mutagens or failure of DNA repair pathways can result in mutations with specific patterns. An example is the light-associated skin cancers, in which C>T and CC>TT nucleotide substitutions are very common [30Pfeifer G.P. et al.Mutations induced by ultraviolet light.Mutat. Res. Mol. Mech. Mutagen. 2005; 571: 19-31Crossref PubMed Scopus (527) Google Scholar]. The advent of NGS technologies allowed the systematic study of mutational patterns in large collections of well-annotated cancer genomes [31Nik-Zainal S. et al.Mutational processes molding the genomes of 21 breast cancers.Cell. 2012; 149: 979-993Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar, 32Alexandrov L.B. et al.Signatures of mutational processes in human cancer.Nature. 2013; 500: 415-421Crossref PubMed Scopus (5077) Google Scholar, 33Alexandrov L. et al.The repertoire of mutational signatures in human cancer.bioRxiv. 2019; (Published online July 3, 2019)https://doi.org/10.1101/322859Google Scholar]. In these analyses, the mutational signatures were analyzed by differentiating the six nucleotide substitution types and stratifying by the nucleotides surrounding the affected base pair, resulting in spectra of 96 mutation types. By mathematically deconvoluting archetypical patterns in the spectra of cancer mutations, these efforts identified 49 single-nucleotide-substitution mutational signatures. These signatures are well annotated and a subset could be linked to underlying mutational mechanisms. The mutational signatures are available online (https://cancer.sanger.ac.uk/cosmic/signatures). Analysis of a mutational spectrum and comparison with the known signatures can hint at the underlying mutational mechanisms. According to the genome-replication hypothesis for the paternal age effect, the number of replications should be directly proportional to the number of DNMs in offspring. The established model for estimating the number of genome replications that occurred during development from zygote to sperm cell includes the following factors [10Crow J.F. The origins, patterns and implications of human spontaneous mutation.Nat. Rev. Genet. 2000; 1: 40-47Crossref PubMed Scopus (615) Google Scholar, 34Clermont Y. The cycle of the seminiferous epithelium in man.Am. J. Anat. 1963; 112: 35-51Crossref PubMed Scopus (464) Google Scholar, 35Drost J.B. Lee W.R. Biological basis of germline mutation: comparisons of spontaneous germline mutation rates among Drosophila, mouse, and human.Environ. Mol. Mutagen. 1995; 25: 48-64Crossref PubMed Scopus (191) Google Scholar]. First, during very early embryogenesis, about ten cell divisions occur until germline cells are specified and the germline is established. Second, during sex organ development, the seminiferous ducts become colonized with germ cells, which requires about 24 germline cell divisions after germ cell specification. During the further fetal period and childhood, the seminiferous epithelium rests and no cell divisions occur. Starting in puberty, spermatogonia will divide continuously to generate sperm cell precursors. It has been estimated that the average spermatogonium divides 23 times per year during this period [36Heller C.G. Clermont Y. Spermatogenesis in man: an estimate of its duration.Science. 1963; 140: 184-186Crossref PubMed Scopus (226) Google Scholar]. Finally, an age-independent number of four genome replications is needed to proceed from spermatogonium to sperm cell. To give an example, the number of genome replications of an average sperm cell of a 30-year-old male can be estimated as ten very early embryonic divisions, plus 24 early embryonic divisions, plus 17 years of sperm production each with 23 divisions (assuming a sperm production onset at the age of 13 years [37Nielsen C.T. et al.Onset of the release of spermatozoa (spermarche) in boys in relation to age, testicular growth, pubic hair, and height.J. Clin. Endocrinol. Metab. 1986; 62: 532-535Crossref PubMed Scopus (150) Google Scholar]), and finally four replications for spermatogenesis. This is in total 429 replications. Fathers at the age of 20 and 60 years would generate sperm after 199 and 1119 replications, respectively. However, these estimates for genome replications are not proportional with the observations on DNM accrual (Figure 2). While the number of replications increases by a factor of more than five between the parental ages of 20 and 60 years, the number of DNMs increases by less than a factor of three, as the estimated numbers would be 40 and 91, respectively [28Goldmann J.M. et al.Germline de novo mutation clusters arise during oocyte aging in genomic regions with high double-strand-break incidence.Nat. Genet. 2018; 50: 487-492Crossref PubMed Scopus (27) Google Scholar]. Supporting the observations on DNMs, the epidemiological risk of giving birth to a child affected by a developmental disorder (of which a large fraction is caused by DNMs [38Gilissen C. et al.Genome sequencing identifies major causes of severe intellectual disability.Nature. 2014; 511: 344-347Crossref PubMed Scopus (677) Google Scholar]) increases with paternal age only by a factor of less than two (from 0.24% for fathers and mothers both below 22 years up to 0.47% for fathers and mothers both above 42 years) [39Deciphering Developmental Disorders StudyPrevalence and architecture of de novo mutations in developmental disorders.Nature. 2017; 542: 433-438Crossref PubMed Scopus (584) Google Scholar]. This mismatch of the observations with the genome replication data has been reported several times and remains unresolved [40Ségurel L. et al.Determinants of mutation rate variation in the human germline.Annu. Rev. Genomics Hum. Genet. 2014; 15: 47-70Crossref PubMed Scopus (148) Google Scholar, 41Forster P. et al.Elevated germline mutation rate in teenage fathers.Proc. Biol. Sci. 2015; 282: 20142898Crossref PubMed Scopus (30) Google Scholar, 42Gao Z. et al.Interpreting the dependence of mutation rates on age and time.PLoS Biol. 2015; 14: e1002355Crossref Scopus (46) Google Scholar, 43Scally A. Mutation rates and the evolution of germline structure.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2016; 371: 20150137Crossref PubMed Scopus (20) Google Scholar, 44Gao Z. et al.Overlooked roles of DNA damage and maternal age in generating human germline mutations.Proc. Natl Acad. Sci. U. S. A. 2019; 116: 9491-9500Crossref PubMed Scopus (45) Google Scholar]. A crucial factor for the genome-replication model is the estimate of the annual post-pubertal spermatogonium division rate. This estimate of 23 divisions per year is based on observations in the 1960s by Heller and Clermont [36Heller C.G. Clermont Y. Spermatogenesis in man: an estimate of its duration.Science. 1963; 140: 184-186Crossref PubMed Scopus (226) Google Scholar], who radiolabeled the seminiferous epithelium of the testicular ducts and showed that one division cycle lasts 16 days, which would extrapolate to 23 divisions per year. This extrapolation implicitly assumes that there are no interruptions or resting phases between the divisions. However, more recent studies have observed the nature of spermatogonial self-renewal to not be a unidirectional process but rather be driven by stochastic cell transitions, including cell stages without replicative activity [12Sharma S. et al.Spermatogonial stem cells: updates from specification to clinical relevance.Hum. Reprod. Update. 2019; 25: 275-297Crossref PubMed Scopus (29) Google Scholar, 45Hara K. et al.Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states.Cell Stem Cell. 2014; 14: 658-672Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 46Krieger T. Simons B.D. Dynamic stem cell heterogeneity.Development. 2015; 142: 1396-1406Crossref PubMed Scopus (62) Google Scholar]. Such a nonhierarchical model of spermatogonial self-renewal with stochastic, continuous oscillation between cell stages is supported by recent single-cell studies in two ways. First, analyses of single sp