The paternal-age effect in Apert syndrome is due,in part,to the increased frequency of mutations in sperm

A paternal-age effect and the exclusive paternal origin of mutations have been reported in Apert syndrome (AS). As the incidence of sporadic AS births increases exponentially with paternal age, we hypothesized that the frequency of AS mutations in sperm would also increase. To determine the frequency of two common FGFR2 mutations in AS, we developed allele-specific peptide nucleic acid-PCR assays. Analyzing sperm DNA from 148 men, age 21-80 years, we showed that the number of sperm with mutations increased in the oldest age groups among men who did not have a child with AS. These older men were also more likely to have both mutations in their sperm. However, this age-related increase in mutation frequency was not sufficient to explain the AS-birth frequency. In contrast, the mutation frequency observed in men who were younger and had children with AS was significantly greater. In addition, our data suggest selection for sperm with specific mutations. Therefore, contributing factors to the paternal-age effect may include selection and a higher number of mutant sperm in a subset of men ascertained because they had a child with AS. No age-related increase in the frequency of these mutations was observed in leukocytes. Selection and/or quality-control mechanisms, including DNA repair and apoptosis, may contribute to the cell-type differences in mutation frequency.     

Spontaneous mutation and parental age in humans.

A statistical analysis of parental age and the incidence of new mutation has been performed. Some new data on Apert, Crouzon, and Pfeiffer syndromes is presented and combined with all available data from the literature on parental age and new mutation. Significant heterogeneity among syndromes for the rate of increase in incidence with parental age was found. A parsimonious conclusion is that mutations fall into two groups, one with a high rate of increase with age and the other with a low rate of increase with age. For the high-rate-of-increase group, a linear model relating incidence to age is rejected, while an exponential model is not. In addition, for this group, increased paternal age cannot account for the observed increase in maternal age--that is, increased maternal age also contributes to the incidence of new mutations. For the low-rate-of-increase group, increased paternal age alone can account for the observed increase in maternal ages; also, either a linear or exponential model is acceptable. In addition, there is no evidence for a mixture of parental age-independent cases with parental age-dependent cases for any of the syndromes examined. The curves reflecting incidence of new mutation and paternal age for two syndromes, Apert and neurofibromatosis, have an anomalous shape. In both cases the curve increases up to age 37 and drops at age 42 before increasing again at age 47. The usual explanation for the effect of parental age on new mutations is the mechanism of "copy-error" at mitotic division in male sperone that specifies an increased probability of mutation with time spent by a spermatozoon or ovum in a haploid state, a period of time that may also increase with age of the parent. A firm answer to the question of parental age and new mutation awaits identification of the molecular defect underlying some of these syndromes; we will then be in a position to determine in which parent the mutation occurred and at what age it did so.     

Positive selection for new disease mutations in the human germline: evidence from the heritable cancer syndrome multiple endocrine neoplasia type 2B

Multiple endocrine neoplasia type 2B (MEN2B) is a highly aggressive thyroid cancer syndrome. Since almost all sporadic cases are caused by the same nucleotide substitution in the RET proto-oncogene, the calculated disease incidence is 100-200 times greater than would be expected based on the genome average mutation frequency. In order to determine whether this increased incidence is due to an elevated mutation rate at this position (true mutation hot spot) or a selective advantage conferred on mutated spermatogonial stem cells, we studied the spatial distribution of the mutation in 14 human testes. In donors aged 36-68, mutations were clustered with small regions of each testis having mutation frequencies several orders of magnitude greater than the rest of the testis. In donors aged 19-23 mutations were almost non-existent, demonstrating that clusters in middle-aged donors grew during adulthood. Computational analysis showed that germline selection is the only plausible explanation. Testes of men aged 75-80 were heterogeneous with some like middle-aged and others like younger testes. Incorporating data on age-dependent death of spermatogonial stem cells explains the results from all age groups. Germline selection also explains MEN2B's male mutation bias and paternal age effect. Our discovery focuses attention on MEN2B as a model for understanding the genetic and biochemical basis of germline selection. Since RET function in mouse spermatogonial stem cells has been extensively studied, we are able to suggest that the MEN2B mutation provides a selective advantage by altering the PI3K/AKT and SFK signaling pathways. Mutations that are preferred in the germline but reduce the fitness of offspring increase the population's mutational load. Our approach is useful for studying other disease mutations with similar characteristics and could uncover additional germline selection pathways or identify true mutation hot spots.     

Paternal age effect mutations and selfish spermatogonial selection: causes and consequences for human disease

Advanced paternal age has been associated with an increased risk for spontaneous congenital disorders and common complex diseases (such as some cancers, schizophrenia, and autism), but the mechanisms that mediate this effect have been poorly understood. A small group of disorders, including Apert syndrome (caused by FGFR2 mutations), achondroplasia, and thanatophoric dysplasia (FGFR3), and Costello syndrome (HRAS), which we collectively term "paternal age effect" (PAE) disorders, provides a good model to study the biological and molecular basis of this phenomenon. Recent evidence from direct quantification of PAE mutations in sperm and testes suggests that the common factor in the paternal age effect lies in the dysregulation of spermatogonial cell behavior, an effect mediated molecularly through the growth factor receptor-RAS signal transduction pathway. The data show that PAE mutations, although arising rarely, are positively selected and expand clonally in normal testes through a process akin to oncogenesis. This clonal expansion, which is likely to take place in the testes of all men, leads to the relative enrichment of mutant sperm over time-explaining the observed paternal age effect associated with these disorders-and in rare cases to the formation of testicular tumors. As regulation of RAS and other mediators of cellular proliferation and survival is important in many different biological contexts, for example during tumorigenesis, organ homeostasis and neurogenesis, the consequences of selfish mutations that hijack this process within the testis are likely to extend far beyond congenital skeletal disorders to include complex diseases, such as neurocognitive disorders and cancer predisposition.     

Is there a paternal age effect for aneuploidy?

Finding a positive association between paternal age and the incidence of aneuploidy is not difficult. A cursory analysis however reveals that any association is indirect, brought about by a close correlation between paternal age and maternal age. Approaches for dissecting out the confounding age effects of the mother has led to a lively exchange among epidemiologists, with perhaps a consensus for the absence of a paternal age effect, at least for trisomy 21. Molecular studies revealed the relatively minor contribution of paternal errors to trisomy, but even research on the paternally derived trisomies alone has been inconclusive; thus studies focussed directly on the sperm heads. Human-hamster fusion assays were superseded by FISH for establishing any possible link between age and the proportion of disomic sperm in an ejaculate. Despite innumerable microscope hours however, although convincing studies suggesting an age effect for disomies 1, 9, 18 and 21 and the sex chromosomes are in the literature, others failed to notice any association for these or other chromosomes. It is biologically plausible that chromosomal non-disjunction errors should increase with age. Male reproductive hormone production, testicular morphology and semen parameters all decline slowly with age and paternal age is implicated in congenital birth defects, such as achondroplasia and Apert syndromes and also linked to compromised DNA repair mechanisms. Despite several decades of epidemiological and molecular cytogenetic studies, however, we are still not close to a definitive answer of whether or not there is a paternal age effect for aneuploidy. In this review we conclude by questioning the efficacy of FISH because of difficulties in detecting nullisomy and because of evidence that the centromeres (from which most sperm-FISH probes are derived) cluster at the nuclear centre. Array-based approaches may well supersede FISH in addressing the question of a paternal age effect; for now, however, the jury is still out.     

Acrocentric chromosome disomy is increased in spermatozoa from fathers of Turner syndrome patients

The aim of the present study was to investigate whether there was an increase of aneuploidy in the sperm from fathers of Turner syndrome patients of paternal origin who, in a previous study, showed an elevated incidence of XY meiotic nondisjunction. Sperm disomy frequencies for chromosomes 4, 13, 18, 21 and 22 were assessed by fluorescence in situ hybridisation in four of these individuals. As a group, the Turner syndrome fathers showed a general increase in disomy frequencies for chromosomes 13, 21 and 22, with a statistically significant increase in disomy frequencies for chromosomes 13 and 22 in one of the fathers and for chromosome 21 in two of them. Data from a previous work carried out by us in two fathers of Down syndrome patients of paternal origin also revealed increased sperm disomy frequencies for chromosomes 13, 21 and 22. Pooled as one group, these six fathers of aneuploid offspring of paternal origin had a statistically significant increase in the frequency of nondisjunction for these chromosomes with respect to control individuals. Our findings indicate that there may be an association between fathering aneuploid offspring and increased frequencies of aneuploid spermatozoa. Such increases do not seem to be restricted to the chromosome pair responsible for the aneuploid offspring. Acrocentric chromosomes and other chromosome pairs that usually show only one chiasma during meiosis seem to be more susceptible to malsegregation.     

Reexamination of paternal age effect in Down's syndrome

Summary The recent discovery that the extra chromosome in about 30% of cases of 47, trisomy 21 is of paternal origin has revived interest in the possibility of paternal age as a risk factor for a Down syndrome birth, independent of maternal age. Parental age distribution for 611 Down's syndrome 47,+21 cases was studied. The mean paternal age was 0.16 year greater than in the entire population of live births after controlling for maternal age. There was no evidence for a significant paternal age effect at the 0.05 level. For 242 of these Down's syndrome cases, control subjects were selected by rigidly matching in a systematic manner. Paternal age was the variable studied, with maternal age and time and place of birth controlled. There was no statistically significant association between paternal age and Down's syndrome. After adjustment for maternal age, these two studies were not consistent with an increase of paternal age in Down's syndrome.     

Numerical chromosome abnormalities in the spermatozoa of the fathers of children with trisomy 21 of paternal origin: generalised tendency to meiotic non-disjunction

The purpose of this study was the evaluation of aneuploidy frequencies in the spermatozoa of two fathers (DP-4 and DP-5) who had children with Down syndrome (DS) of paternal origin and in whom a previous sperm analysis by fluoresence in situ hybridisation (FISH) had suggested a generalised tendency to meiotic non-disjunction. Sperm samples were simultaneously hybridised with FISH probes for chromosomes 4, 13 and 22. Disomy frequencies for each of the chromosomes and diploidy frequencies were compared with data obtained from nine control donors. Both DS fathers had a statistically significant increase in the frequency of disomy for chromosomes 13 and 22. DP-5 also had an increased frequency of diploid spermatozoa. Our data suggest that the two DS fathers have a generalised susceptibility to meiotic non-disjunction and that acrocentric chromosomes seem to be more sensitive to such disturbance in the meiotic process.     

Paternal origin of FGFR3 mutations in Muenke-type craniosynostosis

Muenke syndrome, also known as FGFR3-associated coronal synostosis, is defined molecularly by the presence of a heterozygous nucleotide transversion, c.749C>G, encoding the amino acid substitution Pro250Arg, in the fibroblast growth factor receptor type 3 gene (FGFR3). This frequently occurs as a new mutation, manifesting one of the highest documented rates for any transversion in the human genome. To understand the biology of this mutation, we have investigated its parental origin, and the ages of the parents, in 19 families with de novo c.749C>G mutations. All ten informative cases originated from the paternal allele (95% confidence interval 74–100% paternal); the average paternal age at birth overall was 34.7 years. An exclusive paternal origin of mutations, and increased paternal age, were previously described for a different mutation (c.1138G>A) of the FGFR3 gene causing achondroplasia, as well as for mutations of the related FGFR2 gene causing Apert, Crouzon and Pfeiffer syndromes. We conclude that similar biological processes are likely to shape the occurrence of this c.749C>G mutation as for other mutations of FGFR3 as well as FGFR2.S.V. Rannan-Eliya and I.B. Taylor contributed equally to this work.     

Paternal origin of FGFR2 mutations in sporadic cases of Crouzon syndrome and Pfeiffer syndrome

Crouzon syndrome and Pfeiffer syndrome are both autosomal dominant craniosynostotic disorders that can be caused by mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. To determine the parental origin of these FGFR2 mutations, the amplification refractory mutation system (ARMS) was used. ARMS PCR primers were developed to recognize polymorphisms that could distinguish maternal and paternal alleles. A total of 4,374 bases between introns IIIa and 11 of the FGFR2 gene were sequenced and were assayed by heteroduplex analysis, to identify polymorphisms. Two polymorphisms (1333TA/TATA and 2710 C/T) were found and were used with two previously described polymorphisms, to screen a total of 41 families. Twenty-two of these families were shown to be informative (11 for Crouzon syndrome and 11 for Pfeiffer syndrome). Eleven different mutations in the 22 families were detected by either restriction digest or allele-specific oligonucleotide hybridization of ARMS PCR products. We molecularly proved the origin of these different mutations to be paternal for all informative cases analyzed (P=2. 4x10-7; 95% confidence limits 87%-100%). Advanced paternal age was noted for the fathers of patients with Crouzon syndrome or Pfeiffer syndrome, compared with the fathers of control individuals (34. 50+/-7.65 years vs. 30.45+/-1.28 years, P<.01). Our data on advanced paternal age corroborates and extends previous clinical evidence based on statistical analyses as well as additional reports of advanced paternal age associated with paternal origin of three sporadic mutations causing Apert syndrome (FGFR2) and achondroplasia (FGFR3). Our results suggest that older men either have accumulated or are more susceptible to a variety of germline mutations.     

Age paternel et risques pour la descendance

The age at which a male procreates can affect the quality of his offspring. In man, paternal ageing can increase: a) the risk of recurrent spontaneous abortion; b) the risk of aneuploidy responsible for Klinefelter’s syndrome and, with a lower probability, Down syndrome; c) the risk of autosomal dominant mutations, about 25 of which have been identified at the present time and which cause:-various malformations, such as Apert, Marfan, Crouzon syndromes…- tumours, such as Wilms’ tumour, bilateral retinoblastoma, von Recklinghausen’s neurofibromatosis…-metabolic changes, causing retinitis pigmentosa, fibrodysplasia ossificans progressiva, Costello and Lesch-Nyhan syndromes…- neurophysiological disorders, such as athetoid dystonic cerebral palsy, and psychotic disorders, such as schizophrenia, d) certain X-linked recessive mutations, such as haemophilia A and Duchenne’s muscular dystrophy. Paternal age also appears to lower the life expectancy of daughters. Finally, the offspring of ageing men but also very young males appear to have an increased risk of heart and nervous malformation involving autosomal dominant mutations and, in man and in animals, an impaired level of cognitive functioning of the offspring. The abnormalities related to paternal ageing could be due to mutations linked to errors of recopying of the genetic message during spermatogonia replications, especially as the number of replications preceding the formation of spermatozoa increases with age. Decreased efficacy of DNA repair systems and/or in meiotic controls of spermatogenesis could also be involved. In addition to mutations, the ageing male also presents hypermethylation of ribosomal DNA in spermatozoa, which could be harmful for the conceptus. Abnormalities linked to a very young paternal age could be due to a defect of maturation of the genetic message via epigenetic events, such as imprinting and a cytoplasmic influence. Some statistical studies suggest that other factors, particularly maternal factors, could also play a role in the pathogenesis of some of these abnormalities. However, molecular biology demonstrates the essentially paternal origin of certain autosomal dominant mutations, such as bilateral retinoblastoma and Apert syndrome. These data raise the problem of the optimum age for fatherhood, like that of motherhood, for the offspring.     

Frequency of somatic and germ-line mosaicism in retinoblastoma: implications for genetic counseling.

Although mosaicism can have important implications for genetic counseling of families with hereditary disorders, information regarding the incidence of mosaicism is available for only a few genetic diseases. Here we describe an evaluation of 156 families with retinoblastoma; the initial oncogenic mutation in the retinoblastoma gene had been identified in these families. In 15 ( approximately 10%) families, we were able to document mosaicism for the initial mutation in the retinoblastoma gene, either in the proband or in one of the proband's parents. The true incidence of mosaicism in this group of 156 families is probably higher than our findings indicate; in some additional families beyond the 15 we identified, mosaicism was likely but could not be proven, because somatic or germ-line DNA from key family members was unavailable. Germ-line DNA from two mosaic fathers was analyzed: in one of these, the mutation was detected in both sperm and leukocyte DNA; in the other, the mutation was detected only in sperm DNA. Our data suggest that mosaicism is more common than is generally appreciated, especially in disorders such as retinoblastoma, in which a high proportion of cases represent new mutations. The possibility of mosaicism should always be considered during the genetic counseling of newly identified families with retinoblastoma. As demonstrated here, genetic tests of germ-line DNA can provide valuable information that is not available through analysis of somatic (leukocyte) DNA.     

Reproductive compensation and human genetic disease

The effects of reproductive compensation on the population genetics of sex-linked recessive lethal mutations are investigated. Simple equations are presented which describe these effects, and so complement existing population genetic theory. More importantly, this type of mutation is responsible for several severe human genetic diseases such as Duchenne muscular dystrophy. It is argued that the applications of three modern reproductive technologies--effective family planning, in utero diagnosis with termination, and embryo sexing--will lead to reproductive compensation. The adoption of any of these technologies may rapidly elevate the frequencies of those mutations which are lethal in childhood. This increase is large, in the order of 33% upwards, and occurs rapidly over two to five generations. It also depends on the source of mutations, the effect being larger if most mutations are paternal. In utero diagnosis and/or embryo sexing increase the frequency of the mutation, but simultaneously decrease disease incidence by preventing the birth of affected offspring. In contrast, effective family planning may rapidly increase both mutation frequency and disease incidence.     

Meiotic nondisjunction of chromosomes 1, 17, 18, X, and Y in men more than 80 years of Age

In order to evaluate a possible paternal age effect, testicular sperm cells from three men aged 81, 82, and 83 yr were analyzed by two-color- and three-color-fluorescence in situ hybridization for disomy rates of chromosomes 1, 17, 18, X, and Y as well as for diploidy frequencies. A minimum of 1500 sperm cells per donor and probe was evaluated due to the low number of spermatozoa in the preparations. Diploidy and disomy frequencies were in the same range as found in men aged <30 yr, a slight increase only being noticed for XY nuclei.     

The paternal genome in mouse zygotes is less sensitive to ENU mutagenesis than the maternal genome

The two parental genomes lie separate within the zygote and may be differentially affected by environmental influences. We have shown earlier (Russell et al., 1988) that the maternal genome within the mouse zygote is exquisitely sensitive to the induction of point mutations by N-ethyl-N-nitrosourea (ENU), and that the initial lesion probably occurs in one strand of the DNA. The present experiment measured specific-locus mutation induction in the paternal genome. Zygotes containing a multiple-recessive maternal genome (a; b; p cch; d se; s) and the corresponding wild-type alleles in the paternal one were exposed to 50 mg ENU/kg in vivo at one of two stages: the presumed times of sperm entry and early pronuclear stage. At weaning age, the resulting mice were examined for mutations at the marked loci as well as for other mutations producing externally visible phenotypes. At the marked loci, one possible mosaic (for b) was observed among 2113 classified offspring that had been treated with ENU as zygotes; this animal failed to transmit a mutation. By contrast, in the reciprocal cross (which tests the maternal genome) we had observed 8 specific-locus mutations (6 of them mosaics) among 1555 offspring that had received the same dose of ENU during sperm entry (and completion of oocyte meiosis II). In the present experiment, we also found one mutation at other loci (two at other loci in the reciprocal cross). The frequency of offspring with small white belly spots was significantly greater in the treated groups (3.5 and 1.9% at the earlier and later stage, respectively) than in the control (1.0%), the excess being almost entirely due to daughters. Genetic tests of a large number of such offspring failed to find a genetic cause. Instead, it appears that this phenotype may be influenced by factors in the intrauterine environment. It is concluded that shortly after sperm entry, the paternal genome of the zygote is less sensitive than the maternal one to the induction of mutations by ENU.     

The relationship between paternal age, sex ratios, and aneuploidy frequencies in human sperm, as assessed by multicolor FISH.

We studied the frequencies of X- and Y-chromosome-bearing sperm, diploidy and disomy for chromosomes 1, 12, X, and Y in sperm from 10 normal men aged 21-52 years, to determine whether there was any relationship between donor age and any of these variables. Multicolor FISH was used to control for lack of probe hybridization and to distinguish diploid sperm from disomic sperm. A minimum of 10,000 sperm per donor was evaluated for each chromosome, for a total of 225,846 sperm studied. Sperm were considered disomic if two fluorescent signals were separated by a minimal distance of one signal domain. The mean frequencies of X- and Y-bearing sperm were 50.1% and 49.0%, respectively; not significantly different from 50%. There was no correlation between paternal age and "sex ratio" in sperm. Similarly, there was no association between the frequency of diploid sperm (mean, .16%; range, .06-.42%) and donor age. For disomy frequencies, there was no relationship between donor age and disomy 12 (mean, .16%; range, .10%-.25%), XX (mean, .07%; range, .03%-.17%), and XY sperm (mean, .16%; range, .08%-.24%). There was a significant increase in the frequency of YY sperm (P = .04; mean, .18%; range, .10%-.43%) and disomy 1 sperm (P = .01; mean, .11%; range, .05%-.18%) with donor age. In summary, our results do not support a correlation between paternal age and sex ratio or diploidy.     

Analysis of phenotypic features and FGFR2 mutations in Apert syndrome.

A phenotypic and genotypic survey was conducted on 36 Apert syndrome patients. In all but one patient, an FGFR2 mutation, either S252W or P253R, was found in exon IIIa (exon U or 7). The frequency was 71% and 26%, for the mutations S252W and P253R, respectively. These mutations occur in the linker region between immunoglobulin-like domains II and III, which are involved in activation of the receptor by ligand binding and dimerization. The fact that one patient did not have a mutation in the same exon suggests further genetic heterogeneity in Apert syndrome. The frequencies of occurrence or means for measurements of 29 different clinical features (including severity of craniofacial features, syndactyly of the hands and feet, and multisystem involvement) were determined for all patients and for the two subgroups defined by their mutations. Comparison between the subgroups for the different clinical features was performed and suggested no statistically significant differences. These results are not unexpected, because the two common mutations for Apert syndrome alter FGFR2 at adjacent amino acids that are likely to have similar biological, and therefore phenotypic, consequences.     

An analysis for paternal-age effect in Ohio's Down syndrome births, 1970-1980.

The purpose of this study was to analyze Down syndrome (DS) births during 1970-1980 in the State of Ohio for a paternal-age effect independent of maternal age. Birth certificates and chromosome analysis records were used to ascertain 1,244 white DS births, which by capture-recapture methodology were estimated to comprise two-thirds of all white DS births in Ohio for this period. The control data consisted of 1,667,210 white live births in Ohio during the same period. One method of statistical analysis was a case-control comparison, which for each single-year maternal age compares the mean paternal age for controls with each observed DS paternal age. No statistically significant paternal-age effect was found in nine of the 11 years. For two of the years, and for all years combined, the DS fathers were significantly younger than the fathers of controls. When the data were subdivided according to ascertainment, one subpopulation--those DS individuals obtained from birth certificates alone--also showed a statistically significant negative paternal-age effect. The Mantel-Haenszel test was also applied to these data. Assuming no paternal-age effect, a lower rate of DS births than expected was found at paternal ages greater than or equal to 40, but not at greater than or equal to 45, greater than or equal to 50, or greater than or equal to 55. These same methods were used to test for a maternal-age effect. In each of the 11 years and over all 11 years combined, a strong and statistically significant positive maternal-age effect was detected.     

Human sperm aneuploidy after exposure to pesticides

This study examined the effect of paternal environmental exposure to pesticides on the frequency of aneuploidy in human sperm. To determine if the chromosome number in germ cells was altered by paternal exposure, multicolor fluorescence in situ hybridization (FISH) analysis was utilized to measure aneuploidy frequencies in the sperm of 40 men (20 exposed, 20 controls). Samples were coded for "blind analysis" to eliminate scorer bias. Aneuploidy and diploidy frequencies were assessed for chromosomes 13, 21, X, and Y. A minimum of 10,000 sperm was scored per donor per chromosome probe with a total of 809,935 sperm scored. Hybridization efficiency was 99%. There were no significant differences in aneuploidy or diploidy frequencies between exposed and control groups, suggesting that the pesticides did not increase the risk of numerical chromosomal abnormalities in these men.     

Selfish spermatogonial selection: evidence from an immunohistochemical screen in testes of elderly men

The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia-caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively-represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection.