De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5' splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

SMAD4 mutations causing Myhre syndrome are under positive selection in the male germline.

While it is widely thought that de novo mutations (DNMs) occur randomly, we previously showed that some DNMs are enriched because they are positively selected in the testes of aging men. These "selfish" mutations cause disorders with a shared presentation of features, including exclusive paternal origin, significant increase of the father's age, and high apparent germline mutation rate. To date, all known selfish mutations cluster within the components of the RTK-RAS-MAPK signaling pathway, a critical modulator of testicular homeostasis. Here, we demonstrate the selfish nature of the SMAD4 DNMs causing Myhre syndrome (MYHRS). By analyzing 16 informative trios, we show that MYHRS-causing DNMs originated on the paternally derived allele in all cases. We document a statistically significant epidemiological paternal age effect of 6.3 years excess for fathers of MYHRS probands. We developed an ultra-sensitive assay to quantify spontaneous MYHRS-causing SMAD4 variants in sperm and show that pathogenic variants at codon 500 are found at elevated level in sperm of most men and exhibit a strong positive correlation with donor's age, indicative of a high apparent germline mutation rate. Finally, we performed in vitro assays to validate the peculiar functional behavior of the clonally selected DNMs and explored the basis of the pathophysiology of the different SMAD4 sperm-enriched variants. Taken together, these data provide compelling evidence that SMAD4, a gene operating outside the canonical RAS-MAPK signaling pathway, is associated with selfish spermatogonial selection and raises the possibility that other genes/pathways are under positive selection in the aging human testis.

Fine-tuning germline mutation rates across evolution.

The germline mutation rate (GMR) sets the pace at which mutations, the raw material of evolution, are introduced into the genome. By sequencing a dataset of unprecedently broad phylogenetic scope, Bergeron et al. estimated species-specific GMR, offering numerous insights into how this parameter shapes and is shaped by life-history traits.

New analysis of atypical spermatocytic tumours reveals extensive heterogeneity and plasticity of germ cell tumours†.

Testicular germ cell tumours (TGCTs) derived from immature (type I) and pluripotent germ cell neoplasia in situ (GCNIS, type II) are characterised by remarkable phenotypic heterogeneity and plasticity. In contrast, the rare spermatocytic tumour (SpT, type III), derived from mature spermatogonia, is considered a homogenous and benign tumour but may occasionally present as an anaplastic or an aggressive sarcomatoid tumour. While various oncogenic processes had been proposed, the precise mechanism driving malignant progression remained elusive until the molecular characterisation of a series of atypical SpTs described in a recent issue of The Journal of Pathology. The emerging picture suggests the presence of two distinct trajectories for SpTs, involving either RAS/mitogen-activated protein kinase pathway mutations or a ploidy shift with secondary TP53 mutations and/or gain of chromosome 12p, the latter known as pathognomonic for type II GCNIS-derived TGCTs. Here, we discuss the implications of these findings, seen from the perspective of germ cell biology and the unique features of different TGCTs. The evolving phenotype of SpTs, induced by genomic and epigenetic changes, illustrates that the concept of plasticity applies to all germ cell tumours, making them inherently heterogenous and capable of significant transformation during progression. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Providing recurrence risk counselling for parents after diagnosis of a serious genetic condition caused by an apparently de novo mutation in their child: a qualitative investigation of the PREGCARE strategy with UK clinical genetics practitioners.

BACKGROUND: Diagnosis of a child with a genetic condition leads to parents asking whether there is a risk the condition could occur again with future pregnancies. If the cause is identified as an apparent de novo mutation (DNM), couples are currently given a generic, population average, recurrence risk of ~1%-2%, depending on the condition. Although DNMs usually arise as one-off events, they can also originate through the process of mosaicism in either parent; in this instance, the DNM is present in multiple germ cells and the actual recurrence risk could theoretically be as high as 50%. METHODS: Our qualitative interview study examined the views and reflections on current practice provided by UK practitioners working in clinical genetics (n=20) regarding the potential impact of PREcision Genetic Counselling And REproduction (PREGCARE)-a new preconception personalised recurrence risk assessment strategy. RESULTS: Those interviewed regarded PREGCARE as a very useful addition to risk management, especially for cases where it revised the risk downwards or clarified that a couple's personalised recurrence risk meets National Health Service thresholds for non-invasive prenatal testing, otherwise inaccessible based on the generic DNM recurrence risk. CONCLUSION: Participants said it could release some couples requiring reassurance from undergoing unnecessary invasive testing in future pregnancies. However, they regarded mosaicism and PREGCARE as complex concepts to communicate, requiring further training and additional appointment time for pre-test genetic counselling to prepare couples for all the possible outcomes of a personalised risk assessment, including potentially identifying the parental origin of the DNM, and to ensure informed consent.

Professionals' views on providing personalized recurrence risks for de novo mutations: Implications for genetic counseling.

When an apparent de novo (new) genetic change has been identified as the cause of a serious genetic condition in a child, many couples would like to know the risk of this happening again in a future pregnancy. Current practice provides families with a population average risk of 1%-2%. However, this figure is not accurate for any specific couple, and yet, they are asked to make decisions about having another child and/or whether to have prenatal testing. The PREcision Genetic Counseling And REproduction (PREGCARE) study is a new personalized assessment strategy that refines a couple's recurrence risk prior to a new pregnancy, by analyzing several samples from the parent-child trio (blood, saliva, swabs, and father's sperm) using deep sequencing and haplotyping. Overall, this approach can reassure ~2/3 of couples who have a negligible (<0.1%) recurrence risk and focus support on those at higher risk (i.e. when mosaicism is identified in one of the parents). Here we present a qualitative interview study with UK clinical genetics professionals (n = 20), which investigate the potential implications of introducing such a strategy in genetics clinics. While thematic analysis of the interviews indicated perceived clinical utility, it also indicates a need to prepare couples for the psychosocial implications of parent-of-origin information and to support their understanding of the assessment being offered. When dealing with personalized reproductive risk, a traditional non-directive approach may not meet the needs of practitioner and client(s) and shared decision-making provides an additional framework that may relieve some patient burden. Further qualitative investigation with couples is planned.

Selfish mutations dysregulating RAS-MAPK signaling are pervasive in aged human testes.

Mosaic mutations present in the germline have important implications for reproductive risk and disease transmission. We previously demonstrated a phenomenon occurring in the male germline, whereby specific mutations arising spontaneously in stem cells (spermatogonia) lead to clonal expansion, resulting in elevated mutation levels in sperm over time. This process, termed "selfish spermatogonial selection," explains the high spontaneous birth prevalence and strong paternal age-effect of disorders such as achondroplasia and Apert, Noonan and Costello syndromes, with direct experimental evidence currently available for specific positions of six genes (FGFR2, FGFR3, RET, PTPN11, HRAS, and KRAS). We present a discovery screen to identify novel mutations and genes showing evidence of positive selection in the male germline, by performing massively parallel simplex PCR using RainDance technology to interrogate mutational hotspots in 67 genes (51.5 kb in total) in 276 biopsies of testes from five men (median age, 83 yr). Following ultradeep sequencing (about 16,000×), development of a low-frequency variant prioritization strategy, and targeted validation, we identified 61 distinct variants present at frequencies as low as 0.06%, including 54 variants not previously directly associated with selfish selection. The majority (80%) of variants identified have previously been implicated in developmental disorders and/or oncogenesis and include mutations in six newly associated genes (BRAF, CBL, MAP2K1, MAP2K2, RAF1, and SOS1), all of which encode components of the RAS-MAPK pathway and activate signaling. Our findings extend the link between mutations dysregulating the RAS-MAPK pathway and selfish selection, and show that the aging male germline is a repository for such deleterious mutations.

Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer.

BACKGROUND: Clinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues. METHODS: We developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24-240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified. RESULTS: Cisplatin exposure resulted in a significant reduction in the total number of germ cells (- 44%, p < 0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (- 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (- 42%, p < 0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure. CONCLUSIONS: This is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.

amplimap: a versatile tool to process and analyze targeted NGS data.

SUMMARY: amplimap is a command-line tool to automate the processing and analysis of data from targeted next-generation sequencing experiments with PCR-based amplicons or capture-based enrichment systems. From raw sequencing reads, amplimap generates output such as read alignments, annotated variant calls, target coverage statistics and variant allele counts and frequencies for each target base pair. In addition to its focus on user-friendliness and reproducibility, amplimap supports advanced features such as consensus base calling for read families based on unique molecular identifiers and filtering false positive variant calls caused by amplification of off-target loci. AVAILABILITY AND IMPLEMENTATION: amplimap is available as a free Python package under the open-source Apache 2.0 License. Documentation, source code and installation instructions are available at https://github.com/koelling/amplimap.

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis.

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.

The impact of chemo- and radiotherapy treatments on selfish de novo FGFR2 mutations in sperm of cancer survivors.

STUDY QUESTION: What effect does cancer treatment have on levels of spontaneous selfish fibroblast growth factor receptor 2 (FGFR2) point mutations in human sperm? SUMMARY ANSWER: Chemotherapy and radiotherapy do not increase levels of spontaneous FGFR2 mutations in sperm but, unexpectedly, highly-sterilizing treatments dramatically reduce the levels of the disease-associated c.755C > G (Apert syndrome) mutation in sperm. WHAT IS KNOWN ALREADY: Cancer treatments lead to short-term increases in gross DNA damage (chromosomal abnormalities and DNA fragmentation) but the long-term effects, particularly at the single nucleotide resolution level, are poorly understood. We have exploited an ultra-sensitive assay to directly quantify point mutation levels at the FGFR2 locus. STUDY DESIGN, SIZE, DURATION: 'Selfish' mutations are disease-associated mutations that occur spontaneously in the sperm of most men and their levels typically increase with age. Levels of mutations at c.752-755 of FGFR2 (including c.755C > G and c.755C > T associated with Apert and Crouzon syndromes, respectively) in semen post-cancer treatment from 18 men were compared to levels in pre-treatment samples from the same individuals (n = 4) or levels in previously screened population controls (n = 99). PARTICIPANTS/MATERIALS, SETTING, METHODS: Cancer patients were stratified into four different groups based on the treatments they received and the length of time for spermatogenesis recovery. DNA extracted from semen samples was analysed using a previously established highly sensitive assay to identify mutations at positions c.752-755 of FGFR2. Five to ten micrograms of semen genomic DNA was spiked with internal controls for quantification purposes, digested with MboI restriction enzyme and gel extracted. Following PCR amplification, further MboI digestion and a nested PCR with barcoding primers, samples were sequenced on Illumina MiSeq. Mutation levels were determined relative to the spiked internal control; in individuals heterozygous for a nearby common single nucleotide polymorphism (SNP), mutations were phased to their respective alleles. MAIN RESULTS AND THE ROLE OF CHANCE: Patients treated with moderately-sterilizing alkylating regimens and who recovered spermatogenesis within <3 years after therapy (Group 3, n = 4) or non - alkylating chemotherapy and/or low gonadal radiation doses (Group 1, n = 4) had mutation levels similar to untreated controls. However, patients who had highly-sterilizing alkylating treatments (i.e. >5 years to spermatogenesis recovery) (Group 2, n = 7) or pelvic radiotherapy (Group 4, n = 3) exhibited c.755C > G mutation levels at or below background. Two patients (A and B) treated with highly-sterilizing alkylating agents demonstrated a clear reduction from pre-treatment levels; however pre-treatment samples were not available for the other patients with low mutation levels. Therefore, although based on their age we would expect detectable levels of mutations, we cannot exclude the possibility that these patients also had low mutation levels pre-treatment. In three patients with low c.755C > G levels at the first timepoint post-treatment, we observed increasing mutation levels over time. For two such patients we could phase the mutation to a nearby polymorphism (SNP) and determine that the mutation counts likely originated from a single or a small number of mutational events. LIMITATIONS, REASONS FOR CAUTION: This study was limited to 18 patients with different treatment regimens; for nine of the 18 patients, samples from only one timepoint were available. Only 12 different de novo substitutions at the FGFR2 c.752-755 locus were assessed, two of which are known to be disease associated. WIDER IMPLICATIONS OF THE FINDINGS: Our data add to the body of evidence from epidemiological studies and experimental data in humans suggesting that male germline stem cells are resilient to the accumulation of spontaneous mutations. Collectively, these data should provide physicians and health-care professionals with reassuring experimental-based evidence for counselling of male cancer patients contemplating their reproductive options several years after treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was primarily supported by grants from the Wellcome (grant 091182 to AG and AOMW; grant 102 731 to AOMW), the University of Oxford Medical Sciences Division Internal Fund (grant 0005128 to GJM and AG), the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme (to AG) and the US National Institutes of Health (to MLM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. None of the authors has any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NA.

Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes.

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

Quantification of transmission risk in a male patient with a FLNB mosaic mutation causing Larsen syndrome: Implications for genetic counseling in postzygotic mosaicism cases.

We report the case of a male patient with Larsen syndrome found to be mosaic for a novel point mutation in FLNB in whom it was possible to provide evidence-based personalized counseling on transmission risk to future offspring. Using dideoxy sequencing, a low-level FLNB c.698A>G, encoding p.(Tyr233Cys) mutation was detected in buccal mucosa and fibroblast DNA. Mutation quantification was performed by deep next-generation sequencing (NGS) of DNA extracted from three somatic tissues (blood, fibroblasts, saliva) and a sperm sample. The mutation was detectable in all tissues tested, at levels ranging from 7% to 10% (mutation present in ∼20% of diploid somatic cells and 7% of haploid sperm), demonstrating the involvement of both somatic and gonadal lineages in this patient. This report illustrates the clinical utility of performing targeted NGS analysis on sperm from males with a mosaic condition in order to provide personalized transmission risk and offer evidence-based counseling on reproductive safety.

Mutations in PIGY: expanding the phenotype of inherited glycosylphosphatidylinositol deficiencies.

Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data.

The Fourth International Symposium on Genetic Disorders of the Ras/MAPK pathway.

The RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome, Legius syndrome, and capillary malformation-arteriovenous malformation (CM-AVM) syndrome. In combination, the RASopathies are a frequent group of genetic disorders. This report summarizes the proceedings of the 4th International Symposium on Genetic Disorders of the Ras/MAPK pathway and highlights gaps in the field. © 2016 Wiley Periodicals, Inc.

Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline.

The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline.

Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes.

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

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.