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Delayed fatherhood results in a higher risk to inherit a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early or late-onset effects in the offspring. Here, we investigated the mutation frequency in the DNA of human testis and sperm and the activation state of the expressed mutant protein of eight different FGFR3 variants categorized by ClinVar as deleterious, benign, or not reported. Overall, the ligand-independent activation of the mutant protein resulted in a increased number of mutant sperm; although, strong activating mutations did not necessarily result in the highest frequencies. Moreover, only two mutants c.952G>A and c.1620C>A showed an increase with the donor’s age; the latter also forming larger clonal expansions in the testis. We also showed that the prediction of deleteriousness of a mutation is not always accurate, and similar in silico scores can reflect either a gain-of-function or loss-of-function. Our approach led to the discovery of two novel variants c.1261G>A and c.952G>A to have promiscuous FGFR3 activation and increased mutation frequencies in the male germline. The large fraction of donors with mutations suggests a high de novo rate potentially explained by a selective advantage before the maturation of the male germline. This sequence-function study provides important data for the evaluation and interpretation of variants with relevant clinical implications.

Gain‐of‐function mutations in the receptor tyrosine kinase pathway are often lethal; yet, it might be tolerated at very low frequencies as postzygotic mosaics resulting in a vast range of new phenotypes, unknown before the detection of the mosaic disorder. With the advent of ultra‐accurate next‐generation sequencing, these undercharacterized mosaics hidden in a few cells can now be better characterized in terms of their biological relevance in disease., Mutations occurring during embryonic development affect only a subset of cells resulting in two or more distinct cell populations that are present at different levels, also known as postzygotic mosaicism (PZM). Although PZM is a common biological phenomenon, it is often overlooked as a source of disease due to the challenges associated with its detection and characterization, especially for very low‐frequency variants. Moreover, PZM can cause a different phenotype compared to constitutional mutations. Especially, lethal mutations in receptor tyrosine kinase (RTK) pathway genes, which exist only in a mosaic state, can have completely new clinical manifestations and can look very different from the associated monogenic disorder. However, some key questions are still not addressed, such as the level of mosaicism resulting in a pathogenic phenotype and how the clinical outcome changes with the development and age. Addressing these questions is not trivial as we require methods with the sensitivity to capture some of these variants hidden away in very few cells. Recent ultra‐accurate deep‐sequencing approaches can now identify these low‐level mosaics and will be central to understand systemic and local effects of mosaicism in the RTK pathway. The main focus of this review is to highlight the importance of low‐level mosaics and the need to include their detection in studies of genomic variation associated with disease.

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/MAPK pathway by their tyrosine-kinase activation known to determine cell-growth, tissue differentiation, and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here, we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (mGFP-FGFR3) in certain areas of the plasma membrane of living cells. We quantified receptor activation via total internal reflection fluorescence microscopy of FGFR3 signaling at the cell membrane that captured the recruitment of the downstream signal transducer GRB2 tagged with mScarlet (GRB2-mScarlet) to FGFR3 micropatterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) for wildtype (WT) and four FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, and K650E). Our data showed that ligand addition increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared to previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This can be attributed to the different methodology, since micropatterning specifically captures signaling events at the plasma membrane. Collectively, our results provide further insight into the functional effects of mutations to FGFR3.

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/MAPK pathway by their tyrosine-kinase activation known to determine cell-growth, tissue differentiation and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some of these mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (mGFP-FGFR3) in certain areas of the plasma membrane of living cells. Receptor activation was then quantified via the recruitment of the downstream signal transducer GRB2 tagged with mScarlet (GRB2-mScarlet) to FGFR3 patterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) in the wildtype (WT), as well as in different FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, K650E). Our data showed that the addition of ligands increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared to previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This may be explained by the different receptor populations probed, since the micropatterning method specifically reports on signaling events at the plasma membrane.Graphical AbstractSpecifications: The maximum size of the image should be 200 × 500 pixels with a minimum resolution of 300 dpi, using Arial font with a size of 10-16 points; Preferred file types: TIFF, EPS, PDF or MS Office filesResearch HighlightsQuantification of FGFR3 signaling in live cells on micropatterned surfacesAnalysis of GRB2 recruitment to the mature receptor at the plasma membraneLigand-independent kinase activation of FGFR3 mutantsActivation of FGFR3 at the cell surface can be different than in bulk cell extracts

The mammary gland undergoes hormonally stimulated cycles of proliferation, lactation and involution. We hypothesized that these factors increase the mutational burden in glandular tissue and may explain high cancer incidence rate in the general population and recurrent disease. Hence, we investigated the DNA sequence variants in the normal mammary gland, tumor and peripheral blood from 52 reportedly sporadic breast cancer patients, including breast-conserving surgery cases. Targeted resequencing of 542 cancer associated genes revealed mosaic somatic pathogenic variants of: PIK3CA, TP53, AKT1, MAP3K1, CDH1, RB1, NCOR1, MED12, CBFB, TBX3 and TSHR in the normal mammary gland, at considerable allelic frequencies (9×10−2 to 5.2×10−1) indicating clonal expansion. Further evaluation of the frequently damaged PIK3CA and TP53 genes by ultra-sensitive duplex sequencing demonstrated a diversified picture of multiple low level-mosaic (in 10−2 to 10−4 alleles) hotspot pathogenic variants. Our results raise a question about the oncogenic potential in non-tumor mammary gland tissue of breast-conserving surgery patients.

As recombination plays an important role in evolution, its estimation, as well as, the identification of hotspot positions is of considerable interest. We propose a novel approach for estimating historical recombination along a chromosome that involves a sequential multiscale change point estimator. Our method also permits to take demography into account. It uses a composite likelihood estimate and other summary statistics within a regression model fitted on suitable scenarios. Our proposed method is accurate, computationally fast, and provides a parsimonious solution by ensuring a type I error control against too many changes in the recombination rate. An application to human genome data suggests a good congruence between our estimated and experimentally identified hotspots. Our method is implemented in theR-packageLDJump, which is freely available fromhttps://github.com/PhHermann/LDJump.

De novo mutations (DNMs) are an important player in heritable diseases and evolution, yet little is known about the different mutagenic processes in our germline given the difficulty to reliably identify ultra-low frequency variants. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ~4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified highly mutable sites with mutation frequencies 4-5 orders of magnitude higher than the genome average. Multiple mutations were found at a higher frequency, or exclusively, in older donors, suggesting that these mutations are testis exclusive mosaics expanding in the male germline with age. Also, older donors harbored more mutations associated with congenital disorders. Some mutations were found in both age groups with no significant difference, suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independently of age, the frequency and deleteriousness of the mutations in sperm were elevated compared to reports in the population. Our approach is an important strategy to identify mutations that could be associated with aberrant receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultradeep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ∼4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant allele frequencies (VAFs) of 10−4 to 10−5, with an overall mutation frequency of the region of ∼6 × 10−7. Some of the substitutions are recurrent and are found at a higher VAF in older donors than in younger ones or are found exclusively in older donors. Also, older donors harbor more mutations associated with congenital disorders. Other mutations are present in both age groups, suggesting that these might result from a different mechanism (e.g., postzygotic mosaicism). We also observe that independent of age, the frequency and deleteriousness of the mutational spectra are more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain of function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

Duplex sequencing is currently the most reliable method to identify ultra-low frequency DNA variants by grouping sequence reads derived from the same DNA molecule into families with information on the forward and reverse strand. However, only a small proportion of reads are assembled into duplex consensus sequences, and reads with potentially valuable information are discarded at different steps of the bioinformatics pipeline, especially reads without a family. We developed a bioinformatics tool-set that analyses the tag and family composition with the purpose to understand data loss and implement modifications to maximize the data output for the variant calling. Specifically, our tools show that tags contain PCR and sequencing errors that contribute to data loss and lower DCS yields. Our tools also identified chimeras, which result in unpaired families that do not form DCS. Finally, we also developed a tool called Variant Analyzer that re-examines variant calls from raw reads and provides different summary data that categorizes the confidence level of a variant call by a tier-based system. We demonstrate that this tool identified false positive variants tagged by the tier-based classification. Furthermore, with this tool we can include reads without a family and check the reliability of the call, which increases substantially the sequencing depth for variant calling, a particular important advantage for low-input samples or low-coverage regions.

PR domain containing protein 9 (PRDM9) is a meiosis-specific, multi-domain protein that regulates the location of recombination hotspots by targeting its DNA recognition sequence for double-strand breaks (DSBs). PRDM9 specifically recognizes DNA via its tandem array of zinc fingers (ZnFs), epigenetically marks the local chromatin by its histone methyltransferase activity, and is an important tether that brings the DNA into contact with the recombination initiation machinery. A strong correlation between PRDM9-ZnF variants and specific DNA motifs at recombination hotspots has been reported; however, the binding specificity and kinetics of the ZnF domain are still obscure. Using two in vitro methods, gel mobility shift assays and switchSENSE, a quantitative biophysical approach that measures binding rates in real time, we determined that the PRDM9-ZnF domain forms a highly stable and long-lived complex with its recognition sequence, with a dissociation halftime of many hours. The ZnF domain exhibits an equilibrium dissociation constant (K D) in the nanomolar (nM) range, with polymorphisms in the recognition sequence directly affecting the binding affinity. We also determined that alternative sequences (15–16 nucleotides in length) can be specifically bound by different subsets of the ZnF domain, explaining the binding plasticity of PRDM9 for different sequences. Finally, longer binding targets are preferred than predicted from the numbers of ZnFs contacting the DNA. Functionally, a long-lived complex translates into an enzymatically active PRDM9 at specific DNA-binding sites throughout meiotic prophase I that might be relevant in stabilizing the components of the recombination machinery to a specific DNA target until DSBs are initiated by Spo11. Electronic supplementary material The online version of this article (doi:10.1007/s10577-017-9552-1) contains supplementary material, which is available to authorized users.

Meiosis strongly influences the transmission and evolution of heterozygous poly-A repeats as measured experimentally in a large collection of single recombination products in a human hotspot., Meiotic recombination has strong, but poorly understood effects on short tandem repeat (STR) instability. Here, we screened thousands of single recombinant products with sperm typing to characterize the role of polymorphic poly-A repeats at a human recombination hotspot in terms of hotspot activity and STR evolution. We show that the length asymmetry between heterozygous poly-A’s strongly influences the recombination outcome: a heterology of 10 A’s (9A/19A) reduces the number of crossovers and elevates the frequency of non-crossovers, complex recombination products, and long conversion tracts. Moreover, the length of the heterology also influences the STR transmission during meiotic repair with a strong and significant insertion bias for the short heterology (6A/7A) and a deletion bias for the long heterology (9A/19A). In spite of this opposing insertion-/deletion-biased gene conversion, we find that poly-A’s are enriched at human recombination hotspots that could have important consequences in hotspot activation.

Background Duplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies. Its power comes from pooling together multiple descendants of both strands of original DNA molecules, which allows distinguishing true nucleotide substitutions from PCR amplification and sequencing artifacts. This strategy comes at a cost—sequencing the same molecule multiple times increases dynamic range but significantly diminishes coverage, making whole genome duplex sequencing prohibitively expensive. Furthermore, every duplex experiment produces a substantial proportion of singleton reads that cannot be used in the analysis and are thrown away. Results In this paper we demonstrate that a significant fraction of these reads contains PCR or sequencing errors within duplex tags. Correction of such errors allows “reuniting” these reads with their respective families increasing the output of the method and making it more cost effective. Conclusions We combine an error correction strategy with a number of algorithmic improvements in a new version of the duplex analysis software, Du Novo 2.0. It is written in Python, C, AWK, and Bash. It is open source and readily available through Galaxy, Bioconda, and Github: https://github.com/galaxyproject/dunovo.

Meiotic recombination has strong, but poorly understood, effects on short tandem repeat (STR) instability. Here, we screened thousands of single recombinant products to characterize the transmission and evolution of polymorphic poly-A repeats at a human recombination hotspot. We show that length asymmetry between heterozygous poly-As plays a key role in the recombination outcome and their transmission. A difference of 10 As (9A/19A) elevates the frequency of non-crossovers, complex recombination products, and long conversion tracts. Moreover, asymmetry also influences STR transmission: the shorter allele is transmitted more frequently (deletion bias) at the asymmetric STR (9A/19A), while the longer allele is favored (insertion bias) at the site with a small STR length difference (6A/7A). Finally, potentially due to this opposing insertion/deletion driven evolution, we find that poly-As are enriched at human recombination hotspots predominantly with short poly-As, possibly influencing open chromatin regions that in turn can activate hotspots.

Meiosis is initiated by a double-strand break (DSB) introduced in the DNA by a highly controlled process that is repaired by recombination. In many organisms, recombination occurs at specific and narrow regions of the genome, known as recombination hotspots, which overlap with regions enriched for DSBs. In recent years, it has been demonstrated that conversions and mutations resulting from the repair of DSBs lead to a rapid sequence evolution at recombination hotspots eroding target sites for DSBs. We still do not fully understand the effect of this erosion in the recombination activity, but evidence has shown that the binding of trans-acting factors like PRDM9 is affected. PRDM9 is a meiosis-specific, multi-domain protein that recognizes DNA target motifs by its zinc finger domain and directs DSBs to these target sites. Here we discuss the changes in affinity of PRDM9 to eroded recognition sequences, and explain how these changes in affinity of PRDM9 can affect recombination, leading sometimes to sterility in the context of hybrid crosses. We also present experimental data showing that DNA methylation reduces PRDM9 binding in vitro. Finally, we discuss PRDM9-independent hotspots, posing the question how these hotspots evolve and change with sequence erosion. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’.

To study meiotic recombination products, cis- or trans-association of disease polymorphisms, or allele-specific expression patterns, it is necessary to phase heterozygous polymorphisms separated by several kilobases. Haplotyping using long-range polymerase chain reaction (PCR) is a powerful, cost-effective method to directly obtain the phase of multiple heterozygous sites with standard laboratory equipment in a handful of loci for many samples. The method is based on the amplification of large genomic DNA regions (up to ~40 kb) with a reaction mixture that combines a proofreading polymerase with allele-specific primer pairs that preferentially amplify matched templates. The analysis of two heterozygous SNPs requires four reactions, each containing one of the four possible allele-specific primer combinations (two forward and two reverse primers), with the mismatches occurring at the 3' ends of the primers. The two correct primer combinations will more efficiently elongate the matching alleles than the alternative alleles, and the difference in amplification efficiency can be monitored with real-time PCR.

Characterizing polymorphisms on single molecules renders the phase of different alleles, and thus, haplotype information. Here, we describe a high-throughput method to genotype hundreds-of thousands single molecules in parallel using bead-emulsion haplotyping (BEH). Haplotyping via BEH is an emulsion-PCR-based method that was adapted to amplify multiple DNA fragments on paramagnetic, microscopic beads within a compartment formed by an aqueous-oil emulsion. This generates beads covered by thousands of clonal copies from several polymorphic regions of an initial DNA molecule that are then genotyped with fluorescently labeled probes. With BEH, up to three different polymorphisms (or more if several polymorphisms are within an amplicon) can be typed within a fragment of several kilobases in a singleexperiment, rendering haplotype information of a very large number of initial single molecules. The high throughput and digital nature of the method makes it ideal to quantify rare haplotypes or to assess the haplotype diversity in complex samples.

To study meiotic recombination products, cis- or trans-association of disease polymorphisms, or allele-specific expression patterns, it is necessary to phase heterozygous polymorphisms separated by several kilobases. Haplotyping using long-range polymerase chain reaction (PCR) is a powerful, cost-effective method to directly obtain the phase of multiple heterozygous sites with standard laboratory equipment in a handful of loci for many samples. The method is based on the amplification of large genomic DNA regions (up to ~40 kb) with a reaction mixture that combines a proofreading polymerase with allele-specific primer pairs that preferentially amplify matched templates. The analysis of two heterozygous SNPs requires four reactions, each containing one of the four possible allele-specific primer combinations (two forward and two reverse primers), with the mismatches occurring at the 3' ends of the primers. The two correct primer combinations will more efficiently elongate the matching alleles than the alternative alleles, and the difference in amplification efficiency can be monitored with real-time PCR.

Characterizing polymorphisms on single molecules renders the phase of different alleles, and thus, haplotype information. Here, we describe a high-throughput method to genotype hundreds-of thousands single molecules in parallel using bead-emulsion haplotyping (BEH). Haplotyping via BEH is an emulsion-PCR-based method that was adapted to amplify multiple DNA fragments on paramagnetic, microscopic beads within a compartment formed by an aqueous-oil emulsion. This generates beads covered by thousands of clonal copies from several polymorphic regions of an initial DNA molecule that are then genotyped with fluorescently labeled probes. With BEH, up to three different polymorphisms (or more if several polymorphisms are within an amplicon) can be typed within a fragment of several kilobases in a singleexperiment, rendering haplotype information of a very large number of initial single molecules. The high throughput and digital nature of the method makes it ideal to quantify rare haplotypes or to assess the haplotype diversity in complex samples.

Real-time PCR-based genotyping methods, such as TaqMan allelic discrimination assays and allele-specific genotyping, are particularly useful when screening a handful of single nucleotide polymorphisms in hundreds of samples; either derived from different individuals, tissues, or pre-amplified DNA. Although real-time PCR-based methods such as TaqMan are well-established, alternative methods, like allele-specific genotyping, are powerful alternatives, especially for genotyping short tandem repeat (STR) length polymorphisms. Here, we describe all relevant aspects when developing an assay for a new SNP or STR using either TaqMan or allele-specific genotyping, respectively, such as primer and probe design, optimization of reaction conditions, the experimental procedure for typing hundreds of samples, and finally the data evaluation. Our goal is to provide a guideline for developing genotyping assays using these two approaches that render reliable and reproducible genotype calls involving minimal optimization.

Real-time PCR-based genotyping methods, such as TaqMan allelic discrimination assays and allele-specific genotyping, are particularly useful when screening a handful of single nucleotide polymorphisms in hundreds of samples; either derived from different individuals, tissues, or pre-amplified DNA. Although real-time PCR-based methods such as TaqMan are well-established, alternative methods, like allele-specific genotyping, are powerful alternatives, especially for genotyping short tandem repeat (STR) length polymorphisms. Here, we describe all relevant aspects when developing an assay for a new SNP or STR using either TaqMan or allele-specific genotyping, respectively, such as primer and probe design, optimization of reaction conditions, the experimental procedure for typing hundreds of samples, and finally the data evaluation. Our goal is to provide a guideline for developing genotyping assays using these two approaches that render reliable and reproducible genotype calls involving minimal optimization.

Children of a heterozygous parent are expected to carry either allele with equal probability. Exceptions can occur, however, due to meiotic drive, competition among gametes, or viability selection, which we collectively term “transmission distortion” (TD). Although there are several well-characterized examples of these phenomena, their existence in humans remains unknown. We therefore performed a genome-wide scan for TD by applying the transmission disequilibrium test (TDT) genome-wide to three large sets of human pedigrees of European descent: the Framingham Heart Study (FHS), a founder population of European origin (HUTT), and a subset of the Autism Genetic Resource Exchange (AGRE). Genotyping error is an important confounder in this type of analysis. In FHS and HUTT, despite extensive quality control, we did not find sufficient evidence to exclude genotyping error in the strongest signals. In AGRE, however, many signals extended across multiple SNPs, a pattern highly unlikely to arise from genotyping error. We identified several candidate regions in this data set, notably a locus in 10q26.13 displaying a genome-wide significant TDT in combined female and male transmissions and a signature of recent positive selection, as well as a paternal TD signal in 6p21.1, the same region in which a significant TD signal was previously observed in 30 European males. Neither region replicated in FHS, however, and the paternal signal was not visible in sperm competition assays or as allelic imbalance in sperm. In maternal transmissions, we detected no strong signals near centromeres or telomeres, the regions predicted to be most susceptible to female-specific meiotic drive, but we found a significant enrichment of top signals among genes involved in cell junctions. These results illustrate both the potential benefits and the challenges of using the TDT to study transmission distortion and provide candidates for investigation in future studies.

The amplification of millions of single molecules in parallel can be performed on microscopic magnetic beads that are contained in aqueous compartments of an oil-buffer emulsion. These bead-emulsion amplification (BEA) reactions result in beads that are covered by almost-identical copies derived from a single template. The post-amplification analysis is performed using different fluorophore-labeled probes. We have identified BEA reaction conditions that efficiently produce longer amplicons of up to 450 base pairs. These conditions include the use of a Titanium Taq amplification system. Second, we explored alternate fluorophores coupled to probes for post-PCR DNA analysis. We demonstrate that four different Alexa fluorophores can be used simultaneously with extremely low crosstalk. Finally, we developed an allele-specific extension chemistry that is based on Alexa dyes to query individual nucleotides of the amplified material that is both highly efficient and specific.

Meiosis is a potentially important source of germline mutations, as sites of meiotic recombination experience recurrent double-strand breaks (DSBs). However, evidence for a local mutagenic effect of recombination from population sequence data has been equivocal, likely because mutation is only one of several forces shaping sequence variation. By sequencing large numbers of single crossover molecules obtained from human sperm for two recombination hotspots, we find direct evidence that recombination is mutagenic: Crossovers carry more de novo mutations than nonrecombinant DNA molecules analyzed for the same donors and hotspots. The observed mutations were primarily CG to TA transitions, with a higher frequency of transitions at CpG than non-CpGs sites. This enrichment of mutations at CpG sites at hotspots could predominate in methylated regions involving frequent single-stranded DNA processing as part of DSB repair. In addition, our data set provides evidence that GC alleles are preferentially transmitted during crossing over, opposing mutation, and shows that GC-biased gene conversion (gBGC) predominates over mutation in the sequence evolution of hotspots. These findings are consistent with the idea that gBGC could be an adaptation to counteract the mutational load of recombination.

This study compares the relative effects of advancing male age on multiple genomic defects in human sperm [DNA fragmentation index (DFI), chromatin integrity, gene mutations, and numerical chromosomal abnormalities], characterizes the relationships among these defects and with semen quality, and estimates the incidence of susceptible individuals for a well characterized nonclinical nonsmoking group of 97 men (22–80 years). Adjusting for confounders, we found major associations between age and the frequencies of sperm with DFI and fibroblast growth factor receptor 3 gene ( FGFR3 ) mutations associated with achondroplasia ( P < 0.01) with no evidence for age thresholds. However, we found no associations between age and the frequencies of sperm with immature chromatin, aneuploidies/diploidies, FGFR2 mutations (Apert syndrome), or sex ratio in this cohort. There were also no consistent correlations among genomic and semen-quality endpoints, except between DFI and sperm motility ( r = −0.65, P < 0.001). These findings suggest there are multiple spermatogenic targets for genomically defective sperm with substantially variable susceptibilities to age. Our findings predict that as healthy males age, they have decreased pregnancy success with trends beginning in their early reproductive years, increased risk for producing offspring with achondroplasia mutations, and risk of fathering offspring with Apert syndrome that may vary across cohorts, but with no increased risk for fathering aneuploid offspring (Down, Klinefelter, Turner, triple X, and XYY syndromes) or triploid embryos. Our findings also suggest that the burden of genomic damage in sperm cannot be inferred from semen quality, and that a small fraction of men are at increased risk for transmitting multiple genetic and chromosomal defects.

The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 ( FGFR3 ) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay.

Taxonomic affinity of Peromyscus boylii sacarensis was examined using DNA sequences from the mitochondrial cytochrome-b gene. Maximum parsimony and genetic distance analyses were used to examine the phylogenetic relationship of this taxon to other closely related members of the P. boylii species group (P. beatae, P. boylii rowleyi, P. levipes ambiguus, P. l. levipes, and P. simulus). All analyses, despite differential weighting of transitions and nucleotide positions (1st, 2nd, and 3rd) or method of calculating genetic distances, resulted in similar topologies with P. b. sacarensis forming the sister group to P. beatae. Our data suggest that P. b. sacarensis should be aligned taxonomically with P. beatae instead of its current placement in P. boylii and that 3 genetic subunits exist within P. beatae.

Our understanding of the details of mammalian meiotic recombination has recently advanced significantly. Sperm typing technologies, linkage studies, and computational inferences from population genetic data have together provided information in unprecedented detail about the location and activity of the sites of crossing-over in mice and humans. The results show that the vast majority of meiotic recombination events are localized to narrow DNA regions (hot spots) that constitute only a small fraction of the genome. The data also suggest that the molecular basis of hot spot activity is unlikely to be strictly determined by specific DNA sequence motifs in cis. Further molecular studies are needed to understand how hot spots originate, function and evolve.

The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene (FGFR2) is 100–1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 103 to >104 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10−6) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an offspring, thereby unfavorably altering the mutational load in humans. Studying the anatomical distribution of germline mutations can provide new insights into genetic disease and evolutionary change., Author Summary Some human disease mutations occur 100–1,000 times more frequently than would be predicted from genome wide studies of mutation in different species. In Apert syndrome, for example, two-thirds of all new causal mutations occur at only one base in the affected gene. This unusually high frequency suggests that something about that DNA base or its local surroundings makes it highly susceptible to mutation. We studied this hypothesis by examining the location of cells containing this mutation in the testes of normal men. We found that mutant cells were not uniformly distributed throughout the testes, as would be expected for random mutations. Instead, we found 95% of the mutants in small clusters containing only a few percent of the total testes cells. A higher-than-average mutation rate could not explain the data. We propose that these mutations arise at the expected rate, but that mutated cells gain a selective advantage that allows them to increase their frequency compared to nonmutant cells. Our results—which argue against the mutational hot spot model in favor of a selection model—suggest how germline selection in animals can alter the mutational load of a species., The frequency of Apert syndrome mutations is 100-1,000 times higher than expected from average mutation rates, and it is due to positive selection in the testis increasing the frequency of germ cells carrying the defect.

For decades, classical crossover studies and linkage disequilibrium (LD) analysis of genomic regions suggested that human meiotic crossovers may not be randomly distributed along chromosomes but are focused instead in “hot spots.” Recent sperm typing studies provided data at very high resolution and accuracy that defined the physical limits of a number of hot spots. The data were also used to test whether patterns of LD can predict hot spot locations. These sperm typing studies focused on several small regions of the genome already known or suspected of containing a hot spot based on the presence of LD breakdown or previous experimental evidence of hot spot activity. Comparable data on target regions not specifically chosen using these two criteria is lacking but is needed to make an unbiased test of whether LD data alone can accurately predict active hot spots. We used sperm typing to estimate recombination in 17 almost contiguous ~5 kb intervals spanning 103 kb of human Chromosome 21. We found two intervals that contained new hot spots. The comparison of our data with recombination rates predicted by statistical analyses of LD showed that, overall, the two datasets corresponded well, except for one predicted hot spot that showed little crossing over. This study doubles the experimental data on recombination in men at the highest resolution and accuracy and supports the emerging genome-wide picture that recombination is localized in small regions separated by cold areas. Detailed study of one of the new hot spots revealed a sperm donor with a decrease in recombination intensity at the canonical recombination site but an increase in crossover activity nearby. This unique finding suggests that the position and intensity of hot spots may evolve by means of a concerted mechanism that maintains the overall recombination intensity in the region., Synopsis Meiotic crossover events are not randomly distributed across the human genome, but are concentrated in many small regions of a few kb with high recombination rates compared to surrounding regions. How the distribution of recombination events affects the association of different alleles along the chromosome (linkage disequilibrium, or LD) was recently addressed using sperm typing in regions already known or suspected to contain unusually high recombination intensities. In the current paper, the authors used sperm typing to examine recombination in a region not known or suspected of containing recombination hot spots. They first established the crossover distribution pattern within a 103-kb region of human Chromosome 21. Then, they compared their data to predictions of crossover distributions estimated by statistical analyses of polymorphism in the region. They found a good concordance between the two, although it was not perfect. To the authors' knowledge, this work is the first to compare LD-based estimates of recombination to sperm-typing data from regions not previously known or suspected of containing recombination hot spots. In addition, one of the studied hot spots revealed an example of a decrease in recombination intensity with a concurrent increase at a nearby site. This unique observation suggests that the activity of hot spots may evolve in a concerted fashion such that the overall recombination activity of the region is maintained.

Variation in the mitochondrial cytochrome b gene (1143 bp) was examined to estimate the phylogenetic relationships of taxa within the Peromyscus boylii species group. In addition, phylogenetic relationships among the aztecus, boylii, and truei species groups were addressed. Maximum-likelihood, neighbor-joining, and maximum-parsimony (weighted and equally weighted) analyses produced similar topologies with P. boylii, P. beatae, P. simulus, P. stephani, P. madrensis, P. levipes, and three undescribed taxa from western Mexico forming a monophyletic unit. At least two of the undescribed taxa from western Mexico potentially represent species. Members of the P. aztecus species group formed a clade separate from the P. boylii group and should be recognized as a distinct species group. P. sagax, P. polius, and P. pectoralis, formerly placed in the P. boylii species group, generally formed an unresolved polytomy with the P. truei, P. aztecus, and P. boylii species groups. P. attwateri formed a sister taxon relationship with members of the P. truei species group (P. difficilis and P. nasutus) and should be considered a member of this group. Members of the P. truei species group did not form a monophyletic unit, indicating that this species group is not monophyletic and may be composed of two assemblages.