Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite.

BACKGROUND: As a population genetic tool, mitochondrial DNA is commonly divided into the ~ 1-kb control region (CR), in which single nucleotide variant (SNV) diversity is relatively high, and the coding region, in which selective constraint is greater and diversity lower, but which provides an informative phylogeny. In some species, the CR contains variable tandemly repeated sequences that are understudied due to heteroplasmy. Domestic cats (Felis catus) have a recent origin and therefore traditional CR-based analysis of populations yields only a small number of haplotypes. RESULTS: To increase resolution we used Nanopore sequencing to analyse 119 cat mitogenomes via a long-amplicon approach. This greatly improves discrimination (from 15 to 87 distinct haplotypes in our dataset) and defines a phylogeny showing similar starlike topologies within all major clades (haplogroups), likely reflecting post-domestication expansion. We sequenced RS2, a CR tandem array of 80-bp repeat units, placing RS2 array structures within the phylogeny and increasing overall haplotype diversity. Repeat number varies between 3 and 12 (median: 4) with over 30 different repeat unit types differing largely by SNVs. Five SNVs show evidence of independent recurrence within the phylogeny, and seven are involved in at least 11 instances of rapid spread along repeat arrays within haplogroups. CONCLUSIONS: In defining mitogenome variation our study provides key information for the forensic genetic analysis of cat hair evidence, and for the first time a phylogenetically informed picture of tandem repeat variation that reveals remarkably dynamic mutation processes at work in the mitochondrion.

Defining cat mitogenome variation and accounting for numts via multiplex amplification and Nanopore sequencing.

Hair shed by domestic cats is a potentially useful source of forensic evidence. Analysable hair DNA is predominantly mitochondrial, but the recent domestication history of cats means that mtDNA diversity is low. A 402-bp control region segment is usually sequenced, defining only a small number of distinct haplotypes in populations. Previously, we used a long-amplicon approach to sequence whole mitogenomes in a sample of blood DNAs from 119 UK cats, greatly increasing observed diversity and reducing random match probabilities. To exploit this variation for forensic analysis, we here describe a multiplex system that amplifies the cat mitogenome in 60 overlapping amplicons of mean length 360 bp, followed by Nanopore sequencing. Variants detected in multiplex sequence data from unrooted hair completely mirror those from long-amplicon data from blood from the same individuals. However, applying the multiplex to matched blood DNA reveals additional sequence variants which derive from the major feline nuclear mitochondrial insertion sequence (numt), which covers 7.9 kb of the 17-kb mitogenome and exists in multiple tandem copies. We use long-amplicon Nanopore sequencing to investigate numt variation in a set of cats, together with an analysis of published genome sequences, and show that numt arrays are variable in both structure and sequence, thus providing a potential source of uncertainty when nuclear DNA predominates in a sample. Forensic application of the multiplex was demonstrated by matching hairs from a cat with skeletal remains from its putative mother, both of which shared a globally common haplotype at the control region. The random match probability in this case with the CR 402-bp segment was 0.21 and this decreased to 0.03 when considering the whole mitogenome. The developed multiplex and sequencing approach, when applied to cat hair where nuclear DNA is scarce, can provide a reliable and highly discriminating source of forensic genetic evidence from a single hair. The confounding effect of numt co-amplification in degraded samples where mixed sequences are observed can be mitigated by variant phasing, and by comparison with numt sequence diversity data, such as those presented here.

Characterization of integrated Marek's disease virus genomes supports a model of integration by homology-directed recombination and telomere-loop-driven excision.

IMPORTANCE: Marek's disease virus (MDV) is a ubiquitous chicken pathogen that inflicts a large economic burden on the poultry industry, despite worldwide vaccination programs. MDV is only partially controlled by available vaccines, and the virus retains the ability to replicate and spread between vaccinated birds. Following an initial infection, MDV enters a latent state and integrates into host telomeres and this may be a prerequisite for malignant transformation, which is usually fatal. To understand the mechanism that underlies the dynamic relationship between integrated-latent and reactivated MDV, we have characterized integrated MDV (iMDV) genomes and their associated telomeres. This revealed a single orientation among iMDV genomes and the loss of some terminal sequences that is consistent with integration by homology-directed recombination and excision via a telomere-loop-mediated process.

Variation in human herpesvirus 6B telomeric integration, excision, and transmission between tissues and individuals.

Human herpesviruses 6A and 6B (HHV-6A/6B) are ubiquitous pathogens that persist lifelong in latent form and can cause severe conditions upon reactivation. They are spread by community-acquired infection of free virus (acqHHV6A/6B) and by germline transmission of inherited chromosomally integrated HHV-6A/6B (iciHHV-6A/6B) in telomeres. We exploited a hypervariable region of the HHV-6B genome to investigate the relationship between acquired and inherited virus and revealed predominantly maternal transmission of acqHHV-6B in families. Remarkably, we demonstrate that some copies of acqHHV-6B in saliva from healthy adults gained a telomere, indicative of integration and latency, and that the frequency of viral genome excision from telomeres in iciHHV-6B carriers is surprisingly high and varies between tissues. In addition, newly formed short telomeres generated by partial viral genome release are frequently lengthened, particularly in telomerase-expressing pluripotent cells. Consequently, iciHHV-6B carriers are mosaic for different iciHHV-6B structures, including circular extra-chromosomal forms that have the potential to reactivate. Finally, we show transmission of an HHV-6B strain from an iciHHV-6B mother to her non-iciHHV-6B son. Altogether, we demonstrate that iciHHV-6B can readily transition between telomere-integrated and free virus forms.

Massively parallel sequencing and capillary electrophoresis of a novel panel of falcon STRs: Concordance with minisatellite DNA profiles from historical wildlife crime.

Birds of prey have suffered persecution for centuries through trapping, shooting, poisoning and theft from the wild to meet the demand from egg collectors and falconers; they were also amongst the earliest beneficiaries of DNA testing in wildlife forensics. Here we report the identification and characterisation of 14 novel tetramer, pentamer and hexamer short tandem repeat (STR) markers which can be typed either by capillary electrophoresis or massively parallel sequencing (MPS) and apply them to historical casework samples involving 49 peregrine falcons, 30 of which were claimed to be the captively bred offspring of nine pairs. The birds were initially tested in 1994 with a multilocus DNA fingerprinting probe, a sex test and eight single-locus minisatellite probes (SLPs) demonstrating that 23 birds were unrelated to the claimed parents. The multilocus and SLP approaches were highly discriminating but extremely time consuming and required microgram quantities of high molecular weight DNA and the use of radioisotopes. The STR markers displayed between 2 and 21 alleles per locus (mean = 7.6), lengths between 140 and 360 bp, and heterozygosities from 0.4 to 0.93. They produced wholly concordant conclusions with similar discrimination power but in a fraction of the time using a hundred-fold less DNA and with standard forensic equipment. Furthermore, eleven of these STRs were amplified in a single reaction and typed using MPS on the Illumina MiSeq platform revealing eight additional alleles (three with variant repeat structures and five solely due to flanking SNPs) across four loci. This approach gave a random match probability of < 1E-9, and a parental pair false inclusion probability of < 1E-5, with a further ten-fold reduction in the amount of DNA required (~3 ng) and the potential to analyse mixed samples. These STRs will be of value in monitoring wild populations of these key indicator species as well as for testing captive breeding claims and establishing a database of captive raptors. They have the potential to resolve complex cases involving trace, mixed and degraded samples from raptor persecution casework representing a significant advance over the previously applied methods.

Geographical and linguistic structure in the people of Kenya demonstrated using 21 autosomal STRs.

Kenya is a diverse and populous nation that employs DNA evidence in its criminal justice system, and therefore requires reliable information on autosomal STR allele frequency variation across the country and in its many ethnic groups. In order to provide reference data and to assess population structure, we analysed the 21 autosomal STRs in the GlobalFiler multiplex in a sample of 510 indigenous Kenyans representing the country's eight former provinces, 43 of its 47 counties, three main linguistic families and all 29 ethnic groups that each comprise >0.5% of the 2019 census population. The indigenous population originated from successive migrations of Cushitic, Nilotic and Bantu speaking groups who settled in regions that suited their distinctive sustenance lifestyles. Consequently, they now largely reside in a patchwork of communities with strong associations with particular counties and provinces and limited degrees of inter-group marriage, as shown by DNA donors' ancestry details. We found significant genetic differentiation between the three Nilotic language sub-families, with Western Nilotes (the Luo ethnic group) showing greater similarity to the Bantu than the Southern and Eastern Nilotes which themselves showed closer affinity to the Cushitic speakers. This concurs with previous genetic, linguistic and social studies. Comparisons with other African populations also showed that linguistic affiliation is a stronger factor than geography. This study revealed several rare off-ladder alleles whose structure was determined by Sanger sequencing. Among the unusual features that could affect profile interpretation were a deletion of Amelogenin Y but no other forensic marker (autosomal or Y-chromosomal), a triallelic pattern at TPOX and an extremely short SE33 allele falling within the expected size range of D7S820. Compared with the currently implemented Identifiler multiplex, Random Match Probabilities decreased from 6.4 × 10-19 to 3.9 × 10-27. The appreciation of local population structure provided by the geographically and ethnically representative sample in this study highlights the structured genetic landscape of Kenya.

The Circulating Nucleic Acid Characteristics of Non-Metastatic Soft Tissue Sarcoma Patients.

Soft tissue sarcomas (STS) are rare, malignant tumours with a generally poor prognosis. Our aim was to explore the potential of cell free DNA (cfDNA) and circulating tumour DNA (ctDNA) analysis to track non-metastatic STS patients undergoing attempted curative treatment. The analysed cohort (n = 29) contained multiple STS subtypes including myxofibrosarcomas, undifferentiated pleomorphic sarcomas, leiomyosarcomas, and dedifferentiated liposarcomas amongst others. Perioperative cfDNA levels trended towards being elevated in patients (p = 0.07), although did not correlate with tumour size, grade, recurrence or subtype, suggesting a limited diagnostic or prognostic role. To characterise ctDNA, an amplicon panel covering three genes commonly mutated in STSs was first trialled on serial plasma collected from nine patients throughout follow-up. This approach only identified ctDNA in 2.5% (one in 40) of the analysed samples. Next custom-designed droplet digital PCR assays and Ion AmpliSeq™ panels were developed to track single nucleotide variants identified in patients' STSs by whole exome sequencing (1-6 per patient). These approaches identified ctDNA in 17% of patients. Although ctDNA was identified before radiologically detectable recurrence in two cases, the absence of demonstrable ctDNA in 83% of cases highlights the need for much work before circulating nucleic acids can become a useful means to track STS patients.

Recombination hotspots in an extended human pseudoautosomal domain predicted from double-strand break maps and characterized by sperm-based crossover analysis.

The human X and Y chromosomes are heteromorphic but share a region of homology at the tips of their short arms, pseudoautosomal region 1 (PAR1), that supports obligate crossover in male meiosis. Although the boundary between pseudoautosomal and sex-specific DNA has traditionally been regarded as conserved among primates, it was recently discovered that the boundary position varies among human males, due to a translocation of ~110 kb from the X to the Y chromosome that creates an extended PAR1 (ePAR). This event has occurred at least twice in human evolution. So far, only limited evidence has been presented to suggest this extension is recombinationally active. Here, we sought direct proof by examining thousands of gametes from each of two ePAR-carrying men, for two subregions chosen on the basis of previously published male X-chromosomal meiotic double-strand break (DSB) maps. Crossover activity comparable to that seen at autosomal hotspots was observed between the X and the ePAR borne on the Y chromosome both at a distal and a proximal site within the 110-kb extension. Other hallmarks of classic recombination hotspots included evidence of transmission distortion and GC-biased gene conversion. We observed good correspondence between the male DSB clusters and historical recombination activity of this region in the X chromosomes of females, as ascertained from linkage disequilibrium analysis; this suggests that this region is similarly primed for crossover in both male and female germlines, although sex-specific differences may also exist. Extensive resequencing and inference of ePAR haplotypes, placed in the framework of the Y phylogeny as ascertained by both Y microsatellites and single nucleotide polymorphisms, allowed us to estimate a minimum rate of crossover over the entire ePAR region of 6-fold greater than genome average, comparable with pedigree estimates of PAR1 activity generally. We conclude ePAR very likely contributes to the critical crossover function of PAR1.

Circulating tumour-derived DNA in metastatic soft tissue sarcoma.

Following treatment 40% of soft tissue sarcoma (STS) patients suffer disease recurrence. In certain cancers circulating cell free DNA (cfDNA) and circulating tumour-derived DNA (ctDNA) characteristics correlate closely with disease burden, making them exciting potential sources of biomarkers. Despite this, the circulating nucleic acid characteristics of only 2 STS patients have been reported to date. To address this we used an Ion AmpliSeq™ panel custom specifically designed for STS patients to conduct a genetic characterisation of plasma cfDNA, buffy coat (germline) DNA and where available Formalin-Fixed Paraffin-Embedded (FFPE) primary STS tissue DNA in a cohort of 11 metastatic STS patients. We found that total cfDNA levels were significantly elevated in the STS patients analysed, and weakly correlated with disease burden. Using our Ion AmpliSeq™ panel we also successfully detected ctDNA in 4/11 (36%) patients analysed with a wide variety of STS subtypes and disease burdens. This evidence included the presence of cancer associated TP53 / PIK3CA mutations in 2 patients' plasma and matched primary STS tumour tissue, and in the plasma alone for 2 patients. We also identified 2 potential examples of allelic loss of heterozygosity in an additional patient's STS DNA and cfDNA. This is the largest study performed characterising STS patient cfDNA/ctDNA and confirms that the field remains an attractive potential source of novel STS biomarkers. Further work is required to investigate the circulating nucleic acid characteristics of individual STS subtypes, and the potential prognostic or therapeutic roles that cfDNA/ctDNA may hold for patients with these complex tumours.

Inherited Chromosomally Integrated Human Herpesvirus 6 Genomes Are Ancient, Intact, and Potentially Able To Reactivate from Telomeres.

The genomes of human herpesvirus 6A (HHV-6A) and HHV-6B have the capacity to integrate into telomeres, the essential capping structures of chromosomes that play roles in cancer and ageing. About 1% of people worldwide are carriers of chromosomally integrated HHV-6 (ciHHV-6), which is inherited as a genetic trait. Understanding the consequences of integration for the evolution of the viral genome, for the telomere, and for the risk of disease associated with carrier status is hampered by a lack of knowledge about ciHHV-6 genomes. Here, we report an analysis of 28 ciHHV-6 genomes and show that they are significantly divergent from the few modern nonintegrated HHV-6 strains for which complete sequences are currently available. In addition, ciHHV-6B genomes in Europeans are more closely related to each other than to ciHHV-6B genomes from China and Pakistan, suggesting regional variation of the trait. Remarkably, at least one group of European ciHHV-6B carriers has inherited the same ciHHV-6B genome, integrated in the same telomere allele, from a common ancestor estimated to have existed 24,500 ± 10,600 years ago. Despite the antiquity of some, and possibly most, germ line HHV-6 integrations, the majority of ciHHV-6B (95%) and ciHHV-6A (72%) genomes contain a full set of intact viral genes and therefore appear to have the capacity for viral gene expression and full reactivation.IMPORTANCE Inheritance of HHV-6A or HHV-6B integrated into a telomere occurs at a low frequency in most populations studied to date, but its characteristics are poorly understood. However, stratification of ciHHV-6 carriers in modern populations due to common ancestry is an important consideration for genome-wide association studies that aim to identify disease risks for these people. Here, we present full sequence analysis of 28 ciHHV-6 genomes and show that ciHHV-6B in many carriers with European ancestry most likely originated from ancient integration events in a small number of ancestors. We propose that ancient ancestral origins for ciHHV-6A and ciHHV-6B are also likely in other populations. Moreover, despite their antiquity, all of the ciHHV-6 genomes appear to retain the capacity to express viral genes, and most are predicted to be capable of full viral reactivation. These discoveries represent potentially important considerations in immunocompromised patients, in particular in organ transplantation and in stem cell therapy.

Signatures of human European Palaeolithic expansion shown by resequencing of non-recombining X-chromosome segments.

Human genetic diversity in Europe has been extensively studied using uniparentally inherited sequences (mitochondrial DNA (mtDNA) and the Y chromosome), which reveal very different patterns indicating sex-specific demographic histories. The X chromosome, haploid in males and inherited twice as often from mothers as from fathers, could provide insights into past female behaviours, but has not been extensively investigated. Here, we use HapMap single-nucleotide polymorphism data to identify genome-wide segments of the X chromosome in which recombination is historically absent and mutations are likely to be the only source of genetic variation, referring to these as phylogeographically informative haplotypes on autosomes and X chromosome (PHAXs). Three such sequences on the X chromosome spanning a total of ~49 kb were resequenced in 240 males from Europe, the Middle East and Africa at an average coverage of 181 ×. These PHAXs were confirmed to be essentially non-recombining across European samples. All three loci show highly homogeneous patterns across Europe and are highly differentiated from the African sample. Star-like structures of European-specific haplotypes in median-joining networks indicate past population expansions. Bayesian skyline plots and time-to-most-recent-common-ancestor estimates suggest expansions pre-dating the Neolithic transition, a finding that is more compatible with data on mtDNA than the Y chromosome, and with the female bias of X-chromosomal inheritance. This study demonstrates the potential of the use of X-chromosomal haplotype blocks, and the utility of the accurate ascertainment of rare variants for inferring human demographic history.

Identification of the remains of King Richard III.

In 2012, a skeleton was excavated at the presumed site of the Grey Friars friary in Leicester, the last-known resting place of King Richard III. Archaeological, osteological and radiocarbon dating data were consistent with these being his remains. Here we report DNA analyses of both the skeletal remains and living relatives of Richard III. We find a perfect mitochondrial DNA match between the sequence obtained from the remains and one living relative, and a single-base substitution when compared with a second relative. Y-chromosome haplotypes from male-line relatives and the remains do not match, which could be attributed to a false-paternity event occurring in any of the intervening generations. DNA-predicted hair and eye colour are consistent with Richard's appearance in an early portrait. We calculate likelihood ratios for the non-genetic and genetic data separately, and combined, and conclude that the evidence for the remains being those of Richard III is overwhelming.

Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable, facilitating release of the viral genome from the chromosome.

Linear chromosomes are stabilized by telomeres, but the presence of short dysfunctional telomeres triggers cellular senescence in human somatic tissues, thus contributing to ageing. Approximately 1% of the population inherits a chromosomally integrated copy of human herpesvirus 6 (CI-HHV-6), but the consequences of integration for the virus and for the telomere with the insertion are unknown. Here we show that the telomere on the distal end of the integrated virus is frequently the shortest measured in somatic cells but not the germline. The telomere carrying the CI-HHV-6 is also prone to truncations that result in the formation of a short telomere at a novel location within the viral genome. We detected extra-chromosomal circular HHV-6 molecules, some surprisingly comprising the entire viral genome with a single fully reconstituted direct repeat region (DR) with both terminal cleavage and packaging elements (PAC1 and PAC2). Truncated CI-HHV-6 and extra-chromosomal circular molecules are likely reciprocal products that arise through excision of a telomere-loop (t-loop) formed within the CI-HHV-6 genome. In summary, we show that the CI-HHV-6 genome disrupts stability of the associated telomere and this facilitates the release of viral sequences as circular molecules, some of which have the potential to become fully functioning viruses.

Recombination regulator PRDM9 influences the instability of its own coding sequence in humans.

PRDM9 plays a key role in specifying meiotic recombination hotspot locations in humans and mice via recognition of hotspot sequence motifs by a variable tandem-repeat zinc finger domain in the protein. We now explore germ-line instability of this domain in humans. We show that repeat turnover is driven by mitotic and meiotic mutation pathways, the latter frequently resulting in substantial remodeling of zinc fingers. Turnover dynamics predict frequent allele switches in populations with correspondingly fast changes of the recombination landscape, fully consistent with the known rapid evolution of hotspot locations. We found variation in meiotic instability between men that correlated with PRDM9 status. One particular "destabilizer" variant caused hyperinstability not only of itself but also of otherwise-stable alleles in heterozygotes. PRDM9 protein thus appears to regulate the instability of its own coding sequence. However, destabilizer variants are strongly self-limiting in populations and probably have little impact on the evolution of the recombination landscape.

A major recombination hotspot in the XqYq pseudoautosomal region gives new insight into processing of human gene conversion events.

Recombination plays a fundamental role in meiosis. Non-exchange gene conversion (non-crossover, NCO) may facilitate homologue pairing, while reciprocal crossover (CO) physically connects homologues so they orientate appropriately on the meiotic spindle. In males, X-Y homologous pairing and exchange occurs within the two pseudoautosomal regions (PARs) together comprising <5% of the human sex chromosomes. Successful meiosis depends on an obligatory CO within PAR1, while the nature and role of exchange within PAR2 is unclear. Here, we describe the identification and characterization of a typical ~1 kb wide recombination hotspot within PAR2. We find that both COs and NCOs are strongly modulated in trans by the presumed chromatin remodelling protein PRDM9, and in cis by a single nucleotide polymorphism (SNP) located at the hotspot centre that appears to influence recombination initiation and which causes biased gene conversion in SNP heterozygotes. This, the largest survey to date of human NCOs reveals for the first time substantial inter-individual variation in the NCO:CO ratio. Although the extent of biased transmission at the central marker in COs is similar across men, it is highly variable among NCO recombinants. This suggests that cis-effects are mediated not only through recombination initiation frequencies varying between haplotypes but also through subsequent processing, with the potential to significantly intensify meiotic drive of hotspot-suppressing alleles. The NCO:CO ratio and extent of transmission distortion among NCOs appear to be inter-related, suggesting the existence of two NCO pathways in humans.

Variants of the protein PRDM9 differentially regulate a set of human meiotic recombination hotspots highly active in African populations.

PRDM9 is a major specifier of human meiotic recombination hotspots, probably via binding of its zinc-finger repeat array to a DNA sequence motif associated with hotspots. However, our view of PRDM9 regulation, in terms of motifs defined and hotspots studied, has a strong bias toward the PRDM9 A variant particularly common in Europeans. We show that population diversity can reveal a second class of hotspots specifically activated by PRDM9 variants common in Africans but rare in Europeans. These African-enhanced hotspots nevertheless share very similar properties with their counterparts activated by the A variant. The specificity of hotspot activation is such that individuals with differing PRDM9 genotypes, even within the same population, can use substantially if not completely different sets of hotspots. Each African-enhanced hotspot is activated by a distinct spectrum of PRDM9 variants, despite the fact that all are predicted to bind the same sequence motif. This differential activation points to complex interactions between the zinc-finger array and hotspots and identifies features of the array that might be important in controlling hotspot activity.

PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans.

PRDM9 has recently been identified as a likely trans regulator of meiotic recombination hot spots in humans and mice. PRDM9 contains a zinc finger array that, in humans, can recognize a short sequence motif associated with hot spots, with binding to this motif possibly triggering hot-spot activity via chromatin remodeling. We now report that human genetic variation at the PRDM9 locus has a strong effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Subtle changes within the zinc finger array can create hot-spot nonactivating or enhancing variants and can even trigger the appearance of a new hot spot, suggesting that PRDM9 is a major global regulator of hot spots in humans. Variation at the PRDM9 locus also influences aspects of genome instability-specifically, a megabase-scale rearrangement underlying two genomic disorders as well as minisatellite instability-implicating PRDM9 as a risk factor for some pathological genome rearrangements.

Dynamics and processes of copy number instability in human gamma-globin genes.

Copy number variation in the human genome is prevalent but relatively little is known about the dynamics of DNA rearrangement. We therefore used the duplicated gamma-globin genes as a simple system to explore de novo copy number changes. Rearrangements that changed gene number were seen in both germline and somatic DNA, and mainly arose by unequal sister chromatid exchange between homologous sequences, with evidence from recurrent mosaic rearrangements that many, if not all, of these events in sperm arise before meiosis. Unequal exchange frequencies are apparently controlled primarily by the degree of sequence identity shared by the duplicate genes, leading to substantial variation between haplotypes in copy number instability. Additional, more complex rearrangements generated by mechanisms not involving homologous recombination, and in some cases showing DNA transfer between chromosomes, were also detected but were rare. Sequence changes were also seen in gamma-globin DNA molecules, with strong evidence that some were genuine de novo base substitutions. They were present in sperm at a frequency far higher than predicted from current estimates of germline mutation rates, raising interesting questions about base mutation dynamics in the male germline.

The rise and fall of a human recombination hot spot.

Human meiotic crossovers mainly cluster into narrow hot spots that profoundly influence patterns of haplotype diversity and that may also affect genome instability and sequence evolution. Hot spots also seem to be ephemeral, but processes of hot-spot activation and their subsequent evolutionary dynamics remain unknown. We now analyze the life cycle of a recombination hot spot. Sperm typing revealed a polymorphic hot spot that was activated in cis by a single base change, providing evidence for a primary sequence determinant necessary, though not sufficient, to activate recombination. This activating mutation occurred roughly 70,000 y ago and has persisted to the present, most likely fortuitously through genetic drift despite its systematic elimination by biased gene conversion. Nonetheless, this self-destructive conversion will eventually lead to hot-spot extinction. These findings define a subclass of highly transient hot spots and highlight the importance of understanding hot-spot turnover and how it influences haplotype diversity.

Polymorphism in the activity of human crossover hotspots independent of local DNA sequence variation.

Meiotic crossovers in the human genome cluster into highly localized hotspots identifiable indirectly from patterns of DNA diversity and directly by high-resolution sperm typing. Little is known about factors that control hotspot activity and the apparently rapid turnover of hotspots during recent evolution. Clues can, however, be gained by characterizing variation in sperm crossover activity between men. Previous studies have identified single nucleotide polymorphisms within hotspots that appear to suppress crossover activity and which may be involved in hotspot attenuation/extinction. We now analyse a closely spaced pair of hotspots (MSTM1a, MSTM1b) on chromosome 1q42.3, the former being a candidate for a young hotspot that has failed to leave a significant mark on haplotype diversity. Extensive surveys of different men revealed substantial polymorphism in sperm crossover frequencies at both hotspots, but with very different patterns of variation. Hotspot MSTM1b was active in all men tested but with widely differing crossover frequencies. In contrast, MSTM1a was active in only a few men and appeared to be recombinationally inert in the remainder, providing the first example of presence/absence polymorphism of a human hotspot. Haplotype analysis around both hotspots identified active and suppressed men sharing identical haplotypes, establishing that these major variations in the presence/absence of a hotspot and in quantitative activity are not caused by local DNA sequence variation. These findings suggest a role for distal regulators or epigenetic factors in hotspot activity and provide the first direct evidence for the rapid evolution of recombination hotspots in humans.