Transmission distortion affecting human noncrossover but not crossover recombination: a hidden source of meiotic drive.

Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.

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.

The man behind the DNA fingerprints: an interview with Professor Sir Alec Jeffreys.

In this interview we talk with Professor Sir Alec Jeffreys about DNA fingerprinting, his wider scientific career, and the past, present and future of forensic DNA applications.The podcast with excerpts from this interview is available at: http://www.biomedcentral.com/biome/alec-jeffreys.

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.

L1 hybridization enrichment: a method for directly accessing de novo L1 insertions in the human germline.

Long interspersed nuclear element 1 (L1) retrotransposons are the only autonomously mobile human transposable elements. L1 retrotransposition has shaped our genome via insertional mutagenesis, sequence transduction, pseudogene formation, and ectopic recombination. However, L1 germline retrotransposition dynamics are poorly understood because de novo insertions occur very rarely: the frequency of disease-causing retrotransposon insertions suggests that one insertion event occurs in roughly 18-180 gametes. The method described here recovers full-length L1 insertions by using hybridization enrichment to capture L1 sequences from multiplex PCR-amplified DNA. Enrichment is achieved by hybridizing L1-specific biotinylated oligonucleotides to complementary molecules, followed by capture on streptavidin-coated paramagnetic beads. We show that multiplex, long-range PCR can amplify single molecules containing full-length L1 insertions for recovery by hybridization enrichment. We screened 600 µg of sperm DNA from one donor, but no bone fide de novo L1 insertions were found, suggesting a L1 retrotransposition frequency of <1 insertion in 400 haploid genomes. This lies below the lower bound of previous estimates, and indicates that L1 insertion, at least into the loci studied, is very rare in the male germline. It is a paradox that L1 replication is ongoing in the face of such apparently low 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.

Analysis of meiotic recombination products from human sperm.

Traditional methods for surveying meiotic recombination in humans are limited to pedigree and linkage disequilibrium analyses. We have developed assays that allow the direct detection of crossover and gene conversion molecules in batches of sperm DNA. To date, we have characterized 26 recombination hotspots by allele-specific PCR and selectively amplified recombinant DNA molecules from these regions. These analyses have revealed that meiotic crossover hotspots in humans are highly localized and flanked by DNA segments where recombination is suppressed. The centers of crossover hotspots are also active in noncrossover recombination, displaying short conversion tracts.

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.

Minisatellite MS32 alleles show population specificity among Thai, Chinese, and Japanese.

Lineages of structurally related alleles at minisatellite MS32 in human populations show considerable differentiation at the continental level. However, the regional specificity of these lineages remains unknown. We now describe the comparison of allele structures in Thai, Han Chinese, and Japanese populations with lineages previously established for North Europeans and Africans. The great majority of alignable Asian alleles showed their closest structural relative in Asia, with few instances of preferential alignment of Asian with European alleles and only one isolated incident showing a best match with an African allele. Further, there was a strong tendency, most marked for Japanese, for Asian alleles to align preferentially with other alleles from the same population, indicating strong regional specificity of allele lineages. This rapidly evolving minisatellite can therefore serve as a lineage marker for exploring recent events in human population history and dissecting population structure at the fine-scale level, as well as being an extremely informative DNA marker for personal identification.

Processes of de novo duplication of human alpha-globin genes.

Ectopic recombination between repeated but nonallelic DNA sequences plays a major role in genome evolution, creating gene families and generating copy number variation and pathological rearrangements in human chromosomes. Previous studies on the alpha2- and alpha1-globin genes have shown that de novo deletions common in alpha(+)-thalassemics can be directly accessed in human DNA and provide an informative system for studying deletion dynamics and processes. However, nothing is known about the reciprocal products of ectopic recombination, namely gene duplications. We now show that molecules carrying three alpha-globin genes can be detected in human DNA by using physical enrichment plus an inverse PCR strategy. These de novo duplications are common in blood and sperm and appear to arise by two distinct mechanisms: meiotic exchanges between homologous chromosomes that generate a minority of sperm duplications, plus mitotic ectopic exchanges that occur in the soma and germ line and can show erratic fluctuations in frequency most likely caused by mutational mosaicism. The dynamics and processes of duplication are very similar to those of deletion, particularly for meiotic exchanges. This result suggests rearrangement pathways dominated by fully reciprocal ectopic exchange, with nonreciprocal pathways such as intramolecular recombination and single-strand annealing playing at best only a minor role in the generation of deletions. Finally, the high level of instability at the alpha-globin locus contrasts with the rarity in most populations of chromosomes carrying duplications or deletions, pointing to strong selective constraints that maintain alpha-globin gene copy number in human populations.

Species-wide distribution of highly polymorphic minisatellite markers suggests past and present genetic exchanges among house mouse subspecies.

BACKGROUND: Four hypervariable minisatellite loci were scored on a panel of 116 individuals of various geographical origins representing a large part of the diversity present in house mouse subspecies. Internal structures of alleles were determined by minisatellite variant repeat mapping PCR to produce maps of intermingled patterns of variant repeats along the repeat array. To reconstruct the genealogy of these arrays of variable length, the specifically designed software MS_Align was used to estimate molecular divergences, graphically represented as neighbor-joining trees. RESULTS: Given the high haplotypic diversity detected (mean He = 0.962), these minisatellite trees proved to be highly informative for tracing past and present genetic exchanges. Examples of identical or nearly identical alleles were found across subspecies and in geographically very distant locations, together with poor lineage sorting among subspecies except for the X-chromosome locus MMS30 in Mus mus musculus. Given the high mutation rate of mouse minisatellite loci, this picture cannot be interpreted only with simple splitting events followed by retention of polymorphism, but implies recurrent gene flow between already differentiated entities. CONCLUSION: This strongly suggests that, at least for the chromosomal regions under scrutiny, wild house mouse subspecies constitute a set of interrelated gene pools still connected through long range gene flow or genetic exchanges occurring in the various contact zones existing nowadays or that have existed in the past. Identifying genomic regions that do not follow this pattern will be a challenging task for pinpointing genes important for speciation.

Processes of copy-number change in human DNA: the dynamics of {alpha}-globin gene deletion.

Ectopic recombination between locally repeated DNA sequences is of fundamental importance in the evolution of gene families, generating copy-number variation in human DNA and often leading to pathological rearrangements. Despite its importance, little is known about the dynamics and processes of these unequal crossovers and the degree to which meiotic recombination plays a role in instability. We address this issue by using as a highly informative system the duplicated alpha-globin genes in which ectopic recombination can lead to gene deletions, often very prevalent in populations affected by malaria, as well as reduplications. Here we show that spontaneous deletions can be accessed directly in genomic DNA by using single-DNA-molecule methods. These deletions proved to be remarkably common in both blood and sperm. Somatic deletions arise by a strictly intrachromosomal pathway of homologous exchange that also operates in the germ line and can generate mutational mosaicism, whereas sperm deletions frequently involve recombinational interactions between homologous chromosomes that most likely occur at meiosis. Ectopic recombination frequencies show surprisingly little requirement for long, identical homology blocks shared by paralogous sequences, and exchanges can occur even between short regions of sequence identity. Finally, direct knowledge of germ-line deletion rates can give insights into the fitness of individuals with these alpha-globin gene deletions, providing a new approach to investigating historical levels of selection operating in human populations.

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.

Allelic recombination and de novo deletions in sperm in the human beta-globin gene region.

Meiotic recombination is of fundamental importance in creating haplotype diversity in the human genome and has the potential to cause genomic rearrangements by ectopic recombination between repeat sequences and through other changes triggered by recombination-initiating events. However, the relationship between allelic recombination and genome instability in the human germline remains unclear. We have therefore analysed recombination and DNA instability in the delta-, beta-globin gene region and its associated recombination hotspot. Sperm typing has for the first time accurately defined the hotspot and shown it to be the most active autosomal crossover hotspot yet described, although unusually inactive in non-exchange gene conversion. The hotspot just extends into a homology block shared by the delta- and beta-globin genes, within which ectopic exchanges can generate Hb Lepore deletions. We developed a physical selection method for recovering and validating extremely rare de novo deletions in human DNA and used it to characterize the dynamics of these Hb Lepore deletions in sperm as well as other deletions not arising from ectopic exchanges between homologous DNA sequences. Surprisingly, both classes of deletion showed breakpoints that avoided the beta-globin hotspot, establishing that it possesses remarkable fidelity and does not play a significant role in triggering these DNA rearrangements. This study also provides the first direct analysis of de novo deletion in the human germline and points to a possible deletion-controlling element in the beta-globin gene separate from the crossover hotspot.

Seventh international meeting on single nucleotide polymorphism and complex genome analysis: 'ever bigger scans and an increasingly variable genome'.

In September 2005, the seventh international meeting on single nucleotide polymorphism (SNP) and complex genome analysis was held in Hinckley, near Leicester, UK and the meeting was organised by Anthony Brookes, Stephen Chanock, Ivo Gut, Alec Jeffreys and Pui-Yan Kwok. Similar to prior meetings, the 3-day meeting focused on new trends and methods in the analysis of SNPs and complex human disease. A substantial portion of the meeting was devoted to preliminary analyses of data emerging from the International HapMap Consortium and addressed key issues in patterns of recombination, linkage disequilibrium and population genetics. Of great interest were the sessions that addressed SNP analysis in other species and the emerging field of copy number variation. Overall, there have been a number of recent advances in genomics that promise to accelerate the pace of dissecting the genetic basis of many complex diseases in humans-and perhaps in other species.

Factors influencing recombination frequency and distribution in a human meiotic crossover hotspot.

Little is known about the factors that influence the frequency and distribution of meiotic recombination events within human crossover hotspots. We now describe the detailed analysis of sperm recombination in the NID1 hotspot. Like the neighbouring MS32 hotspot, the NID1 hotspot is associated with a minisatellite, suggesting that hotspots predispose DNA to tandem repetition. Unlike MS32, crossover resolution breakpoints in NID1 avoid the minisatellite, producing a cold spot within the hotspot. This avoidance may be related to the palindromic nature of the minisatellite interfering with the generation and/or processing of recombination intermediates. The NID1 hotspot also contains a single nucleotide polymorphism (SNP) close to the centre, which appears to directly influence the frequency of crossover initiation. Quantitative gene conversion assays show that this SNP affects the frequency of gene conversion and crossover to a very similar extent, providing evidence that conversions and crossovers are triggered by the same recombination initiating events. The recombination-suppressing allele is over-transmitted to recombinant progeny, and provides the most dramatic example to date of recombination-mediated meiotic drive, of a magnitude sufficient to virtually guarantee that the recombination suppressor will eventually replace the more active allele in human populations.