Rita Neumann

Repeat unit sequence variation in minisatellites: A novel source of DNA polymorphism for studying variation and mutation by single molecule analysis

Variation in internal minisatellite structure can be analyzed by mapping variant repeat units within amplified alleles. A system capable of distinguishing greater than 10(70) allelic states at the human hypervariable locus D1S8 has been developed. Population surveys of internal allelic structure indicate that D1S8 alleles evolve rapidly along haploid chromosome lineages. Internal mapping of deletion mutant alleles physically selected from genomic DNA provides further evidence that germline and somatic mutations altering the number of allelic repeat units seldom if ever arise by unequal exchange between alleles. The existence of low level germline mosaicism for new mutants further indicates that many germline mutation events are premeiotic. Physical selection of new mutants also allows minisatellite mutation rates to be estimated directly in human DNA.

Reciprocal crossover asymmetry and meiotic drive in a human recombination hot spot

Human DNA diversity arises ultimately from germline mutation that creates new haplotypes that can be reshuffled by meiotic recombination. Reciprocal crossover generates recombinant haplotypes but should not influence the frequencies of alleles in a population. We demonstrate crossover asymmetry at a recombination hot spot in the major histocompatibility complex, whereby reciprocal exchanges in sperm map to different locations in the hot spot. We identify a single-nucleotide polymorphism at the center of the hot spot and show that, when heterozygous, it seems sufficient to cause this asymmetry, apparently by influencing the efficiency of highly localized crossover initiation. As a consequence, crossovers in heterozygotes are accompanied by biased gene conversion, most likely occurring by gap repair, that can also affect nearby polymorphisms through repair of an extended gap. The result is substantial over-transmission of the recombination-suppressing allele and neighboring markers to crossover products. Computer simulations show that this meiotic drive, although weak at the population level, is sufficient to favor eventual fixation of the recombination-suppressing variant. These findings provide an explanation for the relatively uniform widths of human crossover hot spots and suggest that hot spots may be generally prone to extinction by meiotic drive.

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.

High-resolution mapping of crossovers in human sperm defines a minisatellite-associated recombination hotspot.

Little is known about the fine-scale distribution of meiotic crossovers in human chromosomes. Methods have therefore been developed for detecting and mapping recombination products directly in human sperm DNA. Analysis of crossovers adjacent to the GC-rich minisatellite MS32, which is known to mutate by conversion and crossover within the repeat array, revealed an intense and highly localized recombination hotspot centered upstream of the locus and extending into the beginning of the minisatellite. Allele-specific cosuppression of crossovers and repeat instability suggests that the hotspot is responsible for driving repeat turnover at MS32 and thus that minisatellites might evolve as by-products of localized meiotic recombination in the human genome.

Human recombination hot spots hidden in regions of strong marker association.

The fine-scale distribution of meiotic recombination events in the human genome can be inferred from patterns of haplotype diversity in human populations but directly studied only by high-resolution sperm typing. Both approaches indicate that crossovers are heavily clustered into narrow recombination hot spots. But our direct understanding of hot-spot properties and distributions is largely limited to sperm typing in the major histocompatibility complex (MHC). We now describe the analysis of an unremarkable 206-kb region on human chromosome 1, which identified localized regions of linkage disequilibrium breakdown that mark the locations of sperm crossover hot spots. The distribution, intensity and morphology of these hot spots are markedly similar to those in the MHC. But we also accidentally detected additional hot spots in regions of strong association. Coalescent analysis of genotype data detected most of the hot spots but showed significant differences between sperm crossover frequencies and historical recombination rates. This raises the possibility that some hot spots, particularly those in regions of strong association, may have evolved very recently and not left their full imprint on haplotype diversity. These results suggest that hot spots could be very abundant and possibly fluid features of the human genome.

Minisatellite mutation rate variation associated with a flanking DNA sequence polymorphism.

Human minisatellite mutation in the male germline frequently involves complex inter-allelic gene conversion events restricted to one end of the tandem repeat array. Some alleles at minisatellite MS32 show reduced variability in human populations and are associated with a G to C transversion upstream of the array. Analysis of single sperm demonstrated a frequently profound reduction in mutation rate at alleles carrying the C variant. This mutation suppression acts in cis, but does not affect the ability of an allele to act as sequence donor during gene conversion. This mutation rate polymorphism provides strong evidence for elements near the minisatellite that regulate tandem repeat instability.

Repeat instability at human minisatellites arising from meiotic recombination

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC‐rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion‐like transfer of repeats between alleles. Analysis shows that inter‐allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co‐suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by‐product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm‐spot, even between sequence‐heterologous alleles.

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.

Human minisatellites, repeat DNA instability and meiotic recombination.

Minisatellites include some of the most variable loci in the human genome and are superb for dissecting processes of tandem repeat DNA instability. Single DNA molecule analysis has revealed different mutation processes operating in the soma and germline. Low‐level somatic instability results in simple intra‐allelic rearrangements. In contrast, high frequency germline instability involves complex gene conversions and is therefore recombinational in nature, almost certainly occurring at meiosis. To determine whether true meiotic crossovers occur at human minisatellites, we have used polymorphisms near the repeat array to recover recombinant DNA molecules directly from sperm DNA. Analysis of minisatellite MS32 has revealed an intense and highly localised meiotic crossover hotspot centred upstream of the array, the first example of a human hotspot defined at the molecular level. This hotspot extends into the beginning of the repeat array, resulting in unequal and equal crossovers. Array crossovers occur much less frequently than array conversions but appear to arise by a common process, most likely by alternative processing of a recombination initiation complex. The location of MS32 at the boundary of a recombination hotspot suggests that this locus has evolved as a by‐product of localised meiotic recombination activity, and that minisatellites might in general mark recombinationally proficient hotspots or hot domains in the genome. Finally, sperm crossover analysis makes it possible to explore the molecular rules that govern human meiotic recombination, and to detect phenomena such as meiotic drive that could provide a possible connection between recombination and DNA sequence diversity itself.

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.