Evaluation of PRDM9 variation as a risk factor for recurrent genomic disorders and chromosomal non-disjunction

Recent studies have identified PRDM9, a zinc finger (ZF) protein, as a key regulator of meiotic recombination. As both recurrent genomic disorders and chromosomal non-disjunction are known to be associated with specific unusual patterns of recombination, we hypothesized a possible link between PRDM9 ZF variation and susceptibility to microdeletion syndromes and/or trisomy. We sequenced the PRDM9 ZF domain in 271 parents of patients with de novo microdeletions of known parental origin (velocardiofacial syndrome, the 17q21.31 microdeletion syndrome, Prader-Willi/Angelman syndrome and Williams-Beuren syndrome), and in 61 parents of individuals with a supernumerary X chromosome. We compared PRDM9 ZF genotype frequencies between parents in whose germ line the de novo rearrangement occurred and their spouses. We observed a significantly increased frequency (p?=?0.006) of PRDM9 variants in parents who transmitted de novo 7q11.23 deletions to their offspring. These data suggest that certain PRDM9 alleles may be associated with an increased susceptibility to recurrent 7q11.23 microdeletions that cause Williams-Beuren syndrome. However, as the majority of parents who transmitted a de novo microdeletion/supernumerary X chromosome to their offspring have the common AA genotype, we conclude that none of the rearrangements we have studied are dependent on specific non-A PRDM9 alleles.     

What are the genomic drivers of the rapid evolution of PRDM9?

Mammalian Prdm9 has been proposed to be a key determinant of the positioning of chromosome double-strand breaks during meiosis, a contributor to speciation processes, and the most rapidly evolving gene in human, and other animal, genomes. Prdm9 genes often exhibit substantial variation in their numbers of encoded zinc fingers (ZFs), not only between closely related species but also among individuals of a species. The near-identity of these ZF sequences appears to render them very unstable in copy number. The rare sequence differences, however, cluster within ZF sites that determine the DNA-binding specificity of PRDM9, and these substitutions are frequently positively selected. Here, possible drivers of the rapid evolution of Prdm9 are discussed, including selection for efficient pairing of homologous chromosomes or for recombination of deleterious linked alleles, and selection against depletion of recombination hotspots or against disease-associated genome rearrangement.     

High diversity at PRDM9 in chimpanzees and bonobos

Background

The PRDM9 locus in mammals has increasingly attracted research attention due to its role in mediating chromosomal recombination and possible involvement in hybrid sterility and hence speciation processes. The aim of this study was to characterize sequence variation at the PRDM9 locus in a sample of our closest living relatives, the chimpanzees and bonobos.

Methodology/Principal Findings

PRDM9 contains a highly variable and repetitive zinc finger array. We amplified this domain using long-range PCR and determined the DNA sequences using conventional Sanger sequencing. From 17 chimpanzees representing three subspecies and five bonobos we obtained a total of 12 alleles differing at the nucleotide level. Based on a data set consisting of our data and recently published Pan PRDM9 sequences, we found that at the subspecies level, diversity levels did not differ among chimpanzee subspecies or between chimpanzee subspecies and bonobos. In contrast, the sample of chimpanzees harbors significantly more diversity at PRDM9 than samples of humans. Pan PRDM9 shows signs of rapid evolution including no alleles or ZnFs in common with humans as well as signals of positive selection in the residues responsible for DNA binding.

Conclusions and Significance

The high number of alleles specific to the genus Pan, signs of positive selection in the DNA binding residues, and reported lack of conservation of recombination hotspots between chimpanzees and humans suggest that PRDM9 could be active in hotspot recruitment in the genus Pan. Chimpanzees and bonobos are considered separate species and do not have overlapping ranges in the wild, making the presence of shared alleles at the amino acid level between the chimpanzee and bonobo species interesting in view of the hypothesis that PRDM9 plays a universal role in interspecific hybrid sterility.     

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9 ^+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.     

Characterization of Prdm9 in Equids and Sterility in Mules

Prdm9 (Meisetz) is the first speciation gene discovered in vertebrates conferring reproductive isolation. This locus encodes a meiosis-specific histone H3 methyltransferase that specifies meiotic recombination hotspots during gametogenesis. Allelic differences in Prdm9, characterized for a variable number of zinc finger (ZF) domains, have been associated with hybrid sterility in male house mice via spermatogenic failure at the pachytene stage. The mule, a classic example of hybrid sterility in mammals also exhibits a similar spermatogenesis breakdown, making Prdm9 an interesting candidate to evaluate in equine hybrids. In this study, we characterized the Prdm9 gene in all species of equids by analyzing sequence variation of the ZF domains and estimating positive selection. We also evaluated the role of Prdm9 in hybrid sterility by assessing allelic differences of ZF domains in equine hybrids. We found remarkable variation in the sequence and number of ZF domains among equid species, ranging from five domains in the Tibetan kiang and Asiatic wild ass, to 14 in the Grevy’s zebra. Positive selection was detected in all species at amino acid sites known to be associated with DNA-binding specificity of ZF domains in mice and humans. Equine hybrids, in particular a quartet pedigree composed of a fertile mule showed a mosaic of sequences and number of ZF domains suggesting that Prdm9 variation does not seem by itself to contribute to equine hybrid sterility.     

Diversity of Prdm9 Zinc Finger Array in Wild Mice Unravels New Facets of the Evolutionary Turnover of this Coding Minisatellite

In humans and mice, meiotic recombination events cluster into narrow hotspots whose genomic positions are defined by the PRDM9 protein via its DNA binding domain constituted of an array of zinc fingers (ZnFs). High polymorphism and rapid divergence of the Prdm9 gene ZnF domain appear to involve positive selection at DNA-recognition amino-acid positions, but the nature of the underlying evolutionary pressures remains a puzzle. Here we explore the variability of the Prdm9 ZnF array in wild mice, and uncovered a high allelic diversity of both ZnF copy number and identity with the caracterization of 113 alleles. We analyze features of the diversity of ZnF identity which is mostly due to non-synonymous changes at codons −1, 3 and 6 of each ZnF, corresponding to amino-acids involved in DNA binding. Using methods adapted to the minisatellite structure of the ZnF array, we infer a phylogenetic tree of these alleles. We find the sister species Mus spicilegus and M. macedonicus as well as the three house mouse (Mus musculus) subspecies to be polyphyletic. However some sublineages have expanded independently in Mus musculus musculus and M. m. domesticus, the latter further showing phylogeographic substructure. Compared to random genomic regions and non-coding minisatellites, none of these patterns appears exceptional. In silico prediction of DNA binding sites for each allele, overlap of their alignments to the genome and relative coverage of the different families of interspersed repeated elements suggest a large diversity between PRDM9 variants with a potential for highly divergent distributions of recombination events in the genome with little correlation to evolutionary distance. By compiling PRDM9 ZnF protein sequences in Primates, Muridae and Equids, we find different diversity patterns among the three amino-acids most critical for the DNA-recognition function, suggesting different diversification timescales.     

Zinc Finger Binding Motifs Do Not Explain Recombination Rate Variation within or between Species of Drosophila

In humans and mice, the Cys₂His₂ zinc finger protein PRDM9 binds to a DNA sequence motif enriched in hotspots of recombination, possibly modifying nucleosomes, and recruiting recombination machinery to initiate Double Strand Breaks (DSBs). However, since its discovery, some researchers have suggested that the recombinational effect of PRDM9 is lineage or species specific. To test for a conserved role of PRDM9-like proteins across taxa, we use the Drosophila pseudoobscura species group in an attempt to identify recombination associated zinc finger proteins and motifs. We leveraged the conserved amino acid motifs in Cys₂His₂ zinc fingers to predict nucleotide binding motifs for all Cys₂His₂ zinc finger proteins in Drosophila pseudoobscura and identified associations with empirical measures of recombination rate. Additionally, we utilized recombination maps from D. pseudoobscura and D. miranda to explore whether changes in the binding motifs between species can account for changes in the recombination landscape, analogous to the effect observed in PRDM9 among human populations. We identified a handful of potential recombination-associated sequence motifs, but the associations are generally tenuous and their biological relevance remains uncertain. Furthermore, we found no evidence that changes in zinc finger DNA binding explains variation in recombination rate between species. We therefore conclude that there is no protein with a DNA sequence specific human-PRDM9-like function in Drosophila. We suggest these findings could be explained by the existence of a different recombination initiation system in Drosophila.     

Polymorphism of fecundity genes (BMPR1B, BMP15 and GDF9) in the Indian prolific Black Bengal goat

The Black Bengal is a prolific goat breed in India. Natural mutations in prolific sheep breeds have shown that the transforming growth factor beta (TGF-β) super family ligands such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor, BMPR1B) are crucial for ovulation and as well as for increasing litter size. Mutations in any of these genes increased prolificacy in sheep. Based on the known mutation information in sheep PCR primers were designed to screen known polymorphism in 88 random Black Bengal goats. Only the BMPR1B gene was polymorphic. Three genotypes of animals were detected in tested animals with mutant (FecB^B) and wild type (FecB⁺) alleles were 0.57 and 0.43, respectively. Non-carrier, heterozygous carrier and homozygous carrier Black Bengal does had 2.7, 3.04 and 3.11 kids, respectively. All known point mutations of BMP15 and GDF9 genes were monomorphic in the animals tested. These results preliminarily showed that the BMPR1B gene might be a major gene that influences prolificacy of Black Bengal goats.     

Ovar-MHC Polymorphism in Malpura and Avikalin Sheep Vaccinated for Peste des Petits Ruminants (PPR) Virus

India harbors a vast diversity of sheep (40 breeds). The study was carried out to assess the genetic diversity of DRB1 and DQA2 locus of the ovar-MHC and their possible association with Peste des petits ruminants (PPR) virus vaccine response in Malpura and Avikalin sheep breeds maintained at an organized institute flock in the semi-arid region of India. Genetic analysis revealed the rich diversity of DRB1 locus with 23 alleles in Malpura and 21 alleles in Avikalin sheep that included 9 new alleles. DQA2 locus also had rich diversity with 19 alleles in Malpura and 20 alleles in Avikalin sheep that included 7 new alleles. At the protein level, high variability alike at the nucleotide level was observed. A marker for footrot susceptibility, DQA2*1101 was absent in both breeds. Genotypic association of DRB1 and DQA2 with PPR vaccine response was statistically non-significant. Vaccine response being a multifactorial (polygenic and influenced by environment) variable, could not show statistically significant association with MHC genotypes in the present study. However, rich genetic diversity of DRB1 and DQA2 gene reflects the importance of this locus for future selection programs.     

PRDM9在哺乳动物基因重组热点中的作用

PRDM9(PR domain containing 9)是一种可催化组蛋白H3的4位赖氨酸(H3K4)发生三甲基化修饰的甲基转移酶,还拥有转录因子活性．PRDM9主要在生殖细胞的减数分裂初期表达,它的功能异常可导致不育的发生．哺乳动物PRDM9在C端的锌指结构具有进化快速的特征,而这个特征与基因重组热点的多样性相对应,一系列证据表明,PRDM9参与了基因重组热点的结合和重组的起始．这些进展对深入理解物种进化和基因重组机制具有十分重要的意义．     

Polymorphisms of caprine <Emphasis Type="Italic">GDF</Emphasis>9 gene and their association with litter size in Jining Grey goats

The exons 1, 2 and flanking region of growth differentiation factor 9 (GDF9) gene in five randomly selected does of Jining Grey, Boer and Liaoning Cashmere goats were amplified and analyzed. Thirteen nucleotide differences were identified in GDF9 gene between sheep (AF078545) and goats. Four SNPs (G3288A in intron 1, G423A, A959C [Gln320Pro] and G1189A [Val397Ile] in exon 2) were detected in four goat breeds with different prolificacy, in which G3288A was a new SNP in goats. The results showed that loci 3288, 423 and 1189 in Boer goats, loci 3288 and 423 in Guizhou White goats, loci 423 and 1189 in Liaoning Cashmere goats were all in complete linkage disequilibrium (D′ = 1, r ² = 1), respectively. In moderate (Boer goat) and low prolificacy (Liaoning Cashmere goat) breeds, linkage analysis indicated that there were more fervent linkage disequilibrium among loci 3288, 423 and 1189 than high prolificacy (Jining Grey and Guizhou White goats) breeds. For the 959 locus, the genotype distribution showed obvious difference between high prolificacy breeds and moderate or low prolificacy breeds (P < 0.05 or P < 0.01). The Jining Grey goat does with genotype CC or AC had 0.81 (P < 0.01) or 0.63 (P < 0.01) kids more than those with genotype AA, respectively. The present study preliminarily showed an association between allele C at 959 locus of GDF9 gene and high litter size in Jining Grey goats. These results provide further evidence that the GDF9 gene may be significantly correlated with high prolificacy in goats.     

Genome-wide cross-amplification of domestic sheep microsatellites in bighorn sheep and mountain goats

We tested for cross‐species amplification of microsatellite loci located throughout the domestic sheep (Ovis aries) genome in two north American mountain ungulates (bighorn sheep, Ovis canadensis, and mountain goats, Oreamnos americanus). We identified 247 new polymorphic markers in bighorn sheep (≥ 3 alleles in one of two study populations) and 149 in mountain goats (≥ 2 alleles in a single study population) using 648 and 576 primer pairs, respectively. Our efforts increased the number of available polymorphic microsatellite markers to 327 for bighorn sheep and 180 for mountain goats. The average distance between successive polymorphic bighorn sheep and mountain goat markers inferred from the Australian domestic sheep genome linkage map (mean ± 1 SD) was 11.9 ± 9.2 and 15.8 ± 13.8 centimorgans, respectively. The development of genomic resources in these wildlife species enables future studies of the genetic architecture of trait variation.     

Prion gene (PRNP) haplotype variation in United States goat breeds (Open Access publication)

Scrapie eradication efforts cost 18 million dollars annually in the United States and rely heavily upon PRNP genotyping of sheep. Genetic resistance might reduce goat scrapie and limit the risk of goats serving as a scrapie reservoir, so PRNP coding sequences were examined from 446 goats of 10 breeds, 8 of which had not been previously examined at PRNP. The 10 observed alleles were all related to one of two central haplotypes by a single amino acid substitution. At least five of these alleles (M142, R143, S146, H154, and K222) have been associated with increased incubation time or decreased odds of scrapie. To the best of our knowledge, neither S146 nor K222 has been found in any goats with scrapie, though further evaluation will be required to demonstrate true resistance. S146 was more common, present in several breeds at widely varying frequencies, while K222 was observed only in two dairy breeds at low frequency. Overall, this study provides frequency data on PRNP alleles in US goats, shows the pattern of relationships between haplotypes, and demonstrates segregation of multiple scrapieassociated alleles in several breeds not examined before at PRNP.     

Genetic diversity of <Emphasis Type="Italic">Ovis aries</Emphasis> populations near domestication centers and in the New World

Domestic sheep in Kazakhstan may provide an interesting source of genetic variability due to their proximity to the center of domestication and the Silk Route. Additionally, those breeds have never been compared to New World sheep populations. This report compares genetic diversity among five Kazakhstan (KZ) and 13 United States (US) sheep breeds (N = 442) using 25 microsatellite markers from the FAO panel. The KZ breeds had observed and expected measures of heterozygosity greater than 0.60 and an average number of alleles per locus of 7.8. In contrast, US sheep breeds had observed heterozygosity ranged from 0.37 to 0.62 and had an average number of alleles of 5.7. A Bayesian analysis indicated there were two primary populations (K = 2). Surprisingly, the US breeds were near evenly split between the two clusters, while all of the KZ breeds were placed in one of the two clusters. Pooling breeds within country of sample origin showed KZ and US populations to have similar levels of expected heterozygosity and the average number of alleles per locus. The results of breeds pooled within country suggest that there was no difference between countries for these diversity measures using this set of neutral markers. This finding suggests that populations’ geographically isolated from centers of domestication can be more diverse than previously thought, and as a result, conservation strategies can be adjusted accordingly. Furthermore, these results suggest there may be limited need for countries to alter the protocols for trade and exchange of animal genetic resources that are in place today, since no one population has a unique set of private alleles.     

PRDM9 Drives Evolutionary Erosion of Hotspots in Mus musculus through Haplotype-Specific Initiation of Meiotic Recombination

Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9 ^Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9^Cst arose, and M.m. domesticus, into which Prdm9^Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9^Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent''s Prdm9 allele and the opposite parent''s chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.     

The Red Queen Model of Recombination Hotspots Evolution in the Light of Archaic and Modern Human Genomes

Recombination is an essential process in eukaryotes, which increases diversity by disrupting genetic linkage between loci and ensures the proper segregation of chromosomes during meiosis. In the human genome, recombination events are clustered in hotspots, whose location is determined by the PRDM9 protein. There is evidence that the location of hotspots evolves rapidly, as a consequence of changes in PRDM9 DNA-binding domain. However, the reasons for these changes and the rate at which they occur are not known. In this study, we investigated the evolution of human hotspot loci and of PRDM9 target motifs, both in modern and archaic human lineages (Denisovan) to quantify the dynamic of hotspot turnover during the recent period of human evolution. We show that present-day human hotspots are young: they have been active only during the last 10% of the time since the divergence from chimpanzee, starting to be operating shortly before the split between Denisovans and modern humans. Surprisingly, however, our analyses indicate that Denisovan recombination hotspots did not overlap with modern human ones, despite sharing similar PRDM9 target motifs. We further show that high-affinity PRDM9 target motifs are subject to a strong self-destructive drive, known as biased gene conversion (BGC), which should lead to the loss of the majority of them in the next 3 MYR. This depletion of PRDM9 genomic targets is expected to decrease fitness, and thereby to favor new PRDM9 alleles binding different motifs. Our refined estimates of the age and life expectancy of human hotspots provide empirical evidence in support of the Red Queen hypothesis of recombination hotspots evolution.     

中国主要地方品种猪血液蛋白遗传多样性研究

实验采用水平板淀粉凝胶电泳技术,对中国地方猪五大类型９个品种,１个引入品种共２０１个个体进行了遗传多样性分析。共分析遗传座痊３０个,其中只有ＭＤＨ、ＰＥＰＢ、Ａ从位具有多在记民生。中国主要地方猪品种的多态座位百分比Ｐ＝０．２００,平均杂２合度Ｈ＝０．０６５,平均等位基因数Ａ＝１．３００。根据基因频率采用ＰＨＹＬＩＰ３．５ｃ民计算Ｎｅｉ氏遗传距离,然后用“ＮＥＩＧＨＢＯＲ”程序分别构建Ｎｅｉｇｈｂｏ