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Unlike copy number variants (CNVs), inversions remain an underexplored genetic variation class. By integrating multiple genomic technologies, we discover 729 inversions in 41 human genomes. Approximately 85% of inversions

Funder: Wellcome Trust, Joint phylogenetic analysis of ancient DNA (aDNA) with modern phylogenies is hampered by low sequence coverage and post-mortem deamination, often resulting in over-conservative or incorrect assignment. We provide a new efficient likelihood-based workflow, pathPhynder, that takes advantage of all the polymorphic sites in the target sequence. This effectively evaluates the number of ancestral and derived alleles present on each branch and reports the most likely placement of an ancient sample in the phylogeny and a haplogroup assignment, together with alternatives and supporting evidence. To illustrate the application of pathPhynder, we show improved Y chromosome assignments for published ancient DNA sequences, using a newly compiled Y variation dataset (120,908 markers from 2,014 samples) that significantly enhances Y haplogroup assignment for low coverage samples. We apply the method to all published male ancient DNA samples from Africa, giving new insights into ancient migrations and the relationships between ancient and modern populations. The same software can be used to place samples with large amounts of missing data into other large non-recombining phylogenies such as the mitochondrial tree.

Y-chromosome dataset of worldwide populations for ancient DNA sample placement published as part of the paper: "Placing ancient DNA sequences into reference phylogenies".

Male infertility is a prevalent condition, affecting 5–10% of men. So far, few genetic factors have been described as contributors to spermatogenic failure. Here, we report the first re-sequencing study of the Y-chromosomal Azoospermia Factor c (AZFc) region, combined with gene dosage analysis of the multicopy DAZ, BPY2, and CDYgenes and Y-haplogroup determination. In analysing 2324 Estonian men, we uncovered a novel structural variant as a high-penetrance risk factor for male infertility. The Y lineage R1a1-M458, reported at >20% frequency in several European populations, carries a fixed ~1.6 Mb r2/r3 inversion, destabilizing the AZFc region and predisposing to large recurrent microdeletions. Such complex rearrangements were significantly enriched among severe oligozoospermia cases. The carrier vs non-carrier risk for spermatogenic failure was increased 8.6-fold (p=6.0×10−4). This finding contributes to improved molecular diagnostics and clinical management of infertility. Carrier identification at young age will facilitate timely counselling and reproductive decision-making.

During the last decade, large volumes of ancient DNA (aDNA) data have been generated as part of whole-genome shotgun and target capture sequencing studies. This includes sequences from non-recombining loci such as the mitochondrial or Y chromosomes. However, given the highly degraded nature of aDNA data, post-mortem deamination and often low genomic coverage, combining ancient and modern samples for phylogenetic analyses remains difficult. Without care, these factors can lead to incorrect placement.For the Y chromosomes, current standard methods focus on curated markers, but these contain only a subset of the total variation. Examining all polymorphic markers is particularly important for low coverage aDNA data because it substantially increases the number of overlapping sites between present-day and ancient individuals which may lead to higher resolution phylogenetic placement. We provide an automated workflow for jointly analysing ancient and present-day sequence data in a phylogenetic context. For each ancient sample, we effectively evaluate the number of ancestral and derived alleles present on each branch and use this information to place ancient lineages to their most likely position in the phylogeny. We provide both a parsimony approach and a highly optimised likelihood-based approach that assigns a posterior probability to each branch.To illustrate the application of this method, we have compiled and make available the largest public Y-chromosomal dataset to date (2,014 samples) which we used as a reference for phylogenetic placement. We process publicly available African ancient DNA Y-chromosome sequences and examine how patterns of Y-chromosomal diversity change across time and the relationship between ancient and present-day lineages. The same software can be used to place samples with large amounts of missing data into other large non-recombining phylogenies such as the mitochondrial tree.

Male infertility is a prevalent condition, concerning 5-10% of men. So far, only some recurrent genetic factors have been described as confident contributors to spermatogenic failure. Here, we report the first re-sequencing study of the Y-chromosomal Azoospermia Factor c (AZFc) region combined with gene dosage and Y-haplogroup determination. In analysing 2,324 Estonian men, we uncovered a novel structural variant as a high-penetrant risk factor to male infertility. The Y lineage R1a1-M458, reported at >20% frequency in several European populations, carries a fixed ∼1.6 Mb long r2/r3 inversion destabilizing the AZFc region and predisposing to recurrent microdeletions. Such complex rearrangements were significantly enriched among severe oligozoospermia cases. The carrier vs non-carrier risk to spermatogenic failure was increased 8.6-fold (p = 6.0 × 10−4). The finding contributes to improved molecular diagnostics and clinical management of infertility. Carrier identification in young age will facilitate timely counselling and reproductive decision-making.

The influence of Viking-Age migrants to the British Isles is obvious in archaeological and place-names evidence, but their demographic impact has been unclear. Autosomal genetic analyses support Norse Viking contributions to parts of Britain, but show no signal corresponding to the Danelaw, the region under Scandinavian administrative control from the ninth to eleventh centuries. Y-chromosome haplogroup R1a1 has been considered as a possible marker for Viking migrations because of its high frequency in peninsular Scandinavia (Norway and Sweden). Here we select ten Y-SNPs to discriminate informatively among hg R1a1 sub-haplogroups in Europe, analyse these in 619 hg R1a1 Y chromosomes including 163 from the British Isles, and also type 23 short-tandem repeats (Y-STRs) to assess internal diversity. We find three specifically Western-European sub-haplogroups, two of which predominate in Norway and Sweden, and are also found in Britain; star-like features in the STR networks of these lineages indicate histories of expansion. We ask whether geographical distributions of hg R1a1 overall, and of the two sub-lineages in particular, correlate with regions of Scandinavian influence within Britain. Neither shows any frequency difference between regions that have higher (≥10%) or lower autosomal contributions from Norway and Sweden, but both are significantly overrepresented in the region corresponding to the Danelaw. These differences between autosomal and Y-chromosomal histories suggest either male-specific contribution, or the influence of patrilocality. Comparison of modern DNA with recently available ancient DNA data supports the interpretation that two sub-lineages of hg R1a1 spread with the Vikings from peninsular Scandinavia. ispartof: European Journal Of Human Genetics vol:29 issue:3 pages:512-523 ispartof: location:England status: published

INTRODUCTION: Large-scale human genome sequencing studies to date have been limited to large, metropolitan populations or to small numbers of genomes from each group. Much remains to be understood about the extent and structure of genetic variation in our species and how it was shaped by past population separations, admixture, adaptation, size changes, and gene flow from archaic human groups. Larger numbers of genome sequences from more diverse populations are needed to illuminate these questions. RATIONALE: We sequence 929 genomes from 54 geographically, linguistically and culturally diverse human populations to an average of 35x coverage, and analyze the variation among them. We also physically resolve the haplotype phase of 26 of these genomes using linked-read sequencing. RESULTS: We identify 67.3 million single-nucleotide polymorphisms (SNPs), 8.8 million small insertions or deletions (indels) and 40,736 copy number variants (CNVs). This includes hundreds of thousands of variants that had not been discovered by previous sequencing efforts but which are common in one or more population. We demonstrate benefits to the study of population relationships of genome sequences over ascertained array genotypes, particularly when involving African populations. Populations in central and southern Africa, the Americas and Oceania each harbour tens to hundreds of thousands of private, common genetic variants. The majority of these variants arose as novel mutations rather than through archaic introgression, except in Oceanian populations where many private variants derive from Denisovan admixture. While some reach high frequencies, no variants are fixed between major geographical regions. We estimate that the genetic separation between present-day human populations occurred mostly within the last 250,000 years. However, these early separations were gradual in nature and shaped by protracted gene flow. All populations thus still had some genetic contact more recently than this, but there is also evidence that a small fraction of present-day structure might be hundreds of thousands of years older. Most populations expanded in size over the last 10,000 years, but hunter-gatherer groups did not. The low diversity among the Neanderthal haplotypes segregating in present-day populations indicates that, while more than one Neanderthal individual must have contributed genetic material to modern humans, there was likely only one major episode of admixture. In contrast, Denisovan haplotype diversity reflects a more complex history involving more than one episode of admixture. We find small amounts of Neanderthal ancestry in West African genomes, most likely reflecting Eurasian admixture. Despite their very low levels or absence of archaic ancestry, African populations share many Neanderthal and Denisovan variants that are absent from Eurasia, reflecting how a larger proportion of the ancestral human variation has been maintained in Africa. CONCLUSION: The discovery of substantial amounts of common genetic variation that was previously undocumented, and is geographically restricted, highlights the continued value of anthropologically informed study designs for understanding human diversity. The genome sequences presented here are a freely available resource with relevance to population history, medical genetics, anthropology and linguistics. [Figure: see text]

The genomes of present-day humans outside Africa originated almost entirely from a single migration out ~50,000-70,000 years ago[1, 2], followed by mixture with Neanderthals contributing ~2% to all non-Africans [3, 4]. However, the details of this initial migration remain poorly-understood because no ancient DNA analyses are available from this key time period, and interpretation of present-day autosomal data is complicated due to subsequent population movements/reshaping [5]. One locus, however, does retain male-specific information from this early period: the Y-chromosome, where a detailed calibrated phylogeny has been constructed [6]. Three present-day Y lineages were carried by the initial migration: the rare haplogroup D, the moderately rare C, and the very common FT lineage which now dominates most non-African populations [6, 7]. We show that phylogenetic analyses of haplogroup C, D and FT sequences, including very rare deep-rooting lineages, together with phylogeographic analyses of ancient and present-day non-African Y-chromosomes, all point to East/South-east Asia as the origin 50,000-55,000 years ago of all known non-African male lineages (apart from recent migrants). This observation contrasts with the expectation of a West Eurasian origin predicted by a simple model of expansion from a source near Africa [8, 9], and can be interpreted as resulting from extensive genetic drift in the initial population or replacement of early western lineages from the east, thus informing and constraining models of the initial expansion.

The genomes of humans outside Africa originated almost entirely from a single migration out ∼50,000-60,000 years ago1,2, followed closely by mixture with Neanderthals contributing ∼2% to all non-Africans3,4. However, the details of this initial migration remain poorly-understood because no ancient DNA analyses are available from this key time period, and present-day autosomal data are uninformative due to subsequent population movements/reshaping5. One locus, however, does retain extensive information from this early period: the Y-chromosome, where a detailed calibrated phylogeny has been constructed6. Three present-day Y lineages were carried by the initial migration: the rare haplogroup D, the moderately rare C, and the very common FT lineage which now dominates most non-African populations6,7. We show that phylogenetic analyses of haplogroup C, D and FT sequences, including very rare deep-rooting lineages, together with phylogeographic analyses of ancient and present-day non-African Y-chromosomes, all point to East/South-east Asia as the origin 50,000-55,000 years ago of all known non-African male lineages (apart from recent migrants). This implies that the initial Y lineages in populations between Africa and eastern Asia have been entirely replaced by lineages from the east, contrasting with the expectations of the serial-founder model8,9, and thus informing and constraining models of the initial expansion.

Background Large palindromes (inverted repeats) make up substantial proportions of mammalian sex chromosomes, often contain genes, and have high rates of structural variation arising via ectopic recombination. As a result, they underlie many genomic disorders. Maintenance of the palindromic structure by gene conversion between the arms has been documented, but over longer time periods, palindromes are remarkably labile. Mechanisms of origin and loss of palindromes have, however, received little attention. Results Here, we use fiber-FISH, 10x Genomics Linked-Read sequencing, and breakpoint PCR sequencing to characterize the structural variation of the P8 palindrome on the human Y chromosome, which contains two copies of the VCY (Variable Charge Y) gene. We find a deletion of almost an entire arm of the palindrome, leading to death of the palindrome, a size increase by recruitment of adjacent sequence, and other complex changes including the formation of an entire new palindrome nearby. Together, these changes are found in ~ 1% of men, and we can assign likely molecular mechanisms to these mutational events. As a result, healthy men can have 1–4 copies of VCY. Conclusions Gross changes, especially duplications, in palindrome structure can be relatively frequent and facilitate the evolution of sex chromosomes in humans, and potentially also in other mammalian species.

Summary The Middle East region is important to understand human evolution and migrations but is underrepresented in genomic studies. Here, we generated 137 high-coverage physically phased genome sequences from eight Middle Eastern populations using linked-read sequencing. We found no genetic traces of early expansions out-of-Africa in present-day populations but found Arabians have elevated Basal Eurasian ancestry that dilutes their Neanderthal ancestry. Population sizes within the region started diverging 15–20 kya, when Levantines expanded while Arabians maintained smaller populations that derived ancestry from local hunter-gatherers. Arabians suffered a population bottleneck around the aridification of Arabia 6 kya, while Levantines had a distinct bottleneck overlapping the 4.2 kya aridification event. We found an association between movement and admixture of populations in the region and the spread of Semitic languages. Finally, we identify variants that show evidence of selection, including polygenic selection. Our results provide detailed insights into the genomic and selective histories of the Middle East., Graphical abstract, Highlights • Middle Easterners do not have ancestry from an early out-of-Africa expansion • Basal Eurasian and African ancestry in Arabians deplete their Neanderthal ancestry • Populations experienced bottlenecks overlapping aridification events • Identification of recent single and polygenic signals of selection in Arabia, A high-coverage resource of physically phased genomes from eight Middle Eastern populations generated via linked-read sequencing provides insights into a genetically understudied region and enables more comprehensive study of population history and the detection of millions of variants common to the Middle East but outside short-read accessibility masks and not previously cataloged. It enhances our understanding of regional ancestry, the spread of languages, the effects of climate change on populations, and the evolutionary history of genetic variants.

The great apes (orangutans, gorillas, chimpanzees, bonobos and humans) descended from a common ancestor around 13 million years ago, and since then their sex chromosomes have followed very different evolutionary paths. While great-ape X chromosomes are highly conserved, their Y chromosomes, reflecting the general lability and degeneration of this male-specific part of the genome since its early mammalian origin, have evolved rapidly both between and within species. Understanding great-ape Y chromosome structure, gene content and diversity would provide a valuable evolutionary context for the human Y, and would also illuminate sex-biased behaviours, and the effects of the evolutionary pressures exerted by different mating strategies on this male-specific part of the genome. High-quality Y-chromosome sequences are available for human and chimpanzee (and low-quality for gorilla). The chromosomes differ in size, sequence organisation and content, and while retaining a relatively stable set of ancestral single-copy genes, show considerable variation in content and copy number of ampliconic multi-copy genes. Studies of Y-chromosome diversity in other great apes are relatively undeveloped compared to those in humans, but have nevertheless provided insights into speciation, dispersal, and mating patterns. Future studies, including data from larger sample sizes of wild-born and geographically well-defined individuals, and full Y-chromosome sequences from bonobos, gorillas and orangutans, promise to further our understanding of population histories, male-biased behaviours, mutation processes, and the functions of Y-chromosomal genes.

Understanding human genetic diversity in Africa is important for interpreting the evolution of all humans, yet vast regions in Africa, such as Chad, remain genetically poorly investigated. Here, we use genotype data from 480 samples from Chad, the Near East, and southern Europe, as well as whole-genome sequencing from 19 of them, to show that many populations today derive their genomes from ancient African-Eurasian admixtures. We found evidence of early Eurasian backflow to Africa in people speaking the unclassified isolate Laal language in southern Chad and estimate from linkage-disequilibrium decay that this occurred 4,750–7,200 years ago. It brought to Africa a Y chromosome lineage (R1b-V88) whose closest relatives are widespread in present-day Eurasia; we estimate from sequence data that the Chad R1b-V88 Y chromosomes coalesced 5,700–7,300 years ago. This migration could thus have originated among Near Eastern farmers during the African Humid Period. We also found that the previously documented Eurasian backflow into Africa, which occurred ∼3,000 years ago and was thought to be mostly limited to East Africa, had a more westward impact affecting populations in northern Chad, such as the Toubou, who have 20%–30% Eurasian ancestry today. We observed a decline in heterozygosity in admixed Africans and found that the Eurasian admixture can bias inferences on their coalescent history and confound genetic signals from adaptation and archaic introgression.

Manuel, Marc de et al., Our closest living relatives, chimpanzees and bonobos, have a complex demographic history. We analyzed the high-coverage whole genomes of 75 wild-born chimpanzees and bonobos from 10 countries in Africa. We found that chimpanzee population substructure makes genetic information a good predictor of geographic origin at country and regional scales. Multiple lines of evidence suggest that gene flow occurred from bonobos into the ancestors of central and eastern chimpanzees between 200,000 and 550,000 years ago, probably with subsequent spread into Nigeria-Cameroon chimpanzees. Together with another, possibly more recent contact (after 200,000 years ago), bonobos contributed less than 1% to the central chimpanzee genomes. Admixture thus appears to have been widespread during hominid evolution., M.d.M. is supported by a Formació de personal Investigador fellowship from Generalitat de Catalunya (FI_B01111). M.K. is supported by a Deutsche Forschungsgemeinschaft fellowship (KU 3467/1-1). V.C.S., I.D., and L.E. are supported by Swiss National Science Foundation grants 31003A-143393 and 310030B-16660. T.D. is funded by the Gates Cambridge Trust. O.L. is supported by a Ramón y Cajal grant from Ministerio de Economía y Competitividad (MINECO) (RYC-2013-14797) and MINECO grant BFU2015-68759-P [Fondo Europeo de Desarrollo Region (FEDER)]. P.H. is supported by Estonian Research Council grant PUT1036. J.M.S., A.M.A., and S.C. are funded by the Max Planck Society. J.P.-M., C.T.-S., and Y.X. were supported by The Wellcome Trust (098051). J.M.H.-G. is supported by the María de Maeztu Programme (MDM-2014-0370). A.S. is supported by an Isaac Newton Trust/Wellcome Trust Institutional Strategic Support Fund Joint Research Grant. J.N. had support from a U.S. NIH U01CA198933 grant, and B.M.P. is supported by a Swiss National Science Foundation postdoctoral fellowship. A.N. is supported by MINECO grant BFU2015-68649-P. The collection of fecal samples was supported by the Max Planck Society and Krekeler Foundation’s generous funding for the Pan African Programme. T.M.-B. thanks ICREA; the European Molecular Biology Organization Young Investigator Programme 2013; MINECO grants BFU2014-55090-P (FEDER), BFU2015-7116-ERC, and BFU2015-6215-ERCU01; U.S. NIH grant MH106874; Fundacio Zoo Barcelona; and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya for the support to his laboratory.

Human RBMY1 genes are located in four variable-sized clusters on the Y chromosome, expressed in male germ cells and possibly associated with sperm motility. We have re-investigated the mutational background and evolutionary history of the RBMY1 copy number distribution in worldwide samples and its relevance to sperm parameters in an Estonian cohort of idiopathic male factor infertility subjects. We estimated approximate RBMY1 copy numbers in 1218 1000 Genomes Project phase 3 males from sequencing read-depth, then chose 14 for valid ation by multicolour fibre-FISH. These fibre-FISH samples provided accurate calibration standards for the entire panel and led to detailed insights into population variation and mutational mechanisms. RBMY1 copy number worldwide ranged from 3 to 13 with a mode of 8. The two larger proximal clusters were the most variable, and additional duplications, deletions and inversions were detected. Placing the copy number estimates onto the published Y-SNP-based phylogeny of the same samples suggested a minimum of 562 mutational changes, translating to a mutation rate of 2.20 × 10−3 (95% CI 1.94 × 10−3 to 2.48 × 10−3) per father-to-son Y-transmission, higher than many short tandem repeat (Y-STRs), and showed no evidence for selection for increased or decreased copy number, but possible copy number stabilizing selection. An analysis of RBMY1 copy numbers among 376 infertility subjects failed to replicate a previously reported association with sperm motility and showed no significant effect on sperm count and concentration, serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone levels or testicular and semen volume. These results provide the first in-depth insights into the structural rearrangements underlying RBMY1 copy number variation across diverse human lineages.

While many studies have been undertaken of Middle Eastern populations using autosomal STR profiling by capillary electrophoresis, little has so far been published from this region on the forensic use of massively parallel sequencing (MPS). Here, we carried out MPS of 27 autosomal STRs and 91 identity-informative SNPs (iiSNPs) with the Verogen ForenSeq™ DNA Signature Prep Kit on a representative sample of 89 Saudi Arabian males, and analysed the resulting sequence data using Verogen's ForenSeq Universal Analysis Software (UAS) v1.3 and STRait Razor v3.0. This revealed sequence variation in the composition of complex STR arrays, and SNPs in their flanking regions, which raised the number of STR alleles from 238 distinct length variants to 357 sequence sub-variants. Similarly, between one and three additional polymorphic sites were observed within the amplicons of 37 of the 91 iiSNPs, forming up to six microhaplotypes per locus. These further enhance discrimination compared to the biallelic target SNP data presented by the primary UAS interface. In total, we observed twenty-two STR alleles previously unrecognised in the STRait Razor v3.0 default allele list, along with nine SNPs flanking target iiSNPs that were not highlighted by the UAS. Sequencing reduced the STR-based random match probability (RMP) from 2.62E-30 to 3.49E-34, and analysis of the iiSNP microhaplotypes reduced RMP from 9.97E-37 to 6.83E-40. The lack of significant linkage disequilibrium between STRs and target iiSNPs allowed the two marker types to be combined using the product rule, yielding a RMP of 2.39E-73. Evidence of consanguinity was apparent from both marker types. While TPOX was the only locus displaying a significant deviation from Hardy-Weinberg equilibrium, 23 out of 27 STRs and 63 out of 91 iiSNPs showed fewer than expected heterozygotes, demonstrating an overall homozygote excess probably reflecting the high frequency of first-cousin marriages in Saudi Arabia. We placed our data in a global context by considering the same markers in the Human Genome Diversity Panel (HGDP), revealing that the Saudi sample was typical of Middle Eastern populations, with a higher level of inbreeding than is seen in most European, African and Central/South Asian populations, correlating with known patterns of endogamy. Given reduced levels of diversity within endogamous groups, the ability to combine the discrimination power of both STRs and SNPs offers significant benefits in the analysis of forensic evidence in Saudi Arabia and the Middle East region more generally.

We describe the variation in copy number of a ~ 10 kb region overlapping the long intergenic noncoding RNA (lincRNA) gene, TTTY22, within the IR3 inverted repeat on the short arm of the human Y chromosome, leading to individuals with 0–3 copies of this region in the general population. Variation of this CNV is common, with 266 individuals having 0 copies, 943 (including the reference sequence) having 1, 23 having 2 copies, and two having 3 copies, and was validated by breakpoint PCR, fibre-FISH, and 10× Genomics Chromium linked-read sequencing in subsets of 1234 individuals from the 1000 Genomes Project. Mapping the changes in copy number to the phylogeny of these Y chromosomes previously established by the Project identified at least 20 mutational events, and investigation of flanking paralogous sequence variants showed that the mutations involved flanking sequences in 18 of these, and could extend over > 30 kb of DNA. While either gene conversion or double crossover between misaligned sister chromatids could formally explain the 0–2 copy events, gene conversion is the more likely mechanism, and these events include the longest non-allelic gene conversion reported thus far. Chromosomes with three copies of this CNV have arisen just once in our data set via another mechanism: duplication of 420 kb that places the third copy 230 kb proximal to the existing proximal copy. Our results establish gene conversion as a previously under-appreciated mechanism of generating copy number changes in humans and reveal the exceptionally large size of the conversion events that can occur.

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.

9 páginas, 2 figuras, 3 tablas.-- Grupos de trabajo: HYPEST, BRIGHT.-- Putku, Margus et al., Mutations in WNK1 and WNK4 cause familial hypertension, the Gordon syndrome. WNK1 and WNK4 conserved noncoding regions were targeted to polymorphism screening using DHPLC and DGGE. The scan identified an undescribed polymorphic AluYb8 insertion in WNK1 intron 10. Screening in primates revealed that this Alu-insertion has probably occurred in human lineage. Genotyping in 18 populations from Europe, Asia, and Africa (n = 854) indicated an expansion of the WNK1 AluYb8 bearing chromosomes out of Africa. The allele frequency in Sub-Saharan Africa was ∼3.3 times lower than in other populations (4.8 vs. 15.8%; P = 9.7 × 10−9). Meta-analysis across three European sample sets (n = 3,494; HYPEST, Estonians; BRIGHT, the British; CADCZ, Czech) detected significant association of the WNK1 AluYb8 insertion with blood pressure (BP; systolic BP, P = 4.03 × 10−3, effect 1.12; diastolic BP, P = 1.21 × 10−2, effect 0.67). Gender-stratified analysis revealed that this effect might be female-specific (n = 2,088; SBP, P = 1.99 × 10−3, effect 1.59; DBP P = 3.64 × 10−4, effect 1.23; resistant to Bonferroni correction), whereas no statistical support was identified for the association with male BP (n = 1,406). In leucocytes, the expressional proportions of the full-length WNK1 transcript and the splice-form skipping exon 11 were significantly shifted in AluYb8 carriers compared to noncarriers. The WNK1 AluYb8 insertion might affect human BP via altering the profile of alternatively spliced transcripts., This work forms part of the research themes contributing to the translational research portfolio for Barts and the London Cardiovascular Biomedical Research Unit, which is supported and funded by the National Institute of Health Research. K.K. has been supported by personal travel stipends from the Kristjan-Jaak Foundation and EC ECOGENE project.

The follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (HCG) play a critical role in human reproduction. Despite the common evolutionary ancestry and functional relatedness of the gonadotropin hormone beta (GtHB) genes, the single-copy FSHB (at 11p13) and the multi-copy LHB/CGB genes (at 19q13.32) exhibit locus-specific differences regarding their genomic context, evolution, genetic variation and expressional profile. FSHB represents a conservative vertebrate gene with a unique function and it is located in a structurally stable gene-poor region. In contrast, the primate-specific LHB/CGB gene cluster is located in a gene-rich genomic context and demonstrates an example of evolutionary young and unstable genomic region. The gene cluster is shaped by a constant balance between selection that acts on specific functions of the loci and frequent gene conversion events among duplicons. As the transcription of the GtHB genes is rate-limiting in the assembly of respective hormones, the genomic and genetic context of the FSHB and the LHB/CGB genes largely affects the profile of the hormone production.

The origin of completely novel proteins is a significant question in evolution. The luteinizing hormone (LHB)/chorionic gonadotropin (CGB) gene cluster in humans contains a candidate example of this process. Two genes in this cluster (CGB1 and CGB2) exhibit nucleotide sequence similarity with the other LHB/CGB genes, but as a result of frameshifting are predicted to encode a completely novel protein. Our analysis of these genes from humans and related primates indicates a recent origin in the lineage specific to humans and African great apes. While the function of these genes is not yet known, they are strongly conserved between human and chimpanzee and exhibit three-fold lower diversity than LHB across human populations with no mutations that would disrupt the coding sequence. The 5'-upstream region of CGB1/2 contains most of the promoter sequence of hCGbeta plus a novel region proximal to the putative transcription start site. In silico prediction of putative transcription factor binding sites supports the hypothesis that CGB1 and CGB2 gene products are expressed in, and may contribute to, implantation and placental development.

Segmental duplicons (>1 kb) of high sequence similarity (>90%) covering >5% of the human genome are characterized by complex sequence variation. Apart from a few well-characterized regions (MHC, β-globin), the diversity and linkage disequilibrium (LD) patterns of duplicons and the role of gene conversion in shaping them have been poorly studied. To shed light on these issues, we have re-sequenced the human Luteinizing Hormone/Chorionic Gonadotropin β (LHB/CGB) cluster (19q13.32) of three population samples (Estonians, Mandenka, and Han). The LHB/CGB cluster consists of seven duplicated genes critical in human reproduction. In the LHB/CGB region, high sequence diversity, concentration of gene-conversion acceptor sites, and strong LD colocalize with peripheral genes, whereas central loci are characterized by lower variation, gene-conversion donor activity, and breakdown of LD between close markers. The data highlight an important role of gene conversion in spreading polymorphisms among duplicon copies and generating LD around them. The directionality of gene-conversion events seems to be determined by the localization of a predicted recombination “hotspot” and “warm spot” in the vicinity of the most active acceptor genes at the periphery of the cluster. The data suggest that enriched crossover activity in direct and inverted segmental repeats is in accordance with the formation of palindromic secondary structures promoting double-strand breaks rather than fixed DNA sequence motifs. Also, this first detailed coverage of sequence diversity and structure of the LHB/CGB gene cluster will pave the way for studying the identified polymorphisms as well as potential genomic rearrangements in association with an individual's reproductive success.

Wolfram syndrome gene (WFS1) has been suggested to have a role in the susceptibility for mood disorders. A 26-fold increased risk for psychiatric disorders in WFS1 mutation carriers has been suggested. In this study we tested the hypothesis that the WFS1 gene is related to the risk for mood disorders. We analysed 28 single-nucleotide polymorphisms (SNPs) of the WFS1 gene in 224 unrelated patients with major depressive disorder and bipolar disorder and in 160 healthy control subjects. Patients were further stratified according to their comorbidity with anxiety disorders. We applied arrayed primer extension (APEX)-based genotyping technology followed by association and haplotype analysis. Five SNPs in the WFS1 gene were associated with major depressive disorder, and three SNPs with bipolar disorder. Haplotype analysis revealed a common GTA haplotype, formed by SNPs 684C/G, 1185C/T and 1832G/A, conferring risk for affective disorders. Specifically, for major depression the GTA haplotype has an OR of 1.59 (p = 0.01) and for bipolar disorder an OR of 1.89 (p = 0.03). These results support the hypothesis that the WFS1 gene is involved in the genetic predisposition for mood disorders.

Several genes associated with reproduction have been duplicated during primate evolution and evolved under positive selection. Chorionic gonadotropin hormone (HCG) produced by trophoblast cells has emerged in primates and assists maternal physiology to establish pregnancy. CGB gene, coding for the HCG β subunit has evolved through a duplication of the ancestral lutenizing hormone β (LHB) gene 55–35 million years ago. Only a few sequence changes in the duplicate locus were required to result in a novel CGB gene with different tissue-specificity and function. Evolution of the LHB/CGB genomic region has been accompanied by active genome dynamics either as series of additional duplications in Old World monkeys and apes, or as functional modifications of genes in New World monkeys. Duplicated, highly homologous genes with diverged functions are maintained through a balance between selective pressures targeted to their specific functions and evolution in consort facilitated by interlocus gene conversion. Key Concepts Several genes associated with female and male reproduction have been shown to be duplicated during primate evolution and evolve under positive selection. Chorionic gonadotropin hormone (HCG) produced by the trophoblast cells has emerged in primates and acts as a key hormone to assist maternal physiology to establish pregnancy. Primate-specific CGB gene, coding for the β subunit of HCG has emerged through a duplication of the ancestral lutenizing hormone β (LHB) gene approximately 55–35 million years ago. Only a few sequence changes in the duplicate locus were required to result in a novel CGB gene with different tissue-specificity, expression pattern and function. Evolution of the LHB/CGB genomic region in primates has been accompanied by active genome dynamics either as a series of additional duplications in the lineage to OWM and apes, or as functional modifications of the gene loci in NWM. Comparison of the human and chimpanzee LHB/CGB gene clusters identified large species-specific parallel rearrangement events, resulting in lineage-specific number of CGB gene copies (five versus six genes in chimpanzee compared to human). Duplicated genes tend to evolve in consort facilitated by active interlocus gene conversion increasing and preserving sequence similarity among the gene copies. Intraspecies gene conversion events have led to a higher human–chimpanzee divergence between duplicated LHB/CGB genes compared to the available data for unique genomic regions. Duplicated, highly homologous genes with diverged functions are maintained through balancing interlocus gene conversion activity with selective pressure targeting their specific functions. Keywords: chorionic gonadotropin β genes; primates; placenta; gene duplication; concerted evolution

Human chorionic gonadotropin (HCG) is produced by syncytiotrophoblast of placenta. It delays the apoptosis of corpus luteum and functions in implantation. Its possible role in male reproduction has been raised. HCG beta subunit is encoded by CGB, CGB5, CGB7 and CGB8 genes located at 19q13.3 in a common genome cluster with beta subunit non-coding CGB1 and CGB2. We conducted a sensitive quantification and comparison of CGB gene expression in human trophoblastic (blastocysts, n = 6; normal/failed pregnancy, n = 51) and non-malignant non-trophoblastic tissues (15 different tissue types, samples n = 241), by real-time RT-PCR. We showed a wide transcriptional window of CGB genes in normal pregnancy, a significant reduction in recurrent miscarriages, and a high expression (especially CGB1/CGB2) in ectopic and molar pregnancies. Expression was several orders of magnitude lower in the non-placental tissues, with the highest CGB levels being seen in testis, prostate, thymus, skeletal muscle and lung samples. The contribution of CGB1/CGB2 to the summarized expression of six CGB genes was not proportional to their gene dosage: 1/1000 to 1/10,000. An interesting exception was the testis exhibiting a much higher CGB1/CGB2 to total CGB mRNA ratio of approximately one-third, corresponding to gene dosage. In conclusion, the expressional profile of CGB genes, activated already in blastocyst stage, is associated with the status of pregnancy. The presence of CGB transcripts in testes, and in particular CGB1/CGB2 transcripts, may indicate a role in male reproductive tract.

BackgroundLow nucleotide divergence between human and chimpanzee does not sufficiently explain the species-specific morphological, physiological and behavioral traits. As gene duplication is a major prerequisite for the emergence of new genes and novel biological processes, comparative studies of human and chimpanzee duplicated genes may assist in understanding the mechanisms behind primate evolution. We addressed the divergence between human and chimpanzee duplicated genomic regions by using Luteinizing Hormone Beta (LHB)/Chorionic Gonadotropin Beta (CGB) gene cluster as a model. The placentalCGBgenes that are essential for implantation have evolved from an ancestral pituitaryLHBgene by duplications in the primate lineage.ResultsWe shotgun sequenced and compared the human (45,165 bp) and chimpanzee (39,876 bp)LHB/CGBregions and hereby present evidence for structural variation resulting in discordant number ofCGBgenes (6 in human, 5 in chimpanzee). The scenario of species-specific parallel duplications was supported (i) as the most parsimonious solution requiring the least rearrangement events to explain the interspecies structural differences; (ii) by the phylogenetic trees constructed with fragments of intergenic regions; (iii) by the sequence similarity calculations. Across the orthologous regions ofLHB/CGBcluster, substitutions and indels contributed approximately equally to the interspecies divergence and the distribution of nucleotide identity was correlated with the regional repeat content. Intraspecies gene conversion may have shaped theLHB/CGBgene cluster. The substitution divergence (1.8–2.59%) exceeded two-three fold the estimates for single-copy loci and the fraction of transversional mutations was increased compared to the unique sequences (43% versus ~30%). Despite the high sequence identity amongLHB/CGBgenes, there are signs of functional differentiation among the gene copies. Estimates for dn/dsrate ratio suggested a purifying selection onLHBandCGB8, and a positive evolution ofCGB1.ConclusionIf generalized, our data suggests that in addition to species-specific deletions and duplications, parallel duplication events may have contributed to genetic differences separating humans from their closest relatives. Compared to unique genomic segments, duplicated regions are characterized by high divergence promoted by intraspecies gene conversion and species-specific chromosomal rearrangements, including the alterations in gene copy number.

Objective In the present investigation we screened a large number of single nucleotide polymorphisms in the genes relevant to the neurobiology of anxiety for their association with panic disorder (PD). Methods The study sample included 127 patients with PD and 146 healthy control subjects. Using Arrayed Primer Extension technology we genotyped 90 polymorphisms in 21 candidate genes of serotonin, cholecystokinin, dopamine and opioid neurotransmitter systems. The association and haplotype analyses were performed in the whole group (PD-all) and in the subgroups of PD comorbid with major depression (PD-comorbid, n = 60) and without any comorbidity (PD-pure, n = 42). Results From the set of 90 polymorphisms, eight single nucleotide polymorphism markers in eight genes displayed at least a nominal association with any of the studied PD phenotype subgroups. Several polymorphisms of cholecystokinin, serotonin and dopamine systems were associated with PD-all and/or PD-comorbid phenotypes, while pure PD was associated only with HTR2A receptor 102T-C (P = 0.01) and DRD1 receptor -94G-A (P = 0.02) polymorphisms. Haplotype analysis supported an association of the cholecystokinin gene TG haplotype with the PD-all group (P = 0.04), whereas DRD1 receptor CAA and HTR2A receptor AT haplotypes were associated with a lower risk for PD-pure phenotype (P = 0.03 and P = 0.04, respectively). Conclusions The study results suggest that genetic variants of several candidate genes of neurotransmitter systems, each of a minor individual effect, may contribute to the susceptibility to PD. Our data also indicate that genetic variability may have a distinctive influence on pure and comorbid phenotypes of PD.

The present study focused on 91 single-nucleotide polymorphisms (SNPs) in 21 candidate genes to find associations with major depressive disorder (MDD). In total, 160 healthy controls and 177 patients with MDD were studied. We applied arrayed primer extension (APEX) based genotyping technology followed by association and haplotype analysis. SNPs in CCKAR, DRD1, DRD2, and HTR2C genes showed nom- inally significant associations with MDD. None of these associations remained significant after adjustment for multiple testing. Haplotype analysis revealed CCKAR haplotypes to be associated with MDD (global p=0.004). More precisely, we found the GAGT haplotype to be associated with increased risk for MDD (OR 7.42, 95 % CI 2.13-25.85, p=0.002). This haplotype effect remained significant after Bonferroni correction (p=0.04 after Bonferroni's adjustment). Altogether we were able to find some nominal associations, but due to small sample size these results should be taken as exploratory. However, the effect of GAGT haplotype on the CCKAR gene may be considered as increasing the risk for MDD. Received 2 January 2005 ; Reviewed 19 February 2005 ; Revised 9 March 2005 ; Accepted 14 March 2005

The male-specific region of the human Y chromosome (MSY) includes eight large inverted repeats (palindromes) in which arm-to-arm similarity exceeds 99.9%, due to gene conversion activity. Here, we studied one of these palindromes, P6, in order to illuminate the dynamics of the gene conversion process. We genotyped ten paralogous sequence variants (PSVs) within the arms of P6 in 378 Y chromosomes whose evolutionary relationships within the SNP-defined Y phylogeny are known. This allowed the identification of 146 historical gene conversion events involving individual PSVs, occurring at a rate of 2.9–8.4×10−4 events per generation. A consideration of the nature of nucleotide change and the ancestral state of each PSV showed that the conversion process was significantly biased towards the fixation of G or C nucleotides (GC-biased), and also towards the ancestral state. Determination of haplotypes by long-PCR allowed likely co-conversion of PSVs to be identified, and suggested that conversion tract lengths are large, with a mean of 2068 bp, and a maximum in excess of 9 kb. Despite the frequent formation of recombination intermediates implied by the rapid observed gene conversion activity, resolution via crossover is rare: only three inversions within P6 were detected in the sample. An analysis of chimpanzee and gorilla P6 orthologs showed that the ancestral state bias has existed in all three species, and comparison of human and chimpanzee sequences with the gorilla outgroup confirmed that GC bias of the conversion process has apparently been active in both the human and chimpanzee lineages., Author Summary The sex-determining role of the human Y chromosome makes it male-specific, and always present in only a single copy. This solo lifestyle has endowed it with some bizarre features, among which are eight large DNA units constituting about a quarter of the chromosome's length, and containing many genes important for sperm production. These units are called palindromes, since, taking into account the polarity of the DNA strands, the sequence is the same read from either end of the unit. We investigated the details of a process (gene conversion) that transfers sequence variants in one half of a palindrome into the other, thereby maintaining >99.9% similarity between the halves. We analysed patterns of sequence variants within one palindrome in a set of Y chromosomes whose evolutionary relationships are known. This allowed us to identify past gene conversion events, and to demonstrate a bias towards events that eliminate new variants, and retain old ones. Gene conversion has therefore acted during human evolution to retard sequence change in these regions. Analysis of the chimpanzee and gorilla versions of the palindrome shows that the dynamic processes we see in human Y chromosomes have a deep evolutionary history.