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Previous studies suggested that the copy number of the human salivary amylase gene, AMY1 , correlates with starch-rich diets. However, evolutionary analyses are hampered by the absence of accurate, sequence-resolved haplotype variation maps. We identified 30 structurally distinct haplotypes at nucleotide resolution among 98 present-day humans, revealing that the coding sequences of AMY1 copies are evolving under negative selection. Genomic analyses of these haplotypes in archaic hominins and ancient human genomes suggest that a common three-copy haplotype, dating as far back as 800,000 years ago, has seeded rapidly evolving rearrangements through recurrent nonallelic homologous recombination. Additionally, haplotypes with more than three AMY1 copies have significantly increased in frequency among European farmers over the past 4000 years, potentially as an adaptive response to increased starch digestion.

Diverse sets of complete human genomes are required to construct a pangenome reference and to understand the extent of complex structural variation. Here, we sequence 65 diverse human genomes and build 130 haplotype-resolved assemblies (130 Mbp median continuity), closing 92% of all previous assembly gaps 1,2 and reaching telomere-to-telomere (T2T) status for 39% of the chromosomes. We highlight complete sequence continuity of complex loci, including the major histocompatibility complex (MHC), SMN1 / SMN2 , NBPF8 , and AMY1/AMY2 , and fully resolve 1,852 complex structural variants (SVs). In addition, we completely assemble and validate 1,246 human centromeres. We find up to 30-fold variation in α-satellite high-order repeat (HOR) array length and characterize the pattern of mobile element insertions into α-satellite HOR arrays. While most centromeres predict a single site of kinetochore attachment, epigenetic analysis suggests the presence of two hypomethylated regions for 7% of centromeres. Combining our data with the draft pangenome reference 1 significantly enhances genotyping accuracy from short-read data, enabling whole-genome inference 3 to a median quality value (QV) of 45. Using this approach, 26,115 SVs per sample are detected, substantially increasing the number of SVs now amenable to downstream disease association studies., Competing Interests: Competing Interests E.E.E. is a scientific advisory board member of Variant Bio, Inc. C. Lee is a scientific advisory board member of Nabsys and Genome Insight. S.K. has received travel funds to speak at events hosted by Oxford Nanopore Technologies. The following authors have previously disclosed a patent application (No. EP19169090) relevant to Strand-seq: J.O.K., T.M., and D.P. The other authors declare no competing interests.

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess de novo mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 de novo single-nucleotide variants (SNVs), 7.4 non-tandem repeat indels, 79.6 de novo indels or structural variants (SVs) originating from tandem repeats, 7.7 centromeric de novo SVs and SNVs, and 12.4 de novo Y chromosome events per generation. STRs and VNTRs are the most mutable with 32 loci exhibiting recurrent mutation through the generations. We accurately assemble 288 centromeres and six Y chromosomes across the generations, documenting de novo SVs, and demonstrate that the DNM rate varies by an order of magnitude depending on repeat content, length, and sequence identity. We show a strong paternal bias (75-81%) for all forms of germline DNM, yet we estimate that 17% of de novo SNVs are postzygotic in origin with no paternal bias. We place all this variation in the context of a high-resolution recombination map (~3.5 kbp breakpoint resolution). We observe a strong maternal recombination bias (1.36 maternal:paternal ratio) with a consistent reduction in the number of crossovers with increasing paternal (r=0.85) and maternal (r=0.65) age. However, we observe no correlation between meiotic crossover locations and de novo SVs, arguing against non-allelic homologous recombination as a predominant mechanism. The use of multiple orthogonal technologies, near-telomere-to-telomere phased genome assemblies, and a multi-generation family to assess transmission has created the most comprehensive, publicly available "truth set" of all classes of genomic variants. The resource can be used to test and benchmark new algorithms and technologies to understand the most fundamental processes underlying human genetic variation., Competing Interests: Conflicts of interest E.E.E. is a scientific advisory board (SAB) member of Variant Bio, Inc. C.Lee is an SAB member of Nabsys and Genome Insight. D.P. has previously disclosed a patent application (no. EP19169090) relevant to Strand-seq. Z.K., C.N., E.D., C.F., C.Lambert, T.M., W.J.R., and M.A.E. are employees and shareholders of PacBio. Z.K. is a private shareholder in Phase Genomics. The other authors declare no competing interests.

Segmental duplications (SDs) contribute significantly to human disease, evolution, and diversity yet have been difficult to resolve at the sequence level. We present a population genetics survey of SDs by analyzing 170 human genome assemblies where the majority of SDs are fully resolved using long-read sequence assembly. Excluding the acrocentric short arms, we identify 173.2 Mbp of duplicated sequence (47.4 Mbp not present in the telomere-to-telomere reference) distinguishing fixed from structurally polymorphic events. We find that intrachromosomal SDs are among the most variable with rare events mapping near their progenitor sequences. African genomes harbor significantly more intrachromosomal SDs and are more likely to have recently duplicated gene families with higher copy number when compared to non-African samples. A comparison to a resource of 563 million full-length Iso-Seq reads identifies 201 novel, potentially protein-coding genes corresponding to these copy number polymorphic SDs., Competing Interests: COMPETING INTERESTS E.E.E. is a scientific advisory board (SAB) member of Variant Bio, Inc. C.L. is an SAB member of Nabsys and Genome Insight. The other authors declare no competing interests.

Apes possess two sex chromosomes-the male-specific Y chromosome and the X chromosome, which is present in both males and females. The Y chromosome is crucial for male reproduction, with deletions being linked to infertility 1 . The X chromosome is vital for reproduction and cognition 2 . Variation in mating patterns and brain function among apes suggests corresponding differences in their sex chromosomes. However, owing to their repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (bonobo (Pan paniscus), chimpanzee (Pan troglodytes), western lowland gorilla (Gorilla gorilla gorilla), Bornean orangutan (Pongo pygmaeus) and Sumatran orangutan (Pongo abelii)) and a lesser ape (the siamang gibbon (Symphalangus syndactylus)), and untangled the intricacies of their evolution. Compared with the X chromosomes, the ape Y chromosomes vary greatly in size and have low alignability and high levels of structural rearrangements-owing to the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements and satellites. Many Y chromosome genes expand in multi-copy families and some evolve under purifying selection. Thus, the Y chromosome exhibits dynamic evolution, whereas the X chromosome is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of more than 100 individual great apes. These reference assemblies are expected to inform human evolution and conservation genetics of non-human apes, all of which are endangered species., (© 2024. The Author(s).)

The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date 1 and led to its systematic omission from genomic analyses. Here we present de novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes 2 . Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established 1 boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: High sequence identity between segmental duplications (SDs) can facilitate copy number variants (CNVs) via non-allelic homologous recombination (NAHR). These CNVs are one of the fundamental causes of genomic disorders such as the 3q29 deletion syndrome (del3q29S). There are 21 protein-coding genes lost or gained as a result of such recurrent 1.6-Mbp deletions or duplications, respectively, in the 3q29 locus. While NAHR plays a role in CNV occurrence, the factors that increase the risk of NAHR at this particular locus are not well understood., Methods: We employed an optical genome mapping technique to characterize the 3q29 locus in 161 unaffected individuals, 16 probands with del3q29S and their parents, and 2 probands with the 3q29 duplication syndrome (dup3q29S). Long-read sequencing-based haplotype resolved de novo assemblies from 44 unaffected individuals, and 1 trio was used for orthogonal validation of haplotypes and deletion breakpoints., Results: In total, we discovered 34 haplotypes, of which 19 were novel haplotypes. Among these 19 novel haplotypes, 18 were detected in unaffected individuals, while 1 novel haplotype was detected on the parent-of-origin chromosome of a proband with the del3q29S. Phased assemblies from 44 unaffected individuals enabled the orthogonal validation of 20 haplotypes. In 89% (16/18) of the probands, breakpoints were confined to paralogous copies of a 20-kbp segment within the 3q29 SDs. In one del3q29S proband, the breakpoint was confined to a 374-bp region using long-read sequencing. Furthermore, we categorized del3q29S cases into three classes and dup3q29S cases into two classes based on breakpoints. Finally, we found no evidence of inversions in parent-of-origin chromosomes., Conclusions: We have generated the most comprehensive haplotype map for the 3q29 locus using unaffected individuals, probands with del3q29S or dup3q29S, and available parents, and also determined the deletion breakpoint to be within a 374-bp region in one proband with del3q29S. These results should provide a better understanding of the underlying genetic architecture that contributes to the etiology of del3q29S and dup3q29S., (© 2023. The Author(s).)

Perturbing expression is a powerful way to understand the role of individual genes, but can be challenging in important models. CRISPR-Cas screens in human induced pluripotent stem cells (iPSCs) are of limited efficiency due to DNA break-induced stress, while the less stressful silencing with an inactive Cas9 has been considered less effective so far. Here, we developed the dCas9-KRAB-MeCP2 fusion protein for screening in iPSCs from multiple donors. We found silencing in a 200 bp window around the transcription start site in polyclonal pools to be as effective as using wild-type Cas9 for identifying essential genes, but with much reduced cell numbers. Whole-genome screens to identify ARID1A-dependent dosage sensitivity revealed the PSMB2 gene, and enrichment of proteasome genes among the hits. This selective dependency was replicated with a proteasome inhibitor, indicating a targetable drug-gene interaction. Many more plausible targets in challenging cell models can be efficiently identified with our approach., Competing Interests: Conflict of interests L.C. and O.M.D. receive remuneration and stock options from bit.bio., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 <2 kbp form by twin-priming during L1 retrotransposition; 80% of the larger inversions are balanced and affect twice as many nucleotides as CNVs. Balanced inversions show an excess of common variants, and 72% are flanked by segmental duplications (SDs) or retrotransposons. Since flanking repeats promote non-allelic homologous recombination, we developed complementary approaches to identify recurrent inversion formation. We describe 40 recurrent inversions encompassing 0.6% of the genome, showing inversion rates up to 2.7 × 10 -4 per locus per generation. Recurrent inversions exhibit a sex-chromosomal bias and co-localize with genomic disorder critical regions. We propose that inversion recurrence results in an elevated number of heterozygous carriers and structural SD diversity, which increases mutability in the population and predisposes specific haplotypes to disease-causing CNVs., Competing Interests: Declaration of interests E.E.E. is a scientific advisory board (SAB) member of Variant Bio. C.L. is an SAB member of Nabsys. The following authors have previously disclosed a patent application (no. EP19169090) relevant to Strand-seq: A.D.S., J.O.K., T.M., and D.P.; the other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Joint phylogenetic analysis of ancient DNA (aDNA) with modern phylogenies is hampered by low sequence coverage and post-mortem deamination, often resulting in overconservative 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 aDNA sequences, using a newly compiled Y variation data set (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 aDNA 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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 CDY genes 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., Competing Interests: PH, LK, MP, EA, SR, MG, RF, MJ, OP, KP, PK, KR, YX, CT, ML No competing interests declared, (© 2021, Hallast et al.)

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.

The genomes of present-day humans outside Africa originated almost entirely from a single out-migration ~ 50,000-70,000 years ago, followed by mixture with Neanderthals contributing ~ 2% to all non-Africans. 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. One locus, however, does retain male-specific information from this early period: the Y chromosome, where a detailed calibrated phylogeny has been constructed. 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. Here, 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/Southeast Asia as the origin 50,000-55,000 years ago of all known surviving 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, and can be interpreted as resulting from extensive genetic drift in the initial population or replacement of early western Y lineages from the east, thus informing and constraining models of the initial expansion.

Genome sequences from diverse human groups are needed to understand the structure of genetic variation in our species and the history of, and relationships between, different populations. We present 929 high-coverage genome sequences from 54 diverse human populations, 26 of which are physically phased using linked-read sequencing. Analyses of these genomes reveal an excess of previously undocumented common genetic variation private to southern Africa, central Africa, Oceania, and the Americas, but an absence of such variants fixed between major geographical regions. We also find deep and gradual population separations within Africa, contrasting population size histories between hunter-gatherer and agriculturalist groups in the past 10,000 years, and a contrast between single Neanderthal but multiple Denisovan source populations contributing to present-day human populations., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objective: The male-specific region of the Y chromosome (MSY) remains one of the most unexplored regions of the genome. We sought to examine how the genetic variants of the MSY influence male susceptibility to coronary artery disease (CAD) and atherosclerosis. Approach and Results: Analysis of 129 133 men from UK Biobank revealed that only one of 7 common MSY haplogroups (haplogroup I1) was associated with CAD-carriers of haplogroup I1 had ≈11% increase in risk of CAD when compared with all other haplogroups combined (odds ratio, 1.11; 95% CI, 1.04-1.18; P =6.8×10 -4 ). Targeted MSY sequencing uncovered 235 variants exclusive to this haplogroup. The haplogroup I1-specific variants showed 2.45- and 1.56-fold respective enrichment for promoter and enhancer chromatin states, in cells/tissues relevant to atherosclerosis, when compared with other MSY variants. Gene set enrichment analysis in CAD-relevant tissues showed that haplogroup I1 was associated with changes in pathways responsible for early and late stages of atherosclerosis development including defence against pathogens, immunity, oxidative phosphorylation, mitochondrial respiration, lipids, coagulation, and extracellular matrix remodeling. UTY was the only Y chromosome gene whose blood expression was associated with haplogroup I1. Experimental reduction of UTY expression in macrophages led to changes in expression of 59 pathways (28 of which overlapped with those associated with haplogroup I1) and a significant reduction in the immune costimulatory signal., Conclusions: Haplogroup I1 is enriched for regulatory chromatin variants in numerous cells of relevance to CAD and increases cardiovascular risk through proatherosclerotic reprogramming of the transcriptome, partly through UTY .

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved.)

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.

Interpretations of genetic data concerning the prehistory of Europe have long been a subject of great debate, but increasing amounts of ancient and modern DNA data are now providing new and more informative evidence. Y-chromosome resequencing studies in Europe have highlighted the prevalence of recent expansions of male lineages, and focused interest on the Bronze Age as a period of cultural and demographic change. These findings contrast with phylogeographic studies based on mitochondrial DNA (mtDNA), which have been interpreted as supporting expansions from glacial refugia. Here we have undertaken a population-based resequencing of complete mitochondrial genomes in Europe and the Middle East, in 340 samples from 17 populations for which Y-chromosome sequence data are also available. Demographic reconstructions show no signal of Bronze Age expansion, but evidence of Paleolithic expansions in all populations except the Saami, and with an absence of detectable geographical pattern. In agreement with previous inference from modern and ancient DNA data, the unbiased comparison between the mtDNA and Y-chromosome population datasets emphasizes the sex-biased nature of recent demographic transitions in Europe.

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.

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.

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., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016, American Association for the Advancement of Science.)

The distribution of genetic diversity in great ape species is likely to have been affected by patterns of dispersal and mating. This has previously been investigated by sequencing autosomal and mitochondrial DNA (mtDNA), but large-scale sequence analysis of the male-specific region of the Y Chromosome (MSY) has not yet been undertaken. Here, we use the human MSY reference sequence as a basis for sequence capture and read mapping in 19 great ape males, combining the data with sequences extracted from the published whole genomes of 24 additional males to yield a total sample of 19 chimpanzees, four bonobos, 14 gorillas, and six orangutans, in which interpretable MSY sequence ranges from 2.61 to 3.80 Mb. This analysis reveals thousands of novel MSY variants and defines unbiased phylogenies. We compare these with mtDNA-based trees in the same individuals, estimating time-to-most-recent common ancestor (TMRCA) for key nodes in both cases. The two loci show high topological concordance and are consistent with accepted (sub)species definitions, but time depths differ enormously between loci and (sub)species, likely reflecting different dispersal and mating patterns. Gorillas and chimpanzees/bonobos present generally low and high MSY diversity, respectively, reflecting polygyny versus multimale-multifemale mating. However, particularly marked differences exist among chimpanzee subspecies: The western chimpanzee MSY phylogeny has a TMRCA of only 13.2 (10.8-15.8) thousand years, but that for central chimpanzees exceeds 1 million years. Cross-species comparison within a single MSY phylogeny emphasizes the low human diversity, and reveals species-specific branch length variation that may reflect differences in long-term generation times., (© 2016 Hallast et al.; Published by Cold Spring Harbor Laboratory Press.)

The proportion of Europeans descending from Neolithic farmers ∼ 10 thousand years ago (KYA) or Palaeolithic hunter-gatherers has been much debated. The male-specific region of the Y chromosome (MSY) has been widely applied to this question, but unbiased estimates of diversity and time depth have been lacking. Here we show that European patrilineages underwent a recent continent-wide expansion. Resequencing of 3.7 Mb of MSY DNA in 334 males, comprising 17 European and Middle Eastern populations, defines a phylogeny containing 5,996 single-nucleotide polymorphisms. Dating indicates that three major lineages (I1, R1a and R1b), accounting for 64% of our sample, have very recent coalescent times, ranging between 3.5 and 7.3 KYA. A continuous swathe of 13/17 populations share similar histories featuring a demographic expansion starting ∼ 2.1-4.2 KYA. Our results are compatible with ancient MSY DNA data, and contrast with data on mitochondrial DNA, indicating a widespread male-specific phenomenon that focuses interest on the social structure of Bronze Age Europe.

Many studies of human populations have used the male-specific region of the Y chromosome (MSY) as a marker, but MSY sequence variants have traditionally been subject to ascertainment bias. Also, dating of haplogroups has relied on Y-specific short tandem repeats (STRs), involving problems of mutation rate choice, and possible long-term mutation saturation. Next-generation sequencing can ascertain single nucleotide polymorphisms (SNPs) in an unbiased way, leading to phylogenies in which branch-lengths are proportional to time, and allowing the times-to-most-recent-common-ancestor (TMRCAs) of nodes to be estimated directly. Here we describe the sequencing of 3.7 Mb of MSY in each of 448 human males at a mean coverage of 51×, yielding 13,261 high-confidence SNPs, 65.9% of which are previously unreported. The resulting phylogeny covers the majority of the known clades, provides date estimates of nodes, and constitutes a robust evolutionary framework for analyzing the history of other classes of mutation. Different clades within the tree show subtle but significant differences in branch lengths to the root. We also apply a set of 23 Y-STRs to the same samples, allowing SNP- and STR-based diversity and TMRCA estimates to be systematically compared. Ongoing purifying selection is suggested by our analysis of the phylogenetic distribution of nonsynonymous variants in 15 MSY single-copy genes., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2011 Wiley-Liss, Inc.)

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., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: Low 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 placental CGB genes that are essential for implantation have evolved from an ancestral pituitary LHB gene by duplications in the primate lineage., Results: We shotgun sequenced and compared the human (45,165 bp) and chimpanzee (39,876 bp) LHB/CGB regions and hereby present evidence for structural variation resulting in discordant number of CGB genes (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 of LHB/CGB cluster, 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 the LHB/CGB gene 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 approximately 30%). Despite the high sequence identity among LHB/CGB genes, there are signs of functional differentiation among the gene copies. Estimates for dn/ds rate ratio suggested a purifying selection on LHB and CGB8, and a positive evolution of CGB1., Conclusion: If 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.

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.

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 nominally 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.

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, beta-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 beta (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.

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.