Recurrent inversion polymorphisms in humans associate with genetic instability and genomic disorders.

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.

Distinct Classes of Complex Structural Variation Uncovered across Thousands of Cancer Genome Graphs.

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.

CBFß-SMMHC Inhibition Triggers Apoptosis by Disrupting MYC Chromatin Dynamics in Acute Myeloid Leukemia.

The fusion oncoprotein CBFß-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here, we demonstrate that CBFß-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFß-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFß-SMMHC inhibitor with the BET inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFß-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.

A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia.

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

Long-molecule scars of backup DNA repair in BRCA1- and BRCA2-deficient cancers.

Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.

A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization.

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.

The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

The barley pan-genome reveals the hidden legacy of mutation breeding.

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the 'pan-genome'1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.

Complex evolutionary processes maintain an ancient chromosomal inversion.

Genome re-arrangements such as chromosomal inversions are often involved in adaptation. As such, they experience natural selection, which can erode genetic variation. Thus, whether and how inversions can remain polymorphic for extended periods of time remains debated. Here we combine genomics, experiments, and evolutionary modeling to elucidate the processes maintaining an inversion polymorphism associated with the use of a challenging host plant (Redwood trees) in Timema stick insects. We show that the inversion is maintained by a combination of processes, finding roles for life-history trade-offs, heterozygote advantage, local adaptation to different hosts, and gene flow. We use models to show how such multi-layered regimes of balancing selection and gene flow provide resilience to help buffer populations against the loss of genetic variation, maintaining the potential for future evolution. We further show that the inversion polymorphism has persisted for millions of years and is not a result of recent introgression. We thus find that rather than being a nuisance, the complex interplay of evolutionary processes provides a mechanism for the long-term maintenance of genetic variation.

Standing genetic variation and chromosome differences drove rapid ecotype formation in a major malaria mosquito.

Species distributed across heterogeneous environments often evolve locally adapted ecotypes, but understanding of the genetic mechanisms involved in their formation and maintenance in the face of gene flow is incomplete. In Burkina Faso, the major African malaria mosquito Anopheles funestus comprises two strictly sympatric and morphologically indistinguishable yet karyotypically differentiated forms reported to differ in ecology and behavior. However, knowledge of the genetic basis and environmental determinants of An. funestus diversification was impeded by lack of modern genomic resources. Here, we applied deep whole-genome sequencing and analysis to test the hypothesis that these two forms are ecotypes differentially adapted to breeding in natural swamps versus irrigated rice fields. We demonstrate genome-wide differentiation despite extensive microsympatry, synchronicity, and ongoing hybridization. Demographic inference supports a split only ~1,300 y ago, closely following the massive expansion of domesticated African rice cultivation ~1,850 y ago. Regions of highest divergence, concentrated in chromosomal inversions, were under selection during lineage splitting, consistent with local adaptation. The origin of nearly all variations implicated in adaptation, including chromosomal inversions, substantially predates the ecotype split, suggesting that rapid adaptation was fueled mainly by standing genetic variation. Sharp inversion frequency differences likely facilitated adaptive divergence between ecotypes by suppressing recombination between opposing chromosomal orientations of the two ecotypes, while permitting free recombination within the structurally monomorphic rice ecotype. Our results align with growing evidence from diverse taxa that rapid ecological diversification can arise from evolutionarily old structural genetic variants that modify genetic recombination.

Heterozygous inversion breakpoints suppress meiotic crossovers by altering recombination repair outcomes.

Heterozygous chromosome inversions suppress meiotic crossover (CO) formation within an inversion, potentially because they lead to gross chromosome rearrangements that produce inviable gametes. Interestingly, COs are also severely reduced in regions nearby but outside of inversion breakpoints even though COs in these regions do not result in rearrangements. Our mechanistic understanding of why COs are suppressed outside of inversion breakpoints is limited by a lack of data on the frequency of noncrossover gene conversions (NCOGCs) in these regions. To address this critical gap, we mapped the location and frequency of rare CO and NCOGC events that occurred outside of the dl-49 chrX inversion in D. melanogaster. We created full-sibling wildtype and inversion stocks and recovered COs and NCOGCs in the syntenic regions of both stocks, allowing us to directly compare rates and distributions of recombination events. We show that COs outside of the proximal inversion breakpoint are distributed in a distance-dependent manner, with strongest suppression near the inversion breakpoint. We find that NCOGCs occur evenly throughout the chromosome and, importantly, are not suppressed near inversion breakpoints. We propose a model in which COs are suppressed by inversion breakpoints in a distance-dependent manner through mechanisms that influence DNA double-strand break repair outcome but not double-strand break formation. We suggest that subtle changes in the synaptonemal complex and chromosome pairing might lead to unstable interhomolog interactions during recombination that permits NCOGC formation but not CO formation.

Evolution of Chromosomal Inversions across an Avian Radiation.

Chromosomal inversions are structural mutations that can play a prominent role in adaptation and speciation. Inversions segregating across species boundaries (trans-species inversions) are often taken as evidence for ancient balancing selection or adaptive introgression, but can also be due to incomplete lineage sorting. Using whole-genome resequencing data from 18 populations of 11 recognized munia species in the genus Lonchura (N = 176 individuals), we identify four large para- and pericentric inversions ranging in size from 4 to 20 Mb. All four inversions cosegregate across multiple species and predate the numerous speciation events associated with the rapid radiation of this clade across the prehistoric Sahul (Australia, New Guinea) and Bismarck Archipelago. Using coalescent theory, we infer that trans-specificity is improbable for neutrally segregating variation despite substantial incomplete lineage sorting characterizing this young radiation. Instead, the maintenance of all three autosomal inversions (chr1, chr5, and chr6) is best explained by selection acting along ecogeographic clines not observed for the collinear parts of the genome. In addition, the sex chromosome inversion largely aligns with species boundaries and shows signatures of repeated positive selection for both alleles. This study provides evidence for trans-species inversion polymorphisms involved in both adaptation and speciation. It further highlights the importance of informing selection inference using a null model of neutral evolution derived from the collinear part of the genome.

Rapid and Repeated Climate Adaptation Involving Chromosome Inversions following Invasion of an Insect.

Following invasion, insects can become adapted to conditions experienced in their invasive range, but there are few studies on the speed of adaptation and its genomic basis. Here, we examine a small insect pest, Thrips palmi, following its contemporary range expansion across a sharp climate gradient from the subtropics to temperate areas. We first found a geographically associated population genetic structure and inferred a stepping-stone dispersal pattern in this pest from the open fields of southern China to greenhouse environments of northern regions, with limited gene flow after colonization. In common garden experiments, both the field and greenhouse groups exhibited clinal patterns in thermal tolerance as measured by critical thermal maximum (CTmax) closely linked with latitude and temperature variables. A selection experiment reinforced the evolutionary potential of CTmax with an estimated h2 of 6.8% for the trait. We identified 3 inversions in the genome that were closely associated with CTmax, accounting for 49.9%, 19.6%, and 8.6% of the variance in CTmax among populations. Other genomic variations in CTmax outside the inversion region were specific to certain populations but functionally conserved. These findings highlight rapid adaptation to CTmax in both open field and greenhouse populations and reiterate the importance of inversions behaving as large-effect alleles in climate adaptation.

A high-resolution map of small-scale inversions in the gibbon genome.

Gibbons are the most speciose family of living apes, characterized by a diverse chromosome number and rapid rate of large-scale rearrangements. Here we performed single-cell template strand sequencing (Strand-seq), molecular cytogenetics, and deep in silico analysis of a southern white-cheeked gibbon genome, providing the first comprehensive map of 238 previously hidden small-scale inversions. We determined that more than half are gibbon specific, at least fivefold higher than shown for other primate lineage-specific inversions, with a significantly high number of small heterozygous inversions, suggesting that accelerated evolution of inversions may have played a role in the high sympatric diversity of gibbons. Although the precise mechanisms underlying these inversions are not yet understood, it is clear that segmental duplication-mediated NAHR only accounts for a small fraction of events. Several genomic features, including gene density and repeat (e.g., LINE-1) content, might render these regions more break-prone and susceptible to inversion formation. In the attempt to characterize interspecific variation between southern and northern white-cheeked gibbons, we identify several large assembly errors in the current GGSC Nleu3.0/nomLeu3 reference genome comprising more than 49 megabases of DNA. Finally, we provide a list of 182 candidate genes potentially involved in gibbon diversification and speciation.

SST-editing: in silico spatial transcriptomic editing at single-cell resolution.

MOTIVATION: Generative Adversarial Nets (GAN) achieve impressive performance for text-guided editing of natural images. However, a comparable utility of GAN remains understudied for spatial transcriptomics (ST) technologies with matched gene expression and biomedical image data. RESULTS: We propose In Silico Spatial Transcriptomic editing that enables gene expression-guided editing of immunofluorescence images. Using cell-level spatial transcriptomics data extracted from normal and tumor tissue slides, we train the approach under the framework of GAN (Inversion). To simulate cellular state transitions, we then feed edited gene expression levels to trained models. Compared to normal cellular images (ground truth), we successfully model the transition from tumor to normal tissue samples, as measured with quantifiable and interpretable cellular features. AVAILABILITY AND IMPLEMENTATION: https://github.com/CTPLab/SST-editing.

Sheltering of deleterious mutations explains the stepwise extension of recombination suppression on sex chromosomes and other supergenes.

Many organisms have sex chromosomes with large nonrecombining regions that have expanded stepwise, generating "evolutionary strata" of differentiation. The reasons for this remain poorly understood, but the principal hypotheses proposed to date are based on antagonistic selection due to differences between sexes. However, it has proved difficult to obtain empirical evidence of a role for sexually antagonistic selection in extending recombination suppression, and antagonistic selection has been shown to be unlikely to account for the evolutionary strata observed on fungal mating-type chromosomes. We show here, by mathematical modeling and stochastic simulation, that recombination suppression on sex chromosomes and around supergenes can expand under a wide range of parameter values simply because it shelters recessive deleterious mutations, which are ubiquitous in genomes. Permanently heterozygous alleles, such as the male-determining allele in XY systems, protect linked chromosomal inversions against the expression of their recessive mutation load, leading to the successive accumulation of inversions around these alleles without antagonistic selection. Similar results were obtained with models assuming recombination-suppressing mechanisms other than chromosomal inversions and for supergenes other than sex chromosomes, including those without XY-like asymmetry, such as fungal mating-type chromosomes. However, inversions capturing a permanently heterozygous allele were found to be less likely to spread when the mutation load segregating in populations was lower (e.g., under large effective population sizes or low mutation rates). This may explain why sex chromosomes remain homomorphic in some organisms but are highly divergent in others. Here, we model a simple and testable hypothesis explaining the stepwise extensions of recombination suppression on sex chromosomes, mating-type chromosomes, and supergenes in general.

A germline chimeric KANK1-DMRT1 transcript derived from a complex structural variant is associated with a congenital heart defect segregating across five generations.

Structural variants (SVs) pose a challenge to detect and interpret, but their study provides novel biological insights and molecular diagnosis underlying rare diseases. The aim of this study was to resolve a 9p24 rearrangement segregating in a family through five generations with a congenital heart defect (congenital pulmonary and aortic valvular stenosis and pulmonary artery stenosis), by applying a combined genomic analysis. The analysis involved multiple techniques, including karyotype, chromosomal microarray analysis (CMA), FISH, genome sequencing (GS), RNA-seq, and optical genome mapping (OGM). A complex 9p24 SV was hinted at by CMA results, showing three interspersed duplicated segments. Combined GS and OGM analyses revealed that the 9p24 duplications constitute a complex SV, on which a set of breakpoints matches the boundaries of the CMA duplicated sequences. The proposed structure for this complex rearrangement implies three duplications associated with an inversion of ~ 2 Mb region on chromosome 9 and a SINE element insertion at the more distal breakpoint. Interestingly, this genomic structure of rearrangement forms a chimeric transcript of the KANK1/DMRT1 loci, which was confirmed by both RNA-seq and Sanger sequencing on blood samples from 9p24 rearrangement carriers. Altogether with breakpoint amplification and FISH analysis, this combined approach allowed a deep characterization of this complex rearrangement. Although the genotype-phenotype correlation remains elusive from the molecular mechanism point of view, this study identified a large genomic rearrangement at 9p24 segregating with a familial congenital heart defect, revealing a genetic biomarker that was successfully applied for embryo selection, changing the reproductive perspective of affected individuals.

A novel pathogenic germline chromosome 3 inversion in von Hippel-Lindau disease.

von Hippel-Lindau (VHL) is an autosomal-dominant hereditary tumour susceptibility disease associated with pathogenic germline variants in the VHL tumour suppressor gene. VHL patients are at increased risk of developing multiple benign and malignant tumours. Current CLIA-based genetic tests demonstrate a very high detection rate of germline VHL variants in patients with clinical manifestations of VHL. In this report, we describe a large family with canonical VHL manifestations, for which no germline alteration had been detected by conventional germline testing. We identified a novel 291 kb chromosomal inversion involving chromosome 3p in affected family members. This inversion disrupts the VHL gene between exon 2 and exon 3 and is thereby responsible for the disease observed in this family.

Systematic identification of gene-altering programmed inversions across the bacterial domain.

Programmed chromosomal inversions allow bacteria to generate intra-population genotypic and functional heterogeneity, a bet-hedging strategy important in changing environments. Some programmed inversions modify coding sequences, producing different alleles in several gene families, most notably in specificity-determining genes such as Type I restriction-modification systems, where systematic searches revealed cross phylum abundance. Yet, a broad, gene-independent, systematic search for gene-altering programmed inversions has been absent, and little is known about their genomic sequence attributes and prevalence across gene families. Here, identifying intra-species variation in genomes of over 35 000 species, we develop a predictive model of gene-altering inversions, revealing key attributes of their genomic sequence attributes, including gene-pseudogene size asymmetry and orientation bias. The model predicted over 11,000 gene-altering loci covering known targeted gene families, as well as novel targeted families including Type II restriction-modification systems, a protein of unknown function, and a fusion-protein containing conjugative-pilus and phage tail domains. Publicly available long-read sequencing datasets validated representatives of these newly predicted inversion-targeted gene families, confirming intra-population genetic heterogeneity. Together, these results reveal gene-altering programmed inversions as a key strategy adopted across the bacterial domain, and highlight programmed inversions that modify Type II restriction-modification systems as a possible new mechanism for maintaining intra-population heterogeneity.

Low Mutation Load in a Supergene Underpinning Alternative Male Mating Strategies in Ruff (Calidris pugnax).

A paradox in evolutionary biology is how supergenes can maintain high fitness despite reduced effective population size, the suppression of recombination, and the expected accumulation of mutational load. The ruff supergene involves 2 rare inversion haplotypes (satellite and faeder). These are recessive lethals but with dominant effects on male mating strategies, plumage, and body size. Sequence divergence to the wild-type (independent) haplotype indicates that the inversion could be as old as 4 million years. Here, we have constructed a highly contiguous genome assembly of the inversion region for both the independent and satellite haplotypes. Based on the new data, we estimate that the recombination event(s) creating the satellite haplotype occurred only about 70,000 yr ago. Contrary to expectations for supergenes, we find no substantial expansion of repeats and only a modest mutation load on the satellite and faeder haplotypes despite high sequence divergence to the non-inverted haplotype (1.46%). The essential centromere protein N (CENPN) gene is disrupted by the inversion and is as well conserved on the inversion haplotypes as on the noninversion haplotype. These results suggest that the inversion may be much younger than previously thought. The low mutation load, despite recessive lethality, may be explained by the introgression of the inversion from a now extinct lineage.