Genomic Rearrangements Research Articles

CRISPR-directed gene-editing induces genetic rearrangement within the human globin gene locus

CRISPR-Cas is a revolutionary technology but has already demonstrated significant feasibility for clinical and non-clinical applications. While the efficiency and precision of this remarkable genetic tool is unprecedented, unfortunately, a series of collateral genetic rearrangement have been reported in response to double-stranded DNA breakage. Once these molecular scissions occur, the cascade of DNA repair reactions can lead to genomic rearrangements especially if breakage takes place within a family of sequence related genes. Here, we demonstrate that CRISPR- directed gene editing near the sickle cell mutation site generates a curious genetic outcome; a footprint of the δ globin gene proximal to the CRISPR/Cas cut site(s). This rearrangement is not dependent on the presence of an exogenously added DNA template but is apparently dependent on a double strand break. Our results the highlight recombinational capacity of double strand breaks in human chromosomes where the aim is to edit a human gene.

Phage-mediated resolution of genetic conflict alters the evolutionary trajectory of Pseudomonas aeruginosa lysogens.

The opportunistic human pathogen Pseudomonas aeruginosa is naturally infected by a large class of temperate, transposable, Mu-like phages. We examined the genotypic and phenotypic diversity of P. aeruginosa PA14 lysogen populations as they resolve clustered regularly interspaced short palindromic repeat (CRISPR) autoimmunity, mediated by an imperfect CRISPR match to the Mu-like DMS3 prophage. After 12 days of evolution, we measured a decrease in spontaneous induction in both exponential and stationary phase growth. Co-existing variation in spontaneous induction rates in the exponential phase depended on the way the coexisting strains resolved genetic conflict. Multiple mutational modes to resolve genetic conflict between host and phage resulted in coexistence in evolved populations of single lysogens that maintained CRISPR immunity to other phages and polylysogens that lost immunity completely. This work highlights a new dimension of the role of lysogenic phages in the evolution of their hosts.IMPORTANCEThe chronic opportunistic multi-drug-resistant pathogen Pseudomonas aeruginosa is persistently infected by temperate phages. We assess the contribution of temperate phage infection to the evolution of the clinically relevant strain UCBPP-PA14. We found that a low level of clustered regularly interspaced short palindromic repeat (CRISPR)-mediated self-targeting resulted in polylysogeny evolution and large genome rearrangements in lysogens; we also found extensive diversification in CRISPR spacers and cas genes. These genomic modifications resulted in decreased spontaneous induction in both exponential and stationary phase growth, increasing lysogen fitness. This work shows the importance of considering latent phage infection in characterizing the evolution of bacterial populations.

We are doing it wrong: Putting homology before phylogeny in cyanobacterial taxonomy.

The rapid expansion of whole genome sequencing in bacterial taxonomy has revealed deep evolutionary relationships and speciation signals, but assembly methods often miss true nucleotide diversity in the ribosomal operons. Though it lacks sufficient phylogenetic signal at the species level, the 16S ribosomal RNA gene is still much used in bacterial taxonomy. In cyanobacterial taxonomy, comparisons of 16S-23S Internal Transcribed Spacer (ITS) regions are used to bridge this information gap. Although ITS rRNA region analyses are routinely being used to identify species, researchers often do not identify orthologous operons, which leads to improper comparisons. No method for delineating orthologous operon copies from paralogous ones has been established. A new method for recognizing orthologous ribosomal operons by quantifying the conserved paired nucleotides in a helical domain of the ITS, has been developed. The D1' Index quantifies differences in the ratio of pyrimidines to purines in paired nucleotide sequences of this helix. Comparing 111 operon sequences from 89 strains of Brasilonema, four orthologous operon types were identified. Plotting D1' Index values against the length of helices produced clear separation of orthologs. Most orthologous operons in this study were observed both with and without tRNA genes present. We hypothesize that genomic rearrangement, not gene duplication, is responsible for the variation among orthologs. This new method will allow cyanobacterial taxonomists to utilize ITS rRNA region data more correctly, preventing erroneous taxonomic hypotheses. Moreover, this work could assist genomicists in identifying and preserving evident sequence variability in ribosomal operons, which is an important proxy for evolution in prokaryotes.

Annelid Comparative Genomics and the Evolution of Massive Lineage-Specific Genome Rearrangement in Bilaterians.

The organization of genomes into chromosomes is critical for processes such as genetic recombination, environmental adaptation, and speciation. All animals with bilateral symmetry inherited a genome structure from their last common ancestor that has been highly conserved in some taxa but seemingly unconstrained in others. However, the evolutionary forces driving these differences and the processes by which they emerge have remained largely uncharacterized. Here, we analyze genome organization across the phylum Annelida using 23 chromosome-level annelid genomes. We find that while many annelid lineages have maintained the conserved bilaterian genome structure, the Clitellata, a group containing leeches and earthworms, possesses completely scrambled genomes. We develop a rearrangement index to quantify the extent of genome structure evolution and show that, compared to the last common ancestor of bilaterians, leeches and earthworms have among the most highly rearranged genomes of any currently sampled species. We further show that bilaterian genomes can be classified into two distinct categories-high and low rearrangement-largely influenced by the presence or absence, respectively, of chromosome fission events. Our findings demonstrate that animal genome structure can be highly variable within a phylum and reveal that genome rearrangement can occur both in a gradual, stepwise fashion, or rapid, all-encompassing changes over short evolutionary timescales.

Clonal background and routes of plasmid transmission underlie antimicrobial resistance features of bloodstream Klebsiella pneumoniae

Bloodstream infections caused by the opportunistic pathogen Klebsiella pneumoniae are associated with adverse health complications and high mortality rates. Antimicrobial resistance (AMR) limits available treatment options, thus exacerbating its public health and clinical burden. Here, we aim to elucidate the population structure of K. pneumoniae in bloodstream infections from a single medical center and the drivers that facilitate the dissemination of AMR. Analysis of 136 short-read genome sequences complemented with 12 long-read sequences shows the population consisting of 94 sequence types (STs) and 99 clonal groups, including globally distributed multidrug resistant and hypervirulent clones. In vitro antimicrobial susceptibility testing and in silico identification of AMR determinants reveal high concordance (90.44–100%) for aminoglycosides, beta-lactams, carbapenems, cephalosporins, quinolones, and sulfonamides. IncF plasmids mediate the clonal (within the same lineage) and horizontal (between lineages) transmission of the extended-spectrum beta-lactamase gene blaCTX-M-15. Nearly identical plasmids are recovered from isolates over a span of two years indicating long-term persistence. The genetic determinants for hypervirulence are carried on plasmids exhibiting genomic rearrangement, loss, and/or truncation. Our findings highlight the importance of considering both the genetic background of host strains and the routes of plasmid transmission in understanding the spread of AMR in bloodstream infections.

A transposase-derived gene required for human brain development.

DNA transposable elements and transposase-derived genes are present in most living organisms, including vertebrates, but their function is largely unknown. PiggyBac Transposable Element Derived 5 (PGBD5) is an evolutionarily conserved vertebrate DNA transposase-derived gene with retained nuclease activity in human cells. Vertebrate brain development is known to be associated with prominent neuronal cell death and DNA breaks, but their causes and functions are not well understood. Here, we show that PGBD5 contributes to normal brain development in mice and humans, where its deficiency causes disorder of intellectual disability, movement, and seizures. In mice, Pgbd5 is required for the developmental induction of post-mitotic DNA breaks and recurrent somatic genome rearrangements. In the brain cortex, loss of Pgbd5 leads to aberrant differentiation and gene expression of distinct neuronal populations, including specific types of glutamatergic neurons, which explains the features of PGBD5 deficiency in humans. Thus, PGBD5 might be a transposase-derived enzyme required for brain development in mammals.

Structural variants linked to Alzheimer's Disease and other common age-related clinical and neuropathologic traits.

Advances have led to a greater understanding of the genetics of Alzheimer's Disease (AD). However, the gap between the predicted and observed genetic heritability estimates when using single nucleotide polymorphisms (SNPs) and small indel data remains. Large genomic rearrangements, known as structural variants (SVs), have the potential to account for this missing genetic heritability. By leveraging data from two ongoing cohort studies of aging and dementia, the Religious Orders Study and Rush Memory and Aging Project (ROS/MAP), we performed genome-wide association analysis testing around 20,000 common SVs from 1,088 participants with whole genome sequencing (WGS) data. A range of Alzheimer's Disease and Related Disorders (AD/ADRD) clinical and pathologic traits were examined. Given the limited sample size, no genome-wide significant association was found, but we mapped SVs across 81 AD risk loci and discovered 22 SVs in linkage disequilibrium (LD) with GWAS lead variants and directly associated with AD/ADRD phenotypes (nominal P < 0.05). The strongest association was a deletion of an Alu element in the 3'UTR of the TMEM106B gene. This SV was in high LD with the respective AD GWAS locus and was associated with multiple AD/ADRD phenotypes, including tangle density, TDP-43, and cognitive resilience. The deletion of this element was also linked to lower TMEM106B protein abundance. We also found a 22 kb deletion associated with depression in ROSMAP and bearing similar association patterns as AD GWAS SNPs at the IQCK locus. In addition, genome-wide scans allowed the identification of 7 SVs, with no LD with SNPs and nominally associated with AD/ADRD traits. This result suggests potentially new ADRD risk loci not discoverable using SNP data. Among these findings, we highlight a 5.6 kb duplication of coding regions of the gene C1orf186 at chromosome 1 associated with indices of cognitive impairment, decline, and resilience. While further replication in independent datasets is needed to validate these findings, our results support the potential roles of common structural variations in the pathogenesis of AD/ADRD.

Gene content, phage cycle regulation model and prophage inactivation disclosed by prophage genomics in the Helicobacter pylori Genome Project.

Prophages can have major clinical implications through their ability to change pathogenic bacterial traits. There is limited understanding of the prophage role in ecological, evolutionary, adaptive processes and pathogenicity of Helicobacter pylori, a widespread bacterium causally associated with gastric cancer. Inferring the exact prophage genomic location and completeness requires complete genomes. The international Helicobacter pylori Genome Project (HpGP) dataset comprises 1011 H. pylori complete clinical genomes enriched with epigenetic data. We thoroughly evaluated the H. pylori prophage genomic content in the HpGP dataset. We investigated population evolutionary dynamics through phylogenetic and pangenome analyses. Additionally, we identified genome rearrangements and assessed the impact of prophage presence on bacterial gene disruption and methylome. We found that 29.5% (298) of the HpGP genomes contain prophages, of which only 32.2% (96) were complete, minimizing the burden of prophage carriage. The prevalence of H. pylori prophage sequences was variable by geography and ancestry, but not by disease status of the human host. Prophage insertion occasionally results in gene disruption that can change the global bacterial epigenome. Gene function prediction allowed the development of the first model for lysogenic-lytic cycle regulation in H. pylori. We have disclosed new prophage inactivation mechanisms that appear to occur by genome rearrangement, merger with other mobile elements, and pseudogene accumulation. Our analysis provides a comprehensive framework for H. pylori prophage biological and genomics, offering insights into lysogeny regulation and bacterial adaptation to prophages.

Engineering a Sinorhizobium meliloti Chassis with Monopartite, Single Replicon Genome Configuration.

Multipartite bacterial genomes pose challenges for genome engineering and the establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features such as replichore ratios, GC skew, KOPS, and coding sequence distribution. Under standard culture conditions, the growth rates of these strains and the wild type were nearly comparable, and the ability for symbiotic nitrogen fixation was maintained. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes, including the oriVs of pSymA and pSymB from this strain, resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth, aberrant cellular localization of oriC, and deficiency in symbiosis. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth, but only the genomic rearrangement recovered the symbiotic capacity. These strains will facilitate the integration of secondary replicons in S. meliloti and thus be useful for genome engineering applications, such as generating hybrid genomes.

Approaches for Lynch syndrome screening and characteristics of subtypes with mismatch repair deficiency in patients with colorectal carcinoma.

To evaluate different Lynch syndrome (LS) screening approaches and establish an efficient and sensitive strategy are critical for clinical practice. In total, 583 patients with colorectal carcinoma (CRC) at Fudan University Shanghai Cancer Center were enrolled. Patient samples were examined by immunohistochemistry (IHC) and next-generation sequencing (NGS), and MLH1 promoter hypermethylation (MPH) was detected in MLH1-deficient cases. Germline genetic testing was performed in cases with deleterious variants and large genomic rearrangements (LGRs) of tumor MMR genes were detected in cases with dMMR or MSI-H cases with no MMR germline variants. Our results showed that triage with IHC and followed by BRAF/MLH1 methylation testing (Strategy 1) identified 93.3% (70/75) of LS cases. IHC followed by germline NGS (Strategy 2) or direct tumor NGS (Strategy 3) both identified 98.7% (74/75) of LS cases. The proportion of LGRs in LS cases was 16.0% (12/75), while 84.0% (63/75) showed SNV/Indel. The average cost per patient was ¥6010.81, ¥6058.48, and ¥8029.98 for Strategy 1, Strategy 2 and Strategy 3, respectively. The average time spent on different strategies was 4.74 days (Strategy 1), 4.89 days (Strategy 2), and 14.50 days (Strategy 3) per patient, respectively. LS and Lynch-like syndrome (LLS) were associated with an earlier onset age than MPH. In conclusion, we compared different workflows for LS screening and IHC plus germline NGS is recommended for LS screening when taking sensitivity, time, and cost into account. Moreover, multiplex ligation-dependent probe amplification made up for the shortcoming of NGS and should be incorporated into routine screening.

Complete mitochondrial genome of Hippophae tibetana: insights into adaptation to high-altitude environments.

Hippophae tibetana, belonging to the Elaeagnaceae family, is an endemic plant species of the Qinghai-Tibet Plateau, valued for its remarkable ecological restoration capabilities, as well as medicinal and edible properties. Despite being acknowledged as a useful species, its mitochondrial genome data and those of other species of the Elaeagnaceae family are lacking to date. In this study, we, for the first time, successfully assembled the mitochondrial genome of H. tibetana, which is 464,208 bp long and comprises 31 tRNA genes, 3 rRNA genes, 37 protein-coding genes, and 3 pseudogenes. Analysis of the genome revealed a high copy number of the trnM-CAT gene and a high prevalence of repetitive sequences, both of which likely contribute to genome rearrangement and adaptive evolution. Through nucleotide diversity and codon usage bias analyses, we identified specific genes that are crucial for adaptation to high-altitude conditions. Notably, genes such as atp6, ccmB, nad4L, and nad7 exhibited signs of positive selection, indicating the presence of unique adaptive traits for survival in extreme environments. Phylogenetic analysis confirmed the close relationship between the Elaeagnaceae family and other related families, whereas intergenomic sequence transfer analysis revealed a substantial presence of homologous fragments among the mitochondrial, chloroplast, and whole genomes, which may be linked to the high-altitude adaptation mechanisms of H. tibetana. The findings of this study not only enrich our knowledge of H. tibetana molecular biology but also advance our understanding of the adaptive evolution of plants on the Qinghai-Tibet Plateau. This study provides a solid scientific foundation for the molecular breeding, conservation, and utilization of H. tibetana genetic resources.

Genomic diversity, antibiotic resistance, and virulence in South African Enterococcus faecalis and Enterococcus lactis isolates

This study presents the empirical findings of an in-depth genomic analysis of Enterococcus faecalis and Enterococcus lactis isolates from South Africa. It offers valuable insights into their genetic characteristics and their significant implications for public health. The study uncovers nuanced variations in the gene content of these isolates, despite their similar GC contents, providing a comprehensive view of the evolutionary diversity within the species. Genomic islands are identified, particularly in E. faecalis, emphasizing its propensity for horizontal gene transfer and genetic diversity, especially in terms of antibiotic resistance genes. Pangenome analysis reveals the existence of a core genome, accounting for a modest proportion of the total genes, with 2157 core genes, 1164 shell genes, and 4638 cloud genes out of 7959 genes in 52 South African E. faecalis genomes (2 from this study, 49 south Africa genomes downloaded from NCBI, and E. faecalis reference genome). Detecting large-scale genomic rearrangements, including chromosomal inversions, underscores the dynamic nature of bacterial genomes and their role in generating genetic diversity. The study uncovers an array of antibiotic resistance genes, with trimethoprim, tetracycline, glycopeptide, and multidrug resistance genes prevalent, raising concerns about the effectiveness of antibiotic treatment. Virulence gene profiling unveils a diverse repertoire of factors contributing to pathogenicity, encompassing adhesion, biofilm formation, stress resistance, and tissue damage. These empirical findings provide indispensable insights into these bacteria’s genomic dynamics, antibiotic resistance mechanisms, and virulence potential, underlining the pressing need to address antibiotic resistance and implement robust control measures.

A genetic basis for sex differences in Xp11 translocation renal cell carcinoma

Xp11 translocation renal cell carcinoma (tRCC) is a rare, female-predominant cancer driven by a fusion between the transcription factor binding to IGHM enhancer 3 (TFE3) gene on chromosome Xp11.2 and a partner gene on either chromosome X (chrX) or an autosome. It remains unknown what types of rearrangements underlie TFE3 fusions, whether fusions can arise from both the active (chrXa) and inactive X (chrXi) chromosomes, and whether TFE3 fusions from chrXi translocations account for the female predominance of tRCC. To address these questions, we performed haplotype-specific analyses of chrX rearrangements in tRCC whole genomes. We show that TFE3 fusions universally arise as reciprocal translocations and that oncogenic TFE3 fusions can arise from chrXi:autosomal translocations. Female-specific chrXi:autosomal translocations result in a 2:1 female-to-male ratio of TFE3 fusions involving autosomal partner genes and account for the female predominance of tRCC. Our results highlight how X chromosome genetics constrains somatic chrX alterations and underlies cancer sex differences.

Comparative structure and evolution of the organellar genomes of Padina usoehtunii (Dictyotales) with the brown algal crown radiation clade

BackgroundOrganellar genomes have become increasingly essential for studying genetic diversity, phylogenetics, and evolutionary histories of seaweeds. The order Dictyotales (Dictyotophycidae), a highly diverse lineage within the Phaeophyceae, is long-term characterized by a scarcity of organellar genome datasets compared to orders of the brown algal crown radiation (Fucophycidae).ResultsWe sequenced the organellar genomes of Padina usoehtunii, a representative of the order Dictyotales, to investigate the structural and evolutionary differences by comparing to five other major brown algal orders. Our results confirmed previously reported findings that the rate of structural rearrangements in chloroplast genomes is higher than that in mitochondria, whereas mitochondrial sequences exhibited a higher substitution rate compared to chloroplasts. Such evolutionary patterns contrast with land plants and green algae. The expansion and contraction of the inverted repeat (IR) region in the chloroplast correlated with the changes in the number of boundary genes. Specifically, the size of the IR region influenced the position of the boundary gene rpl21, with complete rpl21 genes found within the IR region in Dictyotales, Sphacelariales and Ectocarpales, while the rpl21 genes in Desmarestiales, Fucales, and Laminariales span both the IR and short single copy (SSC) regions. The absence of the rbcR gene in the Dictyotales may indicate an endosymbiotic transfer from the chloroplast to the nuclear genome. Inversion of the SSC region occurred at least twice in brown algae. Once in a lineage only represented by the Ectocarpales in the present study and once in a lineage only represented by the Fucales. Photosystem genes in the chloroplasts experienced the strongest signature of purifying selection, while ribosomal protein genes in both chloroplasts and mitochondria underwent a potential weak purifying selection.ConclusionsVariations in chloroplast genome structure among different brown algal orders are evolutionarily linked to their phylogenetic positions in the Phaeophyceae tree. Chloroplast genomes harbor more structural rearrangements than the mitochondria, despite mitochondrial genes exhibiting faster mutation rates. The position and the change in the number of boundary genes likely shaped the IR regions in the chloroplast, and the produced structural variability is important mechanistically to create gene diversity in brown algal chloroplast.

Comparative Genomic Analysis of Campylobacter rectus and Closely Related Species.

Campylobacter rectus is a gram-negative, anaerobic bacterium strongly associated with periodontitis. It also causes various extraoral infections and is linked to adverse pregnancy outcomes in humans and murine models. C. rectus and related oral Campylobacters have been termed "emerging Campylobacter species" because infections by these organisms are likely underreported. Previously, no comparative methods have been used to analyze more than single C. rectus strains and until recently, very few C. rectus genomes have been publicly available. More sequenced genomes and comparative analyses are needed to study the genomic features and pathogenicity of this species. We sequenced eight new C. rectus strains and used comparative methods to identify regions of interest. An emphasis was put on the type III flagellar secretion system (T3SS), type IV secretion system (T4SS), and type VI secretion system (T6SS) because these protein complexes are important for pathogenesis in other Campylobacter species. RAST, BV-BRC, and other bioinformatics tools were used to assemble, annotate, and compare these regions in the genomes. The pan-genome of C. rectus consists of 2670 genes with core and accessory genomes of 1429 and 1241 genes, respectively. All isolates analyzed in this study have T3SS and T6SS hallmark proteins, while five of the isolates are missing a T4SS system. Twenty-one prophage clusters were identified across the panel of isolates, including four that appear intact. Overall, significant genomic islands were found, suggesting regions in the genomes that underwent horizontal gene transfer. Additionally, the high frequency of CRISPR arrays and other repetitive elements has led to genome rearrangements across the strains, including in areas adjacent to secretion system gene clusters. This study describes the substantial diversity present among C. rectus isolates and highlights tools/assays that have been developed to permit functional genomic studies. Additionally, we have expanded the studies on C. showae T4SS since we have two new C. showae genomes to report. We also demonstrate that unlike C. rectus , C showae does not demonstrate evidence of intact T6SS except for the strain CAM. The only strain of sequenced C. massilensis has neither T4SS or T6SS.

Morphological Structure Identification, Comparative Mitochondrial Genomics and Population Genetic Analysis toward Exploring Interspecific Variations and Phylogenetic Implications of Malus baccata 'ZA' and Other Species.

Malus baccata, a valuable germplasm resource in the genus Malus, is indigenous to China and widely distributed. However, little is known about the lineage composition and genetic basis of 'ZA', a mutant type of M. baccata. In this study, we compared the differences between 'ZA' and wild type from the perspective of morphology and ultrastructure and analyzed their chloroplast pigment content based on biochemical methods. Further, the complete mitogenome of M. baccata 'ZA' was assembled and obtained by next-generation sequencing. Subsequently, its molecular characteristics were analyzed using Geneious, MISA-web, and CodonW toolkits. Furthermore, by examining 106 Malus germplasms and 42 Rosaceae species, we deduced and elucidated the evolutionary position of M. baccata 'ZA', as well as interspecific variations among different individuals. In comparison, the total length of the 'ZA' mitogenome (GC content: 45.4%) is 374,023 bp, which is approximately 2.33 times larger than the size (160,202 bp) of the plastome (GC: 36.5%). The collinear analysis results revealed abundant repeats and genome rearrangements occurring between different Malus species. Additionally, we identified 14 plastid-driven fragment transfer events. A total of 54 genes have been annotated in the 'ZA' mitogenome, including 35 protein-coding genes, 16 tRNAs, and three rRNAs. By calculating nucleotide polymorphisms and selection pressure for 24 shared core mitochondrial CDSs from 42 Rosaceae species (including 'ZA'), we observed that the nad3 gene exhibited minimal variation, while nad4L appeared to be evolving rapidly. Population genetics analysis detected a total of 1578 high-quality variants (1424 SNPs, 60 insertions, and 94 deletions; variation rate: 1/237) among samples from 106 Malus individuals. Furthermore, by constructing phylogenetic trees based on both Malus and Rosaceae taxa datasets, it was preliminarily demonstrated that 'ZA' is closely related to M. baccata, M. sieversii, and other proximate species in terms of evolution. The sequencing data obtained in this study, along with our findings, contribute to expanding the mitogenomic resources available for Rosaceae research. They also hold reference significance for molecular identification studies as well as conservation and breeding efforts focused on excellent germplasms.

Genomic Landscape of Branchio-Oto-Renal Syndrome through Whole-Genome Sequencing: A Single Rare Disease Center Experience in South Korea.

Branchio-oto-renal (BOR) and branchio-otic (BO) syndromes are characterized by anomalies affecting the ears, often accompanied by hearing loss, as well as abnormalities in the branchial arches and renal system. These syndromes exhibit a broad spectrum of phenotypes and a complex genomic landscape, with significant contributions from the EYA1 gene and the SIX gene family, including SIX1 and SIX5. Due to their diverse phenotypic presentations, which can overlap with other genetic syndromes, molecular genetic confirmation is essential. As sequencing technologies advance, whole-genome sequencing (WGS) is increasingly used in rare disease diagnostics. We explored the genomic landscape of 23 unrelated Korean families with typical or atypical BOR/BO syndrome using a stepwise approach: targeted panel sequencing and exome sequencing (Step 1), multiplex ligation-dependent probe amplification (MLPA) with copy number variation screening (Step 2), and WGS (Step 3). Integrating WGS into our diagnostic pipeline detected structure variations, including cryptic inversion and complex genomic rearrangement, eventually enhancing the diagnostic yield to 91%. Our findings expand the genomic architecture of BOR/BO syndrome and highlight the need for WGS to address the genetic diagnosis of clinically heterogeneous rare diseases.

Evolutionary patterns and functional effects of 3D chromatin structures in butterflies with extensive genome rearrangements

Chromosome rearrangements may distort 3D chromatin architectures and thus change gene regulation, yet how 3D chromatin structures evolve in insects is largely unknown. Here, we obtain chromosome-level genomes for four butterfly species, Graphium cloanthus, Graphium sarpedon, Graphium eurypylus with 2n = 30, 40, and 60, respectively, and Papilio bianor with 2n = 60. Together with large-scale Hi-C data, we find that inter-chromosome rearrangements very rarely disrupted the pre-existing 3D chromatin structure of ancestral chromosomes. However, some intra-chromosome rearrangements changed 3D chromatin structures compared to the ancestral configuration. We find that new TADs and subTADs have emerged across the rearrangement sites where their adjacent compartments exhibit uniform types. Two intra-chromosome rearrangements altered Rel and lft regulation, potentially contributing to wing patterning differentiation and host plant choice. Notably, butterflies exhibited chromatin loops between Hox gene cluster ANT-C and BX-C, unlike Drosophila. Our CRISPR-Cas9 experiments in butterflies confirm that knocking out the CTCF binding site of the loops in BX-C affected the phenotypes regulated by Antp in ANT-C, resulting in legless larva. Our results reveal evolutionary patterns of insect 3D chromatin structures and provide evidence that 3D chromatin structure changes can play important roles in the evolution of traits.

De novo assembly and comprehensive analysis of the mitochondrial genome of Taxus wallichiana reveals different repeats mediate recombination to generate multiple conformations

Taxus plants are the exclusive source of paclitaxel, an anticancer drug with significant medicinal and economic value. Interspecies hybridization and gene introgression during evolution have obscured distinctions among Taxus species, complicating their phylogenetic classification. While the chloroplast genome of Taxus wallichiana, a widely distributed species in China, has been sequenced, its mitochondrial genome (mitogenome) remains uncharacterized.We sequenced and assembled the T. wallichiana mitogenome using BGI short reads and Nanopore long reads, facilitating comparisons with other gymnosperm mitogenomes. The T. wallichiana mitogenome spanning 469,949 bp, predominantly forms a circular configuration with a GC content of 50.51%, supplemented by 3 minor configurations mediated by one pair of LRs and two pairs of IntRs. It includes 32 protein-coding genes, 7 tRNA genes, and 3 rRNA genes, several of which exist in multiple copies.We detailed the mitogenome's structure, codon usage, RNA editing, and sequence migration between organelles, constructing a phylogenetic tree to elucidate evolutionary relationships. Unlike typical gymnosperm mitochondria, T. wallichiana shows no evidence of mitochondrial-plastid DNA transfer (MTPT), highlighting its unique genomic architecture. Synteny analysis indicated extensive genomic rearrangements in T. wallichiana, likely driven by recombination among abundant repetitive sequences. This study offers a high-quality T. wallichiana mitogenome, enhancing our understanding of gymnosperm mitochondrial evolution and supporting further cultivation and utilization of Taxus species.

Resolving the 22q11.2 deletion using CTLR-Seq reveals chromosomal rearrangement mechanisms and individual variance in breakpoints

We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.