Genomic Inversions Research Articles

MAPT haplotype-associated transcriptomic changes in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a neurodegenerative movement and cognitive disorder characterized by abnormal accumulation of the microtubule-associated protein tau in the brain. Biochemically, inclusions in PSP are enriched for tau proteoforms with four microtubule-binding domain repeats (4R), an isoform that arises from alternative tau pre-mRNA splicing. While preferential aggregation and reduced degradation of 4R tau protein is thought to play a role in inclusion formation and toxicity, an alternative hypothesis is that altered expression of tau mRNA isoforms plays a causal role. This stems from the observation that PSP is associated with common variation in the tau gene (MAPT) at the 17q21.31 locus which contains low copy number repeats flanking a large recurrent genomic inversion. The complex genomic structural changes at the locus give rise to two dominant haplotypes, termed H1 and H2, that have the potential to markedly influence gene expression. Here, we explored haplotype-dependent differences in gene expression using a bulk RNA-seq dataset derived from human post-mortem brain tissue from PSP (n = 84) and controls (n = 77) using a rigorous computational pipeline, including alternative pre-mRNA splicing. We found 3579 differentially expressed genes in the temporal cortex and 10,011 in the cerebellum. We also found 7214 differential splicing events in the temporal cortex and 18,802 in the cerebellum. In the cerebellum, total tau mRNA levels and the proportion of transcripts encoding 4R tau were significantly increased in PSP compared to controls. In the temporal cortex, the proportion of reads that expressed 4R tau was increased in cases compared to controls. 4R tau mRNA levels were significantly associated with the H1 haplotype in the temporal cortex. Further, we observed a marked haplotype-dependent difference in KANSL1 expression that was strongly associated with H1 in both brain regions. These findings support the hypothesis that sporadic PSP is associated with haplotype-dependent increases in 4R tau mRNA that might play a causal role in this disorder.

The black honey bee genome: insights on specific structural elements and a first step towards pangenomes

BackgroundThe honey bee reference genome, HAv3.1, was produced from a commercial line sample that was thought to have a largely dominant Apis mellifera ligustica genetic background. Apis mellifera mellifera, often referred to as the black bee, has a separate evolutionary history and is the original type in western and northern Europe. Growing interest in this subspecies for conservation and non-professional apicultural practices, together with the necessity of deciphering genome backgrounds in hybrids, triggered the necessity for a specific genome assembly. Moreover, having several high-quality genomes is becoming key for taking structural variations into account in pangenome analyses.ResultsPacific Bioscience technology long reads were produced from a single haploid black bee drone. Scaffolding contigs into chromosomes was done using a high-density genetic map. This allowed for re-estimation of the recombination rate, which was over-estimated in some previous studies due to mis-assemblies, which resulted in spurious inversions in the older reference genomes. The sequence continuity obtained was very high and the only limit towards continuous chromosome-wide sequences seemed to be due to tandem repeat arrays that were usually longer than 10 kb and that belonged to two main families, the 371 and 91 bp repeats, causing problems in the assembly process due to high internal sequence similarity. Our assembly was used together with the reference genome to genotype two structural variants by a pangenome graph approach with Graphtyper2. Genotypes obtained were either correct or missing, when compared to an approach based on sequencing depth analysis, and genotyping rates were 89 and 76% for the two variants.ConclusionsOur new assembly for the Apis mellifera mellifera honey bee subspecies demonstrates the utility of multiple high-quality genomes for the genotyping of structural variants, with a test case on two insertions and deletions. It will therefore be an invaluable resource for future studies, for instance by including structural variants in GWAS. Having used a single haploid drone for sequencing allowed a refined analysis of very large tandem repeat arrays, raising the question of their function in the genome. High quality genome assemblies for multiple subspecies such as presented here, are crucial for emerging projects using pangenomes.

Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota

Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory Tcells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented "OFF" in IBD patients, which correlated with increased B.fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B.fragilis, induction of colitis caused a decline of PSA in the "ON" orientation that reversed as inflammation resolved. Monocolonization of mice with B.fragilis revealed that bacteriophage infection increased the frequency of PSA in the "OFF" orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease.

Cas12a RNP-mediated co-transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated canker-resistant Citrus sinensis cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, CsLOB1, which led to small indels. Here, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs. Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker- resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants. Taken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.

Comparative genomics analysis of Streptococcus iniae isolated from Trachinotus ovatus: novel insight into antimicrobial resistance and virulence differentiation

BackgroundStreptococcus iniae is an important fish pathogen that cause significant economic losses to the global aquaculture industry every year. Although there have some reports on the genotype of S.iniae and its relationship with virulence, no genome-scale comparative analysis has been performed so far. In our previous work, we characterized 17 isolates of S.iniae from Trachinotus ovatus and divided them into two genotypes using RAPD and rep-PCR methods. Among them, BH15-2 was classified as designated genotype A (in RAPD) and genotype 1 (in rep-PCR), while BH16-24 was classified as genotype B and genotype 2. Herein, we compared the differences in growth, drug resistance, virulence, and genome between BH15-2 and BH16-24.ResultsThe results showed that the growth ability of BH16-24 was significantly faster than that of BH15-2 at the exponential stage. Antimicrobial tests revealed that BH15-2 was susceptible to most of the tested antibiotics except neomycin and gentamycin. In contrast, BH16-24 was resistant to 7 antibiotics including penicillin, sulfasomizole, compound sulfamethoxazole tablets, polymyxin B, spectinomycin, rifampin and ceftazidime. Intraperitoneal challenge of T.ovatus, showed that the LD50 value of BH15-2 was 4.0 × 102 CFU/g, while that of BH16-24 was 1.2 × 105 CFU/g. The genome of S.iniae BH15-2 was 2,175,659 bp with a GC content of 36.80%. Meanwhile, the genome of BH16-24 was 2,153,918 bp with a GC content of 36.83%. Comparative genome analysis indicated that compared with BH15-2, BH16-24 genome had a large-scale genomic inversion fragment, at the location from 502,513 bp to 1,788,813 bp, resulting in many of virulence and resistance genes differentially expression. In addition, there was a 46 kb length, intact phage sequence in BH15-2 genome, which was absent in BH16-24.ConclusionComparative genomic studies of BH15-2 and BH16-24 showed that the main difference is a 1.28 Mbp inversion fragment. The inversion fragment may lead to abnormal expression of drug resistant and virulence genes, which is believed to be the main reason for the multiple resistance and weakened virulence of BH16-24. Our study revealed the potential mechanisms in underlying the differences of multidrug resistance and virulence among different genotypes of S.iniae.

Genomic Characterization of 2 Cutibacterium acnes Isolates from a Surgical Site Infection Reveals Large Genomic Inversion.

Cutibacterium acnes is a common commensal of human skin but may also present as an opportunistic pathogen in prosthetic joint and wound infections. Unfortunately, few complete genomes of C. acnes are publicly available, and even fewer are of isolates associated with infection. Here we report the isolation, characterization, and complete genomes of 2 C. acnes isolates from a surgical site infection of an elbow. We used standard microbiological methods for phenotypic characterization and performed whole genome sequencing on 2 C. acnes isolates using a combination of short-read and long-read sequencing. Antibiotic susceptibility testing showed beta-lactamase negative and low minimal inhibitory concentrations to all antibiotics tested, with the exception of metronidazole. We assembled complete genomes of the 2 isolates, which are approximately 2.5 megabases in length. The isolates belong to the single-locus sequence type (SLST) H1 and the multi-locus sequence type (MLST) IB. Both isolates have similar composition of known virulence genes, and we found no evidence of plasmids but did find phage-associated genes. Notably, the 2 genomes are 99.97% identical but contain a large genomic inversion encompassing approximately half of the genome. This is the first characterization of this large-scale genomic inversion in nearly identical isolates from the same wound. This report adds to the limited numbers of publicly available infection-associated complete genomes of C. acnes.

Polymyxin resistance caused by large-scale genomic inversion due to IS26 intramolecular translocation in Klebsiella pneumoniae.

Polymyxin B and polymyxin E (colistin) are presently considered the last line of defense against human infections caused by multidrug-resistant Gram-negative organisms such as carbapenemase-producer Enterobacterales, Acinetobacter baumannii, and Klebsiella pneumoniae. Yet resistance to this last-line drugs is a major public health threat and is rapidly increasing. Polymyxin S2 (S2) is a polymyxin B analogue previously synthesized in our institute with obviously high antibacterial activity and lower toxicity than polymyxin B and colistin. To predict the possible resistant mechanism of S2 for wide clinical application, we experimentally induced bacterial resistant mutants and studied the preliminary resistance mechanisms. Mut-S, a resistant mutant of K. pneumoniae ATCC BAA-2146 (Kpn2146) induced by S2, was analyzed by whole genome sequencing, transcriptomics, mass spectrometry and complementation experiment. Surprisingly, large-scale genomic inversion (LSGI) of approximately 1.1 Mbp in the chromosome caused by IS26 mediated intramolecular transposition was found in Mut-S, which led to mgrB truncation, lipid A modification and hence S2 resistance. The resistance can be complemented by plasmid carrying intact mgrB. The same mechanism was also found in polymyxin B and colistin induced drug-resistant mutants of Kpn2146 (Mut-B and Mut-E, respectively). This is the first report of polymyxin resistance caused by IS26 intramolecular transposition mediated mgrB truncation in chromosome in K. pneumoniae. The findings broaden our scope of knowledge for polymyxin resistance and enriched our understanding of how bacteria can manage to survive in the presence of antibiotics.

Retrospective Evaluation of Cytogenetic Effects Induced by Internal Radioiodine Exposure: A 27-Year Follow-Up Study

Radioiodine (131I) is widely used in the treatment of hyperthyroidism and as an effective ablative therapy for differentiated thyroid cancer. Radioiodine (131I) constitutes 90% of the currently used therapies in the field of nuclear medicine. Here, we report the cytogenetic findings of a long-term follow-up study of 27 years on a male patient who received two rounds of radioiodine treatment within a span of 26 months between 1992 and 1994 for his papillary thyroid cancer. A comprehensive cytogenetic follow-up study utilizing cytokinesis blocked micronucleus assay, dicentric chromosome assay, genome wide translocations and inversions was initiated on this patient since the first administration of radioiodine in 1992. Frequencies of micronuclei (0.006/cell) and dicentric chromosomes (0.008/cell) detected in the current study were grossly similar to that reported earlier in 2019. The mFISH analysis detected chromosome aberrations in 8.6% of the cells in the form of both unbalanced and balanced translocations. Additionally, a clonal translocation involving chromosomes 14p; 15q was observed in 2 of the 500 cells analyzed. Out of the 500 cells examined, one cell showed a complex translocation (involving chromosomes 9, 10, and 16) besides 5 other chromosome rearrangements. Collectively, our study indicates that the past radioiodine exposure results in long-lasting chromosome damage and that the persistence of translocations can be useful for both retrospective biodosimetry and for monitoring chromosome instability in the lymphocytes of radioiodine exposed individuals.

Genetic study reveals local differentiation persisting in the face of high connectivity and a genomic inversion likely linked with sexual antagonism in a common marine fish

Abstract Sustainable harvest of wild populations requires knowledge of the underlying population structure. The focus of this study is on goldsinny wrasse (Ctenolabrus rupestris), a small marine fish inhabiting coastal waters of the north-eastern Atlantic. This species is caught in large numbers to serve as cleaner fish in salmonid aquaculture. We genotyped 2073 goldsinny wrasse from 43 sites along the Scandinavian coastline with 143 SNPs. Seven of the SNPs were linked and likely reside within a large genomic inversion dominated by one haplotype. The heterokaryotype of the putative inversion displayed sex-specific growth patterns, potentially resolving sexual antagonism for this trait. The unlinked 134 SNPs showed modest isolation-by-distance with samples from the northernmost locations showing highest divergence, whereas sites farther south were much more interconnected. Genetic divergence (FST) was highly variable among sites within regions, suggesting a varying degree of connectivity and local divergence. We conclude that despite a high degree of gene-flow mediated through pelagic dispersal in early life stages, regional and some local population structure remains due to limited adult movement in addition to other unidentified factors. Consequently, the species might be more vulnerable to local disturbances than previously anticipated.

Structural genome variants of Pseudomonas aeruginosa clone C and PA14 strains.

Plasticity of Pseudomonas aeruginosa chromosomes is mainly driven by an extended accessory genome that is shaped by insertion and deletion events. Further modification of the genome composition can be induced by chromosomal inversion events which lead to relocation of genes in the affected genomic DNA segments, modify the otherwise highly conserved core genome synteny and could even alter the location of the replication terminus. Although the genome of the first sequenced strain, PAO1, displayed such a large genomic inversion, knowledge on such recombination events in the P. aeruginosa population is limited. Several large inversions had been discovered in the late 1990s in cystic fibrosis isolates of the major clonal lineage C by physical genome mapping, and subsequent work on these examples led to the characterization of the DNA at the recombination breakpoints and a presumed recombination mechanism. Since then, the topic was barely addressed in spite of the compilation of thousands of P. aeruginosa genome sequences that are deposited in databases. Due to the use of second-generation sequencing, genome contig assembly had usually followed synteny blueprints provided by the existing reference genome sequences. Inversion detection was not feasible by these approaches, as the respective read lengths did not allow reliable resolution of sequence repeats that are typically found at the borders of inverted segments. In this study, we applied PacBio and MinION long-read sequencing to isolates of the mentioned clone C collection. Confirmation of inversions predicted from the physical mapping data demonstrated that unbiased sequence assembly of such read datasets allows the detection of genomic inversions and the resolution of the recombination breakpoint regions. Additional long-read sequencing of representatives of the other major clonal lineage, PA14, revealed large inversions in several isolates, from cystic fibrosis origin as well as from other sources. These findings indicated that inversion events are not restricted to strains from chronic infection background, but could be widespread in the P. aeruginosa population and contribute to genome plasticity. Moreover, the monitored examples emphasized the role of small mobile DNA units, such as IS elements or transposons, and accessory DNA elements in the inversion-related recombination processes.

Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.

The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.

Targeted genomic translocations and inversions generated using a paired prime editing strategy

A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.

Genomic architecture and functional effects of potential human inversion supergenes.

Supergenes are involved in adaptation in multiple organisms, but they are little known in humans. Genomic inversions are the most common mechanism of supergene generation and maintenance. Here, we review the information about two large inversions that are the best examples of potential human supergenes. In addition, we do an integrative analysis of the newest data to understand better their functional effects and underlying genetic changes. We have found that the highly divergent haplotypes of the 17q21.31 inversion of approximately 1.5 Mb have multiple phenotypic associations, with consistent effects in brain-related traits, red and white blood cells, lung function, male and female characteristics and disease risk. By combining gene expression and nucleotide variation data, we also analysed the molecular differences between haplotypes, including gene duplications, amino acid substitutions and regulatory changes, and identify CRHR1, KANLS1 and MAPT as good candidates to be responsible for these phenotypes. The situation is more complex for the 8p23.1 inversion, where there is no clear genetic differentiation. However, the inversion is associated with several related phenotypes and gene expression differences that could be linked to haplotypes specific of one orientation. Our work, therefore, contributes to the characterization of both exceptional variants and illustrates the important role of inversions.This article is part of the theme issue ‘Genomic architecture of supergenes: causes and evolutionary consequences’.

The early-life exposome modulates the effect of polymorphic inversions on DNA methylation

Polymorphic genomic inversions are chromosomal variants with intrinsic variability that play important roles in evolution, environmental adaptation, and complex traits. We investigated the DNA methylation patterns of three common human inversions, at 8p23.1, 16p11.2, and 17q21.31 in 1,009 blood samples from children from the Human Early Life Exposome (HELIX) project and in 39 prenatal heart tissue samples. We found inversion-state specific methylation patterns within and nearby flanking each inversion region in both datasets. Additionally, numerous inversion-exposure interactions on methylation levels were identified from early-life exposome data comprising 64 exposures. For instance, children homozygous at inv-8p23.1 and higher meat intake were more susceptible to TDH hypermethylation (P = 3.8 × 10−22); being the inversion, exposure, and gene known risk factors for adult obesity. Inv-8p23.1 associated hypermethylation of GATA4 was also detected across numerous exposures. Our data suggests that the pleiotropic influence of inversions during development and lifetime could be substantially mediated by allele-specific methylation patterns which can be modulated by the exposome.

Flip-flop genomics: Charting inversions in the human population

Detecting large genomic inversions has long been challenging. In a new study, Porubsky etal. resolve these complex rearrangements in 41 individuals and discover wide regions that undergo recurrent inversions, some of which even toggle back and forth (Porubsky etal., 2022). Many of these regions are associated with genomic disorders.

Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals.

Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (αLA-LPH Tg goat, Akr1A1eGFP/eGFP Tg mouse, and NFκB-Luc Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (αLA-AP1 GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5′ homologous arm inverted repeat donor plasmid (5′IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, p < 0.05). However, the KI rate of the 3′ inverted terminal repeat/inverted repeat donor plasmid (3′ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3′-overhang integration into the host genome via homologous recombination machinery.

Genomic Iterative Replacements of Large Synthetic DNA Fragments in Corynebacterium glutamicum.

Synthetic genomics will advance our understanding of life and allow us to rebuild the genomes of industrial microorganisms for enhancing performances. Corynebacterium glutamicum, a Gram-positive bacterium, is an important industrial workhorse. However, its genome synthesis is impeded by the low efficiencies in DNA delivery and in genomic recombination/replacement. In the present study, we describe a genomic iterative replacement system based on RecET recombination for C. glutamicum, involving the successive integration of up to 10 kb DNA fragments obtained in vitro, and the transformants are selected by the alternative use of kanR and speR selectable markers. As a proof of concept, we systematically redesigned and replaced a 54.3 kb wild-type sequence of C. glutamicumATCC13032 with its 55.1 kb synthetic counterpart with several novel features, including decoupled genes, the standard PCRTags, and 20 loxPsym sites, which was for the first time incorporated into a bacterial genome. The resulting strain semi-synCG-A1 had a phenotype and fitness similar to the wild-type strain under various stress conditions. The stability of the synthetic genome region faithfully maintained over 100 generations of nonselective growth. Genomic deletions, inversions, and translocations occurred in the synthetic genome region upon induction of synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE), revealing potential genetic flexibility for C. glutamicum. This strategy can be used for the synthesis of a larger region of the genome and facilitate the endeavors for metabolic engineering and synthetic biology of C. glutamicum.

The spinach YY genome reveals sex chromosome evolution, domestication, and introgression history of the species

BackgroundSpinach (Spinacia oleracea L.) is a dioecious species with an XY sex chromosome system, but its Y chromosome has not been fully characterized. Our knowledge about the history of its domestication and improvement remains limited.ResultsA high-quality YY genome of spinach is assembled into 952 Mb in six pseudo-chromosomes. By a combination of genetic mapping, Genome-Wide Association Studies, and genomic analysis, we characterize a 17.42-Mb sex determination region (SDR) on chromosome 1. The sex chromosomes of spinach evolved when an insertion containing sex determination genes occurred, followed by a large genomic inversion about 1.98 Mya. A subsequent burst of SDR-specific repeats (0.1–0.15 Mya) explains the large size of this SDR. We identify a Y-specific gene, NRT1/PTR 6.4 which resides in this insertion, as a strong candidate for the sex determination or differentiation factor. Resequencing of 112 spinach genomes reveals a severe domestication bottleneck approximately 10.87 Kya, which dates the domestication of spinach 7000 years earlier than the archeological record. We demonstrate that a strong selection signal associated with internode elongation and leaf area expansion is associated with domestication of edibility traits in spinach. We find that several strong genomic introgressions from the wild species Spinacia turkestanica and Spinacia tetrandra harbor desirable alleles of genes related to downy mildew resistance, frost resistance, leaf morphology, and flowering-time shift, which likely contribute to spinach improvement.ConclusionsAnalysis of the YY genome uncovers evolutionary forces shaping nascent sex chromosome evolution in spinach. Our findings provide novel insights about the domestication and improvement of spinach.

EP460: Diagnostic dilemma in a case of suspected fetal skeletal dysplasia

Intrauterine fetal fractures are a rare finding and can represent lethal skeletal dysplasias such as Osteogenesis Imperfecta, or other metabolic or structural abnormalities. More than 400 skeletal dysplasias have been described; some are associated with classical modes of inheritance, while others are associated with imprinting errors, mosaicism, and teratogen exposure. Accurate prenatal diagnosis is critical for appropriate patient counseling, pregnancy management and expected postnatal outcomes. Prenatal ultrasound (US) remains the standard imaging modality for identifying prenatal structural abnormalities; this, coupled with genetic testing, can aid in establishing diagnoses. Available clinical testing includes chromosome microarray, panel testing, exome, and genome sequencing (ES and GS, respectively). Despite the availability of ES and GS, the genetic or molecular mechanism may remain elusive. Here we present a case of suspected campomelic dysplasia which remains a diagnostic dilemma despite thorough genetic evaluation. A 31-year-old Gravida 2 Para 1-0-0-0 at 20 weeks gestation presented for anatomy US at our Maternal Fetal Medicine clinic. This was an IVF pregnancy. Her obstetric history is complicated by one prior emergency Cesarean delivery for a 35-week fetus with decreased fetal movement and non-reassuring fetal heart tones. The neonate passed 5 days after birth with severe encephalopathy of unknown etiology. The patient and partner are otherwise healthy and have no pertinent medical or surgical history. Family history was non-contributory. Initial anatomy US for current pregnancy revealed a singleton female fetus with severely bowed femurs bilaterally with concern for fracture on the right femur, talipes equinovarus of left foot, with all bones showing normal ossification. The fetus was growth restricted at <10th percentile with all shortened long bones. Markers of lethality of skeletal dysplasia including cardiothoracic (CT) ratio was 53% (normal <50%), and femur length (FL): abdominal circumference (AC) ratio was 0.11 (Normal >0.16). These findings prompted diagnostic genetic work up with amniocentesis sent for FISH, chromosome microarray, and a 700 gene skeletal dysplasia panel; all of which returned normal. Sonographic surveillance continued to show a constellation of findings concerning for skeletal dysplasia including bilateral proximal femoral diaphyseal bowing/fractures, left clubfoot, 11 total ribs, micrognathia, chest-to-abdominal circumference CC/AC ratio 0.87, and FL/AC ratio 0.19 (considered severe when CC/AC <0.60 or FL/AC <0.16). Fetal MRI at 32 weeks demonstrated normal fetal brain structure, short femurs, normal lung volumes, unilateral talipes and micrognathia. The patient underwent prenatal ES, which was negative for pathogenic or likely pathogenic variants associated with the prenatal phenotype. The patient underwent a repeat Cesarean Delivery at 38w2d and delivered a liveborn female neonate weighing 2670 grams with Apgar scores of 7 and 9 at 1 and 5 minutes respectively. ENT was available at bedside for possible neonatal airway compromise. On postnatal physical exam, the neonate was normocephalic with age appropriate fontanelles. There was micrognathia with intermittent airway obstruction and evidence of Pierre Robin sequence. The spine was straight without lesions, both femurs were shortened with clubbing of the left foot. All fingers and toes appeared normal. A skeletal survey revealed 12 ossified rib pairs, hip dislocation, lateral bowing/angulation of the mid femoral diaphyses, as well as club feet bilaterally. No fractures were identified. With exception of hip abduction, she had appropriate movement of her extremities. Postnatal expanded GS was pursued; results revealed no pathogenic, likely pathogenic or variants of uncertain significance. Further research -based testing was pursued through Optical Mapping; this revealed several genomic insertion, deletion, inversion, duplication, and translocation events involving multiple genes. However, after review by our Center for Individualized Medicine (CIM) Genomic Odyssey Board (GOB) team, none of the reported genomic events were felt to be a good fit with the infant’s phenotype. This case remains a diagnostic dilemma. Rapid GS and research based optical mapping failed to identify an underlying genetic etiology. This case illustrates challenges associated with prenatal diagnosis of suspected skeletal dysplasias.

Genomic inversions in Escherichia coli alter gene expression and are associated with nucleoid protein binding sites

Genomic reorganization, such as rearrangements and inversions, influences how genetic information is organized within the bacterial genomes. Inversions, in particular, facilitate genome evolution through gene gain and loss, and can alter gene expression. Previous studies have investigated the impact inversions have on gene expression induced inversions targeting specific genes or examine inversions between distantly related species. This fails to encompass a genome-wide perspective of naturally occurring inversions and their post-adaptation impact on gene expression. Here, we used bioinformatic techniques and multiple RNA-seq datasets to investigate the short- and long-range impact inversions have on genomic gene expression within Escherichia coli. We observed differences in gene expression between homologous inverted and non-inverted genes even after long-term exposure to adaptive selection. In 4% of inversions representing 33 genes, differential gene expression between inverted and non-inverted homologs was detected, with greater than two-thirds (71%) of differentially expressed inverted genes having 9.4–85.6-fold higher gene expression. The identified inversions had more overlap than expected with nucleoid-associated protein binding sites, which assist in the regulation of genomic gene expression. Some inversions can drastically impact gene expression, even between different strains of E. coli, and could provide a mechanism for the diversification of genetic content through controlled expression changes.