Single Insertion Research Articles

Genome-Wide Identification and Expression Analysis of the CLAVATA3/ESR-Related Gene Family in Tiger Nut

CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-related (CLE) genes encode a group of peptide hormones, which coordinate cell proliferation and differentiation in plants. Tiger nut (Cyperus esculentus L.) is a perennial monocot plant that produces oil-rich tubers. However, the mechanisms regulating tuber development in tiger nut are poorly understood, and nothing is known about CLE genes in tiger nut. In this study, we identified 34 CLE genes in the genomes, proteomes, and transcriptomes of C. esculentus (CeCLE). We analyzed their gene structures and expression profiles in different parts of the plant, at three stages of tuber development and in roots in response to dehydration stress. We found a relatively high expression level of CeCLE13 in growing tuber and suggested that the corresponding CLE peptide could be involved in the regulation of tuberization. We also analyzed CeCLE gene sequences in the genome of the most productive K-17 variety in the N. I. Vavilov All-Russian Institute of Plant Genetic Resources collection and found many single nucleotide polymorphisms, insertions, and deletions. Our data provides fundamental information for future research on tiger nut growth and tuberization.

Assembly of Mitochondrial Genomes Using Nanopore Long-Read Technology in Three Sea Chubs (Teleostei: Kyphosidae).

Complete mitochondrial genomes have become markers of choice to explore phylogenetic relationships at multiple taxonomic levels and they are often assembled using whole genome short-read sequencing. Herein, using three species of sea chubs as an example, we explored the accuracy of mitochondrial chromosomes assembled using Oxford Nanopore Technology (ONT) Kit 14 R10.4.1 long reads at different sequencing depths (high, low and very low or genome skimming) by comparing them to 'gold' standard reference mitochondrial genomes assembled using Illumina NovaSeq short reads. In two species of sea chubs, Girella nigricans and Kyphosus azureus, ONT long-read assembled mitochondrial genomes at high sequencing depths (> 25× whole [nuclear] genome) were identical to their respective short-read assembled mitochondrial genomes. Not a single 'homopolymer insertion', 'homopolymer deletion', 'simple substitution', 'single insertion', 'short insertion', 'single deletion' or 'short deletion' were detected in the long-read assembled mitochondrial genomes after aligning each one of them to their short-read counterparts. In turn, in a third species, Medialuna californiensis, a 25× sequencing depth long-read assembled mitochondrial genome was 14 nucleotides longer than its short-read counterpart. The difference in total length between the latter two assemblies was due to the presence of a short motif 14 bp long that was repeated (twice) in the long read but not in the short-read assembly. Read subsampling at a sequencing depth of 1× resulted in the assembly of partial or complete mitochondrial genomes with numerous errors, including, among others, simple indels, and indels at homopolymer regions. At 3× and 5× subsampling, genomes were identical (perfect) or almost identical (quasiperfect, 99.5% over 16,500 bp) to their respective Illumina assemblies. The newly assembled mitochondrial genomes exhibit identical gene composition and organisation compared with cofamilial species and a phylomitogenomic analysis based on translated protein-coding genes suggested that the family Kyphosidae is not monophyletic. The same analysis detected possible cases of misidentification of mitochondrial genomes deposited in GenBank. This study demonstrates that perfect (complete and fully accurate) or quasiperfect (complete but with a single or a very few errors) mitochondrial genomes can be assembled at high (> 25×) and low (3-5×) but not very low (1×, genome skimming) sequencing depths using ONT long reads and the latest ONT chemistries (Kit 14 and R10.4.1 flowcells with SUP basecalling). The newly assembled and annotated mitochondrial genomes can be used as a reference in environmental DNA studies focusing on bioprospecting and biomonitoring of these and other coastal species experiencing environmental insult. Given the small size of the sequencing device and low cost, we argue that ONT technology has the potential to improve access to high-throughput sequencing technologies in low- and moderate-income countries.

Tubings, chord diagrams, and Dyson–Schwinger equations

AbstractWe give series solutions to single insertion place propagator‐type systems of Dyson–Schwinger equations using binary tubings of rooted trees. These solutions are combinatorially transparent in the sense that each tubing has a straightforward contribution. The Dyson–Schwinger equations solved here are more general than those previously solved by chord diagram techniques, including systems and noninteger values of the insertion parameter . We remark on interesting combinatorial connections and properties.

High fidelity DNA ligation prevents single base insertions in the yeast genome

Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1Cdc9 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3–5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1Cdc9-dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome.

Viral coexistence and insertional mutations in the ORF8 region of SARS-CoV-2: A possible mechanism of nucleotide insertion

The virus obtained from a swab sample ID: S66 in Hiroshima was reported to have a single T-base insertion in the ORF8 coding region. However, no T insertion was observed when we determined the genomic sequence using another method. We then extracted RNA from the S66 swab sample and sequenced the insertion site using the Sanger method. The resulting waveform was disrupted beyond the insertion site, suggesting the presence of a mixed population of viruses with different sequences. Through plasmid cloning of RT-PCR amplification fragments and virus cloning by limiting dilution, along with TIDE analysis to determine the ratio of components from the Sanger sequencing waveform, it was confirmed that the sample contained a mixture of viruses with varying numbers of T-base insertions. The virus with one T insertion (T1+) was predominant in 70–75 % of the genomes, and genomes with T0, T2+, T3+, T4+, and T5+ were also detected. No T-base insertion mutations were observed in the ORF8 region in three other SARS-CoV-2 samples. In the S66 sample, a C27911T point mutation near the insertion site in the ORF8 region resulted in a sequence of seven or more consecutive T bases, which was the cause of the T-base insertion. When the cloned S66 virus (T1+) was passaged in cultured cells, there was a tendency for viruses with more insertion bases to become dominant with successive generations, suggesting that the T-base insertion was due to polymerase stuttering. The insertion of T bases resulted in synthesis of deletion mutants of the ORF8 protein, but no significant change was observed in the proliferation of the viruses in cultured cells. A search of the GenBank database using NCBI BLAST for viruses similar to S66 with T-base insertion mutations revealed hundreds of viruses widely distributed on the molecular phylogenetic tree. These base insertion viruses were thought to have occasionally arisen during the virus infection process. This study suggests one mechanism of insertion mutations in SARS-CoV-2, and it is important to consider the emergence of future mutant strains.

Promoter Deletion Leading to Allele Specific Expression in a Genetically Unsolved Case of Primary Ciliary Dyskinesia.

Variation in the non-coding genome represents an understudied mechanism of disease and it remains challenging to predict if single nucleotide variants, small insertions and deletions, or structural variants in non-coding genomic regions will be detrimental. Our approach using complementary RNA-seq and targeted long-read DNA sequencing can prioritize identification of non-coding variants that lead to disease via alteration of gene splicing or expression. We have identified a patient with primary ciliary dyskinesia with a pathogenic coding variant on one allele of the SPAG1 gene, while the second allele appears normal by whole exome sequencing despite an autosomal recessive inheritance pattern. RNA sequencing revealed reduced SPAG1 transcript levels and exclusive allele specific expression of the known pathogenic allele, suggesting the presence of a non-coding variant on the second allele that impacts transcription. Targeted long-read DNA sequencing identified a heterozygous 3 kilobase deletion of the 5' untranslated region of SPAG1, overlapping the promoter and first non-coding exon. This non-coding deletion was missed by whole exome sequencing and gene-specific deletion/duplication analysis, highlighting the importance of investigating the non-coding genome in patients with "missing" disease-causing variation. This paradigm demonstrates the utility of both RNA and long-read DNA sequencing in identifying pathogenic non-coding variants in patients with unexplained genetic disease.

Phenotype-driven genomics enhance diagnosis in children with unresolved neuromuscular diseases.

Establishing a molecular diagnosis remains challenging in half of individuals with childhood-onset neuromuscular diseases (NMDs) despite exome sequencing. This study evaluates the diagnostic utility of combining genomic approaches in undiagnosed NMD patients. We performed deep phenotyping of 58 individuals with unsolved childhood-onset NMDs that have previously undergone inconclusive exome studies. Genomic approaches included trio genome sequencing and RNASeq. Genetic diagnoses were reached in 23 out of 58 individuals (40%). Twenty-one individuals carried causal single nucleotide variants (SNVs) or small insertions and deletions, while 2 carried pathogenic structural variants (SVs). Genomic sequencing identified pathogenic variants in coding regions or at the splice site in 17 out of 21 resolved cases, while RNA sequencing was additionally required for the diagnosis of 4 cases. Reasons for previous diagnostic failures included low coverage in exonic regions harboring the second pathogenic variant and involvement of genes that were not yet linked to human diseases at the time of the first NGS analysis. In summary, our systematic genetic analysis, integrating deep phenotyping, trio genome sequencing and RNASeq, proved effective in diagnosing unsolved childhood-onset NMDs. This approach holds promise for similar cohorts, offering potential improvements in diagnostic rates and clinical management of individuals with NMDs.

ASPYRE-Lung: validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in FFPE tissue.

Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions. The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma.

Right ventriculography to guide left bundle branch pacing in pacing-induced cardiomyopathy: a novel case report

Abstract Background There is emerging evidence for the potential utility of left bundle branch area pacing (LBBAP), as an alternative to conventional cardiac resynchronization therapy (CRT). The utility of right ventriculography by way of power injector to facilitate lead placement has not yet been reported in the literature. Case summary A 79-year-old female, with a background of poorly rate-controlled atrial fibrillation, presented with worsening dyspnoea. She had recently undergone single-chamber pacemaker insertion prior to an atrioventricular nodal (AVN) ablation, owing to failure in achieving successful CRT coronary sinus lead placement. She had clinical evidence of volume overload, and her electrocardiogram demonstrated right ventricular pacing. Echocardiography demonstrated left ventricular (LV) impairment, with an ejection fraction (EF) of 35%, and severe functional mitral regurgitation (MR). Her diagnosis was overall consistent with pacing-induced cardiomyopathy (PIC). In this patient, the use of right ventriculography, using power-injector-delivered contrast, successfully facilitated placement of an LBBAP lead, with confirmation of good threshold and sensing parameters. Following an upgrade to conduction system pacing, the patient recovered well. On recent follow-up, repeat echocardiography (24 months post initial presentation) demonstrated improved LV function (EF 45% from 35%) and only mild-to-moderate MR. Discussion In conclusion, we demonstrate the utility of right ventriculography to facilitate placement of an LBBAP lead, successfully treating a patient who developed PIC from chronic right ventricular pacing following AVN ablation.

A comprehensive guide to bioinformatics tools and databases

The interest in the functional aspects of the genome has increased eventually with the expansion of genomic databases. In this review, various genetic alterations within the genome, such as Single Nucleotide Polymorphisms (SNPs), disease-associated mutations, neutral polymorphisms, and small insertions or deletions (INDELs), have been identified and characterized. These polymorphisms, particularly SNPs, have become widely employed in genetic analyses, serving as valuable molecular markers. They are applied across diverse genetic studies, including the reconstruction of haplotypes. In addition to experimental methods for investigating specific genetic variants, there has been a significant surge in bioinformatics research over the past decade, aimed at the molecular consequences of these genetic alterations. Haplotypes, which represent various alleles of a gene, can be reconstructed not only for entire chromosomes but also for specific genes, utilizing the information derived from SNPs. The computational approach to SNP and haplotype discovery has gained prominence due to the continuous expansion of sequence data within public databases. This growth enables more precise identification of SNPs. This article aims to introduce easily accessible and practical online tools for researchers. In summary, SNP and haplotype identification tools are indispensable in bioinformatics for studying genetic variation, population genetics, disease associations, and personalized medicine. They provide valuable insights into the genetic underpinnings of various traits and diseases, helping advance our understanding of genetics and its applications in healthcare.

Genetic evolution analysis of Chinese bayberry germplasm resources in Southern Zhejiang with single nucleotide polymorphism (SNP) and insertion deletion (InDel) markers

Genetic evolution analysis of Chinese bayberry germplasm resources in Southern Zhejiang with single nucleotide polymorphism (SNP) and insertion deletion (InDel) markers

Mutations in the efflux regulator gene oqxR provide a simple genetic switch for antimicrobial resistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.

SIMBA-A Single-Puncture Approach to Lower Limb Block.

Peripheral nerve blocks provide a safe and reliable alternative in the anesthetic management of femur fractures in elderly subpopulations associated with significant comorbidities. Single-Insertion Multiple Nerve Block Anesthesia (SIMBA) is a technique where a single needle insertion is used to block all four nerves that supply the femur shaft: the femoral nerve, obturator nerve, lateral femoral cutaneous nerve, and sciatic nerve. The authors performed this technique in 11 cardiac compromised geriatric patients with midshaft/distal femur fractures, and the surgery was conducted successfully without any significant hemodynamic change and good postoperative analgesia.

Genomic landscape of early-stage prostate adenocarcinoma in Mexican patients: an exploratory study

BackgroundHealth disparities have been highlighted among patient with prostate adenocarcinoma (PRAD) due to ethnicity. Mexican men present a more aggressive disease than other patients resulting in less favorable treatment outcome. We aimed to identify the mutational landscape which could help to reduce the health disparities among minority groups and generate the first genomics exploratory study of PRAD in Mexican patients.MethodsParaffin-embedded formalin-fixed tumoral tissue from 20 Mexican patients with early-stage PRAD treated at The Instituto Nacional de Cancerología, Mexico City from 2017 to 2019 were analyzed. Tumoral DNA was prepared for whole exome sequencing, the resulting files were mapped against h19 using BWA-MEM. Strelka2 and Lancet packages were used to identify single nucleotide variants (SNV) and insertions or deletions. FACETS was used to determine somatic copy number alterations (SCNA). Cancer Genome Interpreter web interface was used to determine the clinical relevance of variants.ResultsPatients were in an early clinical stage and had a mean age of 59.55 years (standard deviation [SD]: 7.1 years) with 90% of them having a Gleason Score of 7. Follow-up time was 48.50 months (SD: 32.77) with recurrences and progression in 30% and 15% of the patients, respectively. NUP98 (20%), CSMD3 (15%) and FAT1 (15%) were the genes most frequently affected by SNV; ARAF (75%) and ZNF419 (70%) were the most frequently affected by losses and gains SNCA’s. One quarter of the patients had mutations useful as biomarkers for the use of PARP inhibitors, they comprise mutations in BRCA, RAD54L and ATM. SBS05, DBS03 and ID08 were the most common mutational signatures present in this cohort. No associations with recurrence or progression were identified.ConclusionsThis pilot study reveals the mutational landscape of early-stage prostate adenocarcinoma in Mexican men, providing a first approach to understand the mutational patterns and actionable mutations in early prostate cancer can inform personalized treatment approaches and reduce the underrepresentation in genomic cancer studies.

Molecular protocol to develop β-carotene-biofortified rice events via molecular optimization

Providing food with nutrition and functionality is crucial for sustaining human life. Rice (Oryza sativa L.) is a representative staple crop with high carbohydrate content but low amounts of essential amino acids, micronutrients, and carotenoids such as provitamin A. To improve the nutritional quality, rice endosperm was biofortified to accumulate carotenoids such as β-carotene through genetic engineering (i.e., using synthetic carotenoid biosynthetic genes, a nonmammalian viral polycistronic sequence, and an optimized promoter and transit peptide) and high-throughput rice transformation (approximately 300 transgenic plants per construct). To facilitate the safety assessment of genetically modified food, molecular characterization was performed to select elite lines equipped with a single intergenic insertion of T-DNA, high transgene expression, in this case leading to high carotenoid content, and with phenotypic and compositional substantial equivalence. In this study, we present β-carotene-biofortified rice event candidate lines eligible for commercial use and a disclosed molecular protocol for the development of biotech rice crops.

BAASIK technique: an innovative single needle technique of performing shoulder corticosteroid injections.

The novel BAASIK (B-Botchu,Bipin, A-Agrawal, A-Ankit, S- Sindhura, I-Iyengar, K- Kapil) technique is a combined ultrasound-guided injection into the subacromial-subdeltoid (SASD) bursa and the biceps tendon sheath to treat shoulder pain associated with biceps tendonitis/tendinopathy, subacromial bursitis, rotator cuff impingement or to determine the source of shoulder pain. This technique aims in reducing shoulder pain, improving functional activities and enhancing treatment delivery. A single-entry supine ultrasound-guided injection of the subacromial-subdeltoid (SASD) bursa and biceps tendon sheath involves using a single needle insertion point to access both structures for therapeutic injection. This technique could become a favoured alternative technique, rather than subjecting patients to two needlesticks and preparing twoseparate injections to address often concomitant pathologies.

SARS-CoV-2 Omicron subvariant genomic variation associations with immune evasion in Northern California: A retrospective cohort study.

The possibility of association between SARS-CoV-2 genomic variation and immune evasion is not known among persons with Omicron variant SARS-CoV-2 infection. In a retrospective cohort, using Poisson regression adjusting for sociodemographic variables and month of infection, we examined associations between individual non-lineage defining mutations and SARS-CoV-2 immunity status, defined as a) no prior recorded infection, b) not vaccinated but with at least one prior recorded infection, c) complete primary series vaccination, and/or d) primary series vaccination and ≥ 1 booster. We identified all non-synonymous single nucleotide polymorphisms (SNPs), insertions and deletions in SARS-CoV-2 genomes with ≥5% allelic frequency and population frequency of ≥5% and ≤95%. We also examined correlations between the presence of SNPs with each other, with subvariants, and over time. Seventy-nine mutations met inclusion criteria. Among 15,566 persons infected with Omicron SARS-CoV-2, 1,825 (12%) were unvaccinated with no prior recorded infection, 360 (2%) were unvaccinated with a recorded prior infection, 13,381 (86%) had a complete primary series vaccination, and 9,172 (58%) had at least one booster. After examining correlation between SNPs, 79 individual non-lineage defining mutations were organized into 38 groups. After correction for multiple testing, no individual SNPs or SNP groups were significantly associated with immunity status levels. Genomic variation identified within SARS-CoV-2 Omicron specimens was not significantly associated with immunity status, suggesting that contribution of non-lineage defining SNPs to immune evasion is minimal. Larger-scale surveillance of SARS-CoV-2 genomes linked with clinical data can help provide information to inform future vaccine development.

Diverse Proteomimetic Frameworks via Rational Design of π-Stacking Peptide Tectons.

Peptide-based frameworks aim to integrate protein architecture into solid-state materials using simpler building blocks. Despite the growing number of peptide frameworks, there are few strategies to rationally engineer essential properties like pore size and shape. Designing peptide assemblies is generally hindered by the difficulty of predicting complex networks of weak intermolecular interactions. Peptides conjugated to polyaromatic groups are a unique case where assembly appears to be strongly driven by π-π interactions, suggesting that rationally adjusting the geometry of the π-stackers could create novel structures. Here, we report peptide elongation as a simple mechanism to predictably tune the angle between the π-stacking groups to produce a remarkable diversity of pore shapes and sizes, including some that are mesoporous. Notably, rapid jumps in pore size and shape can occur with just a single amino acid insertion. The geometry of the π-stacking residues also significantly influences framework structure, representing an additional dimension for tuning. Lastly, sequence identity can also indirectly modulate the π-π interactions. By correlating each of these factors with detailed crystallographic data, we find that, despite the complexity of peptide structure, the shape and polarity of the tectons are straightforward predictors of framework structure. These guidelines are expected to accelerate the development of advanced porous materials with protein-like capabilities.

Abstract Mo075: Cigarette smoking causes somatic mutations in cardiac endothelial cells

Cigarette smoking (CS) is a major contributor to cardiovascular diseases (CVDs), responsible for one in every four CVD-related deaths. Despite this, the precise mechanisms through which smoking induces CVDs remain elusive. The chemicals in cigarette smoke, particularly oxidants, are known to instigate oxidative stress and activate the vessel wall. Alongside the dysregulation of cellular functions, oxidative stress can lead to DNA damage. Accumulation of DNA damage and somatic mutations is a recognized hallmark of aging, a significant risk factor for various age-related diseases, including CVDs, neurodegenerative diseases, and cancer. Recent studies have indicated a substantial increase in somatic mutations, with mutational signatures reflective of oxidative stress, in bronchial epithelial cells from smokers. Given their proximity in the blood circulation, these pathophysiological findings could naturally extend to the coronary system. Therefore, we hypothesized that CS induces somatic mutations and injuries in cardiac endothelial cells (ECs), laying the mechanisms for cigarette smoking-induced CVDs. To test this hypothesis, we conducted single-cell whole-genome sequencing (scWGS) on 105 cardiac endothelial cells isolated from postmortem hearts of 6 normal individuals, 3 smokers, and 4 smokers with atherosclerotic cardiovascular disease (ASCVD). Our results revealed a significant increase in somatic single nucleotide variants (SNVs) and insertions and deletions (indels) in cardiac ECs from smokers and those with ASCVD. These excess somatic mutations exhibited Catalogue Of Somatic Mutations In Cancer (COSMIC) mutational signatures, including SBS4, SBS29, SBS40, SBS92, and ID3, indicative of DNA damage and mutagenesis associated with cigarette smoking, oxidative stress, and formaldehyde exposure. Further analysis of single-nucleus RNA sequencing (snRNA-seq) data from postmortem hearts of 3 normal individuals, 4 smokers, and 1 smoker with ASCVD demonstrated upregulated pathways involved in apoptosis, EC migration, and vascular development. In conclusion, our study demonstrates that chemicals in cigarette smoke exert direct genotoxic effects on cardiac ECs, mirroring their impact on bronchial epithelial cells. Cigarette smoking significantly accelerates the accumulation of somatic mutations in cardiac ECs, resulting in apoptosis and vascular damage. Consequently, this damage leads to a secondary activation of angiogenesis for vascular repair.

A Specialized Reference Panel with Structural Variants Integration for Improving Genotype Imputation in Alzheimer's Disease and Related Dementias (ADRD).

We developed an imputation panel for Alzheimer's disease (AD) and related dementias (ADRD) using whole-genome sequencing (WGS) data from the Alzheimer's Disease Sequencing Project (ADSP). Recognizing the significant associations between structural variants (SVs) and AD, and their underrepresentation in existing public reference panels, our panel uniquely integrates single nucleotide variants (SNVs), short insertions and deletions (indels), and SVs. This panel enhances the imputation of disease susceptibility, including rare AD-associated SNVs, indels, and SVs, onto genotype array data, offering a cost-effective alternative to whole-genome sequencing while significantly augmenting statistical power. Notably, we discovered 10 rare indels nominal significant related to AD that are absent in the TOPMed-r2 panel and identified three suggestive significant (p-value < 1E-05) AD-associated SVs in the genes EXOC3L2 and DMPK, were identified. These findings provide new insights into AD genetics and underscore the critical role of imputation panels in advancing our understanding of complex diseases like ADRD.