Intron Sequences Research Articles

Five novel RB1 gene mutations and genotype–phenotype correlations in Chinese children with retinoblastoma

PurposeTo identify the spectrum of RB1 gene mutations in 114 Chinese patients with retinoblastoma.MethodsGenomic DNA was extracted from the peripheral blood of 114 Rb patients. Polymerase chain reactions (PCRs) followed by direct Sanger sequencing were used to screen for mutations in the RB1 gene, which contains 26 exons with flanking intronic sequences, except exon 15. Clinical data, including gender, age at diagnosis, laterality of ocular lesions, and associated symptoms, were recorded and compared.ResultsWe identified five novel mutations in the RB1 gene. Twenty-five other mutations found in this study have been previously reported. A higher rate of RB1 mutations, with 47.3% of mutations among bilaterally affected patients vs. 6.8% within unilaterally affected patients, was also observed (p < 0.0001). Bilaterally affected patients were diagnosed earlier when compared to unilaterally affected patients (11 ± 7 months versus 20 ± 14 months, p = 0.0002). Furthermore, nonsense mutations were abundant (n = 14), followed by frameshift mutations (n = 8), splicing site mutations (n = 5), while missense mutations were few (n = 3).ConclusionsWe found five novel mutations in RB1 genes, which expands the mutational spectrum of the gene. Children with bilateral Rb exhibited higher mutation rates and were diagnosed earlier than those with unilateral Rb. These findings will inform clinical diagnosis and genetic therapeutic targeting in Rb patients.

Neoantigen-based cancer vaccination using chimeric RNA-loaded dendritic cell-derived extracellular vesicles.

Cancer vaccines critically rely on the availability of targetable immunogenic cancer‐specific neoepitopes. However, mutation‐based immunogenic neoantigens are rare or even non‐existent in subgroups of cancer types. To address this issue, we exploited a cancer‐specific aberrant transcription‐induced chimeric RNA, designated A‐Pas chiRNA, as a possible source of clinically relevant and targetable neoantigens. A‐Pas chiRNA encodes a recently discovered cancer‐specific chimeric protein that comprises full‐length astrotactin‐2 (ASTN2) C‐terminally fused in‐frame to the antisense sequence of the 18th intron of pregnancy‐associated plasma protein‐A (PAPPA). We used extracellular vesicles (EVs) from A‐Pas chiRNA‐transfected dendritic cells (DCs) to produce the cell‐free anticancer vaccine DEXA‐P. Treatment of immunocompetent cancer‐bearing mice with DEXA‐P inhibited tumour growth and prolonged animal survival. In summary, we demonstrate for the first time that cancer‐specific transcription‐induced chimeric RNAs can be exploited to produce a cell‐free cancer vaccine that induces potent CD8+ T cell‐mediated anticancer immunity. Our novel approach may be particularly useful for developing cancer vaccines to treat malignancies with low mutational burden or without mutation‐based antigens. Moreover, this cell‐free anticancer vaccine approach may offer several practical advantages over cell‐based vaccines, such as ease of scalability and genetic modifiability as well as enhanced shelf life.

Effective splicing restoration of a deep-intronic ABCA4 variant in cone photoreceptor precursor cells by CRISPR/SpCas9 approaches

Stargardt disease is an autosomal recessively inherited retinal disorder commonly caused by pathogenic variants in the ABCA4 gene encoding the ATP-binding cassette subfamily A member 4 (ABCA4) protein. Several deep-intronic variants in ABCA4 have been classified as disease causing. By strengthening a cryptic splice site, deep-intronic variant c.5197-557G>T induces the inclusion of a 188-bp intronic sequence in the mature mRNA, resulting in a premature termination codon. Here, we report the design and evaluation of three CRISPR-Cas9 approaches implementing Streptococcus pyogenes Cas9 (single and dual guide RNA) or Streptococcus pyogenes Cas9 nickase (dual guide RNA) for their potential to correct c.5197-557G>T-induced aberrant splicing in minigene splicing assays and patient-derived cone photoreceptor precursor cells. The different strategies were able to rescue correct splicing by up to 83% and increase the overall correctly spliced transcripts by 1.8-fold, demonstrating the successful CRISPR-Cas9-mediated rescue in patient-derived photoreceptor precursor cells of an ABCA4 splicing defect. The results provide initial evidence of possible permanent splicing correction for Stargardt disease, expanding the therapeutic toolbox to counteract deep-intronic pathogenic variants in ABCA4.

Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate "Diseased" Hepatocytes for Accurate Diagnosis.

The diagnosis of inherited metabolic disorders is a long and tedious process. The matching of clinical data with a genomic variant in a specific metabolic pathway is an essential step, but the link between a genome and the clinical data is normally difficult, primarily for new missense variants or alterations in intron sequences. Notwithstanding, elucidation of the pathogenicity of a specific variant might be critical for an accurate diagnosis. In this study, we described a novel intronic variant c.2597 + 5G > T in the donor splice sequence of the PHKA2 gene. To investigate PHKA2 mRNA splicing, as well as the functional consequences on glycogen metabolism, we generated hepatocyte-like cells from a proband’s fibroblasts by direct reprogramming. We demonstrated an aberrant splicing of PHKA2, resulting in the incorporation of a 27 bp upstream of intron 23 into exon 23, which leads to an immediate premature STOP codon. The truncated protein was unable to phosphorylate the PYGL protein, causing a 4-fold increase in the accumulation of glycogen in hepatocyte-like cells. Collectively, the generation of personalized hepatocyte-like cells enabled an unequivocal molecular diagnosis and qualified the sister’s proband, a carrier of the same mutation, as a candidate for a preimplantation genetic diagnosis. Additionally, our direct reprogramming strategy allows for an unlimited source of “diseased” hepatocyte-like cells compatible with high-throughput platforms.

Revealing molecular architecture of FLT3 internal tandem duplication: Development and clinical validation of a web-based application to generate accurate nomenclature.

FLT3 internal tandem duplicate (ITD) is associated with unfavorable prognosis of acute myeloid leukemia; targeted therapy improves clinical outcome. We propose that FLT3-ITD detected by next generation sequencing (NGS) should be reported with the same nomenclature pattern as single nucleotide variants so that the mutation can be better interpreted clinically. A Python-based web application was developed to generate FLT3-ITD nomenclature as recommended by the Human Genome Variation Society (HGVS). Assembled FLT3-ITD sequences from 84 patients and 11 artificially created ITD sequences were used for the validation of this web-based application. Each sequence was inspected manually to confirm that the nomenclature was accurate. Accurate nomenclatures were generated for 113 of 114 sequencing results and 7 artificial sequences. One assembled sequence and four artificial sequences were not named accurately; warning statements were automatically generated to alert further inspection. Of the 105 unique FLT3-ITDs, the ITD lengths range from 18 to 300 bp. Depending whether the ITD involves intron or extends into exon 15, three patterns were recognized. Only 44 (42%) ITDs were pure duplications, and three types of variants were identified at the 5' of ITD. When ITD involves intronic sequence, the protein may comprise inserted amino acids encoded by the intron, due to disrupted RNA splicing. The web application generates accurate FLT3-ITD nomenclature from NGS results except in rare situations. The HGVS nomenclatures provide information on the molecular architecture of FLT3-ITDs and reveal details of complex insertions with partial duplications.

Internal Introns Promote Backsplicing to Generate Circular RNAs from Spinal Muscular Atrophy Gene.

Human survival motor neuron 1 (SMN1) codes for SMN, an essential housekeeping protein involved in most aspects of RNA metabolism. Deletions or mutations of SMN1 lead to spinal muscular atrophy (SMA), a devastating neurodegenerative disease linked to a high rate of infant mortality. SMN2, a near identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 due to predominant skipping of SMN2 exon 7. Restoration of SMN by splicing modulation of SMN2 exon 7 or gene replacement are currently approved therapies of SMA. Human SMN genes produce a vast repertoire of circular RNAs (circRNAs). However, the mechanism of SMN circRNA generation has not yet been examined in detail. For example, it remains unknown if forward splicing impacts backsplicing that generates circRNAs containing multiple exons. Here, we employed SMN as a model system to examine the impact of intronic sequences on the generation of circRNAs. We performed our experiments in HeLa cells transiently transfected with minigenes expressing three abundantly represented circRNAs containing two or more SMN exons. We observed an enhanced rate of circRNA generation when introns joining exons to be incorporated into circRNAs were present as compared to the intronless context. These results underscore the stimulatory effect of forward splicing in the generation of circRNAs containing multiple exons. These findings are consistent with the reported low abundance of SMN circRNAs comprised of single exons. We confirmed our findings using inducible HEK 293 cells stably expressing the SMN circRNAs. Our results support the role of the exon junction complex in the generation of the exon-only-containing circRNAs. We showed that SMN circRNAs were preferentially localized in the cytoplasm. These findings provide new insights regarding our understanding of circRNA generation and open avenues to uncover novel functions of the SMN genes.

Can Introns Stabilize Gene Duplication?

Simple SummaryEukaryotic genes are organized as DNA sequences containing exon and intron regions. Exons relate to sequences that, after transcription, will be maintained in mature mRNA to provide the blueprint for protein translation. Introns, on the other hand, are present in the primary transcript and are then removed by the splicing mechanisms. The evolutionary solutions that maintain and make this complex gene organization functional are only partially known. Here, we speculate that the presence of introns in the gene sequence can stabilize the products of gene duplication, one of the most effective driving forces in genome evolution. The hypothesis we propose is to be considered additional to those currently reported and not as an alternative.Gene duplication is considered one of the most important events that determine the evolution of genomes. However, the neo-duplication condition of a given gene is particularly unstable due to recombination events. Several mechanisms have been proposed to justify this step. In this “opinion article” we propose a role for intron sequences in stabilizing gene duplication by limiting and reducing the identity of the gene sequence between the two duplicated copies. A review of the topic and a detailed hypothesis are presented.

The evolution of pre-mRNA splicing and its machinery revealed by reduced extremophilic red algae.

The Cyanidiales are a group of mostly thermophilic and acidophilic red algae that thrive near volcanic vents. Despite their phylogenetic relationship, the reduced genomes of Cyanidioschyzon merolae and Galdieria sulphuraria are strikingly different with respect to pre-mRNA splicing, a ubiquitous eukaryotic feature. Introns are rare and spliceosomal machinery is extremely reduced in C.merolae, in contrast to G.sulphuraria. Previous studies also revealed divergent spliceosomes in the mesophilic red alga Porphyridium purpureum and the red algal derived plastid of Guillardia theta (Cryptophyta), along with unusually high levels of unspliced transcripts. To further examine the evolution of splicing in red algae, we compared C.merolae and G.sulphuraria, investigating splicing levels, intron position, intron sequence features, and the composition of the spliceosome. In addition to identifying 11 additional introns in C. merolae, our transcriptomic analysis also revealed typical eukaryotic splicing in G. sulphuraria, whereas most transcripts in C. merolae remain unspliced. The distribution of intron positions within their host genes was examined to provide insight into patterns of intron loss in red algae. We observed increasing variability of 5' splice sites and branch donor regions with increasing intron richness. We also found these relationships to be connected to reductions in and losses of corresponding parts of the spliceosome. Our findings highlight patterns of intron and spliceosome evolution in related red algae under the pressures of genome reduction.

Analyses of Pepper Cinnamoyl-CoA Reductase Gene Family and Cloning of CcCCR1/2 and Their Function Identification in the Formation of Pungency

Cinnamoyl-CoA reductases (CCR) have a possible role in pungency formation of pepper because they can convert feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA into lignin, which are also competitive precursors of capsaicin biosynthesis in phenylpropanoid metabolism. In this study, genome-wide CCR gene family, exon–intron structures, sequence homology, phylogenetic characterization, and promoters were analyzed in pepper. Two CCR genes were cloned from Capsicum chinense, their enzymic kinetic parameters and regulatory function were identified by heterologous expression, ectopic expression, and VIGS. In total, 38 genes were found as predicted CCRs or CCR-like proteins and were composed of 2–10 exons. The promoters of pepper CCRs contained growth, stress, hormone, and light-response elements. The affinity and catalytic efficiency of CcCCR1/2 to feruolyl-CoA was the highest. The analysis of metabolic substances showed that capsaicin content was negatively correlated with lignin and positively correlated with flavonoids. The highest expression of CcCCR1 was found in stems, the higher expression of CcCCR2 was found in stem and early fruit than other organs. CCR1, 2 had certain effects on capsaicin content by regulating related enzyme activity, CCR2 played a more important role in regulating pungency formation. Our results clarify the competitive mechanism between lignin and capsaicin biosynthesis and provide an explanation for spice regulation.

Substitution rate heterogeneity across hexanucleotide contexts in noncoding chloroplast DNA.

Substitutions between closely related noncoding chloroplast DNA sequences are studied with respect to the composition of the 3 bases on each side of the substitution, that is the hexanucleotide context. There is about 100-fold variation in rate, among the contexts, particularly on substitutions of A and T. Rate heterogeneity of transitions differs from that of transversions, resulting in a more than 200-fold variation in the transitions: transversion bias. The data are consistent with a CpG effect, and it is shown that both the A + T content and the arrangement of purines/pyrimidines along the same DNA strand are correlated with rate variation. Expected equilibrium A + T content ranges from 36.4% to 82.8% across contexts, while G–C skew ranges from −77.4 to 72.2 and A–T skew ranges from −63.9 to 68.2. The predicted equilibria are associated with specific features of the content of the hexanucleotide context, and also show close agreement with the observed context-dependent compositions. Finally, by controlling for the content of nucleotides closer to the substitution site, it is shown that both the third and fourth nucleotide removed on each side of the substitution directly influence substitution dynamics at that site. Overall, the results demonstrate that noncoding sites in different contexts are evolving along very different evolutionary trajectories and that substitution dynamics are far more complex than typically assumed. This has important implications for a number of types of sequence analysis, particularly analyses of natural selection, and the context-dependent substitution matrices developed here can be applied in future analyses.

Development of Intron Polymorphism Markers and Their Association With Fatty Acid Component Variation in Oil Palm.

Oil palm (Elaeis guineensis Jacq.) is a tropical woody oil crop of the palm family and is known as “the oil king of the world,” but its palm oil contains about 50% palmitic acid, which is considered unhealthy for humans. Intron polymorphisms (IP) are highly efficient and easily examined molecular markers located adjacent to exon regions of functional genes, thus may be associated with targeted trait variation. In order to speed up the breeding of oil palm fatty acid composition, the current study identified a total of 310 introns located within 52 candidate genes involved in fatty acid biosynthesis in the oil palm genome. Based on the intron sequences, 205 primer pairs were designed, 64 of which showed polymorphism among 70 oil palm individuals. Phenotypic variation of fatty acid content in the 70 oil palm individuals was also investigated. Association analysis revealed that 13 IP markers were significantly associated with fatty acid content variation, and these IP markers were located on chromosomes 2, 5, 6, 8, 9, and 10 of oil palm. The development of such IP markers may be useful for the genetic improvement of fatty acid composition in oil palm.

1459-P: Human Islet Cell Transcriptome Analysis Using Single Nucleus RNA-seq Reveals New Beta-Cell Markers and Define Distinct Beta-Cell Subpopulations with Different Transcriptional Activity

Single-cell RNA sequencing (scRNA-seq) of human islets requires cell dissociation and does not provide information on the transcriptional status of islet cells. On the other hand, single-nucleus RNA sequencing (snRNA-seq) does not require cell dissociation and provides abundant information on intronic sequences that can be used to identify actively transcribed genes. Based on this, we seek to compare scRNA-seq and snRNA-seq approaches in human islets to determine: 1) whether similar cell clusters could be detected; 2) whether new gene markers could be discovered in human islet endocrine cells using intronic reads; 3) whether human beta cell subpopulations could be identified based on INS expression dynamics; and 4) whether snRNA-seq could be used in human islet grafts transplanted in immunosuppressed mice. We performed scRNA-seq and snRNA-seq on three pairs of human islets obtained from three healthy adult human donors using exon only or exon plus intron reads, respectively. Analysis of integrated data revealed similar human islet cell clusters. In the snRNA-seq data, however, top differentially expressed genes were identified as new markers of human endocrine cells such as ZNF385D (β-cells) , PTPRT (α-cells) , LRFN5 (δ-cells) and CACNA2D3 (PP cells) . These markers also accurately define endocrine cell populations in human islet grafts. Additionally, we distinguished several beta cell sub-clusters- INS rich cluster, HNRNPA2B1 (vesicle) rich cluster and active INS transcribing cluster. In conclusion, snRNA-seq analysis of human islet cells is a previously unrecognized tool for the identification of human islet cell types in samples where nuclear RNA processing is required. By comparing INS expression between scRNA-seq and snRNA-seq data sets, we can detect different beta cell sub-populations with distinct gene expression patterns representing different biological dynamic states. Disclosure R.Kang: None. Y.Li: None. C.Rosselot: None. D.Scott: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. G.Lu: None. Funding NIH DK105015

A cell-free RNA-based next-generation sequencing (NGS) assay for the detection of actionable gene fusions in patients with non–small cell lung cancer (NSCLC).

3040 Background: Gene fusions and alternate RNA splice forms represent clinically actionable driver and resistance-conferring alterations in NSCLC and other cancers. Where tissue samples are inadequate for molecular testing, liquid biopsies could address the gap in detection of clinically relevant gene fusions. Targeted cell-free (cf) DNA-based NGS methods are typically used for non-invasive blood-based testing of gene fusions, but have limited sensitivity if fusion breakpoints involve long intronic regions or repetitive sequences (e.g. NTRKs and NRG1). RNA-based approaches are not affected by introns, can identify expressed fusion genes and can discriminate splicing. We developed an NGS assay optimized for cfRNA analyte for the detection of actionable gene fusions and exon deletion/skipping events in liquid biopsies. Methods: A highly multiplexed molecular-barcoded primer panel was designed for cfRNA-based detection of actionable fusion genes in NSCLC ( ALK, BRAF, FGFR2, FGFR3, MET (including exon 14 skipping), NRG1, NTRK1/2/3, RET and ROS1) covering &gt; 90% of reported driver and partner gene exons in COSMIC database. The panel also targets housekeeping genes as endogenous sample controls. We developed a custom bioinformatics pipeline to call fusions and exon skipping based on split and spanning reads. Results: In initial analytical validation using fragmented RNA from pre-characterized reference material (representing 32 unique fusions at known copy numbers), the assay could detect as low as 10 fusion copies with a sensitivity of 97.6% and a specificity of 100%. For clinical testing, cfRNA co-eluted with cfDNA in nucleic acid extracts from plasma was used. In plasma samples (n = 103) from advanced NSCLC, 76% (15/21) of fusions (5 ALK, 3 RET, 2 ROS1, 5 MET ex14 skipping, 1 FGFR3-TACC3) detected in a clinically validated cfDNA assay (LiquidHALLMARK) were also detected in corresponding cfRNA. In a subset of samples that were driver-negative untreated or tyrosine kinase-inhibitor treated, the cfRNA assay yielded 9 gene fusions or breakpoints (1 CD74-NRG1, 4 BRAF, 2 MET, 2 FGFR3-TACC3 fusions) that could not be detected by the cfDNA assay. This represents an increase of 8.7% (9/103) of actionable alterations (driver or resistance) identified using cfRNA. Together cfRNA and cfDNA resulted in 30 fusion events to be detected compared to 21 by the cfDNA assay alone, representing a 42.8% (9/21) increase in fusion-specific detection of the combined assay. Conclusions: This novel cfRNA assay can detect actionable gene fusions and exon skipping events in liquid biopsies with high sensitivity. Combining cfRNA with more routine cfDNA testing can increase the total actionable diagnostic information from non-invasive testing in NSCLC patients where tissue samples are lacking, especially for gene fusions not amenable to detection in cfDNA.

Telomere dynamics in female Columbian ground squirrels: recovery after emergence and loss after reproduction.

Telomeres are specialized non-coding DNA sequences locatedat the end of chromosomesand that protect genetic information. Telomere loss over lifespan is generally viewed as a phenomenon associated with aging in animals. Recently, telomere elongation after hibernation has been described in several mammals. Whether this pattern is an adaptation to repair DNA damage caused during rewarming from torpor or if it coevolved as a mechanism to promote somatic maintenance in preparation for the upcoming reproductive effort remains unclear. In a longitudinal study measuring telomere length using buccal swabs, we tested if telomere elongation was related to reproductive success in wild adult female Columbian ground squirrels (Urocitellus columbianus) that were monitored from emergence from hibernation to the end of the reproductive season. We found three key results. First, female telomere length increased at the start of the breeding season, both in breeding and non-breeding individuals. Second, post-emergence telomere lengthening was unrelated to female future reproductive output. Third, telomere length decreased in breeding females during lactation, but remained stable in non-breeding females over a similar period. Within breeders, telomeres shortened more in females producing larger and heavier litters. We concluded that telomere lengthening after hibernation did not constrain immediate female reproductive capacities. It was more likely to be part of the body recovery process that takes place after hibernation. Telomere erosion that occurs after birth may constitute a physiological cost of female reproduction.

Phylogenetic analysis using SCoT markers and chloroplast trnL intron in some Eriobotrya japonica (Thunb.) Lindl. (Rosaceae) populations from the Aegean region of Turkey

In this study, phylogenetic analyses of some Eriobotrya japonica (Thunb.) Lindl. populations based on SCoT marker technique and cpDNA trnL intron sequence analysis were performed. Specimens from the populations were collected from the Aegean region of Turkey and brought to the laboratory for genomic DNA isolation. To determine the phylogenetic analysis of Eriobotrya japonica populations, seven SCoT primers were used. In the SCoT analysis, a total of 54 bands were obtained, 49 of which were polymorphic and 5 were monomorphic. In chloroplast trnL intron region, trnC, trnD primers were used for PCR amplification. Eriobotrya japonica trnL intron sequence lengths were determined to be between 530 and 547 nucleotides. The average nucleotide rate detected as 38.9% for thymine, 18.1% for cytosine, 28.2% for adenine and 14.8% for guanine. Rosaceae family trnL intron sequences belonging to Rhaphiolepis Lindl., Heteromeles M.Roem., Photinia Lindl. Cotoneaster Medik, Sorbus L., Malus Miller, Pyrus L., Prunus L. and Rosa L. were retrieved from NCBI and their phylogenetic relationship with Eriobotrya japonica populations were revealed. As a result of the study, polymorphism rate was determined as 90.74% in SCoT analysis. According to cpDNA trnL intron results, Eriobotrya japonica populations were identified in the same group with Rhaphiolepis indica, a finding supported by past phylogenetic analyzes.

Prevalence and characteristics of a feline parvovirus-like virus in dogs in China

In this study, 192 diarrheal fecal samples were collected from 2019 to 2021 for monitoring the molecular prevalence of canine parvovirus 2 (CPV-2) among dogs in Southwest China, and 113 samples were detected as Carnivore protoparvovirus 1-positive. Surprisingly, 28/113 (24.8%) strains were identified as feline parvovirus (FPV)-like viruses based on the key amino acid (aa) residues in VP2. Further, 6 FPV-like strains were successfully isolated and genome sequenced, and phylogenetic trees based on the genome, VP2 and NS1 sequences showed that the 6 FPV-like strains were most genetically related with FPV instead of CPV-2. Interestingly, the VP2 proteins of the FPV-like virus contained all key aa residues typical for FPV and can be 100% identical to that of FPV, but the VP1 intron and NS1 aa sequences exhibited some unique molecular characteristics. The FPV-like isolate could hemagglutinate swine erythrocyte at pH values between 6 and 8, and replicated efficiently in MDCK cell line; moreover, the virus could cause canine systemic infection via oral administration. Further analysis based on VP2 sequences of FPV and CPV-2 in GenBank revealed that the FPV-like virus had already existed among dogs in 4 Asian countries, and have circulated widely in China. This study first confirmed that the FPV-like isolates could efficiently infect dogs, and has been prevalent among dogs in China. Moreover, this study first reported the genome characteristics of the FPV-like virus in dogs, which may represent a novel evolution pattern involving in the cross-species transmission of the virus from cats to dogs.

Single-nucleus chromatin accessibility profiling highlights regulatory mechanisms of coronary artery disease risk.

Coronary artery disease (CAD) is a complex inflammatory disease involving genetic influences across cell types. Genome-wide association studies have identified over 200 loci associated with CAD, where the majority of risk variants reside in noncoding DNA sequences impacting cis-regulatory elements. Here, we applied single-nucleus assay for transposase-accessible chromatin with sequencing to profile 28,316 nuclei across coronary artery segments from 41 patients with varying stages of CAD, which revealed 14 distinct cellular clusters. We mapped ~320,000 accessible sites across all cells, identified cell-type-specific elements and transcription factors, and prioritized functional CAD risk variants. We identified elements in smooth muscle cell transition states (for example, fibromyocytes) and functional variants predicted to alter smooth muscle cell- and macrophage-specific regulation of MRAS (3q22) and LIPA (10q23), respectively. We further nominated key driver transcription factors such as PRDM16 and TBX2. Together, this single-nucleus atlas provides a critical step towards interpreting regulatory mechanisms across the continuum of CAD risk.

The functions, oncogenic roles, and clinical significance of circular RNAs in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common form of malignancy affecting the kidneys. Circular RNAs (circRNAs) are non-coding RNAs that are derived from exonic or intronic sequences through a selective shearing process. There is growing evidence that these circRNAs can influence a range of biological pathways by serving as protein decoys, microRNA sponges, regulators of transcriptional activity, or templates for protein translation. The dysregulation of circRNA expression patterns is a hallmark of RCC and other cancer types, and there is strong evidence that these RNA species can play central roles in the onset and progression of RCC tumors. In the present review, we summarized recent findings on the functional roles and clinical impacts of circRNAs in RCC. Further, we discussed their potential utility as diagnostic biomarkers or targets for therapeutic intervention.

Characterization and genomic analyses of a novel alphabaculovirus isolated from the black armyworm, Spodoptera cosmioides (Lepidoptera: Noctuidae)

The black armyworm Spodoptera cosmioides is a pest of increasing importance in Cry1Ac-Bt toxin crops and non-Bt crops of soybean and cotton in Brazil. Here we characterized a baculovirus isolated from extracts of S. cosmioides that died with symptoms of nuclear polyhedrosis. The putative novel virus exhibited polyhedral occlusion bodies (OBs) with virions containing multiple rod-shaped nucleocapsids, characteristic of alphabaculoviruses. The virus isolate was named Spodoptera cosmioides nucleopolyhedrovirus isolate CNPSo-72 (SpcoNPV-CNPSo-72). SpcoNPV-CNPSo-72 was lethal to third-instar S. cosmioides caterpillars but not to S. frugiperda under the tested viral concentrations. Moreover, SpcoNPV-CNPSo-72 contained a circular 147,763 bp long genome and a G + C content of 44.8% with 151 annotated ORFs (10 unique for baculovirus) and five homologous regions (hrs). The 38 currently defined baculovirus core genes were found in the SpcoNPV-CNPSo-72 genome. After phylogenetic analysis, the novel virus was found to be closely related to other members of Alphabaculovirus, especially to the Spodoptera-infecting viruses, which included Spodoptera eridania nucleopolyhedrovirus isolate 251, Spodoptera litura nucleopolyhedrovirus isolate II, Spodoptera exigua multiple nucleopolyhedrovirus isolate US-1, Spodoptera eridania nucleopolyhedrovirus isolate CNPSo-165, and Spodoptera frugiperda multiple nucleopolyhedrovirus isolate 19. Surprisingly, the new baculoviral genome was found to code for a putative arginine-associated tRNA gene with a predicted intronic sequence of 105 nt. The gene was found inside the bjdp CDS. Overall, baculoviruses are pathogens that lethally infect insect larvae and their study allows a better understanding of large DNA virus evolution, which provides important insights for the development and improvement of biological control agents.

RNA analysis of the GALNS transcript reveals novel pathogenic mechanisms associated with Morquio syndrome A

Morquio syndrome A (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficiency of N-acetyl-galactosamine-6-sulfatase (GALNS) which catabolizes the glycosaminoglycans (GAG), keratan sulfate and chondroitin-6-sulfate. Homozygous or compound heterozygous pathogenic variants in the GALNS result in the deficiency of the enzyme and consequent GAG accumulations. DNA sequence and copy number analysis of the GALNS coding region fails to identify biallelic causative pathogenic variants in up to 15% of patients with Morquio syndrome A. RNA transcript analysis was performed to identify pathogenic alterations in two unrelated families with Morquio syndrome A in whom a single heterozygous or no pathogenic alteration was detected by standard analysis of the GALNS gene. RNA sequencing and quantitative expression analysis identified the overabundance of an aberrant GALNS transcript isoform and a reduction of the clinically relevant isoform (NM_000512.4) in the Morquio syndrome A patients from both families. The aberrant isoform (ENST00000568613.1) was produced by alternative splicing and contained intronic sequence that was likely a cryptic exon predicted to result in a reading frame shift and generation of a premature termination codon. These findings indicated that the aberrant splicing is likely the novel molecular defect in our patients. RNA transcript analysis could be useful to identify pathogenic alterations and increase the yield of molecular diagnosis in patients with Morquio syndrome A whose genetic variants are not found by standard sequencing or gene dosage analysis.