Targeted Ablation of Exon 2 of the Avian Leukosis Virus-A (ALV-A) Receptor Gene in a Chicken Fibroblast Cell Line by CRISPR Abrogates ALV-A Infection.

The U.S. Department of Agriculture Avian Disease and Oncology Laboratory currently relies on live birds of specific genetic backgrounds for producing chicken-embryo fibroblasts that are used for the diagnosis and subtyping of field isolates associated with avian leukosis virus (ALV) outbreaks. As an alternative to maintaining live animals for this purpose, we are currently developing cell lines capable of achieving the same result by ablation of the entry receptors utilized by ALV strains. We used CRISPR-Cas9 on the cell fibroblast-derived cell line DF-1 to disrupt the tva gene, which encodes the receptor required for binding and entry of ALV-A into cells. We ultimately identified seven DF-1 clones that had biallelic and homozygous indels at the Cas9 target site, exon 2 of tva. When tested in vitro for their ability to host ALV-A, the five clones that had frameshift mutations that disrupted the Tva protein were unable to support ALV-A replication. This result clearly demonstrates that modified cell lines can be used as part of a battery of tests to determine ALV subtype for isolate characterization, thus eliminating the need for live birds.

Nota de investigación- La ablación dirigida del exón 2 del gene del receptor del virus de la leucosis aviar A (ALV-A) en una línea celular de fibroblastos de pollo mediante CRISPR anula la infección por ALV-A. El Laboratorio de Oncología y Enfermedades Aviares del Departamento de Agricultura de los Estados Unidos. actualmente depende de aves vivas con antecedentes genéticos específicos para producir fibroblastos de embrión de pollo que se utilizan para el diagnóstico y la subtipificación de aislamientos de campo asociados con brotes del virus de la leucosis aviar (ALV). Como alternativa al mantenimiento de animales vivos para este propósito, actualmente se están desarrollando líneas celulares capaces de lograr el mismo resultado mediante la ablación de los receptores de entrada utilizados por las cepas ALV. Se utilizó el método repeticiones palindrómicas cortas agrupadas y regularmente interespaciadas o CRISPR-Cas9 en la línea celular DF-1 derivada de fibroblastos para interrumpir el gene Tva, que codifica el receptor requerido para la unión y entrada de ALV-A en las células. Finalmente, se identificaron siete clones de DF-1 que tenían inserciones y deleciones (indeles) bialélicos y homocigóticos en el sitio blanco Cas9, exón 2 del gene tva. Cuando se probó in vitro su capacidad para albergar ALV-A, los cinco clones que tenían mutaciones que involucraban al marco de lectura y que interrumpieron la proteína Tva no pudieron admitir la replicación de ALVA. Este resultado demuestra claramente que las líneas celulares modificadas se pueden utilizar como parte de una batería de pruebas para determinar el subtipo de ALV para la caracterización de los aislamientos, eliminando así la necesidad de aves vivas.

PD-L1 Exon 3 Is a Hidden Switch of Its Expression and Function in Oral Cancer Cells.

The interaction between programmed cell death 1 ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) protects tumor cells from immune surveillance. PD-L1 exon 3 is a potential alternative exon and encodes an Ig variable (IgV) domain. Here, we found that a lack of exon 3 leads to the significant loss of cellular membrane locations and the dramatically reduced protein expression of PD-L1, indicating that PD-L1 exon 3 is essential for its protein expression and translocation to the cell membrane. Notably, oral cancer cells show almost no exon 3 skipping to ensure the expression of the full-length, functional PD-L1 protein. We discovered two key exonic splicing enhancers (ESEs) for exon 3 inclusion. Two efficient antisense oligonucleotides (ASOs) were identified to block these two ESEs, which can significantly trigger exon 3 skipping and decrease the production of full-length, functional PD-L1 on the surface of cancer cells. Treatment of oral cancer cells with these ASOs significantly enhanced immune cells' suppression of cancer cell proliferation. Surprisingly, these two ASOs also significantly inhibited cell growth and induced cell pyroptosis in oral cancer cells. Altogether, the results of our study demonstrate the pivotal roles of exon 3 in PD-L1 expression and provide a novel anti-PD-L1 method.

Novel start codons introduce novel coding sequences in the human genomes.

Start-gain mutations can introduce novel start codons and generate novel coding sequences that may affect the function of genes. In this study, we systematically investigated the novel start codons that were either polymorphic or fixed in the human genomes. 829 polymorphic start-gain SNVs were identified in the human populations, and the novel start codons introduced by these SNVs have significantly higher activity in translation initiation. Some of these start-gain SNVs were reported to be associated with phenotypes and diseases in previous studies. By comparative genomic analysis, we found 26 human-specific start codons that were fixed after the divergence between the human and chimpanzee, and high-level translation initiation activity was observed on them. The negative selection signal was detected in the novel coding sequences introduced by these human-specific start codons, indicating the important function of these novel coding sequences.

Exons 1-3 deletion in FLCN is associated with increased risk of pneumothorax in Chinese patients with Birt-Hogg-Dubé syndrome.

BACKGROUND: The pathogenic variants responsible for Birt-Hogg-Dubé syndrome (BHDS) in folliculin (FLCN) gene mostly consist of point mutations. Although large intragenic deletions/duplications have been reported in several case reports, the relationship between large intragenic deletions/duplications and phenotype in BHDS remains unclear. METHODS: We retrospectively identified and reviewed patients with a large intragenic deletion spanning exons 1-3 and analyzed their phenotypic features to compare with those of point mutation carriers in our hospital from January 1, 2017 to August 31, 2022. RESULTS: Twenty unique point mutations (including 4 novel mutations) were detected in 62 patients from 45 families (90%). Exons 1-3 deletion were identified in 8 patients from 5 families (10%) that resided in the same region, Feidong County of Anhui Province, China. Breakpoint analysis indicated that all the deletion breakpoints were flanked by Alu repeats. The prevalence of exons 1-3 deletion carriers in Feidong County was 8.1-times higher than that for BHDS in Anhui Province, suggesting a clustered phenomenon of exons 1-3 deletion. Significantly increased risk of pneumothorax was observed in those with exons 1-3 deletion compared with point mutations (91% vs. 58%, p value 0.047). The risk of renal cancer may be higher in those with exons 1-3 deletion than for those with point mutations (18% vs. 4%, p > 0.05). CONCLUSIONS: Large intragenic deletion of exons 1-3 in FLCN was identified as a local aggregation phenomenon in Feidong County, China, and was associated with a significantly higher risk of pneumothorax compared to those with point mutations.

Immune gene variation associated with chromosome-scale differences among individual zebrafish genomes.

Immune genes have evolved to maintain exceptional diversity, offering robust defense against pathogens. We performed genomic assembly to examine immune gene variation in zebrafish. Gene pathway analysis identified immune genes as significantly enriched among genes with evidence of positive selection. A large subset of genes was absent from analysis of coding sequences due to apparent lack of reads, prompting us to examine genes overlapping zero coverage regions (ZCRs), defined as 2 kb stretches without mapped reads. Immune genes were identified as highly enriched within ZCRs, including over 60% of major histocompatibility complex (MHC) genes and NOD-like receptor (NLR) genes, mediators of direct and indirect pathogen recognition. This variation was most highly concentrated throughout one arm of chromosome 4 carrying a large cluster of NLR genes, associated with large-scale structural variation covering more than half of the chromosome. Our genomic assemblies uncovered alternative haplotypes and distinct complements of immune genes among individual zebrafish, including the MHC Class II locus on chromosome 8 and the NLR gene cluster on chromosome 4. While previous studies have shown marked variation in NLR genes between vertebrate species, our study highlights extensive variation in NLR gene regions between individuals of the same species. Taken together, these findings provide evidence of immune gene variation on a scale previously unknown in other vertebrate species and raise questions about potential impact on immune function.

Diverse Functions of Multiple Bdnf Transcripts Driven by Distinct Bdnf Promoters.

The gene encoding brain-derived neurotrophic factor (Bdnf) consists of nine non-coding exons driven by unique promoters, leading to the expression of nine Bdnf transcripts that play different roles in various brain regions and physiological stages. In this manuscript, we present a comprehensive overview of the molecular regulation and structural characteristics of the multiple Bdnf promoters, along with a summary of the current knowledge on the cellular and physiological functions of the distinct Bdnf transcripts produced by these promoters. Specifically, we summarized the role of Bdnf transcripts in psychiatric disorders, including schizophrenia and anxiety, as well as the cognitive functions associated with specific Bdnf promoters. Moreover, we examine the involvement of different Bdnf promoters in various aspects of metabolism. Finally, we propose future research directions that will enhance our understanding of the complex functions of Bdnf and its diverse promoters.

Pairwise Engineering of Tandemly Aligned Self-Splicing Group I Introns for Analysis and Control of Their Alternative Splicing.

Alternative splicing is an important mechanism in the process of eukaryotic nuclear mRNA precursors producing multiple protein products from a single gene. Although group I self-splicing introns usually perform regular splicing, limited examples of alternative splicing have also been reported. The exon-skipping type of splicing has been observed in genes containing two group I introns. To characterize splicing patterns (exon-skipping/exon-inclusion) of tandemly aligned group I introns, we constructed a reporter gene containing two Tetrahymena introns flanking a short exon. To control splicing patterns, we engineered the two introns in a pairwise manner to design pairs of introns that selectively perform either exon-skipping or exon-inclusion splicing. Through pairwise engineering and biochemical characterization, the structural elements important for the induction of exon-skipping splicing were elucidated.

[Drugs for Genetic Myopathy].

Specific therapy for genetic myopathy is still limited to enzyme replacement therapy for Pompe disease with alglucosidase alfa and avalglucosidase alfa, and exon skipping therapy with antisense nucleotide viltolarsen for about 7% of patients with Duchenne muscular dystrophy. Corticosteroid treatment (predonisolone 10-15mg/day) to Duchenne muscular dystrophy, from the age of 5-6 years old, regardless of mutations. Continuation of corticosteroid after loss of ambulation is controversial. Becker muscular dystrophy patients and manifesting female carriers of DMD mutation may also benefit from corticosteroid, but adverse effects should be avoided. In other types of muscular dystrophy, usefulness of corticosteroid has been reported but may be more limited. Drug therapy should be added on the basis of appropriate evaluation and fundamental symptomatic treatment including rehabilitation, in genetic myopathy.

[Drug Administration for Childhood Neurological Disorders].

In this review, we summarized drug administration strategies for childhood diseases, such as childhood epilepsy and attention-deficit hyperactivity disorder (ADHD). Therapeutic drug monitoring is recommended for most antiepileptic drugs; however, dosage of these in the clinical setting is usually based solely on body weight or age. Other factors to be considered are dosage form and taste; these are particularly important in infants and toddlers as they affect the adherence to a given medicine and may impose a limitation on drug administration. In addition, we should be cautious about such side-effects as the effect on appetite. Special attention should be paid if there is a history of long-time treatment during childhood, because appetite loss or stimulation might have had a substantial negative impact on growth during childhood. We also briefly summarized newly introduced drug therapies for spinal muscular atrophy. These include gene therapy and exon-skipping drugs, which increase the amount of functioning SMN2 protein in skeletal muscles. In particular, the focus of this treatment is on the age of the patient and copy number of the SMN2 gene, both of which are key parameters.

Increased mutation and gene conversion within human segmental duplications.

Single-nucleotide variants (SNVs) in segmental duplications (SDs) have not been systematically assessed because of the limitations of mapping short-read sequencing data1,2. Here we constructed 1:1 unambiguous alignments spanning high-identity SDs across 102 human haplotypes and compared the pattern of SNVs between unique and duplicated regions3,4. We find that human SNVs are elevated 60% in SDs compared to unique regions and estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with up to 4.3 megabase pairs of SD sequence converted on average per human haplotype. We develop a genome-wide map of IGC donors and acceptors, including 498 acceptor and 454 donor hotspots affecting the exons of about 800 protein-coding genes. These include 171 genes that have 'relocated' on average 1.61 megabase pairs in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are slightly evolutionarily older when compared to unique sequences, probably owing to IGC. SNVs in SDs, however, show a distinct mutational spectrum: a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts and a 7.6% reduction in the frequency of CpG-associated mutations when compared to unique DNA. We reason that these distinct mutational properties help to maintain an overall higher GC content of SD DNA compared to that of unique DNA, probably driven by GC-biased conversion between paralogous sequences5,6.

[Clinical and genetic analysis of three children with Menkes disease due to variants of ATP7A gene].

OBJECTIVE: To explore the clinical characteristics and genetic etiology of three children with Menkes disease. METHODS: Three children who had presented at the Children's Medical Center, the Affiliated Hospital of Guangdong Medical University from January 2020 to July 2022 were selected as the study subjects. Clinical data of the children were reviewed. Genomic DNA was extracted from peripheral blood samples of the children, their parents and sister of child 1. Whole exome sequencing (WES) was carried out. Candidate variants were verified by Sanger sequencing, copy number variation sequencing (CNV-seq), and bioinformatic analysis. RESULTS: Child 1 was a 1-year-and-4-month male, and children 2 and 3 were monozygotic twin males aged 1-year-and-10-month. The clinical manifestations of the three children have included developmental delay and seizures. WES showed that child 1 has harbored a c.3294+1G>A variant of the ATP7A gene. Sanger sequencing confirmed that his parents and sister did not carry the same variant, suggesting that it was de novo. Children 2 and 3 had carried a c.77266650_77267178del copy number variation. CNV-seq results showed that their mother has carried the same variant. By searching the HGMD, OMIM and ClinVar databases, the c.3294+1G>A was known to be pathogenic. No carrier frequency has been recorded in the 1000 Genomes, ESP, ExAC and gnomAD databases. Based on the Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics (ACMG), the ATP7A gene c.3294+1G>A variant was predicted to be pathogenic. The c.77266650_77267178del variant has involved exons 8 to 9 of the ATP7A gene. ClinGen online system score for it was 1.8, which was also considered to be pathogenic. CONCLUSION: The c.3294+1G>A and c.77266650_ 77267178del variants of the ATP7A gene probably underlay the Menkes disease in the three children. Above finding has enriched the mutational spectrum of Menkes disease and provided a basis for clinical diagnosis and genetic counseling.

Transcriptional profiling of long noncoding RNAs associated with flower color formation in Ipomoea nil.

MAIN CONCLUSION: LncRNAs regulate flower color formation in Ipomoea nil via vacuolar pH, TCA cycle, and oxidative phosphorylation pathways. The significance of long noncoding RNA (lncRNA) in diverse biological processes is crucial in plant kingdoms. Although study on lncRNAs has been extensive in mammals and model plants, lncRNAs have not been identified in Ipomoea nil (I. nil). In this study, we employed whole transcriptome strand-specific RNA sequencing to identify 11,203 expressed lncRNA candidates, including 961 known lncRNA and 10,242 novel lncRNA in the I. nil genome. These lncRNAs in I. nil had fewer exons and were generally shorter in length compared to mRNA genes. Totally, 1141 different expression lncRNAs (DELs) were significantly identified between white and red flowers. The functional analysis indicated that lncRNA-targeted genes were enriched in the TCA cycle, photosynthesis, and oxidative phosphorylation-related pathway, which was also found in differentially expressed genes (DEGs) functional enrichments. LncRNAs can regulate transcriptional levels through cis- or trans-acting mechanisms. LncRNA cis-targeted genes were significantly enriched in potassium and lysosome. For trans-lncRNA, two energy metabolism pathways, TCA cycles and oxidative phosphorylation, were identified from positive association pairs of trans-lncRNA and mRNA. This research advances our understanding of lncRNAs and their role in flower color development, providing valuable insights for future selective breeding of I. nil.

Identification of nsLTP family in Chinese white pear (Pyrus bretschneideri) reveals its potential roles in russet skin formation.

MAIN CONCLUSION: Identification of PbLTP genes in pear and functional characterization of PbLTP4 in the transport of suberin monomers of russet skin formation. Non-specific lipid-transfer protein (nsLTP) is an abundant and diverse alkaline small molecule protein in the plant kingdom with complex and diverse biophysiological functions, such as transfer of phospholipids, reproductive development, pathogen defence and abiotic stress response. Up to now, only a tiny fraction of nsLTPs have been functionally identified, and the distribution of nsLTPs in pear (Pyrus bretschneideri) (PbLTPs) has not been fully characterized. In this study, the genome-wide analysis of the nsLTP gene family in the pear genome identified 67 PbLTP proteins, which could be divided into six types (1, 2, C, D, E, and G). Similar intron/exon structural patterns were observed in the same type, strongly supporting their close evolutionary relationship. In addition, PbLTP4 was highly expressed in russet pear skin compared with green skin, which was located in the plasma membrane. Coexpression network analysis showed that PbLTP4 closely related to suberin biosynthetic genes. The biological function of PbLTP4 in promoting suberification has been demonstrated by overexpression in Arabidopsis. Identification of suberin monomers showed that PbLTP4 promotes suberification by regulating 9,12-octadecadienoic acid and hexadecanoic acid transport. These results provide helpful insights into the characteristics of PbLTP genes and their biological function in the transport of suberin monomers of russet skin formation.

The exon junction complex component EIF4A3 is essential for mouse and human cortical progenitor mitosis and neurogenesis.

Mutations in components of the exon junction complex (EJC) are associated with neurodevelopment and disease. In particular, reduced levels of the RNA helicase EIF4A3 cause Richieri-Costa-Pereira syndrome (RCPS) and copy number variations are linked to intellectual disability. Consistent with this, Eif4a3 haploinsufficient mice are microcephalic. Altogether, this implicates EIF4A3 in cortical development; however, the underlying mechanisms are poorly understood. Here, we use mouse and human models to demonstrate that EIF4A3 promotes cortical development by controlling progenitor mitosis, cell fate and survival. Eif4a3 haploinsufficiency in mice causes extensive cell death and impairs neurogenesis. Using Eif4a3;p53 compound mice, we show that apoptosis has the most impact on early neurogenesis, while additional p53-independent mechanisms contribute to later stages. Live imaging of mouse and human neural progenitors reveals that Eif4a3 controls mitosis length, which influences progeny fate and viability. These phenotypes are conserved, as cortical organoids derived from RCPS iPSCs exhibit aberrant neurogenesis. Finally, using rescue experiments we show that EIF4A3 controls neuron generation via the EJC. Altogether, our study demonstrates that EIF4A3 mediates neurogenesis by controlling mitosis duration and cell survival, implicating new mechanisms that underlie EJC-mediated disorders.

Filtration of Gene Trees From 9,000 Exons, Introns, and UCEs Disentangles Conflicting Phylogenomic Relationships in Tree Frogs (Hylidae).

An emerging challenge in interpreting phylogenomic data sets is that concatenation and multi-species coalescent summary species tree approaches may produce conflicting results. Concatenation is problematic because it can strongly support an incorrect topology when incomplete lineage sorting (ILS) results in elevated gene-tree discordance. Conversely, summary species tree methods account for ILS to recover the correct topology, but these methods do not account for erroneous gene trees ("EGTs") resulting from gene tree estimation error (GTEE). Third, site-based and full-likelihood methods promise to alleviate GTEE as these methods use the sequence data from alignments. To understand the impact of GTEE on species tree estimation in Hylidae tree frogs, we use an expansive data set of ∼9,000 exons, introns, and ultra-conserved elements and initially found conflict between all three types of analytical methods. We filtered EGTs using alignment metrics that could lead to GTEE (length, parsimony-informative sites, and missing data) and found that removing shorter, less informative alignments reconciled the conflict between concatenation and summary species tree methods with increased gene concordance, with the filtered topologies matching expected results from past studies. Contrarily, site-based and full-likelihood methods were mixed where one method was consistent with past studies and the other varied markedly. Critical to other studies, these results suggest a widespread conflation of ILS and GTEE, where EGTs rather than ILS are driving discordance. Finally, we apply these recommendations to an R package named PhyloConfigR, which facilitates phylogenetic software setup, summarizes alignments, and provides tools for filtering alignments and gene trees.

Duchenne Muscular Dystrophy Gene Therapy in 2023: Status, Perspective, and Beyond.

Duchenne muscular dystrophy (DMD) was named more than 150 years ago. About four decades ago, the DMD gene was discovered, and the reading frame shift was determined as the genetic underpinning. These pivotal findings significantly changed the landscape of DMD therapy development. Restoration of dystrophin expression with gene therapy became a primary focus. Investment in gene therapy has led to the approval of exon skipping by regulatory agencies, multiple clinical trials of systemic microdystrophin therapy using adeno-associated virus vectors, and revolutionary genome editing therapy using the CRISPR technology. However, many important issues surfaced during the clinical translation of DMD gene therapy (such as low efficiency of exon skipping, immune toxicity-induced serious adverse events, and patient death). In this issue of Human Gene Therapy, several research articles highlighted some of the latest developments in DMD gene therapy. Importantly, a collection of articles from experts in the field reviewed the progress, major challenges, and future directions of DMD gene therapy. These insightful discussions have significant implications for gene therapy of other neuromuscular diseases.

Expression profiles of the CD274 and PLEKHH2 gene and association of its polymorphism with hematologic parameters in sheep.

CD274 and PLEKHH2 genes have been identified as immune- and multiple diseases-related genes, and have recently garnered significant interest. However, their role in regulating immune functions in sheep remains largely unexplored. In this study, we aimed to investigate the effects of polymorphisms in CD274 and PLEKHH2 on hematologic parameters in 915 sheep. Our results showed that the CD274 and PLEKHH2 genes were most highly expressed in the spleen and tail fat, respectively, as determined by qRT-PCR. We also identified a G to A mutation (g 0.11858 G > A) in the exon 4 region of CD274, and a C to G mutation (g 0.38384 C > G) in the intron 8 region of PLEKH2. Association analysis revealed that CD274 g 0.11858 G > A was significantly associated with RBC, HCT, MCHC, and MCV (P < 0.05), while PLEKHH2 g 0.38384 C > G was significantly associated with HCT, MPV, MCHC, and MCV (P < 0.05). These results suggest that CD274 and PLEKHH2 genes may play a role in regulating blood physiological indicators and could be potential functional candidates for influencing immune traits in sheep breeding programs.

satmut_utils: a simulation and variant calling package for multiplexed assays of variant effect.

The impact of millions of individual genetic variants on molecular phenotypes in coding sequences remains unknown. Multiplexed assays of variant effect (MAVEs) are scalable methods to annotate relevant variants, but existing software lacks standardization, requires cumbersome configuration, and does not scale to large targets. We present satmut_utils as a flexible solution for simulation and variant quantification. We then benchmark MAVE software using simulated and real MAVE data. We finally determine mRNA abundance for thousands of cystathionine beta-synthase variants using two experimental methods. The satmut_utils package enables high-performance analysis of MAVEs and reveals the capability of variants to alter mRNA abundance.