Combining full-length gene assay and SpliceAI to interpret the splicing impact of all possible SPINK1 coding variants.

BACKGROUND: Single-nucleotide variants (SNVs) within gene coding sequences can significantly impact pre-mRNA splicing, bearing profound implications for pathogenic mechanisms and precision medicine. In this study, we aim to harness the well-established full-length gene splicing assay (FLGSA) in conjunction with SpliceAI to prospectively interpret the splicing effects of all potential coding SNVs within the four-exon SPINK1 gene, a gene associated with chronic pancreatitis. RESULTS: Our study began with a retrospective analysis of 27 SPINK1 coding SNVs previously assessed using FLGSA, proceeded with a prospective analysis of 35 new FLGSA-tested SPINK1 coding SNVs, followed by data extrapolation, and ended with further validation. In total, we analyzed 67 SPINK1 coding SNVs, which account for 9.3% of the 720 possible coding SNVs. Among these 67 FLGSA-analyzed SNVs, 12 were found to impact splicing. Through detailed comparison of FLGSA results and SpliceAI predictions, we inferred that the remaining 653 untested coding SNVs in the SPINK1 gene are unlikely to significantly affect splicing. Of the 12 splice-altering events, nine produced both normally spliced and aberrantly spliced transcripts, while the remaining three only generated aberrantly spliced transcripts. These splice-impacting SNVs were found solely in exons 1 and 2, notably at the first and/or last coding nucleotides of these exons. Among the 12 splice-altering events, 11 were missense variants (2.17% of 506 potential missense variants), and one was synonymous (0.61% of 164 potential synonymous variants). Notably, adjusting the SpliceAI cut-off to 0.30 instead of the conventional 0.20 would improve specificity without reducing sensitivity. CONCLUSIONS: By integrating FLGSA with SpliceAI, we have determined that less than 2% (1.67%) of all possible coding SNVs in SPINK1 significantly influence splicing outcomes. Our findings emphasize the critical importance of conducting splicing analysis within the broader genomic sequence context of the study gene and highlight the inherent uncertainties associated with intermediate SpliceAI scores (0.20 to 0.80). This study contributes to the field by being the first to prospectively interpret all potential coding SNVs in a disease-associated gene with a high degree of accuracy, representing a meaningful attempt at shifting from retrospective to prospective variant analysis in the era of exome and genome sequencing.

Evolving Spectrum of Adenosine Deaminase Deficiency: Assessing Genotype Pathogenicity Based on Expressed ADA Activity of 46 Variants.

BACKGROUND: Deficiency of adenosine deaminase 1 (ADA) has broad clinical and genetic heterogeneity. Screening techniques can identify asymptomatic infants whose phenotype and prognosis are indeterminate, and who may carry ADA variants of unknown significance. OBJECTIVE: To systematically assess the pathogenic potential of rare ADA missense variants, and to better define the relationship of red cell deoxyadenosine nucleotide (dAXP) content to phenotype. METHODS: We expressed 46 ADA missense variants in the ADA-deficient SØ3834 strain of E. coli and defined Genotype Categories (GC) ranked I - IV by increasing expressed ADA activity. We assessed relationships among GC rank, red cell dAXP, and phenotype in 58 reference patients with 50 different genotypes. We used our GC ranking system to benchmark AlphaMissense for predicting variant pathogenicity, and a minigene assay to identify an exonic splicing variant in ADA exon 9. RESULTS: The 46 missense variants expressed ∼0.001% to ∼70% of WT ADA activity (40% had <0.05% of WT ADA activity and 50% expressed >1%). Red cell dAXP ranged from undetectable to >75% of total adenine nucleotides and correlated well with phenotype. Both red cell dAXP and clinical severity were inversely related to "txADA" (total ADA activity expressed by both inherited variants). Our GC scoring system performed better than AlphaMissense in assessing variant pathogenicity, particularly for less deleterious variants. CONCLUSION: For ADA deficiency, pathogenicity is a continuum and conditional, depending on the total ADA activity contributed by both inherited variants as indicated by GC rank. However, in patients with indeterminate phenotype identified by screening, red cell dAXP measured at diagnosis may have greater prognostic value than GC rank .

5' splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity.

In the human genome, most 5' splice sites (~99%) employ the canonical GT dinucleotide whereas a small minority (~1%) use the noncanonical GC dinucleotide. The functionality and pathogenicity of 5' splice site GT>GC (+2T>C) variants have been extensively studied but we know very little about 5' splice site GC>GT (+2C>T) variants. Herein, we have addressed this deficiency by performing a meta-analysis of reported +2C>T "pathogenic" variants together with a functional analysis of engineered +2C>T substitutions using a cell culture-based full-length gene splicing assay. Our results establish proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and suggest that, in sharp contrast to +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants and understanding the evolutionary switching between GT and GC 5' splice sites in mammalian genomes.

SERK genes identification and expression analysis during somatic embryogenesis and sporogenesis of sexual and apomictic Brachiaria brizantha (Syn. Urochloa brizantha).

MAIN CONCLUSION: In Brachiaria brizantha BbrizSERK1, BbrizSERK2 and BbrizSERK3 were identified. SERK expression marks somatic embryogenesis, sexual MMC, and sexual and apomictic PMC. BbrizSERK3 might have a regulatory role in reproductive development. Somatic embryogenesis receptor-like kinase (SERK) consists of plasma membrane receptor genes that have been characterized in various species, associated with several aspects of plant development, including reproduction. SERK genes are involved in anther development and in early embryo development in sexual and asexual seed formation. To comprehend the complexity of the SERK genes and their function in Brachiaria reproduction, we performed a homology-based search in a genomic database of a sexual B. brizantha and identified sequences of three SERK genes, BbrizSERK1, BbrizSERK2, and BbrizSERK3. RNASeq data showed equivalent abundance of BbrizSERK1 and BbrizSERK2 transcripts in ovaries at early megasporogenesis of sexuals and apomicts, while BbrizSERK3 transcripts were more abundant in ovaries of sexuals than in apomicts. BbrizSERK3 results in three coding sequences due to alternative splicing, among them Variant 1 results in a protein with all the predicted domains of a SERK. BbrizSERK transcripts were detected in male reproductive tissues of both sexual and apomictic plants, suggesting a role in controlling anther development. BbrizSERK transcripts were detected early in ovule development, in the integuments, and in the megaspore mother cell of the sexual plant, but not in the cells that give rise to apomictic embryo sacs, suggesting a role in female reproductive development of sexuals. This paper provides evidences that SERK genes plays a role in the onset and establishment of somatic embryogenesis and in the reproductive development of B. brizantha and suggests a distinct role of BbrizSERK in apomixis initiation.

The Experimentally Obtained Functional Impact Assessments of 5' Splice Site GT'GC Variants Differ Markedly from Those Predicted.

INTRODUCTION: 5' splice site GT>GC or +2T>C variants have been frequently reported to cause human genetic disease and are routinely scored as pathogenic splicing mutations. However, we have recently demonstrated that such variants in human disease genes may not invariably be pathogenic. Moreover, we found that no splicing prediction tools appear to be capable of reliably distinguishing those +2T>C variants that generate wild-type transcripts from those that do not. METHODOLOGY: Herein, we evaluated the performance of a novel deep learning-based tool, SpliceAI, in the context of three datasets of +2T>C variants, all of which had been characterized functionally in terms of their impact on pre-mRNA splicing. The first two datasets refer to our recently described "in vivo" dataset of 45 known disease-causing +2T>C variants and the "in vitro" dataset of 103 +2T>C substitutions subjected to full-length gene splicing assay. The third dataset comprised 12 BRCA1 +2T>C variants that were recently analyzed by saturation genome editing. RESULTS: Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of these variants (and smaller datasets of +2T>A and +2T>G variants) revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue was that the impact of those +2T>C (and +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to an almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. CONCLUSION: Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

First estimate of the scale of canonical 5' splice site GT>GC variants capable of generating wild-type transcripts.

It has long been known that canonical 5' splice site (5'SS) GT>GC variants may be compatible with normal splicing. However, to date, the actual scale of canonical 5'SSs capable of generating wild-type transcripts in the case of GT>GC substitutions remains unknown. Herein, combining data derived from a meta-analysis of 45 human disease-causing 5'SS GT>GC variants and a cell culture-based full-length gene splicing assay of 103 5'SS GT>GC substitutions, we estimate that ~15-18% of canonical GT 5'SSs retain their capacity to generate between 1% and 84% normal transcripts when GT is substituted by GC. We further demonstrate that the canonical 5'SSs in which substitution of GT by GC-generated normal transcripts exhibit stronger complementarity to the 5' end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were capable of reliably distinguishing 5'SS GT>GC variants that generated wild-type transcripts from those that did not. Our findings imply that 5'SS GT>GC variants in human disease genes may not invariably be pathogenic.

Spectrum of Ankyrin Mutations in Hereditary Spherocytosis: A Case Report and Review of the Literature.

BACKGROUND/AIMS: Hereditary spherocytosis (HS) is a common pediatric hemolytic anemia caused by congenital red blood cell defects. HS due to ankyrin 1 (ANK1) mutations is the most common type. We explored an ANK1 mutation from an HS patient and reviewed the literature. METHODS: We detected the mutation in a Chinese family in which 2 members were diagnosed with HS by next-generation sequencing. The proband was diagnosed with HS in the newborn period, based on clinical manifestations, laboratory data, and family history. The mutation spectrum of the ANK1 gene was summarized based on 85 patients diagnosed with HS carrying ANK1 mutations, and the ANK1 mutation spectrum was summarized and analyzed. RESULTS: We identified a novel mutation affecting ANK1 gene splicing (a splicing mutation) in both the patient and her mother, which is a substitution of T>G 2 nt after exon 25 in intron 26. The study expands our knowledge of the ANK1 gene mutation spectrum, providing a molecular basis for HS. CONCLUSION: A novel ANK1 mutation (NM_000037.3, c.2960+2T>G, intron 26) that is potentially associated with HS was identified. To date, 80 ANK1 mutations have been reported to be associated with HS in humans.

Regulating RNA Research and CRISPR Gene Drives to Combat Biosecurity Threats.

Recent technological breakthroughs in ribonucleic acid (RNA) research and the creation of synthetic gene drives using CRISPR/Cas9 have increased attention on the ethical and legal regulation of this field. RNA is now perceived as not merely a passive carrier of DNA information but especially through its propensity to mutate as a computation engine of cell biology, developmental biology and evolution. Synthetic Gene drives have been hailed as a potential strategy to reduce climate-change-mediated biosecurity threats such as spreading malaria and have attracted significant investment, with the Gates Foundation pledging US$75 million and the Defense Advanced Research Projects Agency awarding US$65 million. Calls for a global moratorium on RNA-mediated genetic engineering may overstate the potential risks of the developing technology, but form a background to the contest between "process"- and "product" -based approaches to regulation, the former purportedly favoured by the public and regulatory agencies and the latter favoured by the broad scientific community and corporate investors. At stake may be the democratic legitimacy of and equitable access to a technology that could be important to reduce the incidence of biosecurity threats both globally and in Australia.

Developing new targeting strategy for androgen receptor variants in castration resistant prostate cancer.

The presence of androgen receptor variant 7 (AR-V7) variants becomes a significant hallmark of castration-resistant prostate cancer (CRPC) relapsed from hormonal therapy and is associated with poor survival of CRPC patients because of lacking a ligand-binding domain. Currently, it still lacks an effective agent to target AR-V7 or AR-Vs in general. Here, we showed that a novel class of agents (thailanstatins, TSTs and spliceostatin A analogs) can significantly suppress the expression of AR-V7 mRNA and protein but in a less extent on the full-length AR expression. Mechanistically, TST-D is able to inhibit AR-V7 gene splicing by interfering the interaction between U2AF65 and SAP155 and preventing them from binding to polypyrimidine tract located between the branch point and the 3' splice site. In vivo, TST-D exhibits a potent tumor inhibitory effect on human CRPC xenografts leading to cell apoptosis. The machinery associated with AR gene splicing in CRPC is a potential target for drugs. Based on their potency in the suppression of AR-V7 responsible for the growth/survival of CRPC, TSTs representing a new class of anti-AR-V agents warrant further development into clinical application.

OGS2: genome re-annotation of the jewel wasp Nasonia vitripennis.

BACKGROUND: Nasonia vitripennis is an emerging insect model system with haplodiploid genetics. It holds a key position within the insect phylogeny for comparative, evolutionary and behavioral genetic studies. The draft genomes for N. vitripennis and two sibling species were published in 2010, yet a considerable amount of transcriptiome data have since been produced thereby enabling improvements to the original (OGS1.2) annotated gene set. We describe and apply the EvidentialGene method used to produce an updated gene set (OGS2). We also carry out comparative analyses showcasing the usefulness of the revised annotated gene set. RESULTS: The revised annotation (OGS2) now consists of 24,388 genes with supporting evidence, compared to 18,850 for OGS1.2. Improvements include the nearly complete annotation of untranslated regions (UTR) for 97 % of the genes compared to 28 % of genes for OGS1.2. The fraction of RNA-Seq validated introns also grow from 85 to 98 % in this latest gene set. The EST and RNA-Seq expression data provide support for several non-protein coding loci and 7712 alternative transcripts for 4146 genes. Notably, we report 180 alternative transcripts for the gene lola. Nasonia now has among the most complete insect gene set; only 27 conserved single copy orthologs in arthropods are missing from OGS2. Its genome also contains 2.1-fold more duplicated genes and 1.4-fold more single copy genes than the Drosophila melanogaster genome. The Nasonia gene count is larger than those of other sequenced hymenopteran species, owing both to improvements in the genome annotation and to unique genes in the wasp lineage. We identify 1008 genes and 171 gene families that deviate significantly from other hymenopterans in their rates of protein evolution and duplication history, respectively. We also provide an analysis of alternative splicing that reveals that genes with no annotated isoforms are characterized by shorter transcripts, fewer introns, faster protein evolution and higher probabilities of duplication than genes having alternative transcripts. CONCLUSIONS: Genome-wide expression data greatly improves the annotation of the N. vitripennis genome, by increasing the gene count, reducing the number of missing genes and providing more comprehensive data on splicing and gene structure. The improved gene set identifies lineage-specific genomic features tied to Nasonia's biology, as well as numerous novel genes. OGS2 and its associated search tools are available at http://arthropods.eugenes.org/EvidentialGene/nasonia/ , www.hymenopteragenome.org/nasonia/ and waspAtlas: www.tinyURL.com/waspAtlas . The EvidentialGene pipeline is available at https://sourceforge.net/projects/evidentialgene/ .

Transcriptomic analysis of alternative splicing events for different stages of growth and development in Sistan Yaghooti grape clusters.

Sistan Yaghooti grape variety, despite characteristics such as early ripening, is vulnerable to cluster rot due to small berries and dense clusters. In this regard, AS may serve as a regulatory mechanism during developmental processes and in response to environmental signals. RNA-Seq analysis was performed to measure gene expression and the extent of AS events in the cluster growth and development stages of Sistan Yaghooti grape. The number of AS events increased during stages, suggesting that it contributes to the grapevine's adaptability to various stresses. In addition, DEG and DAS genes showed little overlap in cluster growth stages. Functional analysis of 19,194 DAS -gene sets showed that VIT_06s0004g06670 gene is involved in the activation of calcium channels (Ca2+) through the activation of 5 PLC biosynthetic pathways. Among the 27,229 DEG -sets, VIT_07s0005g05320 gene showed higher expression. Interestingly, this gene is involved in the synthesis of an EF -hand domain-containing protein capable of binding to Ca2+ by activating 4 biochemical pathways. These genes increase cytosolic Ca2+ concentration, enhancing plant stress tolerance and resistance to cracking. These results show that AS can respond independently to different types of stress. Among the other DAS genes, the GA2ox gene (VvGA2ox) showed an increase in AS events during cluster development. This gene is critical for initiating the degradation process of GA and plays a crucial role in different stages of seed development. Therefore, it is very likely that this gene is one of the main factors responsible for the density and seedlessness of Sistan Yaghooti grape.

Malnutrition in Spinal Muscular Atrophy Type I: Case Report of a Novel Nutritional Intervention With Improved Growth and Function While Receiving Parallel Gene Splicing Therapies.

Children with spinal muscular atrophy (SMA) frequently experience feeding intolerance and diminished growth. Although splicing modulators to prevent symptoms are available worldwide, adequate nutrition to support growth, development, and improved quality of life remains essential. We present a case study of a one-year-old malnourished male with SMA type I who achieved improved growth and feeding tolerance with a human milk (HM)-derived nutrition intervention. Despite feeding with appropriately balanced semielemental formula, he remained severely malnourished after two months of hospitalization. Feeds were partially replaced with HM-based diet plus a HM-based fat modular. Feeding tolerance, fecal calprotectin levels, and z scores for weight and length improved while receiving the HM-based intervention. We hypothesize that the HM-based feeding reduced intestinal inflammation by diminishing pathogenic elements of his microbiome. Owing to their aberrant fatty acid metabolism, patients with SMA are uniquely positioned to benefit from HM-based nutrient acquisition even while receiving splicing modulators to stabilize the disease process.

Case report: A novel mutation of the CAPN3 gene in a Chinese family with limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD R1 Calpain 3-Related, LGMD2A/R1), an autosomal recessive disorder, is characterized by progressive muscle weakness with a prominent presentation in the proximal limb girdle muscles. LGMD2A/R1, which is caused by variants in calcium-activated neutral proteinase 3 (CAPN3), is the most common. The present study aimed at identifying the clinically significant variants in a Chinese family with LGMD2A/R1 and exploring the genotype-phenotype correlations. Clinical symptoms, laboratory findings, and physical examinations were obtained. Genomic DNA was extracted from the peripheral blood samples of this family. Whole-exome sequencing (WES) and Sanger sequencing were used to explore and validate the pathogenic genes. In this study, the proband and his sister, who had two identical mutations in the CAPN3 gene sequence, exhibited diverse clinical features, including disease onset and progression. The mutation c.2120 A>G (p. D707G) is pathogenic and has been reported in the Human Gene Mutation Database (HGMD) and the ClinVar database. c.1783-72 C>G may be a novel pathogenic mutation of LGMD2A/R1 based on the American College of Medical Genetics (ACMG) guidelines, which widens the gene variant pool in CAPN3 and improves diagnosis and genetic counseling.

A novel variation in DEPDC5 causing familial focal epilepsy with variable foci.

Background: Disheveled, EGL-10, and pleckstrin (DEP) domain-containing protein 5 (DEPDC5) is a component of GTPase-activating protein (GAP) activity toward the RAG complex 1 (GATOR1) protein, which is an inhibitor of the amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. GATOR1 complex variations were reported to correlate with familial focal epilepsy with variable foci (FFEVF). With the wide application of whole exome sequencing (WES), more and more variations in DEPDC5 were uncovered in FFEVF families. Methods: A family with a proband diagnosed with familial focal epilepsy with variable foci (FFEVF) was involved in this study. Whole exome sequencing (WES) was performed in the proband, and Sanger sequencing was used to confirm the variation carrying status of the family members. Mini-gene splicing assay was performed to validate the effect on the alternative splicing of the variation. Results: A novel variant, c.1217 + 2T>A, in DEPDC5 was identified by WES in the proband. This splicing variant that occurred at the 5' end of intron 17 was confirmed by mini-gene splicing assays, which impacted alternative splicing and led to the inclusion of an intron fragment. The analysis of the transcribed mRNA sequence indicates that the translation of the protein is terminated prematurely, which is very likely to result in the loss of function of the protein and lead to the occurrence of FFEVF. Conclusion: The results suggest that c.1217 + 2T>A variations in DEPDC5 might be the genetic etiology for FFEVF in this pedigree. This finding expands the genotype spectrum of FFEVF and provides new etiological information for FFEVF.

Long read sequencing to reveal the full complexity of a plant transcriptome by targeting both standard and long workflows.

BACKGROUND: Long read sequencing allows the analysis of full-length transcripts in plants without the challenges of reliable transcriptome assembly. Long read sequencing of transcripts from plant genomes has often utilized sized transcript libraries. However, the value of including libraries of differing sizes has not been established. METHODS: A comprehensive transcriptome of the leaves of Jojoba (Simmondsia chinensis) was generated from two different PacBio library preparations: standard workflow (SW) and long workflow (LW). RESULTS: The importance of using both transcript groups in the analysis was demonstrated by the high proportion of unique sequences (74.6%) that were not shared between the groups. A total of 37.8% longer transcripts were only detected in the long dataset. The completeness of the combined transcriptome was indicated by the presence of 98.7% of genes predicted in the jojoba male reference genome. The high coverage of the transcriptome was further confirmed by BUSCO analysis showing the presence of 96.9% of the genes from the core viridiplantae_odb10 lineage. The high-quality isoforms post Cd-Hit merged dataset of the two workflows had a total of 167,866 isoforms. Most of the transcript isoforms were protein-coding sequences (71.7%) containing open reading frames (ORFs) ≥ 100 amino acids (aa). Alternative splicing and intron retention were the basis of most transcript diversity when analysed at the whole genome level and by specific analysis of the apetala2 gene families. CONCLUSION: This suggests the need to specifically target the capture of longer transcripts to provide more comprehensive genome coverage in plant transcriptome analysis and reveal the high level of alternative splicing.

The Role of Tau Proteoforms in Health and Disease.

Tau is a microtubule-associated binding protein in the nervous system that is known for its role in stabilizing microtubules throughout the nerve cell. It accumulates as ß-sheet-rich aggregates and neurofibrillary tangles, leading to an array of different pathologies. Six splice variants of this protein, generated from the microtubule-associated protein tau (MAPT) gene, are expressed in the brain. Amongst these variants, 0N3R, is prominent during fetal development, while the rest, 0N4R, 1N3R, 1N4R, 2N3R, and 2N4R, are expressed in postnatal stages. Tau isoforms play their role separately or in combination with others to contribute to one or multiple neurodegenerative disorders and clinical syndromes. For instance, in Alzheimer's disease and a subset of frontotemporal lobar degeneration (FTLD)-MAPT (i.e., R406W and V337M), both 3R and 4R isoforms are involved; therefore, they are called 3R/4R mix tauopathies. On the other hand, 4R isoforms are aggregated in progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and a majority of FTLD-MAPT and these diseases are called 4R tauopathies. Similarly, Pick's disease has an association with 3R tau isoforms and is thereby referred to as 3R tauopathy. Unlike 3R isoforms, the 4R variants have a faster rate of aggregation that accelerates the associated neurodegenerative mechanisms. Moreover, post-translational modifications of each isoform occur at a different rate and dictate their physiological and pathological attributes. The smallest tau isoform (0N3R) is highly phosphorylated in the fetal brain but does not lead to the generation of aggregates. On the other hand, proteoforms in the adult human brain undergo aggregation upon their phosphorylation and glycation. Expanding on this knowledge, this article aims to review the physiological and pathological roles of tau isoforms and their underlying mechanisms that result in neurological deficits. Physiological and pathological relevance of microtubule-associated protein tau (MAPT): Tau exists as six splice variants in the brain, each differing with respect to expression, post-translational modifications (PTMs), and aggregation kinetics. Physiologically, they are involved in the stabilization of microtubules that form the molecular highways for axonal transport. However, an imbalance in their expression and the associated PTMs leads to a disruption in their physiological function through the formation of neurofibrillary tangles that accumulate in various regions of the brain and contribute to several types of tauopathies.

Novel compound heterozygous variants in EMC1 associated with global developmental delay: a lesson from a non-silent synonymous exonic mutation.

Background: The endoplasmic reticulum-membrane protein complex (EMC) as a molecular chaperone is required for the proper synthesis, folding and traffic of several transmembrane proteins. Variants in the subunit 1 of EMC (EMC1) have been implicated in neurodevelopmental disorders. Methods: Whole exome sequencing (WES) with Sanger sequencing validation was performed for a Chinese family, including the proband (a 4-year-old girl who displayed global developmental delay, severe hypotonia and visual impairment), her affected younger sister and her non-consanguineous parents. RT-PCR assay and Sanger sequencing were used to detect abnormal RNA splicing. Results: Novel compound heterozygous variants in EMC1, including the maternally inherited chr1: 19566812_1956800delinsATTCTACTT[hg19];NM_015047.3:c.765_777delins ATTCTACTT;p.(Leu256fsTer10) and the paternally inherited chr1:19549890G> A[hg19];NM_015047.3:c.2376G>A;p.(Val792=) are identified in the proband and her affected sister. RT-PCR assay followed by Sanger sequencing reveals that the c.2376G>A variant leads to aberrant splicing, with retention of intron 19 (561bp) in the mature mRNA, which is presumed to introduce a premature translational termination codon (p.(Val792fsTer31)). Conclusion: Novel compound heterozygous variants in EMC1 have been identified in individuals with global developmental delay. Non-silent synonymous mutations should be kept in mind in genetic analysis.

GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells.

Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome-wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ∼2-fold. Bulk ASO uptake is 2- to 5-fold higher in GOLGA8-overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.

Subcutaneous and intramuscular fat transcriptomes show large differences in network organization and associations with adipose traits in pigs.

Subcutaneous fat (SCF) and intramuscular fat (IMF) deposition is relevant to health in humans, as well as meat production and quality in pigs. In this study, we generated RNA sequence data for 122 SCF, 120 IMF, and 87 longissimus dorsi muscle (LDM) samples using 155 F6 pigs from a specially designed heterogeneous population generated by intercrossing four highly selected European commercial breeds and four indigenous Chinese pig breeds. The phenotypes including waist back fat thickness and intramuscular fat content were also measured in the 155 F6 pigs. We found that the genes in SCF and IMF differed largely in both expression levels and network connectivity, and highlighted network modules that exhibited strongest gain of connectivity in SCF and IMF, containing genes that were associated with the immune process and DNA double-strand repair, respectively. We identified 215 SCF genes related to kinase inhibitor activity, mitochondrial fission, and angiogenesis, and 90 IMF genes related to lipolysis and fat cell differentiation, displayed a tissue-specific association with back fat thickness and IMF content, respectively. We found that cis-expression QTL for trait-associated genes in the two adipose tissues tended to have tissue-dependent predictability for the two adipose traits. Alternative splicing of genes was also found to be associated with SCF or IMF deposition, but the association was much less extensive than that based on expression levels. This study provides a better understanding of SCF and IMF gene transcription and network organization and identified critical genes and network modules that displayed tissue-specific associations with subcutaneous and intramuscular fat deposition. These features are helpful for designing breeding programs to genetically improve the two adipose traits in a balanced way.

Splicing Outcomes of 5' Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays.

Combining data derived from a meta-analysis of human disease-associated 5' splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15-18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.