Novel splice site and nonsense variants in PKHD1 cause autosomal recessive polycystic kidney disease in a Chinese Zhuang ethnic family.

BACKGROUND: This study aims to report the clinical characteristics of a child with autosomal recessive polycystic kidney disease (ARPKD) within a Chinese Zhuang ethnic family. METHODS: We used whole exome sequencing (WES) in the family to examine the genetic cause of the disease. Candidate pathogenic variants were validated by Sanger sequencing. RESULTS: We identified previously unreported mutations in the PKHD1 gene of the proband with ARPKD through WES: a splice site mutation c.6809-2A > T, a nonsense mutation c.4192C > T(p.Gln1398Ter), and a missense mutation c.2181T > G(p.Asn727Lys). Her mother is a heterozygous carrier of c.2181T > G(p.Asn727Lys) mutation. Her father is a carrier of c.6809-2A > T mutation and c.4192C > T(p.Gln1398Ter) mutation. CONCLUSIONS: The identification of novel mutations in the PKHD1 gene through WES not only expands the spectrum of known variants but also potentially enhances genetic counseling and prenatal diagnostic approaches for families affected by ARPKD.

An effective deep learning-based approach for splice site identification in gene expression.

A crucial stage in eukaryote gene expression involves mRNA splicing by a protein assembly known as the spliceosome. This step significantly contributes to generating and properly operating the ultimate gene product. Since non-coding introns disrupt eukaryotic genes, splicing entails the elimination of introns and joining exons to create a functional mRNA molecule. Nevertheless, accurately finding splice sequence sites using various molecular biology techniques and other biological approaches is complex and time-consuming. This paper presents a precise and reliable computer-aided diagnosis (CAD) technique for the rapid and correct identification of splice site sequences. The proposed deep learning-based framework uses long short-term memory (LSTM) to extract distinct patterns from RNA sequences, enabling rapid and accurate point mutation sequence mapping. The proposed network employs one-hot encodings to find sequential patterns that effectively identify splicing sites. A thorough ablation study of traditional machine learning, one-dimensional convolutional neural networks (1D-CNNs), and recurrent neural networks (RNNs) models was conducted. The proposed LSTM network outperformed existing state-of-the-art approaches, improving accuracy by 3% and 2% for the acceptor and donor sites datasets.

The Effect of Alternative Splicing Sites on Mirtron Formation and Arm Selection of Precursor microRNAs.

Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.

COQ7 splice site variant causing a spastic paraparesis phenotype in siblings.

The COQ7 gene is one of the causative genes for primary COQ10 deficiency-related disorders. OMIM-related phenotypes include severe encephalo-myo-nephrocardiopathy and distal hereditary motor neuronopathy. In the present study, we performed the exome sequencing analysis on the proband of a single family with two siblings affected by hereditary spastic paraparesis (HSP). Segregation analysis was conducted on the affected siblings and parents using the Sanger sequencing. In silico secondary and tertiary pre-mRNA structure analysis and protein modelling were carried out. Exome sequencing identified a homozygous splice site variant in the COQ7 gene (NM_016138.5: c.367+G>A) in the proband. Sanger sequencing confirmed the homozygous status in the affected sibling and heterozygous status in both parents, consistent with autosomal recessive inheritance. In silico secondary and tertiary premRNA structure analysis and protein modelling predicted the deleterious nature of the variant. This case highlights a distinct intermediate phenotype of COQ7 related disorders comprising early-onset spastic paraparesis due to a novel splice site variant in the COQ7 gene. This expands the spectrum of clinical manifestations associated with COQ7 deficiency and underscores the importance of considering COQ7 gene mutations in the differential diagnosis of HSP.

Neuronal Ceroid Lipofuscinosis in a Mixed-Breed Dog with a Splice Site Variant in CLN6.

A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors, seizure activity, ataxia, and pronounced visual impairment. The clinical signs were accompanied by global brain atrophy. Due to progression in the severity of disease signs, the dog was euthanized at 26 months of age. An examination of the tissues collected at necropsy revealed dramatic intracellular accumulations of autofluorescent inclusions in the brain, retina, and cardiac muscle. The inclusions were immunopositive for subunit c of mitochondrial ATP synthase, and their ultrastructural appearances were similar to those of lysosomal storage bodies that accumulate in some neuronal ceroid lipofuscinosis (NCL) diseases. The dog also exhibited widespread neuroinflammation. Based on these findings, the dog was deemed likely to have suffered from a form of NCL. A whole genome sequence analysis of the proband's DNA revealed a homozygous C to T substitution that altered the intron 3-exon 4 splice site of CLN6. Other mutations in CLN6 cause NCL diseases in humans and animals, including dogs. The CLN6 protein was undetectable with immunolabeling in the tissues of the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the disorder in this dog was classified as an NCL resulting from the absence of the CLN6 protein. Screening the dog's genome for a panel of breed-specific polymorphisms indicated that its ancestry included numerous breeds, with no single breed predominating. This suggests that the CLN6 disease variant is likely to be present in other mixed-breed dogs and at least some ancestral breeds, although it is likely to be rare since other cases have not been reported to date.

A systematic screen identifies Saf5 as a link between splicing and transcription in fission yeast.

Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several integrated fluorescence-based in vivo splicing reporter constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5' splicing site (5'SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter showing more intron retention with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants -Δcwf12, a member of the NineTeen Complex (NTC) and Δsaf5, a methylosome subunit that acts together with the survival motor neuron (SMN) complex in small nuclear ribonucleoproteins (snRNP) biogenesis. We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5'SS differs from the consensus. However, although Δsaf5 cells also have some dependency on 5'SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.

An alternative 3' splice site of PeuHKT1;3 improves the response to salt stress through enhancing affinity to K+ in Populus.

Alternative splicing (AS) serves as a crucial post-transcriptional regulator in plants that contributes to the resistance to salt stress. However, the underlying mechanism is largely unknown. In this research, we identified an important AS transcript in Populus euphratica, PeuHKT1:3a, generated by alternative 3' splice site splicing mode that resulted in the removal of 252 bases at the 5' end of the first exon in PeuHKT1:3. Protein sequence comparison showed that the site of AS occurred in PeuHKT1:3 is located at a crucial Ser residue within the first pore-loop domain, which leads to inefficient K+ transport in HKT I-type transporters. Expressing PeuHKT1;3a in an axt3 mutant yeast strain can effectively compensate for the lack of intracellular K+, whereas the expression of PeuHKT1;3 cannot yield the effect. Furthermore, in transgenic Arabidopsis and poplar plants, it was observed that lines expressing PeuHKT1;3a exhibited greater salt tolerance compared to those expressing the PeuHKT1;3 strain. Analysis of ion content and flux demonstrated that the transgenic PeuHKT1;3a line exhibited significantly higher K+ content compared to the PeuHKT1;3 line, while there was no significant difference in Na+ content. In conclusion, our findings revealed that AS can give rise to novel variants of HKT I-type proteins in P. euphratica with modified K+ selectivity to keep a higher K+/Na+ ratio to enhanced salt tolerance.

SpliceAPP: an interactive web server to predict splicing errors arising from human mutations.

BACKGROUND: Splicing variants are a major class of pathogenic mutations, with their severity equivalent to nonsense mutations. However, redundant and degenerate splicing signals hinder functional assessments of sequence variations within introns, particularly at branch sites. We have established a massively parallel splicing assay to assess the impact on splicing of 11,191 disease-relevant variants. Based on the experimental results, we then applied regression-based methods to identify factors determining splicing decisions and their respective weights. RESULTS: Our statistical modeling is highly sensitive, accurately annotating the splicing defects of near-exon intronic variants, outperforming state-of-the-art predictive tools. We have incorporated the algorithm and branchpoint information into a web-based tool, SpliceAPP, to provide an interactive application. This user-friendly website allows users to upload any genetic variants with genome coordinates (e.g., chr15 74,687,208 A G), and the tool will output predictions for splicing error scores and evaluate the impact on nearby splice sites. Additionally, users can query branch site information within the region of interest. CONCLUSIONS: In summary, SpliceAPP represents a pioneering approach to screening pathogenic intronic variants, contributing to the development of precision medicine. It also facilitates the annotation of splicing motifs. SpliceAPP is freely accessible using the link https://bc.imb.sinica.edu.tw/SpliceAPP . Source code can be downloaded at https://github.com/hsinnan75/SpliceAPP .

Unique features of conventional and nonconventional introns in Euglena gracilis.

BACKGROUND: Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns. RESULTS: A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5' ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans. CONCLUSIONS: Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida.

A Mild Presentation of X-Linked Hypophosphatemia Caused by a Non-Canonical Splice Site Variant in the PHEX Gene.

X-linked hypophosphatemia (XLH) is a rare inherited disorder of renal phosphate wasting with a highly variable phenotype caused by loss-of-function variants in the PHEX gene. The diagnosis of individuals with mild phenotypes can be challenging and often delayed. Here, we describe a three-generation family with a very mild clinical presentation of XLH. The diagnosis was unexpectedly found in a 39-year-old woman who was referred for genetic testing due to an unclear childhood diagnosis of a tubulopathy. Genetic testing performed by next-generation sequencing using a kidney disease gene panel identified a novel non-canonical splice site variant in the PHEX gene. Segregation analysis detected that the consultand's father, who presented with hypophosphatemia and decreased tubular phosphate reabsorption, and the consultand's son also carried this variant. RNA studies demonstrated that the non-canonical splice site variant partially altered the splicing of the PHEX gene, as both wild-type and aberrant splicing transcripts were detected in the two male members with only one copy of the PHEX gene. In conclusion, this case contributes to the understanding of the relationship between splicing variants and the variable expressivity of XLH disease. The mild phenotype of this family can be explained by the coexistence of PHEX transcripts with aberrant and wild-type splicing.

Genome-scale exon perturbation screens uncover exons critical for cell fitness.

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.

Generation of an induced pluripotent stem cell line from a patient with arrhythmogenic right ventricular cardiomyopathy harboring a TMEM43 splice-site variant.

Arrhythmogenic cardiomyopathy (ACM) is a cardiomyopathy that is predominantly inherited and characterized by cardiac arrhythmias and structural abnormalities. TMEM43 (transmembrane protein 43) is one of the well-known genetic culprits behind ACM. In this study, we successfully generated an induced pluripotent stem cell (iPSC) line, YCMi010-A, derived from a male patient diagnosed with ACM. Although these iPSCs harbored a heterozygous intronic splice variant, TMEM43 c.443-2A > G, they still displayed normal cellular morphology and were confirmed to express pluripotency markers. YCMi010-A iPSC line is a promising model for investigating the pathomechanisms associated with ACM and exploring potential therapeutic strategies.

The debranching enzyme Dbr1 regulates lariat turnover and intron splicing.

The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of a viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectrometry. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.

hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing.

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

A splice site variant in MADD affects hormone expression in pancreatic ß cells and pituitary gonadotropes.

MAPK activating death domain (MADD) is a multifunctional protein regulating small GTPases RAB3 and RAB27, MAPK signaling, and cell survival. Polymorphisms in the MADD locus are associated with glycemic traits, but patients with biallelic variants in MADD manifest a complex syndrome affecting nervous, endocrine, exocrine, and hematological systems. We identified a homozygous splice site variant in MADD in 2 siblings with developmental delay, diabetes, congenital hypogonadotropic hypogonadism, and growth hormone deficiency. This variant led to skipping of exon 30 and in-frame deletion of 36 amino acids. To elucidate how this mutation causes pleiotropic endocrine phenotypes, we generated relevant cellular models with deletion of MADD exon 30 (dex30). We observed reduced numbers of ß cells, decreased insulin content, and increased proinsulin-to-insulin ratio in dex30 human embryonic stem cell-derived pancreatic islets. Concordantly, dex30 led to decreased insulin expression in human ß cell line EndoC-ßH1. Furthermore, dex30 resulted in decreased luteinizing hormone expression in mouse pituitary gonadotrope cell line LßT2 but did not affect ontogeny of stem cell-derived GnRH neurons. Protein-protein interactions of wild-type and dex30 MADD revealed changes affecting multiple signaling pathways, while the GDP/GTP exchange activity of dex30 MADD remained intact. Our results suggest MADD-specific processes regulate hormone expression in pancreatic ß cells and pituitary gonadotropes.

A rare ACAN non-canonical splicing-site intron variant results in familial short stature.

OBJECTIVE: ACAN gene variants, prevalent monogenic defects linked to short stature, are characterized by impaired cartilage generation in growth plates. We aimed to unravel the genetic basis of short stature in a specific pedigree by investigating the role of a novel non-canonical splicing-site variant, c.630-13G > A, within the ACAN gene. METHOD: Sanger sequencing was used for pedigree verification, and the effects of this variant on mRNA splicing were analyzed through minigene assay. RESULTS: The study revealed that this variant led to the creation of a previously unreported splice site in the fourth intron, resulting in the incorporation of an 11 bp sequence from the intron into the final transcript. This alteration led to a frameshift and formation of a premature termination codon, impacting the structure of the aggrecan protein. CONCLUSIONS: We document the pathogenicity of an ACAN non-canonical splicing-site variant, emphasizing the significance of considering intronic variants during genetic testing.

Structural insights into the cross-exon to cross-intron spliceosome switch.

Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2-5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5' splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10-16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5' splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.

An interpretable model of pre-mRNA splicing for animal and plant genes.

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.

Functional analysis of the zinc finger modules of the Saccharomyces cerevisiae splicing factor Luc7.

Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site (5'ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5'ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5'ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28.

A deep catalogue of protein-coding variation in 983,578 individuals.

Rare coding variants that substantially affect function provide insights into the biology of a gene1-3. However, ascertaining the frequency of such variants requires large sample sizes4-8. Here we present a catalogue of human protein-coding variation, derived from exome sequencing of 983,578 individuals across diverse populations. In total, 23% of the Regeneron Genetics Center Million Exome (RGC-ME) data come from individuals of African, East Asian, Indigenous American, Middle Eastern and South Asian ancestry. The catalogue includes more than 10.4 million missense and 1.1 million predicted loss-of-function (pLOF) variants. We identify individuals with rare biallelic pLOF variants in 4,848 genes, 1,751 of which have not been previously reported. From precise quantitative estimates of selection against heterozygous loss of function (LOF), we identify 3,988 LOF-intolerant genes, including 86 that were previously assessed as tolerant and 1,153 that lack established disease annotation. We also define regions of missense depletion at high resolution. Notably, 1,482 genes have regions that are depleted of missense variants despite being tolerant of pLOF variants. Finally, we estimate that 3% of individuals have a clinically actionable genetic variant, and that 11,773 variants reported in ClinVar with unknown significance are likely to be deleterious cryptic splice sites. To facilitate variant interpretation and genetics-informed precision medicine, we make this resource of coding variation from the RGC-ME dataset publicly accessible through a variant allele frequency browser.