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PubMed2025

OpenSpliceAI: An efficient, modular implementation of SpliceAI enabling easy retraining on non-human species.

The SpliceAI deep learning system is currently one of the most accurate methods for identifying splicing signals directly from DNA sequences. However, its utility is limited by its reliance on older software frameworks and human-centric training data. Here we introduce OpenSpliceAI, a trainable, open-source version of SpliceAI implemented in PyTorch to address these challenges. OpenSpliceAI supports both training from scratch and transfer learning, enabling seamless retraining on species-specific datasets and mitigating human-centric biases. Our experiments show that it achieves faster processing speeds and lower memory usage than the original SpliceAI code, allowing large-scale analyses of extensive genomic regions on a single GPU. Additionally, OpenSpliceAI's flexible architecture makes for easier integration with established machine learning ecosystems, simplifying the development of custom splicing models for different species and applications. We demonstrate that OpenSpliceAI's output is highly concordant with SpliceAI. In silico mutagenesis (ISM) analyses confirm that both models rely on similar sequence features, and calibration experiments demonstrate similar score probability estimates.

Journal Article↗

PubMed2025

Tandem splice acceptor sites: Profiling their relevance to human disease.

PURPOSE: Interpretation of variation, particularly the creation or disruption of tandem splice acceptor sites (NAGNnAG variants), challenges genomic medicine practice. METHODS: We analyzed the creation and disruption of dinucleotide AG sites within ±30 bases of natural splice-acceptor sites in the GRCh37 human reference genome. These results were compared with variant data from the ClinVar and gnomAD databases, as well as with data from 779 National Institutes of Health Undiagnosed Diseases Program study participants. Using RNA sequencing, we assessed the splicing at NAGNnAG variants for 107 of the Undiagnosed Diseases Program participants and compared the empirical data with SpliceAI predictions. RESULTS: Creation or disruption of NAGNnAG sites within 30 bases of the natural splice acceptor are enriched in ClinVar compared with gnomAD; however, such variants in the 2 databases are rarely differentiated by SpliceAI scores. Empirical evaluation via RNA sequencing analysis supported novel acceptor site usage from -21 to +30; splice-altering variants did not predominate in a specific region or have SpliceAI scores invariantly, suggesting increased spliceogenicity. CONCLUSION: NAGNnAG variants within 30 bp of the natural splice acceptor have a high probability of clinical relevance and are poorly contextualized for clinical utility. Their interpretation benefits from empirical evaluation via RNA analysis.

Humans↗

PubMed2025

Utility of genome sequencing and group-enrichment to support splice variant interpretation in Marfan syndrome.

PURPOSE: To quantify the impact of noncanonical FBN1 splice site variants in undiagnosed Marfan syndrome (MFS), a connective tissue disorder associated with skeletal abnormalities and familial thoracic aortic aneurysm disease (FTAAD). METHODS: A systematic analysis of ultrarare FBN1 variants was performed using genome sequencing data from the 100,000 Genomes Project. Variants were annotated with SpliceAI and the significance of enrichment among individuals with FTAAD was assessed using Fisher's exact test. Experimental validation used RNA sequencing, reverse transcriptase polymerase chain reaction, minigene constructs, and replication analysis was with data from UK Biobank. RESULTS: Using aggregate data for 78,195 individuals, we identified 13,864 singleton single-nucleotide variants in FBN1 of which 21 were predicted to affect splicing (SpliceAI > 0.5). Incidence of candidate splice variants in individuals recruited with FTAAD (9/703) was significantly elevated compared with that seen in non-FTAAD participants (12/77,492; odds ratio = 84, P = 9.7 × 10-14). Additional analysis uncovered a further 14 families harboring 11 different FBN1 splice variants. A total of 20 candidate splice variants in 23 families were identified, of which 70% lay beyond the ±8 splice regions. RNA testing confirmed the predicted splice aberration in 16 of 20 and for 9 of 20, pseudoexonization was the likely splicing anomaly. CONCLUSION: Our findings indicate that noncanonical splice variants may account for approximately 3% of families with undiagnosed FTAAD, highlighting the importance of incorporating analysis of introns and confirmatory RNA testing into genetic testing for Marfan syndrome.

Humans↗

PubMed2026

Tackling non-canonical splicing in arrhythmogenic cardiomyopathy to reduce the uncertain significance variants burden.

BACKGROUND: Splice-altering variants (SAVs), particularly those outside canonical splice sites, are an underappreciated contributor to inherited cardiovascular diseases. In arrhythmogenic cardiomyopathy (ACM), these variants frequently remain classified as of uncertain significance (VUS) due to limited predictive power and lack of transcript-level evidence, constraining genetic yield and clinical management. Our study aimed to determine the functional impact of SAVs in ACM genes and refine their classification using ACMG/AMP and ClinGen SVI criteria. METHODS: SAVs identified in 200 ACM probands underwent SpliceAI prediction, GTEx cardiac exon-usage annotation, and functional assessment using pSPL3-based minigene assays. Aberrant transcripts were quantified using Percent Splicing Alteration (PSA). Segregation data and ACMG/AMP criteria refined by ClinGen SVI were applied to integrate functional and clinical evidence for classification. RESULTS: Aberrant splicing was confirmed in 9/20 variants (45%), including synonymous, missense, and non-canonical intronic changes. SpliceAI scores correlated strongly with PSA values (R²=0.86). Case-control burden testing revealed significant enrichment of splice-altering variants in DSP, DSG2, DSC2 and FLNC. Integrating predictive algorithms with experimental validation and segregation analysis markedly enhances reclassification of 16/20 variants (80%). CONCLUSION: Splicing defects beyond canonical sites significantly shape ACM genetic landscape. Integrating predictive models with experimental validation clarifies uncertain variants bridging the gap between genomic uncertainty and clinical decision-making.

Humans↗

PubMed2025

Substitutions of nucleotides at the 3' ends of COL6A1/2/3 exons induce exon skipping associated with collagen VI-related muscular dystrophies and therapeutic strategies.

PURPOSE: Collagen VI-related muscular dystrophies, characterized by proximal muscle weakness and joint contractures, are caused by pathogenic variants in the genes, COL6A1 to COL6A3. A monoallelic variant at the last nucleotide of a COL6A1 exon was initially classified as a missense variant but acted as a splicing variant, resulting in exon skipping. Here, we evaluated whether single-nucleotide variants at the 3'-ends of COL6A1 to COL6A3 exons cause aberrant splicing. METHODS: Ten relevant variants were identified in patients from our repository or public databases, and their muscle COL6A1 to COL6A3 transcripts were analyzed. The effects of the variants on splicing were also analyzed by minigene assay and SpliceAI in silico prediction. RESULTS: Transcripts from muscles of individuals with suspected collagen VI-related phenotypes showed exon skipping (skipping rate >12%). Findings of minigene assay and in silico prediction experiments supported these findings. Two therapeutic approaches, splicing correction of pre-messenger RNA or gene silencing of mature messenger RNA were assessed. Among them, gene silencing using short interfering RNAs targeting the skipped transcripts proved to be effective in restoring collagen VI in cells containing the pathogenic variant. CONCLUSION: Single-nucleotide variants at the 3'-ends of exons can lead to aberrant splicing, and allele-specific gene silencing targeting such variants is a promising therapeutic strategy.

Humans↗

PubMed2025

Genome sequencing reveals the impact of pseudoexons in rare genetic disease.

PURPOSE: Advancements in sequencing technologies have significantly improved clinical genetic testing; yet, the diagnostic yield remains around 30% to 40%. Emerging technologies are now being deployed to address the remaining diagnostic gap. METHODS: We tested whether short-read genome sequencing could increase the diagnostic yield in individuals enrolled into the UCI-GREGoR research study, who had suspected Mendelian conditions and prior inconclusive testing. Two other collaborative research cohorts, focused on aortopathy and dilated cardiomyopathy, consisted of individuals who were undiagnosed but had not undergone harmonized prior testing. RESULTS: We sequenced 353 families (754 participants) and found a molecular diagnosis in 54 (15.3%) of them. Of these diagnoses, 55.5% were previously missed because the causative variants were in regions not originally interrogated. In 5 cases, they were deep intronic variants, all of which led to abnormal splicing and pseudoexons, as directly shown by RNA sequencing. All 5 of these variants had inconclusive spliceAI scores. In 26% of newly diagnosed cases, the causal variant could have been detected by exome sequencing reanalysis. CONCLUSION: Genome sequencing can overcome limitations of clinical genetic testing, such as the inability to call intronic variants. Our findings highlight pseudoexons as a common mechanism via which deep intronic variants cause Mendelian disease.

Humans↗

PubMed2026

Novel splice-site and recurrent p.Arg729* CNKSR2 variants in ESES/CSWS: insights into sex-dependent expression.

PURPOSE: Pathogenic variants in CNKSR2 (Xp22.12) cause an X-linked neurodevelopmental disorder with intellectual disability, language impairment, and a distinctive epilepsy phenotype, including encephalopathy with status epilepticus during slow-wave sleep (ESES/CSWS). Hemizygous males are typically severely affected, whereas symptomatic females remain rare and incompletely characterized. We describe two unrelated patients with de novo CNKSR2 variants to expand the mutational and sex-dependent phenotypic spectrum of this disorder. METHODS: Both patients underwent clinical, electroencephalographic, and neuroimaging evaluation. CNKSR2 variants were identified by whole exome sequencing with parental segregation, and the splice-site variant was assessed in silico (SpliceAI, MaxEntScan, Human Splicing Finder). RESULTS: Patient 1, a 17-year-old female, harbored a novel canonical splice-site variant (c.64+1G>A) in the N-terminal region and presented with a relatively mild phenotype. In silico analysis supported abolition of the canonical donor splice site. Patient 2, an 8-year-old male, carried a de novo nonsense variant (c.2185C>T, p.Arg729*) and exhibited drug-resistant ESES, autism, and severe language impairment. p.Arg729* had previously been reported in one independent male. Our case represents its second independent occurrence, a CGA>TGA transition at a CpG dinucleotide consistent with a mutational hotspot. CONCLUSION: Together, these cases expand the mutational spectrum and provide further evidence for sex-dependent phenotypic variability in CNKSR2-related epilepsy. Our observations support the hypothesis that X-chromosome inactivation may contribute to phenotypic variability in females, although XCI was not assessed here, and support inclusion of CNKSR2 in epilepsy gene panels regardless of sex.

Humans↗

PubMed2026

Beyond in silico prediction: multi-omics to identify a pathogenic deep intronic HNRNPK variant in Au-Kline syndrome.

Pathogenic variants in HNRNPK are associated with autosomal dominant Au-Kline syndrome (AKS, Au-Kline-Okamoto syndrome, OMIM #616580). This syndrome is characterized by developmental delay and intellectual disability, hypotonia, and distinctive facial features. Despite the use of whole-genome sequencing (WGS) as a powerful diagnostic tool, we nearly dismissed a novel intronic variant (NM_031263.4(HNRNPK):c.214-55 T > A) affecting HNRNPK splicing and function. Although commonly used bioinformatic splice prediction tools, including SpliceAI and PDIVAS, yielded inconclusive results, Face2Gene analysis indicated a high phenotypic similarity to AKS. Characteristic facial features described by Choufani et al. [1] supported the clinical diagnosis of AKS. Subsequent functional studies demonstrated aberrant splicing with intron retention, and DNA methylation profiling revealed a positive HNRNPK-specific episignature. These insights and the de novo status support an evaluation as likely pathogenic. This case report supports the relevance of facial analysis and comprehensive variant validation strategies, particularly for deep intronic variants with ambiguous in silico splicing predictions.

Journal Article↗

PubMed2025

Exome sequencing identifies a homozygous splice site variant in RP1 as the underlying cause of autosomal recessive retinitis pigmentosa in a Pakistani family.

BACKGROUND: Mutations in RP1 gene are the third leading cause of inherited retinal dystrophies (IRDs) in Pakistani families. PATIENTS: A two-generation consanguineous Pakistani family underwent both clinical and genetic analyses. Clinical examinations included visual acuity test, visual field, fundoscopy, and ocular coherence tomography (OCT). Whole exome sequencing (WES) was performed on the proband's DNA, and Sanger sequencing was performed to validate the WES findings. Splicing prediction tools such as Human Splicing Finder (HSF), NNSplice predictor, SpliceAI, MaxENTScan, and SpliceRover were used. RESULTS: A nuclear family of seven children, comprising five affected individuals (four males and one female) and two healthy siblings, was recruited from northwestern Pakistan. The proband was a 49-years old male who was presented with complaints of decreased visual acuity and night blindness since early childhood. Upon clinical evaluation, the proband appeared to have severely reduced visual acuity of hand movement (HM), bilateral visual field constriction, a waxy pale disc with vascular attenuation, pigmentary bone spicules at the periphery associated with chorioretinal degeneration, diffuse macular atrophy, and horizontal nystagmus in both of his eyes. Exome sequencing (ES) in the proband identified a homozygous splice site variant (NM_006269.2: c.615 + 1G > A) in RP1 gene. In-silico analysis, genotype-phenotype co-segregation study, and literature survey strongly supported the causality of the detected variant. CONCLUSIONS: We report a previously known pathogenic splice site variant of RP1 as the underlying cause of early-onset autosomal recessive retinitis pigmentosa (arRP) in a Pakistani family. We contemplate that the detected allele might constitute a mutational hotspot in RP1.

Humans↗

PubMed2026

A novel deep intronic EIF2AK3 variant disrupts splicing and causes Wolcott-Rallison syndrome.

AIM: Deep intronic variants can disrupt splicing and cause monogenic disease but are missed by routine genetic testing. This study assessed the contribution of deep intronic variants to Wolcott-Rallison syndrome (WRS), a recessive disorder characterized by early-onset diabetes and progressive multisystem disease caused by loss-of-function EIF2AK3 variants. METHODS: We investigated a cohort of 116 individuals referred to the Exeter Genomics Laboratory for genetic testing who had diabetes diagnosed at ≤2 years and at least one additional feature consistent with WRS: hepatic dysfunction, skeletal abnormalities or developmental delay. No genetic cause had been identified after testing all known early-onset diabetes genes. We screened genome-sequencing data for rare homozygous intronic EIF2AK3 variants. Candidate variants predicted to affect splicing by SpliceAI were assessed using a minigene exon-trapping assay. RESULTS: We identified two rare homozygous intronic EIF2AK3 variants in two siblings. Only one variant, c.1651-180G>T, was predicted to disrupt splicing in silico. The two children, born to consanguineous parents, were diagnosed with early-onset diabetes (diagnosed at 1 year and 21 weeks), hepatic dysfunction, skeletal abnormalities, developmental delay, thyroid dysfunction, hip dysplasia and gait abnormalities. The minigene assay showed that c.1651-180G>T creates a cryptic donor splice site within intron 9, resulting in inclusion of a 79-nucleotide pseudoexon, causing a frameshift and premature stop codon. Using this evidence, the variant was reclassified as likely pathogenic according to ACMG/ACGS guidelines. CONCLUSIONS: We report the first deep intronic EIF2AK3 variant causing WRS, highlighting the need to consider systematic intronic analysis in unresolved cases.

EIF2AK3↗

PubMed2025

Unraveling the complex genetic landscape of OTOF-related hearing loss: a deep dive into cryptic variants and haplotype phasing.

BACKGROUND: Pathogenic variants in OTOF are a major cause of auditory synaptopathy. However, challenges remain in interpreting OTOF variants, including difficulties in confirming haplotype phasing using traditional short-read sequencing (SRS) due to the large gene size, the potential incomplete penetrance of certain variants, and difficulties in assessing variants at non-canonical splice sites. This study aims to revisit the genetic landscape of OTOF variants in a Taiwanese non-syndromic auditory neuropathy spectrum disorder (ANSD) cohort using a combination of sequencing technologies, predictive tools, and experimental validations. METHODS: We performed SRS to analyze OTOF variants in 65 unrelated Taiwanese patients diagnosed with non-syndromic ANSD, complemented by long-read sequencing (LRS) for haplotype phasing. A prediction-to-validation pipeline was implemented to assess the pathogenicity of cryptic variants using SpliceAI software and minigene assays. RESULTS: Biallelic pathogenic OTOF variants were identified in 33 patients (50.8%), while monoallelic variants were found in five patients. Three novel variants, c.3864G > A (p.Ala1288 =), c.4501G > A (p.Ala1501Thr), and c.5813 + 2T > C, were detected. The pathogenicity of two non-canonical mis-splicing variants, c.3894 + 5G > C and c.3864G > A (p.Ala1288 =), was confirmed by minigene assays. LRS-based haplotype phasing revealed that the common missense variant c.5098G > C (p.Glu1700Gln) and the novel variant c.5975A > G (p.Lys1992Arg) are in cis and form a founder pathogenic allele in the Taiwanese population. CONCLUSIONS: Our study highlights the genetic heterogeneity of DFNB9 and emphasizes the importance of population-specific variant interpretation. The integration of advanced sequencing technologies, predictive algorithms, and functional validation assays will improve the accuracy of molecular diagnosis and inform personalized treatment strategies for individuals with DFNB9.

Humans↗