The investigators evaluated four members of a two-generation family recruited through a school for deaf children: an affected proband and three reportedly unaffected first-degree relatives. The proband passed newborn hearing screening at three days old, but subsequent testing at 19 months found no reproducible auditory brainstem responses at stimulus levels up to 97 dB nHL in either ear. Distortion-product otoacoustic emissions had signal-to-noise ratios below 6 dB at most tested frequencies bilaterally. Evaluation at age six confirmed persistent profound impairment, with click-evoked thresholds above 109 dB nHL and severe-to-profound sensorineural hearing loss across all frequencies on unaided pure-tone audiometry.

Targeted sequencing of a 406-gene hereditary hearing-loss panel identified the heterozygous P2RX2 variant NM_174873:c.278_288delinsACGTGAAGCCCCC. High-depth validation yielded mean coverage above 1,000 times, and the variant was absent from population databases including gnomAD. Segregation analysis showed that the proband inherited the P2RX2 allele from the father. The investigators classified it as pathogenic under the American College of Medical Genetics and Genomics and Association for Molecular Pathology framework using PVS1, PS3 and PM2_Supporting evidence.

The proband also carried the recurrent pathogenic GJB2 frameshift NM_004004:c.235delC, or p.Leu79Cysfs*3, inherited from the mother. The mother and younger sister carried this GJB2 allele but were reported to be unaffected. Because heterozygous GJB2 carriers are typically asymptomatic, the pedigree alone cannot establish that the GJB2 variant caused or modified the proband’s hearing loss. The authors instead treated a possible interaction between the two genes as a hypothesis requiring independent validation.

To test the splice defect, the researchers constructed pcMINI-C minigene reporters containing P2RX2 exon 2, flanking intronic sequence and downstream exon 3. The wild-type reporter yielded one RT-PCR product in HEK293T and HeLa cells, whereas the mutant reporter yielded two aberrant products in both cell lines. Sequencing resolved a 19-base-pair deletion in the first transcript and a 34-base-pair deletion in the second. Both were out-of-frame alterations that introduced premature termination codons, rather than in-frame deletions.

The reconstructed isoforms were designated mut1, corresponding to c.278_309delinsACGTGAAGCCCCCGA and predicted p.Trp93Tyrfs*173, and mut2, corresponding to c.276_309del and predicted p.Trp93Glyfs*26. Three computational tools supported the minigene result. SpliceAI assigned a score of 0.36 to loss of the canonical donor and 0.97 to gain of a cryptic donor. RDDCSC predicted a 36-base-pair deletion and an event score that increased from 0.004 in the wild type to 0.9623 in the mutant, with p=0.006. BDGP identified cryptic donor gains with scores of 0.49 and 0.68.

A SpliceVault search documented use of a cryptic donor site 19 base pairs upstream of the canonical site in 116 GTEx and 217 SRA reference samples, where it represented a rare mis-splicing event. The investigators then expressed each reconstructed isoform separately from intronless cDNA vectors in HEK293T cells, using biological triplicates. Relative to wild-type P2RX2 mRNA, mut1 abundance was lower by approximately 48.3% (p<0.05), while mut2 abundance was lower by 69.4% (p<0.01).

Western blotting detected the expected wild-type protein at about 47 kDa and truncated products at approximately 32 kDa for mut1 and 15 kDa for mut2. Densitometry normalized to GAPDH showed respective protein reductions of 92.7% and 61.0% compared with wild type, both at p<0.0001. These cell-based measurements connect the altered splicing with substantially reduced P2X2 output. Murine cochlear single-cell transcriptomic data showed P2RX2 expression mainly in sensory hair cells and spiral ganglion neurons, whereas GJB2 was enriched in supporting-cell populations, including the inner sulcus and Deiters’ cells. Computational enrichment and network analyses identified shared functional themes and nominated RYR3 as a possible connector, but these mouse-data associations did not demonstrate a digenic mechanism in the family.

The proband’s profound hearing loss contrasted with the father’s milder reported phenotype despite their shared P2RX2 allele, which the authors interpreted as consistent with variable expressivity and incomplete penetrance in P2RX2-linked DFNA41. The study was limited by its single affected proband carrying two variants, the absence of quantitative audiometry and a noise-exposure history for the father, and unavailable family-wide longitudinal audiologic tracking; the proband itself was assessed at 19 months and again at age six. Unmeasured environmental exposures and other genetic modifiers could not be excluded. The minigene and heterologous-cell experiments established the splice defect and reduced receptor output in the tested systems but did not fully reproduce the native cochlear environment. The reported evidence therefore supports the P2RX2 loss-of-function effect, while the cause of the marked intrafamilial variation remains unresolved.

Subject of Research: Functional characterization of a P2RX2 exon-2 splice-disrupting indel in a one-family DFNA41 study

Article Title: A P2RX2 exon-2 complex indel disrupts splicing and reduces P2X2 output: implications for DFNA41 and variable expressivity

Article References: Tao, Y., Song, W., Wang, L., Han, T., Han, D., Hao, L., Zhao, C., Ping, J., & Li, X. (2026). A P2RX2 exon-2 complex indel disrupts splicing and reduces P2X2 output: implications for DFNA41 and variable expressivity. Frontiers in Genetics, 17, Article 1934538. https://doi.org/10.3389/fgene.2026.1934538

Image Credits: AI Generated

DOI: 10.3389/fgene.2026.1934538

Keywords: P2RX2, DFNA41, hearing loss, complex indel, aberrant splicing, P2X2 receptor, loss of function, minigene assay, GJB2, variable expressivity, exon-2, complex

CLN7 disease, also known as variant late-infantile neuronal ceroid lipofuscinosis, is a lysosomal storage disorder caused by biallelic defects in MFSD8. The gene encodes major facilitator superfamily domain-containing protein 8, also called the CLN7 protein, a 518-amino-acid lysosomal transmembrane protein whose precise function remains incompletely understood but is considered essential for normal neuronal homeostasis. Current evidence suggests that MFSD8 may function as a chloride channel involved in lysosomal enzyme trafficking. Classic CLN7 disease typically begins between the ages of two and seven years and can involve seizures, motor impairment, progressive mental regression, myoclonus, speech impairment, vision loss and personality disorders. More broadly, neuronal ceroid lipofuscinoses are estimated to occur in one to four people per million live births, with frequencies varying by genetic subtype and population.

The proband was the first and only child of healthy, non-consanguineous Russian parents. She was born at 38 weeks after an uncomplicated pregnancy, apart from a first-trimester threatened miscarriage. Her birth weight was 3,170 g, her length was 52 cm, and her Apgar scores were 8 and 8. Early motor and speech development was initially within normal limits: she walked independently at 13 months and used phrase speech by two years and four months. At approximately four years of age, subtle cognitive and behavioral changes appeared, including reduced attention and memory. Her first atonic seizure occurred during a febrile illness at four years and ten months. Recurrent afebrile atonic seizures followed, and myoclonic seizures began at five years and two months.

As the illness progressed, the child developed progressive intention tremor, gait ataxia, dysarthria and motor incoordination. From the age of six, her neurological deterioration accelerated. She lost expressive speech, regressed in fine and gross motor skills and lost control of her bladder and bowels. By six and a half years, she could no longer walk independently; later, she could not sit or feed herself. A neurological examination at six years and eight months showed severe developmental regression, absent speech, generalized hypotonia with brisk deep tendon reflexes and ankle clonus, marked upper-limb, head and trunk tremor, truncal ataxia, impaired fixation and complete loss of independent ambulation. She was underweight, with a weight of 21 kg and a BMI of −2.9 standard deviations, and had an acquired head circumference of 50 cm, or −1.1 standard deviations, despite a height of 132 cm, or +2.64 standard deviations.

Brain magnetic resonance imaging revealed diffuse cerebral and cerebellar atrophy. Video electroencephalography showed diffuse background slowing with generalized spike/polyspike-and-slow-wave discharges and a posterior photoparoxysmal response during intermittent photic stimulation. Ophthalmological examination found only subtle optic disc pallor, without clinically significant retinal degeneration at the time of evaluation. By eight years and two months, the patient was bedridden, had lost the ability to swallow independently and required gastrostomy feeding. She also developed severe sleep disturbances requiring pharmacological treatment, while her seizures became more frequent and could be terminated only with injectable midazolam. The clinical course therefore combined childhood-onset epilepsy, progressive developmental regression, ataxia, loss of motor and speech function, and cerebral and cerebellar atrophy.

The initial diagnostic evaluation did not reveal a cause. Routine metabolic investigations, lysosomal enzyme assays, echocardiography, nerve-conduction studies, serum creatine kinase analysis and whole-exome sequencing were all unremarkable. Trio whole-genome sequencing subsequently identified the homozygous deep intronic MFSD8 variant c.999-556C>G, with each parent carrying a heterozygous copy. The variant was absent from gnomAD version 4.1.1 and had not previously been reported in ClinVar or the literature. Its intronic location meant that its effect could not be established from DNA sequence alone, particularly because noncoding variants cannot usually be interpreted from the nucleotide change without functional evidence. The investigators therefore tested the variant at the RNA level to determine whether it altered MFSD8 splicing.

SpliceAI predicted that c.999-556C>G would create cryptic splice sites with a delta score of 0.73 for the acceptor and 0.67 for the donor, resulting in inclusion of a 98-nucleotide pseudoexon. To evaluate that prediction, the researchers isolated RNA from cultured skin fibroblasts of the proband and her heterozygous father. They amplified the region spanning exons 6 through 12 by reverse-transcription polymerase chain reaction and analyzed the products through targeted next-generation sequencing, using RNA from a healthy donor fibroblast culture as a control. Targeted sequencing confirmed inclusion of the predicted pseudoexon at chromosome 4 positions 127,922,520–127,922,617 in RNA from the proband and her father. Both individuals had a canonical transcript and an aberrant transcript containing the pseudoexon.

Pseudoexon inclusion caused a frameshift and generated the premature termination-codon allele p.(Ile334Valfs*113). Among the MFSD8 transcript molecules detected in the targeted amplicon from the homozygous proband, 84% contained the pseudoexon. In the heterozygous father, the aberrant isoform accounted for 12% of the detected transcripts. These figures describe relative proportions within the analyzed RNA amplicons, rather than absolute transcript abundance. The lower proportion in the father is compatible with partial degradation of the premature-termination-containing RNA by nonsense-mediated decay, although the contribution of that mechanism could not be directly quantified because no informative heterozygous exonic variants were available for allele-specific analysis. Treating the father’s fibroblasts with a nonsense-mediated-decay inhibitor increased the relative abundance of the pseudoexon-containing transcript by approximately 1.7-fold, but it did not restore the fraction to the roughly 50% expected for a heterozygous genotype.

An independent minigene assay reproduced the splicing defect in an engineered system. The construct carrying the mutant sequence generated predominantly the aberrant transcript containing the 98-nucleotide pseudoexon, which accounted for 84.3% of transcripts, together with a minor canonical population of 15.5%. By contrast, the wild-type construct generated only the canonical transcript. The mutant construct’s continued production of some canonical RNA indicates that the variant strongly promotes aberrant splice-site recognition without completely abolishing recognition of the normal splice sites. The abundance of canonical transcripts in the minigene system cannot, however, be extrapolated directly to the endogenous MFSD8 locus or used to infer the amount of functional MFSD8 protein remaining in the patient. In combination with the clinical concordance and the findings in patient-derived RNA, the minigene result supports a causal relationship between c.999-556C>G and the abnormal splicing event. The authors consequently classified it as the causative mutation and considered other variants found by whole-genome sequencing unlikely to be disease-causing.

The case illustrates why deep intronic variants can escape routine diagnosis. Exon-focused sequencing may miss pathogenic changes located within introns, while DNA analysis alone may not establish what a noncoding variant does to RNA processing. The findings show how genome sequencing can identify a candidate regulatory change and how transcript analysis can directly test its effect at the endogenous gene. The report nevertheless remains a single-patient case: it does not establish how frequently this mechanism occurs in CLN7 disease, how it is distributed across populations or whether the same variant produces the same clinical course in other people. It nevertheless adds a distinct deep intronic allele to an MFSD8 spectrum already known to include missense, nonsense, frameshift, splice-site and copy-number variants. The absence of prominent retinal degeneration in this patient, despite severe neurological progression, also documents variability within the clinical presentation of CLN7 disease. A patient-specific antisense oligonucleotide has previously been developed for pseudoexon inclusion in another MFSD8-related case, and the clearly defined splicing defect reported here may provide a measurable molecular endpoint for individualized therapeutic studies, but no treatment was tested in this case report.

Subject of Research: Functional characterization of a deep intronic MFSD8 variant associated with CLN7 disease

Article Title: Case Report: A novel deep intronic MFSD8 splice-altering variant causes CLN7 disease through pseudoexon inclusion

Article References: Akimova, D., Voskanyan, A., Orlova, A., & Skoblov, M. (2026). Case Report: A novel deep intronic MFSD8 splice-altering variant causes CLN7 disease through pseudoexon inclusion. Frontiers in Genetics, 17, Article 1980180. https://doi.org/10.3389/fgene.2026.1980180

Image Credits: AI Generated

DOI: 10.3389/fgene.2026.1980180

Keywords: MFSD8, CLN7 disease, deep intronic variant, pseudoexon inclusion, RNA splicing, whole-genome sequencing, rare disease genetics, neuronal ceroid lipofuscinosis, patient-derived fibroblasts, minigene assay, Case, Report

Protein S is a cofactor for the activated protein C system, one of the body’s principal brakes on blood coagulation. When PROS1 function is impaired, the clotting cascade runs unchecked, and affected individuals face elevated risks of deep vein thrombosis and pulmonary embolism. Genetic testing for Protein S deficiency routinely encounters variants of uncertain significance, or VUS, and synonymous changes are among the most frequently dismissed, typically labeled benign because the protein sequence appears intact. The new study set out to test whether that dismissal is justified, systematically evaluating fourteen synonymous PROS1 variants with an unusually comprehensive experimental toolkit.

The investigators combined computational prediction with laboratory validation. In silico analyses assessed each variant’s likely impact on pre-mRNA splicing, messenger RNA stability, and codon usage, the preference for particular synonymous codons that can influence how efficiently ribosomes translate a transcript. The team then moved to bench experiments, deploying minigene splicing assays to see how each variant altered RNA processing in a controlled cellular setting, measuring mRNA decay rates to quantify transcript stability, and running protein expression and anticoagulant activity assays to determine the downstream functional consequences.

The results were unambiguous: these silent mutations are anything but silent. Six of the fourteen variants, or 42.9 percent, caused aberrant pre-mRNA splicing, meaning the cellular machinery misprocessed the RNA intermediate and produced faulty transcripts. Two variants, 14.3 percent, reduced mRNA stability, hastening the degradation of the PROS1 message before enough Protein S could be made. Another two variants, also 14.3 percent, impaired translational efficiency, leaving ribosomes less able to convert the message into functional protein. In every affected case, the molecular defect cascaded into decreased Protein S expression and diminished anticoagulant activity in the relevant assays.

The clinical significance of these findings was reinforced by data from three additional patients carrying spliceogenic PROS1 variants. Each of these individuals exhibited reduced Protein S levels and thrombotic manifestations that matched the functional defects identified experimentally, providing a genotype-phenotype correlation that links laboratory mechanism to real-world disease. This convergence of molecular and clinical evidence is precisely what variant interpretation guidelines have long demanded but rarely received for synonymous changes.

That convergence had immediate diagnostic consequences. By integrating the multi-layer functional evidence with clinical data, the researchers were able to reclassify several variants under the American College of Medical Genetics and Genomics and Association for Molecular Pathology framework, the standard rubric for assessing pathogenicity. Variants previously parked in the VUS category were moved to likely pathogenic or, in some cases, benign, sharpening the molecular diagnosis of Protein S deficiency and giving clinicians firmer ground for counseling patients and their families about thrombotic risk.

Perhaps the most forward-looking part of the study is its therapeutic proof of concept. Rather than stopping at diagnosis, the team pursued mechanism-guided RNA correction. For selected spliceogenic variants, they engineered modified U1 small nuclear RNAs, components of the spliceosome that can be tailored to recognize mutated splice sites, and antisense U7 snRNAs, which can block aberrant splice sites or redirect the splicing machinery. Delivered into cells, these engineered molecules partially restored normal splicing patterns for the targeted variants, demonstrating that the molecular defects are not only identifiable but potentially correctable.

The implications extend well beyond a single gene. Synonymous variants populate every protein-coding gene in the genome, and growing evidence indicates they can contribute to disease through splicing disruption, mRNA destabilization, altered microRNA binding, and impaired translation. Yet in routine clinical reporting they are still too often waved through as benign. The PROS1 study offers a template for how to interrogate them properly: pair computational screening with minigene assays, mRNA measurements, and protein-level functional readouts, then anchor the findings in patient phenotype data. Applied more broadly, such pipelines could rescue meaningful diagnoses from the VUS backlog that clogs modern genomics.

For patients with inherited thrombophilia, the therapeutic angle is particularly compelling. Current management of Protein S deficiency relies on anticoagulant drugs and vigilance rather than correction of the underlying defect. RNA-based splice correction, already being explored in other genetic diseases, offers a strategy that is variant-specific yet conceptually scalable: once a patient’s spliceogenic variant is defined, a tailored snRNA could in principle be designed to fix it. The partial restoration achieved in this study is an early step, and translating engineered snRNAs from cell culture to safe, effective human therapy will require substantial further development, but the feasibility argument has now been made for PROS1-related thrombosis.

The study also carries a cautionary message for genetic counseling. A synonymous PROS1 variant reported as benign today could, in a family with unexplained thrombosis, be the culprit. As functional evidence accumulates, guidelines will need to keep pace, and laboratories may need to treat synonymous variants near splice junctions or in conserved codon contexts with greater suspicion. What this research makes clear is that the genome’s apparent redundancy conceals layers of regulation, splicing, transcript stability, and translation, that mutation can disturb without changing a single amino acid. In the case of PROS1, listening to those silent signals revealed both hidden causes of a dangerous clotting disorder and a credible route toward fixing them with RNA.

Subject of Research: Functional and clinical characterization of synonymous PROS1 variants causing Protein S deficiency and their RNA-based splicing correction

Article Title: Comprehensive molecular and clinical characterization of synonymous PROS1 variants: from molecular mechanisms to RNA-based splicing correction

Article References: Zhang, H., Su, X., Yu, Q., Pan, B., Ma, D., Francesco, B., Guo, W., Wang, B., Balestra, D., & Zhao, Y. (2026). Comprehensive molecular and clinical characterization of synonymous PROS1 variants: from molecular mechanisms to RNA-based splicing correction. Journal of Translational Medicine. https://doi.org/10.1186/s12967-026-09005-0

Image Credits: AI Generated

DOI: 10.1186/s12967-026-09005-0

Keywords: PROS1, Protein S deficiency, synonymous variants, RNA splicing, thrombophilia, U1 snRNA, U7 snRNA, ACMG/AMP classification, mRNA stability, variant interpretation, RNA therapeutics, minigene assay