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

VPS41 encodes a component of the HOPS complex, a six-part molecular machine that acts as a tether, physically bringing lysosomes together with late endosomes and autophagosomes so these membrane-bound compartments can fuse. This fusion step is central to autophagy, the cellular recycling program that clears damaged proteins and organelles, and to endolysosomal trafficking, the logistics network that shuttles cargo through the cell’s degradation compartments. When this pathway falters, cellular waste accumulates, and the consequences are felt most severely in neurons, which are long-lived cells with limited capacity for self-renewal. Disruption of lysosomal function has been implicated in lysosomal storage disorders, neurodevelopmental syndromes, and neurodegenerative conditions, making genes like VPS41 prime suspects in unexplained neurological disease. Since VPS41-related disorders were first reported in 2020, the handful of documented patients have shown cerebellar ataxia, cognitive impairment, and dystonia, inherited in an autosomal recessive pattern, meaning both copies of the gene must be impaired for disease to emerge.

The patient at the center of the new report presented a clinical picture that was both characteristic and perplexing. He had cerebellar ataxia, hypotonia, and intellectual disability dating from early childhood, together with a full-scale IQ of 49 on formal cognitive assessment. Brain magnetic resonance imaging revealed anterior-predominant atrophy of the cerebellum, the movement-coordination structure at the back of the brain. Yet he also displayed features never before recorded in this syndrome: progressive swan-neck deformities of the second to fourth fingers on both hands and pes cavus, a high-arched foot deformity. Because pes cavus often signals peripheral nerve disease, clinicians repeatedly investigated that possibility. Motor nerve conduction studies performed at ages ten, fifteen, and twenty years consistently showed preserved conduction velocities in both arms and legs, and spinal cord imaging of the cervical and upper thoracic regions showed no abnormalities suggestive of posterior column degeneration. A 103-gene sequencing panel covering Charcot-Marie-Tooth disease and related inherited neuropathies, along with chromosomal microarray analysis, all came back unrevealing.

Exome sequencing eventually flagged four variants in VPS41, each confirmed by Sanger sequencing. One was a splice-site variant, c.385-2A>G, at a canonical position where the splicing machinery recognizes an exon boundary. The other three were missense variants that each change a single amino acid: p.Val137Met, p.Thr294Met, and p.Arg416His. Segregation analysis in the family added a complication. The healthy older brother carried none of the variants, and the father carried two of them, Thr294Met and Arg416His, on what was presumed to be one chromosome. But the inheritance pattern of the splice-site variant and the Val137Met variant could not be established, because the patient’s mother had died and her DNA was unavailable. Under the American College of Medical Genetics and Genomics classification framework, the splice-site variant was judged likely pathogenic based on its predicted severe effect on splicing and its rarity in population databases, while the three missense variants remained variants of uncertain significance. Without knowing which variants sat on which parental chromosomes, the team could not confirm that the patient had one damaging mutation on each of his two VPS41 copies, the configuration required for recessive disease.

That is where long-read genome sequencing made the decisive difference. Unlike standard short-read sequencing, which chops DNA into fragments of a few hundred bases and struggles to bridge complex haplotypes, long-read technology can span entire haplotype blocks in single DNA molecules, directly revealing which variants travel together on the same chromosome. The analysis showed that Val137Met sat in cis with the splice-site variant, meaning both occupied the same maternal allele, while Thr294Met and Arg416His lay in trans, on the opposite paternal copy. Because the splice-site variant disrupts splicing and the missense variants on the other chromosome were candidates for functional damage, the phasing narrowed the field to two alleles requiring laboratory validation. In addition, computational splicing prediction with SpliceAI assigned the Arg416His variant a high probability of disrupting the adjacent exon boundary, with delta scores of 0.96 for donor loss and 0.93 for acceptor loss, elevating it to the primary suspect on the paternal allele. With maternal DNA absent, this resolution would have been impossible using conventional approaches.

The researchers then examined RNA extracted from patient-derived lymphoblastoid cell lines, immortalized white blood cells that provide a renewable window into the patient’s gene expression. Transcriptome analysis revealed an abnormal splice junction consistent with skipping of exon 7, a defect the team attributed to the c.385-2A>G variant on the maternal copy. Quantitative RT-PCR showed that total VPS41 messenger RNA was significantly reduced in the patient’s cells compared with controls, and targeted RT-PCR with sequencing confirmed the exon 7 skip, which deletes 66 bases while preserving the reading frame. Because the deletion keeps triplets intact, the transcript evades nonsense-mediated decay, the cellular quality-control system that normally destroys messages carrying premature stop codons, allowing a shortened protein to be made. The paternal allele told a different story. A second primer set detected a low-abundance transcript lacking exon 15, and when the researchers treated the cells with cycloheximide, a drug that indirectly blocks nonsense-mediated decay, this aberrant band grew clearly visible. Exon 15 skipping removes 62 bases and shifts the reading frame, creating a premature stop codon, which explains why the defective message is normally degraded almost completely.

The protein-level consequences were equally informative. Western blot analysis showed that VPS41 protein was present in the patient’s cells but reduced to roughly 30 percent of control levels. Simple loss of one allele through nonsense-mediated decay would be expected to halve expression, so the deeper reduction suggests the exon-7-skipped protein produced by the maternal allele is itself partially unstable or targeted for accelerated degradation. To probe that idea, the team built structural models of the HOPS complex with AlphaFold and compared the wild-type machine against a version carrying the 22-amino-acid in-frame deletion, p.Ile129_Lys150del, that the exon 7 skip produces. Although the deleted region falls outside the annotated WD40 repeat domain spanning roughly amino acids 302 to 747, it lies near TPR-like and CHCR motifs that help shape the scaffold-like beta-propeller architecture of VPS41. The modeling showed a reshaped interaction landscape: some intersubunit interfaces, such as the A-E pairing, gained contact area and predicted stability, while the B-E and B-F interfaces weakened substantially, with destabilizing free-energy changes of 6.6 and 4.3 kilocalories per mole. The deletion, in other words, does not simply amputate part of the protein; it redistributes stress across the entire six-subunit complex and may undermine its overall integrity.

Transmission electron microscopy of the patient’s lymphoblastoid cells then provided the cellular corroboration. The images revealed characteristic endolysosomal abnormalities, including multiple multivesicular bodies and multilamellar bodies, precisely the kind of membrane-compartment pileup expected when the HOPS tethering machinery cannot complete fusion events efficiently. Taken together, the RNA findings, the protein reduction, the structural perturbation, and the ultrastructural phenotype supplied functional evidence for both alleles. Applying the American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines alongside Clinical Genome Resource sequence variant interpretation recommendations, the team reclassified the maternal splice-site variant as likely pathogenic based on PVS1-moderate, PM2, and PM3 evidence, and the paternal Arg416His variant as likely pathogenic based on PVS1 and PM2 evidence, formally establishing a compound heterozygous diagnosis of autosomal recessive spinocerebellar ataxia 29.

The case carries lessons that reach well beyond a single family. It demonstrates that long-read sequencing can resolve haplotypes in rare disease diagnostics even when parental samples are unavailable, a situation that arises frequently given the age at which many such patients are evaluated. It also confirms that RNA-based functional assays, performed directly on patient-derived cells, can convert variants of uncertain significance into actionable diagnoses by revealing the exact molecular consequence of each change. The newly reported swan-neck deformities and pes cavus expand the phenotypic spectrum of VPS41-related disease, though the authors caution that, in a single case, it remains uncertain whether these features are specific to the syndrome or coincidental, and additional cases will be needed to settle that question. Limitations acknowledged by the team include the single-patient design, the current cost and limited clinical availability of long-read sequencing, and the absence of updated spinal cord imaging in adulthood. Even so, the study marks a clear demonstration of how third-generation sequencing and transcript-level analysis together can close diagnostic gaps that once seemed permanent, offering a template for the thousands of rare disease patients still waiting for an answer.

Subject of Research: Biallelic VPS41 variants causing autosomal recessive spinocerebellar ataxia 29 resolved by long-read sequencing and RNA analysis

Article Title: Biallelic VPS41 Variants in Autosomal Recessive Spinocerebellar Ataxia 29 Resolved by Long‐Read Sequencing and RNA Analysis

Article References: Nakamura, N., Nishio, Y., Nyuzuki, H., Fukushima, A., Miura, M., Kobayashi, Y., Ishioka, R., Tsukada, K., Oka, Y., Tsujikawa, K., Morinaga, H., Inaba, M., Tohyama, J., Nakazawa, Y., Ikeuchi, T., Ono, T., Saitoh, S., & Ogi, T. (2026). Biallelic VPS41 Variants in Autosomal Recessive Spinocerebellar Ataxia 29 Resolved by Long‐Read Sequencing and RNA Analysis. Molecular Genetics & Genomic Medicine, 14(9), Article e70285. https://doi.org/10.1002/mgg3.70285

Image Credits: AI Generated

DOI: 10.1002/mgg3.70285

Keywords: VPS41, HOPS complex, spinocerebellar ataxia, long-read sequencing, haplotype phasing, lysosomal trafficking, autophagy, splicing defect, nonsense-mediated decay, rare disease genetics, compound heterozygosity, exon skipping