RNA evidence resolves a specific uncertainty
Observed exon 20 skipping provides additional evidence that changes the variant's formal classification from VUS to likely pathogenic.
↳ Results, Long-Read Transcriptomic Analysis
Abstract
BACKGROUND: CSF1R-related disorder (CSF1R-RD) is a severe autosomal dominant leukoencephalopathy characterized by progressive cognitive, neuropsychiatric, and motor decline. Although genetic testing is widely available, numerous likely pathogenic variants in CSF1R frequently remain classified as variants of uncertain significance (VUS), limiting the option of pre-symptomatic genetic testing or prenatal options for at-risk family members. METHODS: We report a male in his early 50s with progressive leukoencephalopathy and a strong multigenerational family history supported by neuropathological findings consistent with CSF1R-RD. Genetic testing identified a heterozygous CSF1R splice-region variant (c.2763 + 4_2763 + 7del) classified as a VUS. Targeted long-read RNA sequencing of peripheral blood was performed to assess transcript-level consequences. RESULTS: Long-read transcriptomic analysis identified a novel exon 20-skipping isoform absent in pooled controls, accounting for approximately 63% of detected transcripts, with a corresponding reduction in the canonical transcript. Exon skipping (109 bp) results in a frameshift and a premature termination codon in the final exon, predicted to escape nonsense-mediated decay and disrupt the tyrosine kinase domain. These findings establish a direct molecular mechanism of pathogenicity. Functional evidence allowed reclassification of the variant using American College of Medical Genetics and Genomics (ACMG) and Association for Clinical Genomic Science (ACGS) guidelines from VUS to likely pathogenic, enabling definitive molecular diagnosis. CONCLUSIONS: This case demonstrates that targeted long-read transcriptomic sequencing can provide decisive functional evidence to resolve non-canonical splice variants in CSF1R-RD. Focused transcript-level assessment represents a practical adjunct to genomic testing in clinically compelling cases where DNA-based interpretation alone is insufficient. © 2026 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Key Claims
Primary findingTentative
Functional evidence allowed reclassification of the variant using American College of Medical Genetics and Genomics (ACMG) and Association for Clinical Genomic Science (ACGS) guidelines from VUS to likely pathogenic, enabling definitive molecular diagnosis.
single-proband RNA evidence supports likely-pathogenic reclassification rather than the definitive diagnostic certainty stated
Abstract, Results
SecondarySupported
Long-read transcriptomic analysis identified a novel exon 20-skipping isoform absent in pooled controls, accounting for approximately 63% of detected transcripts, with a corresponding reduction in the canonical transcript.
direct transcript observation in one proband with incompletely characterized pooled controls
Abstract, Results; Results, Long-Read Transcriptomic Analysis
Derived from the full evaluation — not a separate score.
Strengths · Limitations
Strengths
Variant classification logic is explicitly documented
The report states the ACMG/ACGS criteria, explains why RNA evidence replaces computational evidence, and avoids counting the same functional result again under PS3.
↳ Results, Long-Read Transcriptomic Analysis
Family testing provides a concrete application
The classification change enables consideration of presymptomatic testing for at-risk relatives and supports genetic counselling.
↳ Results, Long-Read Transcriptomic Analysis; Discussion
Limitations
Single proband limits assay performance assessment
RNA is analyzed in one individual, without reported replicate-level results or a stated pooled-control sample count. This limits assessment of the stability and broader performance of the transcript finding.
↳ Methods, Genetic Analysis; Results, Long-Read Transcriptomic Analysis
Diagnostic certainty exceeds the reported classification
The Abstract calls the diagnosis definitive while formally classifying the variant as likely pathogenic. Discussion also describes skipping as reproducible without presenting replicate evidence.
↳ Abstract, Results; Discussion
Comparative methods and boundaries receive limited discussion
The report does not substantively compare alternative splice-validation approaches or examine transfer beyond this variant and peripheral-blood setting.
↳ Introduction; Discussion
Nabu Commentary
The central contribution is a variant-specific RNA finding linked transparently to a likely-pathogenic classification. Detailed clinical and family documentation and explicit avoidance of double-counting functional evidence support the case-level interpretation. The single RNA sample and limited control reporting prevent this result from establishing broader assay validity. Explicit prediction language appropriately qualifies protein consequences, but references to a definitive diagnosis and reproducible skipping warrant more restrained wording. Comparative methods and blood-assay boundary conditions receive limited discussion.
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Author Note / Field Feedback
Nabu’s assessment, alongside the field’s view.
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Dimension breakdown
Study Quality
Sound3.5
Confidence mediumContribution
3.5
The report resolves a specific CSF1R splice-region VUS using observed RNA changes and documents the resulting classification change. Its advance is variant-specific rather than a validation of RNA-based variant resolution in general.
Results, Long-Read Transcriptomic Analysis; Discussion
Methodological Rigour
3.6
Clinical and familial documentation, multi-relative variant confirmation, and explicit ACMG/ACGS reasoning support the case-level conclusion. Single-proband RNA testing, incomplete control characterization, and partly documented diagnostic alternatives reduce execution certainty.
Clinical Description; Family History; Genetic Analysis; Results, Long-Read Transcriptomic Analysis
Reporting
3.5
The clinical-to-molecular argument is readily followed and downstream protein effects are explicitly predicted. Central references to a definitive diagnosis and reproducible skipping convey more certainty than the reported classification and replication evidence warrant.
Abstract, Results; Results, Long-Read Transcriptomic Analysis; Discussion
Positioning
3.3
The disease, classification framework, and clinical implications are explained. Positioning against prior RNA-based VUS resolution and alternative splice-validation methods is limited, as is discussion of assay boundary conditions.
Introduction; Discussion; references 1–10
Reliability
No concerns4 of 4 checks
Results checkClean
The reported numbers and statistics are consistent with the stated results.
Reporting and transparencyClean
Consent and ethics approval are declared, conflicts are disclosed as absent, and data availability is qualified by privacy restrictions. Limited assay reporting affects methodological assessment rather than establishing a conduct concern.
Conflict of interest: declared · Ethics approval: declared · Data availability: declared
Reference IntegrityClean
10 references in manuscript 7 of 8 checkable references found in an index 2 are books, websites or datasets — counted, but not index-checkable
Post-Publication RecordClean
No retraction or expression of concern found in Crossref (includes Retraction Watch).
Last checked: 19 Oct 2026
Sources: Crossref (incl. Retraction Watch) ✓
Readiness
Where this paper’s evidence sits on the path from initial observation to real-world use.
Demonstrated
The assay was applied to a real diagnostic case and the workflow schematic gives a 1–2 working-day turnaround. Validation across additional cases or sites is not reported.
Discussion; Figure 3
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