From Uncertainty to Pathogenicity: Resolving a CSF1R Variant of Uncertain Significance Using Long-Read...

Area of Science:

• Genomics and transcriptomics
• Neurogenetics
• Molecular diagnostics

Background:

• CSF1R-related disorder (CSF1R-RD) is a severe, progressive leukoencephalopathy.
• Many CSF1R variants remain classified as variants of uncertain significance (VUS), hindering genetic testing.
• Accurate variant classification is crucial for pre-symptomatic and prenatal diagnosis.

Purpose of the Study:

• To investigate a CSF1R splice-region variant classified as VUS in a patient with CSF1R-RD.
• To determine the transcript-level consequences of the identified CSF1R variant.
• To establish the pathogenicity of the variant and enable definitive molecular diagnosis.

Main Methods:

• Targeted long-read RNA sequencing of peripheral blood was performed.
• Transcriptomic analysis was used to identify novel isoforms and assess transcript abundance.
• Variant pathogenicity was evaluated using ACMG and ACGS guidelines.

Main Results:

• A novel exon 20-skipping isoform of CSF1R was identified, absent in controls.
• This isoform accounted for approximately 63% of transcripts, leading to reduced canonical transcript levels.
• The exon skipping causes a frameshift and premature termination codon, disrupting the tyrosine kinase domain, leading to reclassification from VUS to likely pathogenic.

Conclusions:

• Targeted long-read transcriptomic sequencing provides decisive functional evidence for non-canonical splice variants in CSF1R-RD.
• Transcript-level assessment is a valuable adjunct to genomic testing for variants of uncertain significance.
• This approach facilitates definitive molecular diagnosis in clinically compelling cases.