Utility of Urine-Derived Cells for Characterizing Aberrant Splicing Caused by a Novel Deep Intronic L1CAM Variant

Pathogenic variants in L1CAM, located at Xq28, cause a spectrum of neurodevelopmental disorders of varying severity, including congenital hydrocephalus, MASA syndrome, agenesis of the corpus callosum, and intellectual disability.

Exome sequencing (ES) and RNA studies using urine‑derived cells were performed in the younger sibling with agenesis of the corpus callosum, ventriculomegaly, and hearing impairment. A minigene assay was performed to quantitively evaluate the splicing impact of the L1CAM variant.

We identified a deep intronic L1CAM variant (NM_001278116.2:c.1124-24T>G) in Intron 10, for which SpliceAI predicts creation of a cryptic acceptor site (score 0.99) via introduction of an AG dinucleotide. The same L1CAM variant was also detected in the older affected brother. RNA studies using of urine‑derived cells UDCs demonstrated retention of a 23‑bp intronic segment in the transcripts, consistent with nonsense‑mediated decay (NMD). Specifically, TA‑cloning of reverse transcription PCR products detected the mutant allele in 2% of colonies, and RNA‑seq recovered the aberrant junction in only 5 of 18 reads. A minigene assay corroborated the mechanism, yielding a variant-specific larger product corresponding to the 23‑bp retained sequence.

This report broadens the molecular spectrum of intronic L1CAM variants and underscores the practical value of non‑invasive, UDC-based RNA testing in combination with complementary minigene assays for interpreting deep intronic variants.