Novel deep intronic variants in NTRK1 underlying congenital insensitivity to pain with anhidrosis

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disorder caused by mutations in NTRK1 that is characterized by pain insensitivity, anhidrosis, and recurrent fever. While genetic testing is the gold standard for CIPA diagnosis, the complexity of NTRK1 variants poses major challenges. Conventional sequencing that is limited to the coding regions of NTRK1 results in misdiagnoses or missed diagnoses in approximately 57% of patients. Accordingly, to improve the diagnostic efficiency of CIPA, we integrated whole-genome sequencing (WGS) with functional assays to identify deep intronic variants in NTRK1.

All 18 probands were initially screened using polymerase chain reaction (PCR) and Sanger sequencing covering all exons and canonical splice sites of NTRK1. For patients with only one identified pathogenic allele, WGS was performed to detect potential deep intronic variants. Candidate variants were functionally validated using reverse transcription PCR (RT-PCR) and T cloning sequencing to evaluate their effects on pre-mRNA splicing.

Total 23 pathogenic variants including 11 novel variants in NTRK1 were identified in 18 unrelated families with CIPA. Functional assays confirmed that five of these variants disrupted the normal splicing of NTRK1, resulting in multiple aberrant splicing patterns, including two exon-skipping events (c.428 + 273A>T, c.850 + 5G>A), three intron retentions (c.2187 + 389C>T, c.2188-459G>T, c.287 + 4A>C), and one pseudoexon insertion (c.2188-459G>T).

This study expands the spectrum of pathogenic variants in NTRK1 and improves the genetic diagnosis of CIPA. The functional characterization of five novel non-canonical splicing variants provides deeper insight into the molecular pathogenesis of this disorder and establishes a foundation for future precision medicine approaches in CIPA.