L1cam alternative shorter transcripts encoding the extracellular domains were overexpressed in the intestine of L1cam knockdown mice.

Hirschsprung disease (HSCR) is a congenital disorder of functional bowel obstruction due to the absence of enteric ganglia in distal bowel. Different L1cam variants were reportedly associated with L1cam syndrome and HSCR, whose phenotypes lacked predictable relevance to their genotypes. Using next-generation sequencing (NGS), we found an L1CAM de novo frameshift mutation in a female with mild hydrocephalus and skip-type HSCR. A nearly identical L1cam variant was introduced into FVB/NJ mice via the CRISPR-EZ method. A silent mutation was created via ssODN to gain an artificial Ncol restriction enzyme site for easier genotyping. Six L1cam protein-coding alternative transcripts were quantitatively measured. Immunofluorescence staining with polyclonal and monoclonal L1cam antibodies was used to characterize L1cam isoform proteins in enteric ganglia. Fifteen mice, seven males and eight females, generated via CRISPR-EZ, were confirmed to carry the L1cam frameshift variant, resulting in a premature stop codon. There was no prominent hydrocephalus nor HSCR-like presentation in these mice, but male infertility was noticed after observation for three generations in a total of 176 mice. Full-length L1cam transcripts were detected at a very low level in the intestinal tissues and almost none in the brain of these mice. Alternative shorter transcripts encoding the extracellular domains were overexpressed in the intestine of L1cam knockdown mice. Immunofluorescence confirmed no fulllength L1cam protein in enteric ganglia. These shorter L1cam isoform proteins might play a role in protecting L1cam knockdown mice from HSCR.