Age‐ and Tissue‐Specific Alternative Splicing of the Neurofibromin Gene in Swine

Abstract

Neurofibromatosis type 1 (NF1) is one of the most common monogenic disorders that affects 1 in 2,500 children. The disorder manifests in varying, complex phenotypes that include cognitive and learning dysfunction, bone and cardiovascular disorders, and an increased risk of developing benign and malignant nervous system tumors. NF1 is caused by mutations in the neurofibromin gene; over 4,000 different mutations have been clinically identified with very little understanding of genotype‐phenotype association in terms of the clinical presentation of the disorder. The human neurofibromin gene contains 61 exons, of which, four are alternatively spliced (optionally included) to produce isoforms of the protein. Over 50% of the mutations in NF1 patients are thought to produce abnormalities in splicing. Yet, only a handful of studies have examined the role of neurofibromin alternative splicing or acknowledged their potential role in cell biology. Recently, several swine models of NF1 have been developed in hopes that these models will recapitulate the disorder in humans better than existing rodent models and thus offer a more translational platform for the development of novel therapies. In order to assess translatability of swine for NF1, we have characterized the tissue specific expression patterns of neurofibromin in newborn and adult swine. Tissue samples were collected from brain (frontal cortex, cerebellar vermis, amygdala, hippocampus, pituitary body, substantia nigra), nerves (brachial plexus, cranial nerve IV, optic nerve), heart (left ventricle apex), lung (lobe apex), muscle (superior trapezius), gastrointestinal tract (distal ileum, proximal large intestine), and skin (epithelium, adipose) of two‐day old (N=5) and eight‐month old (N=6) swine. The inclusion or exclusion of the four known alternatively spliced exons (ASEs) were determined by RT‐qPCR. Generally, inclusion of ASE 12 was relatively higher in cortical brain and muscle tissue, while inclusion of ASE 31, implicated in regulation of RAS activity, was relatively higher in myelin dense tissue. Inclusion of ASE 57 was significantly higher exclusively in muscle tissue. Interestingly, ASE 13 was not detected in any of the tissue and subsequent sequencing of the exon revealed a significant genomic variation compared to human: ASE 13 in swine contains an additional nucleotide that results in an early stop codon in neurofibromin transcription if this ASE was included. Overall neurofibromin expression was similar between age groups but expression of ASE 31 was overall lower in newborn tissue. Tissues from newborn swine also had lower expression of ASE 12 in brachial plexus, muscle, heart, and lung tissues. This study provides the first characterization of neurofibromin ASE expression in swine.Support or Funding InformationThe research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF North Central, NF Network, and NF Team Foundation.