Generation of human induced motor neurons from patients with BVVL harboring novel neurodegeneration causing mutation

Brown-Vialetto-Van Laere syndrome (BVVL) is a rare ALS-like neurodegenerative disorder. BVVL symptoms start with sensorineural deafness and cranial nerve palsies, followed by sensory and cranial neuron degeneration. Currently, mutations in the Riboflavin transporter family (RFVT/SLC52) protein-encoding genes are linked to riboflavin deficiency and BVVL. Interestingly, in our patient group of six siblings, no mutations were detected in RFVT/SLC52 genes. Instead, whole-exome sequencing supported with in silico analysis and haplotype analysis resulted in a novel "Glu1656Lys" mutation in the KIF13A gene, encoding "Kinesinlike protein – KIF13A". KIF13A is known to play a major role in the endosomal – lysosomal system by mediating clathrin-independent endosomal recycling, which is crucial for a healthy, functioning nervous system. In this study, we have generated and characterized patientspecific induced pluripotent stem cells (iPSCs) of KU-BVVL patients carrying the neurodegeneration, causing novel KIF13A mutation. In addition, I have generated and characterized patient-specific induced motor neurons (iMNS) of KU-BVVL patients carrying the KIF13A mutation via a small-molecule-based differentiation method, adapted from previous studies and optimized to suit our experimental requirements. KU-BVVL-iMNs can be used as a valid model cell line that reflects the diseased phenotype as KIF13A protein is abundantly present in iMNs, predominantly in the soma and dendritic regions. Furthermore, I have also designed and generated various plasmid constructs, including mutant and wild type constructs of KIF13A, KIF13A targeting CRISPR/Cas9 expression vectors, and KIF13A-BioID fusion constructs. These molecular reagents, along with patient iPSC-derived motor neurons, will be important tools to understand KIF13A related processes and molecular mechanisms that are crucial for a healthy, functioning nervous system.