PT-010 - ALCOHOL USE DISORDER PHARMACOGENOMICS: ROLE OF FNDC4 IN HUMAN NEURONAL FUNCTION.

X. Zhu¹, D. Liu², A. John², H. Gao², B. Coombes², J. Biernacka², V. Karpyak², R. Weinshilboum²; ¹Mayo Clinic, Mayo Clinic, Rochester, MN, , ²Mayo Clinic.

Background: We performed a genome-wide association study (GWAS) for acamprosate and/or naltrexone treatment response in alcohol use disorder (AUD) patients (n=1083) and identified a genome-wide significant single-nucleotide polymorphism (SNP) signal. The “top” SNP, rs56951679, was a splicing quantitative trait locus (sQTL) for the fibronectin type III domain containing 4 (FNDC4) gene in a series of brain regions, a splicing variant that resulted in the loss of FNDC4 exon 6. FNDC4 encodes a single-pass transmembrane protein which is highly expressed in neurons. However, the function of FNDC4 in brain and molecular mechanism(s) responsible for the GWAS association are unknown.

Methods: Our study began with characterization of the GWAS SNP-related alteration in FNDC4 splicing and its effect on FNDC4 protein and its intracellular localization. To help us understand the molecular function of FNDC4 in brain and AUD drug treatment response, we studied human induced pluripotent stem cell (iPSC)-derived neurons and generated FNDC4 knock-out (KO) iPSCs using CRISPR/cas9. The FNDC4 KO and wildtype (WT) iPSCs were differentiated to generate forebrain organoids, followed by single-nucleus RNA sequencing (snRNA-seq) to identify differences in neural cell types and gene expression affected by FNDC4 KO.

Results: We observed that loss of FNDC4 exon 6 resulted in a truncated protein which was unable to localize to the cell membrane. The truncated FNDC4 protein was localized primarily in the cytosol and was rapidly degraded. Furthermore, in iPSC-derived neurons, FNDC4 expression could be upregulated by ethanol-dependent induction of TGF-β2, a growth factor known to play a role in neuronal differentiation. The snRNA-seq data for early-stage (45 days of differentiation) forebrain organoids identified a unique cluster of “mature neurons” in FNDC4 KO but not in WT organoids. Finally, the number of clustered “neural progenitor cells” was significantly reduced in FNDC4 KO forebrain organoids when compared with WT. These results suggest a possible role for FNDC4 in neuronal differentiation.

Conclusion: We demonstrated that FNDC4 may play a role in neuronal differentiation, a mechanism which may contribute to variation in AUD drug treatment response.