Functional characterisation of the amyotrophic lateral sclerosis risk locus GPX3/TNIP1.
Restuadi R., Steyn FJ., Kabashi E., Ngo ST., Cheng F-F., Nabais MF., Thompson MJ., Qi T., Wu Y., Henders AK., Wallace L., Bye CR., Turner BJ., Ziser L., Mathers S., McCombe PA., Needham M., Schultz D., Kiernan MC., van Rheenen W., van den Berg LH., Veldink JH., Ophoff R., Gusev A., Zaitlen N., McRae AF., Henderson RD., Wray NR., Giacomotto J., Garton FC.
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a complex, late-onset, neurodegenerative disease with a genetic contribution to disease liability. Genome-wide association studies (GWAS) have identified ten risk loci to date, including the TNIP1/GPX3 locus on chromosome five. Given association analysis data alone cannot determine the most plausible risk gene for this locus, we undertook a comprehensive suite of in silico, in vivo and in vitro studies to address this. METHODS: The Functional Mapping and Annotation (FUMA) pipeline and five tools (conditional and joint analysis (GCTA-COJO), Stratified Linkage Disequilibrium Score Regression (S-LDSC), Polygenic Priority Scoring (PoPS), Summary-based Mendelian Randomisation (SMR-HEIDI) and transcriptome-wide association study (TWAS) analyses) were used to perform bioinformatic integration of GWAS data (Ncases = 20,806, Ncontrols = 59,804) with 'omics reference datasets including the blood (eQTLgen consortium N = 31,684) and brain (N = 2581). This was followed up by specific expression studies in ALS case-control cohorts (microarray Ntotal = 942, protein Ntotal = 300) and gene knockdown (KD) studies of human neuronal iPSC cells and zebrafish-morpholinos (MO). RESULTS: SMR analyses implicated both TNIP1 and GPX3 (p < 1.15 × 10-6), but there was no simple SNP/expression relationship. Integrating multiple datasets using PoPS supported GPX3 but not TNIP1. In vivo expression analyses from blood in ALS cases identified that lower GPX3 expression correlated with a more progressed disease (ALS functional rating score, p = 5.5 × 10-3, adjusted R2 = 0.042, Beffect = 27.4 ± 13.3 ng/ml/ALSFRS unit) with microarray and protein data suggesting lower expression with risk allele (recessive model p = 0.06, p = 0.02 respectively). Validation in vivo indicated gpx3 KD caused significant motor deficits in zebrafish-MO (mean difference vs. control ± 95% CI, vs. control, swim distance = 112 ± 28 mm, time = 1.29 ± 0.59 s, speed = 32.0 ± 2.53 mm/s, respectively, p for all