SLCO5A1 and synaptic assembly genes contribute to impulsivity in juvenile myoclonic epilepsy

Abstract

Elevated impulsivity is a key component of attention-deficit hyperactivity disorder (ADHD), bipolar disorder and juvenile myoclonic epilepsy (JME). We performed a genome-wide association, colocalization, polygenic risk score, and pathway analysis of impulsivity in JME (n = 381). Results were followed up with functional characterisation using a drosophila model. We identified genome-wide associated SNPs at 8q13.3 (P = 7.5 × 10^-9) and 10p11.21 (P = 3.6 × 10^-8). The 8q13.3 locus colocalizes with SLCO5A1 expression quantitative trait loci in cerebral cortex (P = 9.5 × 10^-3). SLCO5A1 codes for an organic anion transporter and upregulates synapse assembly/organisation genes. Pathway analysis demonstrates 12.7-fold enrichment for presynaptic membrane assembly genes (P = 0.0005) and 14.3-fold enrichment for presynaptic organisation genes (P = 0.0005) including NLGN1 and PTPRD. RNAi knockdown of Oatp30B, the Drosophila polypeptide with the highest homology to SLCO5A1, causes over-reactive startling behaviour (P = 8.7 × 10^-3) and increased seizure-like events (P = 6.8 × 10^-7). Polygenic risk score for ADHD genetically correlates with impulsivity scores in JME (P = 1.60 × 10^-3). SLCO5A1 loss-of-function represents an impulsivity and seizure mechanism. Synaptic assembly genes may inform the aetiology of impulsivity in health and disease.

Fig. 1. Manhattan plot showing GWAS with BIS-Brief score.

Linear regression was used to test association of each SNP with BIS-Brief. Sex, genotyping batch, age at consent, first three PCs, and the frequency of myoclonus or absence seizures were included as covariates in the model. We found two significant genome-wide associations on chromosome 8 (rs73293634 (G/T)) and 10 (rs75042057 (T/G)) in the analysis of 324 European individuals with JME. Variants below −log₁₀P < 1 were omitted in the plot.

Fig. 2. LocusFocus plot for the GWAS with BIS-Brief in JME (circles) and eQTLs in GTEx brain and tibial nerve tissues for the SLCO5A1 gene (lines).

The Simple Sum 2 and COLOC2 colocalization methods implemented in LocusFocus (v1.4.9) were used to test for colocalization of the BIS-Brief genome-wide peaks with eQTL analyses brain tissues from GTEx v8, PsychENCODE, and fetal brain. a Colocalization figure from LocusFocus for the SLCO5A1 gene. Lines depict the minimum P value trace in a sliding window for SLCO5A1 eQTLs from GTEx, one line per tissue. Circles depict the GWAS with BIS-Brief, with the lead SNP in purple and pairwise LD with the lead SNP marked as shown in the legend, calculated using the 1000 Genomes Project European subset. Significant colocalization is observed for SLCO5A1 eQTLs in GTEx v8 for the cerebral cortex after increasing sample size in a mega-GWAS (n = 367, −log₁₀ Simple Sum 2³⁶P = 9.5 × 10⁻³). Colocalization analysis with only the Europeans is provided in Supplementary Fig. 7. Colocalization was also tested for all other nearby genes shown in the figure, but no other genes’ eQTLs colocalized with BIS-Brief GWAS (not shown). b Colocalization analysis with PsychENCODE eQTLs in the dorsolateral prefrontal cortex (DLPFC) (n = 1866), and eQTLs derived from second trimester fetal brains (n = 120), with GTEx’s brain cortex eQTL as in A provided for reference. Colocalization analysis results suggest no colocalization with either PsychENCODE (Simple Sum 2 P = 0.985) or fetal brain eQTLs (does not pass first stage test in Simple Sum 2 for having significant eQTLs in the region). c Violin plot for the eQTL effect of rs73293634 SNP on SLCO5A1 expression in the cerebral cortex from GTEx v8. d Expression change of SLCO5A1 from brains in various developmental stages from BrainSpan^,. pcw, post conception weeks; preadolescence, 2–12 years old (inclusive); adolescence, 13–19 years old; adult, ≥20 years old (oldest samples are 40 years old). The centre lines represent the 50th percentile (median) and the bounds of the boxes are the 75th and 25th percentiles (interquartile range) with the whiskers being the largest value within 1.5 times the interquartile range above the 75th percentile and smallest values within 1.5 times the interquartile range below the 25th percentile.

Fig. 3. Domain architecture of human SLCO5A1.

a Schematic representation of the protein with the indication of recognised domains. A SMART analysis to identify structural domains confirmed the presence of two modules, Major Facilitator Superfamily (MFS) and a Kazal domain, interspaced with potentially unstructured sequences. The MFS transporters are membrane proteins capable of transporting small solutes in response to chemiosmotic ion gradients^,. They are represented in many organisms from Archaea to Homo sapiens. MFS proteins target a wide range of substrates, including ions, carbohydrates, lipids, amino acids and peptides, nucleosides and other small molecules and transport them in both directions across the membrane. The Kazal domain is an evolutionary conserved module usually acting as a serine-protease inhibitor. b Predicted model of the monomeric form of SLCO5A1 from amino acids 115–766, built using the SwissModel homology server (https://swissmodel.expasy.org) and utilising the template structure pdb:7eeb. Red: alpha helices; Yellow: Beta strands; Green: Loops.

Fig. 4. Startling reaction to trains of vibrations, increased seizure prevalence and increased post-seizure recovery time in flies with Oatp30B knockdown.

a Startling reaction to trains of vibrations. The UAS-Oatp30B^IR (GD12775) transgenic or the control UAS-GFP^IR were driven with nSyb-Gal4 and Ubi-Gal80ts. The w¹¹¹⁸ strain is a control for the genetic background in absence of transgenes. Mean ±SEM **P < 0.01, One Way ANOVA, Tukey’s post-hoc test. Units are the vibration events experienced 6 times for each fly, n = 174–210. b Increased seizure prevalence. The UAS-Oatp30B^IR (GD12775) transgenic or the control UAS-GFP^IR were driven with nSyb-Gal4 and Ubi-Gal80ts. Percent ±SE ****P < 0.0001, Log-rank (Mantel-Cox) test, X² 24.68 for 1 df, n = 34–36. c Increased post-seizure recovery time. The UAS-Oatp30B^IR (GD12775) transgenic or the control UAS-GFP^IR were driven with nSyb-Gal4 and Ubi-Gal80ts. Mean ±SEM *P < 0.05, Mann Whitney non-parametric test, two tails, n = 10–26. Only flies that displayed a seizure within 120 s as in b have been included in the analysis.