Nova2 interacts with a cis-acting polymorphism to influence the proportions of drug-responsive splice variants of SCN1A

Abstract

An intronic polymorphism in the SCN1A gene, which encodes a neuronal sodium-channel alpha subunit, has been previously associated with the dosing of two commonly used antiepileptic drugs that elicit their pharmacologic action primarily at this ion-channel subunit. This study sought to characterize the functional effects of this polymorphism on alternative splicing of SCN1A and to explore the potential for modulating the drug response in the pharmacologically unfavorable genotype by identification of a splice modifier acting on SCN1A. The effects of the genotype at the SCN1A IVS5N+5 G-->A polymorphism on SCN1A splice-variant proportions and the consequences of increased expression of splice modifiers were investigated both in human temporal neocortex tissue and in a cellular minigene expression system. Quantitative real-time polymerase chain reaction was used to quantify the amounts of SCN1A transcripts forms. We show that the polymorphism has a dramatic effect on the proportions of neonate and adult alternative transcripts of SCN1A in adult brain tissue and that the effect of the polymorphism also appears to be modified by Nova2 expression levels. A minigene expression system confirms both the effect of the polymorphism on transcript proportions and the role of Nova2 in the regulation of splicing, with higher Nova2 expression increasing the proportion of the neonate form. A larger Nova2-mediated effect was detected in the AA genotype that is associated with increased dose requirements. The effects of Nova2 on modulation of the alternative splicing of 17 other neuronally expressed genes were investigated, and no effect was observed. These findings emphasize the emerging role of genetic polymorphisms in modulation of drug effect and illustrate both alternative splicing as a potential therapeutic target and the importance of considering the activity of compounds at alternative splice forms of drug targets in screening programs.

Figure 1.

Schematic of exons 4–6 of the SCN1A gene. A, The adult (5A [red]) and neonatal (5N [green]) mRNA transcript forms of exon 5. The location of the SCN1A IVS5N+5 G→A polymorphism (rs3812718) is indicated by the red star located in the 5′ splice-donor site after exon 5N. The blue line stretching from intron 4 to intron 5 indicates the gene region covered by the minigene construct. B, Sequence of the minigene construct with exon 5N (highlighted in green) and exon 5A (highlighted in red). The nine putative Nova-binding sites ([T/C]CA[T/C]) are highlighted in yellow. Highlighted in blue are SNPs that are located in the minigene construct (in order of appearance in the sequence: rs2195144 [A→G], rs2217199 [T→C], rs3812719 [G→T], and rs3812718 [G→A]).

Figure 2.

Evidence of the genetic control of SCN1A exon 5 alternative splicing in human brain tissue. A, Correlation of the SCN1A IVS5N+5 G→A genotype with the percentage of transcripts containing neonatal exon 5N in the temporal neocortex of control tissue (unblackened circles) and mesial temporal lobe epileptic brain tissue (blackened circles). P<.001 by ANOVA for mesial temporal lobe epilepsy and control samples combined, and P<.001 by ANOVA for separate analyses of mesial temporal lobe epilepsy and control samples. By Tukey post hoc statistical analysis, P<.001 for AA versus AG, P<.001 for AG versus GG, and P<.001 for AA versus GG. B, Difference in the percentage of transcripts containing neonatal exon 5N in the temporal neocortex of control samples and mesial temporal lobe epileptic brain tissue. Data are presented as mean ± SEM. P<.05 by Student’s t test for comparison with control.

Figure 3.

Percentage of SCN1A mRNA transcripts containing exon 5N graphed versus NOVA2 mRNA expression (normalized to β-actin expression) for controls and subjects with mesial temporal lobe epilepsy with the AA (triangles), AG (circles), and GG (squares) genotypes at the SCN1A IVS5N+5 G→A polymorphism. NOVA2 mRNA expression correlates with the extent of SCN1A exon 5 splicing for the AA genotype in human brain tissue. Lines indicate the fit of a linear regression model to the data for each genotype subgroup.

Figure 4.

Evidence of the genetic control of SCN1A exon 5 alternative splicing in a minigene construct. Shown is the change in the percentage of exon 5N expression with SCN1A IVS5N+5 G→A genotype in HEK293 cells transfected with the genomic DNA fragment containing the G, A, or A and G alleles (1 μg plasmid in total). Data are presented as the mean of two independent replications (values for independent observations are superimposed by use of dashes [–]).

Figure 5.

Top panels, Change in the percentage of SCN1A transcripts containing exon 5N in the presence of increasing NOVA2 mRNA expression in the HEK293 minigene construct with genotype AA (A) or GG (B) at the SCN1A IVS5N+5 G→A polymorphism. Lines indicate the fit of the E_max model in equation (1) to the data. Bottom panels, Western blot of the change in HA-tagged Nova2 protein expression with increasing amounts of NOVA2 plasmid in the HEK293 cells. Nova2 modulates SCN1A splicing in the AA genotype to a greater extent than in the GG genotype.