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. 2021 Aug 23;13(1):135.
doi: 10.1186/s13073-021-00949-0.

Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse brain

Lindsay Liang  1 , Siavash Fazel Darbandi  1 , Sirisha Pochareddy  2 , Forrest O Gulden  2 , Michael C Gilson  1 , Brooke K Sheppard  1 , Atehsa Sahagun  3 , Joon-Yong An  4 , Donna M Werling  5 , John L R Rubenstein  1 , Nenad Sestan  2   6   7   8   9 , Kevin J Bender  3 , Stephan J Sanders  10   11   12
Affiliations

Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse brain

Lindsay Liang et al. Genome Med. .

Abstract

Background: Genetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human brain.

Methods: RNA-seq data from 783 human brain samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples.

Results: In the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A, a brain-wide synchronized 5N to 5A transition occurs between 24 post-conceptual weeks (2nd trimester) and 6 years of age. In mice, the equivalent 5N to 5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories.

Conclusions: Exon usage in SCN1A, SCN2A, SCN3A, and SCN8A changes dramatically during human brain development. These splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.

Keywords: Autism spectrum disorder; Developmental delay; Epileptic encephalopathy; Exon 5A; Exon 5N; Intellectual disability; Isoform; Seizures; Splicing; Voltage-gated sodium channel.

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Conflict of interest statement

J.L.R.R. is cofounder, stockholder, and currently on the scientific board of Neurona, a company studying the potential therapeutic use of interneuron transplantation. S.J.S. receives research funding from BioMarin Pharmaceutical Inc. The remaining authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Splicing isoforms in voltage-gated sodium channels. A Voltage-gated sodium channels are composed of four similar domains (I, II, III, IV), each of which includes six transmembrane segments with extracellular or intracellular linkers. The fourth transmembrane segment (S4) in each domain acts as a voltage sensor. Between the fifth and sixth transmembrane segment (S5, S6) is a pore loop that forms the ion selectivity filter. The fifth protein-coding exon (5A/5N, CDS 5) encodes a portion of the first domain, while the 20th protein-coding exon (18A/18N, CDS 20) encodes a similar portion of the third domain. B Location, genomic coordinates (GRCh38/hg38), and amino acid sequence of the “5A” and “5N” exons in SCN1A. C–E The data in “B” is repeated for the genes SCN2A, SCN3A, and SCN8A. F Patterns of whole-gene expression of SCN1A in the human dorsolateral prefrontal cortex (DLPFC) across prenatal and postnatal development from the BrainVar dataset [33]. G–I The data in “F” is repeated for the genes SCN2A, SCN3A, and SCN8A. CDS: coding sequence; CPM: counts per million. Genomic coordinates are based on GRCh38/hg38 using GENCODE v31 gene definitions
Fig. 2
Fig. 2
Expression of 5A and 5N in the human cortex across development. A The expression of 5A (red) and 5N (blue) in SCN1A is shown for 176 BrainVar human cortex (DLPFC) samples across development (points). On the left, the colored line shows the Loess smoothed average and 95% confidence interval (shaded region). On the right, boxplots show the median and interquartile range for the same data, binned into fetal, transitional, and mature developmental stages. B The ratio of 5A and 5N expression from panel A is shown across development (left) and in three developmental stages (right). C–H Panels A and B are repeated for the genes SCN2A, SCN3A, SCN8A. For comparison, the 5A/5N ratio is shown on the same y-axis in Additional file 1: Fig. S3, and equivalent plots for CDS four and six are shown in Additional file 1: Fig. S4. CPM: Counts per million; DLPFC: Dorsolateral prefrontal cortex. Statistical tests: B, D, F, H Left panel, linear regression of log2(5A:5N ratio) and log2(post-conceptual days). Right panel, two-tailed Wilcoxon test of log2(5A:5N ratio) values between fetal and mature groups
Fig. 3
Fig. 3
Orthogonal analysis of voltage-gated sodium channel gene splicing in the developing human brain. A Sashimi plot of splicing in prenatal (top, N = 112 samples) and postnatal (bottom, N = 60 samples) DLPFC for SCN2A. Linewidth reflects the proportion of split reads observed for each intron compared to all split reads between CDS 4 and CDS 6, this value is also shown as a percentage. Introns related to 5A inclusion are shown in red, those related to 5N inclusion are shown in blue, and others are in grey. B, C Equivalent plots for SCN3A (a negative-strand gene with the orientation reversed to facilitate comparison to the other two genes) and SCN8A. D The ratio of 5A and 5N expression is shown across development for SCN1A in six human brain regions. For each region, the colored line shows the Loess smoothed average and 95% confidence interval (shaded region). Equivalent data across 11 cortical regions are shown in Additional file 1: Fig. S6. E–G This analysis is repeated for SCN2A, SCN3A, and SCN8A. Statistical tests: A–C P values compare the prenatal and postnatal cluster using a Dirichlet-multinomial generalized linear model, as implemented in Leafcutter [41]
Fig. 4
Fig. 4
Expression of 5A and 5N in the mouse cortex across development. A The expression of 5A (red) in Scn1a is shown for 58 mouse cortex samples across development (points); no functional 5N equivalent is present in the mouse genome. On the left, the colored line shows the Loess smoothed average and 95% confidence interval (shaded region). On the right, boxplots show the median and interquartile range for the same data, binned into fetal, transitional, and mature developmental stages. B The Loess smoothed average expression of the four voltage-gated sodium channels in human cortex (top, Fig. 1) and mouse cortex (bottom). C Panel A is repeated for Scn2a, with the addition of 5N expression (blue). D The ratio of 5A and 5N expression from panel ‘C’ is shown across development (left) and in three developmental stages (right). Values reported previously in mouse cortex are shown on the same scale in green for comparison [20]. E-H Panels C and D are repeated for the genes Scn3a, Scn8a. CPM: Counts per million. Statistical tests: D, F, H Left panel, linear regression of log2(5A:5N ratio) and log2(post-conceptual days). Right panel, two-tailed Wilcoxon test of log2(5A:5N ratio) values between fetal and mature groups
Fig. 5
Fig. 5
Developmental trajectories of CDS 20 (18A/18N) in human cortex in SCN8A. A Location, genomic coordinates (GRCh38/hg38), and amino acid sequence of the 18A and 18N exons in SCN8A. B Sashimi plot of intron splicing in prenatal (top, N = 112 samples) and postnatal (bottom, N = 60 samples) dorsolateral prefrontal cortex. Linewidth reflects the proportion of split reads observed for each intron compared to all split reads between the exon before and after, this value is also shown as a percentage. Introns related to 18A exon inclusion are shown in green, those related to 18N exon inclusion are shown in purple, and others are in grey. C Expression of the 18A (green) and 18N (purple) for 176 BrainVar human dorsolateral prefrontal cortex samples across development (points). On the left, the colored line shows the Loess smoothed average with the shaded area showing the 95% confidence interval. On the right, boxplots show the median and interquartile range for the same data, binned into fetal, transitional, and mature developmental stages. D The 18A:18N ratio is shown for each sample from panel C across development (left) and binned into three groups (right). CPM: Counts per million; Statistical analyses: B Dirichlet-multinomial generalized linear model, as implemented in Leafcutter [41]. D Left panel, linear regression of log2(18A:18N ratio) and log2(post-conceptual days). Right panel, two-tailed Wilcoxon test of log2(18A:18N ratio) values between fetal and mature groups
Fig. 6
Fig. 6
Identification of protein-coding exons with complex developmental trajectories. A The correlation between the ratio of CPM expression between pairs of exons (log-scaled) and developmental stage (post-conceptual days, log-scaled) for SCN1A was assessed with a linear model (e.g., Fig. 2B). The R2 value of each exon pair is shown as a heat map with ‘hot’ colors representing exon pairs with high R2 values for which variation in the ratio is correlated with developmental age, i.e., pairs of exons that show substantially different expression across development. Exon numbers from DEXSeq (Additional file 3: Table S2) are shown on the bottom and right and equivalent CDS numbers are shown on the top and left (see Additional file 3: Table S2). B–D The analysis is repeated for SCN2A, SCN3A, and SCN8A
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References

    1. Heyne HO, Singh T, Stamberger H, Abou Jamra R, Caglayan H, Craiu D, et al. De novo variants in neurodevelopmental disorders with epilepsy. Nat Genet [Internet]. 2018;1. Available from: http://www.nature.com/articles/s41588-018-0143-7 - PubMed
    1. Satterstrom FK, Kosmicki JA, Wang J, Breen MS, De Rubeis S, An J-Y, et al. Large-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism. Cell. 2020 Jan; - PMC - PubMed
    1. Kaplanis J, Samocha KE, Wiel L, Zhang Z, Arvai KJ, Eberhardt RY, et al. Integrating healthcare and research genetic data empowers the discovery of 49 novel developmental disorders. bioRxiv [Internet]. 2019 Jan 1;797787. Available from: http://biorxiv.org/content/early/2019/10/16/797787.abstract
    1. Catterall WAWA, Marban E, Catterall WAWA, Cestèle S, Catterall WAWA, Wood JN, et al. Voltage-gated sodium channels. J Physiol [Internet] 2001;1(1):17–21.
    1. Bender KJ, Trussell LO. The physiology of the axon initial segment. Annu Rev Neurosci. 2012;35(1):249–265. doi: 10.1146/annurev-neuro-062111-150339. - DOI - PubMed

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