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Long-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain

Clark, Michael B., Wrzesinski, Tomasz, Garcia, Aintzane B., Hall, Nicola A. L., Kleinman, Joel E., Hyde, Thomas, Weinberger, Daniel R., Harrison, Paul J., Haerty, Wilfried, Tunbridge, Elizabeth M.·2020·Molecular Psychiatry
DOI 10.1038/s41380-019-0583-1PMC PMC6906184

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Abstract

RNA splicing is a key mechanism linking genetic variation with psychiatric disorders. Splicing profiles are particularly diverse in brain and difficult to accurately identify and quantify. We developed a new approach to address this challenge, combining long-range PCR and nanopore sequencing with a novel bioinformatics pipeline. We identify the full-length coding transcripts of CACNA1C in human brain. CACNA1C is a psychiatric risk gene that encodes the voltage-gated calcium channel Ca V 1.2. We show that CACNA1C ’s transcript profile is substantially more complex than appreciated, identifying 38 novel exons and 241 novel transcripts. Importantly, many of the novel variants are abundant, and predicted to encode channels with altered function. The splicing profile varies between brain regions, especially in cerebellum. We demonstrate that human transcript diversity (and thereby protein isoform diversity) remains under-characterised, and provide a feasible and cost-effective methodology to address this. A detailed understanding of isoform diversity will be essential for the translation of psychiatric genomic findings into pathophysiological insights and novel psychopharmacological targets.

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Fig. 1 Amplicon sequencing of CACNA1C. a UCSC genome browser screenshot of CACNA1C isoforms annotated in GENCODE V27. All transcripts in “Basic” annotated set shown. Black arrow shows direction of transcription. TSS transcription start site. Position of forward and reverse long-range PCR primers in first exon and universal portion of final exon shown. Length vs Quality of all 2D pass reads from b Run 1 c Run 2. Most reads are the full-length CACNA1C CDS. The presence of the 3.5 kb positive control CDS spike-in can be seen in Run1. Visualisation limited to reads between 2 and 8 kb, encompassing >98% of pass reads in each run
Fig. 1 Amplicon sequencing of CACNA1C. a UCSC genome browser screenshot of CACNA1C isoforms annotated in GENCODE V27. All transcripts in “Basic” annotated set shown. Black arrow shows direction of transcription. TSS transcription start site. Position of forward and reverse long-range PCR primers in first exon and universal portion of final exon shown. Length vs Quality of all 2D pass reads from b Run 1 c Run 2. Most reads are the full-length CACNA1C CDS. The presence of the 3.5 kb positive control CDS spike-in can be seen in Run1. Visualisation limited to reads between 2 and 8 kb, encompassing >98% of pass reads in each runCC BY · PubMed Central
Fig. 2 a Annotation and read count for novel exonic sequences within CACNA1C. Red arrows indicate exons that have been validated (Supplementary Table 2). b, c Top 10 most abundant CACNA1C isoforms identified in brain using exon-level approach. b UCSC genome browser screenshot of top isoforms. Colours denote transcript type. c Proportion of high-confidence transcripts reads from the ten most abundant transcripts
Fig. 2 a Annotation and read count for novel exonic sequences within CACNA1C. Red arrows indicate exons that have been validated (Supplementary Table 2). b, c Top 10 most abundant CACNA1C isoforms identified in brain using exon-level approach. b UCSC genome browser screenshot of top isoforms. Colours denote transcript type. c Proportion of high-confidence transcripts reads from the ten most abundant transcriptsCC BY · PubMed Central
Fig. 3 Comparison of CACNA1C isoform expression between individuals and tissues. a Transcript expression levels (TPM) across tissues and individuals. “One” and “Two” denote sequencing runs. b Principal Component Analysis based on normalised transcript expression. c Isoform switching of ENST00000399641 and CACNA1C n2199 in cerebellum. Left panel: box plots show minimum to maximum values with line at mean value. Right panel: the sequences of ENST00000399641 (blue) and CACNA1C n2199 (black) differ in the sequences of their 30th exons. The detail shows the amino acid sequences of this exon in the two transcripts
Fig. 3 Comparison of CACNA1C isoform expression between individuals and tissues. a Transcript expression levels (TPM) across tissues and individuals. “One” and “Two” denote sequencing runs. b Principal Component Analysis based on normalised transcript expression. c Isoform switching of ENST00000399641 and CACNA1C n2199 in cerebellum. Left panel: box plots show minimum to maximum values with line at mean value. Right panel: the sequences of ENST00000399641 (blue) and CACNA1C n2199 (black) differ in the sequences of their 30th exons. The detail shows the amino acid sequences of this exon in the two transcriptsCC BY · PubMed Central
Fig. 4 Impact of novel splicing events on the CACNA1C protein model. a CACNA1C encodes the primary pore-forming subunit of the CaV1.2. CaV1.2 is formed of four domains (I–IV), each comprised of six transmembrane domains (S1–S6), which are linked by intracellular loops. The obligate beta subunit binds to the I-II intracellular loop, as shown. Grey boxes indicate the location of novel, in-frame insertions and deletions, discussed in the main text. Values indicate the mean proportion of reads containing each variant. Where variants were identified using both analysis approaches, exon-level counts were used to derive abundance (red text); variants identified only using the splice-site-level approach are indicated with blue text. b Number of protein isoforms containing three microdeletions: (i) in the I-II linker, (ii) in the IV4–5 linker and (iii) the previously reported microdeletion in the IV3–4 linker
Fig. 4 Impact of novel splicing events on the CACNA1C protein model. a CACNA1C encodes the primary pore-forming subunit of the CaV1.2. CaV1.2 is formed of four domains (I–IV), each comprised of six transmembrane domains (S1–S6), which are linked by intracellular loops. The obligate beta subunit binds to the I-II intracellular loop, as shown. Grey boxes indicate the location of novel, in-frame insertions and deletions, discussed in the main text. Values indicate the mean proportion of reads containing each variant. Where variants were identified using both analysis approaches, exon-level counts were used to derive abundance (red text); variants identified only using the splice-site-level approach are indicated with blue text. b Number of protein isoforms containing three microdeletions: (i) in the I-II linker, (ii) in the IV4–5 linker and (iii) the previously reported microdeletion in the IV3–4 linkerCC BY · PubMed Central

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Cited by

Metadata

Authors
Clark, M. B., Wrzesinski, T., Garcia, A. B., Hall, N. A. L., Kleinman, J. E., Hyde, T., Weinberger, D. R., Harrison, P. J., Haerty, W., & Tunbridge, E. M.
Venue
Molecular Psychiatry
Publisher
Springer Science and Business Media LLC
Published
2019-11-06
Volume
25
Issue
1
Pages
37-47
ISSN
  • 1359-4184
  • 1476-5578
DOI
10.1038/s41380-019-0583-1
PMC
PMC6906184
Cited by
148
References
13
Language
en
Licence
Funders
RCUK | Biotechnology and Biological Sciences Research Council (BB/J004669/1, BB/P016774/1)

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