Nicola Hall: How nanopore sequencing is changing psychiatry
Dr. Nicola Hall, of the University of Oxford, described how she and her team are using nanopore sequencing to investigate the schizophrenia-linked gene CACNA1C.
Nicola began by asking: why does psychiatry need to change? How can nanopore cDNA sequencing be used to do this, and how can this lead to new treatments for patients? She explained that the diagnosis and treatment of psychiatric conditions is difficult; multiple overlapping symptoms between conditions make correct diagnosis, and therefore identifying suitable treatments, a hit-and-miss process, as current methods target the symptoms rather than the cause. She stresses that “we need to understand the underlying biology” behind psychiatric conditions. Genome-wide association studies have identified multiple genetic risk loci for the condition; most of these are not protein-coding, leading to the hypothesis that genetic risk loci alter gene expression via the regulation of mRNA splicing.
Nicola focuses on the gene CACNA1C, which encodes a voltage-gated calcium channel. There is strong supporting evidence that Ca2+ is dysregulated in psychiatric disorders; however, the gene is very long, with over 50 exons and multiple splice isoforms that impact protein function – to really understand splicing of the gene, she stressed, requires data from full-length transcripts. In order to characterise full-length isoforms with long-range sequencing, Nicola and her team purified RNA from six regions of 3 regions of the brain – they used human brain samples, as splicing is not conserved between humans and rodents. They then reverse transcribed the samples and performed targeted amplification of CACNA1C, barcoded each sample and sequenced them in multiplex; the team’s targeted cDNA method will soon be published. The sequencing data revealed that CACNA1C splicing is “much more diverse than was previously known”, with 38 novel exons and 83 high-confidence novel isoforms. Nicola showed data for the 10 most abundant isoforms identified: the topmost abundant isoform is annotated, but the other 9 are all novel, and 8 are protein-coding. Analysis of differences between the three individuals revealed minimal variation; however, variation was seen between regions of the brain: this is the ideal outcome for developing targeted treatments.
The next steps will be to compare splicing between healthy individual and schizophrenia patients and between brain tissue vs heart tissue, to ensure targeted treatments would not produce off-target effects, followed by functional studies into voltage sensitivity, drug binding properties and protein interactions. This could then be taken further into rodent models and human iPSCs, and next on to treatment.
Nicola concluded by stressing that psychiatry needs to change as we don’t understand the cellular causes of psychiatric disorders. Using long-range nanopore cDNA sequencing enabled the characterisation of splice isoform expression patterns, leading to potential identification of therapeutic targets.