Surveying alternative transcripts of disease-associated genes and their cell type specificity – a case study with ANK3

The ANK3 gene has been conclusively linked to bipolar disorder, but its role in disease etiology remains unclear: both the specific transcript and its expressing cell type remain unclear. This is a near-universal problem for genes associated with polygenic neuropsychiatric disorders. One of the causes of this unfortunate stasis is that human transcript databases have an incomplete list of alternative isoforms of genes and that it is technically challenging to identify the cell-types in which they are expressed.

Here, we address this knowledge gap for ANK3. ANK3 is a useful case study because of its rich alternative splicing and important neurobiological role in neurons and oligodendrocytes. We used Oxford nanopore sequencing of full-length transcripts to identify all human and mouse ANK3 exons.

Then, we isolate and sort brain nuclei and use droplet-digital PCR to measure cell-type specific expression of alternatively spliced exons. We confirm that RefSeq transcript annotations of ANK3 lack many of the alternatively spliced exons and we show that, despite being lowly expressed, these exons are highly evolutionarily conserved and have strong cell type specificity. Elucidating psychiatric disorder etiology will require an overview of the transcripts of all the disorder-associated genes and the cell types in which these transcripts are expressed.

This work on ANK3 illustrates the extent to which such information is currently lacking, ways in which the information can be acquired, and the technical challenges that are encountered in the process. The sequencing service was provided by the Norwegian Sequencing Centre (www.sequencing.uio.no), a national technology platform hosted by the University of Oslo and supported by the Functional Genomics and Infrastructure programs of the Research Council of Norway and the Southeastern Regional Health Authorities. The flow cytometry core facility at OUS-Ullevål (www.ous-research.no/flow/) provided the sorting service.

We thank Hanne Hjorthaug and Elena Kondratskaya for valuable comments and discussion. This study was supported by funding from the Research Council of Norway (295679). Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway Martin Falck, Denis Reis de Assis, Asbjørn Holmgren, Ragnhild Aaløkken, Sarah Åsheim, Srdjan Djurovic & Timothy Hughes FYSCELL - Spatial Immunology and Nanomedicines (Comparative immunology), University of Oslo, Oslo, Norway Centre for Precision Psychiatry, Division of Mental Health and Addiction, Oslo University Hospital and Institute of Clinical Medicine, University of Oslo, Oslo, Norway Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway Department of Nutrition, Institute of Basic Medical Science, University of Oslo, Oslo, Norway The Norwegian Transgenic Center, Institute of Basic Medical Science, University of Oslo, Oslo, Norway The authors declare no competing interests.

All methods were performed in accordance with the relevant guidelines and regulations. The collection of mouse brain samples was approved by the Norwegian Animal Research Authority under the Ministry of Agriculture (FOTS ID 27485). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.