Somatic deletions implicated in functional diversity of brain cells of individuals with schizophrenia and unaffected controls

Abstract

While somatic DNA copy number variations (CNVs) have been identified in multiple tissues from normal people, they have not been well studied in brain tissues from individuals with psychiatric disorders. With ultrahigh depth sequencing data, we developed an integrated pipeline for calling somatic deletions using data from multiple tissues of the same individual or a single tissue type taken from multiple individuals. Using the pipelines, we identified 106 somatic deletions in DNA from prefrontal cortex (PFC) and/or cerebellum of two normal controls subjects and/or three individuals with schizophrenia. We then validated somatic deletions in 18 genic and in 1 intergenic region. Somatic deletions in BOD1 and CBX3 were reconfirmed using DNA isolated from non-pyramidal neurons and from cells in white matter using laser capture microdissection (LCM). Our results suggest that somatic deletions may affect metabolic processes and brain development in a region specific manner.

Figure 1. Procedures for calling somatic deletions in whole genome sequencing data from multiple tissues from one individual or from a single tissue from multiple individuals.

* All deletion candidates and selected candidates (read count ≤6) used for downstream filtering in sequencing data from multiple tissues and single tissue respectively.

Figure 2. Validation of somatic deletions in brain DNA of an individual with schizophrenia.

(a), PFC specific deletion in PRKRA and annotated genes were visualized using the UCSC genome browser. (b), 844 bp DNA fragment was amplified by nested PCR using amplified DNA from PFC as template. (c), The 1309 bp DNA fragment was amplified by first round PCR with nested primers using unamplified DNA from all three tissues as templates (top). The 299 bp somatic deletion specific DNA fragment was amplified with breakpoint specific primers using unamplified DNA from PFC only as template (bottom). (d). Validation of breakpoints in PFC DNA by Sanger sequencing of 845 bp DNA fragment amplified by nested PCR amplification. NC: no template control, PFC: prefrontal cortex, Cere: cerebellum. Gel images are cropped to highlight relevant bands and images of original full gels are presented in Supplementary Figure S5.

Figure 3. Revalidation of a somatic deletion in PFC of an individual with schizophrenia using cells isolated by laser capture microdissection.

(a), PFC specific deletions in BOD1 and annotated coding regions were visualized using the UCSC genome browser. (b), 275 bp and 685 bp DNA fragment were amplified by nested PCR using DNA from PFC and cerebellum as templates respectively. (c), Validation of breakpoints of somatic deletion in cerebellum DNA (685 bp fragment) by Sanger sequencing. (d). Validation of breakpoints of somatic deletion in PFC DNA (275 bp fragment) by Sanger sequencing (e). Microscopic images showing a pyramidal neuron, a non-pyramidal cell and a cell in white matter in PFC after firing laser. (f). 143 bp and 275 bp DNA fragment were amplified by nested PCR using DNA from non-pyramidal cells and cells in white matter as templates respectively. (g), Validation of breakpoints of somatic deletion in non-pyramidal cells (143 bp fragment) by Sanger sequencing. (h), Validation of breakpoints of somatic deletion in cells in white matter (275 bp fragment) by Sanger sequencing. NC: no template control, PFC: prefrontal cortex, Cere: cerebellum, BP: break point, Ins: insertion, non-Py; non-pyramidal cells, WM; cells in white matter. Gel images are cropped to highlight relevant bands (images of entire original gels are presented in Supplementary Figure S6).

Figure 4. Total number of somatic deletions in PFC of two unaffected controls and two schizophrenia cases and the biological processes associated with somatic deletions in the schizophrenia and unaffected controls.

(a), Number of somatic deletions in genic and intergenic chromosomal regions in PFC. (B), Biological processes related to genes disrupted by somatic DNA deletion candidates in the PFC. Classification of the Gene Ontology biological processes was done by using Panther software.

Figure 5. Validation of somatic deletions in PFC of two individuals with schizophrenia and two unaffected controls.

PFC specific somatic deletions in BCLAF1, CBX3, PRKRA, SUCLG2 were confirmed by PCR validation. Two independent somatic deletions in PFC and cerebellum were validated in TDG and TYRO3. *The deletions with the same break points in TDG and intergenic region were validated in PFC and cerebellum. However, the deletions were considered somatic deletions because the read depth analysis indicated there was no clear decline in depth of coverage and deleted fragments were not amplified in our first PCR. Neg: no template control, PFC: prefrontal cortex, Cere: cerebellum. Gel images are cropped to highlight relevant bands (images of entire original gels are presented in Supplementary Figure S9).