Ide Copy Number Variant Does Not Influence Stroke Severity in 2 C57BL/6J Mouse Models nor in Humans: An Exploratory Study

Abstract

Background: Contrary to the common belief, the most commonly used laboratory C57BL/6J mouse inbred strain presents a distinctive genetic and phenotypic variability, and for several traits, the genotype-phenotype link remains still unknown. Recently, we characterized the most important stroke survival factor such as brain collateral plasticity in 2 brain ischemia C57BL/6J mouse models (bilateral common carotid artery stenosis and middle cerebral artery occlusion) and observed a Mendelian-like fashion of inheritance of the posterior communicating artery (PcomA) patency. Interestingly, a copy number variant (CNV) spanning Ide locus was reported to segregate in an analogous Mendelian-like pattern in the C57BL/6J colonies of the Jackson Laboratory. Given IDE critical role in vascular plasticity, we hypothesized Ide CNV may have explained PcomA variability in C57BL/6J inbred mice.

Methods: We applied a combination of techniques (T2-weighted magnetic resonance imaging, time-of-flight angiography, cerebral blood flow imaging, and histology) to characterize the collaterome in 77 C57BL/6J bilateral common carotid artery stenosis, middle cerebral artery occlusion, naive, and sham mice and performed on these Taqman genotyping, exome sequencing, and RNA sequencing. We then investigated the hypothesis that IDE structural variants (CNVs, gain/loss of function mutations) may have influenced the cerebrovascular phenotype in a large cohort of 454 040 cases and controls (UK Biobank, Genomics England).

Results: We detected an Ide CNV in a bilateral common carotid artery stenosis mouse with 2 patent PcomAs (minor allele frequency, 1.3%), not segregating with the PcomA patency phenotype. In addition, 2 heterozygous IDE CNVs, resulting in loss of function were found in 1 patient with hereditary ataxia, a patient with hereditary congenital heart disease, and 2 healthy individuals (minor allele frequency 9×10^-6). Moreover, we report 4 IDE loss of function point mutations (p.Leu5X, p.Met394ValfsX29, p.Pro14SerfsX26, p.Leu889X, minor allele frequency 0.02%) present also in controls or inherited from healthy parents.

Conclusions: Ide CNV and loss of function variants are rare, do not crucially influence PcomA variability in C57BL/6J inbred mice, and do not cause a vascular phenotype in humans.

Keywords: carotid stenosis; genetics; mice; phenotype; stroke.

Graphic abstract

Figure 1. Pipeline followed in our study.

We used 2 acute brain ischemia mouse models (BCCAS [bilateral common carotid artery stenosis] and MCAO [middle cerebral artery occlusion]) and studied the posterior communicating artery phenotype (PcomA) using T2 weighted MRI, cerebral blood flow measurement through arterial spin labeling and time-of-flight-angiography (TOF MRA). We then selected BCCAS and MCAO mice with different PcomA phenotype for Ide rs30920120 Taqman genotyping, exome sequencing and RNA sequencing. We identified one BCCAS mouse with 2 prominent PcomAs carrying a Ide CNV. We screened also the IDE gain and loss of function mutations in a human cohort of cases and controls from 2 databases (UK-Brain Bank and Genomics England), where IDE CNV and loss of function mutations are very rare and not likely to play a critical influence on vascular phenotypes. D indicates day/s; wks, weeks; CSVD, cerebral small vessel disease; OP, surgery.

Figure 2. Ide rs30920120 (C/G) Taqman genotyping in a cohort of bilateral common carotid artery stenosis (BCCAS), middle cerebral artery occlusion (MCAO) and naïve C57BL/6J mice with different posterior communicating artery (PcomA) phenotype.

Figure 3. Ide copy number variant (CNV) detection in the C57BL/6J BCCAS and MCAO exome sequencing mouse cohort and Ide CNV carrier phenotype.

A. CNV analysis of Ide locus, based on exome sequencing data on 12 BCCAS and MCAO mice with different PcomA phenotype. In the red rectangle is underlined the Ide CNV detected in a BCCAS mouse. B-F. Neuroradiological phenotype of the BCCAS mouse carrying Ide CNV. B Schematic representation of the circle of Willis, displaying a complete circle with 2 prominent PcomAs (blue arrows). ACA, anterior communicating artery; MCA, middle cerebral artery; PCA, posterior cerebral artery; SCA, superior cerebellar artery; BA, basilar artery; PcomA, posterior communicating artery. C. Time of Flight (TOF) angiography showing 2 patent PcomAs (red arrows) 7 days post surgery and secondary collateralization from the external carotid artery (blue arrows). D. Gross anatomy of the BCCAS mouse carrying the Ide CNV, 7 days post surgery and after Ivans Blue injection, presenting 2 very prominent PcomAs (red arrows). E. Graph showing initial cerebral blood flow drop 24 hours post surgery and progressive recovery 7 days post surgery, through brain collaterals. The blue segment represents the right circulation and the red segment the left circulation measured by arterial spin labelling. D, day; CBF, cerebral blood flow. F. Atlas registration overlap at different time points (2 days and 7 days post surgery) of the BCCAS mouse carrying Ide CNV, showing no ischemic lesions.

Figure 4. Ide gene expression analysis in BCCAS and MCAO mice during acute (2 days) and subacute (7 days) hypoperfusion with different PcomA patency pattern, hippocampal lesion sizes and cerebral blood flow recovery.

A. RNA sequencing Ide brain expression analysis in BCCAS and SHAM mouse brain (B; prefrontal cortex, hippocampus and striatum) during acute and subacute hypoperfusion. B. Real-TIME PCR Ide expression analysis in BCCAS, MCAO and naïve mice with different PcomA patency in blood during acute hypoperfusion. C-E. Hippocampal percentage of lesion volume (C), CBF measurements (CBF, ml/min/100g) (D) and Ide gene expression (RNA sequencing, E) in BCCAS mice 7 days post-surgery. H, hippocampus; TPM, transcript per million and d, days.