Genetic polymorphisms and transcription profiles associated with intracranial aneurysm: a key role for NOTCH3

Results

Preliminary screening of high-throughput data for IA-related genes

We preliminarily screened IA-related genes from high throughput genomic and transcriptome data. In order to screen IA-related mutant genes, we performed high-throughput genome sequencing for 20 Chinese subjects (19 were familial cases, while 1 was a sporadic case) with IA as a discovery cohort (Figure 1A and 1B). A total of 6649 deleterious SNPs in 4906 genes was discovered from profiling our next generation sequencing data, which was annotated and filtered based on functional changes by SAMtools, ANNOVAR, PolyPhen-2 and SIFT software (Figure 1C). Transcriptome sequencing data from GEO database revealed 1422 DEGs, of which 147 were upregulated and 1275 were downregulated in IA compared to healthy cerebral artery (Supplementary Table 1). The screened DEGs are shown in the volcano plot (Figure 2A).

Figure 1. (A) Pedigrees of the 20 cases with intracranial aneurysm (IA). (B) IA reconstruction from digital subtraction angiography, magnetic resonance angiography, and computed tomography angiography. The red arrows indicate the location of the aneurysm. (C) Workflow for filtering deleterious single-nucleotide polymorphisms (SNPs).

Figure 2. (A) Volcano plot of significantly up-regulated (red) and down-regulated (green) DEGs. (B) Venn diagram showing the number of genes from next-generation sequencing (NGS) that were considered mutant (mutagenesis), the number of genes from transcriptome sequencing data in the GEO database considered differentially expressed genes (DEGs), and the number of genes common to the NGS and transcriptome sequencing datasets. (C) Heat map showing expression of genes overlapping between the NGS and transcriptome sequencing datasets.

Determining key IA-related gene(s)

There were 369 genes shared between the mutant genes identified in our next-generation sequencing and the DEGs identified from GEO transcriptome sequencing data (Figure 2B and 2C) [22]. These shared genes were classified based on their functionality according to GO categories tool BiNGO (Figure 3A) [23]. Next, we screened the most relevant GO classifications. As previously published work, IA onset and development can be promoted due to abnormal hemodynamic stress, immune response, or cerebrovascular hypoplasia [7–10, 24]. Considering this, we focused on four GO categories that seemed to be IA-related: response to stress (FDR-adjusted P-value = 6.93×10^-3), immune system process (FDR-adjusted P-value = 3.57×10^-2), anatomical structure development (FDR-adjusted P-value = 3.97×10^-2), and nervous system development (FDR-adjusted P-value = 1.49×10^-2) (Table 1). From the intersection of the genes of their GO categories, we further narrowed our focus to three genes (TACC3, TRPM2, and NOTCH3) that were shared across all four groups (Figure 3B). However, we found that only NOTCH3 of these screened three genes was associated with cerebral artery disease in previous work [25]. Therefore, we hypothesized that NOTCH3 might be the key IA-related gene. Indeed, mRNA expression data from the GEO database showed NOTCH3 to be significantly downregulated in IA tissue compared to cerebral artery tissue (Figure 3C).

Table 1. Genes classified under Gene Ontology (GO) terms related to intracranial aneurysm

GO-ID and GO-terms	p value
	unadjusted	False discovery rate-adjusted	Genes in intracranial aneurysm-related GO-terms
6950 response to stress	2.14E-05	6.93E-03	SLC23A2,AVIL,HFE,BRCA1,TRH,CXCL16,CHAF1A,CDH3,DMBT1, CREB3L4,EXO1,ADORA3,KYNU,GTSE1,IKBKE,TNFRSF4,AP3B1, NRG1,AVPR1A,DGKZ,TLR1,ATRN,CCKBR,SMO,RTN4RL1,ADAM9, MASP2,TLR4,SCG2,PPARD,NOTCH3,PNKP,PROZ,AGER,C2,CYP27B1,TRPM2,LMAN1,CLN3,ADAMTS13,APOL1,NGFR,POLQ,GCKR,VDR, F12,XRCC3,HPS1,LY86,XRCC1,USP28,DEF6,SYT7,CRYGD,RAD51, NEDD4,TRPV4,POLE2,TACC3,FOXA3,FOXA2
7399 nervous system development	7.19E-05	1.49E-02	RET, NOTCH3, LAMA1, AVIL, CPNE6,CHRD,SEMA3E,PPP1R9A,AGER,IGSF9,CELSR3,NPAS2, TRPM2,SALL1,SIX4,CHL1,SLIT1,GPC2, NKX6-1, ZNF488,SH3GL1,NGFR,SEMA6A,EDN3,UNC5B,LRRN4,LIMK1,NRG1, SEMA4F,MYO7A,AVPR1A,SYNJ2,SEMA4G,TAL2,SMO,DOK5,RTN4RL1, NEDD4,KCNQ2,TACC3,EPHA2,PPARD,FOXA2
2376 immune system process	4.14E-04	3.57E-02	NOTCH3,FLT3,HFE,STXBP2,CTSW,TREM2,CD1B,CXCL14,RASGRP4, C2,RELB,CXCL16,CYP27B1, TRPM2,DMBT1,SIX4,EXO1,KYNU, APOL1,CD34,IKBKE,TNFRSF4,EDN3,VDR,F12,LY86,AP3B1,TLR1,PC, TACC3,ADAM9,MASP2,TLR4,SCG2,BCAR1
48856 anatomical structure development	4.79E-04	3.97E-02	RET,FLT3,AVIL,TREH,CPNE6,PPP1R9A,CELSR3,TGM1,ADAMTS2, CYP26B1,SALL1,DMBT1,SIX4,CHL1,EXO1,SALL4,TRPS1,COL10A1, BTRC,ZNF488,SH3GL1,FOXD1,SEMA6A,EDN3,UNC5B,NRG1,MYO7A, AVPR1A,COL2A1,SPINT1,TAL2,SMO,ANGPTL6,DOK5,RTN4RL1, KCNQ2,MMP19,ADAM9,SCG2,EPHA2,PPARD,NOTCH3,TMPRSS6, LAMA1,CHRD,SEMA3E,PPL,AGER,IGSF9,NPAS2,RASGRP4,RELB, CYP27B1,TRPM2,CHST11,RAB26,SLIT1,GPC2,NKX61,ARSE, NGFR,CDSN,VDR,LRRN4,LIMK1,PCDH8,SEMA4F,SYNJ2,SEMA4G, ALOX15B,DAB2,PC,NEDD4,ITGA11,TACC3,HOXD4,FOXA2

Figure 3. (A) Schematic showing enrichment of functions from intersections of filtered mutant genes and DEGs. The color of the node indicates the significance of gene representation, and its size corresponds to the number of genes in that gene ontology (GO) category. The IA-related GO-terms that were carried forward are indicated by green arrows. (B) Diagram showing unique and shared genes from the selected IA-related GO terms. (C) NOTCH3 mRNA expression data from IA and cerebral artery (CA) tissue, as deposited in the GEO database.

Association of IA with NOTCH3 SNPs

Our analysis identified three low-frequency NOTCH3 SNPs related to IA in our discovery cohort: allele T of rs779314594, allele A of rs200504060, and allele T of rs2285981. Bioinformatic software tools predicted that allele T of rs779314594, allele A of rs200504060, and allele T of rs2285981 lead to loss-of-function. Allele T of rs779314594 and allele A of rs200504060, both in the conserved domain of NOTCH3, were predicted to alter an amino acid residue and therefore potentially the protein structure (Figure 4) [26]. These SNPs was predicted be deleterious by bioinformatics tools GERP++ [47], SIFT [46], CADD [48], PolyPhen-2 [49], and MutationTaster [29] (Table 2). All three risk alleles were enrichment in the IA cases of our discovery cohort compared to the different populations of large genetic variant databases (Genome Aggregation Database, gnomAD) (http://gnomad.broadinstitute.org/) (Table 3) [27]. When we used the gnomAD data as controls, statistical analyses confirmed that IA was associated with allele T of rs779314594 (P = 0.001), allele A of rs200504060 (P = 0.004), and allele T of rs2285981 (P = 0.004). However, no significant enrichment was detected in the sporadic IA cases (Supplementary Table 2).

Figure 4. Sequence query of NOTCH3 conserved domains. Reprinted from https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=AAB91371.1. Data show allelic variants that result in amino acid changes and protein structure, as modeled by SWISS-MODEL (https://www.swissmodel.expasy.org/).

Table 2. Functional annotation of variations at the NOTCH3 locus

ID	REF	ALT	Func	ExonicFunc	SIFTa	MutationTasterb	gerp++gt2c	CADDd
rs779314594	C	T	exonic	missense SNV	0.183,T	1.000,D	3.92	16.94
rs200504060	G	A	exonic	missense SNV	0.006,D	1.000,N	2.93	16.86
rs2285981	C	T	exonic	missense SNV	0.002,D	1.000,D	4.41	15.22
ALT, Sample genome base type; REF, Reference genome base type; SNV, single nucleotide variant
a SIFT score indicates whether the variation is likely to cause changes in protein structure or function: “D”, deleterious (sift ≤ 0.05); “T”, tolerated (sift > 0.05);
b MutationTaster represents the effect of the mutation on the protein sequence: “A”, “disease_causing_automatic”; “D”, “disease_causing”; “N”, “polymorphism”; “P”, “polymorphism_automatic”
c Variations with a gerp++gt2 score > 2 are considered conservative.
d CADD score >15 means that the variation affects protein function.

NOTCH3 expression in IA and healthy cerebral artery

We tested NOTCH3 expression in the IA and cerebral artery by immunohistochemistry. Staining showed significantly lower NOTCH3 expression in the IA (Figure 5A and 5C). The Imaging diagnosis of IA specimens was provided in Figure 5B. The decreased mRNA and protein expression of NOTCH3 in IA tissue suggests that NOTCH3 can protect against IA. We further confirmed that the genotypes of these samples did not contain risk variants allele T of rs779314594 allele A of rs200504060, and allele T of rs2285981 (Supplementary Table 3).

Figure 5. (A) Representative pictures of NOTCH3 immunohistochemistry staining or hematoxylin and eosin staining of IA and cerebral artery tissues. (B) Reconstructed images from diagnostic CTA and DSA scans. The red arrows indicate the location of the IA. (C) Percentage of positively stained cells in panel (A) as measured using Image J. ACoA: anterior communicating artery, MCA: middle cerebral artery. p<0.05.

NOTCH3 expression in neutrophils

Due to the role of the immune response in IA rupture, we retrieved data on NOTCH3 expression in peripheral blood neutrophils from individuals with IA and healthy controls from the GEO database (https://www.ncbi.nlm.nih.gov/geo, GSE106520) [14]. NOTCH3 mRNA expression was significantly higher in IA neutrophils (Figure 6A).

Figure 6. (A) Quantification of NOTCH3 transcription in peripheral blood neutrophils from samples with or without IA. (B) Western blot and RT-qPCR of whole cell lysate from HUVECs transduced with negative control shRNA or NOTCH3-shRNA. (C) Quantification of IA-related factor transcripts from HUVECs transduced with negative control shRNA or NOTCH3-shRNA.

NOTCH3 knockdown down-regulates angiogenesis factors in HUVEC

IA has been associated with IL-1β, IL-6, TNF-α, MMP9, MMP2, NF-κB, MCP-1, and VCAM1 [8, 11, 12, 18–20]. To determine whether these factors are regulated by NOTCH3, we transfected HUVECs with shRNA targeting NOTCH3 or negative control shRNA, and we verified NOTCH3 knockdown in HUVECs using Western blotting and RT-qPCR (Figure 6B). Levels of mRNAs encoding all these factors tended to decrease in the presence of NOTCH3 knockdown, but the decrease was significant only in the case of IA-related and angiogenesis factors MMP9, NF-κB, MCP1, IL-6 and VCAM-1 (Figure 6C). However, no noticeable differences in these significantly decreased angiogenesis factors were found between IA and healthy artery (Supplementary Figure 1).

Discussion

In this study, we identified NOTCH3 as a key IA-related gene through a series of bioinformatic analyses followed by validation with IA patients’ samples and cell culture. First, we analyzed next-generation sequence data and discovered 6649 deleterious SNPs in 4537 genes in IA patients. Second, we collected RNA-sequencing data from GEO datasets and identified 1403 DEGs associated with IA. Third, we focused on 369 genes shared between the genes that were mutated in our next-generation sequencing data and the DEGs from the GEO data. Finally, we took the intersection of four highly relevant IA-related GO-term genes and decided to focus on NOTCH3 because of its previously defined role in the cerebral artery. We then performed immunohistochemical studies on IA and cerebral artery tissue and showed NOTCH3 down-regulation in IA. Further, we found that NOTCH3 knockdown down-regulated angiogenesis molecules in HUVECs. However, the mRNA expression of NOTCH3 increases in peripheral blood neutrophil.

In this study, we found that IA may be associated with allele T of rs779314594 (P = 0.001), allele A of rs200504060 (P = 0.004), and allele T of rs2285981 (P = 0.004) in discovery cohort (Table 3). However, no significant enrichment was detected in the sporadic IA cases (Supplementary Table 2). It indicates that the detected SNPs of NOTCH3 remain familial accumulations. They locate in the exons regions of NOTCH3 gene, and may affect the function of protein, because the variations alter the amino acid residue of original protein so that the structure of the NOTCH3 protein was changed [28] (Figure 4). Additionally, this alteration was predicted be deleterious by bioinformatics tools GERP++ [47], SIFT [46], CADD [48], PolyPhen-2 [49], and MutationTaster [29] (Table 2).

Table 3. Single-nucleotide polymorphisms in NOTCH3 that are associated with intracranial aneurysm

ID	Polymorphism	our study (risk allele/ normal allele)	GnomADa (risk allele/ normal allele)	Fisher's Exact Test	OR	95% CI
						Lower	Upper
rs779314594	T	T/C = 1/19	T/C = 14/223582	P<0.001	841	105	6715
rs200504060	A	A/G = 1/19	A/G = 237/245087	P<0.004	269	35	2048
rs2285981	T	T/C = 1/19	T/C = 48/245638	P<0.004	256	37	1765
a Genome Aggregation Database (http://gnomad.broadinstitute.org/)

Damage to the cerebral artery is the induction factor for IA [30], and NOTCH3 plays a vital role in cerebral blood vessels. NOTCH3 is essential for the structural integrity of small distal elastic arteries. In NOTCH3-null mice, myogenic tone significantly decreases, and isolated cerebral and tail caudal arteries show increased flow-mediated dilation. NOTCH3 governs the reactivity of vessels to pressure, flow, and other mechanical factors via the RhoA/ROCK signaling pathway [31]. Recently, our team identified the guanine exchange factor ARHOGEF17 in the RhoA/ROCK signaling pathway as a risk factor for IA [32]. Furthermore, the Notch pathway cross-talks with important signaling networks involving angiotensin-2 (AngII), [33] TGFβ, [34] ALK1, [35] and VEGF [36]. All these pathways play essential roles in IA onset and formation. Therefore, our findings support the importance of NOTCH3 in IA development. Further studies should be performed to determine how reduced expression of NOTCH3 affects the cerebral artery as well as the downstream IA-related signaling pathways mentioned above. Our findings here are consistent with previous studies linking NOTCH3 to IA [37], and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy [25].

NOTCH3 can promote inflammation in some biological processes [38], and inflammatory cells play a crucial role in IA formation and rupture. We examined NOTCH3 mRNA expression in neutrophils from the GEO database and found mRNA expression to be higher in IA neutrophils than in control neutrophils (Figure 6A). It may be that higher levels of NOTCH3 in neutrophils promote inflammation in the cerebral artery, injuring it and thereby leading to IA. We did not find direct connection between the expression level of NOTCH3 and the patient prognosis in our study. Nevertheless, rupture of IA is the most severe prognostic indicator for IA patient, and the risk of aneurysm rupture is associated with the degree of inflammation in the arterial wall, which in turn could be aggravated by upregulated NOTCH3 in neutrophils [39, 40]. In addition to inflammation, altered NOTCH3 expression may contribute to IA by influencing angiogenesis damaging cerebrovascular endothelial repair. We found that NOTCH3 knockdown in HUVECs significantly down-regulated MMP9, NF-κB, MCP1, IL-6 and VCAM-1. These factors help regulate angiogenesis under homeostatic conditions [41], and an imbalance of angiogenesis factors is associated with IA [42]. We extracted the expression levels of impacted genes by NOTCH3 knockdown in HUVEC of from the GEO dataset but no noticeable difference was found between IA and healthy artery (Supplementary Figure 1). The authors deem that the possible cause is that these decreased changes of IA-related factors are mainly in cerebral vessel endothelium but not smooth muscle, and these changes cannot be shown in the sequencing of the complete IA and healthy artery. In short, distinct expression of NOTCH3 in IA tissue vs. neutrophils may have differential implication in IA: down-regulated NOTCH3 in IA tissue disrupts angiogenesis and cerebrovascular endothelial repair [43]; up-regulated NOTCH3 in neutrophils promotes inflammation and damage cerebral vessels directly.

In conclusion, we think that variations in NOTCH3 and dysregulation of its expression (down-regulation in cerebral artery, up-regulation in neutrophils) contribute to IA, perhaps directly as well as indirectly by affecting the levels of downstream IA-related factors (Figure 7). For precision medicine [44], our work identifies NOTCH3 as a novel target for treating or even preventing IA. Further research is needed to clarify how NOTCH3 participates in IA formation and development.

Figure 7. Schematic illustrating a possible role for NOTCH3 in IA. NOTCH3 is altered at the genetic polymorphisms which were predicted to alter an amino acid residue and therefore potentially the protein structure. Down-regulated expression of NOTCH3 in the cerebral artery influences angiogenesis damaging cerebrovascular endothelial repair. Up-regulated expression of NOTCH3 in neutrophil activate and promote inflammation in the cerebral artery. In a word, these abnormities in NOTCH3 cause damage to blood vessels in the brain, which can lead to the onset and development of intracranial aneurysm. Image modified from ScienceSlides (VisiScience Corp., North Carolina, USA).

Author Contributions

Xinyu Yang, Xiaozhi Liu and Weidong Yang performed and designed the research. Mengqi Li and Xinlong Dong collected data and wrote the manuscript. Bioinformatics analysis and immunohistochemistry were performed by Mengqi Li. IA and cerebral artery specimens as well as the patients' clinical data were collected and analyzed by Shi Chen. Chao Yang and Weihan Wang were responsible for cell culture maintenance and RT-qPCR. Bochuan Li reviewed and revised the part of cell biology experiments. Degang Liang reviewed the clinical data of this study.

Acknowledgments

The authors would like to thank Senior Service Engineer Wenxing Li from OE Biomedical Technology (Shanghai, China).

Conflicts of Interest

The authors have no conflicts of interest to declare.

Funding

We thank the National Natural Science Foundation of China (81571144), the Natural Science Foundation of Tianjin City (16JCZDJC35700), and the Natural Science Foundation of Fujian province (2018J01359) for supporting this work. The National Natural Science Foundation of China (81571144) and the Natural Science Foundation of Tianjin City (16JCZDJC35700) provided funding for performing NGS for 20 IA patients, data mining, and other biological experiments. The Natural Science Foundation of Fujian province (2018J01359) provided financial support for the collection of human specimens and samples genotyping by Sanger sequencing.

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