1

Gene Expression Profiling and Pathway Analysis of Ruptured Intracranial

Aneurysm (GSE36791)

Marial Aizen C. Alinsug1, Emmanuel B. Dulman1, Riatries Y. Saavedra1

and Leana Rich M. Herrera1*

1
Department of Physical Sciences, College of Science, Polytechnic University of the

Philippines, Sta. Mesa, Manila

*
corresponding author: leanaherrera@yahoo.com
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Abstract

Ruptured intracranial aneurysm (RIA) causes subarachnoid hemorrhage (SAH)

that may result to death or serious disability of some survivors. Knowledge of the

molecular mechanisms associated to ruptured intracranial aneurysm may give

therapeutic remedies for the SAH patients or prevention of this disease. Such

mechanisms could be provided by microarray analysis. Samples used in this study

were gathered from series GSE36791 of NCBI GEODataSets. Comparison of gene

expression of 43 RIA samples and 18 control samples was executed using XLStat 2016

with its feature Differential Expression. Gene ontology terms found upon entering the

probe ID of each differentially expressed genes to DAVID were used for functional

analysis of the said genes. In determining which pathways the differentially expressed

genes were associated to RIA, KEGG pathway was employed while STRING was for

determining protein-protein interactions. Upon interpretation of data, most of the

downregulated genes were associated to immune system specifically memory cell, T

cell differentiation, T cell receptor signalling pathway and hematopoietic cell lineage.

This explains why immune system process is the most significantly enriched biological

process. On the other hand, some upregulated genes were involved in inflammatory

response. Future researchers should conduct a study on quantitative real-time

polymerase chain reaction to validate the results.

Key words: intracranial aneurysm, subarachnoid hemorrhage, microarray, Gene Ontology, T cell
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Introduction

Intracranial aneurysm (IA) is a dilating part of an artery that carries blood to the

brain. Unruptured IA is considered as asymptomatic, a person is a carrier but not

exhibiting any symptoms. However, once it has ruptured, it causes subarachnoid

hemorrhage (SAH) wherein 50% of the cases are fatal and some survivors suffer from

serious disability (Tromp et al., 2014). Risk factors include polycystic kidney disease,

smoking, increasing age, female gender, hypertension, and family history. Both ruptured

and unruptured intracranial aneurysms can be treated by surgical clipping and

endovascular coiling (Chalouhi et al., 2013). These two treatments, however, are

invasive processes that may hasten the condition of a patient. Moreover, majority of the

Filipino patients could not afford the cost of these surgical operations.

Performing microarray technique and pathway analysis could reduce risks of

medical operations in treating a disease with high morbidity.

Microarray technique has been used in the field of medicine to determine gene

expression patterns. Functional analysis of the differentially expressed genes shows

which Gene Ontology terms are overrepresented. Compared to a group of control,

upregulation and downregulation of particular genes from an individual with a disease

are observed. Biological processes associated to a disease are known by executing

pathway analysis and network mapping revealing protein-protein interactions. Moreover,

variants of a gene in a given human population could give insights on the susceptibility

of an individual to a certain disease.

Microarray data for this study were gathered from GEO DataSets with accession

number GSE36791, wherein samples were collected from peripheral blood of SAH
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patients and control group. Tools available online were used in performing analyses of

the data.

Knowledge of the molecular mechanisms associated to rupture of an intracranial

aneurysm (IA) and its causation could provide a system for administering SAH patients

and even create drugs to prevent the disease from transpiring.

Materials and Methods

Raw Data Collection

Samples were collected from series GSE36791 of NCBI GEODataSets. The

platform used in this series was GPL10558: Illumina HumanHT-12 v4 Expression

BeadChip with more than 47000 probes, each representing oligonucleotide chain of 50

bases. The aneurysm group was composed of 43 samples and the control group had 18

samples.

Microarray Data Analysis

Comparison of the two groups was performed using an add-in tool for MS Excel,

XLStat 2016, with its feature Differential Expression. Benjamini-Hochberg method under

nonparametric test was chosen and the genes with p-value less than 0.05 were

generated. The fold change of each gene was calculated by log 2(RIA/control). Genes

with p-value < 0.05 and absolute log2FC 0.585 were considered as differentially

expressed. A positive log2(FC) value means a gene from RIA sample is upregulated

and a negative value denotes downregulation.

Database for Annotation, Visualization and Integrated Discovery (DAVID)

DAVID (david.ncifcrf.gov) is an online database developed by the Laboratory of

Immunopathogenesis and Bioinformatics that provides functional annotation tools. This

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helps researchers to comprehend biological terms from a vast list of genes and

visualize them on pathway maps.

Probe ID of each differentially expressed gene was entered to DAVID. For

identifying these probes, ILLUMINA_ID was selected before submitting the gene list to

the functional annotation tool. GO terms were categorized to molecular functions,

biological processes, and cellular compartment. Differentially expressed genes involved

in each term were included in the functional annotation chart. GO terms with Benjamini

p-value < 0.05 and Fold change > 2 were denoted as significantly enriched or

overrepresented.

Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database of pathway

maps characterizing information on various cell functions. KEGG pathway mapping has

enabled researchers in examining which pathways the differentially expressed genes

are associated to.

The pathway map may explain further the biological processes related to

ruptured intracranial aneurysm and its complications. Same threshold values for the

Gene Ontology terms were used for the pathways to be considered as significantly

enriched or overrepresented.

Network Analysis

The database Search Tool for the Retrieval of Interacting Genes/Proteins

(STRING) was used in this analysis. Gene symbol of each differentially expressed gene

was entered as multiple proteins to STRING for determining protein-protein interactions.

The URL for online tool is string-db.org.

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A network map is composed of nodes and edges. Nodes represent proteins

produced by protein-coding genes. Colored nodes denote proteins that are included in

first shell of interactors, and white nodes are for second shell of interactors. Edges

represent protein-protein associations. An interaction between proteins means they

cooperatively contribute to a shared function. This however, does not necessarily mean

they are physically attached to each other.

Genes with Benjamini-Hochberg corrected p-values and absolute log2FC 1.

This more astringent fold change value was used to make the analysis easier.

Genetic variants and the differentially expressed genes with more astringent

threshold were entered in the STRING database. Interactions of proteins encoded by

these genes were analyzed.

Table 1 Most significant associations with IA

In Candidate Gene In GWAS
Studies
ACE CDKN2B
COL1A2 CNNM2
COL3A1 EDNRA
ELN FGD6
HSPG2 RRBP1
IL6 SOX17
JDP2 STARD13
KLK8
LIMK1
SERPINA3
TCN2
TNFRSF13B
VCAN
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Results and Discussions

This study assessed the gene expression profiles of 43 ruptured intracranial

aneurysm patients. From 47234 genes, 259 of which have p-value<0.05 and absolute

log2FC 0.585. The log2FC was also used to determine whether a gene is upregulated

or downregulated. A total of 132 genes have increased expression or upregulated and

127 genes were downregulated.

Functional analysis of the 259 differentially expressed genes revealed that these

were involved in several Gene Ontology (GO) terms: 29 biological processes, 8 cellular

components, and 3 molecular functions.

Immune system process was the most significantly enriched biological process

having the smallest p-value (1.54E-09) based on Benjamini method. However, only T

cell differentiation showed a 100% downregulation. T cell is a type of white blood cell

that is an essential component of the immune system and when activated, it can kill a

foreign cell. T cells arise from bone marrow and differentiate in the thymus gland.

Differentiation means a T cell undergoes changes depending on the specific immune

response the body requires. (Karp, 2013)

T cell receptor complex was the most overrepresented cellular component and

had 100% downregulation. Its child term, alpha-beta T cell receptor complex, also had a

100% downregulation. These two were associated with the aforementioned biological

process, T cell differentiation. For a T cell to be activated, it has to interact with an

antigen displayed on the surface of an antigen-presenting cell (APC). This interaction

can be achieved by receptors of the T cell and the APC. With this decreased expression

of genes associated to T cells, the immune system declined.

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Cytokine binding, cytokine receptor activity, and receptor activity were the

overrepresented molecular functions associated with ruptured intracranial aneurysm.

Approximately half of the genes in each molecular function were downregulated.

Cytokine is a protein released by the immune system that serves as a chemical

messenger. Its subtype interleukins are involved in the regulation of T cell and some are

for inflammatory action. (Gillaspy, 2015)

The downregulated genes in the mentioned molecular functions were linked to T

cell differentiation and T cell receptor complex. The remaining genes, those with

increased expression, were involved in the binding of inflammatory mediators.

The most significantly enriched pathways generated by KEGG database were

hematopoietic cell lineage and T cell receptor signaling pathway, both were associated

with the adaptive immune response. In contrast to innate immune response, adaptive

immune response is highly specific to a foreign body it destroys.

Hematopoietic stem cell gives rise to two different progenitor cells: myeloid and

lymphoid. Myeloid progenitor cells differentiate to red blood cells, macrophages, and

dendritic cells. On the other hand, lymphoid progenitor cells differentiate to natural killer

cells, B, and T lymphocytes. Only interleukin 1 receptor types I and II were upregulated.

These two genes encode receptor for proteins that participate in inflammatory response.

The only upregulated gene in the T cell receptor signaling pathway was the

mitogen-activated protein kinase 14. The kinase encoded by this gene is activated by

environmental stress and pro-inflammatory cytokines.

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The upregulated pro-inflammatory genes in these enriched pathways inhibited

the expression of the remaining genes. The same mechanism could also explain why

most of the gene ontology terms do not exhibit 100 % downregulation or upregulation.

Figure 1. Network of DE genes with p<0.05 and log2FC = 1. It shows

the interaction of proteins: interleukin 18 receptor 1, and interleukin 18
receptor accessory protein, both of which were upregulated. Interleukin
18 is a pro-inflammatory cytokine involved in the induction of
inflammatory mediators, control of natural killer cell and T cell activities.
This cytokine binds to target cells by its receptor, which has two
subunits: IL18R1 and IL18RAP. The receptor complex formed by these
subunits has high affinity to its target cells. (Alboni,2010)
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Figure 2 Network of DE genes and the genetic variants. The genetic variant
interleukin-6 had the most number of protein interactions, having 4 nodes connected
to it. Two differentially expressed genes from this study, arginase 1 and CD163
interact with IL-6. Interleukin-6 is a pro-inflammatory cytokine released by the brain
during harmful stimuli. However, uncontrolled or prolonged inflammation can lead to
cell damage. This response is then shifted to an anti-inflammatory action of arginase
1, which also functions in converting arginine into prolines and polyamines in tissue
remodeling. (Cherry,2014) Arginase 1 also had the highest log2FC value in this study.
Furthermore, CD163 is an interleukin-6-regulated receptor for hemoglobin
scavenging and has an anti-inflammatory function. (Moller, 2002) The IL-6 is a
multifunctional cytokine which also exerts anti-inflammatory action on monocytes and
induces the upregulation of CD163. (Buechler, 2000). The CD163 was found to be
upregulated, with log2FC value of 1.015.Another genetic variant TNFRSF13B was
found to interact with a differentially expressed gene, CD27. The TNFRSF13B protein
directs the expression of another protein called TACI on B cell, producer of
antibodies. The highest expression of TACI was found on CD27 + B cell (memory
cell). (Salzer, 2005) The log2FC value of CD27 was found low at -1.025, thus it was
downregulated. This decreased expression of a memory cell marker could result to
re-occurrence of an aneurysm.
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Increased expression of anti-inflammatory genes possibly resulted in wound

repair. However, the rest of the genes associated to immune system have decreased

expression which could possibly be associated to the high incidence of intracranial

aneurysm cases.

Conclusions and Recommendations

Comparison of ruptured intracranial aneurysm and the control revealed that

immune system specifically T cell differentiation, T cell receptor signaling pathway, and

hematopoietic cell lineage were weakened. Increased activity was observed on

inflammatory response, which was then reversed by anti- inflammatory mediators.

These mediators were possibly working for wound repair. However, this repair was not

adequate for an aneurysm patient to immediately regain an efficient immune system.

The genetic variants from literature were not observed to be differentially

expressed in this study. However, genes found to interact with these variants play

significant association with the disease, especially anti-inflammatory response and

memory. With the downregulation of gene associated to memory cell, aneurysm could

form again.

A study to validate the results through quantitative real-time polymerase chain

reaction (qPCR) must be conducted. Also, mechanisms directing to aneurysm growth

should be presented to stop it from re-occurring.

Different threshold values could significantly alter the data due to the limited

number of samples. Nevertheless, larger number of samples must be collected to

increase the reproducibility of the results.

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References

Barrett, J., & Kawasaki, E. S. (2003). Microarrays: The use of oligonucleotides and
cDNA for the analysis of gene expression. Drug Discovery Today, 8(3), 134-141.
doi:10.1016/s1359-6446(02)02578-3

Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful
approach to multiple testing. J R Stat Soc Series B.1995;57:289 300.

Bergeron, B. P. (2003). Bioinformatics Computing. Upper Saddle River, NJ: Prentice

Hall/Professional Technical Reference.

Bing, N. (2004). Statistical analysis of gene expression profile: Transcription network

inference and sample classification (Unpublished doctoral dissertation). Virginia
Polytechnic Institute and State University.

Cerebral Aneurysms Fact Sheet. (2013). Retrieved August 17, 2016, from
http://www.ninds.nih.gov/disorders/cerebral_aneurysm/detail_cerebral_aneu
rysms.htm

Chalouhi, N., Hoh, B. L., & Hasan, D. (2013). Review of Cerebral Aneurysm Formation,
Growth, and Rupture. Stroke, 44(12), 3613-3622.
doi:10.1161/strokeaha.113.002390

Chyatte D, Bruno G, Desai S, Todor DR. Inflammation and intracranial aneurysms.

Neurosurgery. 1999;45:11371146.

Du P, Kibbe WA, Lin SM. Lumi: a pipeline for processing illumine microarray.
Bioinformatics. 2008;24:15471548.

Etminan N et al. The Unruptured Intracranial Aneurysm Treatment Score, A

Multidisiplinary Concensus. Neurology 2015: 881-888.

Ewens, W. J., & Grant, G. R. (2005). Statistical Methods in Bioinformatics: An

Introduction. New York: Springer.

Huang DW, Sherman BT, Tan Q, Collins JR, Alvord WG, Roayaei J et al. The DAVID
Gene Functional Classification Tool: a novel biological module-centric algorithm
to functionally analyze large gene lists. Genome Biol 2007; 8: R183.

Karp, G. (2013). Cell and Molecular Biology: Concepts and Experiments. Hoboken, NJ:
John Wiley & Sons.

Korja M, Lehto H, Juvela S. Lifelong rupture risk of intracranial aneurysms depends on

risk factors: a prospective Finnish cohort study. Stroke 2014;45:19581963.
 13

Krischek, B., Kasuya, H., Tajima, A., Akagawa, H., Sasaki, T., Yoneyama, T., . . . Inoue,
I. (2008). Network-based gene expression analysis of intracranial aneurysm
tissue reveals role of antigen presenting cells. Neuroscience, 154(4), 1398-1407.
doi:10.1016/j.neuroscience.2008.
04.049

Kurki, M. I., Hkkinen, S., Frsen, J., Tulamo, R., Fraunberg, M. V., Wong, G., . . . Yl-
Herttuala, S. (2011). Upregulated Signaling Pathways in Ruptured Human
Saccular Intracranial Aneurysm Wall: An Emerging Regulative Role of Toll-Like
Receptor Signaling and Nuclear Factor-B, Hypoxia-Inducible Factor-1A, and
ETS Transcription Factors. Neurosurgery, 68(6), 1667-1676.
doi:10.1227/neu.0b013e318210f001

Kwoon J, Lavine, S, Vega C. Intracranial Aneurysms: Current Evidence and Clinical

Practice. American Family Physician 2002: 601.

Lin SM, Du P, Huber W, Kibbe WA. Model-based variance-stabilizing transformation for

Illumina microarray data. Nucleic Acids Res. 2008; 36:e11.

Nacu, S., Critchley-Thorne, R., Lee, P., & Holmes, S. (2007). Gene expression network
analysis and applications to immunology. Bioinformatics, 23(7), 850-858.
doi:10.1093/bioinformatics/btm019

Nahed BV, Bydon M, Ozturk AK, Bilguvar K, Bayrakli F, Gunel M. Genetics of

intracranial aneurysms. Neurosurgery 2007, 60: 213225 discussion 225226.

Nakaoka, H., Tajima, A., Yoneyama, T., Hosomichi, K., Kasuya, H., Mizutani, T., &
Inoue, I. (2014). Gene Expression Profiling Reveals Distinct Molecular
Signatures Associated With the Rupture of Intracranial Aneurysm. Stroke, 45 (8),
2239-2245. doi:10.1161/strokeaha.114.005851

Pera, J., Korostynski, M., Krzyszkowski, T., Czopek, J., Slowik, A., Dziedzic, T., . . .
Szczudlik, A. (2009). Gene Expression Profiles in Human Ruptured and
Unruptured Intracranial Aneurysms: What Is the Role of Inflammation?. Stroke,
41 (2), 224-231. doi:10.1161/strokeaha.109.562009

Pera, J., Korostynski, M., Piechota, M., Golda, S., Krupa, M., Moskala, M., . . . Slowik,
A. (2012). Gene Expression Profiling in Peripheral Blood in Ruptured Intracranial
Aneurysm (S23.006). Neurology, 78. doi:10.1212/w
nl.78.1_meetingabstracts.s23.006
Rinkel GJ, Algra A. Long-term outcomes of patients with aneurysmal subarachnoid
hemorrhage. Lancet Neurol 2011; 10: 349356.

Rinkel GJ, Djibuti M, Algra A, van Gijn J. Prevalence and risk of rupture of intracranial
aneurysms: a systematic review. Stroke. 1998;29:251256.
 14

Shi, C., Awad, I. A., Jafari, N., Lin, S., Du, P., Hage, Z. A., . . . Bendok, B. R. (2009).
Genomics of Human Intracranial Aneurysm Wall. Stroke, 40(4), 1252-1261.
doi:10.1161/strokeaha.108.532036

Speed, T. P. (2014). Gene Expression Analysis. Wiley StatsRef: Statistics Reference

Online. doi:10.1002/9781118445112.stat05379

Tarca, A. L., Romero, R., & Draghici, S. (2006). Analysis of microarray experiments of
gene expression profiling. American Journal of Obstetrics and Gynecology, 195
(2), 373-388. doi:10.1016/j.ajog.2006.07.001

Tromp, G., Weinsheimer, S., Ronkainen, A., & Kuivaniemi, H. (2014). Molecular basis
and genetic predisposition to intracranial aneurysm. Annals of Medicine, 46 (8),
597-606. doi:10.3109/07853890.2014.949299

Unruptured intracranial aneurysmsrisk of rupture and risks of surgical intervention.

International Study of Unruptured Intracranial Aneurysms Investigators. N Engl J
Med. 1998;339:17251733.

Vlak MH, Algra A, Brandenburg R, Rinkel GJ. Prevalence of unruptured intracranial

aneurysms, with emphasis on sex, age, comorbidity, country, and time period: a
systematic review and meta-analysis. Lancet Neurol 2011;10:626636

Wardlaw JM, White PM. The detection and management of unruptured intracranial
aneurysms. Brain 2000;123(pt 2):205-21.

Wiebers DO, Whisnant JP, Huston J III, et al. Unruptured intracranial aneurysms:
natural history, clinical outcome, and risks of surgical and endovascular
treatment. Lancet 2003;362:103110.888.

Weinsheimer S, Lenk GM, van der Voet M, Land S, Ronkainen A, Alafuzoff I,

Kuivaniemi H, Tromp G. Integration of expression profiles and genetic mapping
data to identify candidate genes in intracranial aneurysm. Physiol Genomics.
2007;32:4557.
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