Increased expression of genetically-
regulated FLT3 implicated in Tourette’s
Syndrome
Calwing Liao
1,2
*, Veikko Vuokila
2
*, Hélène Catoire
2
, Fulya Akçimen
1,2
, Jay P. Ross
1,2
, Cynthia
V. Bourassa
2
, Patrick A. Dion
2,3
, Inge A. Meijer
4
, Guy A. Rouleau
1,2,3
*Equally contributing first authors
1
Department of Human Genetics, McGill University, Montréal, Quebec, Canada
2
Montreal Neurological Institute, McGill University, Montréal, Quebec, Canada.
3
Department of Neurology and Neurosurgery, McGill University, Montréal, Quebec, Canada
4
Department of Neurosciences and CHU Sainte-Justine, University of Montréal, Montreal,
Quebec, Canada.
Corresponding Author:
Dr. Guy A. Rouleau, MD, PhD, FRCPC, OQ
Department of Neurology and Neurosurgery
McGill University
Montréal, Québec, Canada
H3A 2B4
E-mail: guy.rouleau@mcgill.ca
Keywords: FLT3, Tourette’s Syndrome
Conflict of Interests: All authors report no conflicts of interest.
Funding source: Canadian Institutes of Health Research
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 21, 2019. ; https://doi.org/10.1101/812420doi: bioRxiv preprint
Abstract
Tourette’s Syndrome (TS) is a neurodevelopmental disorder that is characterized by
motor and phonic tics. A recent TS genome-wide association study (GWAS) identified a
genome-wide significant locus. However, determining the biological mechanism of GWAS
signals remains difficult. Here, we conduct a TS transcriptome-wide association study (TWAS)
consisting of 4,819 cases and 9,488 controls and found that increased expression of FLT3 in the
dorsolateral prefrontal cortex (DLPFC) is associated with TS. We further showed that there is
global dysregulation of FLT3 across several brain regions and probabilistic causal fine-mapping
of the TWAS signal prioritizes FLT3 with a posterior inclusion probability of 0.849. We
validated the gene’s expression in 100 lymphoblastoid cell lines, establishing that TS cells had a
1.72 increased fold change compared to controls. A phenome-wide association study points
towards FLT3 having links with immune-related pathways such as monocyte count. We also
identify several splicing events in MPHOSPH9, CSGALNACT2 and FIP1L1 associated with TS,
which are also implicated in immune function. This analysis of expression and splicing begins to
explore the biology of TS GWAS signals.
Introduction
Tourette’s Syndrome (TS) is a neuropsychiatric disorder that is characterized by motor
and phonic tics
1
. The onset of the disorder is typically between the age of 5-7 years. TS has been
shown to have a large genetic component, in which the first-degree relatives of TS patients have
a 10- to 100-fold higher rate of TS compared to the general population
2,3
. Past genetic studies of
TS have identified several implicated genes such as CELSR3, a gene where recurrent do novo
variants are found in probands
4
. Furthermore, a recent genome-wide association study (GWAS)
identified a genome-wide significant hit on chromosome 13, rs2504235, which is within the
FLT3 (Fms Related Tyrosine Kinase 3) gene
5
. Although GWAS is a powerful method for
identifying associated genetic loci, it is often difficult to interpret the biological effects of
significant hits.
Recently, a transcriptomic imputation (TI) methodology was developed to allow for the
integration of genetic and expression data from datasets such as the CommonMind (CMC) and
Genotype-Tissue Expression (GTEx) consortia
6,7
. The derivation of panels involves a machine-
learning approach to characterize the relationship between gene expression and genotypes,
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 21, 2019. ; https://doi.org/10.1101/812420doi: bioRxiv preprint
making tissue-specific predictive models. The TI methodology can leverage these reference
imputation panels from these consortia and identifies the genetic correlation between imputed
expression and GWAS data
6
. Ultimately, TI allows for better characterization of GWAS data by
prioritizing tissue-specific genes associated with disease
8
. This methodology has already been
used to prioritize genes in many different traits
9–11
.
To identify genetically regulated genes associated with TS, we conducted a
transcriptome-wide association study (TWAS) of the current largest TS cohort of 4,819 cases
and 9,488 controls
5
. Brain-specific panels were derived from the CMC and GTEx 53 v7. The
TWAS revealed the expression of FLT3 to be increased across many brain tissues in TS, with the
largest effect in the dorsolateral prefrontal cortex (DLPFC). This is consistent with previous
studies that also implicated the DLPFC region in TS. Given that FLT3 is expressed in
lymphoblasts, we additionally measured the RNA expression of FLT3 in 100 lymphoblastoid cell
lines (LCL;50 cases and 50 controls). There was an increased expression in FLT3 in LCL
derived from TS cases, consistent with TWAS results. The top hit for the omnibus test also
identified ATP6V0A2 as a putative gene associated with TS, however, this was not genome-wide
significant. In conclusion, increased expression of FLT3 was implicated through TWAS across
several brain tissues and expression in lymphoblastoid cell lines.
Results
Transcriptome-wide significant hits
To identify genes associated with TS, a TWAS was conducted using FUSION. The
strongest significant hit was FLT3, with increased expression (Z=4.67, P=2.98E-06) in the
DLPFC (Table 1). Interestingly, the gene also had increased expression in the brain cortex,
hippocampus, anterior cingulate cortex, frontal cortex, cerebellum, and cerebellar hemispheres
suggesting a global dysregulation across brain tissue types. The gene DHRS11 was also
implicated (Z=4.26, P=2.01E-05), although not genome-wide significant. An omnibus test also
identified the top two genes: ATP6V0A2 (P=3.70E-05) and NEB (P=1.72E-04) (Table 1).
Splicing in Tourette’s Syndrome
To identify splicing events associated with TS, a TWAS was done using CMC splicing
data. There were no significant hits, however, there were several significant genes after
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 21, 2019. ; https://doi.org/10.1101/812420doi: bioRxiv preprint
permutation. The top three hits were MPHOSPH9 (Z=-4.32, P=1.58E-05), FIP1L1 (Z=4.21, P=
2.55E-05) and CSGALNACT2 (Z=4.14, P=3.39E-05) (Table 1). However, we also caution on the
interpretability of the effect direction given that alternatively spliced exons are typically
negatively correlated
9
.
Fine-mapping of FLT3 locus
To determine whether FLT3 is the putatively causal gene on the DLPFC, FOCUS was
used to assign a probabilistic inclusion probability for genes at the TWAS region
12
. For the
region 13:27284583-13:29257379 (hg19 coordinates), the FLT3 gene had the highest posterior
inclusion probability (PIP) of 0.849 and was included in the 90% credible gene set (Figure 1).
RT-qPCR of FLT3
To test normality of qPCR data, a Shapiro-Wilk test was done. It was found that the
∆
CT
values (a measure of expression) were normally distributed (W=0.99, P=0.70). Next, an ANOVA
was done and determined that the disease status explained a significant proportion of variance in
the dataset (F=7.06, P=0.0095). A Tukey test showed that TS patients had significantly higher
expression of FLT3 compared to controls, with a
∆
CT difference 0.780 (P=0.009) (Figure 2).
The corresponding fold change is +1.72 higher in TS than controls. The effect size was
determined to be moderate-large (Cohen’s F, 0.30).
Phenome-wide association study of FLT3
To identify phenotypes associated with the FLT3 gene, a regional phenome-wide
association study (pheWAS) was done. The pheWAS identified several immunological traits
associated with FLT3 such as monocyte count (3.87E-40) and percentage of white blood cells
(1.42E-21) (Figure 3).
Discussion
While recent GWAS for TS have successfully identified risk loci, the biological
relevance of these associations remains unknown. Here, we conduct a TWAS using the summary
statistics of over 14,000 individuals from the most recent TS GWAS
5
. This approach allows for
imputation of expression by leveraging genotype-expression reference panels. From this, we
identified an increased FLT3 expression as the top hit in the DLPFC and additionally found an
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 21, 2019. ; https://doi.org/10.1101/812420doi: bioRxiv preprint
increase in expression across most brain tissue types, suggesting global dysregulation. Validation
of expression in LCL prepared from TS cases found an increased or an increase in RNA
expression compared to LCL from control individuals. The FLT3 gene encodes for a tyrosine-
protein kinase and it has been associated with inflammation and immune function
13,14
. This could
point towards immunity in TS as a putative biological mechanism. Furthermore, the pheWAS
identified that FLT3 is associated with immunological traits such as monocyte count and white
blood cell counts. Interestingly, previous studies have demonstrated that TS patients have
significantly higher levels of monocytes compared to healthy controls
15
. This could suggest
dysregulation of monocytes partially due to increased expression of FLT3, which may contribute
towards pathogenicity of TS. Furthermore, fine-mapping the TWAS hit demonstrated that FLT3
was in the 90% causal credible-set with a PIP of 0.849 for the DLPRC. This further stipulates
that FLT3 is the correctly prioritized gene at this locus.
The splicing TWAS identified several putatively associated genes (MPHOSPH9,
FIP1L1, and CSGALNACT2) associated with TS, suggesting that both splicing and genetically
regulated genes are implicated in TS. The MPHOSPH9 gene encodes for a protein that regulates
cell cycling
16
. This gene has been implicated in multiple sclerosis, which is an inflammatory
disease of the central nervous system
16
. Additionally, FIP1L1 is associated with pre-mRNA 3’-
end formation and has been implicated in immunological function by cooperating with IL-5
17,18
.
These findings further support the hypothesis that the pathophysiology of TS may include or
involve the immune system. Understanding the role of immunity in TS may elucidate the link
between streptococcal infections and tic exacerbations as proposed in the pediatric autoimmune
neuropsychiatric disorders associated with streptococcal infections (PANDAS) hypothesis
1,19
.
The CSGALNACT2 gene encodes for chondroitin sulfate protein, which is involved in the brain
matrix
20
. A previous meta-analysis of ADHD and TS showed implication of sulfuration of
chondroitin, suggesting potential relevance
21
.
We conclude this study with some caveats and potential future directions. First, TWAS
signals can putatively be confounded due to expression imputation from weighted linear
combinations of SNPs. Because of this, some of these SNPs may be associated with non-
regulatory mechanisms that inflate the test statistic. A second caveat is that there is currently no
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 21, 2019. ; https://doi.org/10.1101/812420doi: bioRxiv preprint
