Journal of Clinical Neuroscience xxx (2018) xxx–xxx

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Journal of Clinical Neuroscience

journal homepage: www.elsevier.com/locate/jocn

Experimental study

Gene variations in PBX1, LMX1A and SLITRK1 are associated with

obsessive-compulsive disorder and its clinical features
Fernanda B. Melo-Felippe a, Leonardo F. Fontenelle b,c,d, Fabiana B. Kohlrausch a,⇑
a
Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal Fluminense (UFF), Niterói, Brazil
b
Programa de Transtornos Obsessivo-Compulsivos e de Ansiedade, Instituto de Psiquiatria, Universidade Federal do Rio de Janeiro (UFRJ), Universidade Federal do Rio de
Janeiro (UFRJ), Rio de Janeiro, Brazil
c
Instituto D’Or de Pesquisa e Ensino (IDOR), Rio de Janeiro, Brazil
d
School of Psychological Sciences, MONASH University, Melbourne, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Genetic factors probably influence OCD development and a current hypothesis proposes that genes
Received 14 May 2018 involved in the development of the central nervous system (CNS) are related to OCD. The aim of this study
Accepted 7 October 2018 was to analyze six Single Nucleotide Polymorphisms (SNPs) in five genes with functions related to neu-
Available online xxxx
rodevelopment in OCD. A total of 203 patient and 203 control samples were genotyped using the
TaqManÒ methodology. Statistically significant associations between OCD and PBX1 (rs2275558) in total
Keywords: sample (P = 0.002) and in males (P = 0.0003) were observed. Concerning symptom dimensions, the
Brazil
expression of neutralization showed a statistical significant association with LMX1A (rs4657411,
Genetics
Polymorphism
P = 0.004) in total sample. We also observed significant association between LMX1A (rs4657411) and
OCD washing dimension in females (P = 0.01). Additionally, SLITRK1 (rs9593835) was significantly associated
Symptom dimensions with checking dimension in male patients (P = 0.04). Our results indicate an important influence of neu-
rodevelopment genes in the OCD susceptibility. Additional studies with larger samples are needed to con-
firm these results.
Ó 2018 Elsevier Ltd. All rights reserved.

1. Introduction important role in neurodevelopment and highly expressed in CSTC

Obsessive-Compulsive Disorder (OCD) is characterized by
uncontrollable, unwanted thoughts and repetitive, ritualized
behaviors [1]. OCD is a clinically heterogeneous disorder with a
large variety of symptomatic expressions [2]. Traditionally, its
symptoms has been grouped into four to six symptom dimensions
that may include checking, washing, ordering, obsession and
neutralization [3,4], with hoarding now representing a different
disorder. The etiology of OCD is currently unknown but there are
evidences suggesting that genetic and environmental factors may
play a significant role in OCD symptoms [2,5].
The cortico-striato-thalamo-cortical circuitry (CSTC) is the pre-
vailing neural and pathophysiology model of OCD [6]. Currently,
there is a hypothesis proposing that genes involved in the Central
Nervous System (CNS) development are related to OCD [7–13].
Some studies observed that knockout (KO) mice for genes with
⇑ Corresponding author at: Departamento de Biologia Geral, Instituto de Biologia,
Universidade Federal Fluminense, 24061-970 Niterói, RJ, Brazil.
E-mail address: fabianabk@id.uff.br (F.B. Kohlrausch).
circuit (such as SAPAP3 and SLITRK1) showed similar OCD symp-
toms and behaviors [14,15].
Synapse-associated protein 90/postsynaptic density protein
95-associated protein 3 (SAPAP3) is a protein localized in post-
synaptic excitatory synapses density, highly expressed in the stria-
tum. Sapap3 (Dlgap3) gene KO mice show several abnormalities,
such as increased anxiety and compulsive grooming behavior
[15], and striatal abnormalities that may reflect striatal hyperme-
tabolism [16]. Polymorphic variations in SAPAP3 were previously
associated with conditions from the obsessive-compulsive spec-
trum, such as grooming disorders/trichotillomania (TTM) [17,18]
and Tourette Syndrome (TS) [19]. Boardman et al. investigated
seven polymorphisms in SAPAP3 in South African white OCD and
linked some variants to the development of earlier onset OCD [20].
The SLITRK1 (Slit and Trk-like family member 1) gene encodes a
member of a neuronal transmembrane protein family, which
promotes neurite outgrowth [21] and is expressed in the CSTC
[22]. Variations in SLITRK1 have been previously associated with
OCD [23], TTM [24], and TS [25]. Moreover, Slitrk1-deficient mice
exhibit anxiety-like behavior and an elevated level of nore-
pinephrine in the prefrontal cortex [14].

https://doi.org/10.1016/j.jocn.2018.10.042
0967-5868/Ó 2018 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Melo-Felippe FB et al. Gene variations in PBX1, LMX1A and SLITRK1 are associated with obsessive-compulsive disorder
and its clinical features. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.10.042
2 F.B. Melo-Felippe et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx

Two Genome Wide Association Studies (GWAS) suggested a role 2.2. Laboratory analyses
for other important neurodevelopment genes in OCD, including
PBX1, LMX1A, and RYR3 [10,26]. LMX1A (LIM homeobox DNA extraction from saliva samples was performed according
transcription factor 1 alpha) is a transcription factor, and PBX1 to a modified protocol previously published [37]. Genotyping of
(Pre-B-cell leukemia homeobox 1) is closely related to these tran- the polymorphisms in RYR3 (rs2229116 and rs1044129), SAPAP3
scription factors. PBX1 gene is intimately involved as a cofactor in (rs6662980), SLITRK1 (rs9593835), LMX1A (rs4657411), and
homeobox genes and myogenic differentiation [27], showing to be PBX1 (rs2275558) was performed by allelic discrimination
important in neurodevelopment [26]. Some polymorphisms in using real-time polymerase chain reaction predesigned and
LMX1A gene (e.g. rs4657411) have been associated with disorders validated TaqManÒ assays (C__31568815_10, C__31568815_10,
related to dopaminergic transmission, such as Parkinson’s disease C__42520836_10, C___1797114_10, C__15879055_30, C__
[28]. The ryanodine receptor is induced by calcium channel and 15964080_10, respectively).
RYR3 appears to be high expressed primarily in brain areas impli-
cated on OCD [29]. Knockout mice for Rir3 had impaired spatial
2.3. Statistical analyses
learning, hippocampal synaptic plasticity, and deficiencies in fear
conditioning and social interactions [30]. In addition, this gene is
The Hardy-Weinberg equilibrium was assessed using v2 tests.
located at 15q14 region, which showed a strong linkage signal with
Statistical analyzes were performed by comparing allele and geno-
OCD [10].
type distribution between OCD patients and healthy controls,
Since these genes have not been evaluated in OCD Brazilian
using the two-tailed v2 or Fisher test. The influence of other vari-
patients, the aim of this case-control study was to assess whether
ables (gender, age of onset and six OCD symptom dimensions) was
six Single Nucleotide Polymorphisms (SNPs) in five genes with
also evaluated through the same tests. Multivariate logistic regres-
functions related to neurodevelopment (SAPAP3, SLITRK1, PBX1,
sions were performed to assess the odds ratio (OR) and 95% confi-
LMX1A, and RYR3) could influence susceptibility to OCD and OCD
dence intervals (95% CI), controlling by independent variables
clinical features in Brazilians. Our hypothesis is that since these
(gender and early onset, when applicable). Differences in quantita-
genes have a role on neurodevelopment, variations in their
tive variables were assessed using Mann-Whitney U test. Analyses
sequence may have some implication in the expression of OCD
were performed using the SPSS version 20.0 and P values <0.05
and its clinical characteristics.
were considered significant.

2. Material and methods 3. Results

2.1. Participants 3.1. Demographic and clinical data

The sample consisted of 203 unrelated Brazilian Obsessive
Compulsive patients attending the Anxiety, Obsessions and Com-
pulsions Research Program of the Federal University of Rio de
Janeiro and/or the Association of Family, Friends and People with
Obsessive Compulsive Disorder in Rio de Janeiro (‘‘RIOSTOC”)
[31]. Patients fulfilled the diagnostic criteria for current OCD
according to the Diagnostic and Statistical Manual of Mental Disor-
ders, 4th edition revised (DSM-IV-TR) [32].
The diagnosis of OCD was confirmed through the means of the
Mini International Neuropsychiatric Interview (MINI) [33].
Obsessive-compulsive inventory-revised (OCI-R) [34] was applied
to evaluate the presence of specific obsessive-compulsive
symptoms dimensions (checking, washing, ordering, hoarding,
obsession, and neutralization). OCI-R is an 18-item questionnaire
that allows to rate the degree to which participants are bothered
or distressed by OCD symptoms on a 5-point scale from 0 (not at
all) to 4 (extremely). Therefore, we categorized the six symptom
dimensions as present (1–4) or absent (0). Age at onset of each
OCD symptom was assessed by means of the Florida Obsessive-
Compulsive Inventory (FOCI) [35], and patients were classified
into those exhibiting early onset (<18 years) and late onset
The main characteristics and distribution of symptom dimen-
sions in our OCD sample are summarized in Table 1. The sample
included OCD patients aged between 18 and 73 years
(37.89 ± 13.24 years) and healthy controls aged between 18 and
75 years (32.38 ± 13.45 years). The mean age of symptoms onset
in male patients was 13.95 (±6.20) years, and in females 15.66
(±8.37) years, both ranging between 3 and 44 years. No statistically
significant difference was observed in the mean age of onset of
symptoms between men and women (z = 0.94, P = 0.35). Consid-
ering early (<18 years) and late (>18 years) onset, we also did not
observe significant difference between males and females
(P = 0.07). Regarding symptoms dimensions, obsession showed
predominance over the other five dimensions (94% of patients),
and there were no significant differences between men and
women in all symptom dimensions manifestation (0.07 < P < 0.95).
Table 1
Sample characteristics of OCD patients.
Characteristics OCD
Males (99) Females (1 0 4) Total (2 0 3)

(18 years) OCD according to the symptom showing the earliest Agea 34.79 (±11.93) 40.85 (±13.80) 37.89 (13.24)
appearance [31]. Age of illness onsetb 13.95 (± 6.20) 15.66 (± 8.37) 14.84 (7.41)
Early onsetc 73 (0.74) 67 (0.64) 140 (0.69)
A sample of healthy control subjects consisted of 203 unrelated
Symptom dimensionsd
individuals from the Brazilian general population, recruited from Checking 70 (0.71) 84 (0.81) 154 (0.76)
hospital staff, administrative personal and university students. Washing 61 (0.62) 71 (0.68) 132 (0.65)
These individuals were assessed with the Modified Mini Screen Ordering 72 (0.73) 76 (0.73) 148 (0.73)
Hoarding 71 (0.72) 62 (0.60) 133 (0.65)
(MMS) [36] for the exclusion of severe psychiatric disorders and
Obsession 90 (0.91) 100 (0.96) 190 (0.94)
suicidal behavior. Neutralization 59 (0.60) 63 (0.61) 122 (0.60)
All procedures performed in this study were approved and are
a
in accordance with the ethical standards of the Brazilian National In years, mean ± SD (standard deviation), Wilcoxon-Mann-Whitney test,
z = -3.20, P = 0.001.
Research Ethics Committee. Informed consent was obtained b
In years, mean ± SD, z = -0.94, P = 0.35.
from all participants after the study protocol had been fully c
Number of individuals (frequency), P = 0.07.
d
explained. number of individuals (frequency), 0.065 < P < 0.985.

Please cite this article in press as: Melo-Felippe FB et al. Gene variations in PBX1, LMX1A and SLITRK1 are associated with obsessive-compulsive disorder
and its clinical features. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.10.042
 F.B. Melo-Felippe et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx 3

Table 2
Genotype and allele frequency comparisons between patients and controls.

Total sample Females Males

SNPs OCD OCD Total P OCD Controls Total P OCD Controls Total P
Controls
RYR3 rs2229116 201 201 402 104 124 228 97 77 174
AA 122 (0.61) 135 (0.67) 257 0.39 71 (0.68) 85 (0.69) 156 0.59 51 (0.53) 50 (0.65) 101 0.20
AG 71 (0.35) 60 (0.30) 131 29 (0.28) 37 (0.29) 66 42 (0.43) 23 (0.30) 65
GG 8 (0.04) 6 (0.03) 14 4 (0.04) 2 (0.02) 6 4 (0.04) 4 (0.05) 8
A 315 (0.78) 330 (0.82) 645 0.17 171 (0.82) 207 (0.83) 378 0.82 144 (0.74) 123 (0.80) 267 0.27
G 87 (0.22) 72 (0.18) 159 37 (0.18) 41 (0.17) 78 50 (0.26) 31 (0.20) 81
RYR3 rs1044129 201 203 404 102 125 227 99 78 177
AA 25 (0.12) 24 (0.12) 49 0.60 13 (0.13) 18 (0.14) 31 0.54 12 (0.12) 6 (0.08) 18 0.61
AG 90 (0.45) 101 (0.50) 191 43 (0.42) 60 (0.48) 102 47 (0.48) 41 (0.53) 88
GG 86 (0.43) 78 (0.39) 164 46 (0.45) 47 (0.38) 94 40 (0.40) 31 (0.40) 71
A 140 (0.35) 149 (0.37) 289 0.67 69 (0.34) 96 (0.38) 165 0.36 71 (0.36) 53 (0.34) 124 0.80
G 262 (0.65) 257 (0.63) 519 135 (0.66) 154 (0.62) 289 127 (0.64) 103 (0.66) 230
SAPAP3 rs6662980 199 200 399 102 124 226 97 76 173
AA 93 (0.47) 98 (0.49) 191 0.89 47 (0.46) 60 (0.48) 107 0.53 46 (0.48) 38 (0.50) 84 0.66
AG 89 (0.45) 86 (0.43) 175 50 (0.49) 54 (0.44) 104 39 (0.40) 32 (0.42) 71
GG 17 (0.09) 16 (0.08) 33 5 (0.05) 10 (0.08) 15 12 (0.12) 6 (0.08) 18
A 275 (0.69) 282 (0.71) 557 0.69 144 (0.71) 174 (0.70) 318 0.92 131 (0.68) 108 (0.71) 239 0.58
G 123 (0.31) 118 (0.29) 241 60 (0.29) 74 (0.30) 134 63 (0.32) 44 (0.29) 107
SLITRK1 rs9593835 192 184 376 99 113 212 93 71 164
CC 99 (0.52) 85 (0.46) 184 0.39 53 (0.54) 61 (0.54) 114 0.58 46 (0.49) 24 (0.34) 70 0.12
TC 70 (0.36) 80 (0.43) 150 31 (0.31) 40 (0.35) 71 39 (0.42) 40 (0.56) 79
TT 23 (0.12) 19 (0.10) 42 15 (0.15) 12 (0.11) 27 8 (0.09) 7 (0.10) 15
C 268 (0.70) 250 (0.68) 518 0.58 137 (0.69) 162 (0.72) 299 0.65 131 (0.70) 88 (0.62) 219 0.13
T 116 (0.30) 118 (0.32) 234 61 (0.31) 64 (0.28) 125 55 (0.30) 54 (0.38) 109
LMX1A rs4657411 200 200 400 103 124 227 97 76 173
AA 160 (0.80) 149 (0.75) 309 0.20 80 (0.78) 94 (0.76) 174 0.76 80 (0.82) 55 (0.72) 135 0.12
AG 40 (0.20) 49 (0.25) 89 23 (0.22) 30 (0.24) 53 17 (0.18) 19 (0.25) 36
GG 0 (0.00) 2 (0.01) 2 0 (0.00) 0 (0.00) 0 0 (0.00) 2 (0.03) 2
A 360 (0.90) 347 (0.87) 707 0.18 183 (0.89) 218 (0.88) 401 0.86 177 (0.91) 129 (0.85) 306 0.07
G 40 (0.10) 53 (0.13) 93 23 (0.11) 30 (0.12) 53 17 (0.09) 23 (0.15) 40
PBX1 rs2275558 197 201 398 101 123 224 96 78 174
GG 123 (0.62) 153 (0.76) 276 0.08 67 (0.66) 87 (0.71) 154 0.73 56 (0.58) 66 (0.85) 122 <0.001
AG 65 (0.33) 41 (0.20) 106 32 (0.32) 33 (0.27) 65 33 (0.34) 8 (0.10) 41
AA 9 (0.05) 7 (0.03) 16 2 (0.02) 3 (0.02) 5 7 (0.07) 4 (0.05) 11
G 311 (0.79) 347 (0.86) 658 0.006a 166 (0.82) 207 (0.84) 373 0.61 145 (0.76) 140 (0.90) 285 0.001c
A 83 (0.21) 55 (0.14) 138 36 (0.18) 39 (0.16) 75 47 (0.24) 16 (0.10) 63
GG 123 (0.62) 153 (0.76) 276 0.003b 67 (0.66) 87 (0.71) 154 0.56 56 (0.58) 66 (0.85) 122 <0.001d
AA + AG 74 (0.38) 48 (0.24) 122 34 (0.34) 36 (0.29) 70 40 (0.42) 12 (0.15) 52

The P-values are bold where they are less than the significance level cut-off of 0.05.
a
A allele: OR 1.68, 95% CI 1.16–2.45.
b
Dominant model AA + AG: OR 1.92, 95%CI 1.24–2.96.
c
A allele: OR 2.84, 95%CI 1.54–5.24.
d
Dominant model AA + AG: OR 3.93, 95%CI 1.88–8.20.

3.2. Case-control study
The allele and genotype frequency distribution of the six ana-
lyzed SNPs are shown in Table 2. Genotype distributions in total
sample, patients and controls were in Hardy-Weinberg
equilibrium.
A statistically significant difference between patients and con-
trols was observed for rs2275558 in PBX1 (Table 2). When we
pooled homozygous and heterozygous individuals for the A allele,
considering all A-carriers, we observed significant differences
between patients and controls (P = 0.002, OR = 1.91 [1.24–2.96]).
Analyses of allele frequencies also showed a significant association
between the A allele and OCD (P = 0.004, OR = 1.68 [1.16–2.45]).
Analyzing by gender, we observed these associations to be exclu-
sive to males. There was a statistically significant predominance
of the A allele in the male patient group compared to the male
healthy control group (P = 0.001, OR = 2.84 [1.54–5.23]), and also
a dominant effect of the A allele (P = 0.0003, OR = 3.93
[1.88–8.21]) (Table 2). After controlling for confounders in logistic
regression analyses, the rs2275558 in PBX1 was confirmed to be
significantly associated with OCD in males (Table 4). No statisti-
cally significant differences in allelic and genotypic distributions
of the remaining SNPs were observed (Table 2).
3.3. OCD clinical features
We did not observe significant differences in genotype and
allele distributions of the six SNPs between early-onset and
late-onset OCD (data not shown). However, we found statistically
significant associations between polymorphisms and specific
OCD symptom dimensions (Table 3).
The expression of neutralization dimension showed a trend for
association with rs6662980 in SAPAP3 (P = 0.05, A-allele OR = 1.96
[1.07–3.62]) and a statistical significant association with
rs4657411 in LMX1A (Table 3). Neutralization was significantly
associated with the AA genotype of rs4657411 in the total sample
(P = 0.004, OR = 2.78 [1.36–5.67]) and a trend to this association
was observed in male patients (P = 0.05; OR 3.34 95%CI
1.12–9.99). The A allele also showed a significant association with
neutralization in total sample (P = 0.01, OR = 2.46 [1.26–4.80]).

Please cite this article in press as: Melo-Felippe FB et al. Gene variations in PBX1, LMX1A and SLITRK1 are associated with obsessive-compulsive disorder
and its clinical features. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.10.042
4 F.B. Melo-Felippe et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx

Table 3
Genotype and allele distribution among three OCD dimensions.

Total sample Females Males

SNPs Present Absent Total P Present Absent Total P Present Absent Total P
CHECKING
SLITRK1 rs9593835 148 43 191 81 17 98 67 26 93
CC 79 (0.53) 19 (0.44) 98 0.27 42 (0.52) 10 (0.59) 52 0.74 37 (0.55) 9 (0.35) 46 0.04
TC 54 (0.36) 16 (0.37) 70 27 (0.33) 4 (0.24) 31 27 (0.40) 12 (0.46) 39
TT 15 (0.10) 8 (0.19) 23 12 (0.15) 3 (0.18) 15 3 (0.04) 5 (0.19) 8
C 212 (0.72) 54 (0.63) 266 0.15 111 (0.69) 24 (0.71) 135 0.97 101 (0.75) 30 (0.58) 131 0.02a
T 84 (0.28) 32 (0.37) 116 51 (0.31) 10 (0.29) 61 33 (0.25) 22 (0.42) 55
TC + CC 133 (0.90) 35 (0.81) 168 0.22 69 (0.85) 14 (0.82) 83 0.94 64 (0.96) 21 (0.81) 85 0.04b
TT 15 (0.10) 8 (0.19) 23 12 (0.15) 3 (0.18) 15 3 (0.04) 5 (0.19) 8
WASHING
LMX1A rs4657411 128 71 199 69 33 102 59 38 97
AA 107 (0.84) 52 (0.73) 159 59 (0.86) 20 (0.61) 79 48 (0.81) 32 (0.84) 80
AG 21 (0.16) 19 (0.27) 40 0.06 10 (0.14) 13 (0.39) 23 0.005c 11 (0.19) 6 (0.16) 17 0.79
GG 0 (0.00) 0 (0.00) 0 0 (0.00) 0 (0.00) 0 0 (0.00) 0 (0.00) 0
A 235 (0.92) 123 (0.87) 358 0.14 128 (0.93) 53 (0.80) 181 0.01d 107 (0.91) 68 (0.92) 177 0.80
G 21 (0.08) 19 (0.13) 40 10 (0.07) 13 (0.20) 23 11 (0.09) 6 (0.08) 17
NEUTRALIZATION
LMX1A rs4657411 118 80 198 61 40 101 57 40 97
AA 102 (0.86) 56 (0.70) 158 0.005e 51 (0.84) 27 (0.68) 78 0.59 51 (0.89) 29 (0.73) 80 0.03g
AG 16 (0.14) 24 (0.30) 40 10 (0.16) 13 (0.33) 23 6 (0.11) 11 (0.27) 17
GG 0 (0.00) 0 (0.00) 0 0 (0.00) 0 (0.00) 0 0 (0.00) 0 (0.00) 0
A 220 (0.93) 134 (0.85) 356 0.007f 112 (0.92) 67 (0.84) 179 0.12 108 (0.95) 67 (0.86) 175 0.03h
G 16 (0.07) 24 (0.15) 40 10 (0.08) 13 (0.16) 23 6 (0.05) 11 (0.14) 17
SAPAP3 rs6662980 117 80 197 60 40 100 57 40 97
AA 59 (0.50) 34 (0.43) 93 0.39 27 (0.45) 20 (0.50) 47 0.88 32 (0.56) 14 (0.35) 46 0.10
AG 50 (0.43) 37 (0.46) 87 30 (0.50) 18 (0.45) 48 20 (0.35) 19 (0.48) 39
GG 8 (0.07) 9 (0.11) 17 3 (0.05) 2 (0.05) 5 5 (0.09) 7 (0.18) 12
A 168 (0.72) 104 (0.66) 273 0.25 84 (0.70) 58 (0.73) 142 0.82 84 (0.74) 46 (0.59) 131 0.03i
G 66 (0.28) 54 (0.34) 121 36 (0.30) 22 (0.28) 58 30 (0.26) 32 (0.41) 63
AA 59 (0.50) 34 (0.42) 93 0.38 27 (0.45) 20 (0.50) 47 0.77 32 (0.56) 14 (0.35) 46 0.05
AG + GG 58 (0.50) 45 (0.58) 104 33 (0.55) 20 (0.50) 53 25 (0.44) 25 (0.65) 51

The P-values are bold where they are less than the significance level cut-off of 0.05.
a
C Allele: OR 2.24, 95%CI 1.14–4.41.
b
TC + CC: OR 5.08, 95%CI 1.12–23.08.
c
AA: OR 3.83, 95%CI 1.46–10.09.
d
A Allele: OR 3.14, 95%CI 1.30–7.60.
e
AA: OR 2.73, 95%CI 1.34–5.57.
f
A Allele: OR 2.46, 95%CI 1.26–4.80.
g
AA: OR 3.22, 95% 1.08–9.63.
h
A allele: OR 2.96, 95%CI 1.04–8.36.
i
A Allele: OR 1.95, 95%CI 1.05–3.60.

Table 4
Logistic regression analyses.

Variables b SE Wald P OR (95% CI)

Case-Control
Gender (male) 0.43 0.21 4.39 0.036 1.54 (1.03–2.30)
PBX1 (AA) 0.67 0.22 9.08 0.003 1.96 (1.26–3.03)
Checking
Gender (male) -0.71 0.38 3.47 0.06 0.49 (0.23–1.04)
Early onset (<18 y) 0.17 0.41 0.17 0.68 1.19 (0.53–2.68)
SLITRK1 (CC) 0.87 0.53 2.68 0.10 2.39 (0.84–6.78)
Neutralization
Gender (male) -0.22 0.31 0.51 0.48 0.80 (0.43–1.47)
Early onset (<18 y) 0.50 0.35 2.12 0.15 1.65 (0.84–3.26)
LMX1A (AA) 0.77 0.38 4.14 0.04 2.17 (1.02–4.56)
SAPAP3 (AA) 0.19 0.31 0.40 0.53 1.24 (0.66–2.22)
Washing
Gender (female) -0.37 0.32 1.34 0.25 0.69 (0.37–1.30)
Early onset (<18y) 0.74 0.35 4.49 0.03 2.10 (1.06–4.16)
LMX1A (AA) 0.69 0.48 3.24 0.72 1.99 (0.94–4.24)

The P-values are bold where they are less than the significance level cut-off of 0.05.
b = estimated coefficient; SE = standard error; Wald = test the statistical significance of each coefficient (b) in the model (Z statistic); d = degrees of freedom; OR = odds ratio;
CI = confidence interval.
The P-values are bold where they are less than the significance level cut-off of 0.05.

Please cite this article in press as: Melo-Felippe FB et al. Gene variations in PBX1, LMX1A and SLITRK1 are associated with obsessive-compulsive disorder
and its clinical features. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.10.042
 F.B. Melo-Felippe et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx
Logistic regression analysis confirmed the importance of rs4657411
on neutralization, after controlling for confounders (Table 4).
In relation to washing dimension (Table 3), females who
express this symptom dimension were more likely to be AA
homozygous for rs4657411 (LMX1A) than those who did not
express (P = 0.01, OR = 3.83 [1.46–10.09], when the A allele was
considered recessive). Additionally, the A allele was also associated
to washing in females (P = 0.02, OR = 3.14 [1.30–7.60]).
Checking dimension was significantly associated with
rs9593835 in SLITRK1 in male patients (Table 3). Genotype distribu-
tion showed significant differences between patients who
expressed checking symptoms than those who did not (P = 0.04).
In a dominant model, considering C-carriers, we also observed an
association in OCD males (P = 0.04, OR = 5.08 [1.12–23.08]). The C
allele was also observed more frequently in male patients who
expressed checking symptoms (P = 0.03; OR 2.24 95% CI 1.14–4.41).
4. Discussion
In this study, we evaluated the influence of six SNPs in neurode-
velopment genes on the etiology of OCD. Since SAPAP3, SLITRK1,
LMX1A, PBX1 and RYR3 have shown involvement in neurodevelop-
ment, and OCD begins early in life, we predicted that genetic vari-
ations at these genes could play roles on OCD etiology.
A previous GWAS reported association between OCD and SNPs
in homeobox genes [26]. One of these SNPs is located between
PBX1 and LMX1A genes. PBX1 is involved in the development of
the striatum in rats [38,39], a region previously associated with
OCD and obsessive-compulsive behaviors related to the CSTC dys-
function [6,40]. We found that presence of at least one copy of the
A allele of rs2275558 in PBX1 was related to OCD in total sample
and in OCD males. However, no functional study was conducted
with this SNP to confirm its influence on PBX1 expression and OCD.
To the best of our knowledge, there are no previous studies to
corroborate our association of LMX1A with neutralization and
washing. The activation of the LMX1A transcription factor is
involved in the differentiation and maturation of dopamine produc-
ing neurons in midbrain [41]. The use of small interfering RNA to
silence the expression of Lmx1a in chick embryos resulted in loss
of mesencephalic dopamine neurons [42]. According to previous
results of our group, significant associations between Catechol-O-
Methyltransferase gene (COMT) SNPs and OCD were observed in
Brazilians. The COMT enzyme is one of the major enzymes to
degrade dopamine in the prefrontal cortex; therefore, dopaminer-
gic hipoactivity due to COMT polymorphisms might be related to
OCD development [31] and symptom dimensions occurrence. How-
ever, the precise relationship between LMX1A rs4657411 polymor-
phism and dopamine dysfunction is still unknown.
A previous study provided evidence that the expression of
SLITRK1 is dynamic and shows a developmentally regulated
expression pattern in projection neurons of the CSTC circuit [22].
In this study, we observed the SLITRK1 gene associated with check-
ing dimension in male OCD patients. Ozomaro et al. performed a
genetic and functional analysis of rare variants in SLITRK1 and
observed that important functional differences in the N400I vari-
ant may contribute to OCD and OC spectrum symptoms [23]. A
study examined the association between different regional brain
volumes in pediatric OCD patients and nine candidate genes,
including SLITRK1 [43], and no SNP was significantly associated
with volumetric changes in orbitofrontal cortex (OFC), anterior cin-
gulate cortex (ACC), thalamus, caudate, putamen, globus pallidus
and pituitary. Additionally, SLITRK1 gene variations have been also
associated with disorders in the OC spectrum, as trichotillomania
[24] and TS [44–49], disorders that probably share common etio-
logic factors with OCD.
Please cite this article in press as: Melo-Felippe FB et al. Gene variations in PBX1, LMX1A and SLITRK1 are associated with obsessive-compulsive disorder
and its clinical features. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.10.042
5
Previous studies observed significant associations of polymor-
phisms in SAPAP3 with earlier onset OCD [20] and grooming behav-
iors [17]. Besides, knockout mice for Sapap3 showed similar
characteristics to OCD patients, such grooming, facial lesions,
increased anxiety, response to fluoxetine therapy and a reduction
of the cortical-striatal synaptic transmission [15]. SAPAP3 was clo-
sely associated with neutralization dimension in our sample of
OCD patients. It is difficult to speculate on the significance of this
finding, as the evidence supporting the existence of a neutraliza-
tion dimension is still meager. For instance, in other factor-
analytic studies, the items that compose OCI-R neutralization
dimension have load on ordering (such as counting) and miscella-
neous (i.e. ‘‘I feel that there are good and bad numbers”) symptoms
[50]. Therefore, our results may contribute to the biological valida-
tion of the neutralization dimension.
Our study has some methodological limitations. The sample
size was not large, so we cannot exclude Type I errors. The clinical
heterogeneity of OCD coupled with the use different assessment
instruments (such as the use of Yale-Brown Obsessive-
Compulsive Symptom Checklist) makes the comparability of our
results with previous studies difficult. In fact, to the best of our
knowledge, our series of studies are the first to assess the genetic
correlates of different OCI-R dimensions [31,51,52]. As an addi-
tional limitation, we can mention the Brazilian racial makeup,
which results from a mix of ethnicities and a population whose
genetics can be considered unique [53]. Unfortunately, we were
also not able to evaluate the influence of environmental factors,
which may have a large influence on OCD development and symp-
tomatology. Finally, all SNPs in the present study are of unknown
function, therefore the hypothesis that they are associated with
reduced function and abnormal neurodevelopment in OCD need
to be tested in functional studies.
In conclusion, our study supports the hypotheses that genes
involved in neurodevelopment are related to OCD and its clinical
features in our Brazilian sample. The current analysis is unprece-
dented in samples of Brazilian population, and as an exploratory
study our results may help identify genes involved in OCD patho-
genesis. Although replication of these findings is required, PBX1,
LMX1A, SLITRK1 and SAPAP3 seem to be promising candidate genes
for OCD.
Acknowledgments
We wish to thank all patients and heathy controls who volun-
teered to participate in this study, and to all their relatives. We also
thank the support of Isabele Gomes Giori and Juliana Braga de Sal-
les Andrade in sample collection.
Funding disclosure
This work was supported by the Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq, Brazil; grant number
477658/2009-1 and 472033/2012-3), Fundação de Amparo à Pes-
quisa do Estado do Rio de Janeiro (FAPERJ, Brazil; grant number
E-26/110.080/2010 and E-26/111.751/2012), and Pró-Reitoria de
Pesquisa, Pós-Graduação e Inovação from Universidade Federal
Fluminense (Proppi/PDI/UFF). The funding source(s) had no
involvement in study design; in the collection, analysis and inter-
pretation of data; in the writing of the report; and in the decision
to submit the article for publication.
Conflict of interest
The authors declared no conflict of interest.
6 F.B. Melo-Felippe et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx

References [28] Bergman O, Hakansson A, Westberg L, Belin AC, Sydow O, Olson L, et al. Do
polymorphisms in transcription factors LMX1A and LMX1B influence the risk
for Parkinson’s disease? J Neural Transm 2009;116:333–8.
[1] American Psychiatric Association. Diagnostic and Statistical Manual of Mental
[29] Rauch SL, Whalen PJ, Curran T, Shin LM, Coffey BJ, Savage CR, et al. Probing
Disorders 5 th2013.
striato-thalamic function in obsessive-compulsive disorder and Tourette
[2] Pauls DL, Abramovitch A, Rauch SL, Geller DA. Obsessive-compulsive disorder:
syndrome using neuroimaging methods. Adv Neurol 2001;85:207–24.
an integrative genetic and neurobiological perspective. Nat Rev Neurosci
[30] Matsuo N, Tanda K, Nakanishi K, Yamasaki N, Toyama K, Takao K, et al.
2014;15:410–24.
Comprehensive behavioral phenotyping of ryanodine receptor type 3 (RyR3)
[3] Foa EB, Huppert JD, Leiberg S, Langner R, Kichic R, Hajcak G, et al. The
knockout mice: decreased social contact duration in two social interaction
Obsessive-Compulsive Inventory: development and validation of a short
tests. Front Behav Neurosci 2009;3:3.
version. Psychol Assess 2002;14:485–96.
[31] Melo-Felippe FB, de Salles Andrade JB, Giori IG, Vieira-Fonseca T, Fontenelle LF,
[4] Mataix-Cols D, Rosario-Campos MC, Leckman JF. A multidimensional model of
Kohlrausch FB. Catechol-O-methyltransferase gene polymorphisms in specific
obsessive-compulsive disorder. Am J Psychiatry 2005;162:228–38.
obsessive-compulsive disorder patients’ subgroups. J Mol Neurosci: MN
[5] Taylor S, Jang KL, Asmundson GJ. Etiology of obsessions and compulsions: a
2016;58:129–36.
behavioral-genetic analysis. J Abnorm Psychol 2010;119:672–82.
[32] American Psychiatric Association. Diagnostic and Statistical Manual of Mental
[6] Burguiere E, Monteiro P, Mallet L, Feng G, Graybiel AM. Striatal circuits, habits,
Disorders 4th, 2000.
and implications for obsessive-compulsive disorder. Curr Opin Neurobiol
[33] Amorim P. Mini International Neuropsychiatric Interview (MINI): validation of
2015;30:59–65.
a short structured diagnostic psychiatric interview. Rev Bras Psiquiatr
[7] Dennis C. Psychiatric disease: all in the mind of a mouse. Nature
2000;22:106–15.
2005;438:151–2.
[34] Souza FF, Silva EB, Meyer EM, NiederauerI IE, RaffinI KG, Cordiol AL, et al.
[8] Grados M, Wilcox HC. Genetics of obsessive-compulsive disorder: a research
Tradução e adaptação transcultural das escalas Obsessive Compulsive
update. Expert Rev Neurother 2007;7:967–80.
Inventory – OCI e do Obsessive Compulsive Inventory Revisado – OCI-R. Rev
[9] Hyman SE. A bone to pick with compulsive behavior. Cell 2010;141:752–4.
Bras de Psiquiatria 2007;30.
[10] Ross J, Badner J, Garrido H, Sheppard B, Chavira DA, Grados M, et al.
[35] Storch EA, Kaufman DA, Bagner D, Merlo LJ, Shapira NA, Geffken GR, et al.
Genomewide linkage analysis in Costa Rican families implicates
Florida Obsessive-Compulsive Inventory: development, reliability, and
chromosome 15q14 as a candidate region for OCD. Hum Genet
validity. J Clin Psychol 2007;63:851–9.
2011;130:795–805.
[36] Spotts J. Utility of the Modified Mini Screen (MMS) for screening mental health
[11] Yang XW, Lu XH. Molecular and cellular basis of obsessive-compulsive
disorders in a prison population. University of Iowa; 2008.
disorder-like behaviors: emerging view from mouse models. Curr Opin
[37] Aidar M, Line SR. A simple and cost-effective protocol for DNA isolation from
Neurol 2011;24:114–8.
buccal epithelial cells. Braz Dent J 2007;18:148–52.
[12] Gasso P, Ortiz AE, Mas S, Morer A, Calvo A, Bargallo N, et al. Association
[38] Toresson H, Parmar M, Campbell K. Expression of Meis and Pbx genes and their
between genetic variants related to glutamatergic, dopaminergic and
protein products in the developing telencephalon: implications for regional
neurodevelopment pathways and white matter microstructure in child and
differentiation. Mech Dev 2000;94:183–7.
adolescent patients with obsessive-compulsive disorder. J Affect Disord
[39] Cecconi F, Proetzel G, Alvarez-Bolado G, Jay D, Gruss P. Expression of Meis2, a
2015;186:284–92.
Knotted-related murine homeobox gene, indicates a role in the differentiation
[13] Cappi C, Brentani H, Lima L, Sanders SJ, Zai G, Diniz BJ, et al. Whole-exome
of the forebrain and the somitic mesoderm. Dev Dyn 1997;210:184–90.
sequencing in obsessive-compulsive disorder identifies rare mutations in
[40] Nestadt G, Grados M, Samuels JF. Genetics of obsessive-compulsive disorder.
immunological and neurodevelopmental pathways. Transl Psychiatry 2016;6:
Psychiatr Clin North Am 2010;33:141–58.
e764.
[41] Friling S, Andersson E, Thompson LH, Jonsson ME, Hebsgaard JB, Nanou E, et al.
[14] Katayama K, Yamada K, Ornthanalai VG, Inoue T, Ota M, Murphy NP, et al.
Efficient production of mesencephalic dopamine neurons by Lmx1a expression
Slitrk1-deficient mice display elevated anxiety-like behavior and
in embryonic stem cells. Proc Natl Acad Sci USA 2009;106:7613–8.
noradrenergic abnormalities. Mol Psychiatry 2010;15:177–84.
[42] Andersson E, Tryggvason U, Deng Q, Friling S, Alekseenko Z, Robert B, et al.
[15] Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, et al. Cortico-striatal
Identification of intrinsic determinants of midbrain dopamine neurons. Cell
synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature
2006;124:393–405.
2007;448:894–900.
[43] Wu K, Hanna GL, Rosenberg DR, Arnold PD. The role of glutamate signaling in
[16] Mintzopoulos D, Gillis TE, Robertson HR, Dalia T, Feng G, Rauch SL, et al.
the pathogenesis and treatment of obsessive-compulsive disorder. Pharmacol
Striatal magnetic resonance spectroscopy abnormalities in young adult
Biochem Behav 2012;100:726–35.
SAPAP3 knockout mice. Biol Psychiatry: Cogn Neurosci Neuroimaging
[44] Abelson JF, Kwan KY, O’Roak BJ, Baek DY, Stillman AA, Morgan TM, et al.
2016;1:39–48.
Sequence variants in SLITRK1 are associated with Tourette’s syndrome.
[17] Bienvenu OJ, Wang Y, Shugart YY, Welch JM, Grados MA, Fyer AJ, et al. Sapap3
Science 2005;310:317–20.
and pathological grooming in humans: results from the OCD collaborative
[45] Miranda DM, Wigg K, Kabia EM, Feng Y, Sandor P, Barr CL. Association of
genetics study. Am J Med Genetics Part B, Neuropsychiatr Genet
SLITRK1 to Gilles de la Tourette Syndrome. Am J Med Genet Part B,
2009;150B:710–20.
Neuropsychiatr Genet 2009;150B:483–6.
[18] Zuchner S, Wendland JR, Ashley-Koch AE, Collins AL, Tran-Viet KN, Quinn K,
[46] Zimprich A, Hatala K, Riederer F, Stogmann E, Aschauer HN, Stamenkovic M.
et al. Multiple rare SAPAP3 missense variants in trichotillomania and OCD. Mol
Sequence analysis of the complete SLITRK1 gene in Austrian patients with
Psychiatry 2009;14:6–9.
Tourette’s disorder. Psychiatr Genet 2008;18:308–9.
[19] Crane J, Fagerness J, Osiecki L, Gunnell B, Stewart SE, Pauls DL, et al. Family-
[47] Speed WC, O’Roak BJ, Tarnok Z, Barta C, Pakstis AJ, State MW, et al. Haplotype
based genetic association study of DLGAP3 in Tourette Syndrome. Am J Med
evolution of SLITRK1, a candidate gene for Gilles de la Tourette syndrome. Am J
Genet Part B, Neuropsychiatr Genet 2011;156B:108–14.
Med Genet Part B, Neuropsychiatr 2008;147B:463–6.
[20] Boardman L, van der Merwe L, Lochner C, Kinnear CJ, Seedat S, Stein DJ, et al.
[48] O’Roak BJ, Morgan TM, Fishman DO, Saus E, Alonso P, Gratacos M, et al.
Investigating SAPAP3 variants in the etiology of obsessive-compulsive disorder
Additional support for the association of SLITRK1 var321 and Tourette
and trichotillomania in the South African white population. Compr Psychiatry
syndrome. Mol Psychiatry 2010;15:447–50.
2011;52:181–7.
[49] Inai A, Tochigi M, Kuwabara H, Nishimura F, Kato K, Eriguchi Y, et al. Analysis
[21] Aruga J, Mikoshiba K. Identification and characterization of Slitrk, a novel
of SLITRK1 in Japanese patients with Tourette syndrome using a next-
neuronal transmembrane protein family controlling neurite outgrowth. Mol
generation sequencer. Psychiatr Genet 2015;25:256–8.
Cell Neurosci 2003;24:117–29.
[50] Rosario-Campos MC, Miguel EC, Quatrano S, Chacon P, Ferrao Y, Findley D,
[22] Stillman AA, Krsnik Z, Sun J, Rasin MR, State MW, Sestan N, et al.
et al. The Dimensional Yale-Brown Obsessive-Compulsive Scale (DY-BOCS): an
Developmentally regulated and evolutionarily conserved expression of
instrument for assessing obsessive-compulsive symptom dimensions. Mol
SLITRK1 in brain circuits implicated in Tourette syndrome. J Comp Neurol
Psychiatry 2006;11:495–504.
2009;513:21–37.
[51] Kohlrausch FB, Giori IG, Melo-Felippe FB, Vieira-Fonseca T, Velarde LG, de
[23] Ozomaro U, Cai G, Kajiwara Y, Yoon S, Makarov V, Delorme R, et al.
Salles Andrade JB, et al. Association of GRIN2B gene polymorphism and
Characterization of SLITRK1 variation in obsessive-compulsive disorder. PLoS
Obsessive Compulsive disorder and symptom dimensions: a pilot study.
ONE 2013;8:e70376.
Psychiatry Res 2016;243:152–5.
[24] Zuchner S, Cuccaro ML, Tran-Viet KN, Cope H, Krishnan RR, Pericak-Vance MA,
[52] Gomes CKF, Vieira-Fonseca T, Melo-Felippe FB, de Salles Andrade JB,
et al. SLITRK1 mutations in trichotillomania. Mol Psychiatry 2006;11:887–9.
Fontenelle LF, Kohlrausch FB. Association analysis of SLC6A4 and HTR2A
[25] Paschou P, Fernandez TV, Sharp F, Heiman GA, Hoekstra PJ. Genetic
genes with obsessive-compulsive disorder: Influence of the STin2
susceptibility and neurotransmitters in Tourette syndrome. Int Rev
polymorphism. Compr Psychiatry 2018;82:1–6.
Neurobiol 2013;112:155–77.
[53] Pena SD, Di Pietro G, Fuchshuber-Moraes M, Genro JP, Hutz MH, Kehdy Fde S,
[26] Nestadt G, Wang Y, Grados MA, Riddle MA, Greenberg BD, Knowles JA, et al.
et al. The genomic ancestry of individuals from different geographical regions
Homeobox genes in obsessive-compulsive disorder. Am J Med Genet Part B,
of Brazil is more uniform than expected. PLoS ONE 2011;6:e17063.
Neuropsychiatr Genet 2012;159B:53–60.
[27] Berkes CA, Bergstrom DA, Penn BH, Seaver KJ, Knoepfler PS, Tapscott SJ. Pbx
marks genes for activation by MyoD indicating a role for a homeodomain
protein in establishing myogenic potential. Mol Cell 2004;14:465–77.

Please cite this article in press as: Melo-Felippe FB et al. Gene variations in PBX1, LMX1A and SLITRK1 are associated with obsessive-compulsive disorder
and its clinical features. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.10.042