Variants in Doublecortin- and Calmodulin Kinase Like 1, a Gene Up-Regulated by BDNF, Are Associated with Memory and General Cognitive Abilities
Are Associated with Memory and General Cognitive Abilities. PLoS ONE 4(10): e7534. doi:10.1371/journal.pone.0007534
Variants in Doublecortin- and Calmodulin Kinase Like 1, a Gene Up-Regulated by BDNF, Are Associated with Memory and General Cognitive Abilities
Clive R. Bramham 0
Ian J. Deary 0
Ivar Reinvang 0
Vidar M. Steen 0
Bernhard T. Baune, James Cook University, Australia
0 1 Bergen Mental Health Research Center, Department of Clinical Medicine, University of Bergen , Bergen , Norway , 2 Dr Einar Martens' Research Group for Biological Psychiatry, Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital , Helse Bergen HF, Bergen , Norway , 3 Center for the Study of Human Cognition, Department of Psychology, University of Oslo , Blindern, Oslo , Norway , 4 Centre for Cognitive Ageing and Cognitive Epidemiology, Department of Psychology, University of Edinburgh , Edinburgh , United Kingdom , 5 Centre for Cognitive Ageing and Cognitive Epidemiology, Medical Genetics Section, University of Edinburgh Centre for Molecular Medicine, Western General Hospital , Edinburgh , United Kingdom , 6 Department of Biomedicine and Bergen Mental Health Research Center, University of Bergen , Bergen , Norway , 7 Department of Biological and Medical Psychology, University of Bergen , Bergen , Norway
Background: Human memory and general cognitive abilities are complex functions of high heritability and wide variability in the population. The brain-derived neurotrophic factor (BDNF) plays an important role in mammalian memory formation. Methodology / Principal Finding: Based on the identification of genes markedly up-regulated during BDNF-induced synaptic consolidation in the hippocampus, we selected genetic variants that were tested in three independent samples, from Norway and Scotland, of adult individuals examined for cognitive abilities. In all samples, we show that markers in the doublecortin- and calmodulin kinase like 1 (DCLK1) gene, are significantly associated with general cognition (IQ scores) and verbal memory function, resisting multiple testing. DCLK1 is a complex gene with multiple transcripts which vary in expression and function. We show that the short variants are all up-regulated after BDNF treatment in the rat hippocampus, and that they are expressed in the adult human brain (mostly in cortices and hippocampus). We demonstrate that several of the associated variants are located in potential alternative promoter- and cis-regulatory elements of the gene and that they affect BDNF-mediated expression of short DCLK1 transcripts in a reporter system. Conclusion: These data present DCLK1 as a functionally pertinent gene involved in human memory and cognitive functions.
Funding: This work was supported by the Research Council of Norway, the Helse Vest Regionalt helseforetak, the Dr Einar Martens Fund and the University of
Bergen. Funding from the Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council, Economic and Social
Research Council and Medical Research Council is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The results presented in this article are part of a patent application: 09005576.51222.
. These authors contributed equally to this work.
" These authors also contributed equally to this work.
Empirical evidence for genetic factors underlying cognitive
variation is strong, but identification of specific genetic variants has
proved challenging. The study of differences in human mental
abilities, measured with psychometric tools, has defined a
hierarchical structure of human cognition. General cognitive
ability (g) stands at the pinnacle, accounting for about 50% of the
total variance in test performances. Correlated with g, there are
additional, separable group factors representing distinct cognitive
domains, with memory as a prominent example [1,2]. Behavioural
studies suggest that the impact of genetic factors on specific
cognitive domains, e.g. memory, derives from both inherited
contributions to general cognitive ability and from genetic variation
that more selectively influences memory. This heritability is
thought to be polygenic but, to date, few molecular genetic studies
have provided replicated genetic associations with cognitive
Variations in the gene for the brain-derived neurotrophic factor
(BDNF) have been examined for associations between cognitive
traits and psychiatric disorders. Several studies have identified
association of a single nucleotide polymorphism (SNP - rs6265)
which corresponds to an amino acid change (Val66Met) and
verbal memory, general cognitive ability, age related change in
reasoning skills and hippocampal functions, but others have failed to
replicate these findings [4,5,6,7,8,9]. The same Val66Met genetic
variant of BDNF has been largely studied as a risk factor for bipolar
affective disorder, schizophrenia and other psychiatric disorders (for
review see [10,11]). Since several of the cognitive traits associated
with BDNF variants have been reported potentially deficient in
major psychosis (e.g. verbal memory and general cognition in
bipolar disorder and/or schizophrenia [12,13]), these common
associations might reflect genetic associations to clinical
endophenotypes of these disorders. Indeed, neurocognitive traits have been
proposed as endophenotypes for psychosis that, because they are
less clinically heterogeneous and have high heritability, would be
more powerful in identifying genetic factors of susceptibility
Functional convergent genomics approaches, which use a set of
candidate genes identified in gene expression-based relevant
models [17,18,19], have been successful to identify new genes
for schizophrenia, antipsychotic induced weight gain and bipolar
disorders [20,21,22]. In order to broaden the study of BDNF in
cognitive traits, we chose to develop a functional convergent
genomics approach to characterise genes up-regulated by BDNF,
and their implication in cognition. BDNF plays a critical role as a
trigger of memory formation and transcription-dependent
enhancement of synaptic strength , via neuronal activity induced
gene expression . Previously, we used a rat model where
BDNF is infused in the dentate gyrus region of the hippocampal
formation, and identified a panel of 14 genes that were
upregulated following BDNF treatment that induces long-term
potentiation LTP, in a similar way as the immediate early gene
Arc . Among these genes, the five most strongly differentially
expressed genes were further examined by in situ hybridization and
also found to be up-regulated in another LTP inducing model
(afferent high frequency stimulation of the synaptic strength in the
dentate gyrus). In the present study, we selected haplotype-tagging
SNPs to screen those genes (ARL4L, NEURITIN, DCLK1, KLF10
and NPTX2) together with BDNF and ARC for influence on human
cognitive functioning in samples of healthy individuals who
volunteered for testing of their memory and general intellectual
Materials and Methods
1. Samples descriptions
All Norwegian participants read an information sheet and
signed a statement of informed consent approved by the regional
ethical committee for medical research (Project ID: S-03116).
Permission to obtain and store blood samples for genotyping
together with cognitive and MRI data in a biobank was given by
the Department of Health, and permission to establish a registry
with relevant information for a time period of 10 years was given
by the Department of Health. For the Scottish samples, ethics
permission for the study protocol was obtained from the
MultiCentre Research Ethics Committee for Scotland (MREC/01/0/
56) and from Lothian Research Ethics Committee (LREC/2003/
2/29). The research was carried out in compliance to the Helsinki
Declaration. All subjects gave written, informed consent.
The Norwegian Cognitive NeuroGenetics (NCNG)
sample. consists of 271 participants aged 4675 years (mean
age of 62.6 years, s.d. 7.9) that were recruited through
advertisements in local newspapers in the Oslo and Bergen urban
areas (see previous report in Espeseth et al., 2006 ). Candidates
were first interviewed by phone according to a check list about
health and previous illness or injuries. Exclusion criteria were
previously diagnosed neurological or psychiatric illness, any other
chronic illness that might influence test performance, or sensory or
motor impairments. Participants with a history of alcohol or
substance abuse, or current addictive disorders, were also
excluded. Participants had to be native speakers of Norwegian
and have completed obligatory basic education (7 years for this age
group) without diagnosed reading or learning disorders. Persons
on adequate medication for hypertension, diabetes or
hypercholesterolemia were not excluded. Participants were not allowed to
consume nicotine or caffeine during the test period or in the lab
premises, but were not required to abstain from these substances
prior to attendance. There were 195 females (72%) and the
average total years of education was 13.9 (s.d. 3.0). The
Vocabulary and Matrix Reasoning subscales of the Wechsler
Abbreviated Scale of Intelligence (WASI, ) were used to
estimate IQ, and the California Verbal Learning Test II
(CVLTII, ) to test verbal episodic memory.
The Lothian Birth Cohort 1921 (LBC1921). are surviving
participants of the Scottish Mental Survey of 1932 (SMS1932).
They were recruited in Edinburgh and the surrounding areas
either through the Community Health Index a list of individuals
registered with a General Practitioner or as volunteers replying
to media calls . The first wave of follow-up ran from 1999
2001, and 550 individuals (234 men and 316 women) were tested
individually at the Wellcome Trust Clinical Research Facility
(WTCRF) at the Western General Hospital, Edinburgh. The
mean age of the LBC1921 participants was 10.9 years (s.d. 0.3) at
the time of the SMS1932 and 79.1 years (s.d. 0.6) at wave 1
In the SMS1932, all participants completed the Moray House
Test (MHT) Number 12 which was re-administered at age 79. The
Moray House Test is a well-validated IQ-type test with a
predominance of verbal reasoning items, though there are also
some other types of item. The raw MHT scores were corrected for
age in days at the time of testing and then converted into IQ
scores. At wave 1, a further battery of cognitive tests was also
completed, including the Ravens Progressive Matrices to assess
non-verbal reasoning , Verbal Fluency to assess executive
function , and Logical Memory to assess verbal declarative
memory . The Mini-Mental State Examination as a brief
screen for dementia  and the Hospital Anxiety and Depression
Scale  were also administered. Further description can be
found in Deary et al. (2004) .
In the present study, only the association with IQ score, logical
memory (immediate, delayed and total), verbal fluency and
Ravens matrices were analyzed as these variables were the most
comparable to the variables analyzed in the NCNG sample.
The Lothian Birth Cohort 1936 (LBC1936). comprises
1091 participants who were born in 1936 and tested on a general
measure of verbal reasoning (Moray House Test No. 12; MHT) at
age 11 in the Scottish Mental Survey of 1947 (Scottish Council for
Research in Education [SCRE], ). They were recruited in
Edinburgh and the surrounding areas either through the
Community Health Index a list of individuals registered with
a General Practitioner or as volunteers replying to media calls
. All participants lived independently in the community and
were able to travel to the clinical research facility for testing. They
undertook medical and cognitive testing at age 70 (mean age of
69.6 years, s.d. 0.8) as reported in detail previously , including
the same MHT test as they had taken at age 11. The raw MHT
scores were corrected for age in days at the time of testing and
then converted into IQ scores. The Mini-Mental State
Examination (MMSE) was used to screen for possible dementia . The
battery of cognitive tests sampled a variety of specific cognitive
abilities, with an emphasis on memory and processing speed.
Memory domains were assessed by the following subtests of the
Wechsler Memory Scale-IIIUK (WMS-IIIUK; ): Logical
Memory I (immediate verbal declarative memory), Logical
Memory II (delayed verbal declarative memory), Verbal paired
associates (immediate and delayed verbal memory and learning)
and Spatial span (non-verbal, spatial memory). The information
processing speed battery comprised two psychometric tests from
the WAIS-IIIUK (Digit symbol coding and Symbol search) and
two elementary cognitive tasks, Reaction Time (simple and choice
conditions) and Inspection Time. Other cognitive tests which
tapped diverse abilities included: Backward digit span (working
memory) from the Wechsler Memory Scale-IIIUK; and
Letternumber sequencing (working memory), Matrix reasoning
(nonverbal reasoning) and Block design (constructional ability) from the
WAIS-IIIUK . The Verbal fluency test provided a measure of
executive function .The g factor was calculated via principal
components analysis of the following Wechsler tests: Backward
digit span, Letter-number sequencing, Matrix reasoning, Block
design, Digit Symbol and Symbol Search subtests. A full
description of these tests can be found in the Lothian Birth
Cohort 1936 protocol article . The final sample for analysis
(i.e., those who also had genotype data) was N = 1077, and
included 535 females and 542 males.
2. Selection, genotyping and analysis of genetic markers
Markers selection, genotyping and analysis in the NCNG
sample. Haplotype tagging markers (single nucleotide
polymorphisms SNP) were selected using the Phase I (16c.1,
June05, based on NCBI B34 assembly, dbSNP b124) version of
hapmap_phaseI/)., and according to the protocol described
in Christoforou et al. . Briefly, Hapmap data for the CEU trios
were downloaded and analysed in Haploview v2.5, using the
following criteria: pair-wise comparisons of markers more than
500 kb apart were ignored, minor allele frequency $0.10, a
HardyWeinberg (HW) P-value $0.001, genotyping success rate
$0.75. The haplotype blocks were defined using the solid spine of
LD approach, using Haploviews internal tagging program.
Haplotype tagging SNPs were selected on a block-by-block basis
to represent haplotypes of frequencies higher than or equal to
0.10. The markers were genotyped on a Sequenom Massarray
platformTM (http://www.sequenom.com/, Sequenom Inc., San
Diego, CA, USA) at CIGENE, Center for Integrative Genetics
(Universitetet for milj- og biovitenskap, As, Norway, http://
www.umb.no/), which is the national FUGE platform for
genotyping (www.fuge.no), supported by the Research Council
Markers selection, genotyping and analysis in the
LBC1921 and LBC1936 samples. In the replication sample
we decided to concentrate only on the DCLK1 gene. Seven
markers that showed association in the NCNG sample (p-value
,0.01: rs4591003, rs1926467, rs943220, rs10507435, rs7323560,
rs7334245, rs9315383) were selected for replication in the LBC
samples. In addition, we chose to include another eight markers
for genotyping in these Scottish samples, since they show
association to psychiatric disorders in preliminary studies
(unpublished information): rs12430800, rs2296645, rs10492555,
rs872060, rs9545332, rs7989245, rs7989807 and rs9315390. The
marker rs4391923 was also included for its potential functional
Assays were developed for the ABI PRISMH 7900HT Sequence
Detection System using TaqMan technology (Applied Biosystems,
CA, USA), with genotyping at the Welcome Trust Clinical
Research Facility, Genetics Core (http://www.wtcrf.ed.ac.uk/
genetics/default%20genetics.htm, University of Edinburgh, UK).
Our discovery sample (the NCNG, N = 271) provides 93%,
73% and 30% power to detect an additive QTL effect explaining
5%, 3% and 1% of the trait variance, respectively (uncorrected
P = 0.05; two-tailed), calculated with the Genetic Power
Calculator, http://pngu.mgh.harvard.edu/,purcell/gpc/ .
Genotypes were quality controlled with the following criteria:
individual samples with genotype call rate ,90% and markers
with call rate ,96% or Hardy Weinberg P-value ,0.001 were
excluded from analysis (see supplementary material SOM, for
summary of locations, markers tested and analysed).
Genotyping data were analyzed using the Helix Tree software
for linear regression, genotypic association and haplotype trend
regression of 2- and 3- markers sliding windows, similarly to the
analysis described in .
CNV considerations. Copy Number Variants (CNVs) were
not considered at the time of the markers selection (few data were
available at this time, September 2005). Retrospectively, the
Toronto database (http://projects.tcag.ca/variation/) was
screened for CNVs in the genes investigated. Markers in the
NPTX2, BDNF and DCLK1 genes are not affected by any known
CNVs. ARC is located within the rare variation_30296 seen in 1 in
1086 chromosomes by Jakobsson et al. , KLF10 is located
within the CNV region of variation_7659 described by de Smith
et al.  observed in 1 in 100 chromosomes, and NRN1 is located
on the same clone as the variation_0072 seen in 1 in 110
chromosomes by Iarfate et al.  which has not been further
refined. Considering their relatively low frequency, and the Hardy
Weinberg equilibrium observed for genetic variants of this region
in our genotyping, we consider that none of these known CNVs
are likely to affect the associations reported in this study.
3. RT-PCR of human DCLK1 transcripts
cDNA samples were synthesized from human brain
regionspecific total RNA samples (Clontech Laboratories, CA, USA)
(200 ng RNA input) using the SuperScript III First-Strand
Synthesis System (Life Technologies, St Paul, MN, USA),
according to manufacturers protocol (20 ml reaction volume).
0,5 ml cDNA was used as template in 10 ml real-time PCR assays
(50uC/2 min; 95uC/10 min; 406(95uC/15 sec; 60uC/1 min)).
Real-time PCR analyses were performed with an ABI Prism
7900HT sequence detector system (Applied Biosystems, CA, USA)
using SYBR-green (Eurogentec, Belgium) as detector, AmpliTaq
Gold DNA polymerase (Applied Biosystems, CA, USA), 2x
SYBRgreen mix, and the following PCR primer sets:
CARP: 59-GGATGACTTGGATTCAGTAGGAGACT, 59-C
long DCLK1: 59-GGAGTGGTGAAACGCCTGTAC, 59- GG
short DCLK1: 59-ACACTAAGACTGTGTCCATGTTAGAA
The specificity of all RT-PCR assays was verified by
DNAsequencing of the amplified PCR products.
4. Semi-quantitative RT-PCR of BDNF-induced expression of Dclk1 in vivo
The expression of Dclk1 transcripts in the rat hippocampus were
measured by semi-quantitative RT-PCR on cDNA samples
obtained from our previous study on BDNF-mediated induction
of Long-Term Potentiation in the rat hippocampus . PCR
were performed as described above with the following primer sets:
Dclk1 long: 59-GGTGTGGTGAAGCGTCTGTAC, 59-CAAAA
5-GGCCATCGTTCTCATCCATT. The primer sets for Carp and Neuritin has been described
5. Sequencing of intron 5 of human DCLK1
Genomic DNAs of 23 individuals selected for their genotypes at
the markers rs943220 and rs10507435 from the NCNG sample
were amplified to sequence the DCLK1 intron 5. Nineteen pairs of
primers for PCR amplification (sequences can be made available
upon request) of the intron 5 were designed, using the Primer3 tool
 (http://fokker.wi.mit.edu/primer3/input.htm). The PCR
amplicons were designed to have a sufficient (100150 bp) overlap
to obtain contiguous sequences. PCR fragments were amplified
using AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA)
according to the manufacturers instructions with 25 cycles: 94uC
for 10 s, 55uC for 30 s, 72uC for 30 s (initial denaturation 94uC for
10 min). The PCR products were sequenced with BigDye v3.1
(Applied Biosystems) according to the manufacturers instructions
and the sequences were aligned with phredPhrap program and
read in Consed39  (http://bozeman.mbt.washington.edu/
All new polymorphism identified have been deposited in dbSNP
(http://www.ncbi.nlm.nih.gov/sites/entrez?db = snp, build 130)
under the following IDs: rs61949282, rs61949292, rs66492553,
rs67014603, rs72652874, rs72652875, rs72652876, rs72652877,
rs72652878, rs72652879, rs72652880, rs72652881, rs72652882,
6. In silico prediction of promoter and cis-regulatory regions in intron 5 of human DCLK1
Candidate gene-regulatory regions were identified on the basis
of clustering of transcription factor (TF) DNA-binding sites, as
identified by the TF-search engine (www.cbrc.jp/research/db/
TFSEARCH) and the Cis-element Cluster Finder
(Cister)program (http://zlab.bu.edu/%7Emfrith/cister.shtml) using
matrixes from TRANSFAC (http://www.gene-regulation.com). TFs
predicted to bind proximal to SNPs verified by sequencing in the
human DCLK1 intron 5 were selected for the Cister-analyses.
The Neuronal Network Promoter Prediction program was used
to rank candidate TATA-boxes in intron 5 (http://www.fruitfly.
org/seq_tools/promoter.html). Probability scores of clustering
between promoter elements and TATA-boxes were investigated
by the Cister-program and the TF-search engine.
7. Construction, transfections and assays of the Luciferase reporter vectors
Construction of luciferase reporter vectors. The predicted
promoter fragments were amplified from genomic DNA (from 2
individuals homozygous for the C or T allele of rs4391923/marker
m5.3) by PCR with AmpliTaq Gold DNA polymerase (Applied
Biosystems), using the forward primer
59-CTAGACTCGAGCCTCCTGAAGATAGCTTTGC and the reverse primer 59-
CAGACAAGCTTCAGTCTCAGGAATACCTTGC (XhoI and HindIII
sites introduced, underlined). Purified PCR fragments were cloned
into the XhoI/HindIII (all restriction enzymes from New England
Biolabs, Ipswich, MA, USA) sites of pGL4.11[luc2P] (Promega,
Madison, WI, USA), generating the pGL4.11-Cprom and
pGL4.11Tprom luciferase reporters.
Three haplotypes of the intron 5 cis2 element (hap1a, hap1b and
hap2, see section 6 and supplementary online material - SOM) were
PCR amplified from genomic DNA (from 3 individuals
homozygous for the three haplotypes identified by sequencing), using the
59-GTCACGGATCCTTGGAAACTCAAGAAGATAGGC and the reverse primer 59-
GATCAGTCGACCCACAGGAAACAAAGCAACC (BamHI and Sal1 sites introduced,
underlined). Amplified DNA was cloned into the BamH1/Sal1 sites
of pGL4.11-Cprom, pGL4.11-Tprom and pGL4.11[luc2P],
generating haplotype specific cis2-C/Tprom luciferase reporters and
promoter-less control vectors. In the final plasmids, the Cis2
element locates 2216 bp upstream of the promoter-region, the two
being separated by a synthetic poly(A) sequence for the reduction of
background signals. All plasmid constructions were verified by DNA
sequencing. See supplementary material SOM, for a list of
commercial and constructed plasmids used in the study.
Cell cultures, differentiation and plasmid transfection.
SH-SY5Y cells (ATCC, LGC Promochem, UK) were grown in
RPMI supplemented with 10% (v/v) horse fetal serum, 5% (v/v)
fetal bovine serum and 2 mM L-glutamine (Cambrex Biosciences,
Cambrex Corporation, Charles City, IA, USA). Cultures were
incubated at 37uC in a 5 vol-% CO2/air incubator.
Plasmid transfections were performed over-night using
MetafecteneTM Pro (Biontex, Munchen, Germany) under conditions
optimized according to the manufacturers guidelines. Neuronal
differentiation was initiated the day after transfection by replacing
the transfection media with fresh media containing BDNF (50
ng/ml)/RA (10 mM all-trans retinoic acid) (Sigma-Aldrich, St
Louis, MO, USA), as described by Holback et al. . Control
cells (non-differentiated) were treated similarly but exposed to
media with vehicle only (0.01% DMSO). Cell viability was
monitored using WST-1 (F. Hoffmann-La Roche Ltd, Germany).
All transfections were performed in quadruplicates on 96
multiwell plates. Each experiment was controlled for transfection
efficiency by transfecting a green fluorescent protein (GFP)
expressing vector (pSIREN-RetroQ-ZsGreen) into a separate set
of cells. GFP expression was analyzed two days post transfection
on a FACSCaliburTM Flow Cytometer (BD systems, BD Europe,
Luciferase Reporter Assays. The transcriptional activity of
each reporter plasmid was measured two days post transfection
using the Dual-Luciferase Reporter Assay system (Promega Biotech
AB, Madison, WI, USA), according to manufacturers protocol.
Luciferase signal intensities were recorded on a Chameleon plate
reader (Reactionlab Sverige AB, Sweden). All plasmid transfections
and luciferase measurements were performed in 96 multiwell plates,
with quadruplicates of each sample. Each experiment was repeated
5 times for the pGL4-C/Tprom plasmids, and 7 times for plasmids
harboring cis2 elements. All experiments were performed blind to
the vectors haplotypes. Luciferase signal intensities were normalized
for transfection efficiency on the basis of renilla luciferase signal
intensities from the co-transfected pGL4-73[hRluc/SV40] plasmid.
The transcriptional activity of a given reporter plasmid is listed
relative to its respective promoter-less control vector. All cis2
promoter-less control vectors showed similar background activity
(data not shown).
Association to verbal memory and general cognition in
the NCNG sample
In the original study design, seven genes were selected:
BDNF,ARC, ARL4L, NEURITIN, DCLK1, KLF10 and NPTX2.
During quality check of the genotyping results, we noticed that the
official annotation of the rat Arl4l had been updated to Arf4l and
that the human homolog we selected (ARL4L) was erroneous and
should have been ARL4D. We therefore decided to exclude the
ARL4L markers from further analysis as this gene is apparently not
up-regulated during LTP in rat brain.
We excluded DNA samples with a genotyping success below 0.9,
and markers with call rate ,96% or Hardy Weinberg P-value
,0.001 from analysis. For the 6 remaining genes studied, a total of
48 markers were genotyped in the Norwegian Cognitive
NeuroGenetics NCNG sample. This sample consists of 271
individuals (mean age: 62.6 years, range 5075), which had been
recruited via media advertisements and who were subjected to
cognitive testing, i.e. verbal memory and general cognition (IQ
score). In this sample, we found strong associations between several
markers in the DCLK1 gene (doublecortin- and calmodulin kinase
like 1, a.k.a. DCAMKL1) and aspects of verbal memory function and
IQ score, which resisted Bonferroni correction (see Table 1, Table 2
and SOM for all results of markers tested). The most significant
associations were observed for intron 5 markers m5.1 (rs10507435)
and m5.2 (rs943220), where the less frequent genotypes were
significantly associated with reduced verbal memory performance
(see Table 1). Marker m5.1 was also associated with IQ score, like
several other DCLK1 SNPs (see Table 2).
For the other genes tested we found some significant effects, but
no strong association (p,0.01) or associations across several
cognitive traits (see SOM). However, in a 2-loci analysis for
genegene interaction effects, there were significant interactions between
DCLK1 intron 5 markers (m5.1 and m5.2) and markers tagging
BDNF and ARC on the association with verbal memory
(pvalue = 0.0320.003, after Bonferroni correction, see SOM). At
the genetic level, this finding is consistent with the co-upregulation
of Dclk1 transcription by Arc and Bdnf, as observed in a rat
hippocampal model of synapse consolidation.
Replication in the Lothian Birth Cohorts
Sixteen DCLK1 SNPs were selected for replication testing in two
independent samples (see SOM). The Scottish Lothian Birth Cohort
(LBC) studies of individuals born in 1921 and in 1936 (LBC1921
and LBC1936) are two independent cohorts of individuals who
underwent an IQ test at age 11 (IQ11), and who at the age of 79
years for the LBC1921, and 70 years for the LBC1936, participated
in follow-up testing with examination of IQ (IQ79 and IQ70,
respectively) and other cognitive functions [29,36].
In the LBC1921, we found an association between verbal memory
and m11, an exon 11 synonymous marker (see Table 1). For IQ79,
especially when regressed for IQ11, we replicated and further
strengthened the association to markers in the intron 15 and intron
19 (which are located in the same haplotype block), and to intron 19
3-markers haplotype (resisting permutation testing, see Table 2).
In the LBC1936, we observed associations between an intron 5
marker (m5.3) and several memory-specific and general cognition
traits at age 70 (see Table 1, and 2). Also in this sample, the
association with intron 5 was even stronger (resisting permutation
testing), for both memory and general cognition traits, at the
haplotype level (3-markers haplotype covering the intron 5, see
Table 2). In this sample, the intron 15 intron 19 markers (except
for m19.1, rs12430800) were associated with childhood cognitive
variables (IQ11). Further association testing with other cognitive
abilities (see SOM) showed an effect of markers in both the intron
5 and intron 1519, but also an effect of the age at testing. This
age-dependent association is especially pronounced for the general
cognitive ability factor, g factor, which was associated with both
intron 5 (m5.3, rs4391923) and intron 15 - intron 19, but only the
intron 5 association remained significant when IQ11 was adjusted
for in the regression model.
Expression of rat Dclk1 variants after BDNF treatment in
the dentate gyrus
The rodent Dclk1 gene is expressed as several transcripts, e.g.,
long (exons 120 except 6 and 8), short (exons 620 except 8) and
Carp (exons 68; see figure 1A), which vary in expression (spatial
and developmental) and in function (see discussion). In rat, we
previously demonstrated that the Carp variant is induced by BDNF
Analyses were performed with Helix Tree software. Only p-values ,0.01 are reported (see SOM for regression analysis, marker details, results and description of
cognitive traits tested). In the NCNG sample, verbal learning and delayed recall were assessed with the California Verbal Learning Test (CVLT-II ). In total, six
BDNFLTP related genes were tested and 48 markers were analyzed. In the LBC samples, verbal memory was tested with the Wechsler memory scale test (LM- delayed and
learning [32,37]) and the samples were genotyped for 16 DCLK1 markers only, with no screening of the other genes (n.t: not tested). P-values below Bonferroni corrected
p-value threshold ( = 0.001 for NCNG), are highlighted in bold.
Both single marker linear regression (LR) and haplotype trend regression of 3-markers sliding windows analyses (HTR3) are presented. Only p-values below 0.05 are
displayed. All analyses were performed using sex and age as covariates. The NCNG was assessed with the Wechsler Abbreviated Scale of Intelligence . LBC1921 and
LBC1936 were assessed for IQ with the Moray House Test (see SOM). For LBC1921, no association below p = 0.05 was observed for IQ11. In the LBC samples, all
regression analyses were performed both with and without IQ11 as a covariate. P-values highlighted in bold are resisting a 10,000 permutations testing.
exposure, but at this time we did not further look at the other
transcripts. Further examination of the samples from that study,
show that all short DCLK1 variants are induced by BDNF but not
the long variants (see figure 1B).
Expression of DCLK1 variants in human brain tissues
In the rodent, the short variants show higher expression in adult
brains whereas long variants dominate during embryonic stages in
rat . In humans, the expression of DCLK1 variants has not
been fully documented, but expression of long DCLK1 has been
shown in both embryonic and adult brain tissues . Using
transcript-specific PCR assays, we found expression of both long
and short transcripts of DCLK1 in the fetal brain (2640 weeks) as
well as in specific regions of the adult human brain (see Figure 1C).
The expression of short variants was especially high in regions
involved in memory performance (hippocampus, occipital pole,
frontal lobe and temporal lobe). The expression of CARP was low
in all human brain tissues, consistent with previous observations in
rodents of low basal CARP expression and robust induction by
specific treatments [25,49].
In silico characterisation of regulatory elements and
identification of additional genetic variants in the intron
5 of DCLK1
Considering that one of the main signals of association was
observed for markers in the intron 5 of DCLK1 and that short
transcripts of DCLK1 all start from the exon 6, we hypothesised that
the intron 5 might harbour alternative promoter and regulatory
regions. We searched for transcriptional cis-regulatory elements and
promoters in the human intron 5 with the aim of analyzing the effect
of these in luciferase reporter assays. The search predicted a
TATAbox promoter and three specific cis-regulatory elements (cis1-3, see
Figure 2A). To identify additional sequence variants that could
affect the regulatory elements, we sequenced the entire intron 5
from genomic DNA of 23 individuals from the NCNG sample. In
the putative alternative promoter region in intron 5, the marker
m5.3 (C/T variant) was located 2 bp from the potential
transcription start. For the adjacent regulatory elements we
observed several variants in the cis2 that could affect the regulatory
properties, defining three haplotypes: the related hap1a and hap1b,
and the hap2 (see SOM for details).
Assessment of the functional effect of SNPs in potential
regulatory regions on the expression of DCLK1 short
Luciferase reporter assays were constructed by cloning the
promoter C (C-prom) or T allele (T-prom) of m5.3, from genomic
DNAs of NCNG individuals. Additional reporter assays were
constructed to mimic in vitro the potential effect of the 3 cis2
haplotypes on the promoter. The assays were analysed for
expression of the reporter luciferase protein under control
conditions and under BDNF/RA differentiation (retinoic acid,
protocol for BDNF induced differentiation as described by
Holback et al. ).
In luciferase reporter assays, both the C-prom and T-prom
constructs displayed basic promoter reporter-activity in the
undifferentiated SH-SY5Y control cells. However, during
BDNFinduced neuronal differentiation, only the T-prom construct
demonstrated increased expression of the luciferase reporter (see
Figure 2C). The addition of a single hap1a or hap1b variant of the
cis2 element into the non-inducible C-prom reporter-vector
rendered the C-prom inducible by BDNF (see Figure 2D), whereas
the hap2 variant of cis2 did not show this effect on C-prom. None of
the cis2 variants had any significant additional effect on the activity
and inducibility of the T-prom sequence. These reporter assays thus
demonstrated that the predicted promoter in intron 5 displays an
allele-specific promoter activity and inducibility which can be
further influenced by regulatory elements, such as cis2, in an
Our data show that genetic variants in DCLK1 significantly
influence the performance on tests of memory and intellectual
function in three independent samples. Although the size of our
discovery sample is small, we detected association in this sample.
We observed further associations between genetic variants in
DCLK1 and cognitive abilities in the replication samples but with
differences regarding the panel of markers that were associated.
These differences could be due to type I errors, but also to type II
errors by lack of power for either the discovery or the replication
samples to detect association. However it is probable that these
differences are due to allelic heterogeneity that reflects the
variation between the samples. Even though all subjects have
been phenotyped for similar traits (e.g. verbal memory and general
cognition), they are different in the geographical origin, age, or
mode of recruitment, as well as in the specific tests used to assess
Comparison of the associations in the three samples highlights
three main regions of interest in the DCLK1 gene that could have an
influence on the heritability of cognitive traits. The first region
extends from the 59UTR to the intron 3 of the gene, where the
markers m3.1 and m3.2 in single marker allelic analysis or certain
3markers haplotypes in haplotype trend regression are associated to
verbal memory or to general cognition in the NCNG sample (see
Tables 1 and 2), to IQ at age 79 in the LBC1921 (see SOM) and to
several cognitive traits at the genotypic level in the LBC1936. The
region spanned by these markers is large and covers the promoter
region and the first exons and introns. Notably, it includes a large
proportion of the wide intron 3, which contains a large number of
highly conserved non-coding elements as seen in the UCSC browser
The second region of association is localised in intron 5 of the
gene. This area is associated to verbal memory and IQ at the
single marker level in the NCNG sample (see Table 1 and 2), to
verbal memory and several other cognitive traits at the single
marker level (m5.3, see Table 1 and SOM) and to IQ at age 70 at
the 3-markers haplotype level in the LBC1936 (see Table 2 and
SOM). Several short transcripts for the DCLK1 start from the exon
6 (as further detailed below). We characterised a potential
promoter in intron 5, which may be used for transcription of the
short variants, as well as three regions with potential regulatory
effect on this transcription. Our reporter assay studies of this
putative promoter and one of the regulatory regions (chosen
because it encompasses associated genetic variants) displayed that
the efficiency of these regulatory elements was influenced by the
alleles of the associated markers and by the treatment with BDNF.
In this context, it is interesting to note that the BDNF- inducible
C-prom/cis2 haplotypes (hap 1a/b), equivalent to 2-marker
m5.1m5.3_GA haplotype of intron 5, shows the strongest association to
IQ score and verbal memory (p-value = 0.0027, see SOM and
0.0019, data not shown) in the LBC1936, and that the
noninducible constructs corresponds to the m5.1-m5.3 GG haplotype
that is the rarest, found only in 2.5% of the chromosomes.
The third region of association points to a haplotype block of
markers covering intron 15 to intron 19. This region is associated
to IQ score in the NCNG sample at the single marker and
3markers haplotype level (see Table 2), to IQ79 in the LBC1921
(single marker and three markers haplotype, see Table 2) and to
IQ11 (see Table 2) and to other cognitive variables (see SOM) at
the single marker level in the LBC1936. This association calls for
additional studies, but it is interesting to note that the associated
markers are located near exon 19 that is alternatively spliced in
several transcripts, affecting the DCLK1 kinase activity ,
within a region of high inter-species conservation (as seen in the
In addition, in both the LBC1921 and LBC1936, single marker
association to the marker rs2296645 were observed for verbal
memory and cognitive abilities. This genetic variant is located in
the exon 11 but does not affect the amino acid sequence.
The thorough examination of the Lothian Birth Cohorts,
especially the LBC1936, shows that the signal of association
depends on the cognitive variable studied and on the age at
examination, which points to similarities and differences in genetic
contributions to cognitive abilities across the lifespan. Several
cognitive associations in old age were affected by the integration of
childhood IQ as a covariate, which can suggest that these are
genetic associations to lifelong cognitive change.
In addition, we show that there might be interaction between
variants in DCLK1 and variants in BDNF and ARC for an effect on
verbal memory and general cognition. This finding is coherent at
the genetic level with the in vivo observation that Dclk1 is
coupregulated with Arc by BDNF in the rat hippocampus . In
addition to the known implication of BDNF in long term
potentiation, the interactions with Arc are also noteworthy as this
immediate early gene is required for multiple forms of synaptic
plasticity and long-term memory formation, including synaptic
potentiation induced by BDNF infusion [51,52,53].
Considering the conflicting results reported regarding the
genetic association of BDNF with human cognition and with
psychiatric disorders [5,6,7,8,9,10], we suggest that re-analysis of
the possible BDNF-DCLK1 interaction might improve the
interpretation of these studies. In this perspective, it is also
interesting to notice that in two recent studies of functional
convergent genomics for bipolar affective disorder  and a
genome wide scan for personality traits , both BDNF and
DCLK1 (a.k.a. DCAMKL1 in these reports) were ranked as strong
The rodent Dclk1 gene is expressed as several transcripts, e.g.,
long (exons 120 except 6 and 8), short (exons 620 except 8) and
Carp (exons 68; see figure 1A), which vary in expression (spatial
and developmental) and in function. The long transcripts encode
an N-terminal domain similar to the lissencephalia-related
doublecortin gene, sharing its microtubule-binding and -stabilizing
properties [55,56]. N-terminal Dclk1 transgenic and Dcl
knockdown mice develop brain abnormalities that affect the
organization of hippocampal neurons, cortical neurogenesis, neuronal
migration and axonal wiring [56,57]. The C-terminal domain,
present in both long- and short-Dclk1, contains a domain similar to
the Ca2+/calmodulin-dependent protein kinase which may
phosphorylate the myelin basic protein [47,50,58]. The function
of Carp remains largely unknown but it is expressed in response to
diverse stimuli, and may be involved in both neuronal
activityinduced strengthening of synaptic transmission as well as apoptosis
. We now show that the short variants are, similarly to Carp,
up-regulated by BDNF treatment in the rat hippocampus, and
that in human these variants are expressed in brain structures
relevant to cognition.
In conclusion, in this study we report DCLK1, a gene
upregulated BDNF, as being a novel candidate gene associated to
cognitive traits, in three different samples, but with allelic
heterogeneity between the samples. Further work will be needed
to better understand the function(s) of DCLK1 in cognitive
processes, and its role in synaptic plasticity, especially since other
related genes (CAMK2G and CAMTA1) have also been associated
to human memory performance [60,61]. Drawing on the example
of this study, it will be interesting to mine ongoing genome wide
studies of large samples characterised for cognitive abilities with
further gene sets identified from micro-array gene expression
experiments on models relevant to synaptic plasticity.
Supplementary information SOM
with additional material and methods and complementary
tables and figures, is linked to the online version of the paper (see
Supporting Information Material S1).
Supporting Information Material S1 Methods and Tables.
Revised version of the supplementary information, as accepted
after resubmission 1.
Found at: doi:10.1371/journal.pone.0007534.s001 (1.52 MB
We thank the NCNG, LBC1921 and LBC1936 members, CIGENE
(FUGE/Research Council of Norway-funded national SNP platform) and
the Wellcome Trust Clinical Research Facility Genetics Core for
genotyping the samples.
Conceived and designed the experiments: SLH BH TE RL KW CB IR
VS. Performed the experiments: SLH BH HB. Analyzed the data: SLH
BH. Contributed reagents/materials/analysis tools: SLH BH TE ML AJG
SH JMS AL DP ID IR VS. Wrote the paper: SLH BH TE CB ID IR VS.
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