Neuropharmacology 80 (2014) 83e94

Contents lists available at ScienceDirect

Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Invited review

The genetics of cognitive epigenetics

Tjitske Kleefstra a, Annette Schenck a, Jamie M. Kramer a, Hans van Bokhoven a, b, *
a
Radboud University Medical Center, Department of Human Genetics, Nijmegen Center for Molecular Life Sciences (NCMLS), Nijmegen, The Netherlands
b
Radboud University Medical Center, Department of Cognitive Neurosciences, Donders Institute for Brain, Cognition and Behaviour, Nijmegen,
The Netherlands

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

Article history: Cognitive disorders (CDs) are a heterogeneous group of disorders for which the genetic foundations are
Received 16 November 2013 rapidly being uncovered. The large number of CD-associated gene mutations presents an opportunity to
Received in revised form identify common mechanisms of disease as well as molecular processes that are of key importance to
29 December 2013
human cognition. Given the disproportionately high number of epigenetic genes associated with CD,
Accepted 30 December 2013
Available online 13 January 2014
epigenetic regulation of gene transcription is emerging as a process of major importance in cognition.
The cognate protein products of these genes often co-operate in shared protein complexes or pathways,
which is reected in similarities between the neurodevelopmental phenotypes corresponding to these
Keywords:
Intellectual disability
mutant genes. Here we provide an overview of the genes associated with CDs, and highlight some of the
Cognitive disorder epigenetic regulatory complexes involving multiple CD genes. Such common gene networks may provide
Genetic a handle for designing therapeutic interventions applicable to a number of cognitive disorders with
Epigenetic variable genetic etiology.
Mutation This article is part of the Special Issue entitled Neuroepigenetic Disorders.
Chromatin Crown Copyright 2014 Published by Elsevier Ltd. All rights reserved.

1. Introduction promising strategy for identifying networks of interacting proteins

and novel regulatory pathways that play a role in CDs. Moreover, it
Cognitive disorders (CDs) are a large and heterogeneous is a prerequisite for understanding the underlying pathogenic
collection of disorders, which collectively impose a large burden to mechanisms and for the development of therapies.
health care systems. The 2013 update to the authoritative manual of Severe ID (IQ < 50) affects 0.3e0.4% of the general population
the American Psychiatric Association (APA), the Diagnostic and (Leonard and Wen, 2002). Approximately half of these cases are
Statistical Manual of Mental Disorders Fifth Edition (DSM-5; APA, caused by chromosomal aberrations and single gene mutations,
2013) has 18 diagnostic groups, which each contain a variety of whereas borderline ID (IQ 70e85) can be expected to be the
subgroups. This grouping is a subject of much criticism since clin- consequence of multiple genetic variants (Coe et al., 2012). To date,
ical and genetic research increasingly suggests that mental illnesses mutations in more than 500 genes have been implicated in ID (van
are cannot be so clearly dened, and rather are spread along a Bokhoven, 2011; Kochinke et al., in preparation). Due to the advent
spectrum with a high degree of overlapping characteristics (Adam, of next generation sequencing (NGS) technology, the number of
2013). Disorders that have a major genetic predisposition are found known ID genes is steeply increasing. Based on current number of
in the diagnostic groups Neurodevelopmental Disorders, well over 100 known ID genes on the X chromosome, an estimated
comprising intellectual disability (ID), autism spectrum disorders total number of around 2500 genes might be involved in mono-
(ASD), communication disorders and motor disorders, and the genic causes of ID. Likewise, NGS has been performed in large co-
group schizophrenia (SCZ) and other psychotic disorders. horts of patients diagnosed with ASD, SCZ, and epilepsy, with or
Regarding the associated direct and indirect costs, ID presents a without associated ID (ORoak et al., 2012; Neale et al., 2012; Carvill
large burden to our society and in the Dutch health care system it et al., 2013). These studies have revealed large numbers of de novo
ranks highest in the costs of all disease categories (Slobbe et al., mutations, which might be causative or contributory to the neu-
2011). The large-scale identication of ID and other CD genes is a rodevelopmental phenotype of these patients. Here we will discuss
exclusively the genes, collectively referred to as CD genes, for which
compelling evidence exist about their contribution to the CD
* Corresponding author. Radboud University Medical Center, Department of
phenotype, based on the identication of recurrent mutations in
Human Genetics 855, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31
24 3614017; fax: 31 24 3668752. the respective gene in independent patients or on supporting evi-
E-mail address: [email protected] (H. van Bokhoven). dence from functional studies.

0028-3908/$ e see front matter Crown Copyright 2014 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuropharm.2013.12.025
84 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94

1.1. CD genes operate in common molecular networks 1.2. Epigenetic genes and mechanisms

Causative mutations and variants in genes contributing to CDs are The genetic information encoded by our genomes is found in
rapidly being elucidated by next generation sequencing approaches, highly ordered chromatin structures that facilitate the packaging of
which has great benets for diagnostic and prognostic purposes. The DNA into the nucleus and are critical to control basic cellular pro-
identication of causative and contributory genetic defects provide a cesses such DNA replication, recombination, transcription, and DNA
stepping stone towards gaining mechanistic insight into patholog- repair. The core organizational structure is the nucleosome, con-
ical pathways involved in CDs. Analysis of the biological functions of sisting of 147 basepairs of DNA wrapped around an octamer of
large groups of proteins has revealed key cellular processes under- histone proteins. Most nuclear DNA is found in this beads-on-a-
lying CD pathogenesis (Chelly et al., 2006). For example, impaired string structure and has varying degrees of poorly understood
proliferation of neural progenitors is often seen in CDs with micro- local compaction and long-range contacts between loci. Tradi-
cephaly, and mutations affecting cues involved in neural migration tionally, a distinction was made between two types of chromatin,
and axonal guidance are characteristic of disorders involving cortical euchromatin and heterochromatin, which were thought to reect
malformations. Disruptions of synaptogenesis and control of syn- regions of active and inactive transcription. However, this gener-
aptic activity are characteristic of ID, ASD, and schizophrenia (Grant, alization appears to be an oversimplication of the true genomic
2012). Indeed, many of the mutated genes associated with these organization. Recently, combinatorial analysis of different histone
disorders affect genes involved in core synaptic processes (van modications and chromatin binding proteins has revealed several
Bokhoven, 2011). In addition, a disproportionately high number of novel models for chromatin organization that include anywhere
ID genes encode for proteins involved in chromatin-mediated con- from 5 to 51 different chromatin states, depending on the param-
trol of transcription, further alluded to as epigenetic transcription eters and organism being analyzed (Bernstein et al., 2012; Zentner
regulation. In the human genome there are 572 genes currently and Henikoff, 2013; Kundaje et al., 2012; de Graaf and van Steensel,
annotated with the GO terms chromatin binding, chromatin 2013). The regional chromatin structure, as determined by epige-
remodeling, and chromatin modication, representing about 2.9% of netic factors such as DNA modication, histone modication, pro-
all human genes. Amongst a catalog of 519 ID genes (Kochinke et al., tein complexes, non-coding RNA molecules (ncRNA), and
in preparation) there are 40 genes annotated with these GO terms nucleosome positioning, helps to dene the gene expression prole
(7.7% of ID genes). Consequently, chromatin-related genes are of a given cell.
enriched in ID by a factor of about 2.7 fold (Fig. 1). This is similar to DNA methylation is known for a long time as an important
the enrichment seen for synapse-related genes (3 fold), despite the regulator of transcription. Methylation of cytosines (mC) at CpG sites,
likely bias towards analysis of synaptic function for genes that are which are often enriched in islands at promoter regions, is generally
implicated in neurological disorders (Hamdan et al., 2009). In com- associated with gene silencing (for review see Smith and Meissner,
bination with this gene ontology analysis we further curated a list of 2013). However, abundant DNA methylation is also seen at non-
55 ID genes involved in chromatin-related regulation of transcrip- CpG islands, which may have an important role in the tissue-
tion (Table 1). Mutations in these 55 genes are seen in families with specic regulation of gene expression. Indeed, whole-genome sin-
an autosomal dominant, recessive, or X-linked mode of inheritance. gle base proling of the DNA methylome has revealed profound
Notably, a high proportion of the associated disorders are caused by changes in the epigenomic proles during brain development in
heterozygous loss of function mutations, suggesting that gene mice and humans (Lister et al., 2013). Besides cytosine methylation
dosage is critically important for many epigenetic genes. The also other cytosine modications have been identied, such as 5-
importance of correct dosage is illustrated by the X-linked MECP2 hydroxymethylcytosine (hmC), 5-formylcytosine (fmC) and 5-
gene, for which loss of function mutations are incompatible with life carboxylcytosine (caC) (Kriaucionis and Heintz, 2009; reviewed by
in males and associated with RETT syndrome in females, whereas Song et al. (2012)). Moreover, the recently discovered reversible
gene duplications give rise to syndromic ID in males and females do methylation of N6-methyladenosine in RNA may uncover a totally
not usually have any symptoms (Van Esch, 2008). new eld of RNA epigenetics (Zheng et al., 2013).
The post-translational modication of amino acid residues in the
tails of histone proteins is another major epigenetic mechanism of
transcription regulation. A variety of modications can occur: acet-
ylation, methylation, ubiquitylation, phosphorylation, sumoylation
and numerous less well-studied modications (Tan et al., 2011). The
covalent binding of acetyl groups to lysines residues removes the
positive charge on the histone tails, thereby decreasing the inter-
action of the N-termini of histones with the negatively charged
phosphate groups of DNA. As a consequence, acetylated chromatin is
less condensed, which facilitates transcription by making the DNA
more accessible to transcription promoting protein complexes and
the basic transcription machinery. In contrast, histone methylation
can serve as both an activator and repressor of transcription,
depending on the residue being modied and the degree of modi-
cation (i.e. mono-, di-, or tri-methylation). For several histone
modications a specic activity towards transcription has been
Fig. 1. Enrichment of Synapse-related and Chromatin-related genes in ID. All human
genes associated with the GO terms synapse (GO:0045202), synapse organization established: e.g. histone H3 lysine 9 di- and tri-methylation
(GO:0050808), and regulation of synaptic plasticity (GO:0048167) were combined to (H3K9me2/3; repression), histone H3 lysine 4 trimethylation
form the group Synapse Related (N 636). All genes associated with the terms (H3K4me3; activation), histone H3 lysine 27 acetylation (H3K27Ac,
chromatin binding (GO:0003682), chromatin modication (GO:0016568), and chro- active enhancer), histone H3 lysine 27 trimethylation (H3K27me3,
matin remodeling (GO:0006338) were combined to form the group Chromatin
Related (N 572). Enrichment was based the proportion of genes present amongst all
repression). However, there is still much to be learned about the
ID genes (N 519) compared to all human genes (N 20,687, Pennisi, 2012). Chro- effects of specic modications, let alone about the effects imposed
matin related, p 5.9  109; Synapse related, p 3.4  1012, hypergeometric test. by combinatorial effects of possible DNA and histone modications.
 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94 85

Table 1
Genes with epigenetic functions that are involved in cognitive disorders. There are 55 genes in total which are distributed over four categories: 1. writers (n 17), 2. Erasers
(n 5), 3. Chromatin remodelers of the DEAD/H-ATPase family (n 13), and 4. Other readers and chromatin remodelers (n 20). Gene names are provided by the ofcial HGCN
symbol (HUGO Gene Nomenclature Committee). In some cases alternative names are provided. The column OMIM Gene lists the number by which information of the gene
can be assessed in the OMIM database (Online Mendelian inheritance in Man; http://www.ncbi.nlm.nih.gov/omim/). The cognitive disorders associated with mutations in the
respective genes are listed and more information about these disorders can be found by following the number provided in the column OMIM Morbid. The lasts colums list the
epigenetic role of the respective protein and the mode of inheritance of the CD caused by mutations. AR, autosomal recessive; AD, autosomal dominant; XL, X-linked.

Gene symbol OMIM gene Phenotype OMIM Morbid Function (chromatin) Inheritance

1. Writers
DNA methylation
DNMT1 126375 Hereditary sensory neuropathy 614116 DNA methylation AD
type IE (HSN1E)
Cerebellar ataxia, deafness, 604121 AD
and narcolepsy,
DNMT3B 602900 ICF syndrome 242860 DNA methylation AR
FTO 610966 Growth retardation, developmental delay, 612938 RNA demethylation AR
coarse facies, and early death
Histone modication
CREBBP 600140 RubinsteineTaybi syndrome 1 (RSTS1) 180849 Histone acetyltransferase (HAT) AD, de novo
CUL4B 300304 Mental retardation, X-linked, 300354 Histone ubiquitination (H2AK119Ub1) XL
syndromic 15 (Cabezas type)
EHMT1 607001 Kleefstra syndrome 610253 Histone methyltransferase AD, de novo
(KMT1D; H3K9me1/2)
EP300 602700 RubinsteineTaybi syndrome 2 (RSTS2) 613684 Histone acetyltransferase (HAT) AD, de novo
EZH2 601573 Weaver syndrome 277590 Lysine N-methyltransferase 6 AD, de novo
(KMT6A; H3K27me3)
HLCS 609018 Holocarboxylase synthetase deciency 253270 Histone biotinylation (H4K16bio) AR
HUWE1 300697 Mental retardation, X-linked syndromic, 300706 Histone ubiquitination XL
Turner type
KAT6B 605880 Ohdo syndrome, SBBYS variant 603736 Histone acetyltransferase (MYST4) AD, de novo
Genitopatellar syndrome 606170 AD, de novo
KMT2A 159555 WiedemanneSteiner syndrome 605130 Histone methylation (MLL) AD, de novo
KMT2D 602113 Kabuki syndrome 147920 Histone methylation (MLL2) AD, de novo
KMT2C 607001 Kleefstra Syndrome spectrum 610253 Histone methylation (MLL3) AD, de novo
NSD1 606681 Sotos syndrome 117550 Histone methylation (KMT3B) AD, mostly de novo
BeckwitheWiedemann syndrome 130650 AD, de novo
WHSC1 602952 WolfeHirschhorn syndrome 194190 Histone methyltransferase (NSD2) AD, deletions
(H3K36me3, H4K20me2)
UBE2A 312180 X-Linked ID, Nascimento type 300860 Histone ubiquitination XL
2. Erasers
HDAC4 605314 Brachydactyly-mental retardation syndrome 600430 Histone deacetylase AD, de novo
HDAC8 300269 WilsoneTurner syndrome 309585 Histone deacetylase XL
Cornelia de Lange syndrome 5 300882 XL
KDM5C 314690 X-linked syndromic mental retardation; 300534 Histone demethylase XL
Claes-Jensen type (H3K4 tridemethylase)
KDM6A 300128 Kabuki syndrome 2 300867 Histone demethylase (H3K27me3/2) XL
PHF8 300560 Siderius X-Linked Mental 300263 Histone demethylase XL
Retardation Syndrome (H4K20me1, H3K9me1/me2)
3. Chromatin remodelers (DEAD/H ATPase family)
ACTB 102630 BaraitsereWinter syndrome 243310 SWI/SNF, INO80 and ISWI complex AD, de novo
Dystonia, juvenile onset 607371 AD* (single family, caution)
ARID1A 603024 Mental retardation, Autosomal dominant 14 614607 DEAD/H ATPase helicase family, AD, de novo
SWI/SNF subfamily
CofneSiris syndrome 135900 AD, de novo
ARID1B 614556 Mental retardation, Autosomal dominant 12 614562 DEAD/H ATPase helicase family, AD, de novo
SWI/SNF subfamily
CofneSiris syndrome 135900 AD, de novo
ATRX 300032 Alpha thalassemia mental retardation 301040 DEAD/H ATPase helicase family XL
syndrome, X-linked (ATRX)
Mental retardation-hypotonic facies syndrome 309580 XL
CHD2 602119 Epileptic encephalopathy, childhood-onset 615369 DEAD/H ATPase helicase family, AD, de novo
CHD subfamily
CHD7 608892 CHARGE syndrome 214800 DEAD/H ATPase helicase family, AD, de novo
CHD subfamily
CHD8 610528 Autism, susceptibility to, AUTS18 615032 DEAD/H ATPase helicase family, AD, de novo
CHD subfamily
SMARCA2 600014 NicolaideseBaraitser syndrome 601358 DEAD/H ATPase helicase family, AD, de novo
SWI/SNF subfamily
CofneSiris syndrome 135900 AD, de novo
SMARCA4 603254 Mental retardation, Autosomal dominant 16 614609 DEAD/H ATPase helicase family, AD, de novo
SWI/SNF subfamily
CofneSiris 135900
SMARCB1 601607 Mental retardation, Autosomal dominant 15 614608 DEAD/H ATPase helicase family, AD
SWI/SNF subfamily
CofneSiris syndrome 135900
SMARCE1 603111 CofneSiris syndrome
(continued on next page)
86 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94

Table 1 (continued )

Gene symbol OMIM gene Phenotype OMIM Morbid Function (chromatin) Inheritance

DEAD/H ATPase helicase family,

SWI/SNF subfamily
SRCAP 611421 Floating-Harbor syndrome 136140 DEAD/H ATPase helicase family, AD, de novo
INO80/SWR subfamily
SS18L1 606472 Amyotrophic lateral sclerosis (ALS) SWI/SNF complex AD, de novo
4. Other readers and chromatin remodelers
ASXL1 612990 BohringeOpitz syndrome 605039 PR-DUB complex, histone H2A AD, de novo
deubiquitination
BCOR 300485 Microphthalmia, syndromic 300166 Polycomb complexes XL
containing Ring1B
CHMP1 164010 pontocerbellar hypoplasia 8 614961 Targets polycomb protein BMI to AR
condensed chromatin
CTCF 604167 ID, microcephaly and growth retardation Chromatin binding factor, insulator AD, de novo
GATAD2B 614998 Mental retardation, autosomal dominant 18 615074 NuRD complex AD, de novo
HCFC1 300019 Mental retardation, X-linked; MRX3/ 309541 Found in repressor and XL
activator complexes
Methylmalonic acidemia and homocysteinenia XL
(Cobalamin disorder)
KANSL1 612452 KooleneDe Vries syndrome 610443 NSL1 histone acetyltransferase complex AD, de novo
MBD5 611472 Mental retardation, autosomal dominant 1 156200 Associated with heterochromatin AD, de novo
(2q deletion syndrome)
Kleefstra Syndrome spectrum 610253 AD, de novo
MECP2 300005 RETT syndrome; RTT 312750 Binds to methylated DNA XL, mainly females
RETT syndrome, atypical; Angelman 105830 XL
syndrome-like
Autism, susceptibility to, 300496 XL
X-linked 3; AUTSX3
Encephalopathy, neonatal severe 300673 Xl
Mental retardation, X-linked, 300055 XL
syndromic 13; MRXS13
Duplication MECP2; Lubs X-linked 300260 XL
mental retardation syndrome
PHF6 300414 BorjesoneForssmaneLehmann syndrome 301900 NuRD complex XL
CofneSiris-like 135900 XL
POGZ 614787 Autism spectrum disorder Pogo transposable element AD, de novo
with ZNF domain
SKI 164780 ShprintzeneGoldberg syndrome 182212 HDAC recruiting complexes AD
MED12 300188 LujaneFryns syndrome 309520 Mediator complex XL
OpitzeKaveggia syndrome 305450 Mediator complex XL
OHDO syndrome MaateKieviteBrunner 300895 Mediator complex XL
MED17 603810 Microcephaly, postnatal progressive, 613668 Mediator complex AR
with seizures and brain atrophy
MED23 605042 Mental retardation, autosomal recessive 18 614249 Mediator complex AR
NIPBL 608667 Cornelia de Lange syndrome 1; CDLS1 122470 Cohesin complex AD
RAD21 606462 Cornelia de Lange syndrome 4; CDLS4 614701 Cohesin complex AD, de novo
SALL1 602218 TowneseBrocks syndrome 107480 Member of NuRD histone AD
deacetylase complex
SMC1A 300040 Cornelia de Lange syndrome 2; CDLS2 300590 Cohesin complex XL
SMC3 606062 Cornelia de Lange syndrome 3; CDLS3 610759 Cohesin complex AD, de novo

The role of ncRNAs in epigenetic regulation of transcription is 2011). A maternal mutation in the UBE3A gene can cause Angel-
suspected to be very large given that most genes overlap with or man syndrome, a classic example of an imprinting disorder char-
can bind to multiple ncRNAs. These can affect transcription, acterized by severe ID, sleep disturbance, seizures, jerky
translation and RNA breakdown. The important role of ncRNA in movements, and frequent laughter. In neurons, but not in most
epigenetic regulation is most evident during inactivation of the X other cell lineages, this results in the total absence of UBE3A pro-
chromosome in females, where expression of the large ncRNA gene tein. Mutations affecting ncRNAs have been associated with genetic
XIST initiates the condensation of the respective X chromosome disorders, including CDs (Berdasco and Esteller, 2013; Willemsen
into a barr body that is decorated with XIST transcripts (Augui et al., et al., 2011). However, to our knowledge these mutations do not
2011). The initiation of X inactivation occurs randomly, leading to a directly impact other modications of the chromatin structure
roughly equal silencing of the maternal and paternal X chromo- (DNA and histones). Below we focus on neurodevelopmental dis-
somes. Skewing of the X-inactivation ratio can occur as a conse- orders with known causal gene disruptions, which result in aber-
quence of an X-chromosomal aberration or mutation that induces a rant modulation of chromatin structure.
selective disadvantage for cells in which the mutation-carrying X
chromosome is active. Genomic imprinting also appears to be co- 1.3. Epigenetic genes underlying cognitive disorders
regulated by ncRNAs. An intriguing example is the silencing of
the paternally derived UBE3A gene due to the paternal expression The epigenetic genes that we discuss are presented in Table 1
of a UBE3A-antisense transcript (UBE3A-ATS). This transcript is and have been divided into four major categories: 1. Writers of
silenced on the maternal chromosome by parent-of-origin-specic epigenetic modications, involved in enzymatic addition of side
DNA methylation in the UBE3A-ATS promoter, which allows for groups (DNA methylation, RNA methylation and histone modi-
expression of UBE3A from the maternal chromosome (Mabb et al., cation); 2. Erasers of epigenetic modications, the enzymes that
 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94 87

remove these side groups; 3. Chromatin Remodelers of the DEAD/ acetylate a variety of lysine residues of histone proteins (H2A, H2B,
H ATPase helicase family, which are involved in the regulation of H3 and H4) and non-histone proteins. They bind specically to
nucleosome positioning; 4. Other readers and chromatin phosphorylated CREB (cAMP response element-binding protein)
remodelers, containing proteins that recognize and bind to their and enhance its transcriptional activity toward cAMP-responsive
cognate chromatin marks, as well as proteins that are found in genes. In the nervous system this cAMP-mediated response is
transcription repressor and activator complexes and other com- involved in the formation of long-term memories (Dash et al., 1990;
plexes that regulate DNA accessibility by the basic transcription Bourtchuladze et al., 1994). Interestingly, de novo mutations in
machinery. Below we summarize the characteristics of the epige- these closely related HATs give rise to RubinsteineTaybi syndrome,
netic genes and associated disorders. For additional extensive de- characterized by ID, postnatal growth deciency, microcephaly,
scriptions we refer to other comprehensive reviews (Kramer and broad thumbs and halluces, and characteristic facial appearance
van Bokhoven, 2009; van Bokhoven and Kramer, 2010; Berdasco (Petrij et al., 1995; Roelfsema et al., 2005). The notion that CBP and
and Esteller, 2013; Millan, 2013; Ronan et al., 2013). p300 mutations give rise to similar phenotypes suggests that these
paralogous proteins have shared functions, which is supported by
2. Writers their high sequence similarity and similar domain structure,
including a bromodomain, cysteineehistidine-rich regions, and a
2.1. DNA/RNA methylation histone acetyltransferase domain.
The Lysine acetyltransferase 6B (KAT6B, also known as MYST4)
In mammals, there are three active DNA methyltransferases, underlies two distinct CDs: the SayeBarbereBieseckereYounge
DNMT1, DNMT3A and DNMT3B, and one related protein that lacks Simpson variant of Ohdo syndrome, which is usually associated
catalytic activity (DNMT3L). DNMT3A/B are de novo DNMTs, with severe ID, delayed motor milestones, and signicantly
whereas DNMT1 secures maintenance of DNA methylation. Muta- impaired speech (Clayton-Smith et al., 2011), and Genitopatellar
tions in two of the three active DNMTs have been linked to CDs. syndrome, with severe psychomotor retardation and microcephaly.
Dominant mutations in DNMT1 are associated with hereditary The different phenotypic features of these two conditions and the
sensory neuropathy type IE (HSN1E; Klein et al., 2011) and with location of the underlying mutations suggest that distinct molec-
autosomal dominant cerebellar ataxia, deafness, and narcolepsy ular mechanisms account for the respective phenotypes (Campeau
(Winkelmann et al., 2012). Recessive mutations in DNMT3B cause et al., 2012). Of interest, the MaateKieviteBrunner variant of Ohdo
immunodeciency-centromeric instability-facial anomalies syn- syndrome is caused by a mutation in the MED12 subunit of the
drome 1 (ICF1; Xu et al., 1999). DNA methylation is known to be mediator complex, which bridges chromatin modications to the
important for brain development, as well as memory consolidation basal transcription machinery (see below).
(Miller et al., 2010). Hence, it is not surprising that disruption of the In contrast to acteyltransferases, histone methyltransferases
methylation machinery can cause a cognitive disorder. (HMTs) have a higher degree of specicity for catalyzing the
Analogous to DNA modications, numerous different kinds of modication of specic lysine residues. This is reected by the large
RNA modications can occur. Although these modications are not number of histone methyltransferases (at least 27 according to the
strictly epigenetic (i.e. on top of the genetic code), such modi- HUGO Gene Nomenclature Committee; http://www.genenames.
cations regulate gene activity and hence are popularly referred to as org/genefamilies/KDM-KAT-KMT; Allis et al., 2007) that are enco-
RNA-epigenetics (Zheng et al., 2013). Cellular RNAs contain more ded in the mammalian genome. Mutations in seven HMTs are
than a hundred structurally distinct post-transcriptional modi- currently linked to CDs. Mutations are almost exclusively de novo
cations at thousands of sites. Some RNA modications are dynamic and give rise to loss of one functional copy of the gene. Three of
and may have critical regulatory roles analogous to those of protein these genes are implicated in so-called overgrowth syndromes.
and DNA modications. The internal N6-methyladenosine (m6A) Mutations in the NSD1 (Nuclear receptor binding SET domain) gene
modication in messenger RNA is one of the most abundant RNA cause Sotos syndrome, which is characterized by pre- and postnatal
modications in eukaryotes. Demethylation of m6A is mediated by overgrowth, macrocephaly, advanced bone age, typical facial fea-
FTO (fat mass and obesity-associated protein). Polymorphisms in tures, and variable degrees of ID (Kurotaki et al., 2002). Interest-
the FTO gene are associated with variability in body mass index, but ingly, NSD1 mutations were also observed in patients with another
the underlying mechanism has not been studied (Yang et al., 2012). overgrowth disorder, BeckwitheWiedeman syndrome (BWS). BWS
In addition, a homozygous missense mutation in the FTO gene, is an imprinting disorder involving a cluster of genes on chromo-
resulting in an Arg316Gln substitution co-segregated with an ID some 11p15, including H19, CDKN1C and KCNQ1OT1. Therefore, it
phenotype in a consanguineous family with growth retardation, has been suggested that NSD1 is involved in imprinting of the
developmental delay, coarse facies, and early death (Boissel et al., chromosome 11p15 region (Baujat et al., 2004). Also of interest is
2009). that there is considerable phenotypic overlap between Sotos and
Weaver syndrome, and NSD1 mutations have been identied in
2.2. Histone modications three patients initially diagnosed with Weaver syndrome (Douglas
et al., 2003; Tatton-Brown and Rahman, 2013). Mutations in the
The importance of post-translation histone modications in related gene NSD2, which is not listed among the 27 HMTs in the
gene regulation has been established in many studies. Last year, HUGO gene nomenclature overview, is located in the minimal
comprehensive genome-wide histone modication proles related critical region dened by hemizygous deletions of chromosome
to gene expression and chromatin structure were established by 4p16.3 that are associated with WolfeHischhorn syndrome (Stec
the Encyclopedia of DNA Elements (ENCODE) project (Bernstein et al., 1998). WolfeHirschhorn syndrome is a congenital malfor-
et al., 2012). These studies suggested that up to 80% of the mation syndrome characterized by pre- and postnatal growth
genome outside the coding regions may have a function and not deciency, intellectual disability (usually severe), characteristic
reect junk DNA. Fourteen epigenetic genes associated with ID craniofacial features, cardiac features, seizures, and midline defects.
phenotypes encode proteins engaged in the post-translational NSD2, also known as WHSC1, is a histone H3 lysine 36 trimethyl-
modication of histone tails (Table 1). Histone acetyltransferases transferase which is associated with active transcription. In-
(HATs) such as CBP (encoded by CREBBP) and p300 (EP300) are the teractions between NSD2 and EZH2, which is mutated in an
least specic of these histone-modifying enzymes as they can overgrowth syndrome (see below), have been reported in
88 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94

oncogenesis (Asangani et al., 2013). In addition, NSD2 interacts associates with the E3 enzyme BRE1 and catalyzes the mono-
with NKx2-5 in heart development and Sall1, a cell-type specic ubiquitination of histone H2B (H2BK120ub1), a specic tag for
transcriptional co-regulator of the NuRD complex, which is activation of transcription (Kim et al., 2005). Interestingly, the
mutated in syndromic ID (TowneseBrock syndrome, see below and ASXL1 protein, involved in Boring Opitz syndrome, is a core
Table 1) (Nimura et al., 2009). component of the PR-DUB protein complex involved in histone
Weaver syndrome is an overgrowth syndrome for which de deubiquitination, as discussed below. Finally, the Holocarboxylase
novo mutations were recently identied in the EZH2 gene synthetase (HLCS) catalyzes the covalent binding of biotin to lysines
(Enhancer of Zeste Homolog 2) (Gibson et al., 2012; Tatton Brown in histones H3 and H4, thereby creating rare gene repression marks
et al., 2011). The observed phenotypic overlap seen for NSD1 and such as K16-biotinylated histone H4 (H4K16bio) that promote
EZH2 mutations suggest that the corresponding proteins act in nucleosome condensation (Singh et al., 2013). Furthermore, HLCS
similar epigenetic regulatory pathways. NSD1 preferentially interacts physically with both DNMT1 and MeCP2 in the tran-
methylates lysine residue 36 of histone 3 (H3K36) and lysine 20 of scriptional repression of long-terminal repeats (Xue et al., 2013).
histone H4 (H4K20), which are primarily associated with active
transcription. In contrast, EZH2 shows specicity for trimethylation 3. Erasers
of histone H3 on lysine 27 (H3K27me3), which is associated with
transcriptional repression. These apparent opposite activities might Five erasers of epigenetic marks have been linked to CD,
be differently affected by the mutations identied in the respective including two histone deacetylases (HDAC) and three histone
genes as NSD1 mutations in Sotos syndrome typically exhibit loss of demethylases (Table 1). HDAC4 is a class II HDAC expressed in the
function effects whereas EZH2 mutations in Weaver syndrome are cranial neural crest, osteoblasts, heart, skeletal muscle, and brain. It
expected to give rise to the synthesis of mutant EZH2 protein, with acts to inhibit a variety of transcription factors, including MEF2C
possible gain of function effects (Tatton-Brown and Rahman, 2013). and RUNX2, both of which are essential for proper skeletal devel-
The four other HMT proteins affected by mutations in CDs opment. HDAC4 expression and activities are in line with the
include three members of the mixed-lineage leukemia protein phenotype observed in mouse models and in human individuals
(MLL) protein family and the euchromatic histone-lysine N-meth- carrying de novo deletions and mutations affecting the HDAC4 gene
yltransferase 1 (EHMT1). EHMT1 preferentially mono- and di- that have brachydactyly-mental retardation syndrome (Williams
methylates histone H3 at lysine 9 (H3K9me1/me2). This is et al., 2010). HDAC8 is a class I HDAC that is expressed in the ner-
thought to contribute to epigenetic transcriptional repression by vous and alimentary system and controls patterning of the skull by
recruiting HP1 proteins (Ogawa et al., 2002). MLL proteins are class repressing transcription factors in the neural crest (Haberland et al.,
2 lysine-specic methyltransferases (KMT2 proteins, their ofcial 2009). Mutations in the X-linked HDAC8 gene have been identied
name), which preferentially mono- and tri-methylate lysine 4 of in males with WilsoneTurner syndrome, a syndromic form of ID,
histone H3 (H3K4me1/me3). H3K4me1 and H3K4me3, are thought characterized by severe intellectual disability, dysmorphic facial
to be specic tags for enhancers and for transcriptional activation, features, gynecomastia, hypogonadism, short stature, and truncal
respectively (Del Rizzo and Trievel, 2011; Goldsworthy et al., 2013; obesity in males, whereas females have a milder phenotype
Herz et al., 2012). Chimeric protein fusions involving KMT2 proteins (Haraklova et al., 2012). These features resemble those of Borjesone
as well as somatic mutations are frequently found leukemia, lym- ForssmaneLehmann syndrome, another X-linked disorder caused
phomas and other tumors, such as medulloblastoma. Strikingly, by mutation of the PHF6 gene which is a component of the nucle-
germline mutations in these genes do not give rise to cancer-prone osome remodeling and deacetylase complex (NuRD complex, see
phenotypes, but rather to three syndromes with multiple congen- below). Interestingly, HDAC8 mutations were recently shown to be
ital anomalies that show little phenotypic overlap beside their responsible for a small subgroup of patients with Cornelia de Lange
neurodevelopmental defects. De novo mutations in KMT2A (MLL) syndrome. The majority of cases with this syndrome have a mu-
cause WiedemanneSteiner syndrome, characterized by hairy el- tation in components of the cohesin complex (see Table 1, subgroup
bows, short stature, facial dysmorphism, ID, and speech delay 4): NIPBL, SMC1A, SMC3 and RAD21. Interestingly, it was demon-
(Jones et al., 2012). De novo mutations in KMT2D (MLL2) are seen in strated that HDAC8 also acts as a deacetylase of SMC3, thus linking
Kabuki syndrome, a congenital syndrome with ID and additional HDAC8 to the cohesin acetylation cycle (Deardorff et al., 2007,
features, such as postnatal dwarsm and a typical facial appear- 2012a, 2012b).
ance, reminiscent of the make-up of traditional Japanese Kabuki The histone demethylases KDM5C (JARID1C), KDM6A (UTX),
actors (Ng et al., 2010). Finally, de novo mutations in KMT2C (MLL3) and PHF8 are involved in the removal of methyl groups from spe-
have been identied in a patient with a phenotype resembling cic lysines. KDM5C and KDM6A are members of the Jumonji C
Kleefstra syndrome (Kleefstra et al., 2012) and in a patient with family of proteins. Mutations in the X-linked KDM5C gene cause
autism spectrum disorder and a nonverbal IQ of 82 (ORoak et al., syndromic ID with variable severity of cognitive and dysmorphic
2012). Mutations in the EHMT1 gene give rise to the archetypal phenotypes in males and females. There is some correlation be-
Kleefstra syndrome, suggesting a functional relationship between tween phenotypic severity and the predicted disruptive effect of
EHMT1 and KMT2C. Indeed these two proteins and several others the KDM5C mutation (Rujirabanjerd et al., 2010). KDM5C specif-
are part of an epigenetic underlying the Kleefstra syndrome ically acts as a demethylase for di- and trimethylated H3K4me3,
phenotypic spectrum (Kleefstra et al., 2012), as further discussed thus converting an activating histone mark into a repressive one by
below. recruiting histone deacetylases and RE1-silencing transcription
The nal three epigenetic writers implicated in CD mediate factor (REST) to neuron-restrictive silencer elements. This activity is
histone modications with poorly dened effects on transcription, antagonistic to the H3K4 trimethylase activity of the KMT2 pro-
ubiquitination and biotinylation. Two proteins with a role in his- teins. KDM6A occupies the promoters of HOX gene clusters and
tone ubiquitination are involved in unrelated X-linked ID syn- regulates their transcription by modulating the recruitment of the
dromes. Cullin 4B (CUL4B) is the main component of the Cullin4B- Polycomb Repressive Complex PRC1 and the monoubiquitination of
Ring E3 ligase complex, which physically associates with histone H2A. KDM6a demethylates H3K27me3, which through
polycomb-repressive complex 2. It was shown that CUL4B re- association with KMT2C/D complexes, is concomitant with
presses gene expression through its intrinsic monoubiquitination methylation of H3K4 (Lee et al., 2007). The phenotype associated
activity towards histone H2A (H2AK19ub1) (Hu et al., 2012). UBE2A with KDM6A mutations resembles classical Kabuki syndrome,
 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94 89

which is usually caused by mutations in KMT2D/MLL2 (Lederer functions, found to be an important constituent of DEAD/H ATPase
et al., 2012). The PHD nger protein 8 (PHF8) is a demethylase complexes, including the nBAF, INO80 and ISWI complexes
that acts on repressive marks such as H3K9me/me2, H3K27me2, (Hargreaves and Crabtree, 2011).
and H4K20me1, thus promoting transcription. Mutations in this
gene cause ID with cleft lip/palate (Laumonnier et al., 2005).
5. Other readers and chromatin remodelers
4. Readers of the DEAD/H ATPase helicase family
This category includes genes that encode integral members of
Proteins involved in reading of the epigenetic code and in protein complexes that recognize and bind to chromatin and help
remodeling of the chromatin structure constitute the largest and to reshape the chromatin structure to regulate accessibility to the
most variable category of CD-associated proteins. We have sub- transcription machinery (Table 1).
divided these in essentially two classes. Group 3 contains proteins The MECP2 protein, which is involved in a variety of neurolog-
of the DEAD/H ATPase helicase family, which are key to positioning ical disorders including Rett syndrome, is an archetypal reader of
of nucleosomes. The removal and exchange of nucleosomes re- the epigenetic code as it mediates binding of protein complexes to
quires energy, which is provided by the intrinsic ATPase activity of methylated CpG sites. Because of its interaction with histone
proteins of the DEAD/H family of helicase. This family contains four deacetylases and co-repressor complexes, and because CpG
subfamilies, SWI/SNF, INO80/SWR1, ISWI, and CHD ATPases methylation at promoters is associated with transcriptional
(Hargreaves and Crabtree, 2011). Several members of the SWI/SNF repression, it was initially thought that MECP2 acts strictly in
family have been implicated in CofneSiris syndrome, which is transcriptional repression. However, it is now well established that
characterized by variable phenotypic manifestations including ID, MECP2 has a much broader regulatory role. It binds to methylated
coarse facial features, hypertrichosis, and hypoplastic or absent CpG sites globally and is also engaged in transcription activation
fth ngernails or toenails. CofneSiris syndrome is most often (Charour et al., 2008). The methyl-CpG binding domain protein 5
caused by mutation of the ARID1B gene, but de novo mutations (MBD5) is another potential reader of DNA methylation, although
have also been identied in other genes: ARID1A, SMARCA2, its direct binding to DNA has not been demonstrated. MBD5 is
SMARCA4, SMARCB1 and SMARCE1 encoding subunits of the associated with heterochromatin (Laget et al., 2010). Deletions of
neuronal chromatin remodeling complex nBAF (Tsurusaki et al., chromosome 2q23.1 encompassing the MBD5 gene are relative
2012). The disruption of individual constituents of the same pro- common and haploinsufciency of MBD5 appears to be causative
tein complex underlying a group of similar phenotypes is in line for ID in these patients as MBD5-specic mutations give rise to
with the general notion that similar phenotypes share a common similar phenotypes (Wagenstaller et al., 2007; Kleefstra et al., 2012;
molecular and cellular etiology (Oti and Brunner, 2007). Interest- Hodge et al., 2013).
ingly, mutations affecting the chromatin remodeler PHF6 were Several of the genes dened in this last category do not have a
originally identied in BorjessoneForssmaneLehmann syndrome, clear enzymatic function, but are consistently found as core com-
but more recently also in CofneSiris syndrome (Wieczorek et al., ponents in various different transcriptional repressor (ASXL1,
2013). PHF6 appears to be part of the NuRD complex, that also in- BCOR, SKI) or activator (HCFC1, KANSL1) complexes. Loss of func-
cludes CHD4 and HDAC1 (Todd and Picketts, 2012), but no func- tion mutations in ASXL1 (Additional sex combs-like) are found in
tional interactions have been reported with members of the nBAF BohringeOpitz syndrome, a severe developmental and malforma-
complex that could explain the similarities to the Cofn-Siris tion disorder characterized by intrauterine growth retardation,
phenotype. It should be noted that the Cofn-Siris phenotype is poor feeding, profound ID, and various other congenital anomalies
highly variable and that mutations in the associated genes can lead (Hoischen et al., 2011). ASXL1 is a core component of the Polycomb
to other phenotypes, such as NicolaideseBaraitser syndrome repressive deubiquitinase (PR-DUB) complex, a dimeric protein
(SMARCA2), Kleefstra syndrome spectrum (SMARCB1) or unspeci- complex that removes monoubiquitin from histone H2A. The
ed dominant ID (several genes). Moreover, de novo mutations chromatin-modifying protein, charged multivesicular body protein
were recently identied in SS18L1 (also known as CREST), which (CHMP1) is also involved in polycomb-mediated gene silencing by
encodes another subunit of the nBAF complex (Chesi et al., 2013). targeting the Polycomb group (PcG) protein BMI1/PCGF4 to regions
The respective patients had no similarity with CofneSiris syn- of condensed chromatin (Stauffer et al., 2001). Recessive mutations
drome, but had developed amyotrophic lateral sclerosis, a devas- in CHMP1 cause pontocerbellar hypoplasia 8, characterized by se-
tating neurodegenerative disorder. vere psychomotor retardation, abnormal movements, hypotonia,
Three members of the chromodomain (CHD) subfamily of spasticity, and variable visual defects (Mochida et al., 2012).
ATPase helicases have been associated with various neuro- Mutations in the X-linked BCL6 corepressor gene, BCOR, are
developmental disorders. Mutation of CHD7 is the most common found in Lenz microphthalmia and Oculofaciocardiodental syn-
cause of CHARGE syndrome, an acronym for the most prominent drome (OFCD), which typically features eye anomalies (micro-
clinical features: Coloboma, Heart anomaly, choanal Atresia, phthalmia or anophthalmia), microcephaly and structural brain
Retardation, Genital and Ear anomalies (Vissers et al., 2004). More anomalies, but usually no cognitive impairments. BCOR acts as a
recently, de novo mutations in CHD2 and CHD8 were identied in corepressor by recruiting BCL6, MLLT3 and several HDACs (Huynh
several patients with epileptic encephalopathy (Carvill et al., 2013) et al., 2000). Also the SKI protein is a transcriptional corepressor,
and ASD (Neale et al., 2012), respectively. The latter study and in particular for repressing TGFb target genes through interaction
several other large studies involving exome sequencing in ASD trios with PRDM16. This promotes recruitment of the SMAD3-HDAC1
have also revealed single de novo mutations in other CHD genes, complex to the promoter of these target genes (Harada et al., 2003).
but these remain to be conrmed in further patients. The ATRX GATAD2B is a core component of the NuRD complex that seems to
gene, involved in alpha-thalassemia-mental retardation (Gibbons regulate the localization of this complex to specic subnuclear foci
et al., 1995) and SRCAP (SNF2-related CBP activator protein), via interactions with MBD2 (Brackertz et al., 2006). PHF6 was
mutated in oating harbor syndrome (Hood et al., 2012), are also shown to interact with members of the NuRD complex in a prote-
members of the DEAD/H ATPase family. Finally, BaraitsereWinter omic analysis, however its function in this complex is not known
syndrome is caused by mutations affecting the ubiquitous b-actin (Todd and Picketts, 2012). Mutations in these NuRD complex
protein (Rivire et al., 2012), which is, among many other cellular components are associated with differential types of syndromic ID
90 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94

(Lower et al., 2002; de Ligt et al., 2012; Wieczorek et al., 2013; CD, for example MEF2C, YY1, Sall1 and members of the KRAB-
Willemsen et al., 2013). domain Zinc nger family (ZNFs) to name a few.
Mutations in HCFC1 are associated with ID and specic meta-
bolic effects (cobalamin disorder; Yu et al., 2013). HCFC1 is engaged 5.1. Similar CD phenotypes reect functional relationships between
in a large number of proteineprotein interactions both in tran- epigenetic CD genes
scription repressor (SIN3A/HDAC) and activator complexes such as
the highly conserved COMPASS (Shilatifard, 2012; Michaud et al., The phenotypic similarity that is seen for a number of genetic
2013) and NSL complexes (Wysocka et al., 2003). The COMPASS disorders despite the involvement of mutations in distinct genes is
complexes activate transcription via H3K4 methylation, while the a reection of common molecular pathways and cellular processes
NSL complex activates transcription through acetylation of H4K16. that are affected by such mutations. There are many examples for
Another core component of the NSL complex, KANSL1, is the culprit which a common clinical phenotype is caused by mutations in
in Koolen-de Vries syndrome, a recognizable form of syndromic ID different genes that have a known functional relationship, for
formerly known as chromosome 17q21.31 microdeletion syndrome example proteineprotein interactions, complementary enzymatic
(Koolen et al., 2012). activities, or co-regulation of target genes. This is also the case for
Other CD-associated genes encode proteins with various roles in CD genes with an epigenetic function. Construction of a protein
chromatin remodeling during transcription and other cellular interaction network based on currently annotated proteineprotein
processes. For example, POGZ, which has recurrent de novo mu- interactions reveals a striking degree connectivity amongst the
tations in ASD patients (Iossifov et al., 2012), is essential for normal epigenetic ID genes (Fig. 2). Several examples of such interactions
mitotic progression. The CCTC-binding factor CTCF is involved in have already been discussed above: the cohesion mutations in
several transcriptional mechanism: repression, activation, and Cornelia de Lange syndrome, nBAF mutations in CofneSiris syn-
enhancer blocking (Weth and Renkawitz, 2011) respectively. drome, and mutations affecting the interacting proteins KMT2D
Moreover, CTCF regulates the three-dimensional chromatin struc- and KDM6A in Kabuki syndrome. We have also discussed cases
ture by binding to insulator elements. The binding to insulators where the same phenotype involves different proteins for which
prevents interactions between promoter and nearby enhancers and functional or even molecular relationships are still to be elucidated,
silencer elements, which makes CTCF a key regulator of transcrip- e.g. KAT6B and MED12, which underlie different variants of Ohdo
tion through control of long-range chromatin-mediated in- syndrome. It is possible that the respective genes are involved in
teractions (Phillips-Cremins and Corces, 2013). Mutations in the parallel pathways that eventually converge onto similar cellular
CTCF gene are associated with ID, microcephaly, and growth processes. However, it is also likely that in such cases our knowl-
retardation (Gregor et al., 2013). edge of the activities is currently insufcient to recognize their
Besides the epigenetic co-regulator proteins, category 4 con- shared activities or protein networks. The identication of muta-
tains proteins that are found in large complexes bridging chro- tions in distinct genes underlying a similar disorder can be an
matin remodeling complexes to basic transcription factors. One incentive to look for interactions between these genes. For Kleefstra
such complex is the cohesion complex, for which mutations of syndrome spectrum phenotypes, genetic interaction studies in
several of its components (NIPBL, SMC1A, SMC3, RAD21, HDAC8) Drosophila combined with knowledge about proteineprotein in-
give rise to a clinically similar phenotype known as Cornelia de teractions has uncovered a chromatin modication module
Lange syndrome (Deardorff et al., 2007, 2012a, 2012b). Cohesin is a encompassing EHMT1 and four other genes that were found to
conserved multi-protein complex that plays a crucial role in carry de novo mutations associated with this recognizable pheno-
keeping sister chromatids together from S-phase until mitosis or type (Fig. 2; Kleefstra et al., 2012). Undoubtedly, this module will be
meiosis. Cohesion facilitates DNA damage repair that occurs dur- extended, as a signicant number of patients with the Kleefstra
ing replication, and enforces faithful segregation of chromosomes syndrome phenotypic spectrum do not have mutations in any of
during cell division. In addition, cohesin regulates gene expression these ve genes. Moreover, the proteins contained in the module,
by stabilizing the chromatin loops formed by CTCF (Mehta et al., e.g. SMARCB1 and KMT2C are engaged in a wide variety of other
2013). Another bridging complex that is involved in ID is the proteineprotein interactions, including proteins involved in CD
mediator complex, which links chromatin modifying complexes to that are presented in Table 1 (e.g. MED12 and KDM5C via EHMT2/
the basal transcription machinery. Three mediator components, G9a. and with other SWI/SNF complex members via SMARCB1).
MED12, MED17 and MED23, are implicated in various unrelated Thus, our network of annotated proteineprotein interactions be-
forms of ID. The Mediator complex consists of 25e30 subunits, tween chromatin remodeling CD genes will likely expand in size
and plays a central role in the regulation of RNA Pol II mediated and medical importance with the emergence of additional func-
transcription (Ansari and Morse, 2013). Different subunits of tional connections (Fig. 2).
Mediator are clustered in modules (head, middle, tail and CDK
domain) that mediate contacts to other proteins and complexes 5.2. Variable phenotypes can be due to mutation of the same
involved in various activities including transcriptional co- epigenetic gene
regulators, general transcription factors, subunits of RNA Pol II,
and chromatin. In contrast to mutations in various members of the The opposite of the shared pathway-shared phenotype para-
cohesion complex, the phenotypes associated with mutations in digm can be seen when mutations in the same gene give rise to
individual mediator genes is variable aside from the occurrence of different clinical phenotypes. For example, mutations in SMARCB1
ID. This may be a reection of the wide variety of different in- have been identied both in the Kleefstra syndrome phenotypic
teractions that each of the mediator protein has. spectrum and in CofneSiris syndrome. The reason for the differ-
Our compilation of epigenetic regulators in CD does not include ential phenotype could be due to the differential consequences of the
transcription factors that are known to interact with remodeling underlying mutations. This seems to be the case for MECP2 muta-
complexes, because by denition, transcription factors (TF) are not tions, where highly disruptive mutations give rise to female-
epigenetic regulators. In general, by binding to specic target DNA restricted Rett syndrome and mutations that predict residual pro-
sequences these TFs provide specicity in the range of controlled tein function give rise to ID in males and no features in female mu-
target genes controlled by repressor and activator complexes. tation carriers. In addition, gene duplications predicting enhanced
Mutations in a variety of TFs give rise to phenotypes that include MECP2 activity give rise to yet another recognizable CD phenotype in
 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94 91

Fig. 2. Epigenetic networks in cognitive disorders. A) Protein network of the epigenetic regulators that are listed in Table 1 shows that they are highly connected. The network was
generated using Genemania (www.genemania.org) and displays direct physical protein interactions. B) Magnication of the epigenetic network that comprises the SWI/SNF
complex and genes causing the Kleefstra syndrome spectrum. The associated human disorders and the type of interaction are color-coded. Manual curation reveals further links
between ID genes, illustrating that further connections can be expected to be uncovered in the future.

male patients. Several other mechanisms can attribute to contribute pre- and postnatal environmental stressors might affect the pheno-
the clinical variability observed for mutations in single genes. The type as well. Finally, some caution is warranted in the diagnostic
genetic context of individual patients may introduce clinical vari- labels associated with the clinical classication of phenotypes. The
ability due to the presence of additional independent mutations that clinical diagnosis appears to be highly dependent on the medical
add more features or increase the severity of the CD phenotype. The specialist who is making the diagnosis (i.e. psychiatrist, neurologist,
genetic background can also modulate the core phenotype by genetic pediatrician, clinical geneticist). In addition, we must recognize that
(SNPs) and genomic polymorphisms (CNVs) that enhance or sup- mental illnesses are spread along a spectrum, and that splitting them
press features of the core phenotype (Coe et al., 2012). In addition, into subcategories ignores their substantial clinical overlap.
92 T. Kleefstra et al. / Neuropharmacology 80 (2014) 83e94

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