American Journal of Medical Genetics Part C (Seminars in Medical Genetics)

R E S E A R C H R E V I E W

Angelman Syndrome: Current and Emerging

Therapies in 2016
WEN-HANN TAN AND LYNNE M. BIRD*

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a loss of the maternally-inherited
UBE3A; the paternal UBE3A is silenced in neurons by a mechanism involving an antisense transcript (UBE3A-AS) at
the unmethylated paternal locus. We reviewed all published information on the clinical trials that have been
completed as well as the publicly available information on ongoing trials of therapies in AS. To date, all clinical trials
that strove to improve neurodevelopment in AS have been unsuccessful. Attempts at hypermethylating the
maternal locus through dietary compounds were ineffective. The results of an 8-week open-label trial using
minocycline as a matrix metalloproteinase-9 inhibitor were inconclusive, while a subsequent randomized placebo-
controlled trial suggested that treatment with minocycline for 8 weeks did not result in any neurodevelopmental
gains. A 1-year randomized placebo-controlled trial using levodopa to alter the phosphorylation of calcium/
calmodulin-dependent kinase II did not lead to any improvement in neurodevelopment. Topoisomerase inhibitors
and antisense oligonucleotides are being developed to directly inhibit UBE3A-AS. Artificial transcription factors are
being developed to “super activate” UBE3A or inhibit UBE3A-AS. Other strategies targeting specific pathways are
briefly discussed. We also reviewed the medications that are currently used to treat seizures and sleep disturbances,
which are two of the more common complications of AS. © 2016 Wiley Periodicals, Inc.

KEY WORDS: Angelman syndrome; clinical trial; mouse model; therapy; therapeutic

How to cite this article: Tan W-H, Bird LM. 2016. Angelman syndrome: Current and emerging therapies in
2016. Am J Med Genet Part C Semin Med Genet 9999C:1–18.

INTRODUCTION AND
BACKGROUND
Angelman syndrome (AS) is a rare neuro-
developmental disorder with recent esti-
mates of its prevalence being between
1:22,000 and 1:52,000 [Oiglane-Shlik
et al., 2006; Mertz et al., 2013; Luk and
Lo, 2016], although previous studies had
suggested that the prevalence was about 1
in 10,000 to 1 in 20,000 [Kyllerman, 1995;
Petersen et al., 1995; Buckley et al., 1998].
Individuals with AS have global develop-
mental delay that evolves to severe
intellectual disability, with their language
skills being more delayed than their motor
skills, and their expressive language being
far more delayed than their receptive
language, usually having only minimal
speech. Their behavioral profile is charac-
terized by a happy demeanor with easily-
provoked laughter, fascination with water,
mouthing behaviors, hyperkinetic behav-
iors, and maladaptive behaviors such as
hair-pulling. In addition, most individuals
with AS go through prolonged periods of
sleep disturbance in the form of increased
sleep latency and multiple awakenings
throughout the night. Other neurologic
features include generalized hypotonia in
younger individuals with the development
of peripheral hypertonia in older individ-
uals, ataxia, epilepsy, as well as resting and
intention tremorsin adolescents and adults.
Additional clinical features have been
described in recent reviews, and clinical
diagnostic criteria for AS have been
proposed [Williams et al., 2006; Thibert
et al., 2013; Bird, 2014; Larson et al., 2015].
Lack of expression of the
maternally-inherited UBE3A
gene in the brain results in

Conflict of interest: Wen-Hann Tan and Lynne M. Bird declare that they have no conflict of interest.
Wen-Hann Tan is a clinical geneticist at Boston Children's Hospital and an assistant professor of pediatrics at Harvard Medical School. He has been
actively involved in a nationwide natural history study and clinical trials in Angelman syndrome since 2006. He also serves on the Angelman Syndrome
Foundation Scientific Advisory Committee.
Lynne M. Bird is a professor of clinical pediatrics at University of California, San Diego, and a clinical geneticist/dysmorphologist at Rady Children's
Hospital San Diego with more than 15 years of experience in the clinical care and research of patients with Angelman syndrome. She served as the
Principal Investigator for the Angelman Syndrome Natural History Study sponsored by the Rare Diseases Clinical Research Network.
This is an updated version of a manuscript entitled “Pharmacological therapies for Angelman syndrome” that was previously published by the same
authors in Wiener Medizinische Wochenschrift 2016 Jan 12 [Epub ahead of print]. It is reproduced in part with permission from Springer.
*Correspondence to: Lynne M. Bird, M.D., Rady Children's Hospital San Diego, Division of Genetics/Dysmorphology, 3020 Children's Way, #5031
San Diego, CA 92123. E-mail: lbird@rchsd.org
DOI 10.1002/ajmg.c.31536
Article first published online in Wiley Online Library (wileyonlinelibrary.com).

ß 2016 Wiley Periodicals, Inc.

2 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) RESEARCH REVIEW

AS. Unlike most genes, the
paternally-inherited copy of
UBE3A is silenced (i.e.,
imprinted) in neurons, so
neurons are entirely dependent
on UBE3A expressed from the
maternal allele.
Lack of expression of the mater-
nally-inherited UBE3A gene in the
brain results in AS. Unlike most genes,
the paternally-inherited copy of
UBE3A is silenced (i.e., imprinted) in
neurons, so neurons are entirely depen-
dent on UBE3A expressed from the
maternal allele. Located on chromo-
some 15q11.2, UBE3A encodes ubiq-
uitin-protein ligase E3A, which is also
known as E6AP ubiquitin-protein ligase
or human papillomavirus E6-associated
protein. UBE3A is a member of the
HECT (Homologous to the E6AP
Carboxyl Terminus) E3 ligase family. It
catalyzes the last step in the ubiquitina-
tion pathway in which activated ubiq-
uitin is transferred from the E2
conjugating enzyme and covalently
ligated to lysine residues of proteins
designated for degradation in the pro-
teasome. A given protein can be either
monoubiquitinated (i.e., only one ubiq-
uitin molecule attached to a single lysine
residue) or polyubiquitinated (i.e., mul-
tiple ubiquitin molecules attached to
multiple lysine residues). In general,
monoubiquitination regulates intracel-
lular transport and transcription,
whereas polyubiquitination regulates
the concentrations of specific intracel-
lular proteins temporally and spatially
through proteasome-mediated protein
degradation [Dagli et al., 2012; Bird,
2014; Buiting et al., 2016]. Notwith-
standing its role in the ubiquitination of
specific proteins, studies of gene expres-
sion in the cerebellum of maternal
Ube3a knockout mice and in the heads
of Drosophila with Dube3a null muta-
tions have suggested that loss of this
protein results in either the up-regula-
tion or down-regulation of specific
genes; in the maternal Ube3a knockout
mice, seven genes were up-regulated
and 57 were down-regulated. Most of
these genes have been implicated in
cellular signaling, regulation of cell
death, neurogenesis, and neurotransmis-
sion. Among the proteins encoded by
these genes are transcription factors for
the differentiation of dopaminergic
neurons, dopamine receptors, cytoskel-
etal proteins, heat shock proteins, and a
sodium-potassium ATPase transporter
[Low and Chen, 2010; Jensen et al.,
2013]. However, as noted in a recent
review on the putative substrates of
UBE3A, there are inter-species differ-
ences in the substrates that interact with
UBE3A, and different substrates may
interact with UBE3A at different stages
in development [Sell and Margolis,
2015]. Nonetheless, these data suggest
that although AS is due to the loss of a
single gene, its pathogenesis involves the
dysregulation of many genes involved
in multiple cellular and physiological
pathways.
The imprinting of UBE3A is regu-
lated by a bipartite imprinting center on
chromosome 15q11.2 about one mega-
base (Mb) centromeric to UBE3A
comprising a centromeric AS imprinting
center (AS-IC) and a telomeric Prader-
Willi syndrome imprinting center
(PWS-IC) about 35 kilobases (kb) apart
(Fig. 1) [Buiting et al., 1999]. This
imprinting center is flanked by NPAP1
(previously known as C15ORF2) on the
centromeric side and by SNURF-
SNRPN on the telomeric side. The
PWS-IC extends from the SNURF-
SNRPN promoter through the first
exon of SNRPN and includes a differen-
tially methylated region (DMR) that is
methylated on the maternal allele and
unmethylated on the paternal allele
[Dubose et al., 2011]. SNURF-SNRPN
is bicistronic and encodes two over-
lapping transcripts, SNURF (SNRPN
upstream reading frame) and SNRPN
(Small nuclear ribonucleoprotein poly-
peptide N); the first three exons of
SNRPN encode SNURF, while the start
codon for the SNRPN protein resides in
the fourth exon [Dubose et al., 2011]. In
addition, SNURF-SNRPN encodes a
long non-coding transcript covering
about 600 kb of genomic DNA that
forms multiple long non-coding RNAs
(lncRNAs) such as IPW, small nucleolar
RNAs (snoRNAs) such as SNORD116/
HBII-85, and small nucleolar long non-
coding RNAs (snolncRNAs). The ex-
pression of these non-coding RNAs may
be regulated by the multiple alternative
promoters and exons 50 (i.e., “upstream”)
of SNURF-SNRPN exon 1. These
upstream exons, some of which are
differentially methylated between the
paternal and maternal allele, are used
primarily in neurons [Dittrich et al.,
1996; Farber et al., 1999]. The mecha-
nism of action of the AS-IC remains
unclear. Recent data suggest that in
oocytes, a SNURF-SNRPN upstream
exon within the AS-IC initiates (i.e.,
promotes) transcription of the DNA
sequence into SNRPN exon 2 by
splicing out exon 1. This process results
in DNA methylation at the PWS-IC by
means that are still poorly understood,
perhaps through chromatin modification
[Lewis et al., 2015]. In most human cell
types, the SNURF-SNRPN non-coding
transcript extends to the IPW gene,
which itself encodes a lncRNA. How-
ever, in neurons, the transcript extends
beyond IPW, resulting in the generation
of other lncRNAs, including UBE3A-
AS (UBE3A anti-sense transcript),
which is also known as SNHG14
(snoRNA host gene 14). UBE3A-AS
represses the expression of paternal
UBE3A in cis, but the exact mechanism
remains unclear [Chamberlain, 2013].
Since only the paternally-inherited
SNURF-SNRPN and UBE3A-AS tran-
scripts are expressed due to the methyl-
ation of the PWS-IC on the maternal
allele, only the paternally-inherited
UBE3A allele is silenced. In fact, deletion
of the PWS-IC on the paternal allele in a
mouse model resulted in the re-activa-
tion of the paternal Ube3a in neurons
[Chamberlain and Brannan, 2001].
There are four mechanisms that can
result in the loss of UBE3A expression
from the maternal allele, viz. deletion on
the maternal copy of chromosome
15q11.2q13.1 (about 70% of AS indi-
viduals), paternal uniparental disomy
(about 9%), imprinting defect due to
either a deletion in the maternal AS-IC
or an epimutation that results in the
RESEARCH REVIEW
Figure 1. Genomic region of human chromosome 15q11q13 in neurons: Blue rectangles represent imprinted transcripts that are
paternally expressed, red rectangles represent imprinted transcripts that are maternally expressed, black rectangles represent the repressed
alleles of imprinted genes, and gray rectangles represent transcripts that are not imprinted. Reproduced with permission from:
Chamberlain SJ. RNAs of the human chromosome 15q11-q13 imprinted region. Wiley Interdiscip Rev RNA. 2013;4:155-66.
maternal allele having the “imprint” of
the paternal allele (about 8%), and a
pathogenic variant in the maternal
UBE3A allele (about 11%) [Bird,
2014]. Unless the paternal UBE3A allele
harbors a pathogenic variant, the pater-
nal UBE3A allele should remain intact,
albeit silenced, regardless of the mecha-
nism by which expression of the mater-
nal UBE3A allele is lost.
Although the genetic etiology of
AS has been elucidated, the pathogene-
sis remains poorly understood at the
cellular level. UBE3A localizes to the
nucleus, and to a lesser extent the
dendrites, of neurons as well as to the
pre- and post-synaptic compartments.
Mice that lacked maternal expression of
Ube3a have reduced dendritic spine
length and density, suggesting that
UBE3A may be responsible for synaptic
formation at a structural level [Dindot
et al., 2008]. Moreover, these mice have
deficits in long-term potentiation
(LTP), which is a cellular model for
memory formation, in hippocampal-
dependent associative learning, and in
the experience-dependent maturation
AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
of neocortical synapses [Jiang et al.,
1998; Yashiro et al., 2009].
In addition to treating clinical
symptoms empirically, attempts at treat-
ing AS have focused primarily on one of
three strategies, viz. (i) activation of the
paternal UBE3A allele that is normally
silenced, (ii) direct introduction of a
functional copy of UBE3A, or (iii)
correcting specific cellular pathways
that are disrupted by the lack of
UBE3A. Although a few clinical trials
have been conducted as described
below, a cure for AS remains elusive.
METHODS
For this review of therapies in Angelman
syndrome, we searched the PubMed
database (http://www.ncbi.nlm.nih.
gov/pubmed/) using the search terms:
“Angelman AND (therapy OR therapies
OR therapeutic OR therapeutics OR
treatment OR treatments).” We also
searched the ClinicalTrials.gov registry
(https://www.clinicaltrials.gov/) using
the term “Angelman” to identify un-
published and ongoing clinical trials.
3
RESULTS
Activation of Paternal UBE3A
In theory, the imprinted paternal UBE3A
allele may be expressed through one of
three mechanisms: (i) Hypermethylation
of the SNURF-SNRPN DMR in the
paternally-inherited copy of PWS-IC,
thereby repressing the expression of the
SNURF-SNRPN transcript that includes
UBE3A-AS on the paternal chromosome
15; (ii) Direct repression of UBE3A-AS by
interfering with the binding of UBE3A-
AS to the UBE3A DNA bases; or (iii)
Either “super activation” of UBE3A
promoter or repression of UBE3A-AS
using an artificial transcription factor.
Hypermethylation of SNURF-SNRPN
DMR
Studies in rodent models of carcinogen-
esis have suggested that a severely
“methyl-deficient diet” resulted in hy-
pomethylation of genomic DNA, lead-
ing to the overexpression of specific
genes and repression of other genes
[Dizik et al., 1991]. The major methyl
4 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) RESEARCH REVIEW

donor for DNA methylation is S-
adenosylmethionine (SAM), which is
synthesized directly from methionine.
The methionine-homocysteine cycle
that generates SAM is shown in Figure 2.
Methionine synthase transfers a methyl
group from 5-methyltetrahydrofolate
(5-MTHF) to homocysteine, thereby
converting homocysteine to methio-
nine, and 5-MTHF to tetrahydrofolate
(THF). Methylcobalamin, a form of
vitamin B12, is the cofactor for methi-
onine synthase. In the liver and probably
in the kidneys, methionine can also be
synthesized by the hydrolysis of betaine
by betaine-homocysteine methyltrans-
ferase to form dimethylglycine. The
main source of THF is dietary folic acid,
which is converted to THF by dihy-
drofolate reductase. After SAM has
donated its methyl group, it is converted
to S-adenosylhomocysteine (SAH).
SAH inhibits DNA methyltransferases,
which are the enzymes that methylate
genomic DNA, and the “SAM/SAH
ratio” is termed the “methylation
index” [Van den Veyver, 2002].
Based on this hypothesis, two
clinical trials, both with the
overall goal of activating the
paternal UBE3A by
hypermethylating the paternal
SNURF-SNRPN locus
through the administration of
these “pro-methylation” oral
supplements, have been
published.
It has been hypothesized that “pro-
methylation” dietary supplements such
as folic acid derivatives and betaine
would lead to an increase in the
concentration of SAM, which in turn,
would result in a global increase in DNA
methylation and hence activation of the
paternal UBE3A allele [Peters et al.,
2010]. Based on this hypothesis, two
clinical trials, both with the overall goal
of activating the paternal UBE3A by
hypermethylating the paternal SNURF-
SNRPN locus through the administra-
tion of these “pro-methylation” oral
supplements, have been published. In
the first study, 48 children with AS aged
between 5 months and 14 years old
completed a 1-year double-blind pla-
cebo-controlled trial of folic acid and
betaine (n ¼ 28 in placebo group;
n ¼ 20 in the treatment group).
Although the levels of betaine, dime-
thylglycine, and red blood cell (RBC)
folate were higher in the treatment
group than in the control group at the
end of the 1-year study, there were no
statistically significant differences in the
levels of methionine and homocysteine
between the two groups. The develop-
mental outcomes as assessed by the
Bayley Scales of Infant Development
(2nd edition) and the Preschool Lan-
guage Scale (3rd edition) were not
significantly different between the two
groups either [Peters et al., 2010].
In a follow-up clinical trial (Clin-
icalTrials.gov identifier: NCT00348933),
65 children with AS aged between
5 months and 5 years old completed a
1-year open-label trial in which all
subjects received a combination treatment
comprising L-5-methyltetrahydrofolate,
vitamin B12, betaine, and creatine. The
rationale for this combination was to try to
enhance the availability of SAM by
providing the cofactor for methionine
synthase (i.e., vitamin B12) and reducing
the utilization of SAM in the synthesis of
creatine by supplying exogenous creatine
(although SAM is also used for the
synthesis of many other methylated
compounds). L-5-methyltetrahydrofolate
was used instead of folic acid due to
concerns that excess “unmetabolized”

Figure 2. The methionine-homocysteine cycle. The enzymes are in italics and the cofactor is in square brackets. The compounds that
were administered in the two clinical trials involving “pro-methylation” compounds, as described in the text, are shown in the boxes. SAM,
S-adenosylmethionine; SAH, S-adenosylhomocysteine; THF, tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; MTHFR,
methylene tetrahydrofolate reductase.
RESEARCH REVIEW
folic acid might be harmful in the long
term [Bird et al., 2011]. For the analysis of
this open-label trial, the control group was
composed of the 22 subjects in the
placebo arm of the previous folic acid-
betaine trial described above [Peters et al.,
2010] who were between 15 and
59 months old. The only biochemical
changes in the treatment group that were
statistically significant were increases in
betaine, dimethylglycine, and creatine;
there were no statistically significant
changes in the levels of RBC folate,
methionine, and homocysteine. There
were no detectable changes in the
methylation patterns at 27,578 CpG
dinucleotide sites as measured by the
Infinium HumanMethylation27 Bead-
Chip array in the whole blood samples
of 16 subjects who were treated for 1 year
compared to their baseline level of
methylation. Although the daily living
skills and motor skills as measured by the
Vineland Adaptive Behavior Scales (2nd
edition) (VABS-II) were better in the
treatment group compared to the control
group, there were no statistically signifi-
cant differences in the developmental
domains measured by the Bayley Scales
of Infant Development (3rd edition)
(BSID-III); of note, the VABS-II is based
on parental reports through a semi-
structured interview while the BSID-III
is based on objective assessments by a
psychologist. The authors felt that overall,
this treatment cocktail did not have any
appreciable impact on the developmental
outcome of children with AS [Bird et al.,
2011]. The findings from these two
clinical trials suggest that the paternal
UBE3A allele cannot be activated to any
clinically significant degree, if at all,
through the use of oral “pro-methylation”
compounds, although the authors did not
assess the degree of UBE3A activation in
the participants in each of these trials.
Inhibiting transcription of Snurf-Snrpn and
Ube3a-As by topoisomerase inhibitors
In an attempt to discover compounds
that might activate paternal UBE3A,
Huang et al. screened 2,306 small
molecules in cultured mouse cortical
neurons containing an engineered
Ube3a-YFP (yellow fluorescent protein)
gene on the paternal allele and identified
AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
one molecule, irinotecan, that produced
a positive signal. Irinotecan is an
inhibitor of the topoisomerase type I
enzyme, a complex that relaxes the
supercoiling of DNA. On further
screening of topoisomerase inhibitors,
the authors identified 15 other topo-
isomerase type I and II inhibitors that
were also capable of activating paternal
Ube3a in vitro in their cultured neurons.
In particular, topotecan, a topoisomer-
ase type I inhibitor, which is approved
by the Food and Drug Administration
(FDA) as a chemotherapeutic agent in
specific cancers, was found to be far
more potent than irinotecan, which was
the only other FDA-approved topo-
isomerase inhibitor identified through
this assay. The extent to which top-
otecan was able to promote the expres-
sion of paternal Ube3a in mouse models
when administered through intracere-
bral infusion directly into the lateral
ventricles was dependent on the dose,
with the lower doses resulting in the
activation of paternal Ube3a only in the
cerebral cortex, hippocampus, and stri-
atum, and the higher doses resulting in
the activation of paternal Ube3a in the
cerebellum as well. Moreover, paternal
Ube3a expression, at least in the spinal
cord neurons, was sustained for up to
12 weeks after intrathecal administration
of topotecan. The in vitro experiments
also demonstrated that topotecan re-
pressed the expression of Snurf-Snrpn
and Ube3a-As in cultured neurons
[Huang et al., 2012]. Subsequent ex-
periments have suggested that topotecan
represses the expression of paternal
Ube3a-As by stabilizing R-loops at the
paternal Snord116 locus [Powell et al.,
2013]. The mechanism of R-loop
formation remains unclear, but R-loops
may be formed during transcription and
comprise a triple-stranded nascent
mRNA-template DNA hybrid with
the displaced single-stranded non-tem-
plate DNA. R-loops can result in
“genomic instability” due to various
putative mechanisms, and they can also
prevent the elongation of transcription.
One of the means through which R-
loop formation is suppressed is through
the activity of topoisomerases [Skourti-
Stathaki and Proudfoot, 2014].
5
Inhibition of topoisomerase results in
the accumulation of R-loops, increased
chromatin decondensation (through an
unknown mechanism), and inhibition
of transcription elongation at the loci
where the R-loops have accumulated; it
remains unknown whether chromatin
decondensation is a cause or effect of R-
loop formation. The consequence of R-
loop stabilization at the paternal
Snord116 locus is the cessation of
transcription beyond this locus, resulting
in the lack of Ube3a-As and expression of
paternal Ube3a [Powell et al., 2013;
Skourti-Stathaki and Proudfoot, 2014].
In cultured human and mouse
neurons, topoisomerases promote the
expression of many genes longer than
67 kb and almost all genes longer than
200 kb (i.e., “long genes”), and the
repression of these genes by topotecan
and a topoisomerase II inhibitor was
both dose-dependent and gene-length-
dependent (longer genes being more
susceptible to repression than shorter
genes). Interestingly, the expression of
most genes shorter than 67 kb was
increased, albeit to a “very small” extent,
by the application of topotecan [King
et al., 2013]. More recently, the gene
expression profile of cultured mouse
neurons treated with topotecan was
compared to that of mouse neurons
with a homozygous deletion of Top1,
the gene that encodes topoisomerase I.
Interestingly, far more genes were both
upregulated and downregulated in the
topotecan treated wild-type neurons
than in those neurons with the Top1
deletion; in particular, many immediate
early genes were downregulated in the
topotecan treated neurons but not in the
neurons with the Top1 deletion, al-
though the authors suggested that this
might be due to “reduced spontaneous
neuronal activity” following treatment
with topotecan. Moreover, Ube3a ex-
pression was restored in the topotecan
treated but not in the Top1-deleted
neurons. The data from these authors
also suggest that topotecan inhibits
topoisomerase type I by binding to
both topoisomerase type I and DNA,
forming a topoisomerase type I cleavage
complex [Mabb et al., 2016]. These data
raise the concern that the systemic use of
6 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
topoisomerase inhibitors such as top-
otecan for the treatment of AS may
result in unintended off-target effects
due to the repression of many different
long genes and possibly over-expression
of the shorter genes [Plasschaert and
Bartolomei, 2013].
Inhibiting transcription of Ube3a-As by
antisense oligonucleotides
Since the expression of paternal UBE3A
is normally repressed by UBE3A-AS, it
has been hypothesized that loss of
UBE3A-AS would result in transcrip-
tion of the paternal UBE3A allele. To
evaluate this hypothesis in vivo, Meng
et al. engineered a mouse model that had
a poly(A) “stop cassette” inserted be-
tween Snord115 and Ube3a that should
theoretically prevent the expression of
Ube3a-As downstream of the cassette
without affecting transcription of the
other non-coding RNAs that are all
upstream of Snord115. As expected, this
reduction in Ube3a-As expression was
observed only when the stop cassette
was on the paternally-inherited chro-
mosome, but not when it was on the
maternally-inherited chromosome since
Ube3a-As is not normally expressed on
the maternal allele. In mice with the
poly(A) cassette on the paternal allele,
the amount of Ube3a protein was
increased to between 50% and 70% of
wild type (WT) levels in different
regions of the brain. Mice engineered
with maternal Ube3a knockout and
paternal poly(A) stop cassette demon-
strated improved performance in motor
coordination (as assessed by the acceler-
ating rotarod test) and long-term mem-
ory (as assessed by the fear conditioning
test). Obesity, which is typically seen in
AS mice, was not observed in these
engineered mice. Deficits in LTP were
also restored in these mice. However,
some other phenotypic features typically
seen in AS mice such as hypoactivity
were not significantly different between
the engineered and WT mice. The
authors hypothesized that paternal
Ube3a-As mediates the imprinting of
Ube3a through “transcriptional colli-
sion” whereby the RNA polymerases
that transcribe Ube3a and Ube3a-As in
opposite directions collide with each
other, resulting in the premature termi-
nation of both transcripts, and the
incomplete Ube3a transcript is then
degraded [Meng et al., 2013]. These
data suggest that direct repression of
Ube3a-As could be a potential thera-
peutic strategy for AS.
Since the expression of
paternal UBE3A is normally
repressed by UBE3A-AS, it
has been hypothesized that
loss of UBE3A-AS would
result in transcription of the
paternal UBE3A allele. To
evaluate this hypothesis in
vivo, Meng et al. engineered a
mouse model that had a
poly(A) “stop cassette”
inserted between Snord115
and Ube3a that should
theoretically prevent the
expression of Ube3a-As
downstream of the cassette
without affecting transcription
of the other non-coding RNAs
that are all upstream of
Snord115.
The use of antisense oligonucleo-
tides (ASOs) to modulate gene expres-
sion in vivo has been attempted for a
variety of systemic diseases and malig-
nancies. ASOs are synthetic single-
stranded oligonucleotides that bind to
specific target RNAs in the nucleus and
the ASO-RNA duplexes are then
cleaved by ribonuclease H (RNase H),
with the resulting RNA strands being
degraded by exonucleases. Thus, ASOs
can be used to target and degrade RNAs
with specific sequences. This approach
has been found to be effective in treating
some types of hypercholesterolemia and
inflammatory bowel disease when
RESEARCH REVIEW
administered intravenously [Agarwala
et al., 2015; Marafini et al., 2015; Santos
et al., 2015], as well as some monogenic
neuromuscular and neurodegenerative
conditions.
In September 2016, the FDA
approved the use of an intravenous
ASO, eteplirsen (EXONDYS 51), for
the treatment of Duchenne muscular
dystrophy (DMD), although the evi-
dence for efficacy was weak [Food and
Drug Administration, 2016]. Eteplirsen
binds to exon 51 of dystrophin pre-
mRNA, resulting in the deletion of this
exon from the mature mRNA. In
patients with specific frameshift muta-
tions, this results in the restoration of the
reading frame, thereby converting the
clinical phenotype of DMD to that of
the less severe Becker muscular dystro-
phy [Jacobson and Feldman, 2016;
Shimizu-Motohashi et al., 2016]. Nu-
sinersen is an ASO that binds to exon 7
of the paralogous SMN2 pre-mRNA
and promotes the formation of a full-
length mature mRNA by preventing the
normal skipping of exon 7 in SMN2,
hence allowing the presence of SMN2
to compensate for the loss of SMN1
[Hua et al., 2008]. Based on the interim
analysis of a phase 3 trial of nusinersen in
infantile-onset SMA (ClinicalTrials.gov
identifier: NCT02193074), a New
Drug Application was filed with the
FDA in September 2016 for the use of
nusinersen in SMA. ASOs are also being
developed to treat Huntington disease
by reducing expression of the mutant
HTT (huntingtin) gene [Godinho et al.,
2015] (ClinicalTrials.gov identifier:
NCT02519036), familial amyotrophic
lateral sclerosis (ClinicalTrials.gov iden-
tifier: NCT02623699), and some forms
of frontotemporal dementia [Mis et al.,
2016; Picher-Martel et al., 2016]. All of
these clinical trials will help provide
evidence for the potential safety and
efficacy of ASO therapy administered
intrathecally for the treatment of a wide
variety of neurologic disorders due to
mutations in a single gene, thereby
raising the hope that this may also be a
potentially effective therapeutic strategy
for AS. It should be noted, however, that
the cognitive impairment in AS is
congenital in onset, so it remains
RESEARCH REVIEW
unknown whether postnatal treatment
would be effective.
In order to target and repress
Ube3a-As directly, Meng et al., in
collaboration with Ionis Pharmaceuti-
cals, developed a set of ASOs designed to
bind to a 113 kb sequence in mouse
Ube3a-As. One ASO injected directly
into the lateral cerebral ventricle of adult
WT mice resulted in a reduction in
paternal Ube3a-As by 60–70%, and an
upregulation of Ube3a protein in the
cortex, hippocampus, and spinal cord by
33–82% 4 weeks after injection; expres-
sion of Ube3a-As remained suppressed
for 16 weeks after injection of a single
dose of ASO. These data suggested that
this ASO was able to partially unsilence
paternal Ube3a. Importantly, the expres-
sion of Snrpn, Snord115, Snord116, and
five of the long genes that they
specifically examined was not down-
regulated by treatment with this ASO.
When this ASO was subsequently
injected into the lateral ventricle of
adult AS mice, Ube3a was expressed in
the cortex, hippocampus, and cerebel-
lum to about 35–47% of that seen in WT
mice. Behavioral assays on these AS
mice 4 weeks after treatment suggested a
partial restoration of the WT phenotype
with normalized “contextual freezing”
behavior (indicative of long-term mem-
ory), but no improvement in other
neurologic assays such as the “marble
burying” and accelerating rotarod tests.
The authors postulated that a complete
reversal of the AS phenotype may not be
possible unless the mice were treated
“before a critical developmental win-
dow” or a greater amount of Ube3a is
produced [Meng et al., 2015]. Never-
theless, these data suggest that the use of
ASOs might be a viable form of therapy
for AS if a sufficient amount of paternal
UBE3A can be expressed throughout
the brain in early infancy.
Repression of Ube3a-As by zinc finger
artificial transcription factor
Artificial transcription factors (ATFs) are
synthetic compounds that regulate tran-
scription of genes by binding to specific
target DNA sequences. At a minimum,
each ATF comprises a specific DNA-
binding protein (domain), an effector
AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
domain that either activates or represses
the target gene, and a nuclear localiza-
tion signal that guides the ATF into the
nucleus. The DNA-binding protein
may be a zinc finger protein (ZFP),
transcription activator-like effectors
(TALEs), or a clustered regularly inter-
spaced short palindromic repeat
(CRISPR)-derived protein. ZFP-
ATFs are being developed for the
treatment of Huntington disease, retini-
tis pigmentosa [Agustin-Pavon and
Isalan, 2014], amyotrophic lateral scle-
rosis (ClinicalTrials.gov identifier:
NCT00748501) and diabetic neuropa-
thy (ClinicalTrials.gov identifier:
NCT01079325). Bailus et al. (2016)
created 11 different ZFP-ATFs targeting
Ube3a and its regulatory regions, with a
mixture of activation and repressor
domains. The ATF that had the stron-
gest activity and could be produced in
sufficient amount was TAT-S1, a repres-
sor ATF that binds between the AS-IC/
PWS-IC and SNURF/SNRPN locus.
The authors found that both subcutane-
ous and intraperitoneal administration
of TAT-S1 into adult AS mice resulted in
an increase in Ube3a throughout the
brain, especially in the hippocampus and
cerebellum [Bailus et al., 2016].
Direct Injection of UBE3A Into the
Brain Using Recombinant Adeno-
Associated Virus (rAAV)
Another therapeutic approach that has
been attempted in a mouse model is the
direct injection of Ube3A into the
hippocampi bilaterally using a rAAV
vector (serotype 9). When AS mice were
so treated, it was found that Ube3a
expression in the hippocampus was up-
regulated to a level comparable to that in
the WT mice, but Ube3a was not over-
expressed; however, Ube3a expression in
the cerebellum was not increased, at least
not after a single injection of the rAAV-
Ube3a vector. The rAAV-Ube3a treated
mice demonstrated normalization in
fear conditioning as measured by appro-
priate contextual freezing 24 hr after
training, which suggested an improve-
ment in associative learning. However,
the apparent improvement in spatial
memory assessed through the use of
7
the Morris water maze was not con-
vincing, and there was no improvement
in rotarod performance. Moreover,
deficits in LTP were only partially
restored in the treated mice [Daily
et al., 2011]. As such, viral gene therapy
is promising, but it is not a feasible
therapeutic strategy yet. Recently, Agilis
Biotherapeutics, Inc. (Cambridge, MA)
licensed this technology from these
authors and it is now developing DNA
constructs and novel vectors in an
attempt to develop a gene transfer
product for AS [Agilis Biotherapeutics
Inc., 2016].
Treating Disruptions in Specific
Cellular Pathways Caused by Lack
of UBE3A
As discussed in the Introduction, loss of
UBE3A leads to both the up-regulation
and down-regulation of multiple genes,
which results in disruption of various
signaling pathways and synaptic trans-
missions, some of which may be
modulated by specific therapeutic
compounds.
Dopaminergic pathways and
phosphorylation of calcium/calmodulin-
dependent kinase II (CaMKII)
CaMKII, an enzyme in the post-synap-
tic density of neurons, is activated when
it binds to the calcium-calmodulin
complex and it is essential for LTP in
the cerebral cortex and hippocampus.
Activated CaMKII phosphorylates the
GluR1 subunit of the AMPA (a-amino-
3-hydroxy-5-methylisoxazole-4-pro-
prionic acid) glutamate receptor, in-
creasing the conductance of the sodium
ion channel. CaMKII also autophos-
phorylates its own threonine residues at
amino acid positions 286, 305, and 306
(Thr286, Thr305/306). Phosphoryla-
tion at Thr286 results in prolonged
activation of CaMKII, the inhibition of
which impairs LTP in the hippocampus.
In contrast, phosphorylation at Thr305/
306 prevents the binding of substrates to
CaMKII and regulates the cellular
localization of CaMKII in the post-
synaptic density. The regulation of
CaMKII activity and translocation by
phosphorylation of these specific
8 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
threonine residues is critical for “cogni-
tive flexibility” and fine-tuning of spatial
learning [Giese et al., 1998; Elgersma
et al., 2002; Lisman et al., 2002; Blitzer
et al., 2005].
In a study on LTP in AS mice, it was
found that these mice had increased
phosphorylation at the Thr286 and
Thr305/306 residues in CaMKII in
the hippocampus and hence reduced
CaMKII kinase activity, resulting in
impaired LTP induction and memory
formation [Weeber et al., 2003]. Impor-
tantly, when AS mice were crossed with
mice that had a specific mutation in the
CaMKII-Thr305/306 residues that pre-
vented these residues from becoming
phosphorylated, the double mutants
(i.e., AS mice with these mutated
Thr305/306 residues) had normal
CaMKII activity in the hippocampus,
normal hippocampal-dependent learn-
ing, normal spatial learning, improve-
ment in gait ataxia, and a decrease in
seizure tendency [van Woerden et al.,
2007]. These data suggest that thera-
peutic interventions that prevent the
excessive phosphorylation of Thr305/
306 residues in CaMKII may be
beneficial in individuals with AS.
It has previously been found in a rat
model of Parkinson disease that levo-
dopa (L-dopa) can reverse the excessive
phosphorylation of threonine residues
in specific synaptic and neuronal en-
zymes including CaMKII [Brown et al.,
2005]. Preliminary data suggest that
treatment of AS mice with L-dopa leads
to reduced phosphorylation at both
Thr286 and Thr305/306 residues in
CaMKII, and an improvement in motor
skills [Tan et al., 2016]. It was therefore
hypothesized that the use of L-dopa in
AS individuals may result in an improve-
ment in their motor and cognitive
development.
To evaluate the potential
efficacy of levodopa for the
treatment of AS, a double-
blind placebo-controlled trial
of levodopa/carbidopa was
initiated in children with AS
aged between 4 and 12 years.
Among the 55 children who
completed this 1-year study,
no clinically or statistically
significant differences were
found in any of the outcome
measures, which included the
BSID-III or Mullen Scales of
Early Learning, Vineland
Adaptive Behavior Scales, and
Aberrant Behavior Checklist.
To evaluate the potential efficacy of
levodopa for the treatment of AS, a
double-blind placebo-controlled trial of
levodopa/carbidopa was initiated in
children with AS aged between 4 and
12 years. Among the 55 children who
completed this 1-year study, no clini-
cally or statistically significant differ-
ences were found in any of the outcome
measures, which included the BSID-III
or Mullen Scales of Early Learning,
Vineland Adaptive Behavior Scales, and
Aberrant Behavior Checklist. These
findings suggest that L-dopa is not an
effective therapy for AS, at least at the
dose used in this study (i.e., 15 mg/kg/
day); as noted by the authors, it is
unknown whether use of a much lower
dose could have been effective since in
theory, L-dopa inhibits the phosphory-
lation of the threonine residues in a non-
specific manner, and inhibition of
Thr286 in CaMKII could impair LTP
[Tan et al., 2016].
Dendritic spine maturation and matrix
metalloproteinases
UBE3A localizes to the nuclei, growth
cones, and dendrites, as well as the pre-
and post-synaptic compartments of
neurons. Dendrites are usually lined
with short protrusions (i.e., dendritic
spines) that form synapses with axons.
Mature dendrites usually have long
dendritic spines with relatively large
“mushroom-shaped” heads instead of
the “filopodia-like” spines seen in
RESEARCH REVIEW
immature dendrites. In AS mice, the
density of the dendritic spines was found
to be lower than that in wild-type mice,
and the spines that were present were
shorter with more variable head sizes
[Dindot et al., 2008].
Matrix metalloproteinases (MMPs)
are a family of zinc-binding endopepti-
dases that catalyze the proteolysis of
specific proteins in the extracellular
matrix, cell adhesion molecules, and
cell surface receptors. MMPs are also
synthesized and secreted by neurons,
and to a lesser extent, glia, in the central
nervous system. Some MMPs are ex-
pressed more highly in specific brain
regions. In particular, MMP9 localizes
to the hippocampal dendrites and
dendritic spines, as well as the cerebral
cortex and cerebellum [Dziembowska
and Wlodarczyk, 2012; Huntley, 2012].
MMP3 and MMP9 are believed to be
important in both associative and non-
associative learning and in the formation
of memory [Huntley, 2012]. Although
genetically-engineered mice that over-
express Mmp9 in neurons had increased
dendritic spine density, prolonged main-
tenance and greater potentiation during
LTP, and improvement in the “non-
spatial novel object recognition” task,
which suggests greater neuronal plastic-
ity [Fragkouli et al., 2012], hippocampal
neurons that were chronically exposed
to MMP9 in a “bath” (i.e., affecting
entire neuron rather than dendritic
spines only) in vitro formed long and
thin immature dendritic spines [Dziem-
bowska and Wlodarczyk, 2012; Hunt-
ley, 2012]. Moreover, in vivo and in
vitro data suggest that pharmacologic
inhibition of MMP9 results in arrest of
dendritic spine growth and maturation
[Bilousova et al., 2009; Dziembowska
and Wlodarczyk, 2012]. There are no
published reports on the activity of any
MMP in AS. However, increased
MMP9 activity has been found in the
cerebral cortex of post-mortem fetuses
and adults with Down syndrome [Iulita
et al., 2014; Vafadari et al., 2015] and in
the cerebral cortex and hippocampus in
Fragile X mice and in humans with
Fragile X syndrome. There are data from
Fragile X mice suggesting that normali-
zation of MMP9 activity using a double
RESEARCH REVIEW
knockout model was beneficial [Sidhu
et al., 2014]; however, attempts to alter
MMP9 activity using minocycline in
humans to treat Fragile X syndrome did
not produce convincing results [Dziem-
bowska et al., 2013].
Minocycline is a tetracycline anti-
biotic that has been used to treat acne for
almost 50 years [Cullen and Cohan,
1976]. Tetracyclines bind to bacterial
ribosomes and divalent cations, and the
chelation of zinc by tetracyclines may
inhibit the activity of MMPs. Tetracy-
clines may also modulate pro-inflamma-
tory cytokines, resulting in secondary
inhibition of MMP transcription. Min-
ocycline is one of the most lipophilic
tetracyclines and it crosses the blood-
brain barrier well [Jonas and Cunha,
1982; Griffin et al., 2010]. Despite the
lack of studies showing altered MMP9
activity, a trial of minocycline in AS was
undertaken based on an in vitro small
molecule screen and preliminary mouse
data. Twenty five children with AS
between the age of 4 and 12 years
were treated with minocycline for
8 weeks in an open-label trial. Devel-
opmental assessments were performed at
the end of the 8-week trial and at
16 weeks after initiation of therapy (i.e.,
8 weeks after discontinuation of mino-
cycline). The authors found slight
overall improvement, as measured by
the Clinical Global Impressions—Se-
verity scale. There was also an improve-
ment in receptive communication on
the VABS-II, which is based on parental
report, at the end of the 8-week trial, but
this improvement was not sustained
8 weeks after discontinuation of mino-
cycline [Grieco et al., 2014]. However,
multiple flaws in the study design,
including the lack of a control group,
have made it impossible to draw any
conclusions regarding the efficacy of
minocycline in AS from this trial.
Moreover, MMP9 activity was not
measured in this study, so it is unknown
whether minocycline therapy had any
impact on MMP9 activity in AS.
Subsequently, an 8-week cross-over
double-blind placebo-controlled trial
of minocycline in 32 children and adults
with AS, randomized 2:1 to minocy-
cline or placebo, (ClinicalTrials.gov
AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
identifier: NCT02056665) was con-
ducted by a different group of inves-
tigators, and no differences in cognitive
and motor skills were detected between
the minocycline and placebo cohorts.
These results were presented at the 12th
Congress of the European Association
for Clinical Pharmacology and Thera-
peutics in 2015 [Ruiz-Antor
a n et al.,
2015], but the full manuscript has not
been published yet.
Synaptic signaling pathways
There is evidence from animal studies
that several synaptic signaling pathways
are altered in AS. Presumably, defective
Ube3a activity impairs ubiquitination
and subsequent degradation of its sub-
strates, resulting in increased abundance
of its target proteins. Several Ube3a
targets have been identified, including
Arc [Greer et al., 2010] and Ephexin5
[Margolis et al., 2010]. Ube3a regulates
Arc levels either by regulating estradiol-
induced transcription of Arc [Kuhnle
et al., 2013] or by direct ubiquitination
of Arc [Greer et al., 2010]. Arc in turn
regulates surface expression of a-amino-
3-hydroxy-5-methyl-4-isoxazole-pro-
pionic acid receptors (AMPARs). When
Ube3a is deficient, Arc levels are
excessive, and the excitatory postsynap-
tic AMPARs are internalized, which
impairs synaptic transmission [Greer
et al., 2010]. Using a Ube3a/Arc
double knockout mouse model, Man-
del-Brehm et al. showed that reducing
the levels of Arc decreases abnormal
brain spiking in AS mice and rescues the
increased duration of freezing (seizure-
like) behavior in response to an audio-
genic stimulus. However, other behav-
ioral measures (ultrasonic vocalizations,
hind limb-clasp phenotype, hypoactiv-
ity) in the double knockout mice
remained unchanged compared with
AS mice [Mandel-Brehm et al., 2015].
Ephexins are guanine nucleotide
exchange factors (GEFs), which modu-
late the development of excitatory
synapses. Ephexin5 (E5) is one such
GEF, which functions to suppress excit-
atory synapse development. Ube3a
mediates the degradation of E5, so
when Ube3a is absent, E5 levels are
elevated and excitatory synapse
9
development is impaired [Margolis
et al., 2010]. Reducing E5 levels did
not alter the audiogenic stimulus-in-
duced seizure like freezing behavior in
AS mice [Mandel-Brehm et al., 2015].
Because the common deletion
causing AS includes three g-amino-
butyric acid type A (GABAA) receptor
subunits: b3, a5, and g3, GABAergic
dysfunction is thought to account for
the observation that the deletion sub-
type of AS has a more severe phenotype
than AS caused by other molecular
mechanisms. GABA, the main inhibi-
tory neurotransmitter in the mammalian
central nervous system, acts to regulate
muscle tone and reduce neuronal excit-
ability. In frozen post-mortem cortical
samples of four human AS brains,
Roden et al. [2010] found alterations
in GABAA receptor subunit expression
that were consistent with impaired
cortical extra-synaptic GABAergic ac-
tivity. GABA transporter 1 (GAT1)
activity is increased in AS mice, pre-
sumably because Ube3a deficiency de-
creases GAT1 ubiquitination. Since
GAT1 takes up GABA from the extra-
synaptic space, tonic inhibition, a
GABAergic form of continuous inhibi-
tory transmission mediated through
specialized GABAA receptors, is de-
creased in the cerebellar granule cells of
AS mice due to reduced availability of
GABA in the extra-synaptic space
[Egawa et al., 2012]. Tonic inhibition
regulates neuronal excitability in the
entire brain, including the neocortex,
hippocampus, thalamus, hypothalamus,
basal ganglia, and cerebellum, as well as
the spinal cord, and in retinal ganglion
cells. In various mouse models, loss of
tonic inhibition results in increased
seizure susceptibility and in some in-
stances, sleep disturbances, depending
on the GABAA receptor subtype that is
knocked out [Lee and Maguire, 2014].
Egawa et al. also found that cerebellar
ataxia, as demonstrated by gait analysis
and the clasping reflex, improved when
the AS mice were treated with 4,5,6,7-
tetrahydroisothiazolo-[5,4-c]pyridin-3-
ol (THIP), an extra-synaptic GABAA
receptor agonist selective for those
receptors that contain the d subunit.
These results suggested that the
10 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
cerebellar ataxia in AS is mediated
through reduced tonic inhibition
[Egawa et al., 2012]. In light of these
findings, Ovid Therapeutics Inc. (New
York, NY) will be conducting a phase 2
randomized double-blind placebo-con-
trolled trial of THIP, also known as
gaboxadol, in adults with AS to assess its
safety and potential efficacy in this
population [Ovid Therapeutics Inc.,
2016]. However, a recent study sug-
gested that the “locomotor deficits” in
AS mice, as determined by the acceler-
ating rotarod test, are not due to a
cerebellar defect but may be due to
loss of Ube3a in the “extracerebellar
motor circuits” instead [Bruinsma et al.,
2015].
As previously indicated, dendritic
spine abnormalities are seen in mouse
models of AS [Dindot et al., 2008].
Shortly after LTP induction, dendritic
spine actin polymerization occurs, and
this is tightly correlated with LTP
stabilization, consolidation and memory
[Lynch et al., 2008; Simmons et al.,
2009; Baudry et al., 2011]. The induc-
tion of LTP in the hippocampal slices of
AS mice is apparently normal, but LTP
stabilization, which requires dendritic
spine actin polymerization in response
to the inductive stimulus, is severely
impaired in AS mice. Deficits in LTP
stabilization and spine actin polymeri-
zation are reversed in the hippocampal
slices by short-term treatment with an
ampakine, which also reversed deficits in
contextual fear conditioning in AS mice
[Baudry et al., 2012]. Ampakines work
by binding to the glutamatergic AMPA
receptor, enhancing fast excitatory
transmission, facilitating LTP and upre-
gulating brain-derived neurotrophic
factor (BDNF) (reviewed in [Chang
et al., 2012; Panja and Bramham, 2014]).
AS mice have defective BDNF signaling
(NOT necessarily concentration) due to
elevated Arc levels [Cao et al., 2013],
and BDNF is required for proper
postsynaptic signaling [Yoshii and Con-
stantine-Paton, 2007; Yoshii et al.,
2011]. Interestingly, in 12 children and
adults with AS, it was found that the
plasma BDNF concentrations were
elevated compared to typically develop-
ing controls. Although the clinical
implications of this finding are unclear,
the authors postulated that this may be
related to the unusually happy personal-
ity in AS since individuals who are
depressed have reduced BDNF concen-
trations [Wink et al., 2015].
In a pilot study of 11 children and
adults with AS, it was found that the
plasma concentrations of amyloid pre-
cursor protein (APP) and its break-
down products were higher in those
with AS compared to age- and gender-
matched controls, and the levels of total
APP and APP alpha were positively
correlated with plasma BDNF concen-
trations, but the levels of total APP and
its breakdown products did not correlate
with the two neurodevelopmental out-
come measures that were used in the
study [Erickson et al., 2016]. The
clinical implications of these findings
remain unclear, but the authors sug-
gested that APP (or its breakdown
products) might be a therapeutic target
in the future.
Because of perceived clinical and
neuropathological similarities between
AS and schizophrenia, Kaphzan et al.
[2012] examined AS mice for dysregu-
lation of the neuregulin 1 (Nrg1)-
ErbB4 pathway, which is implicated in
the pathophysiology of schizophrenia.
NRG1 signals through its receptor
tyrosine kinase ErbB4 (Erb-B2 Recep-
tor Tyrosine Kinase 4). Nrg1 concen-
trations are significantly increased in the
hippocampi of AS mice compared to
wild-type littermates; high levels of
Nrg1 result in constitutive activation
(phosphorylation) of ErbB4. Constitu-
tive activation of ErbB4 results in
impaired LTP [Kwon et al., 2005;
Pitcher et al., 2008], and ErbB inhibitors
can rescue LTP impairments in AS mice
hippocampal slices [Kaphzan et al.,
2012]. Deficits in contextual fear con-
ditioning, a hippocampus-dependent
form of associative memory, were
rescued in AS mice by post-training
injection of an ErbB inhibitor into both
hippocampi. Neither Nrg1 nor ErbB4
interacts directly with Ube3a. More-
over, there were no differences in the
expression or phosphorylation status of
AMPA receptors or N-methyl-D-aspar-
tate (NMDA) receptors in the AS mice
RESEARCH REVIEW
compared to the wild type mice, so it is
unlikely that the increased Nrg1-ErbB4
signaling is mediated through these
ionotropic glutamate receptors [Kaph-
zan et al., 2012].
The mechanistic target of rapamy-
cin, mTOR, plays critical roles in brain
development and synaptic plasticity.
mTOR is the catalytic subunit of two
complexes, mTORC1 and mTORC2
(mechanistic target of rapamycin com-
plexes 1 and 2, respectively). Sun et al.
found increased activity of mTORC1
with increased phosphorylation of some
of its substrates including 40S ribosomal
protein S6 and ribosomal protein S6
kinase beta-1 (S6K1), but decreased
phosphorylation of mTORC2 sub-
strates such as AKT in the cerebellum
of AS mice. These changes in
mTORC1 and mTORC2 activity
were mediated by an increase in the
concentration of TSC2 (tuberous scle-
rosis complex 2), which is usually
degraded by UBE3A. Treatment with
intraperitoneal rapamycin (also known
as sirolimus), an mTOR inhibitor,
corrected the activity of both complexes
and improved motor function and
dendritic spine morphology in the AS
mice [Sun et al., 2015a]. These authors
subsequently reported that treatment of
AS mice with intraperitoneal rapamycin
also led to an improvement in LTP and
in their learning and memory as deter-
mined by the fear conditioning assay
[Sun et al., 2016].
Reelin is a ligand of the apo-
lipoprotein receptor family, which is
primarily secreted by GABAergic in-
terneurons. Reelin regulates AMPA
receptor function and NMDA receptor
insertion, and it is necessary for proper
neuronal migration as well as adult
neuronal synaptic plasticity. Purified
recombinant Reelin injected into the
cerebral ventricles of WT [Rogers et al.,
2011] and AS [Hethorn et al.,
2015] adult mice enhances LTP, in-
creases dendritic spine density, and
improves associative learning and
memory. Reelin did not rescue motor
deficits in the AS mice [Hethorn et al.,
2015].
Another substrate that is ubiquiti-
nated by UBE3A for endocytosis is the
RESEARCH REVIEW
small conductance calcium-acti-
vated potassium channel protein 2
(SK2). SK2 is a postsynaptic potassium
channel that regulates LTP and long-
term depression (LTD), and hence
synaptic plasticity. As expected, there
was an increase in the density of SK2
channels in the postsynaptic membrane
of hippocampal neurons derived from
AS mice compared to wild type mice
[Lizarraga and Morrow, 2015; Sun et al.,
2015b]. Interestingly, treatment of wild
type mice and rats with apamin (a SK
antagonist) resulted in an improvement
in their learning and memory [Moreno
and Giralt, 2015]. When hippocampal
slices from AS mice were incubated with
apamin, there was an improvement in
LTP and LTD; when AS mice were
treated with intraperitoneal apamin,
there was an improvement in their
learning and memory, as determined
by the fear-conditioning assay [Sun
et al., 2015b]. These data suggest that
an excessive concentration of SK2
channels on the postsynaptic membrane
in neurons contributes to the patho-
physiology of AS, including the LTP and
LTD deficits. Fluoxetine, a serotonin
selective reuptake inhibitor (SSRI), has
been found to block all three SK
channels in vitro [Terstappen et al.,
2003]. Treatment of adult AS mice
with subcutaneous fluoxetine partially
restored hippocampal neurogenesis, but
learning and behavioral assays were not
reported for these fluoxetine-treated
mice [Godavarthi et al., 2015], and
there are no reports on the outcome of
any individual with AS who has received
fluoxetine.
Mitochondrial Dysfunction
AS mice exhibit deficits in hippocam-
pal LTP, and Su et al. [2011] observed
that the hippocampal mitochondria of
AS mice are small with disorganized
cristae and reduced complex III activ-
ity. Coenzyme Q10, a cofactor in the
electron transport chain, is frequently
prescribed as a supplement in patients
with mitochondrial disease. Adminis-
tration of a coenzyme Q10 analogue
increased mitochondrial complex III
and complex IV expression in
AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
hippocampal neurons of AS mice,
normalized the electroencephalogram
(EEG) pattern, and improved motor
coordination and anxiety [Llewellyn
et al., 2015]. More recently, it was
found that mitochondrial superoxide
production was increased in AS mice
compared to wild type mice, but
treatment with intraperitoneal MitoQ
10-methanesuflonate, an antioxidant
that contains coenzyme Q10, normal-
ized the superoxide concentration as
well as hippocampal LTP and contex-
tual fear conditioning. These data
suggest that oxidative stress may con-
tribute to the learning and memory
deficits seen in AS [Santini et al., 2015].
Strategies Addressing Specific
Symptoms
Seizures
Seizures occur in about 80–95% of
individuals with AS with the age of
onset being less than 3 years old in about
75% of them (reviewed in [Thibert et al.,
2013]). The epilepsy of AS is considered
a generalized epilepsy, but partial seiz-
ures are seen commonly as well. The
mechanism of epileptogenesis in AS is
not well understood (reviewed in [Pelc
et al., 2008a]), but a recent mouse study
suggested that susceptibility to seizures is
caused by loss of Ube3a in cortical
GABAergic, but not glutamatergic,
neurons resulting in neuronal hyperex-
citability [Santini and Klann, 2016].
Based on a questionnaire survey of
461 respondents (of which 86% indi-
cated a family member with AS had
experienced seizures), Thibert et al.
[2009] reported that the most frequently
reported types of seizure were atonic,
generalized tonic-clonic, atypical ab-
sence, and partial complex seizures; focal
motor, tonic, secondarily generalized
and myoclonic seizures as well as
infantile spasms and Lennox–Gastaut
syndrome were also reported. Some
authors report myoclonic seizures as one
of the most common seizure types
[Valente et al., 2013]. The average age
of onset of seizures was 2.9 years (earlier
in those with deletions) [Thibert et al.,
2009]. Both convulsive and non-con-
vulsive status epilepticus are reported
11
with an unknown prevalence, but in
probably no less than 10%. Those with
deletions had the highest rates of
epilepsy, and those with imprinting
defects the lowest. Pelc et al.
[2008a] reported that approximately
half of patients with AS have multiple
seizures types. Those with the larger
deletions tend to have more frequent
and disabling seizures and are more
likely to need polytherapy compared to
those with the smaller deletions [Valente
et al., 2013]. There are no obvious
candidate genes in the non-overlapping
deletion region (NIPA1, NIPA2,
CYF1P1, GCP5) that readily explain
this observation.
Although valproic acid, and to
a lesser extent, clonazepam,
were the most commonly-
prescribed anti-epileptic
medications in AS,
levetiracetam, lamotrigine,
and clobazam were perceived
to be more efficacious,
especially when compared
with clonazepam.
Although valproic acid, and to a
lesser extent, clonazepam, were the most
commonly-prescribed anti-epileptic
medications in AS, levetiracetam, lamo-
trigine, and clobazam were perceived to
be more efficacious, especially when
compared with clonazepam. More im-
portantly, compared to the newer anti-
epileptic medications, valproic acid was
associated with more side effects, in-
cluding thrombocytopenia, pancreatitis,
tremor, balance, and gait difficulties
[Thibert et al., 2013; Shaaya et al.,
2016]. Carbamazepine and phenobarbi-
tal had the lowest rates of efficacy, the
highest rates of seizure exacerbation, and
the highest rates of side effects [Franz
et al., 2000; Dion et al., 2007; Thibert
et al., 2009]. Vigabatrin and oxcarbaze-
pine are also associated with seizure
12 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) RESEARCH REVIEW

exacerbation [Ostergaard and Balslev,
2001; Valente et al., 2006].
Non-pharmacological therapies for
epilepsy in AS include the ketogenic diet
(KD), low glycemic index treatment
(LGIT) and vagal nerve stimulator. KD
is a highly limited carbohydrate diet,
with approximately 90% of calories
coming from fat, intending to mimic
the fasting state. There are few random-
ized controlled trials of KD in children
[Neal et al., 2008; Neal et al., 2009], and
none in AS specifically [Levy et al.,
2012]. KD is associated with a significant
reduction in seizure frequency in a
pharmaco-resistant population (38%
vs. 6% in controls) [Neal et al., 2008],
and a recent systematic review by the
Cochrane Epilepsy Group agreed that
KD could be a viable therapeutic option
for children, but its efficacy has not been
demonstrated in adults with epilepsy
[Martin et al., 2016]. KD has been used
anecdotally in AS with great benefit in
some cases and remains a valuable
treatment modality for patients with
refractory epilepsy [Valente et al., 2006;
Evangeliou et al., 2010; Stein et al.,
2010]. The main side effects of KD are
gastrointestinal disturbances such as
constipation, metabolic acidosis, hyper-
lipidemia, and occasionally renal calculi
[Kossoff et al., 2009; Bergqvist, 2012].
There may be substantial difficulties
adhering to the diet, and in one series,
only about 10% of individuals remained
on it for an extended period of time
[Hemingway et al., 2001].
In order to address issues with
palatability leading to reduced adher-
ence, modifications to the KD have
been made. LGIT limits the amount of
carbohydrates to 10% of the diet and the
type of carbohydrates to those with a
glycemic index less than 50, while
permitting up to 30% protein. In a small
prospective study of LGIT in AS, five of
six patients experienced a more than
80% reduction in seizures (including
one patient who did not achieve the
target carbohydrate reduction), and 50%
became seizure free [Thibert et al.,
2012].
Given the challenges of adhering to
a low glycemic index diet and to
explore alternative means of inducing
ketosis for the treatment of epilepsy in
AS, Ciarlone et al. [2016] fed 4–6
week-old AS mice a ketone ester, R,S-
1,3-butanediol acetoacetate diester, in
addition to their regular rodent chow ad
lib for 8 weeks, after they had initially
fasted for 8 hr. The authors found that
the mice whose feeds were supple-
mented with ketone ester had lower
blood glucose and higher beta-hydrox-
ybutyrate levels compared to the con-
trol mice. Although the treated mice
did not show any improvement in their
anxiety and general locomotion, their
performance on the accelerating ro-
tarod and “wire hang” was better than
the untreated AS mice, suggesting an
improvement in motor coordination
and motor learning, although not as
well as the wild type mice. The treated
AS mice were also less likely to develop
seizures in response to audiogenic and
chemical (kainic acid) stimulation
[Ciarlone et al., 2016]. These findings
suggest that ketone ester supplementa-
tion may be an alternative to treatment
with KD or LGIT, although some
clinical experts opined that this strategy
might not work unless the “regular
diet” is at least somewhat ketogenic
[Ronald L. Thibert, Personal Commu-
nication, July 2016].
Corticosteroids have been used to
treat various forms of epilepsy, most
notably infantile spasms. Forrest et al.
reported four children with AS (all with
a deletion) who responded to predniso-
lone with cessation of seizures and
improvement in sleep disturbance.
When prednisolone was tapered, seiz-
ures frequently recurred, but were less
severe [Forrest et al., 2009].
Cannabinoids are being used clini-
cally to treat refractory epilepsy [dos
Santos et al., 2015; Fasinu et al., 2016].
Anecdotal evidence and preclinical
studies support this as a potentially
useful modality in some situations, but
no specific reports on the use of
cannabinoids in AS are available. Cur-
rently, there are three ongoing open-
label trials of cannabinoids in children
with epilepsy, but none are specific for
AS (ClinicalTrials.gov identifiers:
NCT02447198, NCT02397863,
NCT02523183).
Sleep
Sleep disturbance is one of the most
vexing problems in AS. Sleep disruption
in children with AS correlates positively
with sleep disturbance in adult care-
takers and with some measures of
parental stress [Didden and Sigafoos,
2001; Goldman et al., 2012]. There
appears to be a reduced need for sleep in
individuals with AS, and problems with
both the initiation and maintenance of
sleep have been reported [Goldman
et al., 2012]. Severe sleep problems
affect a significant portion of individuals
with AS. A questionnaire study of sleep
problems in 49 Italian individuals with
AS showed that 70% of those children
slept for less than 8 hr per night, and 32%
of them took longer than 30 min to fall
asleep. Sixty-two percent had more than
two awakenings per night, and 57%
reported difficulty in resuming sleep
after awakening at night. Poor sleep
quality was reported by 70% of re-
spondents. Only 24% reported daytime
somnolence and only 8% reported
falling asleep at school, indicating that
despite the sleep disruption, most indi-
viduals with AS do not exhibit daytime
somnolence [Bruni et al., 2004]. An-
other questionnaire study of 109 Dutch-
speaking individuals found 40% had
severe sleep problems, mostly consisting
of night-time wakening rather than
prolonged sleep latency [Didden et al.,
2004]. Walz et al. examined the sleep
patterns of 339 individuals registered
with the Angelman Syndrome Founda-
tion using a validated sleep question-
naire. Difficulty falling asleep and
sleeping less than peers were reported
in 48% and 42% of respondents respec-
tively [Walz et al., 2005]. Sleep problems
did not appear to abate with age,
although the sleep issues may be most
severe between the ages of 2 and 6 years
[Bruni et al., 2004; Didden et al., 2004;
Walz et al., 2005; Robinson-Shelton
and Malow, 2016].
In the only study to examine sleep
objectively in AS, Miano et al.
[2004] performed polygraphy on 15
children with AS and found decreased
sleep efficiency and REM sleep, as well
as an increased percentage of time spent
in slow wave sleep in those with AS
RESEARCH REVIEW
under the age of 8 years compared to a
control group of children, and decreased
REM sleep in those more than 8 years of
age compared with controls. Sleep-
disordered breathing and periodic leg
movements did not occur more fre-
quently in AS than in other disorders
with intellectual disability with or
without epilepsy [Miano et al., 2005].
The etiology of the sleep problems in
AS is unknown, but it is speculated to be
related to thalamocortical dysfunction
(reviewed in [Pelc et al., 2008b]). In
addition, pathophysiological processes
unrelated to the neurologic regulation
of sleep such as epilepsy, gastrointestinal
discomfort, and the use of certain
medications, may adversely impact the
quality or duration of sleep in any
individual. In a small study, melatonin
levels were found to be low in those with
AS [Takaesu et al., 2012], corroborating
the prior results of small open-label and
randomized placebo-controlled trials.
Use of melatonin shortened the time to
sleep onset, decreased sleep latency,
increased total sleep time, and reduced
night awakenings [Zhdanova et al., 1999;
Braam et al., 2008]. The effects of
melatonin are not unique to AS; melato-
nin has been shown to be effective in
diverse conditions associated with devel-
opmental disability [Braam et al., 2009;
Schwichtenberg and Malow, 2015]. Clo-
nidine was effective in a small cohort of
patients with severe intellectual disability
[Ingrassia and Turk, 2005] and it seems to
be effective in some patients with AS
(Bird & Tan, personal observations), but it
has not been studied specifically in the AS
population. Other medications that are
used less often in the AS population but
could be considered in specific circum-
stances include: (i) oral iron supplemen-
tation—if there is periodic limb
movement disorder or serum ferritin
levels are low, (ii) ramelteon (melatonin
agonist)—clinical benefit over melatonin
remains to be determined, (iii) gabapen-
tin—this is a precursor of GABA and
increases synaptic GABA concentration,
(iv) clonazepam—as a benzodiazepine,
there is an increased risk for daytime
somnolence and habituation, (v) zolpi-
dem, and (vi) trazodone [Blackmer and
Feinstein, 2016].
AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
Behavior modification can be ef-
fective in some patients with AS [Allen
et al., 2013], as it can be in other
conditions with developmental disabil-
ity [Grigg-Damberger and Ralls, 2013].
The importance of sleep hygiene,
including the imposition of fixed bed-
times, and a consistent bedtime routine
and sleep schedule in the management
of sleep disturbance is crucial. Allen et al.
[2013] showed that a behavioral “pack-
age” could result in independent sleep
initiation, modest increase in total sleep
time, and reduction of disruptive be-
haviors (both sleep- and non-sleep-
related). Severity of epilepsy correlates
with sleep problems, but whether more
severe seizures create sleep disturbance
or whether poor sleep patterns exacer-
bate epilepsy remains unclear [Conant
et al., 2009].
ANIMAL MODELS OF
ANGELMAN SYNDROME
AND FUTURE CHALLENGES
Much of what we know about the
pathogenesis of AS has come from
studies in animal models of AS that
could not possibly be conducted in
humans, and it is only through a better
understanding of the basic biology of AS
that investigators have been able to
develop potential therapies for AS that
are safe, specific, and potentially effec-
tive for AS such as some of those
described above. Animal models have
also been helpful in enabling investi-
gators to assess the efficacy and safety of
potential therapeutic compounds before
they are tested in human clinical trials.
The mouse Ube3a protein shares about
99% similarity with human UBE3A,
and murine Ube3a is imprinted just as
human UBE3A is. A few different AS
mouse models have been developed, all
of which recapitulate some, but not all,
of the human AS phenotypic findings.
Among the more commonly used
mouse models are one that harbors a
mutation in exon 2 of the maternally-
inherited Ube3a only resulting in a
complete loss of Ube3a expression [Jiang
et al., 1998], and one with a deletion
including part of exon 15 and all of exon
16 [Miura et al., 2002]. The phenotypic
13
features that are present in most of these
mouse models include impaired associa-
tive and contextual learning, seizures/
abnormal EEG, and gait ataxia [Jana,
2012]. The only other animal model
that has been widely used in studies of
AS has been Drosophila whose UBE3A
homolog, Dube3a, is about 62% identi-
cal to human UBE3A; these flies had
deficits in motor activities and “associa-
tive olfactory memory” [Gatto and
Broadie, 2011]. However, the substrates
of mouse Ube3a and Drosophila Dube3a
are not identical, so the findings from
Drosophila are not always translatable to
mice, much less to humans.
While animal models have been
useful in advancing our understanding of
AS, they have limitations. Some of these
mouse models have been incompletely
characterized [Margolis et al., 2015]. It
has been found that the genetic back-
ground and the age of the AS mouse
model greatly impacts the phenotype.
Huang et al. found that the AS 129S7/
SvEvBrd-Hprtb-m2 mice exhibited no
significant differences from their WT
counterparts in phenotypic assays that
assessed motor skills, but they were
deficient in their “reversal learning” skills
compared to the WT mice. In contrast,
the AS C57BL/6J mice demonstrated
marked deficiencies in various motor and
cognitive skills compared to their WT
counterparts. However, even among the
C57BL/6J mice, some of the differences
between the AS and WT mice were only
observed in mice in certain age groups
[Huang et al., 2013]. Similarly, “cued fear
conditioning” is only seen in the mutant
exon 15/16 deletion mouse on a C57
background strain, but not in the exon 2
deletion mouse on a 129 background
strain [Margolis et al., 2015]. As such, the
genetic background, the age of the mice,
and possibly the sex, should be taken into
consideration when interpreting the
results of studies using mouse models of
AS. Another major limitation of both
mouse and Drosophila models of AS is the
inherent inability to capture one of the
most striking phenotypes in AS—the
lack of expressive language. There is
therefore an urgent need to develop new
animal models of AS that recapitulate
more fully the human phenotype.
14 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
Since AS is a congenital disorder, it
has long been suspected that there are
“windows of opportunity” for the ame-
lioration of the phenotype beyond which
some phenotypic manifestations will not
be amenable to treatment. This suspicion
was further reinforced by findings from
the studies in which AS mice treated with
topotecan, an ASO, or viral gene therapy,
failed to normalize the phenotype
completely even though there were
improvements in some aspects of their
deficits [Daily et al., 2011; Huang et al.,
2012; Meng et al., 2015]. In a recent
study using an AS mouse model in which
the maternally-inherited copy of the
Ube3a gene is inactivated by a floxed
cassette that can be reactivated by Cre
recombinase, it was found that reactiva-
tion of Ube3a during embryonic devel-
opment resulted in phenotypically
normal offspring. In contrast, when
Ube3a was reactivated at different postna-
tal ages, viz. 3 weeks, 6 weeks, and
14 weeks of age, corresponding very
approximately to human childhood,
adolescence, and adulthood, the pheno-
typic deficits that can be rescued were
different. For example, reactivation of
Ube3a at 3 weeks old led to the complete
resolution of the “motor coordination”
deficit, whereas reactivation at 6 weeks
old resulted in only a partial rescue of the
same deficit, and reactivation at 14 weeks
of age failed to rescue this deficit.
Similarly, other phenotypic features
such as autism and anxiety could only
be rescued if Ube3a were reactivated prior
to a certain age. Interestingly, the epilepsy
phenotype could not be rescued postna-
tally [Silva-Santos et al., 2015]. These
findings underscore the critical impor-
tance of identifying and treating children
with AS as early as possible since a cure
may only be possible up to a certain (as yet
undetermined) age.
In a recent study using an AS
mouse model in which the
maternally-inherited copy of
the Ube3a gene is inactivated
by a floxed cassette that can be
reactivated by Cre
recombinase, it was found that
reactivation of Ube3a during
embryonic development
resulted in phenotypically
normal offspring.
The development of new thera-
peutic compounds for AS is currently
limited by the lack of valid and reliable
biomarkers that would allow for the
rapid detection of potential clinical
efficacy, rather than having to await
the outcomes of neurodevelopmental
assessments, which are generally not
valid if conducted at less than 6-monthly
intervals. Given the slow developmental
trajectories typical of most individuals
with AS, we do not believe that
meaningful changes can be detected by
assessments that are performed less than
1 year apart. Our knowledge of the
clinical presentation and phenotypes of
adults with AS is limited because most
clinical studies on AS have traditionally
focused on children. Hence, the natural
history of AS in adults needs to be
further explored in order to identify the
medical needs in that population.
Nevertheless, with the progress that
we have made in our understanding of
AS and the development of new genetic
engineering tools over the last 10 years,
an effective therapy for AS is an exciting
prospect for the near future. It is possible
that successful treatment of AS may
require a therapeutic “cocktail” com-
prising compounds that activate the
normally imprinted paternal UBE3A,
compounds that target some of the
pathways that underlie the pathophysi-
ology of AS, and compounds that
address some of the specific symptoms
as discussed above.
ACKNOWLEDGMENTS
The authors would like to thank Stormy
J. Chamberlain, Ph.D., (University of
Connecticut Health Center) for review-
ing and revising the sections in this
manuscript that describe imprinting in
Angelman syndrome.
RESEARCH REVIEW
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