REVIEWS

DISEASE MECHANISMS

Down syndrome and the complexity

of genome dosage imbalance
Stylianos E. Antonarakis
Abstract | Down syndrome (also known as trisomy 21) is the model human phenotype for all
genomic gain dosage imbalances, including microduplications. The functional genomic exploration
of the post-sequencing years of chromosome 21, and the generation of numerous cellular and
mouse models, have provided an unprecedented opportunity to decipher the molecular
consequences of genome dosage imbalance. Studies of Down syndrome could provide knowledge
far beyond the well-known characteristics of intellectual disability and dysmorphic features, as
several other important features, including congenital heart defects, early ageing, Alzheimer
disease and childhood leukaemia, are also part of the Down syndrome phenotypic spectrum. The
elucidation of the molecular mechanisms that cause or modify the risk for different Down syndrome
phenotypes could lead to the introduction of previously unimaginable therapeutic options.

Department of Genetic
Medicine and Development,
University of Geneva Medical
School and University
Hospitals of Geneva, 1 rue
Michel-Servet, 1211 Geneva,
Switzerland.
Stylianos.Antonarakis@
unige.ch
doi:10.1038/nrg.2016.154
Published online 28 Dec 2016
Down syndrome (DS) is the most commonly known
disorder of intellectual disability, with a frequency of
one in approximately 700 births worldwide. DS was
first clinically described in 1866 by Langdon Down in
London1. Almost a century later, in 1959, trisomy 21
(T21) was reported as the underlying genomic abnor-
mality in Down syndrome by Lejeune–Gautier–Turpin
at Necker Hospital in Paris, France2. The most useful
mouse model for T21 was generated by Davisson in 1990
(REF. 3), and the long arm of chromosome 21 (HSA21)
was fully sequenced in 2000 through an international
collaborative effort4. Owing to its role in DS and its small
size, HSA21 is the most-studied human chromosome
(Supplementary information S1 (figure)).
In the 16 years since HSA21 genome sequencing,
there has been considerable progress in understanding
the protein-coding gene content of HSA21, the content
of other functional genomic elements and the individ-
ual variability of the sequences of HSA21. In addition,
the development of several different mouse models of
T21 has facilitated many studies that have enhanced our
understanding of this syndrome. There has also been
substantial progress in understanding the effect of T21
on the cellular transcriptome and on cellular differenti-
ation, which has been studied using induced pluri­potent
stem cells (iPSCs). Important advances have been made
in understanding the phenotypic impact of the over​
expression of some HSA21 genes, the molecular basis
of the early onset Alzheimer disease (EOAD) that is seen
in DS, the molecular basis of the leukaemias that fre-
quently occur in DS and the identification of genomic
regions of HSA21 that harbour functional elements or
causative genetic variation for certain phenotypes, such
as congenital heart defects. Importantly, the past decade
has generated a cautious enthusiasm for endeavouring
to treat individuals with DS using drugs; this enthu-
siasm and encouragement originated from studies in
mouse models.
All these advances in elucidating the molecular
mechanisms that underlie the phenotypes and diseases
associated with DS are discussed in this Review. The pro-
posed formation of an international Down Syndrome
Genomes Project is also briefly discussed in order to
initiate discussions and debate around this audacious
project, which aims to understand the genomic vari­
ability that contributes to the phenotypic variability of
DS. Several previous reviews have covered many aspects
of T21 and DS5–15; this Review discusses the most recent
developments and prospects for future research. The
widespread implementation of non-invasive pre­natal
testing 16,17 as a screening test for the fetal diagnosis
of T21 has been reviewed elsewhere18 and is thus not
discussed here.
HSA21 functional elements and variation
Since the complete DNA sequence of the long arm of
HSA21 was reported4, considerable progress has been
made in understanding the functional genomic units of
the chromosome (see FIG. 1 and Supplementary infor-
mation S2 (table)). This has been achieved by large
international projects such as the Encyclopedia  of
DNA Elements (ENCODE)19 and through the work
of individual laboratories that are interested in spe-
cific research questions. A complete understanding of

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a
Exons 3.6%
Protein coding (2.4%)
lncRNA, miRNA, antisense (0.7%)
Pseudogene (0.3%)
Other (0.2%)
Regulatory 12.4%
Promoter (2.4%)
Enhancer (5.7%)
Insulator (1.3%)
Open chromatin (3%)
Repeats 36%
DNA (2.4%)
LINE (14.7%)
SINE (9.4%)
LTR (7.7%)
Other (1.8%)
Not annotated (48%)

b p13 p12 p11.2 q11.2 q21.1 q21.2 q21.3 q21.11 q22.12 q22.2 q22.3

GRC37/hg19
chr21:26,651,798-27,651,798 (1Mb)

26,700,000 26,800,000 26,900,000 27,000,000 27,100,000 27,200,000 27,300,000 27,400,000 27,500,000 27,600,000

Chromatin–chromatin interactions (HiC)

Gene
RPL13AP7 MIR155HG JAM2 APP MARCKSP1
LINC00158 MIR155 RNGTTP1 ATP5J
LINC00515 GABPA
FDX1P2
MRPL39
Promoter (ENCODE CHMM, 7 cell lines)

Enhancer (ENCODE CHMM, 7 cell lines)

Insulator (ENCODE CHMM, 7 cell lines)

DNase I DGF (UW 7 cell lines)

Linkage disequilibrium (Phased Genotypes, CEU)

eQTL (GTEx, 44 tissues)

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Expression quantitative the functional nucleotides of HSA21 is of paramount
trait loci importance for the elucidation of the molecular basis
(eQTLs). Genetic variation and ­pathophysiology of the various phenotypes of DS.
that is associated with gene HSA21 is the smallest human chromosome, and
expression variation.
its overall gene density per megabase is average for
Copy number variants the human genome at approximately 15 genes per Mb
(CNVs). One form of genetic (REFS 4,9). Gene density for protein-coding and pseudo­
variation in which sections of genes per Mb is also average; however, HSA21 is among
the genome are repeated and
the richest chromosomes for long non-coding RNA
the number of repeats varies
between individuals.
(lncRNA)-encoding genes but, by contrast, among
the poorest for microRNA (miRNA)-encoding genes
Indels per Mb (REF. 9). Some of these lncRNAs and mi­RNAs
A short name for insertions have emerged as important post-transcriptional regu-
and deletions in the genome.
lators in stem cells20,21. HSA21 is also relatively poor in
Acrocentric other non-coding RNAs (ncRNAs), as well as in long
Mammalian chromosomes in interspersed nuclear elements (LINEs) and short inter-
which the centromere is close spersed nuclear elements (SINEs) (see Supplementary
to one end. Chromosomes 13,
information S3 (figure) for numbers and types of tran-
14, 15, 21 and 22 are
acrocentric in humans.
scripts per functional category). Overall, and as per the
ENCODE project results, HSA21 is among the poorest
chromosomes in terms of functional DNA elements per
Mb. It is easy to speculate that this is one of the reasons
that T21 is viable in the postnatal period. Alternatively,
the viability of T21 could be due to the absence of any
genes on HSA21 for which trisomy is not compatible
with life after birth. Another alternative explanation is
that the presence of an extra HSA21 does not substan-
tially disturb the mitotic mechanism and thus somatic
cells with T21 (and without additional abnormalities)
could be continuously produced in vivo.
Interestingly, HSA21 shows significant enrichment
for encoded proteins that are located in cytoskeletal
structures compared with the rest of the genome, as
can be seen when using the tool gostat (see Further
information). These cytoskeletal proteins are known
to be involved in neurological disorders, in particu-
lar, Alzheimer neuropathology 22. Among the pro-
teins known to be encoded by HSA21, 23 proteins are
involved in signal transduction and 31 proteins are
◀ Figure 1 | Functional elements of HSA21. a | Hierarchical annotation of the functional
fraction of the long arm of HSA21 (21q). The functional fraction of 21q (comprising gene,
regulatory regions and open chromatin) includes approximately 16% of the total
sequence. The non-annotated portion is likely to also contain functional elements.
This fraction includes all introns and intergenic sequences (data from build hg19 and
REFS 156–159). The short arm of HSA21 is not included here because its sequences are
shared with those of the short arms of all other acrocentric chromosomes, and the exact
sequences have not yet been completely elucidated. b | A representative 1 Mb segment
of HSA21 sequence. The chromatin interaction domains (topologically associating
domains; TADs) as determined by Hi‑C are shown below the sequence. Genes,
promoters, enhancers, insulators, DNase hypersensitivity (all likely to be functional
elements) are listed in the tracks below. The linkage disequilibrium domains (from
genomes of European ancestry) and the expression quantitative trait loci (eQTLs;
genomic variation associated with the gene expression variation) in 44 tissues identified
by the GTEx (genotype tissue expression) project are shown at the bottom. The figure has
been created with the data from the genome browsers USCS and ENSEMBL. The seven
Encyclopedia of DNA Elements (ENCODE) cell lines are GM12878, H1hESC, HepG2,
HSMM, HUVEC, K562 and NHLF. CEU, samples of European ancestry from the Centre
d’Etude du Polymorphisme Humain; CHMM, continuous hidden Markov model; DGF,
digital genomic footprinting; LINE, long interspersed nuclear element; lncRNA, long
non-coding RNA; LTR, long terminal repeat; miRNA, microRNA; SINE, short interspersed
nuclear element; UW, University of Washington.
transcription factors that may influence the expression
of other genes in the genome. Studying the overexpres-
sion of transcription factor genes on HSA21 will pro-
vide important information about the proportion of the
entire transcriptome that is influenced by each of these
genes, and by their combined action.
In one such recent study, which utilized the power of
mouse cellular models, chromatin immunoprecipi­tation
sequencing (ChIP-seq) analysis detected the binding of
the HSA21‑encoded transcription factor single-minded 2
(SIM2) to more than 1,000 high-confidence DNA sites in
mouse embryonic stem cells following the overexpres-
sion of the SIM2 gene. These sites overlap with regions
of occupancy of master transcription factors, which
define a particular sub-category of enhancers known as
the superenhancers23, which provides an explanation for
how SIM2 can regulate its gene network and can result
in some of the DS phenotypes.
HSA21 encodes several classes of ncRNAs, the
overexpression of some of which may contribute to
different phenotypes of DS. A recent study of the non-­
ribosomal transcriptome of endothelial progenitor cells
in T21 provided initial evidence for the dysregulation
of the non-coding transcriptome in DS24. For example,
HSA21 miR-155 was found to be overexpressed, which
may be associated with the lower blood pressure that is
usually observed in DS. miR-155 downregulates angio­
tensin receptor 1 (AGTR1) in an allele-specific man-
ner by binding the 1166A allele of AGTR1 but not the
­hypertension-associated 1166C allele25.
The genomic variability of HSA21 is of considerable
clinical importance, as a fraction of this chromosome is
functional and could contribute to the phenotypic vari­
ability of DS. Some single nucleotide variants (SNVs)
alter codons in important functional parts of certain
proteins, whereas other SNVs occur in transcription
factor-binding sites and could alter levels of gene expres-
sion. Some SNVs have been identified as expression
quantitative trait loci (eQTLs) for gene expression26, and
as controlling differential methylation, splicing or chro-
matin marks27–30. Furthermore, there are close to 1,000
copy number variants (CNVs), approximately 500 indels
and 25 inversions that have been mapped to HSA21 in
different individuals. All of these variants are candi-
dates for contributing to the phenotypic variability of
T21. I argue that the sequence of HSA21 and the rest
of the exome or genome should be determined in every
individual with DS in order to define the genomic vari­
ants that are associated with or that cause the different
phenotypic features of the syndrome.
Gene expression dysregulation domains
T21 is probably a disorder of gene expression, as the
basic genetic abnormality is the presence of an additional
copy of the 45 Mb acrocentric HSA21. The overexpres-
sion of the protein-coding genes and ncRNAs is likely
to disturb several cellular functions and developmental
processes. In addition, the supernumerary HSA21 may
result in a global disturbance of the transcriptome owing
to the additional chromosomal material, regardless of its
genic content or regulatory repertoire.

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Lamina-associated domains
(LADs). Regions of the
chromatin that interact with
the nuclear lamina.
Replication domains
Regions of the genome that are
copied (replicated) at the same
time during the S phase of cell
division.
Thus, the study of the transcriptomic differences
between normal cells and T21 cells and tissues is of
fundamental importance in understanding the molec-
ular basis of the different phenotypic features of T21.
Studies using microarray and RNA-Seq (see REFS 8,31
for additional references) have confirmed the increased
expression of the majority of the HSA21‑encoded genes
in DS individuals and have also indicated transcript
differences for genes on other chromosomes. There is,
however, a substantial problem regarding these tran-
scriptome comparisons, as the gene expression variation
among individuals introduces considerable ‘noise’. The
abundance of genomic variants identified as eQTLs in
the genome substantially alters allelic expression and
thus for most genes there is wide variation in transcript
levels across individuals. In an early study by Prandini
et al.32 on HSA21 gene expression in cell lines from
unrelated individuals, only 39% and 62% of the studied
HSA21 genes in lymphoblastoid and fibroblast cell lines
respectively showed a significant difference between T21
and normal samples. More than 50% of the transcripts
studied showed substantial overlap of the distribution
of expression levels between T21 and normal samples in
both cell types. This problem is even more pronounced
in transcriptomic comparisons for genes outside of
HSA21. Therefore, the natural variation of gene expres-
sion masks the effect of the extra chromosome on the
overall transcriptome.
One possible solution to this problem is to study the
differences in gene expression between samples from
monozygotic twins discordant for T21; in this case, the
whole genome sequence is identical and thus all
the regu­latory variation is shared between the samples,
with the only differences originating from the super-
numerary HSA21. An alternative solution is to sepa-
rate the cells from mosaic T21 tissues and to establish
pure populations of cells; in this case, the genomes are
also identical except for the presence of an extra copy
of HSA21 in one of the two cell populations. Our lab­
oratory was fortunate to identify a pair of monozygotic
twin fetuses that were discordant for T21 (REF. 33). The
extra HSA21 in the T21 fetus was maternal in origin
and had a recombination 4 Mb from the telomere of
21q. RNA-Seq analysis revealed the expected over-
all increase in gene expression of HSA21 genes; there
were also numerous differentially expressed genes on
other chromosomes. Surprisingly, however, the over-
all genome-wide differential expression in fibroblasts
between the normal and T21 monozygotic twin sam-
ples was organized along all chromosomes into domains
that were either upregulated or downregulated in T21.
These were termed gene expression dysregulation
domains (GEDDs)34. The GEDDs were well conserved
in iPSCs derived from the twins’ fibroblasts. The com-
parison of the transcriptome of the fibroblasts from the
Ts65Dn mouse model of DS and normal littermates also
showed GEDDs along the mouse chromosomal regions
that were syntenic in humans. Overexpression domains
in humans were also overexpressed in the syntenic chro-
mosomal regions in the mouse. The GEDDs correlated
with lamina-associated domains (LADs), as defined by
the van Steensel laboratory 35, and replication domains in
mammalian cells. The overall position of LADs was not
altered in trisomic cells; however, the active promoter
chromatin mark profile of H3K4me3 (REF. 19) in T21
fibroblasts was modified and accurately followed the
GEDD pattern.
These results indicate that the nuclear compartments
of T21 cells undergo chromatin modifications that influ-
ence the overall transcriptome and that GEDDs may,
therefore, contribute to some phenotypes of T21. The
relation of GEDDs to specific phenotypes has, however,
not yet been studied, and it would be challenging to
prove which phenotypes are linked to GEDDs. These
GEDDs could not be observed in transcriptome compar-
isons of unrelated T21 fibroblasts and normal fibroblasts
(as would be expected due to variation in gene expres-
sion); however, when only genes with low coefficients of
variation of expression are considered a highly statistical
difference is observed in the fold-change in expression
between T21 samples and normal samples for genes at
the overexpressed GEDDs compared with those at the
underexpressed GEDDs.
These results provide preliminary evidence that T21
could be considered a disorder that affects chromatin
function and that the resulting phenotypes may be due
to two reasons: first, the overexpression of HSA21 genes
(coding and non-coding); and second, the dysregula-
tion of the expression of genes that are not on HSA21.
In addition, the chromatin alterations observed in T21
may not be T21 specific but may also occur in other
aneuploidies or in normal cells under specific types
of stress.
The hypothesis that could be immediately tested
is that the overexpression of certain genes on HSA21
causes GEDDs: such candidate genes include HMGN1
(high mobility group nucleosome binding domain 1),
the product of which influences the structure and func-
tion of chromatin through histone modifications and
is considered to be an important modulator of gene
expression36. Other candidates are genes such as HLCS
(holocarboxylase synthetase), DYRKIA (dual specificity
tyrosine-phosphorylation-regulated kinase 1A), BRWD1
(bromodomain and WD repeat-containing protein 1)
and RUNX1 (Runt-related transcription factor 1),
which encode proteins that are involved in epigenetic
mechanisms and which may also influence chromatin
structure. The competing hypothesis is that any extra
chromosomal material of sufficient length may lead to
GEDDs; in this scenario, several trisomies, including
T13 and T18, may result in a similar general dysfunc-
tion of gene expression. This could also be extended to
­cancer cells with different aneuploidies. Current mouse
models of T21 will provide excellent experimental bases
to test these alternative hypotheses.
A recent study compared the transcriptomes of 58
paired tissue samples from high-quality post-mortem
human brains from individuals with DS and euploid
controls. The transcriptomes were assessed using an
exome array platform. The results of this study once
again showed that most of the dysregulated protein-­
coding genes do not map on HSA21, and that a

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Partial T21
A genomic alteration in which
a portion of chromosome 21
is triplicated.
Driver mutations
A mutation in a gene or other
functional genomic element
that provides a selective
advantage to a clone of cells.
Variant allele frequency
The frequency of a variant (not
reference) allele in each human
population.
substantial fraction of these differentially expressed
genes are associ­ated with oligodendrocyte differenti­ation
and myelination31. The involvement of the oligodendro-
glia was also observed in several iPSC differentiation
studies34,37,38. Another recent transcriptome study using
fibroblasts and blood cells from T21 and normal controls
showed the increased induction of interferon-stimulated
genes39, confirming an old idea that the interferon path-
way is a major signalling cascade that is dysregulated in
T21 (REF. 40).
DS and Alzheimer disease
Almost all patients with T21 develop neuropathology
that is similar to that of Alzheimer disease (AD), and
the majority of them develop dementia by the age of
60 years10,41. Individuals with DS now live longer than
they used to thanks to improvements in health care
and in care for people with disabilities; a recent study
in England and Wales estimated that the median life
expectancy of people with DS who were born in 2011
is 58 years42. Thus, a considerable number of individ-
uals with DS now reach the age of onset of AD. In fact,
T21 individuals constitute the largest fraction of people
with EOAD.
The proteins and cleavage peptides encoded by APP
(amyloid beta precursor protein) on HSA21 have been
extensively studied as important components of the
neuropathology of AD43,44. The deposition of the cleav-
age peptide amyloid beta (Aβ) in extracellular plaques
occurs decades earlier in DS than in non‑DS individu-
als. In DS–AD there are more and earlier plaques and
tangles in the hippocampus compared with EOAD
and late-onset Alzheimer disease (LOAD) in non‑DS;
most of the deposition in non-DS LOAD is in the cor-
tex 11. Therefore, it is important to determine whether
the presence of three copies of the APP gene in T21 con-
tributes to EOAD. Several lines of evidence support this
hypothesis: first, rare duplications of the APP locus (in
non‑DS individuals) causes autosomal dominant EOAD
with cerebral amyloid angiopathy 11,45, which is not
observed in individuals with full T21; second, genomic
variation within the promoter of APP that increases its
expression also increases the risk for AD46; and third, a
78‑year-old individual with partial T21 but with only two
copies of APP did not show the symptoms and neuro-
pathology of AD47. Conversely, in all the genome-wide
association studies reported so far no signal that has a
significant association with EOAD and that maps near
the APP locus has been detected. The expression of
APP (as for most genes) is variable across individuals
and different tissues (FIG. 2a), and thus the expression of
APP in different tissues per individual should be con-
sidered32. In addition, this expression variation results
in an extensive overlap between trisomy and normal
tissues (FIG. 2b). Recent publicly available data from
the GTEx project (see Further information)48 on the
gene expression in different tissues from a considera-
ble number of subjects have shown that APP expres-
sion is poorly correlated among blood, skin and brain
(FIG. 2c–d); therefore, the reported correlations between
APP expression and AD in T21 and non‑T21 patients
may not be reliable, as these correlations are based on
expression in skin fibroblasts and blood rather than in
brain. However, the GTEx project has revealed several
genetic variants (eQTLs) that control the expression of
APP in different tissues, including several brain regions;
these eQTLs could be used in well-powered studies as
risk biomarkers for APP expression levels and Aβ depo­
sition. The expression variation of APP (and the other
genes involved in this pathway) due to cis-regulatory
variation could explain the observation that levels of
the soluble Aβ42 peptide (an Aβ isoform that is 42 amino
acids in length) are increased in approximately 50% of
T21 fetal brains49. Other HSA21 genes that are involved
in the processing of APP include DYRK1A, SUMO3
(small ubiquitin-like modifier 3), BACE2, ETS2 and
miR155 (REF. 17).
Increased APP expression in the Ts65Dn mouse
model has been shown to disrupt transport of nerve
growth factor (NGF) and may thus cause the degener-
ation of basal forebrain cholinergic neurons (BFCNs)50
that occurs in T21 and AD51–53. These observations were
linked to abnormal endocytosis, a process that is essen-
tial for neuronal function and the traffic of neurotrophic
factors54. It has recently been shown that APP increases
the activation of RAB5 to cause the enlargement of
endosomes and that it disrupts the retrograde axonal
trafficking of NGF signals55; thus, dysregulation of RAB5
in response to APP overexpression may contribute to
EOAD in DS. HSA21 genes other than APP that affect
the secretory-endosomal function, and which thus may
also be implicated in EOAD in DS, include DOPEY2,
cystatin B (CSTB) and synaptojanin 1 (SYNJ1)56,57.
DS and leukaemia
Children with T21 are at an increased risk of two dif-
ferent types of leukaemia. The presence of a super-
numerary HSA21 confers a 20‑fold increased risk of
B‑lymphoblastic acute leukaemia58,59, and a 500‑fold
increased risk of acute megakaryoblastic leukaemia
(AMKL) 60 compared with the aged-matched gen-
eral population. Thus, T21 provides the opportunity
to better understand the molecular mechanisms of
­leukaemo​genesis58,61,62 (FIG. 3).
Acute B‑lymphoblastic leukaemia in DS. Children with
acute B-lymphoblastic leukaemia in DS (DS‑ALL) have
poorer survival rates than non‑DS children with ALL;
this is predominantly due to a much higher incidence
of relapses63. The current understanding of tumori­
genesis in DS‑ALL is that the primary somatic events
are mutations or rearrangements of CRLF2 (cytokine
receptor-like factor 2) or genes encoding other lympho-
cyte differentiation factors, which are detected in the vast
majority of DS-ALLs64–66. The progression of the disease
is associated with secondary activating driver ­mutations
in either JAK2 or RAS (NRAS and KRAS) genes, which
coexist with those in CRLF2 and other primary driver
genes but which are frequently characterized by a
smaller variant allele frequency. Mutations in JAK2 or RAS
are present in roughly one-third of DS‑ALLs, and are
almost completely mutually exclusive66. Preliminary data

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a 500

400

300

b 4
APP
RPKM

Normalized expression values

200 3

(qPCR)
2

100

0
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am
isp e

bl
be
eb

cu uc
m eb
fr

ex
en

le
oc
re

ac N
at

he er
n

ho
m
Ce
ai

in
pp
ud

Lymphoblastoid Fibroblasts
ta

W
Br

Sk
Hi

Pu

cell lines
Nucleus accumbens

Putamen basal
Caudate basal

Hippocampus

Skin exposed

Whole blood
Brain frontal

hemisphere
Cerebellum
Cerebellar
ganglia

ganglia

ganglia
cortex

c d
1
Brain frontal 1 0.57 0.52 0.13 0.2 0.65 0.42 ‑0.08 ‑0.13
cortex 0.8
APP expression, hippocampus (RPKM)

Caudate basal 1 0.52 0.44 0.55 0.61 0.76 ‑0.15 ‑0.04 300
ganglia 0.6
Cerebellar 1 0.49 0.29 0.48 0.46 ‑0.14 ‑0.27
hemisphere 0.4

Cerebellum 1 0.38 0.13 0.32 0.16 ‑0.08 0.2

1 0.46 0.45 ‑0.04 0.17 0 200

Hippocampus
Nucleus accumbens 1 0.57 ‑0.15 ‑0.05 −0.2
ganglia
Putamen basal −0.4
1 ‑0.06 0.02
ganglia
−0.6 100
Skin exposed 1 0.17
−0.8
Whole blood 1
−1
80 100 120 140 160
APP expression, skin (RPKM)
Figure 2 | Variation in expression of amyloid precursor protein. (Spearman correlation values are shown). Note the low correlation of
a | Example of expression variation of the amyloid precursor protein (APP) Nature
expression between the blood, skin and regions of Reviews
the brain. | Genetics
APP expression
gene in different tissues from samples collected and analysed in the GTEx is also poorly correlated among the different regions of the brain. Thus, for
project (REF. 48 and GTEx project). b | Example of gene expression variation APP transcription studies, it is not possible to use one tissue as a proxy
for the APP gene on HSA21. The expression levels of several samples from for another, and it is necessary to study the appropriate tissue for the
T21 and normal cells in lymphoblastoid cell lines and fibroblasts are shown. hypothesis that is being tested. d | The poor correlation of APP expression
Note the extensive overlap of gene expression variation among the 62 between the skin and hippocampus. Data from the GTEx project. qPCR,
samples tested. Data from REF. 32. c | Correlation of expression of the APP quantitative polymerase chain reaction; RPKM, reads per kilobase of
gene in different tissues from the GTEx (genotype tissue expression) project transcript per million mapped reads.

152 | MARCH 2017 | VOLUME 18 www.nature.com/nrg

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JAK2

B-lymphocyte • CRLF2
B-lymphocyte • Chromatin remodellers
proliferation • Haemato lineage TFs RAS DS-ALL
• Tumour suppressors
HMGN1 • Cohesin complex
Lymphoid
stem cell

Haematopoietic
stem cell

GATA1 (X-chr)
+

AMKL
Myeloid +
stem cell Additional
driver mutations
GATA1 (X-chr)
+
TMD
Megakaryoblast

Disappearance
of clones
Figure 3 | Leukaemogenesis in Down syndrome. Mechanisms of leukaemogenesis in the two types of leukaemia in
Down syndrome, acute B-lymphoblastic leukaemia (DS-ALL) and acute megakaryoblastic leukaemia Nature Reviews
(AMKL), Genetics
are| depicted.
In DS‑ALL (top part of the figure) the overexpression of the HMGN1 gene on HSA21 contributes to B‑lymphocyte
proliferation, and subsequent somatic driver mutations in either JAK2 or RAS genes cause the progression to DS‑ALL.
Additional somatic mutations that contribute to the leukaemogenesis are shown and include cytokine receptor-like
factor 2 (CRLF2), chromatin remodellers, haematopoietic-related transcription factors (TFs), tumour suppressors and
proteins of the cohesin complex. In transient myeloproliferative disorder (TMD) and AMKL (bottom part of figure) the
somatic mutation of GATA1 on the X‑chromosome (X-chr) together with the overexpression of genes on HSA21 leads to
TMD. Additional driver somatic mutations convert some of the TMD clones to AMKL. Alternatively, the TMD clones may be
eliminated, resulting in spontaneous remission.

Megakaryoblasts
Precursor cell to a
promegakaryocyte,
which in turn becomes a
megakaryocyte during
haematopoiesis.
suggest that the secondary driver mutation in DS‑ALL
may switch between the primary tumour and relapse,
for example, from JAK2 or PTPN11 to RAS but that the
primary driver mutation remains the same66.
Notably, none of the above-mentioned driver muta-
tions maps to HSA21. What then is the importance of the
extra HSA21 in DS‑ALL? The study of transgenic mice
(mouse model Ts1Rhr) revealed that a triplication of a
genomic interval orthologous to HSA21 that contains 31
genes confers self-renewal of mouse progenitor B cells.
Among these genes, overexpression of HMGN1, which
encodes a nucleosome remodelling protein, is associated
with altered chromatin marks, such as the suppression
of the repressive mark H3K27me3, and the proliferation of
B lymphocytes. HMGN1 overexpression remarkably links
the DS‑ALL and the GEDD observations discussed above,
providing a rationale for further ­investigations of the role
of this gene in phenotypes of DS67.
Transient myeloproliferative disorder and AMKL.
The occurrence of transient myeloproliferative dis­order
(TMD) in newborns with DS and the occurrence of
AMKL in children with DS shed light on a leukaemic
process that begins in the fetal period and that evolves
for several years. Approximately 10–15% of newborns
with DS present with TMD, that is, with circulating
­ egakaryoblasts, leukocytosis, anaemia, hepatomegaly
m some years later, usually between 1 and 4 years of age76.
and splenomegaly 68,69. The clonal expansion of mega­
karyoblasts is exclusively associated with somatic trun-
cating mutations in the GATA1 transcription factor gene
that maps on the X‑chromosome. These mutations elimi­
nate the full-length GATA1 protein and leave only the
short isoform, GATA1s. Interestingly, GATA1 mutations
are very rarely observed in leukaemic cells lacking T21
(REF. 70), further suggesting the necessity of ­interaction
between T21 and GATA1s in AMKL in DS.
The contribution of T21 to AMKL may be related to
the triplicate overexpression of the RUNX1 (REF. 71) and
ERG genes72, as the somatic triplication (focal amplifica-
tion) of the HSA21 region that contains these genes in
non‑DS individuals is associated with myeloblastic leu-
kaemia73,74. Recent work using iPSCs from T21 with or
without GATA1 mutations and with or without deletion
of a 4‑Mb region of HSA21 showed that T21 resulted
in the accelerated production of aberrantly differenti-
ated cells through the increased production of early
haematopoietic progenitors and the upregulation of
mutated GATA1 (REF. 75). These effects were mediated by
­dosage alterations of RUNX1, ETS2 and ERG genes that
are encoded within the 4 Mb deleted region on HSA21.
Remarkably, most infants with DS‑TMD spontan­
eously regress to complete remission. However, 20–30%
of the cases with regressed DS‑TMD develop AMKL

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REVIEWS

The leukaemic clones in these DS‑AMKL cases contain
the initial GATA1‑truncating mutations, but have also
acquired, through continuous mutagenesis, additional
driver mutations in known cancer genes70,77, including
cohesin component gene products and epigenetic regu-
lators. These third-hit driver gene mutations are similar
to those observed in non‑DS leukaemia genes (such as
FLT3, EZH2, APC, JAK and RAS).
Open questions on the fascinating molecular mech-
anism underlying the TMD to AMKL progression in
DS include the exact mechanism of the cooperation
between the GATA1 and HSA21 genes, the mutation
rate in cellular clones with GATA1 mutations, and the
sequential genomic and epigenomic changes that drive
leukaemogenesis78.
Congenital heart defects and T21
Among the most notable and relatively easily scor­
able phenotypes of DS are the various congenital heart
defects (CHDs). However, all of these display incomplete
penetrance, as only approximately 40% of DS individ-
uals present with some form of CHD79. The most fre-
quent forms of CHDs in DS are the atrioventricular
septal defects (AVSDs) constituting 43% of CHD cases,
whereas ventricular septal defects (VSDs), atrial septal
defects (ASDs) and tetralogy of Fallot constitute 32%,
19% and 6%, respectively. In fact, AVSDs are almost
exclusively seen in DS79,80. Attempts have been made
to map the genomic determinants of the CHDs in DS
on HSA21. By analysing rare partial T21 cases with
CHDs, investigators have identified minimal CHD can-
didate regions. One study identified a candidate region
Box 1 | The Down Syndrome Genomes Project
The current working hypothesis in the field is that the phenotypic heterogeneity and
variability in Down syndrome (DS) is primarily due to: first, the three copies of
functional genomic elements on HSA21; second, the genetic variation of HSA21; and
third, the genetic variation on non‑HSA21 loci. Thus, the variable phenotypes are likely
to be multifactorial, caused by variation at multiple loci and interactions between them
and with non-genetic factors.
I propose the formation of a new international collaborative effort, the Down
Syndrome Genomes Project, to collect samples, to establish and monitor the phenotype,
and to identify the genomic variation of a large number of DS samples from all geoethnic
groups in order to unravel genomic contribution to the phenotypic variability. The
genome exploration may include the generation of a high depth sequence of the entire
HSA21 (more than 100x coverage), an exome or genome sequence of more than 50x
coverage, and at a later stage a long-read sequence of HSA21.
The objective will be to identify not only the single nucleotide variants (SNVs) but
also the copy number variants (CNVs) and any other variability that may contribute to
the phenotypes of DS. The required number of samples needs to be determined but a
rough estimate of 5,000 would probably suffice to study the common phenotypic
features of DS. For example, in the case of the atrioventricular septal defects (AVSDs)
in DS with a prevalence of 16%, a genotype relative risk of a causative variant of 2,
and a variant allele frequency (VAF) of 0.3, a sample size of 300 cases (DS with
AVSDs) and 300 controls (DS without any congenital heart defects) provides 90%
statistical power to detect the risk allele154,155. Similarly, for the early onset Alzheimer
disease (EOAD) in DS a sample size of 1,000 cases and an equal number of controls
(DS without EOAD) would be required. Of paramount importance is the accurate
phenotypic characterization of the participating subjects and appropriate follow-up.
Cohorts of different ages will also be important in order to capture the entire
spectrum of the phenotypic variability.
of approximately 5.27 Mb on 21q22.3 (REF. 81) that was
later narrowed down to 1.77 Mb (REF. 82) (between genes
DSCAM and ZBTB21). In a similar study with differ-
ent partial T21 samples, the CHD candidate region was
mapped to an overlapping region of 15.4 Mb (REF. 83).
The mouse models Ts65Dn, Ts1Cje and Dp(16)2Yey,
which have partial T21 and which include CHDs in
their phenotypic manifestations, provided a candidate
interval that is compatible with the conclusions from the
human partial T21 (REFS 84–87). Recently, studies using
the mouse model Dp3Tyb further narrowed down the
CHD interval to a 5‑Mb region88. The use of a combined
approach that included Drosophila melanogaster, mouse
transgenesis and a cardiac myoblast H9C2 cell model
suggested that the combined overexpression of DSCAM
and COL6A2 contributes to ASD89.
The example of CHDs in DS illustrates the necessity
of a multidisciplinary approach to identify the tripli-
cated functional genomic elements that are necessary
for each phenotypic feature of DS. A detailed analysis
of the variation in HSA21 and the rest of the genome
that modifies the risk for CHDs was previously lack-
ing. However, recently, such a study was carried out
using cohorts of approximately 250 DS individuals
with or without CHDs. This study identified SNPs and
CNVs on HSA21 that significantly modify the risk for
CHDs and, in particular, AVSDs through genotyping
and comparative genomic hybridization using a tiling
array for the entire HSA21 (REF. 90). These findings
were not confirmed by a separate study, although the
genotyping methodology was different between the
two studies91. The search for the genomic variability
underlying CHDs in DS provides a proof‑of‑principle
for the large-scale sequence analysis of a sufficiently
large number of DS individuals that is needed in order
to identify the genomic determinants of the DS pheno-
types. The results of these analyses will advance not only
understanding of the molecular pathophysiology of DS,
but also understanding of similar phenotypes (includ-
ing AD, CHDs and intellectual disability) in non‑DS
­individuals (see BOX 1).
Candidate genes by haploinsufficiency
Some genes in the genome are categorized as haplo­
insufficient, as loss‑of‑function variants of one allele
result in a recognizable phenotype. An illustrative exam-
ple is the DYRK1A gene on HSA21; truncating mutations
in one allele cause one form of dominant intellectual
disability 92,93. It has been argued that haplo­insufficient
genes are also sensitive to three copies and are thus good
candidates for contributing to some of the phenotypes
in full or partial trisomies94. Indeed, several studies have
shown that the overexpression of DYRK1A and other
genes contributes to the phenotypes of DS94,95. Recent
studies have resulted in haploinsufficiency prediction
scores for most protein-coding genes96,97. The curated
and publicly available data from exome sequencing of
approximately 60,000 samples from the Broad Institute
(ExAC; see Further information)98, provide valuable
information on the tolerance of loss‑of‑function vari-
ants in each gene; genes intolerant to loss‑of-function

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7.5

GRIK1−AS2

7.0
S100B

KCNE1 SMIM11
TFF1
LRRC3 DSCR4
KCNE2 CLDN8 PCP4
CSTB SUMO3
TFF3 OLIG1
PIGP C21orf62
CLDN14 OLIG2 SOD1
ATP5J
6.5 CBR3 B3GALT5
YBEY HMGN1
N6AMT1 MRPS6
SH3BGR TFF2 CYYR1 CRYAA
−log 10 (P value)

TCP10L CBR1
AP000695.1 C21orf128 KCNJ15 WRB TMEM50B
CLDN17 MIS18A
SPATC1L
FAM207A PTTG1IP RCAN1
MAP3K7CL RIPPLY3 MRAP
ATP5O
C21orf91 CHODL BTG3
IL10RB C21orf33 HSF2BP RWDD2B UBE2G2 DSCR3
C21orf2 KCNJ6
FAM3B
RBM11 HSPA13 PSMG1 PDXK U2AF1
SLC5A3
LIPI ICOSLG EVA1C
RSPH1 IGSF5 IFNAR2 CXADR BACE2
C21orf59 IFNGR2 ETS2
6.0 DNAJC28 CLIC6 SAMSN1 RUNX1 ERG
C21orf58 AGPAT3
LCA5L WDR4 PKNOX1
POFUT2 SLC19A1 CHAF1B PCBP3
NDUFV3 CRYZL1 PRMT2
TMPRSS3 CBS JAM2 ADARB1 CCT8
SETD4 POTED
DNMT3L BACH1 MRPL39 IFNAR1 GABPA HUNK
HLCS ABCG1
FTCD
RRP1 TMPRSS2 SIM2 ZBTB21
SIK1
DONSON RIPK4 C2CD2
AIRE ADAMTS5 NRIP1
DYRK1A
MX1 TSPEAR ITGB2
ADAMTS1 PWP2
UBASH3A MX2 RRP1B GART NCAM2
USP16 MORC3
PFKL
GRIK1 PDE9A SLC37A1 APP
LSS TRAPPC10
TPTE PAXBP1
5.5 TMPRSS15 SCAF4
COL6A2 DIP2A COL6A1
UMODL1 PRDM15 USP25
TRPM2 TIAM1
COL18A1 MCM3AP
SON
LTN1
DOPEY2 SYNJ1
DSCAM
TTC3 ITSN1
BRWD1

PCNT

5.0
0 0.25 0.50 0.75 1.00
Haploinsufficiency (pLI)
Figure 4 | Haploinsufficiency of genes on HSA21. Plot of the haploinsufficiency (intolerability of loss‑of‑function
variants) index of HSA21 genes. The x‑axis contains the pLI metric from the EXaC database (see FurtherNature Reviews | Genetics
information),
which is a metric of the number of observed loss‑of‑function variants over that expected. The y‑axis contains the negative
log 10 (P value) for the estimated metric. Genes on the right of the plot, close to pLI of 1 (in the darker orange area) are
more likely to be haploinsufficient, whereas genes on the left, close to pLI of 0 (in the lighter orange area) are less likely to
be haploinsufficient. In red are genes that are causative for recessive disorders in which carriers with non-functional
alleles are unaffected. As expected, these genes lie mostly within the non-haploinsufficient area. Genes with the highest
haploinsufficiency index are most likely to contribute to a Down syndrome (DS) phenotype, whereas those with the
lowest haploinsufficiency index are least likely to contribute to a DS phenotype. More details on the calculation of the pLI
are provided in the EXaC FAQ page (see Further information).

variants in heterozygosity are likely to be haploinsuffi­
cient, whereas genes that tolerate such variants in
hetero­zygosity are more likely to be involved in reces-
sive Mendelian phenotypes. HSA21 protein-­coding
genes with high haploinsufficiency scores could then
be considered as good candidates for contribution to
DS phenotypes. Notably, when HSA21 protein-coding
genes are ranked according to haploinsufficiency (that is,
intolerability to loss‑of‑function variants), several of the
genes that rank highly (such as DYRK1A) have already
been associated with the pathophysiology of certain
­clinical features of DS (FIG. 4).

NATURE REVIEWS | GENETICS VOLUME 18 | MARCH 2017 | 155

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Mouse model studies of DS The continuation of studies in laboratory mice of

The elucidation and comparative analysis of the
genomes of human and laboratory mice revealed that the
long arm of human HSA21 is homologous to portions
of three mouse chromosomes: MMU16, MMU17 and
MMU10. It thus became clear that it would be difficult
to create a mouse model for trisomy of the entire HSA21.
Mouse models for T21 that have been introduced over
the years3,85,86,88,99–106 are described in BOX 2.
partial T21 is crucial for understanding DS, and cross-
ing existing mice could provide additional models.
For example, crossing mice in which single genes on
HSA21 have been disrupted could be used to system-
atically delete the extra copy of specific genes from the
trisomic genome, thus enabling the contribution of
each gene to be assessed. In addition, the introduction
of newer gene-editing methods could, in theory, make

Box 2 | Mouse models of T21

A mouse trisomic for mouse chromosome 16 (MMU16) was created in 1985,
but it was embryonic lethal. Myriel Davisson at the Jackson Laboratory in
1990 made a viable partial trisomy 16 mouse3 equivalent to a large segment
of HSA21. This mouse, Ts65Dn, has been extensively used for the
understanding of the molecular pathophysiology of Down syndrome as it
had abnormal cognitive phenotypes and neurophysiology findings. Ts65Dn
also carries a substantial segment of MMU17 not syntenic to HSA21.
Another mouse trisomic for a smaller segment of MMU16 (and a partial
monosomy for MMU12) was subsequently generated86 and the comparison
of its phenotypes was instructive in order to begin to dissect the genomic
segments that are involved in different phenotypes. The advancement of
techniques for the manipulation of the mouse genome has since resulted in
a substantial number of additional mouse models of partial ‘human T21’.
The figure schematically shows the different partial trisomy mouse models
(for genomic regions syntenic to HSA21) published to date. A similar
number of partial monosomies for regions syntenic to HSA21 have also
been made.
A whole ‘human T21’ mouse model was produced in 2010 through
appropriate crosses of partial trisomy mice for all syntenic regions of
HSA21 (REF. 85) (combination of models Dp(16)1Yey, Dp(17)1Yey and
Dp(10)1Yey; see the figure). There is currently an effort to create mouse
models for smaller regions of partial trisomy and to study the resulting
phenotypes. Most of these newer mouse models have not yet been
extensively studied but their availability and the collaborative spirit within
the T21 scientific community provides an enthusiasm for many discoveries
in the future.
In parallel, a transchromosomic mouse has been generated by introducing
a copy of the human HSA21 in mice105. Human HSA21 in the mouse has
several small deletions, duplications and rearrangements owing to previous
cell irradiation, but this Tc1 mouse continues to serve as a useful model,
particularly in the study of Alzheimer neuropathology that is seen in T21.
In general, mice with partial triplication of MMU16 show cognitive
phenotypes similar to those in T21 (using the Morris water maze, T‑maze,
fear condition and tests of synaptic plasticity), although partial trisomy
MMU10 mice do not show cognitive impairment. Partial MMU17 trisomic
mice show abnormal hippocampal long-term potentiation. Detailed
descriptions of the phenotypes in each particular mouse model can be
found in the original literature.
See the figure for a schematic representation of the mouse models of T21.
The syntenic fraction of MMU16 is show in blue, that of MMU17 in green,
and that of MMU10 in red. The extent of the partial triplication per mouse
model is shown below. The bordering genes for these triplicated regions are
also shown. The human HSA21 inserted in mouse, Tc1 (REFS 107,146), is
shown at the top of the figure. Duplicated regions are shown with thick
lines, and deletions are shown with thin lines. The references for these
mouse models are: Ts65Dn3, Ts1Cje86, Ts1Rhr101, Dp1Yah102, Dp2Yah103,
Dp3Yah108, Dp1Tyb–Dp9Tyb88, Dp(16)1Yey104, Dp(16)2Yey105, Dp(16)3Yey and
Dp(16)4Yey106, and Dp(17)1Yey and Dp(10)1Yey85.

Tc1

HSA21

MMU16 MMU17 MMU10

Hspa13 Il10rb mir802 Dyrk1a B3galt5 Zbtb21 U2af1 Cstb Prmt2

Lipi mir155 App Sod1 mir18a Cbr1 Kcnj6 Fam3b Rrp1b Pdxk
Hunk Ifnar1 Runx1 Dscr3 Igsf5 Abcg1
Tiam1 Umodl1
Dp9Tyb
Dp1Tyb
Dp3Yah
Dp(16)1Yey
Dp(10)1Yey
Dp2Yah
Dp(17)1Yey
Ts65Dn Dp1Yah
Dp(16)2Yey
Dp(16)3Yey
Ts1Cje
Dp7Tyb
Dp2Tyb
Dp8Tyb
Dp(16)4Yey
Dp4Tyb
Dp3Tyb
Ts1Rhr
Dp5Tyb
Dp6Tyb

Nature Reviews | Genetics

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Table 1 | iPSCs from trisomy 21

Samples Differentiation Conclusions Refs
DS and control 109
DS and control Neurons Cortical neurons derived from T21 iPSCs exhibited greater secretion 110
of amyloid peptides, tau protein phosphorylation and cell death
DS (free T21 and 111
translocation)
DS and control Neurons • Reduced number of neurons 112
• NPCs produced more APP
DS (isogenic, the extra Haematopoietic Higher colony-forming potential for erythroid, myeloid and 113
HSA21 was lost in one progenitors megakaryocytic lineages
iPSC)
DS and control Haematopoietic Increased propensity for erythropoiesis 114
progenitors
DS (isogenic) Neurons Impairment of neurogenesis 115
DS and control Neurons Defective early glial development 38
Mosaic DS (isogenic) Neurons Significant synaptic deficit of excitatory glutamatergic synapses 116
and inhibitory GABAergic synapses
DS Silencing of one HSA21 through insertion of XIST gene 117
Monozygotic twins DS Neurons • Reduced neurogenesis, more astroglia and oligodendroglia 34
and normal (isogenic) • DYRK1A inhibition corrected these defects 37
DS and normal Neurons • Astrocytes had higher ROS and lower synaptic molecules; oxidative 118
(isogenic and stress-mediated cell death and abnormal maturation of neurons
non-isogenic) • Partial correction with minocycline
Mosaic DS (isogenic) • Neurons • Reduced neurogenesis, mitochondrial dysfunction, increased 119
• Haematopoietic secretion of amyloid peptides
progenitors • Reduced and faster expansion of haematopoietic lineage
Full T21, partial T21 Haematopoietic Model for TMD; T21 perturbs haematopoietic development through 75
(del 4 Mb) and normal progenitors increased production of early haematopoietic progenitors and
with and without upregulation of the mutant GATA1; this in turn results in accelerated
GATA1 mutations production of aberrantly differentiated cells
APP, amyloid precursor protein; DS, Down syndrome; iPSCs, induced pluripotent stem cells; NPCs, neuronal progenitor cells;
ROS, reactive oxygen species; T21, trisomy 21; TMD, transient myeloproliferative disorder.

it possible to silence one allele of the triplicated genes
in these mouse models.
When studying genotype–phenotype correlations,
the specific regulation of gene expression in different
mouse strains and the polymorphic variability of extra
chromosomal material should be taken into account.
Mouse model studies may also be limited by the differ-
ential expression of mouse and human genes that results
from differences in the regulatory landscapes between
the two species.
Current mouse models are useful for studying not
only the cognitive defects of DS, but also additional
phenotypes, including CHDs and dysmorphic features.
For example, the most recent results of studies using
the mouse models in BOX 2 have established that the
region between miR-802 and Zbtb21 that contains 39
genes is sufficient to cause CHDs when present in three
copies88; further analyses of these models suggests that
three copies of at least two causative genes in this inter-
val are required for the appearance of this developmental
defect88.
Finally, mouse models can be used for the initial
evaluation of treatment modalities before clinical trials
in humans; for example, the epigallocatechin‑3‑gallate
(EGCG) inhibitor of DYRK1A107 and the GABAA antag-
onist picrotoxin108 were used for the treatment of mouse
phenotypes due to partial trisomy 16.
iPSCs with T21
An emerging cellular model approach for the study of
the developmental effects of T21 involves the generation
of iPSCs and subsequent differentiation to various cell
types and tissues. This experimental approach could
provide information on the impact of the supernumer-
ary HSA21 in neural, haematopoietic and cardiac devel-
opment, and in other processes, such as trophoblast
formation. In addition, iPSC lines upon differentiation
could be used to test drugs or other chemicals that could
correct the developmental defects of T21. All T21 iPSC
lines thus far were generated using fibroblasts from
patients, or amniotic fluid cells from pregnancies, and
appropriate controls were used in most studies. The first
such T21 iPSC line was reported in 2008; since then, 12
additional iPSC lines have been published34,37,38,75,109–119
(see TABLE 1 for a list of published iPSC lines and the
main observations per study). The continuation of
development of T21 iPSCs, central banking of these

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cells and their distribution to researchers with appro-
priate projects will certainly enhance our understanding
of some of the developmental processes that are affected
in T21. Because of the variability of iPSC lines produced
even from a single sample, more than one such iPSC
line per sample should be produced in order to partially
account for this limitation.
Therapy for DS?
Pharmacotherapy
The prospect of therapy for DS was unimaginable a dec-
ade ago. However, the initial knowledge of the protein-­
coding gene content of HSA21, the continuous efforts
to understand the signalling and other metabolic and
developmental pathways that are dysregulated in DS,
the availability of mouse models, the generation of T21
iPSCs and their differentiation to various cell types
and tissues, and the understanding of the numerous
neurodevelopmental alterations, now provide some
enthusiasm for potential therapeutic modalities. In
addition, the EOAD that develops in the majority of
DS individuals (who survive long enough) could serve
as a model to understand non‑T21 AD, and provides
a strong rationale for the pharmaceutical industry
to focus some efforts on the therapy of the different
­neurodevelopmental manifestations of DS.
The Ts65Dn mouse model has been extensively used
for preclinical studies of experimental pharmaceutical
therapies. It is likely that more recently described novel
mouse models will be used in the future, as the Ts65Dn
model carries an additional partial trisomy for MMU17,
and also has the problem of male sterility, which makes
it somewhat difficult to maintain a colony.
The current focus of the pharmacological treatment
of DS is on the improvement of the cognitive impairment
that is probably due to neurodevelopmental alterations,
neurotransmitter alterations and neuro­degeneration,
and is also targeted to the overexpression of selected
genes on HSA21.
Ideally, future therapeutic efforts should target the
embryonic and/or fetal period, as the neurogenesis and
neuronal migration in the neocortex takes place dur-
ing this period. Importantly, however, neuro­genesis
and neuronal migration in the hippocampal dentate
gyrus and the cerebellum continue to occur after birth;
remarkably, neurogenesis in the hippocampus120 contin-
ues at a slow rate throughout life. Thus, treatment may be
beneficial not only after birth, but also later in life. This
is particularly relevant to hippocampal neuro­genesis, as
most of the mouse therapies have so far been focused
on types of learning and memory that are related to the
hippocampal circuit physiology. A detailed description
of the mouse preclinical t­ herapeutic studies in general is
provided in REF. 121.
Some of these therapies were directed at antagoniz-
ing GABAergic transmission to diminish the excessive
inhibition of neurotransmission that is seen in DS,
whereas others were aimed at enhancing the function of
the NMDA receptor, serotonergic signalling, the endo-
cannabinoid system, noradrenergic transmission and
cholinergic transmission. Other therapeutic objectives
were to reduce neurodegeneration using antioxidants,
interfere with dysfunctional signalling pathways, and
to reduce the level or function of proteins encoded by
HSA21 genes.
As postnatal therapies are of more practical impor-
tance than prenatal therapies, I briefly discuss below five
different approaches for cognitive impairment therapy
that have generated considerable interest in the past
couple of years.
The inhibitory GABAergic system. Ts65Dn mice dis-
play excessive GABA-mediated inhibition, so drugs
that affect the GABAA receptors are a logical choice for
the therapy of cognitive impairment in DS. Negative
modu­lators (antagonists) of GABAA receptors (picro-
toxin (PTX) and phenyltetrazole) were first used in
2007 in the Ts65Dn DS mouse model, and resulted
in the recovery of cognition and long-term potenti-
ation108. These compounds were known to have pro-
convulsant and anxio­genic effects, and attention has
subsequently focused instead on selective negative
allosteric regulators of the GABAAa5 receptor 122, such
as compound RO4938581, which reduces GABAAa5
receptor-mediated inhibition and rescues functional
and neuromorphological deficits in Ts65Dn mice123.
A related compound, RO5186582 (Basmisanil; Roche)
has now completed phase II clinical studies in humans.
The preliminary results of these ­trials were recently
announced (Statement on CLEMATIS trial; see Further
information), but unfortunately there was no differ-
ence observed in primary and secondary end points on
improvement in cognition between adults (18–30 years
of age) and adolescents (12–17  years of age). The
­ongoing clinical trial in children (6–11 years of age) has
been discontinued. More detailed information is needed
to evaluate this clinical trial and to plan for the future of
this type of approach.
Kinase inhibitors of DYRK1A. The DYRK1A gene on
HSA21 has been implicated in several phenotypic fea-
tures of DS and Ts65Dn mice. DYRK1A is a tyrosine
phosphorylation-regulated kinase, the overexpression
of which is involved in neuronal deficits, atrophy of
dendritic spines, spine dysgenesis, precocious AD-like
neurodegeneration and cognitive deficits. It has thus
become an attractive target for therapy, as several com-
pounds could inhibit its kinase activity with differ-
ent specificity 95. The kinase inhibitors that have been
­studied so far in Ts65Dn mice include the polyphenols
in green tea124 and the purified polyphenol epigallo-
catechin‑3‑gallate (EGCG), both of which have shown
promising results125. Furthermore, EGCG reverses the
developmental abnormalities of iPSC differentiation in
T21 (REF. 126). Two clinical trials using EGCG in par-
ticipants 14–29 (REF. 127) and 16–34 (REF. 128) years of
age have been completed. The results of the latter trial,
which was carried out in Catalonia, Spain, have recently
been published129, and reported that EGCG combined
with 12 months of cognitive training was significantly
more effective than placebo and cognitive training
at improving visual recognition memory, inhibitory

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control and adaptive behaviour; there were positive
results in two of the 15 tests in the cognitive evaluation
battery of tests and one of nine adaptive skills in the
Adaptive Behaviour Assessment System II129. It is not yet
clear whether these results are over and above the null
expectation. As the authors suggest, phase III trials will
be needed to assess and confirm the long-term efficacy
of EGCG and cognitive training.
Activators of the Sonic Hedgehog pathway. The cere­
bellum in DS and Ts65Dn mice is smaller than in nor-
mal controls and contains a significantly decreased
number of granule and Purkinje cell neurons. This
pheno­typic characteristic has been functionally linked
to reduced mitogenic response to Sonic Hedgehog
(Shh) signalling 130. Treatment of the Ts65Dn trisomic
mice with SAG 1.1, a synthetic activator of the Shh path-
way restored the number of granule cell precursors and
mitotic cells to the levels found in euploid littermates130.
A follow-up study 131 using a single treatment of new-
born mice with SAG 1.1 resulted in normal cerebellar
morphology in adults; remarkably, this treatment also
resulted in behavioural improvements and normal-
ized performance in tasks for learning and memory.
However, despite the restored cerebellar morphology,
this treatment did not rescue the cerebellum-dependent
motor learning deficits. Currently, there are no known
clinical trials in DS using Shh activators.
Serotonergic system, neurogenesis and dendritic spines.
The neurogenesis defect and reduced dendritic spine
size in DS and Ts65Dn mice132 have also been linked to
the serotonergic system and reduced expression of the
serotonin 5‑HT1A receptor (HTR1A)133. Treatment of
these mice with fluoxetine (a selective serotonin reup-
take inhibitor) resulted in the complete rescue of defec-
tive proliferation in the hippocampal dentate gyrus,
subventricular zone, striatum and neocortex, and a
complete recovery of memory performance134. Several
additional anatomical and physio­logical deficiencies
were fully rescued 1–3 months after treatment 135–137.
These results provide a substantial body of prelimi-
nary evidence for the initiation of clinical trials in DS.
As fluoxetine is an approved drug for the treatment of
major depressive disorder, obsessive-­compulsive dis­
order and panic disorder, it would be faster and easier
to plan and perform such clinical trials in DS compared
with trials of unapproved or novel therapies.
Cholinergic system degeneration. BFCN degenera-
tion is a common finding in DS and AD, as well as in
the Ts65Dn mouse model; this degeneration is linked
to defects in explicit memory function and attention
and working memory. Protection of BFCN degenera-
tion by a high concentration of choline in pregnancy
has been proposed as a potential therapeutic inter-
vention. Prenatal treatment with choline in pregnant
mice resulted in the partial enhancement of cognitive
function and hippocampal neurogenesis in Ts65Dn off-
spring 138–140. The mechanism underlying these obser-
vations remains unclear. However, a clinical trial in
non‑T21 adults with AD using formoterol, a long-acting
β2 adrenergic agonist based on these preclinical results
is under way 141.
In conclusion, there are now several theoretical and
practical therapeutic scenarios, and undoubtedly addi-
tional ones will be proposed in the future. A scientifically
rigorous review of the existing data, and cautious enthu-
siasm will probably lead to well thought out clinical
trials with defined outcomes and objectives. The meas-
urable and objective outcomes need to be standardized
and debated for the different studies to be comparable.
International collaborations among scientists and physi-
cians, as well as the participation of patient groups, and
coordination among the funding agencies and industry
will benefit the patients and families on this exciting
road ahead.
Silencing of the extra chromosome 21: the XIST effect
The therapeutic dream in DS is to be able to silence, in all
cells, the expression of all genes on the supernumerary
HSA21 and to eliminate the trans-regulatory influence
of this extra chromosome. One extraordinary labora-
tory step towards this audacious goal was the silencing
of most of the protein-coding genes on one of the three
copies of chromosome 21 in T21 pluripotent stem cells,
which was achieved by the introduction of the XIST
gene into one HSA21 in the cultured trisomic cells.
XIST, the X‑inactivation non-coding gene, is located
on the X‑chromosome and is exclusively expressed on
the inactive X‑chromosome in female cells. The RNA
product of XIST accumulates across the interphase
inactive X‑chromosome and, through numerous hetero­
chromatin modifications, silences the transcription of
most of the genes located there142. Remarkably, the
introduction of a single copy of XIST into the DYRK1A
locus of one of the three copies of chromosome 21 in T21
pluripotent stem cells resulted in the silencing of most
protein-­coding genes on the XIST-bearing HSA21 with-
out affecting the expression of genes elsewhere on the
genome117. These results provide the technical possibility
of correcting the consequences of T21 (and potentially
other full-chromosome trisomies) by embryo manipu-
lation. It is not known whether the introduction of XIST
could rescue the phenotypic effects in mouse models of
DS as no such studies have yet been published. Even if
such mouse experiments are successful, and are with-
out considerable side effects, genome editing in human
embryos using the current CRISPR‑Cas9 technology
is far from acceptable, and a societal debate needs to
focus on this potential therapeutic approach that could
­theoretically be used in preimplantation embryos.
Deep brain stimulation?
As mentioned above, studies on Ts65Dn mice revealed
that one of the problems of DS lies with defects in
hippocampus-dependent learning and memory 143–147.
Experimental studies in humans with AD and rats with
amnesia have shown that deep brain stimulation (DBS)
to the fimbria-fornix could improve this phenotype148,149.
In a recent study 150, forniceal DBS rescued spatial learn-
ing and memory, and restored in vivo hippocampal

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initiated a vast field of research, and created a new

Environment Chromosome
21 DNA
Functional
Stochastic elements
events of Chr21
Down
syndrome
phenotypes
Epigenetic
modifications Variability
(non-genetically of Chr21
controlled)
Variability
Chromatin of other
structure
chromosomes
Figure 5 | Determinants contributing to the Down
syndrome phenotypes. The relative Nature Reviews
weight | Genetics
of these
determinants, and their interactions are not shown as
these are not fully known. Chromosome 21 DNA: the
presence of additional chromosomal material in the
nucleus that is approximately 45 Mb long. Functional
elements of Chr21: all the genomic regions that contain
a function, including protein-coding genes, non-coding
RNAs and regulatory elements. Variability of Chr21: the
nucleotide variability of this chromosome that could
influence the function of each element. Variability of
other chromosomes: the nucleotide variability
throughout the genome that has functional
importance. Chromatin structure: the particular
chromatin topology and composition. Epigenetic
modifications: particularly those that are not determined
by the primary DNA sequence.
long-term potentiation and hippo­campal neuro­genesis
in a mouse model of Rett syndrome (a genetic dis­order
with intellectual disability due to loss-of-function vari­
ants in the MECP2 gene on the X-chromosome151).
This successful, albeit preliminary, non-gene-specific
intervention in the Rett mouse model, now raises the
question: could forniceal DBS be successful in treat-
ing similar hippocampal-related defects in the mouse
­models of DS?
A vaccine against the Aβ peptides of APP?
An immunotherapy to reduce the levels of the Aβ amy-
loid peptides has been tested in the Ts65Dn mouse
model. The anti‑Aβ vaccine was given at 5 months of
age, and resulted in a modest, nonsignificant reduction
in brain Aβ levels152; however, there was an improve-
ment of memory deficits, and reduction of cholinergic
neuron atrophy. These initial results provide yet another
­therapeutic option that could be further explored.
Perspectives
The phenotypic expression and variability of T21 is
complex and complicated (as shown schematically in
FIG. 5). The discovery of the extra HSA21 in DS has
medical specialty, but the search for understanding
and treatment is far from completion. The advances in
the functional exploration of the genome, the reduc-
tion of the cost of sequencing DNA and RNA, the new
methods to study the brain, the ability to study the
genomics of single cells, and the interest of the pharma­
ceutical industry, provide renewed excitement for a
renaissance of translational and fundamental research
in DS.
Future research objectives that reflect the opinion
of the author are presented below (note that several
other research objectives could be added, and thus the
list is not exhaustive). These objectives include: first,
to understand the function of all functional elements
of HSA21, and to identify which functional genomic
elements in three copies have a phenotypic impact.
Second, to identify the genomic determinants of the
phenotypic variability in DS. The proposed Down
Syndrome Genomes Project (see BOX 1) aims to accom-
plish this task. Third, to identify the influence of T21
on the different cell types and tissues. Mouse models are
the most appropriate proxies for such purposes. Fourth,
to understand the impact of genome dosage imbalance
at the proteome level, as there is considerable ‘buffering’
from the transcriptome to proteome. Fifth, to intensify
research for drug therapies; the molecular targets for DS
therapies are expanding and single drug trials, or com-
binations of drugs that act on different targets, could
be tested. Sixth, to develop in vitro tissues (organoids
such as those described in REF. 153) from T21, and to
explore them as experimental models. Seventh, to use
iPSCs from T21 and to differentiate them to different
cell types. A considerable number of iPSC lines ori­
ginating from different individuals are needed to also
study the impact of genetic variation and epigenetic
modifications on the phenotypic variability. The iPSC
lines complement the mouse models, as they could help
us to understand phenotypic consequences that cannot
be studied in mice. Eighth, to use all the genomic and
chromatin biochemical and epigenetic marks and to
study the chromatin structure and topology in order
to understand the genome regulation in the different
cells or tissues. This genomic regulation is likely to be
determined by the disomic genome-wide variation,
and the trisomic genomic variation on HSA21. Ninth,
the continuous study of the brain development and
function is important to understand the intellectual
disability of DS, and the frequent EOAD. Tenth, the
metabolomes of different tissues (that is, the chemicals
with qualitative and quantitative alterations) in DS need
to be studied not only to identify biomarkers for differ-
ent phenotypes but also to understand the pathophysi­
ology of the various phenotypes of DS. An unbiased
approach may be necessary, as the tissue of primary
interest is not always obvious. The prime example of
phenylketonuria in which the resulting intellectual dis-
ability is due to the abnormality of an enzyme deficiency
in the liver and not in the brain is compelling; thus,
out‑of‑the-box hypotheses may provide unexpected
breakthroughs in DS.

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Acknowledgements
The author thanks A. Fort, Y. Hibaoui, S. Nikolaev, M.
Garieri, C. Borel, F. Santoni, Y. Herault, E. Yu, V. Tybulewicz,
X. L. d’Ardhuy for information and assistance in the prepara-
tion of this manuscript. The author also thanks the GTEx
investigators for data access, and the funding agencies SNF,
EU, ERC, Lejeune, and ChildCare for the support of research
in our laboratory. Furthermore, the author thanks all past
and present members of the laboratory, collaborators, and
the patients and their families for their inspiration
and support.
Competing interests statement
The author declares no competing interests.
DATABASES
ExAC: http://exac.broadinstitute.org/
GTEx project: http://www.gtexportal.org
gostat: http://gostat.wehi.edu.au/
FURTHER INFORMATION
Broad Institute: http://exac.broadinstitute.org/faq
Statement on CLEMATIS trial: http://www.roche.com/media/
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NATURE REVIEWS | GENETICS VOLUME 18 | MARCH 2017 | 163

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