European Journal of Human Genetics (2013) 21, 1253–1259

& 2013 Macmillan Publishers Limited All rights reserved 1018-4813/13

www.nature.com/ejhg

ARTICLE
The intellectual disability of trisomy 21: differences in
gene expression in a case series of patients with lower
and higher IQ
André Mégarbané*,1,2, Florian Noguier3, Samantha Stora1, Laurent Manchon3, Clotilde Mircher1,
Roman Bruno3, Nathalie Dorison1, Fabien Pierrat3, Marie-Odile Rethoré1, Bernadette Trentin3, Aimé Ravel1,
Marine Morent3, Gerard Lefranc4 and David Piquemal3

Trisomy 21 (T21), or Down syndrome (DS), is the most frequent and recognizable cause of intellectual disabilities. The level of
disability, as evaluated by the intelligence quotient (IQ) test, varies considerably between patients independent of other factors.
To determine the genetic or molecular basis of this difference, a high throughput transcriptomic analysis was performed on
twenty T21 patients with high and low IQ, and 10 healthy controls using Digital Gene Expression. More than 90 millions of
tags were sequenced in the three libraries. A total of 80 genes of potential interest were selected for the qPCR experiment
validation, and three housekeeping genes were used for normalizing purposes. HLA DQA1 and HLA DRB1 were significantly
downregulated among the patients with a low IQ, the values found in the healthy controls being intermediate between those
noted in the IQ þ and IQ  T21 patients. Interestingly, the intergenic region between these genes contains a binding sequence
for the CCCTC-binding factor, or CTCF, and cohesin (a multisubunit complex), both of which are essential for expression of HLA
DQA1 and HLA DRB1 and numerous other genes. Our results might lead to the discovery of genes, or genetic markers, that are
directly involved in several phenotypes of DS and, eventually, to the identification of potential targets for therapeutic
interventions.
European Journal of Human Genetics (2013) 21, 1253–1259; doi:10.1038/ejhg.2013.24; published online 20 February 2013

Keywords: Down syndrome; Trisomy 21; intellectual disability; gene expression; HLA

INTRODUCTION expression or transcriptome differences between them and healthy

Trisomy 21 (T21) or Down syndrome (DS) is a chromosomal controls using the Digital Gene Expression (DGE) technique. This
disorder resulting from the triplication of all or part of a chromosome pilot description may suggest a genetic indicator of better intellectual
21. It is a common birth defect, the most frequent and recognizable prognosis in DS patients.
form of intellectual disabilities (ID), appearing in about one out of
every 700 newborns. The average intelligence quotient (IQ) of
MATERIALS AND METHODS
children with DS is around 50, ranging between 30 and 70.
Subjects
Remarkably, a small number of patients have a profound degree of
After the analysis of nearly 1000 clinical files of DS patients at the Jérôme
ID, whereas others have a mild degree despite the absence of any Lejeune Institute, a series of 20 patients with a free and homogeneous T21,
genetic, cultural or familial favoring or disfavoring causes.1 aged between 18 and 40 years were enrolled in this study. Patients were
Recent progress in studies of patients with partial T21 and classified into two groups: those with a relatively lower IQ (IQ o20 or IQ ;
mouse models of T21 suggest that it will be soon possible to link four males and one female), and those with a higher one (IQ 470 or IQ þ ; six
characteristic phenotype changes with differential gene expression of males and nine females). The IQ was measured using the Columbia test, a tool
specific genes and help to decipher the molecular basis for these validated and largely used in similar settings in France.
abnormalities, which may lead to treatment of the most distressing Patients were not taking medications, had no neurological problems
aspects of this disorder.2 Such optimism is based on recent success (epilepsy, seizures, west syndromes, and so on), no changes suggestive of
with high-throughput genomic approaches in human medicine, early dementia, no autism, no endocrinological problems (hypo or hyperthyr-
oidism, diabetes, and so on), no sleep-disordered breathing problems,
gene expression signatures, and gene profiling studies that have
no hearing impairment or vision impairment, no heart problems, no
linked specific gene regulation to specific phenotypic abnormalities, immune deficiency, and no cancer. In addition, in none of the families of
and aided the diagnosis, the treatment, or the prevention of diseases the patients serious events were noted (death, frequent hospitalization, child
in DS.3,4 abuse, and so on).
The purpose of this paper is to identify a case-series of patients A third group of 10 individuals without T21 (four males and six females),
with lower (IQ ) and higher IQ (IQ þ ) and to describe gene aged between 26 and 39 years were added as controls.

1InstitutJérôme Lejeune, Paris, France; 2Unité de Génétique Médicale et Laboratoire Associé INSERM UMR_S910, Beirut, Lebanon; 3Skuldtech, Montpellier, France;
4Université Montpellier 2 et CNRS UPR 1142, Institut de Génétique Humaine, Montpellier, France
*Correspondence: Professor A Mégarbané, Institut Jérôme Lejeune. 37, rue des Volontaires, Paris 75015, France. Tel: +33 1 56 58 63 00; Fax: +9611421632;
E-mail: [email protected]
Received 21 August 2012; revised 27 December 2012; accepted 22 January 2013; published online 20 February 2013
 Gene expression and IQ in trisomy 21
A Mégarbané et al
1254

This study was granted approval from the Institute Jérôme Lejeune (Roche Applied Science, Meylan, France) and 4 ml of Forward and Reverse
Committee on Clinical Investigation and conformed to the tenets of the primers mix (final concentration in PCR reaction of 0.5 mM each).
Declaration of Helsinki. To discriminate specific from non-specific products and primer dimers, a
melting curve was obtained by gradual increase in temperature from 65 to
RNA samples 95 1C. PCR efficiency was measured on standard curves performed for each
Blood samples were collected for genetic studies after informed written consent primer pairs using a pool of all cDNA diluted as following: 1/10, 1/100, 1/1000,
was obtained from all parents or guardians on behalf of the participants 1/10000 dilution factor. Primer pairs with PCR efficiency o1.8 were excluded
because of their inability to provide consent, and from the healthy controls. of the analysis. The qPCR data were analyzed using the 2 (DDCT) method.6
RNA samples were extracted from lymphoblastoid cell lines. RNA samples Data were normalized using three housekeeping genes: LIMK1 (NM_002314),
were obtained from 4  106 cells pellet with RNeasy Plus Mini kit (Qiagen APEH (BC000362) and TUFM (NM_003321) (Table 1). The Partial Least
Courtaboeuf, France) and QIAshredder (Qiagen). Control of RNA integrity Squares Discriminant Analysis regression (PLSDA) analysis was performed
was performed with the 2100 Bioanalyzer (Agilent Technologies, Lesulis, with the R package mixOmics.7 The PLSDA was used to select the most
France) using Eukaryotic Total RNA 6000 Nano Chip (Agilent Technologies). discriminant genes. According to the mixOmics vignette and from the variance
RNA quantity was controlled using NanoDrop ND-1000 spectrophotometer importance plot, a score 41 represent a strong weight in the patient
(LABTECH, Palaiseau, France). discrimination.7

DGE library construction and tag-to-gene mapping RESULTS

Three DGE libraries were constructed from pooled RNA samples of patients By using Next-Generation Sequencing, we performed a transcrip-
IQ þ and IQ , and healthy controls. The libraries were constructed with tomic study using the open method, DGE, to reveal genes differen-
Illumina’s DGE Tag Profiling kit (ILLUMINA; SanDiego, CA, USA) according tially expressed in the two different selected phenotypes of DS. More
to the manufacturer’s protocol (version 2.1B), using 5 mg of total RNA (mixing than 90 million tags were sequenced in the two libraries. By taking
equal amounts of RNA from each individual). Sequencing analysis and base into consideration the DGE-tags with a minimal occurrence of two
calling were performed using the Illumina Pipeline, and sequence tags were
times, the two libraries revealed 68 046 unique tags. Raw and treated
obtained after purity filtering. Data from each DGE library were analyzed with
BIOTAG software (Skuldtech, Montpellier, France) for tag detection, tag
data were integrated in a database with associated tools for annota-
counting and for assessing DGE library quality.5 Raw and treated data are tion, in silico PCR, tag prediction and data visualization. This database
available on http://www.skuldtech.com/trisomie21. is accessible via a user-friendly website on http://www.skuldtech.
com/trisomie21 and can incorporate future data from the Next-
Tag annotation and selection Generation Sequencing.
A local database compiling Homo sapiens sequences and related information After data were filtered and classified according to the statistical
from well-annotated sequences of UniGene clusters (NCBI) was generated. For approach by DESeq and the fold induction among the well-annotated
each sequence of this database, the expected DGE tag (canonical tag) located tags, 80 genes were selected (Table 2). To explore the individual
upstream of the 30 -nearest NlaIII restriction site (CATG) of the sequence (R1), variability, individual qPCR analysis was performed.
as well as putative tags located in inner positions (labeled as R2, R3 and R4 The qPCR data analysis showed that two genes were sufficient
starting from the 30 end of the transcript) were extracted.5 Experimental tags enough based on our selection criteria (fold change expression 42.5
obtained from DGE libraries were matched and annotated (exact matches for
and SDo0.1) to discriminate our entire population between IQ 
the 17 bp) using this collection of virtual tags. First, a correspondence for each
and IQ þ . The PLSDA run on these data with the R package
experimental tag with the virtual canonical tags (R1) was looked for. Then,
unmatched experimental tags with the R2 tags, then with R3, and R4 were mixOmics7 showed that the genes HLA-DQA1 (NM_002122, major
annotated. Targeted tags were selected using R package DESeq (Anders S, histocompatibility complex (MHC), class II, DQ-a 1) and HLA-DRB1
Bioconductor) for processing data without replicates. The analyzed genes were (NM_002124, MHC, class II, DR-b 1) obtained the highest values
selected according to mathematic filters with the highest differential Fold (HLA-DQA1 got 2.84 and 2.22 for component 1 and 2, respectively,
Change (41.5), FDR adjusted P-value criterion (o10%, Benjamini-Hochberg and HLA-DRB1 got 2.56 and 1.90 for component 1 and 2,
Method) based on the type I (ar5%) error. respectively, of the PLSDA) from the variance importance plot.
Both expressions appeared to be less frequent in the IQ  group
cDNA synthesis and real-time PCR (Figure 1). Interestingly, the values found in the healthy controls were
Reverse transcriptions were performed for each of the 30 RNA samples in 20 ml between both those noted in the IQ  and IQ þ T21 patients. A
final reaction volume with 1.5 mg of total RNA using 200 units of SuperScript Monte–Carlo test on a factorial discriminate analysis gave a sig-
II enzyme (M-MLV RT Type, Invitrogen, St Aubin, France) and 250 ng of nificant P-value of 0.02.
random primers according to manufacturer’s instructions (25 1C 10 min, 42 1C
50 min, 70 1C 15 min), the same day with the same pipettor set and the same
manipulator. qPCR experiments were carried out using SYBR Green chemistry DISCUSSION
on LightCycler480 qPCR apparatus system I (Roche, Meylan, France). The T21 is a direct consequence of either an additional copy of protein-
reaction mix was prepared in a final volume of 10 ml as follows: 1 ml of cDNA coding genes that are dosage sensitive or an additional copy of non-
matrix (1/30 diluted in H2O) was added to 5 ml of SYBR Green I Master Mix protein-coding sequences that are regulatory or otherwise functional.

Table 1 PCR primers list for the two targeted genes and the three housekeeping genes (*)

Gene Gb id. Primer forward Primer reverse

0 0
HLA-DQA1 NM_002122 5 -GACCACGTCGCCTCTTATGG-3 50 -ACGTAGAACTGCTCATCTCCA-30
HLA-DRB1 AK290388 50 -TGTTCTCCAGCATGGTGTGT-30 50 -AGAAACGTGGTCTGGTGTCC-30
LIMK1* NM_002314 50 -ATGAGGTTGACGCTACTTTGTTG-30 50 -CCTCTCCCATACGTTCTTCCC-30
APEH* BC000362 50 -CTGGAACGCATGGAGAACATT-30 50 -CCGTCATGGAACACCAGGTA-30
TUFM* NM_003321 50 -GGGGCTAAGTTCAAGAAGTACG-30 50 -CACATGAGCCGCATTGATGG-30

European Journal of Human Genetics

 Table 2 Expression data from DGE analysis of the 80 selected transcripts in whole-blood cells in IQ þ versus IQ  patients

DGE-tag IQ þ IQ  P-value Sequence i.d. Cyt loc Hugo name Description GO annotation
0 0
5 -CAAATGAGGAGTGCCAG-3 0 541 1E 160 NM_001130523 1p13.2 CSDE1 Neuroblastoma RAS viral (v-ras) oncogene P:regulation of transcription,
homolog DNA-dependent
0 0
5 -TCCAAAGTAATGGAGAT-3 0 247 1E 72 BC000301 1p34 HDAC1 Histone deacetylase 1 F:histone deacetylase activity
50 -ATCAGTGGCTTTGAATG-30 0 239 3E 70 BC000331 1q21 PSMB4 Proteasome (prosome, macropain) subunit, P:small molecule metabolic process
b-type 4
50 -GAAGCCCCAGCTCAGCT-30 842 3 1E 124 BC066343 2p12 IGKC Immunoglobulin k-constant P:regulation of immune response
50 -ATATTTTAAATGTTAAG-30 0 183 2E 53 BC047933 2p15 B3GNT2 UDP-GlcNAc: b-Gal-b-1,3-N-acetylglucosa- F:transferase activity, transferring
minyltransferase 2 glycosyl groups
50 -TTCATACACCTATCCCC-30 432 27556 3E 118 BC001797 2p16.1 CCDC104 Coiled-coil domain-containing 104
50 -TTAACAACATTAAAAAC-30 22 64 2E 06 NM_001139488 2p25.1- RASGRP3 RAS guanyl releasing protein 3 (calcium and F:Ras GTPase binding
p24.1 DAG-regulated)
50 -TCATTGTAATGATTTCA-30 0 237 1.46E 69 BC014274 2q11.2 STARD7 StAR-related lipid transfer (START) domain
containing 7
50 -GAAAAATGGTTGATGGA-30 0 4692 3E 174 BC010054 3p22.2 RPSA Ribosomal protein SA P:mRNA metabolic process
50 -CGTGTTAATGGCTGTTC-30 0 171 7E 50 BC000288 3q21 CNBP CCHC-type zinc finger, nucleic acid-binding P:positive regulation of transcription from
protein RNA polymerase II promoter
50 -TCTTAATGAAGTTTGAA-30 0 286 3E 84 CR612348 3q28 EIF4A2 Eukaryotic translation initiation factor 4A2 F:helicase activity
50 -ATGTCATCAAATGGGTG-30 0 217 1E 63 NM_004068 3q28 AP2M1 Adaptor-related protein complex 2, P:intracellular protein transport
mu 1 subunit
50 -TGTGCTAAATGTGTTCG-30 0 673 2E 200 BC070208 4q25 RPL34 Ribosomal protein L34 F:structural constituent of ribosome
50 -ATACTTTAATCAGAAGC-30 0 186 2E 54 NM_001154 4q26- ANXA5 Annexin A5 F:receptor tyrosine kinase binding
q28|4q27
50 -TCTGCAATGAAGAGATT-30 0 372 1E 109 BC005288 5q13.3 NSA2 NSA2 ribosome biogenesis homolog P:rRNA processing
(S. cerevisiae)
0 0
5 -TTACTAAATGGTGTTAC-3 0 428 8E 127 NM_001746 5q35 CANX Calnexin P:protein secretion
50 -ACACTAAAATGGCAGAG-30 0 191 8E 56 BC011676 6p21.1 NFKBIE Nuclear factor of k-light polypeptide gene P:D-serine transport
enhancer in B-cells inhibitor, epsilon
50 -TTGATTTCTTAGCTGAC-30 425 1 6E 126 AF533900 6p21.3 HLA-DQA1 Major histocompatibility complex, class II, P:T-cell receptor-signaling pathway
DQ-a 1
0 0
5 -GCAGTTCTGACAGTGAC-3 1616 1 1E 119 AK290388 6p21.3 HLA-DRB1 Major histocompatibility complex, class II, F:peptide antigen binding
DR-b 1
0 0
5 -TAGATAATGGCCATCAT-3 0 372 5E 110 AK098772 6p21.33 TUBB Tubulin-b F:structural constituent of cytoskeleton
50 -GTCTTAAAGTGAGATTT-30 4 16 4E 03 NM_000636 6q25.3 SOD2 Superoxide dismutase 2, mitochondrial P:negative regulation of fat cell
differentiation
50 -TACATCCGAATGCTAAA-30 0 212 4E 62 NM_001128619 7q33 LUZP6 Myotrophin P:positive regulation of macromolecule
biosynthetic process
A Mégarbané et al

50 -AACAGAAGCAAATGATG-30 0 396 3E 117 BG480804 8q13|8q13 SNHG6 Small nucleolar RNA host gene 6 (non-
protein coding)
0 0
5 -ATCAAATGCAACCTCAC-3 0 364 1E 107 NM_002467 8q24.21 MYC V-myc myelocytomatosis viral oncogene F:sequence-specific DNA-binding transcrip-
homolog (avian) tion factor activity
0 0
5 -GACAATGCCAGCAAGAA-3 0 165 4E 48 NM_001001973 10p15.1 ATP5C1 ATP synthase, H þ transporting, mitochon- P:small molecule metabolic process
drial F1 complex, gamma polypeptide 1
50 -GCCATAAAATGGCTTTA-30 0 745 5E 222 NM_002727 10q22.1 SRGN Serglycin P:mast cell secretory granule organization
Gene expression and IQ in trisomy 21

50 -CTAAATATTCTTTCCTA-30 22 55 6E 05 NM_005445 10q25 SMC3 Structural maintenance of chromosomes 3 P:chromosome organization
50 -GGGACGGCGCGGAGGAG-30 29 51 4E 03 NM_030930 11q13 UNC93B1 Unc-93 homolog B1 (C. elegans) P:toll-like receptor-3 signaling pathway
50 -GTACAGGCTTGGAGCTT-30 85 32 3E 07 NM_012296 11q14.1 GAB2 GRB2-associated binding protein 2 P:phosphatidylinositol-mediated signaling
50 -GACTTTTCTGGGAAATG-30 0 179 3E 52 Z49194 11q23.1 POU2AF1 POU class 2 associating factor 1 P:regulation of transcription, DNA-
dependent
50 -AATAGGTCCAACCAGCT-30 9081 577 5E 123 NM_001028 11q23.3 RPS25 Ribosomal protein S25 P:mRNA metabolic process
50 -CCAGGAGGAATGCCTGG-30 0 243 2E 71 BC042163 11q24.1 HSPA8 Heat shock 70 kDa protein 8 F:ATPase activity, coupled
50 -TTTCTGTATGTTAATGA-30 0 415 6E 123 NM_001013699 12p11.21 H3F3C H3 histone, family 3C F:DNA binding
50 -GAAATGATGAGTCAGAA-30 0 250 3E 73 BM562920 12q12 PFDN5 Prefoldin subunit 5 P:cellular protein metabolic process
50 -GAAATGTAAGAGTGGAA-30 0 357 1E 105 AK123458 12q13.12- PCBP2 Poly(rC)-binding protein 2 F:RNA binding
q13.13
50 -GTGCTGAATGGCTGAGG-30 0 1162 9E 173 BC017455 12q13.2 MYL6 Myosin, light chain 6, alkali, smooth muscle P:skeletal muscle tissue development
and non-muscle
0 0
5 -TCACAAGCAAATGTGTC-3 0 5532 9E 206 NM_001113203 12q23-q24.1 NACA Nascent polypeptide-associated complex-a P:translation
subunit
50 -TTTCTGTGAAAATGTAT-30 0 232 4E 68 NM_004592 12q24.33 SFSWAP Splicing factor, suppressor of white-apricot P:regulation of transcription, DNA-
homolog (Drosophila) dependent
50 -CTGTTGATTGCTAAATG-30 0 334 1E 98 AK126454 13q14.3 HNRNPA1L2 Heterogeneous nuclear ribonucleoprotein F:RNA binding
A1-like 2

European Journal of Human Genetics

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 1256
Table 2 (Continued )

DGE-tag IQ þ IQ  P-value Sequence i.d. Cyt loc Hugo name Description GO annotation

50 -TCAAATGCATCCTCTAG-30 0 702 4E 209 AK126950 14q11.2 HNRNPC Heterogeneous nuclear ribonucleoprotein C F:nucleic acid binding
(C1/C2)
0 0
5 -GAGATCCGCAATGCTTA-3 0 745 5E 222 NM_006263 14q11.2 PSME1 Proteasome (prosome, macropain) activator P:small molecule metabolic process
subunit 1 (PA28-a)
0 0
5 -GAGATGCCTTTGGTGCT-3 17 3 1E 03 NM_005132 14q11.2-q12 REC8 REC8 homolog (yeast) P:meiosis
50 -GAAATAAAGCACCCACC-30 8003 739 1E 96 BC016381 14q32.33 IGHM Immunoglobulin heavy constant gamma 1 F:antigen binding

European Journal of Human Genetics

(G1m marker)
50 -TTTAATACATAGGTGAT-30 0 189 3E 55 NM_001014812 15q22.31 FAM96A Family with sequence similarity 96, F:molecular_function
member A
0 0
5 -TAATGGTAACTTGGACT-3 0 177 1E 51 NM_004255 15q24.1 COX5A Cytochrome c oxidase subunit Va F:metal ion binding
50 -TTTGTATTGCACAGATC-30 29 49 6E 03 NM_144572 15q24.3- TBC1D2B TBC1 domain family, member 2B F:phospholipid binding
q25.1
50 -AGACCTGTAATAAAATA-30 6 20 3E 03 NM_006565 16q21-q22.3 CTCF CCCTC-binding factor (zinc finger protein) P:regulation of histone methylation
50 -AAAAAGATAATGGAGAC-30 0 215 5E 63 AK295346 18q21.32 SEC11C SEC11 homolog C (S. cerevisiae) P:cellular protein metabolic process
50 -GCCTTATCTGTTCCAGT-30 33 9 1E 04 NM_002918 19p13.1 RFX1 Regulatory factor X, 1 (influences HLA class F:RNA polymerase II distal enhancer
II expression) sequence-specific DNA-binding transcrip-
tion factor activity
50 -TGTTAATGTTACGATGT-30 0 243 2E 71 AB209074 19p13.3 MKNK2 MAP kinase-interacting serine/threonine F:protein serine/threonine kinase activity
kinase 2
0 0
5 -CACGCAATGCTAGCTGC-3 0 654 1E 194 AK095154 19p13.3 AES Amino-terminal enhancer of split P:response to interleukin-1
50 -CCCCCAATGCTGAGGCC-30 0 684 1E 203 NM_007165 19p13.3- SF3A2 Splicing factor 3a, subunit 2, 66 kDa F:metal ion binding
p13.2
50 -GCCCAGCTCAAATGCTA-30 0 189 3E 55 AK308809 19q13.2 PSMD8 Proteasome (prosome, macropain) 26S P:proteolysis
subunit, non-ATPase, 8
0 0
5 -AAATGTTTGGCCTTAGT-3 0 368 8E 109 NM_012068 19q13.3 ATF5 Activating transcription factor 5 F:sequence-specific DNA binding
50 -CCTTCGAGATCATACAC-30 147 5856 2E 189 BG165682 19q13.4 RPS5 Ribosomal protein S5 P:mRNA metabolic process
50 -CCAGGAACAATGTCTCC-30 0 309 4E 91 AK054634 20p11 SNX5 Sorting nexin 5 P:cell communication
A Mégarbané et al
Gene expression and IQ in trisomy 21

50 -ACAAATCCTTGAATGTT-30 0 290 1E 85 NM_000801 20p13 FKBP1A FK506-binding protein 1A, 12 kDa F:transforming growth factor-b-activated
receptor activity
50 -AAGGAAGCAATGGTTCA-30 0 163 3E 47 NM_006392 20p13 NOP56 NOP56 ribonucleoprotein homolog (yeast) F:RNA binding
50 -TAAATAAATACATTCTT-30 0 6 5E 03 NM_000484 21q21.2 APP Amyloid-b (A4) precursor protein P:axon cargo transport
50 -GACTTGGCCCAAAAGAA-30 64 5 1E 14 NM_000819 21q22.11 GART Phosphoribosylglycinamide formyltransfer- P:small molecule metabolic process
ase, phosphoribosylglycinamide synthetase,
phosphoribosylaminoimidazole synthetase
50 -GCTTCCTAAATGGCCCT-30 0 6 500E 03 NM_000071 21q22.3 CBS Cystathionine-b-synthase P:cartilage development involved in endo-
chondral bone morphogenesis
0 0
5 -CTCAGGAAATAAATGTG-3 0 192 4E 56 BC005402 22cen-q12.3 UQCR10 Ubiquinol-cytochrome c reductase, complex P:small molecule metabolic process
III subunit X
50 -AACGCGGCCAATGTGGG-30 0 623 4E 185 BC036909 22q11.23 LOC284889 Hypothetical LOC284889 F:phenylpyruvate tautomerase activity
50 -AAACTAGAAATGTCATC-30 0 433 2E 128 AF230904 Xp22.1- SH3KBP1 SH3-domain kinase-binding protein 1 P:regulation of cell shape
p21.3
50 -CAATGTGTTATGTAGTG-30 0 197 1E 57 NM_004541 Xq24 NDUFA1 NADH dehydrogenase (ubiquinone) 1-a F:NADH dehydrogenase (ubiquinone)
subcomplex, 1, 7.5 kDa activity
0 0
5 -GTTAATAACAAATGAAT-3 0 35 2E 11 NM_006603 Xq25 STAG2 Stromal antigen 2 P:negative regulation of DNA
endoreduplication
50 -TGAAGGATGCCAATGGC-30 0 220 1E 64 NM_001008 Yp11.3 RPS4Y1 Ribosomal protein S4, Y-linked 1 P:mRNA metabolic process
50 -GAATCCAACTGCTTCGA-30 3597 176 5E 105 AJ712551 — — Transcribed locus, moderately similar to P:small molecule metabolic process
NP_061929.2 NADH dehydrogenase
(ubiquinone) 1-b subcomplex subunit 11,
mitochondrial isoform 1 (Homo sapiens)
50 -CTGCTATACGAGAGAAT-30 22 2699 2E 192 AV755966 — — Transcribed locus, strongly similar to P:mRNA metabolic process
XP_003086526.1 PREDICTED: 60S ribo-
somal protein L5-like isoform 3 (Mus
musculus)
50 -AGGGCAGGGAATGTGTC-30 0 175 4E 51 AW967735 — — Transcribed locus
50 -ACTTTTTCAAAAAAAAA-30 5286 213 3E 159 BE877281 — — Transcribed locus, strongly similar to F:helicase activity
NP_001460.1 X-ray repair cross-
complementing protein 6 (Homo sapiens)
50 -GGGACGGCGCGGAGGAG-30 29 51 4E 03 BG469659 — — Transcribed locus, moderately similar to P:toll-like receptor 3-signaling pathway
XP_001721426.3 PREDICTED: protein
unc-93 homolog B1-like (Homo sapiens)
 Gene expression and IQ in trisomy 21
A Mégarbané et al
1257

1.0 IQ –

0.8 IQ +

F:ubiquitin protein ligase binding

Control
0.6

The 17 bp DGE-tags are indicated in the first column. The 2nd and 3rd columns indicate the exact occurrences of the DGE-tags sequenced. The ‘0’ value indicate that a DGE-tag was not identified in one of the 2 groups. Some GO (http://
P:lamellipodium assembly
0.4

F:metal ion binding

P:apoptotic process
0.2
GO annotation

0.0
HLA-DQA1 HLA-DRB1
HLA-DQA1 HLA-DRB1
3.70 E-3 ± 4.4 E-3 0.384 ± 0.09
domain-containing E1, RNA binding (Rattus
XP_002729159.1 PREDICTED: cold shock

IQ –
tein RAD23 homolog A (Rattus norvegicus)
NP_001013208.1 UV excision repair pro-
Transcribed locus, moderately similar to

MRNA adjacent to 3- end of integrated

IQ + 1 1
NP_004885.1 6.8 kDa mitochondrial
proteolipid isoform 1 (Homo sapiens)
Transcribed locus, strongly similar to

Transcribed locus, strongly similar to

Control 0.129 ± 0.19 0.641 ± 0.18

CDNA clone IMAGE:6385453

Figure 1 HLA-DRB1 and HLA-DQA1 genes expression profiles in the two

conditions of IQ and one in Control patients for DS patients using the
2 (DDCT) method from qPCR data. The arbitrary value of 1 is given to the
IQ þ patient values. ± indicates the SD values.
Transcribed locus
Transcribed locus
Transcribed locus

HPV16 (INT290)
Description

norvegicus)

The effect of some dosage-sensitive genes on the phenotypes might be

allele-specific, and could also depend on the polymorphism of coding
and non-coding (regulatory, mRNAs, and so on) nucleotide
sequences and on the combination and interaction of all these
Hugo name

variants coding for proteins alleles with qualitative (alleles with

WASF2
SPOP

amino-acid variation) or quantitative (alleles with variation in gene

—

—
—

expression level) traits. Dosage sensitive genes could act directly to

induce pathogenesis or indirectly by interacting with genes or gene
www.geneontology.org/) annotations are listed in the last column with ‘P:*’ for Biological process and ‘F:*’ for molecular function.

products of either aneuploid or non-aneuploid genes or gene

Cyt loc

products.8 It is a reasonable speculation that the genetic

background of individuals has an important role in the variability
—

—
—
—
—
—

of phenotypic severity that is seen in DS.2

NM_001007226
NM_001098576

In order to understand why some T21 patients present a wide

Sequence i.d.

NM_006990
BM541359

BM553029

difference in their intelligence despite the absence of any known

BU566862
BG498681

BG680089

favoring or disfavoring factors, we initiate the construction of three

transcriptomic libraries, in T21 IQ  and IQ þ patients and in
healthy controls using the SAGE technique. Indeed, searching for
transcriptional alterations is an easier (or more effective) means to
1E 160

6E 223

8E 230
4E 118
1E 65

3E 70

2E 68
2E 10
P-value

find relevant loci than would be complete sequencing. Such approach

was already tested in DS and different pathologies.9–12
The low number of patients, especially in the IQ  group, was
secondary to the fact that we wanted to restrict this study only to the
224

541

239

1496
233
73
771
399

T21 patients that we could discriminate by IQ test. Interestingly, the

IQ 

group IQ  was predominantly males, whereas the IQ þ group was

frequently females with a sex ratio of 2/3. Few reports already pointed
the fact that boys with DS are more affected than girls but without
any reason.13
0

0
0
16
0
0
IQ þ

Two genes with major expression differences were found: HLA-

DQA1 and HLA-DRB1. Both genes were less expressed in the T21
IQ  population (Figure 1). HLA loci were already reported as
50 -CAAATGAGGAGTGCCAG-30

50 -CCACCCCGAATGGCTCA-30
50 -CTTCGGATGTCTTGGAG-30
50 -AATAAATGGATCTGTGA-30

50 -AATACTTAAATGCCAAC-30

leading to significant risk factor for celiac disease in DS patients,14 but

50 -TTTATTTAAGAAATGGA-30

50 -CAATAAATGTTCTGGTT-30
50 -TTTTTAATGTTGTCTGT-30
Table 2 (Continued )

never for ID. Nevertheless, HLA has been associated with cognitive
ability. For example, Cohen et al.15 showed that a proportion of
patients with primary neuronal degeneration of the Alzheimer type
have the HLA-B7 antigen, and that patients with HLA-B7 antigens
had selective attention span significantly lower than Alzheimer
DGE-tag

patients without the antigen. Recently, HLA-DRB1 has been

associated with cognitive ability in both demented and non-

European Journal of Human Genetics

 Gene expression and IQ in trisomy 21
A Mégarbané et al
1258

demented individuals,16 and a positive association of HLA-DR4 with overexpressed in the IQ  group v/s IQ þ group. The glycinamide
attention deficit and hyperactivity disorder and the role of the HLA- phosphoribosyltransferase (GART) gene, located on 21q22.11 was
DRB1 in the etiology of some types of childhood neuropsychiatric found overexpressed in the IQ þ group. GART is an essential
illnesses were reported.17 Future qualitative and quantitative studies at enzyme in de novo purine biosynthesis (OMIM 138440). In 1993,
the level of the HLA-DQA1 and HLA-DRB1 proteins on the PBLs of Peeters et al.34 studied the variations in mitotic index of lymphocyte
more IQ þ and IQ  DS patients might further validate our results cultures to which various metabolites of purine synthesis (inosine,
found at the transcriptional level. Likewise, the comparison between adenosine and guanosine) were added. They unexpectedly found a
the SNPs located within the DS critical region on chromosome 21 in significant decrease in mitotic index in DS patients without psychiatric
the two groups of DS patients might be informative. complications when compared with normal controls, and opposite
Interestingly, the intergenic region between HLA-DQA1 and HLA- reactions in DS patients presenting psychotic features. They concluded
DRB1, is characterized by the presence of a CTCF-binding CCCTC that T21 patients may have different purine metabolism depending on
sequence, XL9, of high histone acetylation and of particular impor- whether or not they have associated psychiatric complications. Purine-
tance for the control of the expression of both HLA genes and for the rich diet or the prescription of exogenous inosine, and serotonine-rich
chromatin architecture of the MHC class II locus.18–21 The fact that diet were used to treat psychotic T21 patients and some results in
CTCF expression is increased and transcription of HLA DQA1 and reduction of self-injurious behavior were reported.34,35 Our results
HLA DRB1 is decreased in the IQ  group could be in favor to a tend to give a beginning of explanation to the latter observations. The
repressor role for CTCF. On the other hand, the RFX1 gene, coding overexpression of GART in IQ þ patients might prevent the
for a protein that binds to the X-boxes of MHC class II genes and apparition of any abnormal behavior in T21 patients leading to a
which is essential in their expression, shows a three-fold decreased better IQ. Supplementing such patients with a purine-rich diet might
level of expression in the IQ  group in comparison to the IQ þ not be beneficial (as seen by the decrease mitotic index) on the
group. In contrast, in the IQ þ group, the downregulated CTCF gene contrary of patients with a lower expression of GART.
expression and the increased RFX1 gene expression could explain the Interestingly, DYRK1A (Dual-specificity tyrosine-(Y)-phosphoryla-
upregulation of the HLA-DQA1 and HLA-DRB1 genes. Furthermore, tion-regulated kinase 1A), located on chromosome 21q22.2 and
it was found recently that cohesin and CTCF-binding sites have a high known to have a significant role in developmental brain defects and
degree of overlap, and interacts with each other.20,22–24 Moreover, in early onset neurodegeneration, neuronal loss and dementia in DS36
often cohesin and CTCF have been found to colocalize at several did not show a significantly different expression between the IQ 
thousand sites in non-repetitive sequences in the human and IQ þ groups.
genome.22,24,25 In addition to the regulation of gene expression, In conclusion, we established a transcriptome of DS patients with
cohesin has also other important functions: it forms a huge tripartite IQ  and IQ þ and found that HLA-DQA1 and HLA-DRB1 may
ring, mediates the sister chromatid cohesion, and facilitates the repair discriminate between the two populations. However, the pools of
of damaged DNA.26,27 From our analysis, we can postulate that HLA- eligible patients available for such studies in particular centers are
DQA1 and HLA-DRB1 may represent a genetic biomarker for usually small, and therefore findings remain limited in their power to
predicting differences in ID conditions, but also that significantly rule in or out a marker that discriminates early between
polymorphisms or mutations of the cohesin subunits might have DS who will be IQ  and who will be IQ þ . Larger multicenter series
an important role for the non-disjunction of the chromosomes 21 will allow to determine in a valid way the presence of such markers
and/or for the dysregulation of the expression of many genes. and whether they are sex-specific. Beyond providing evidence to
Other genes present a different transcriptional pattern between the support the hypothesis that has directed much of the work discussed,
IQ  and IQ þ groups. Although this difference is not as obvious as the importance of determining valid markers would have major
with HLA, it might be interesting to be carefully looked at (Table 2). consequences for informing pathogenesis and for providing defined
For instance, APP (Amyloid-b (A4) precursor protein), located on targets to combat pathogenesis. This may lead to the discovery of
chromosome 21q21.3, was overexpressed in the IQ  group. Duplica- genes that are ‘directly’ involved in several phenotypes of DS and
tion of the APP gene was found to lead to early-onset Alzheimer eventually to the identification of potential targets for therapeutic
disease (AD) and prominent cerebral amyloid angiopathy.28 interventions. For example, the genetic association with HLA
Triplication of the APP gene accelerates the APP expression, leading supports the involvement of the immune system in ID and offers
to cerebral accumulation of APP-derived amyloid-b peptides, early- new targets for drug development.
onset AD neuropathology, and age-dependent cognitive sequelae.29 At Continued and increasing investments in research on the genetic
relatively early ages, DS patients develop progressive formation and and molecular basis of T21 promise to transform the lives of these
extracellular aggregation of amyloid-b peptide, considered as one of individuals and the communities in which they live.
the causal factors in the pathogenesis of AD.30 The cystathionine
b-synthase (CBS) gene, located on human chromosome 21q22.3 CONFLICT OF INTEREST
encodes a key enzyme of sulfur-containing amino acid metabolism, a The authors declare no conflict of interest.
pathway involved in several brain physiological processes. It is
overexpressed in the brain of individuals with DS,31 and thus was
ACKNOWLEDGEMENTS
considered as a good candidate in having a role in the DS cognitive This work was supported by the Jérôme Lejeune Foundation.
profile.32 Recently, Régnier et al.33 studied the neural consequences of
CBS overexpression in a transgenic mouse line expressing the human
CBS gene. They observed that the transgenic mice showed normal
behavior, and that hippocampal synaptic plasticity was facilitated. 1 Carr J: Six weeks to 21 Years: A longitudinal study of children with Down’s syndrome
Thus, they raised the possibility that CBS overexpression might have and their families. J Child Psychol Psychiatry 1991; 29: 407–431.
2 Mégarbané A, Ravel A, Mircher C et al: The 50th anniversary of the discovery of trisomy
an advantageous effect on some cognitive functions in DS. Our results 21: the past, present, and future of research and treatment of Down syndrome. Genet
do not confirm the latter hypothesis as we found that CBS was Med 2009; 11: 611–616.

European Journal of Human Genetics

 Gene expression and IQ in trisomy 21
A Mégarbané et al
1259

3 Lima FA, Moreira-Filho CA, Ramos PL et al: Decreased AIRE expression and global 19 Majumder P, Gomez JA, Chadwick BP, Boss JM: The insulator factor CTCF controls
thymic hypofunction in Down syndrome. J Immunol 2011; 187: 3422–3430. MHC class II gene expression and is required for the formation of long-distance
4 Loudin MG, Wang J, Eastwood Leung HC et al: Genomic profiling in Down syndrome chromatin interactions. J Exp Med 2008; 205: 785–798.
acute lymphoblastic leukemia identifies histone gene deletions associated with altered 20 Majumder P, Boss JM: CTCF controls expression and chromatin architecture of the
methylation profiles. Leukemia 2011; 25: 1555–1563. human major histocompatibility complex class II locus. Mol Cell Biol 2010; 30:
5 Piquemal D, Commes T, Manchon L et al: Transcriptome analysis of monocytic 4211–4223.
leukemia cell differentiation. Genomics 2002; 80: 361–371. 21 Majumder P, Boss JM: DNA methylation dysregulates and silences the HLA-DQ locus
6 Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time by altering chromatin architecture. Genes Immun 2011a; 12: 291–299.
quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402–408. 22 Parelho V, Hadjur S, Spivakov M et al: Cohesins functionally associate with CTCF on
7 Lê Cao KA, Boitard S, Besse P, Sparse PLS: discriminant analysis: biologically relevant mammalian chromosome arms. Cell 2008; 132: 422–433.
feature selection and graphical displays for multiclass problems. BMC Bioinformatics 23 Majumder P, Boss JM: Cohesin regulates MHC class II genes through interactions with
2011; 12: 253. MHC class II insulators. J Immunol 2011; 187: 4236–4244.
8 Antonarakis SE, Lyle R, Dermitzakis ET, Reymond A, Deutsch S: Chromosome 21 and 24 Wendt KS, Yoshida K, Itoh T et al: Cohesin mediates transcriptional insulation by
down syndrome: from genomics to pathophysiology. Nat Rev Genet 2004; 5: 725–738. CCCTC-binding factor. Nature 2008; 451: 796–801.
9 Booij BB, Lindahl T, Wetterberg P et al: A gene expression pattern in blood for the early 25 Wendt KS, Peters JM: How cohesin and CTCF cooperate in regulating gene expression.
detection of Alzheimer’s disease. J Alzheimers Dis 2011; 23: 109–119. Chromosome Res 2009; 17: 201–214.
10 Chrast R, Scott HS, Papasavvas MP et al: The mouse brain transcriptome by SAGE: 26 Kudo NR, Wassmann K, Anger M et al: Resolution of chiasmata in oocytes requires
differences in gene expression between P30 brains of the partial trisomy 16 mouse separase-mediated proteolysis. Cell 2006; 126: 135–146.
model of Down syndrome (Ts65Dn) and normals. Genome Res 2000; 10: 27 Kudo NR, Anger M, Peters AH et al: Role of cleavage by separase of the Rec8
2006–2021. kleisin subunit of cohesin during mammalian meiosis I. J Cell Sci 2009; 122:
11 Costa V, Angelini C, D’Apice L et al: Massive-scale RNA-Seq analysis of non ribosomal 2686–2698.
transcriptome in human trisomy 21. PLoS ONE 2011; 6: e18493. 28 Rovelet-Lecrux A, Hannequin D, Raux G et al: APP locus duplication causes autosomal
12 Sommer CA, Pavarino-Bertelli EC, Goloni-Bertollo EM, Henrique-Silva F: Identification dominant early-onset Alzheimer disease with cerebral amyloid angiopathy. Nat Genet
of dysregulated genes in lymphocytes from children with Down syndrome. Genome 2006; 38: 24–26.
2008; 51: 19–29. 29 Moncaster JA, Pineda R, Moir RD et al: Alzheimer’s disease amyloid-beta links lens
13 Kittler P, Krinsky-McHale SJ, Devenny DA: Sex differences in performance over 7 years and brain pathology in Down syndrome. PLoS ONE 2010; 5: e10659.
on the Wechsler Intelligence Scale for Children-Revised among adults with intellectual 30 Azkona G, Amador-Arjona A, Obradors-Tarragó C et al: Characterization of a mouse
disability. J Intellect Disabil Res 2004; 48: 114–122. model overexpressing beta-site APP-cleaving enzyme 2 reveals a new role for BACE2.
14 Book L, Hart A, Black J, Feolo M, Zone JJ, Neuhausen SL: Prevalence and clinical Genes Brain Behav 2010; 9: 160–172.
characteristics of celiac disease in Down’s syndrome in a US study. Am J Med Genet 31 Lockstone HE, Harris LW, Swatton JE et al: Gene expression profiling in the adult Down
2001; 98: 70–74. syndrome brain. Genomics 2007; 90: 647–660.
15 Cohen D, Eisdorfer C, Walford RL: Histocompatibility antigens (HLA) and patterns of 32 Lejeune J: Pathogenesis of mental deficiency in trisomy 21. Am J Med Genet 2000; 7:
cognitive loss in dementia of the Alzheimer type. Neurobiol Aging 1981; 2: 277–280. 20–30.
16 Payton A, van den Boogerd E, Davidson Y et al: Influence and interactions of cathepsin 33 Régnier V, Billard JM, Gupta S et al: Brain phenotype of transgenic mice over-
D, HLA-DRB1 and APOE on cognitive abilities in an older non-demented population. expressing cystathionine b-synthase. PLoS ONE 2012; 7: e29056.
Genes Brain Behav 2006; 5: 23–31. 34 Peeters MA, Megarbane A, Cattaneo F, Rethore MO, Lejeune J: Differences in purine
17 Aureli A, Sebastiani P, Del Beato T et al: Investigation on the possible relationship metabolism in patients with Down’s syndrome. J Intellect Disabil Res 1993; 37:
existing between the HLA-DR gene and attention deficit hyperactivity disorder and/or 491–505.
mental retardation. Int J Immunopathol Pharmacol 2008; 21: 985–991. 35 Gedye A: Dietary increase in serotonine reduces self-injurious behavior in a Down’s
18 Majumder P, Gomez JA, Boss JM: The human major histocompatibility complex class syndrome adult. J Ment Defic Res 1990; 34: 247–258.
II HLA-DRB1 and HLA-DQA1 genes are separated by a CTCF-binding enhancer- 36 Wegiel J, Gong CX, Hwang YW: The role of DYRK1A in neurodegenerative diseases.
blocking element. J Biol Chem 2006; 281: 18435–18443. FEBS J 2011; 278: 236–245.

European Journal of Human Genetics