Simple detection of genomic microdeletions and
microduplications using QMPSF in patients with
idiopathic mental retardation
Pascale Saugier-Veber*,1,2, Alice Goldenberg1, Vale ´rie Drouin-Garraud1,
Ce ´line de La Rochebrochard3, Vale ´rie Layet4, Nathalie Drouot1,2, Nathalie Le Meur5,
Brigitte Gilbert-Du-ssardier6, Ge ´raldine Joly-He ´las3, He ´le `ne Moirot3, Annick Rossi5,
Mario Tosi2and Thierry Fre ´bourg1,2
1Department of Genetics, Rouen University Hospital, Rouen, France;2Inserm U614, Faculty of Medicine, 22 boulevard
Gambetta, Rouen, France;3Department of Cytogenetics, Rouen University Hospital, Rouen, France;4Department of
Cytogenetics, Le Havre Hospital, Le Havre, France;5Department of Genetics, Etablissement Franc¸ais du Sang, Bois-
Guillaume, France;6Department of Genetics, Jean Bernard University Hospital, Poitiers, France
In contrast to the numerous well-known microdeletion syndromes, only a few microduplications have been
described, and this discrepancy may be due in part to methodological bias. In order to facilitate the detection
of genomic microdeletions and microduplications, we developed a new assay based on QMPSF (Quantitative
Multiplex PCR of Short fluorescent Fragments) able to explore simultaneously 12 candidate loci involved in
mental retardation (MR) and known to be the target of genomic rearrangements. We first screened 153
patients with MR and facial dysmorphism associated with malformations, or growth anomalies, or familial
history, with cytogenetically normal chromosomes, and the absence of FRAXA mutation and subtelomeric
rearrangements. In this series, we found a 5q35 deletion removing the NSD1 gene in a patient with severe
epilepsy, profound MR and, retrospectively, craniofacial features of Sotos syndrome. In a second series, we
screened 140 patients with MR and behaviour disturbance who did not fulfil the de Vries criteria for
subtelomeric rearrangements and who had a normal karyotype and no detectable FRAXA mutation. We
detected a 22q11 deletion in a patient with moderate MR, obesity, and facial dysmorphism and a 4Mb 17p11
duplication in a patient with moderate MR, behaviour disturbance, strabismus, and aspecific facial features.
This new QMPSF assay can be gradually upgraded to include additional loci involved in newly recognised
microduplication/microdeletion syndromes, and should facilitate wide screenings of patients with idiopathic
MR and provide better estimates of the microduplication frequency in the MR population.
European Journal of Human Genetics (2006) 14, 1009–1017. doi:10.1038/sj.ejhg.5201661; published online 14 June 2006
Keywords: QMPSF; microdeletion; microduplication
Mental retardation (MR) occurs in 2–3% of the general
population, but its aetiology can be established only in
approximately 50% of cases, limiting therefore consider-
ably the efficiency of genetic counselling, detection of
carriers, and prenatal diagnosis.1In this context, the
detection and characterisation of deleterious genomic
rearrangements, such as microdeletions and microduplica-
tions, represents an important challenge. These rearrange-
ments, resulting mainly from abnormal pairing and
nonallelic homologous recombination mediated by repeat
elements such as Alu repeats and low-copy repeats (LCRs),
Received 1 December 2005; revised 6 April 2006; accepted 13 April 2006;
published online 14 June 2006
*Correspondence: Dr P Saugier-Veber, Inserm U614, Faculty of Medicine,
22 boulevard Gambetta, 76183 Rouen, France.
Tel: þ33 2 32 88 88 58; Fax: þ33 2 32 88 80 80;
European Journal of Human Genetics (2006) 14, 1009–1017
& 2006 Nature Publishing GroupAll rights reserved 1018-4813/06 $30.00
are the cause of many Mendelian diseases, contiguous gene
syndromes, or chromosomal disorders.2–4Other unchar-
acterised recombinational hotspots may also key roles,
especially in subtelomeric regions where chromosomal
rearrangements are found in about 5% of the patients with
idiopathic MR.5–7Thus, genome architectural features are
involved in the origin of recurrent deleterious DNA
rearrangements.3,8,9The use of FISH has significantly
improvedthe diagnosis of microdeletion
suggested by clinical evidence. Nevertheless, recent de-
scriptions of microduplication syndromes in patients with
MR have highlighted the wide phenotypic variability
complicating their clinical recognition. Although non-
allelic homologous recombination is supposed to generate
microdeletions as well as microduplications, in the field of
MR only four microduplications have clearly been related
to phenotypes: a 15q11–q13 duplication has been detected
in patients presenting autistic features and its frequency
has been estimated to 1/200–600 among patients with
developmental delay.10,11A 17p11.2 duplication has been
associated with moderate MR and behavioural distur-
bance.12,13The 22q11 duplication, initially identified in
patients with a clinical presentation similar to the classical
22q11 deletion, has recently been shown to result into a
highly variable phenotype.14,15A 7q11.23 duplication has
been related to severe expressive language delay.16Among
the possible explanations for the lower frequency of
observed duplications, compared to deletions, one can
speculate that duplications often result in a different or less
severe phenotype and/or that a methodological bias
contributes to this discrepancy. Therefore, systematic
molecular screenings of patients ascertained indepen-
dently of the clinical presentation should facilitate the
characterisation of the clinical spectrum of microduplica-
CGH-array at a 1Mb resolution will probably represent
in a near future the most attractive tool for genomewide
screening to investigate patients with idiopathic MR.
Nevertheless, the recent findings highlighting the pre-
viously unsuspected extend of the copy-number poly-
morphisms in the human genome17–19hampers, at the
present time, its use on a routine basis in molecular
genetics laboratories.20–24Therefore, we considered that
molecular assays focused on regions that have already been
identified as targets for microdeletions and microduplica-
tions should be more effective in detecting selectively
In order to facilitate the detection of microrearrange-
ments and especially duplications in MR patients, we
developed a simple assay based on QMPSF (Quantitative
Multiplex PCR of Short Fluorescent Fragments), a method in
amplified under quantitative conditions using dye-labelled
primers. QMPSF has been shown to be a sensitive method
for the detection of both deletions and duplications25–28
and is currently used in numerous molecular diagnostic
laboratories. The QMPSF assay that we developed for the
present study explores simultaneously 12 candidate loci
known to be the target of genomic rearrangements and
involved in MR. Here, we report the results obtained on
two series of patients with idiopathic MR, the first series
consisting of 153 patients referred for subtelomeric re-
arrangement screening, and the second series consisting of
140 patients referred for fragile X syndrome testing.
Patients and methods
A total of 293 patients was analysed in this study. For each
patient, blood samples were collected after having ob-
tained written informed consent. Each patient had been
examined by a clinical geneticist or a experienced pedia-
trician and had been diagnosed with developmental delay.
The study population was divided into two groups: the first
series was composed of 153 patients (92 males and 61
females) with MR and facial dysmorphism associated with
malformations, or growth anomalies, or familial history.
The additional inclusion criteria were cytogenetically
normal chromosomes and no detectable subtelomeric
rearrangement. In this series, 69 cases (45.5%) presented
with a familial history of MR, whereas 83 cases (54%) were
sporadic and the familial history of one patient was
unknown because of adoption. The second series was
composed of 140 consecutive files of patients (104 males
and 36 females) with MR and behaviour disturbance
referred for Fragile X syndrome testing. These patients
did not fulfil the de Vries criteria for subtelomeric
did not harbour expansions within the FMR1 gene.29In
this second series, 35 cases (25%) presented with a familial
history of MR, whereas 104 cases (74%) were sporadic and
one patient was adopted. Genomic DNA was extracted
from peripheral blood lymphocytes using the QIAamp
DNA blood mini kit (Qiagen, Courtaboeuf, France).
Short exonic fragments (170–240pb) of 12 candidate loci
(Table 1) were simultaneously PCR amplified, in a single
tube, using dye-labelled primers corresponding to unique
sequences (Table 2). An additional fragment, correspond-
ing to exon 13 of the HMBS gene located on chromosome
11, was coamplified, as a control. PCR was performed in a
final volume of 25ml containing 100ng of genomic DNA,
0.3–0.9mM of each primer, 200mM dNTPs, 1.5mM MgCl2,
10% of DMSO, and 1U of Taq DNA polymerase (ABgene,
Courtaboeuf, France). The PCR consisted of 22 cycles of
941C for 10s, 521C for 15s, and 721C for 20s, preceded by
an initial denaturation step of 5min at 941C and followed
by a final extension of 5min at 721C. One ml of the PCR
product was resuspended in a mix containing 1.25ml of
QMPSF detection of microrearrangements
P Saugier-Veber et al
European Journal of Human Genetics
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QMPSF detection of microrearrangements
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European Journal of Human Genetics