Screening for genomic rearrangements and methylation abnormalities of the 15q11-q13 region in autism spectrum disorders.

Abstract

Maternally derived duplications of the 15q11-q13 region are the most frequently reported chromosomal aberrations in autism spectrum disorders (ASD). Prader-Willi and Angelman syndromes, caused by 15q11-q13 deletions or abnormal methylation of imprinted genes, are also associated with ASD. However, the prevalence of these disorders in ASD is unknown. The aim of this study was to assess the frequency of 15q11-q13 rearrangements in a large sample of patients ascertained for ASD.
A total of 522 patients belonging to 430 families were screened for deletions, duplications, and methylation abnormalities involving 15q11-q13 with multiplex ligation-dependent probe amplification (MLPA).
We identified four patients with 15q11-q13 abnormalities: a supernumerary chromosome 15, a paternal interstitial duplication, and two subjects with Angelman syndrome, one with a maternal deletion and the other with a paternal uniparental disomy.
Our results show that abnormalities of the 15q11-q13 region are a significant cause of ASD, accounting for approximately 1% of cases. Maternal interstitial 15q11-q13 duplications, previously reported to be present in 1% of patients with ASD, were not detected in our sample. Although paternal duplications of chromosome 15 remain phenotypically silent in the majority of patients, they can give rise to developmental delay and ASD in some subjects, suggesting that paternally expressed genes in this region can contribute to ASD, albeit with reduced penetrance compared with maternal duplications. These findings indicate that patients with ASD should be routinely screened for 15q genomic imbalances and methylation abnormalities and that MLPA is a reliable, rapid, and cost-effective method to perform this screening.

Full text

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Biol Psychiatry . Author manuscript
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Screening for genomic rearrangements and methylation abnormalities of
the 15q11-q13 region in autism spectrum disorders
Christel Depienne 1 2 , Daniel Moreno-De-Luca 3 , Delphine Heron 2 , Delphine Bouteiller 1 , Aur lie Gennetier
Pauline Chaste 4 , Jean-Pierre Siffroi 5 , Sandra Chantot-Bastaraud 5 , Baya Benyahia 2 , Oriane Trouillard 2 , Gudrun Nygren 6 ,
Svenny Kopp 6 , Maria Johansson 6 , Maria Rastam 6 , Lydie Burglen 5 , Eric Leguern 1 2 , Alain Verloes 7 , Marion Leboyer 8 9 , Alexis
Brice 1 2 , Christopher Gillberg 6 10 , Catalina Betancur 3 *
é
3 , Richard Delorme 4 ,
Neurologie et th rapeutique exp rimentale
é
Boulevard de LHopital 75651 PARIS CEDEX 13,FR
'

1
é
INSERM : U679 , IFR70 , Universit Pierre et Marie Curie - Paris VI
é
, GH Piti -Salpetri re 47,
éè
D partement de G n tique Cytog n tique et Embryologie
2 éé é
47-83, boulevard de lH pital 75651 PARIS Cedex 13,FR
' ô

é é
AP-HP , H pital Piti -Salp tri re
ôéêè
, Universit Pierre et Marie Curie - Paris VI
é
,
Neurobiologie et Psychiatrie
05,FR
4
rurier 75019 PARIS,FR
5
é é
avenue du Docteur Arnold-Netter 75571 PARIS Cedex 12,FR
6

3
INSERM : U513 , Universit Pierre et Marie Curie - Paris VI
é
, 9 quai Saint Bernard 75252 Paris Cedex
Service de psychopathologie de lenfant et de ladolescent
''
AP-HP , H pital Robert Debr
ôé , Universit Paris-Diderot - Paris VII
é
, 48, Bd Sé
Service de g n tique et embryologie m dicales
é
AP-HP , H pital Armand Trousseau
ô
, Universit Pierre et Marie Curie - Paris VI
é
, 26,
Department of Child and Adolescent Psychiatry Gothenburg University , Institute of Neuroscience and Physiology, Gothenburg,SE
Unit fonctionnelle de g n tique clinique
é

7
é é
AP-HP , H pital Robert Debr
ôé , Universit Paris-Diderot - Paris VII
é
, Paris,FR
D partement de Psychiatrie
8 é
AP-HP , H pital Albert Chenevier
ô
, 40 rue de Mesly 94000 Cr teil,FR
é
IMRB, Institut Mondor de recherche biom dicale
mal de lattre de tassigny 94010 CRETEIL CEDEX,FR
10

9
é
INSERM : U841 , Universit Paris XII Val de Marne
é
, H pital Henri Mondor 51, av du
ô
Institute of Child Health London
[] University College London , University College London Gower Street London WC1E 6BT,GB
* Correspondence should be adressed to: Catalina Betancur <Catalina.Betancur@inserm.fr >
Abstract
Background
Maternally-derived duplications of the 15q11-q13 region are the most frequently reported chromosomal aberrations in autism
spectrum disorders (ASD). Prader-Willi and Angelman syndromes, caused by 15q11-q13 deletions or abnormal methylation of
imprinted genes, are also associated with ASD. However, the prevalence of these disorders in ASD is unknown. The aim of this study
was to assess the frequency of 15q11-q13 rearrangements in a large sample of patients ascertained for ASD.
Methods
A total of 522 patients belonging to 430 families were screened for deletions, duplications and methylation abnormalities involving
15q11-q13 using multiplex ligation-dependent probe amplification (MLPA).
Results
We identified four patients with 15q11-q13 abnormalities: a supernumerary chromosome 15, a paternal interstitial duplication, and
two subjects with Angelman syndrome, one with a maternal deletion and the other with a paternal uniparental disomy.
Conclusions
Our results show that abnormalities of the 15q11-q13 region are a significant cause of ASD, accounting for approximately 1 of
cases. Maternal interstitial 15q11-q13 duplications, previously reported to be present in 1 of patients with ASD, were not detected in
our sample. Although paternal duplications of chromosome 15 remain phenotypically silent in the majority of patients, they can give
rise to developmental delay and ASD in some subjects, suggesting that paternally-expressed genes in this region can contribute to
ASD, albeit with reduced penetrance compared to maternal duplications. These findings indicate that patients with ASD should be
routinely screened for 15q genomic imbalances and methylation abnormalities and that MLPA is a reliable, rapid and cost-effective
method to perform this screening.
%
%
MESH Keywords Adolescent ; Adult ; Angelman Syndrome/ genetics ; Autistic Disorder/ genetics ; Child ; Child, Preschool ; Chromosome Aberrations ; Chromosomes,
Human, Pair 15, genetics ; DNA Methylation/ genetics ; Female ; Gene Deletion ; Gene Dosage ; Humans ; Male ; Microsatellite Repeats/ genetics ; Prader-Willi Syndrome/
genetics ; Uniparental Disomy
Author Keywords autism ; chromosome 15 ; deletion ; duplication ; Angelman syndrome ; MLPA

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Introduction
Autism is a neurodevelopmental disorder characterized by impaired social interaction and communication, and a restricted range of
interests and activities, with onset during the first three years of life. Autism spectrum disorders (ASDs), which include autism, pervasive
developmental disorder not otherwise specified (PDD-NOS) and Asperger syndrome, have a typical male preponderance and their
estimated prevalence is 6/1000 ( ). ASDs are etiologically heterogeneous, with an underlying genetic disorder identified in 101
cases. Monogenic disorders such as fragile X syndrome, tuberous sclerosis, and Rett syndrome are found in a small percentage of patients;
in addition, rare mutations in other genes (e.g., NLGN3, NLGN4X, PTEN, SHANK3
individuals ( ). Cytogenetically visible chromosomal aberrations are identified in 32
higher-resolution whole-genome analyses using array-based technologies have revealed genomic imbalances in at least 10 of cases (
).
25 of
%%–
) have been reported in a small number of
6 of affected individuals ( ,
%– %
), while recent 3 4
%
5 –7
Duplications of the 15q11-q13 region are the most frequently reported chromosomal aberration in individuals with ASDs ( ). This
region includes the Prader-Willi syndrome/Angelman syndrome (PWS/AS) critical region, which is subject to genomic imprinting. Most
duplications of this interval are caused by supernumerary chromosomes formed by the inverted duplication of proximal 15q, known as
isodicentric chromosome 15 idic() (). Interstitial duplications of this region are less frequent (
[
15 ] 9 –13
reported in association with autism ( , , ). The majority of cases are associated with maternally-derived duplications, whereas10 13 15 –17
paternal inheritance usually leads to normal phenotypes ( , , , 10 15 16 18
8
), but many cases have been 14
).
Deletions or methylation abnormalities of chromosome 15 result in either Prader-Willi syndrome or Angelman syndrome, depending
on whether they arise on the paternal or maternal chromosome. Both syndromes have been described in patients with ASD or autistic
behavior, and recent studies estimate that over half of the patients with Angelman syndrome have ASD ().19 –21
The genomic instability of proximal chromosome 15 is mediated by low-copy repeats, resulting in five recurrent breakpoints (BP)
involved in deletions, duplications and idic( ) (). The deletions involve either the proximal BP1 or BP2 and share the same distal15 22
breakpoint (BP3), whereas the duplications can extend more distally to BP4 or BP5 (
the 15q11-q13 duplication syndrome lies between BP2 and BP3. Nonallelic homologous recombination between flanking low-copy repeats
is also involved in other microdeletion/microduplication syndromes associated with mental retardation and ASDs, including 17p11.2,
22q11.2 and 7q11.23 ( ).23
). The critical region involved in PWS/AS andFig. 1
Although the frequency of 15q11-q13 duplications in autism is widely assumed to be 1
series of patients. In a study of 140 subjects with autism, two were found with a maternal interstitial duplication of 15q11-q13 (
Schroer studied 100 patients with autism and identified two idic(et al.
deletion (). The small numbers of patients studied makes it difficult to draw conclusions on the real contribution of such duplications to 10
ASD. The prevalence of proximal 15q deletions in ASDs is also unknown. Recent genome-wide studies of copy number variants in large
ASD samples included an unknown proportion of patients who had been previously screened for chromosomal abnormalities and
15q11-q13 rearrangements (and excluded if positive), thus precluding the estimation of prevalence ( , ).
3 , this estimate is based on two small
%– %
).24
), one maternally-derived interstitial duplication and one maternal 15
5 6
The aim of this study was to assess the frequency of 15q11-q13 abnormalities in 522 individuals with ASDs. We screened the
PWS/AS region for gene dosage alterations using multiplex ligation-dependent probe amplification (MLPA) and quantitative
microsatellite analysis. In addition, we searched for methylation abnormalities of chromosome 15, including uniparental disomies and
imprinting center defects, using methylation-sensitive MLPA (). Epigenetic defects have long been suspected in autism ( 25
no systematic screening of methylation abnormalities of chromosome 15 had been performed.
, ), but26 27
Methods and Materials
Patients
A total of 522 patients with ASD belonging to 430 families were studied. Subjects were recruited by the Paris Autism Research
International Sibpair (PARIS) study at specialized clinical centers in Europe and the United States. The patients included 393 males and
129 females (ratio 3:1), with a mean age at the last evaluation of 11 7.5 years (range 2.5 43); 187 belonged to 95 multiplex families (with
±
two or more affected siblings) and 335 were sporadic cases. All patients were evaluated by psychiatrists or child neurologists and
diagnosed based on clinical evaluation and DSM-IV criteria. Patients were assessed with the Autism Diagnostic Interview-Revised
(ADI-R) () and the Asperger Syndrome Diagnostic Interview (28 29
for Asperger syndrome and 24 for PDD-NOS. Three-hundred fifty-six patients (68 ) had mental retardation, 261 (50 ) had very limited
or no language, and 66 (13 ) had a history of epilepsy. Most patients were Caucasian (89 ). Laboratory tests included karyotype, fragile
%
X and metabolic screening; brain imaging and EEG were performed when possible. Patients with known genetic disorders were excluded.
Chromosome analysis identified one female harboring a supernumerary isodicentric derivative chromosome 15; we report here the
cytogenetic, molecular and phenotypic characterization of this patient. No other patients had been excluded from the sample prior to this
–
). Four-hundred seventy-two individuals met criteria for autism, 26
%
%
%

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study because of cytogenetic abnormalities of chromosome 15q11-q13. The study was approved by the research ethics boards of the
collaborating institutions. Informed consent was obtained from all families participating in the study.
Multiplex ligation-dependent probe amplification (MLPA)
Patients were screened for rearrangements involving the 15q11-q13 region using the ME028 PWS/AS and/or the P064 MR1 MLPA
kits (n 351 and 522, respectively) (MRC-Holland). The ME028 kit contains 25 probes specific for sequences in or near the PWS/AS
=
critical region, as well as 5 probes to assess methylation status (). The P064 kit contains 5 probes in the PWS/AS region (one probe in 25
and and two in MLPA data were analyzed using GeneMarker 1.70 software (SoftGenetics). After MKRN3, NDN, GABRB3, UBE3A).
population normalization, the peak height from each sample was compared to a synthetic control, which represents the median of all
normal samples in each experiment. Peak heights below 0.75 were considered as deletions and values above 1.3 as duplications. Cases
with apparent deletions or duplications were confirmed with quantitative PCR (qPCR) and fluorescent in situ hybridization (FISH).
Apparent deletions of a single probe were sequenced to rule out single-base changes within the probe-binding region. For further details
see Supplement 1.
Quantitative microsatellite analysis
Five microsatellite markers in the 15q11-q13 region (D15S11, D15S817, D15S1513, D15S1019 and D15S815) were used to assess
DNA copy number and follow the transmission of the alleles ( Fig. 1
up so as to perform allele dosage. PCR products were quantified on an ABI3730 sequencer (Applied Biosystems).
). PCR conditions (in particular the number of PCR cycles) were set
Results
Five quantitative assays using microsatellites were used to screen the 15q11-q13 region in 217 patients. However, analysis of the
markers D15S11 and D15S817 revealed three alleles in 10 of patients, a proportion comparable to that in a control population, indicating
%
that this region is duplicated in healthy individuals. We therefore decided to screen all patients using MLPA.
Overall, we identified four patients with 15q11-q13 abnormalities considered to be pathogenic: an idic(
a deletion and an uniparental disomy. Their clinical features are summarized in
), an interstitial duplication, 15
and are described in more detail in Supplement 1. Table 1
Supernumerary isodicentric chromosome 15
Patient 1 had an idic(
, UBE3A
++
D15S11, D15S817 and D15S815 (
by the increased dosage of the methylated probes in
in the 15q11-q13 region (between BP1 and BP3) but did not affect two genes located distally, at 15q26,
Further analysis of the 15q11-q13 interval with 15 microsatellites showed six triallelic markers, with one allele of paternal origin and two
of maternal origin ( ). One informative microsatellite between BP4 and BP5, D15S1031, had a single maternal allele, suggestingFig. 2D
the breakpoint lies proximal to this marker (). Detailed investigation of the distal breakpoint with qPCR showed increased dosage Fig. 2D
of and located between BP4 and BP5, but two distal genes in the same interval, MTMR10, TRPM1 KLF13,
showed a normal copy number (). These findings suggest that the distal breakpoint of the idic( Fig. 2E
agreement with the microsatellite results. The probeb showed an increase in dosage to the hexasomy range, suggesting a complexKLF13
rearrangement at the site of the breakpoint. We could not differentiate between one or two breakpoints, because of the inability to estimate
the precise copy number of individual probes on the basis of qPCR ( Fig. 2E
) revealed by the karyotype and confirmed by FISH, described as 47,XX, mar.ish der(
). Microsatellite analysis of the patient and her parents showed that she had a
). Methylation-sensitive MLPA revealed that the idic(Fig. 2B
and SNRPN NDN Fig. 3
)(D15Z1
copy number gain of
, SNRPN 15
+
15
++
) (
++
Fig. 2A de novo
) was maternally-derived, as indicated
) involved all the probes15
and BLM
15
( ). MLPA confirmed that the idic(
().IGF1R Fig. 2C
and OTUD7A
) lies between BP4 and BP5, in
CHRNA7,
15
).
Interstitial 15q11-q13 duplication
Patient 2 had three alleles for both D15S11 and D15S817. He was homozygous for D15S1513 and D15S1019, which were therefore
uninformative, and heterozygous without imbalance for D15S815, located between BP3 and BP4 (not shown). MLPA confirmed the
presence of a ~4.6 Mb duplication between BP2 and BP3, corresponding to the critical region deleted in PWS/AS (
Methylation-sensitive MLPA showed normal dosage of the methylated maternal probes in
duplication was paternally derived ( ). MLPA of the parents showed that the duplication was inherited from the unaffected father ( Fig. 3
). Methylation analysis of the father showed a paternal origin of the duplication (not shown), but no DNA was available from the Fig. 4
grandfather to determine if the duplication was inherited or had arisen
an increased signal in Patient 2 (not shown).
). Fig. 4
and indicating that the SNRPN NDN,
FISH using the UBE3A probe confirmed the presence ofde novo.
Angelman syndrome: deletion and uniparental disomy
Patients 3 and 4 had only one allele for three markers located in the PWS/AS region (D15S11, D15S817 and D15S1513), suggesting
that they could be either homozygous or deleted in this region ( Fig. 5A ). Examination of the parents haplotypes showed that both patients
’

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had only one allele from the father and none from the mother (
extending ~5.6 Mb from BP1 to BP3 that arose
Patient 4, MLPA showed a normal copy number of the 15q11-q13 region (
absence of maternal methylated DNA sequences, indicating a paternal uniparental disomy (
probes SNRPN and D15S10 (not shown), consistent with the diagnosis of uniparental disomy.
). MLPA confirmed that Patient 3 had a maternal class I deletion
). FISH with probe D15S10 confirmed the deletion (not shown). In
) but methylation-sensitive MLPA revealed a completeFig. 5C
). FISH showed normal hybridization ofFig. 3
Fig. 5A
, (de novo Fig. 3 5C
Abnormal copy number of the BP1-BP2 region
MLPA also detected three patients with a deletion (Patients 5, 6 and 7) and two with a duplication (Patients 8 and 9) of the BP1-BP2
interval, which spans 254 kb and encompasses four genes, TUBGCP5, CYFIP1, NIPA1,
inherited from parents who were unaffected or had unrelated psychiatric disorders (
and (). All the rearrangements were NIPA2 Fig. 1
).Table 1
The cytoplasmic FMR1 interacting protein 1 (CYFIP1) interacts with FMRP, encoded by the fragile X mental retardation 1 (FMR1)
gene. Since fragile X syndrome is often associated with autism, CYFIP1
possibility that point mutations in this gene, either inherited from the other parent or
BP1-BP2 imbalance, we sequenced the whole coding region of CYFIP1
Patients 5 and 8 we extended the search of point mutations to the coding regions of
and which encode proteins of the FMRP complex. No mutations were identified in any of the genes screened. FMR1, FXR1 FXR2,
constitutes a candidate gene for autism. In order to test the
could lead to autism in association with the de novo,
in the five probands with abnormal dosage of this region. In
and NIPA1, NIPA2 TUBGCP5, as well as those of
Other deletions or sequence variants considered non pathogenic
We identified one patient with a paternally-inherited deletion of exon 1 in one of the transcript variants of the
the deleted exon is noncoding and is maternally expressed, this genomic imbalance is unlikely to have clinical consequences. InUBE3A
another patient, MLPA showed an apparent deletion of one probe in the
nucleotide change previously reported as a rare neutral variant (see Supplement 1 for further details on these two patients).
gene. Because UBE3A
promoter/exon1 region, due to a maternally-inherited SNRPN
Methylation abnormalities
Aside from the uniparental disomy identified in Patient 4, no other methylation abnormalities were observed in 331 patients screened
with methylation-sensitive MLPA. In the remaining subjects, uniparental disomy was ruled out with microsatellite analysis.
Discussion
In this study, we found a pathogenic rearrangement of the 15q11-q13 region in 4 out of 430 families. Thus, proximal 15q
abnormalities, including deletions, supernumerary isodicentric chromosomes and interstitial duplications, are found in about 1 of patients
with ASD, representing one of the most common genetic causes of ASD, together with fragile X syndrome.
%
Supernumerary chromosome 15 and ASD
Supernumerary marker chromosomes are a relatively common cytogenetic finding, with an estimated incidence of 0.8/1000 prenatal
diagnoses; those derived from chromosome 15 account for about half of all marker chromosomes (
with developmental delay, with 10 idic() identified in 2000 such cases in one study (15
critical region have no clinical effect ( ), whereas those including the PWS/AS region lead to a neurobehavioral phenotype including31
autism or autistic-like behavior, cognitive deficits, hypotonia, mild dysmorphic features, and seizures (
reports of idic() associated with autism ( ). As in Patient 1, the vast majority of idic(15 9 –13
maternally derived and arise ( ). Almost two-thirds extend beyond BP3 and the majority are asymmetrical, with two distinct de novo 33
breakpoints, at BP4 and BP5 ( , ). Analysis of the number of copies across the 15q11-q13 region in Patient 1 with qPCR and14 33
microsatellites predicted an atypical distal breakpoint between BP4 and BP5. A similar breakpoint was reported in a patient with an idic(15
), between D15S1013 andD S1031 ( ).
!
33
). The frequency is higher in children
) that do not include the PWS/AS15
30
). Idic(11
). There have been numerous
) that encompass the PWS/AS locus are
32
15
Although genotype/phenotype correlation studies show that the segmental tetrasomy of idic(
phenotype in terms of developmental outcome compared to interstitial triplications or duplications, indicating a dosage effect, no clear
correlation has been observed between gene dosage and autism phenotype (
between rearrangement size and clinical severity ( , ). 11 34
) is associated with a more severe 15
). Furthermore, no consistent relationship has been observed 34
Interstitial duplications and ASD
At least 33 cases of interstitial duplications of the 15q11-q13 region have been reported in association with ASDs, according to the
Autism Chromosome Rearrangement Database (http://projects.tcag.ca/autism/
include the PWS/AS critical region have no clinical effect, are usually familial and may be considered normal variants (
interstitial duplications of the PWS/AS locus have an abnormal phenotype that includes developmental delay, particularly affecting speech
). Interstitial duplications of proximal 15q that do not
). Patients with 18

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and language, varying degrees of mental retardation, autism or autistic features, motor coordination difficulties, and mild or no dysmorphic
features ( ). The phenotype is highly variable, even among members of the same family carrying identical rearrangements ( 16
in some cases manifest as developmental language disorder and dyspraxia, without autism (
similar to those described in patients with maternal 15q11-q13 duplications, although he lacks the hypotonia, motor coordination problems
and joint laxity observed in other cases (, ).16 17
) and can 16
). The clinical findings in Patient 2 are 35
The frequency of 15q11-q13 interstitial duplications is estimated at 1:600 individuals referred with developmental delay (
Although previous studies in small samples of patients had suggested a frequency of 1 for maternally-derived interstitial duplications in
autism (, ), we did not detect any such cases in our sample but found one patient harboring a paternal 15q11-q13 duplication. In 10 24
agreement with our findings, Bolton screened 181 patients with autism and found only one interstitial duplication of paternal origin (et al.
). Similarly, Thomas studied 327 samples referred for a suspected ASD as well as 87 patients with confirmed autistic disorder and37 et al.
found no 15q duplications (). More recently, a genome-wide microarray study of 427 subjects with ASD identified two 36
maternally-derived 15q11-q13 duplications ( ). Collectively, these results suggest that maternal interstitial duplications are less frequent7
than previously assumed, with an estimated ~0.3 frequency (4/1369) in subjects with ASD. Based on the same studies, the estimated
%
frequency of idic( ) is ~0.2 (3/1369) and that of paternal duplications ~0.1 (2/1369). Note however, that the frequency of idic(15
%
may be underestimated because previous studies included an unknown proportion of subjects that had been screened for cytogenetic
abnormalities prior to inclusion. It also should be noted that none of these studies were based on epidemiological samples and the
possibility of ascertainment biases cannot be excluded.
).36
%
)
%
15
Maternal versus paternal duplications
Interstitial duplications of 15q11-q13 of maternal origin are associated with developmental delay and/or autistic behavior, whereas
paternally-derived duplications usually lead to a normal phenotype (10 15 16 18
least six reports of paternal duplications encompassing the PWS/AS region associated with an abnormal phenotype, including mental
retardation, delayed or absent speech, and ASD or autistic behavior (
triplication of paternal origin have been reported (, ). Together with the patient with the paternal duplication described here, these42 43
findings suggest that paternally-derived duplications may lead to phenotypic effects, including autism. Thus, there may be other genetic
and/or environmental/epigenetic factors operating to modify the penetrance and expressivity of paternal duplications. Similar as yet
unknown factors could come into play to modify the phenotypic expression of maternal 15q11-q13 duplications, which are also associated
with a wide range of developmental problems and show marked clinical variability among members of the same family, including
individuals who appear unaffected (). 16
, , , ). However, as shown in , there have been atTable 2
). In addition, two patients with an interstitial 15q11-q1337 –41
It is interesting to note that several of the patients with paternally-inherited duplications or triplications described previously had
Prader-Willi syndrome-like features ( ) (, , ), suggesting a role of paternally-expressed genes mapping within the PWS/ASTable 2 38 40 43
region (e.g., and in the phenotypic manifestations. Several recent studies have shown that MKRN3, MAGEL2, NDN, SNRPN-SNURF)
ASD is more prevalent in Prader-Willi syndrome patients than was previously thought (
paternally-expressed genes to the ASD phenotype. Further research is needed to examine the effect of parental origin on phenotypic
outcomes of 15q11-q13 duplications and their contribution to ASD. In particular, studies of gene expression in tissues from patients with
paternal duplications may improve our understanding of the effect of these rearrangements on phenotype.
), in agreement with a contribution of 44
Angelman syndrome and ASD
The prevalence of Angelman syndrome is estimated at 1/12,000 (
retardation, profound speech impairment, ataxia, and typical behavior including happy disposition with frequent laughter/smiling and
hand-flapping. Seizures, microcephaly and distinctive physical traits (large open mouth, widely-spaced teeth, prognathism) are also
common (). It is caused by deficiency of the maternally-inherited 46
region (70 ), paternal uniparental disomy (2 ), mutations (10 ), or imprinting defects (5 ), with no molecular abnormality
%%
UBE3A
identified in the remaining patients ( ). 13
). Angelman syndrome is characterized by severe mental 45
gene, resulting from deletion of the maternal 15q11.2-q13
%
UBE3A
%
The association between Angelman and autism has been known for more than a decade (
range 5081 ) of subjects with Angelman syndrome meet criteria for autism or ASD (
%–%
behavioral phenotype of Angelman syndrome. Although the diagnosis of ASD in Angelman syndrome is complicated by the severe
intellectual disability usually observed in these patients, there is clear evidence for ASD in a subgroup of Angelman patients who exhibit
social and communication deficits that are disproportionate to their overall cognitive function. There is little information about the
prevalence of Angelman syndrome in ASD. Schroer found one maternal 15q11-q13 deletion among 100 patients with autism (et al.
this study, we identified two subjects with Angelman syndrome, one with a maternal deletion and the second with a uniparental disomy,
for a ~0.5 frequency.
%
). According to recent data, 62 (38/61,
), suggesting that ASD may be part of the
45
%
19 –21
). In10

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Because certain features of autism overlap with those seen in Angelman syndrome, including severe mental retardation, absence of
speech or very limited language, epilepsy and/or abnormal EEG, and stereotyped behaviors, the diagnosis of Angelman syndrome may be
overlooked in patients with ASD, as happened with our two patients. Although neither patient exhibited the classical phenotype, their
clinical presentation is within the normal range of phenotypic variability observed in Angelman syndrome. In common with the majority
of patients with Angelman, they exhibited severe cognitive impairment and limited or absent language, but had no ataxia or microcephaly,
and seizures and unprovoked laughter (during infancy only) were observed only in one.
BP1-BP2 microdeletions/microduplications
We identified three deletions and two duplications of the region between BP1 and BP2, all inherited from unaffected parents. A
BP1-BP2 deletion was reported recently in a boy with mental retardation, speech delay and neurological deficits, but the interpretation was
complicated by the presence of the same deletion in the father, who also showed cognitive impairment (
losses of this region have also been identified in genome-wide array analyses of control individuals (Database of Genomic Variants,
), with a population frequency estimated at 1 (http://projects.tcag.ca/variation/
polymorphism. In agreement with this interpretation, individuals carrying small idic(
the BP1-BP2 interval but that do not carry extra genomic material distal to BP2 are phenotypically normal (
genes do not exert clinically significant dosage effects.
). Copy number gains and47
), suggesting that this may be a copy number
) with extra copies of the four genes included in
31
% 48
15
), suggesting that these
Nevertheless, it is interesting to note that
phenotype. Angelman patients with class I deletions (including the BP1-BP2 interval) are more likely to meet criteria for autism and lack
vocalizations compared with patients having smaller class II deletions (
associated with more obsessive-compulsive behaviors and lower intellectual ability (
positively correlated with better behavioral outcomes ( ). More recently, BP1-BP2 deletions were shown to be significantly associated50
with schizophrenia ( ), suggesting that these variants may increase risk for various neuropsychiatric phenotypes, albeit with low51
penetrance and/or variable expressivity.
and have been suggested to modulate the PWS/ASNIPA1, NIPA2, CYFIP1 TUBGCP5
). Similarly, in Prader-Willi syndrome, class I deletions are
), and mRNA levels of these four genes are 49
20
One of the genes situated in this interval,
mental retardation protein (FMRP) as well as with the Rho GTPase Rac1, which plays a role in the regulation of axonal migration and
dendritic spine morphology. CYFIP1 has been proposed as a potential molecular link between fragile X syndrome and 15q11-13
duplication, since both disorders result in excess free CYFIP1 ( ). In order to test the possibility that the BP1-BP2 deletion unmasked a 52
recessive mutation on the other chromosome we sequenced CYFIP1,
X-related proteins and in patients carrying BP1-BP2 deletions, identifying no mutations. Clearly, more research is(FMR1, FXR1 FXR2),
required to determine the phenotype correlations of BP1-BP2 deletions and duplications and the role of
, if any, in neurodevelopmental disorders. TUBGCP5
is particularly interesting in the context of ASD. CYFIP1 interacts with fragile XCYFIP1,
as well as and genes encoding fragile NIPA1, NIPA2, TUBGCP5,
, , and NIPA1 NIPA2 CYFIP1
Conclusion
The present findings confirm that genetic abnormalities of the 15q11-q13 region are an important cause of ASD, accounting for
approximately 1 of cases. Our results suggest that patients with ASD, particularly those with mental retardation, should be systematically
%
screened for duplications, deletions and methylation abnormalities of the PWS/AS region. Routine chromosomal analysis, currently
recommended in the etiological evaluation of individuals with autism, would miss many of the deletions and duplications in the 15q11-q13
region, as well as cases resulting from uniparental disomy. Traditional cytogenetic tests such as FISH are expensive and would miss some
duplications and all cases of uniparental disomy. Furthermore, array-based methods are also expensive and currently are not in wide use
for detecting methylation changes. Methylation-sensitive MLPA allows simple, rapid, accurate and economic screening of dosage
imbalances and imprinting defects in this region, providing detailed information on the parental origin and the extent of the rearrangement,
and appears thus as a method of choice to screen chromosome 15 abnormalities in patients with ASD.
Ackowledgements:
We thank the families who participated in this research and the members of the Paris Autism Research International Sibpair (PARIS) Study
for patient ascertainment (Supplement 1). We also thank the DNA and cell bank of the INSERM U679 (H pital Piti -Salp tri re) and the
Centre d Investigations Cliniques-H pital Robert Debr for processing the samples from the French families. This research was supported by
’ôé
Fondation de France, INSERM, Fondation pour la Recherche M dicale, Fondation France T l com, Cure Autism Now, Assistance
é
Publique-H pitaux de Paris, and the Swedish Science Council.
ô
ôéêè
é é
Footnotes:
Financial Disclosures

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The authors report no biomedical financial interests or potential conflicts of interests.
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–
San Diego, California






Figure 1
Map of chromosome 15 showing the Prader-Willi/Angelman syndrome critical region. Genes are shown above the map and microsatellites
below. The five microsatellite markers used in the quantitative analysis are indicated in bold. Paternally and maternally expressed genes are
indicated in yellow and pink, respectively. The recurrent breakpoints (BP) are indicated as purple boxes. The imprinting center (IC), located in
the 5 untranslated region of , is indicated in green. The horizontal red bars indicate the regions deleted in Angelman and Prader-Willi
′
SNRPN
syndromes (class I and class II deletions), as well as the recently described copy number variant between BP1 and BP2 and the 15q13.3
microdeletion syndrome. The horizontal green bars indicate the regions involved in typical interstitial duplications and supernumerary
isodicentric chromosome 15. The critical region for Prader-Willi, Angelman and 15q11-q13 duplication syndromes lies between BP2 and
BP3. The distance (expressed in Mb from pter) is shown at the top of the map.

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Figure 2
Supernumerary isodicentric chromosome 15 in Patient 1. A. Metaphase FISH of lymphoblastoid cell lines from Patient 1 using probes
UBE3A (red) for the region of interest and D15S936 (green) as a control probe in 15q26.3 showed two normal signals on chromosomes 15 as
well as additional hybridization spots of the duplicated 15q11-q13 region (red) on a supernumerary chromosome (arrow). B. Quantitative
microsatellite analyses showed increased dosage of D15S11, D15S817 and D15S815 in the proband, but not in her parents. C. MPLA
confirmed increased copy number of all the probes in the 15q11-q13 region between BP1 and BP4 (from TUBGCP5 to APBA2), and normal
dosage of BLM and IGF1, located at the distal end of the chromosome, at 15q26. The peak heights above 1.5 are consistent with tetrasomy of
the region. D. Genotyping of microsatellites in the 15q11-q13 region revealed three alleles for informative markers between BP1 and BP4,
with one allele of paternal origin and two of maternal origin. The presence of one informative microsatellite, D15S1031, with a single
maternal allele indicates that the breakpoint lies proximal to this marker. E. Genomic qPCR showed a transition from increased copy number
to normal dosage between KLF13 and OTUD7A, indicating that the distal breakpoint lies between BP4 and BP5.

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10 13
Figure 3
Methylation-sensitive MLPA of the chromosome 15q region. Histograms show the dosage of probes located in the
promoter regions in undigested and digested DNA. In undigested DNA, both the maternal and paternal copies of the genes are visible.
Because and are paternally imprinted, after digestion with the methylation-sensitive enzyme SNRPN NDN
methylated DNA sequences are amplified, indicated by the light-colored bars. In a normal subject (control), a 50 reduction in peak height is
observed after digestion. Patient 1, who has an isodicentric chromosome 15, shows 4 copies of the probes in the PWS/AS region, and a
twofold increase in peak height of methylated DNA sequences, corresponding to a maternal origin of the extra chromosome. Patient 2 shows
no increase in amplification of methylated DNA sequences after digestion, as expected with a duplication of paternal origin. Patient 3, with an
Angelman syndrome resulting from a maternal deletion of chromosome 15q11-q13, shows a 50 reduction in peak height of probes in the
PWS/AS region, and absent methylated DNA peaks after digestion. Patient 4, with Angelman syndrome resulting from a paternal uniparental
disomy, shows normal peak heights before digestion with and an absence of methylated DNA peaks afterwards, indicating the presence HhaI,
of two paternal chromosomes.
and SNRPN NDN
only the maternally HhaI
%
%
Figure 4
Paternally inherited interstitial duplication of chromosome 15 in family ASD 2. MPLA showed increased gene dosage of all the genes from
to between BP2 and BP3, while the genes before BP2 (MKRN3 OCA2,
dosage.
and ) and after BP3 () showed normalTUBGCP5 CYFIP1 APBA2

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11 13
Figure 5
Two patients with ASD and Angelman syndrome, one secondary to a maternal deletion (family ASD 3) and the other to a paternal uniparental
disomy (family ASD 4). A. Microsatellites D15S817 and D15S1019 show loss of heterozygosity, with apparently only one allele inherited
from the father in both probands. B. Genotyping of five microsatellites in the 15q11-q13 region confirmed the presence of only one allele
inherited from the father in Patient 3, indicating a maternal deletion. Patient 4 showed two alleles inherited from the father, indicating a
paternal uniparental disomy. C. MPLA showed normal gene dosage in Patient 4, as expected in an uniparental isodisomy, and a 50%
reduction in probes ranging from to in Patient 3, showing a class I deletion of the PWS/AS region.TUBGCP5 OCA2

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Table 1
Clinical features of patients with rearrangements of the 15q11-q13 region
Patient Rearrangement
Pathogenic rearrangements
 
Patient
1
distal BP between
BP4/BP5)
Origin
(chromosome) Gender
Age
last
evaluation ASD
at
Developmental
delayLanguage EpilepsyOther features
idic(15)
(pter-15q13.3,
de
(maternal)
novo F30 yAutism Severe MRN o
language
Absence
seizures,
resistant
treatment (onset
13 y)
No
to
Feeding difficulties, psychomotor delay, dysmorphic features (large bulbous nose, dental
malposition, prominent jaw), short stature
 
Patient
2
 
Patient
3
 
Patient
4
interstitial
duplication
(BP2-BP3)
deletion BP2-BP3
(Class I)
paternalM 6 yAutism Moderate MRSpeech
delay, no
phrases
N o
language
Frequent otitis, mastication/swallowing difficulties, no dysmorphic features
de
(maternal)
novo M 24 yAutism Severe MRNo Sleep difficulties, delayed motor development, gait difficulties and inappropriate laughter
during infancy, unilateral renal hypoplasia, strabismus, tongue protrusion, no dysmorphism,
normal head circumference, normal brain CT, normal EEG
Normal motor development, hyperactivity, aggressiveness, sleep difficulties, minor
dysmorphic features (slightly bulbous nose, dysplastic ears, widely spaced teeth), tapering
fingers, hyperextension of finger joints, bilateral genu valgum, pyramidal syndrome, slightly
spastic gait, no inappropriate laughter, normal head circumference
paternal
uniparental
disomy
de
(paternal)
novo M 18 yAutism Severe MR Limited
language,
no phrases
Absence seizures
since
childhood;
seizure-free
present
early
at
Copy number variants likely non pathogenic
 
Patient
5
 
Patient
6
 
Patient
7
 
Patient
8
 
Patient
9
 
Patient
10
ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder; BP, breakpoint; CT, computed tomography; EEG, electroencephalogram; idic(
mental retardation; MRI, magnetic resonance imaging
deletion BP1-BP2 paternalM5 y Autism Severe MRN o
language
No
abnormal EEG)
(but Hypotonia, motor delay, frequent respiratory infections during infancy, macrocephaly,
retrognathia, normal brain MRI
deletion BP1-BP2 maternalF13 yAutism Normal IQ No speech
delay
No Developmental coordination disorder, depression. The mother, who also carries the deletion,
has panic disorder and depression
deletion BP1-BP2 paternalF9 yAutism Normal IQNo speech
delay
NoADHD combined type. Two older sisters also carry the deletion, one with ADHD and the
other with dyslexia, while the younger healthy brother did not inherit the deletion.
duplication
BP1-BP2
maternalM5 y Autism Severe MRFunctional
language
Yes (onset 9 m) Normal brain MRI
duplication
BP1-BP2
paternalM 15 yAutism Severe MR N o
language
1
seizure at 11 y
absenceNeonatal overgrowth, macrocephaly and increased height persist, long narrow hands and feet
deletion ex 1
UBE3A
paternalM6 yAutism Mild MRSevere
language
impairment
No No dysmorphic features, normal neurological exam
), isodicentric chromosome 15; MR,
15

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Table 2
Patients with paternally-derived interstitial duplications or triplications of chromosome 15q11-q13 with an abnormal phenotype reported in the literature
StudyRearrangementSex Age Clinical characteristics
39 duplication (at least
BP2-BP3)4
mo
38 duplication (at least
BP2-BP3)
Mohandas 1999 ()et al.,
M 2 y, developmental delay, absent speech, partial agenesis of the corpus callosum and heterotopic gray matter in the right hemisphere; no
dysmorphic features
Engelen ., 1999 () et al
M 12 y features similar to Prader-Willi syndrome, including developmental delay, obesity starting during the first year of life, mild mental
retardation, excessive eating, skin picking, minor dysmorphic features (high forehead, upslanting palpebral fissures), myopia, and
small hands
M 16 y developmental and speech delay, behavioral problems (mood liability, social immaturity), uncontrolled appetite, food stealing,
self-injurious behavior, depression and anxiety, low average IQ, short stature, obesity, no dysmorphic features
F 5 y proband: motor and speech delay delay, PDD-NOS, borderline mental retardation, clumsy and uncoordinated, stiff gait, mild
hypotonia, joint laxity, slight down-slanting palpebral fissures
Mao 2000 ()et al.,
40 duplication
BP2-BP3)
duplication BP1-BP3
(at least
Roberts
2004, Veltman
)
2002, Bolton
2005 (et al.,
et al., et al.,
, ,
37 53
54
duplication BP1-BP3F8 y older sister: language and speech delay, developmental motor coordination disorder, oppositional defiant behavior, encopresis, autistic
behavior when younger but no ASD at 8 y, low average IQ; clumsy, mild hypotonia, joint laxity*
17 y hypotonia, development and speech delay, autism, dyscalculia, short stature, minimal dysmorphism and motor coordination problems;
normal IQ
6 y neonatal hypotonia, difficulty feeding, motor and speech delay (words 2.5 y, phrases 6 y), violent and repetitive behavior, high pain
tolerance, short stature, wide mouth, normal neurological exam
Smith 2004 ()et al.,
41 duplication BP2-BP3F
Cassidy 1996 ()et al.,
42 triplication
(BP1-2 not studied) (BP3-4
normal)
triplication BP1-BP4
BP2-BP3F
Ungaro ., 2001 () et al
43 F 12 y features similar to Prader-Willi syndrome, including mild mental retardation, cleft palate, obesity, compulsive eating, small hands and
feet, and short stature
All rearrangements included the Prader-Willi syndrome/Angelman syndrome critical region. All duplications/triplications were characterized molecularly and parental transmission was assessed with
methylation studies, except Engelen ( ) who determined parental origin based on cytogenetic polymorphisms, and Smith
) observed the duplication with FISH but microsatellite analysis showed no duplication and the methylation pattern was normal, thus making this case questionable.
*Another sibling, who did not carry the duplication, also had mild hypotonia and a history of marked speech delay and articulatory dyspraxia, while the mother had delayed speech and troubles with
reading and writing in school.
Abbreviations: ASD, autism spectrum disorder; BP, breakpoint, PDD-NOS, pervasive developmental disorder not otherwise specified
( ), who used microsatellite analysis. In addition, Engelen (et al. 38 et al. 41 et al.
38