Quelqu'un aurait il le coeur à l'ouvrage ?? 
Brief Research Communication
*
Correspondence to Guy A. Rouleau, Centre hospitalier de l'Université de Montréal, 2099, Alexandre De-Seve Street, Room Y-3633,
Montreal, Quebec, Canada.
Please cite this article as follows: Gauthier J, Spiegelman D, Piton A, Lafrenière RG, St-Onge J, Lapointe L, Hamdan FF, Cossette P,
Mottron L, Fombonne É, Joober R, Marineau C, Drapeau P, Rouleau GA. 2008. Novel De Novo SHANK3 Mutation in Autistic Patients. Am
J Med Genet Part B.
Received: 14 March 2008; Accepted: 28 May 2008
10.1002/ajmg.b.30822 About DOI
Autism spectrum disorder (ASD) is a neurodevelopmental disease characterized by complex behavioral and cognitive deficits. A number of
studies have confirmed that genetic factors play an important role in ASD [Muhle et al., [2004]]. Our gene discovery strategy for ASD is to
systematically sequence a large number of genes in autistic individuals, focusing on genes encoding synapse proteins. This strategy is
based on two hypotheses: (1) that de novo mutations in different genes may account for a significant portion of ASD and (2) that mutations
in synaptic genes are responsible for the ASD phenotype. It is known that ASD is highly heterogeneous and cannot be explained by few
Novel de novo SHANK3 mutation in autistic patients
Julie Gauthier 1, Dan Spiegelman 1, Amélie Piton 1, Ronald G. Lafrenière 1, Sandra Laurent 1, Judith St-Onge 1, Line Lapointe 1, Fadi F.
Hamdan 2, Patrick Cossette 1, Laurent Mottron 3, Éric Fombonne 4, Ridha Joober 5, Claude Marineau 1, Pierre Drapeau 6, Guy A.
Rouleau 1 *
1
Centre of Excellence in Neuromics of Université de Montréal, CHUM Research Centre, Notre-Dame Hospital, Université de Montréal,
Montreal, QC, Canada
2
Division of Medical Genetics, CHU Sainte-Justine, Université de Montréal, Montreal, QC, Canada
3
Department of Psychiatry, Université de Montréal, Hôpital Rivière-des-Prairies, Montreal, Canada
4
Department of Psychiatry, McGill University and Montreal Children's Hospital, Montreal, QC, Canada
5
Department of Psychiatry, McGill University and Douglas Hospital Research Centre, Montreal, QC, Canada
6
Department of Pathology and Cellular Biology, and Groupe de recherche sur le système nerveux central, Université de Montréal,
Montreal, QC, Canada
email: Guy A. Rouleau (guy.rouleau@umontreal.ca)
KEYWORDS
Splice site • Autism spectrum disorder • SHANK • de novo • Pervasive developmental disorder
ABSTRACT
A number of studies have confirmed that genetic factors play an important role in autism spectrum disorder (ASD). More recently de novo
mutations in the SHANK3 gene, a synaptic scaffolding protein, have been associated with the ASD phenotype. As part of our gene
discovery strategy, we sequenced the SHANK3 gene in a cohort of 427 ASD subjects and 190 controls. Here, we report the identification
of two putative causative mutations: one being a de novo deletion at an intronic donor splice site and one missense transmitted from an
epileptic father. We were able to confirm the deleterious effect of the splice site deletion by RT-PCR using mRNA extracted from cultured
lymphoblastoid cells. The missense mutation, a leucine to proline at amino acid position 68, is perfectly conserved across all species
examined, and would be predicted to disrupt an alpha-helical domain. These results further support the role of SHANK3 gene disruption
in the etiology of ASD. © 2008 Wiley-Liss, Inc.
DIGITAL OBJECT IDENTIFIER (DOI)
ARTICLE TEXT
Novel de novo SHANK3 mutation in autistic patients
Julie Gauthier 1, Dan Spiegelman 1, Amélie Piton 1, Ronald G. Lafrenière 1, Sandra Laurent 1, Judith St-Onge 1, Line Lapointe 1, Fadi F.
Hamdan 2, Patrick Cossette 1, Laurent Mottron 3, Éric Fombonne 4, Ridha Joober 5, Claude Marineau 1, Pierre Drapeau 6, Guy A.
Rouleau 1 *
1
Centre of Excellence in Neuromics of Université de Montréal, CHUM Research Centre, Notre-Dame Hospital, Université de Montréal,
Montreal, QC, Canada
2
Division of Medical Genetics, CHU Sainte-Justine, Université de Montréal, Montreal, QC, Canada
3
Department of Psychiatry, Université de Montréal, Hôpital Rivière-des-Prairies, Montreal, Canada
4
Department of Psychiatry, McGill University and Montreal Children's Hospital, Montreal, QC, Canada
5
Department of Psychiatry, McGill University and Douglas Hospital Research Centre, Montreal, QC, Canada
6
Department of Pathology and Cellular Biology, and Groupe de recherche sur le système nerveux central, Université de Montréal,
Montreal, QC, Canada
email: Guy A. Rouleau (guy.rouleau@umontreal.ca)
KEYWORDS
Splice site • Autism spectrum disorder • SHANK • de novo • Pervasive developmental disorder
ABSTRACT
A number of studies have confirmed that genetic factors play an important role in autism spectrum disorder (ASD). More recently de novo
mutations in the SHANK3 gene, a synaptic scaffolding protein, have been associated with the ASD phenotype. As part of our gene
discovery strategy, we sequenced the SHANK3 gene in a cohort of 427 ASD subjects and 190
common variants. Furthermore, the few genes that have been found to definitely predispose to ASD explain only a small fraction of cases
[Jamain et al., [2003]; Durand et al., [2007]]. The similar ASD incidence across different populations, in spite of a low reproductive fitness,
argues in favor of a novel mutation scenario. A large body of neurobiological studies indicate that synaptic dysfunctions occur in ASD:
reduced neuronal size and shortened dendritic patterns are a few examples [Raymond et al., [1996]; Kemper and Bauman, [1998]]. In
addition, the identification of mutations in the postsynaptic cell adhesion neuroligin three and four genescommon variants. Furthermore, the few genes that have been found to definitely predispose to ASD explain only a small fraction of cases
[Jamain et al., [2003]; Durand et al., [2007]]. The similar ASD incidence across different populations, in spite of a low reproductive fitness,
argues in favor of a novel mutation scenario. A large body of neurobiological studies indicate that synaptic dysfunctions occur in ASD:
reduced neuronal size and shortened dendritic patterns are a few examples [Raymond et al., [1996]; Kemper and Bauman, [1998]]. In
addition, the identification of mutations in the postsynaptic cell adhesion neuroligin three and four genes in autistic patients further support
this hypothesis [Jamain et al., [2003]].
A cohort of 427 ASD subjects (66 females: 361 males) and 190 ethnically matched controls were screened for the entire coding region and
intronic splice junctions of the SHANK3 gene except exon 11, which was problematic even after numerous primer redesigns and PCR
amplification conditions, as reported by others [Wilson et al., [2003]; Moessner et al., [2007]]. Diagnostic and selection criteria for the ASD
subjects are described in detail elsewhere [Gauthier et al., [2005]]. Briefly, all subjects were diagnosed using the Diagnostic and Statistical
Manual of Mental Disorders criteria. Depending on the recruitment site, Autism Diagnostic Interview-Revised and the Autism Diagnostic
Observation Schedule were used. In addition, the Autism Screening Questionnaire (ASQ) was also completed for all our subjects. We
excluded patients with an estimated mental age <18 months, a diagnosis of Rett syndrome or Childhood Disintegrative Disorder and
patients with evidence of any psychiatric and neurological conditions including: birth anoxia, rubella during pregnancy, fragile-X disorder,
encephalitis, phenylketonuria, tuberous sclerosis, Tourette and West syndromes. Primer sequences were designed using ExonPrimer from
UCSC Genome Browser. Sequence determination and base pair variant detection were performed at the McGill University and Genome
Quebec Innovation Centre in Montreal, Canada (www.genomequebecplatforms.com/mcgill/) on a 3730XL DNA Analyzer System. The
human SHANK3 cDNA sequence was kindly provided by Stephen Scherer [Moessner et al., [2007]]. RT-PCR was performed on mRNA
isolated from cultured lymphoblastoid cells by using a forward primer targeting the exon 15/16 junction: 5-TTCCTCATCGAGGTGAACG-
3 and a reverse primer targeting the exon 20/21 junction: 5-AGCTTCTCGTCCTCCCCTAC-3. Approximately 106 lymphoblastoid cells
were lysed in TRIZOL® Reagent (Invitrogen, Carlsbad, CA) and total RNA was extracted according to the protocol of manufacturer . We
obtained 150 µg of total RNA. As SHANK3 is not well expressed in lymphoblastoid cells [Durand et al., [2007]], we purified mRNA from
150 µg of total RNA before the reverse transcription by using Oligotex protocol (Qiagen, Valencia, CA). After this purification, the mRNA
was reverse transcribed into cDNA using M-MLV RT (Moloney murine leukemia virus reverse transcriptase, Invitrogen): 29 µl of mRNA (
2-5 µg), 1 µl random hexamer 1 µg/µl (GE Healthcare, Piscataway, NJ), 65°C for 3 min, 10 µl first strand buffer 5X (Invitrogen), 5 µl of
DTT 0.1 M (Invitrogen), 1 µl of RNAguardTM Rnase inhibitor porcine 32.2 mU/µl (GE Healthcare), 1 µl of M-MLV RT enzyme 200 U/µl
(Invitrogen), 2 µl of dNTP 25 mM (Invitrogen), 37°C for 1 hr. 50 µl of RT are diluted in a final volume of 100 µl.
Eight missense variants and one potential splice site mutation were identified during the screen (Table I). Four of the missenses were
identified in both patient and control samples: the I245T missense was previously reported as rs9616915, at allelic frequencies
comparable to our study; A721T was found in 5-6% of samples; P1654T was previously identified in normal control samples [Durand et al.,
[2007]]; and R1298K was found in one ASD and one control sample. Two missenses were found only in single control samples (R215C,
which maps within the third Ankyrin repeat and A1324T). The A224T missense identified in one of our ASD samples was previously
identified in one control sample [Durand et al., [2007]]. The number of variants identified is consistent with previous studies, considering
we did not screen these samples for CNVs. The L68P missense was identified in one female patient with pervasive developmental
disorder not otherwise specified, and was inherited from the father of patient who was diagnosed with epilepsy. The proband did not have
seizures. There is also a paternal cousin diagnosed with dysphasia. The mother had pre-eclampsia at 38 weeks of pregnancy and the
pregnancy was provoked at 39 weeks. The ASQ score for this patient was 16 (score > 15 = ASD). The proband's development was
considered normal from birth to age one. She walked at 13-14 months. She stopped saying the few words learned at one and a half years
of age. Suspicion of ASD came around 18 months of age when parents noted a language deficiency.
Table I. SHANK3 Non-Synonymous Variants Identified in ASD and Control Subjects
Exona
Nucleotide
changeb
Amino acid
changeb
Occurrence
Transmission
from
Known
SNP
ASD
(n = 427)
CTRL
(n = 190)
2 c.203T>C L68P 1 0 Father No
6 c.643C>T R215C 0 1 Mother No
6 c.670G>A A224T 1 0 Not done Durand et
al.
6 c.734T>C I245T 270 124 Not done rs9616915
19 c.2161G>A A721T 28 10 Not done No
19 c.2265C +1delG S755Sfs × 1 1 0 de novo No
21 c.3893G>A R1298K 1 1 Mother No
21 c.3970G>A A1324T 0 1 Mother No
24 c.4960C>A P1654T 2 3 Not done Durand et
al.
common variants. Furthermore, the few genes that have been found to definitely predispose to ASD explain only a small fraction of cases
[Jamain et al., [2003]; Durand et al., [2007]]. The similar ASD incidence across different populations, in spite of a low reproductive fitness,
argues in favor of a novel mutation scenario. A large body of neurobiological studies indicate that synaptic dysfunctions occur in ASD:
reduced neuronal size and shortened dendritic patterns are a few examples [Raymond et al., [1996]; Kemper and Bauman, [1998]]. In
addition, the identification of mutations in the postsynaptic cell adhesion neuroligin three and four genes in autistic patients further support
this hypothesis [Jamain et al., [2003]].
A cohort of 427 ASD subjects (66 females: 361 males) and 190 ethnically matched controls were screened for the entire coding region and
intronic splice junctions of the SHANK3 gene except exon 11, which was problematic even after numerous primer redesigns and PCR
amplification conditions, as reported by others [Wilson et al., [2003]; Moessner et al., [2007]]. Diagnostic and selection criteria for the ASD
subjects are described in detail elsewhere [Gauthier et al., [2005]]. Briefly, all subjects were diagnosed using the Diagnostic and Statistical
Manual of Mental Disorders criteria. Depending on the recruitment site, Autism Diagnostic Interview-Revised and the Autism Diagnostic
Observation Schedule were used. In addition, the Autism Screening Questionnaire (ASQ) was also completed for all our subjects. We
excluded patients with an estimated mental age <18 months, a diagnosis of Rett syndrome or Childhood Disintegrative Disorder and
patients with evidence of any psychiatric and neurological conditions including: birth anoxia, rubella during pregnancy, fragile-X disorder,
encephalitis, phenylketonuria, tuberous sclerosis, Tourette and West syndromes. Primer sequences were designed using ExonPrimer from
UCSC Genome Browser. Sequence determination and base pair variant detection were performed at the McGill University and Genome
Quebec Innovation Centre in Montreal, Canada (www.genomequebecplatforms.com/mcgill/) on a 3730XL DNA Analyzer System. The
human SHANK3 cDNA sequence was kindly provided by Stephen Scherer [Moessner et al., [2007]]. RT-PCR was performed on mRNA
isolated from cultured lymphoblastoid cells by using a forward primer targeting the exon 15/16 junction: 5-TTCCTCATCGAGGTGAACG-
3 and a reverse primer targeting the exon 20/21 junction: 5-AGCTTCTCGTCCTCCCCTAC-3. Approximately 106 lymphoblastoid cells
were lysed in TRIZOL® Reagent (Invitrogen, Carlsbad, CA) and total RNA was extracted according to the protocol of manufacturer . We
obtained 150 µg of total RNA. As SHANK3 is not well expressed in lymphoblastoid cells [Durand et al., [2007]], we purified mRNA from
150 µg of total RNA before the reverse transcription by using Oligotex protocol (Qiagen, Valencia, CA). After this purification, the mRNA
was reverse transcribed into cDNA using M-MLV RT (Moloney murine leukemia virus reverse transcriptase, Invitrogen): 29 µl of mRNA (
2-5 µg), 1 µl random hexamer 1 µg/µl (GE Healthcare, Piscataway, NJ), 65°C for 3 min, 10 µl first strand buffer 5X (Invitrogen), 5 µl of
DTT 0.1 M (Invitrogen), 1 µl of RNAguardTM Rnase inhibitor porcine 32.2 mU/µl (GE Healthcare), 1 µl of M-MLV RT enzyme 200 U/µl
(Invitrogen), 2 µl of dNTP 25 mM (Invitrogen), 37°C for 1 hr. 50 µl of RT are diluted in a final volume of 100 µl.
Eight missense variants and one potential splice site mutation were identified during the screen (Table I). Four of the missenses were
identified in both patient and control samples: the I245T missense was previously reported as rs9616915, at allelic frequencies
comparable to our study; A721T was found in 5-6% of samples; P1654T was previously identified in normal control samples [Durand et al.,
[2007]]; and R1298K was found in one ASD and one control sample. Two missenses were found only in single control samples (R215C,
which maps within the third Ankyrin repeat and A1324T). The A224T missense identified in one of our ASD samples was previously
identified in one control sample [Durand et al., [2007]]. The number of variants identified is consistent with previous studies, considering
we did not screen these samples for CNVs. The L68P missense was identified in one female patient with pervasive developmental
disorder not otherwise specified, and was inherited from the father of patient who was diagnosed with epilepsy. The proband did not have
seizures. There is also a paternal cousin diagnosed with dysphasia. The mother had pre-eclampsia at 38 weeks of pregnancy and the
pregnancy was provoked at 39 weeks. The ASQ score for this patient was 16 (score > 15 = ASD). The proband's development was
considered normal from birth to age one. She walked at 13-14 months. She stopped saying the few words learned at one and a half years
of age. Suspicion of ASD came around 18 months of age when parents noted a language deficiency.
Table I. SHANK3 Non-Synonymous Variants Identified in ASD and Control Subjects
Exona
Nucleotide
changeb
Amino acid
changeb
Occurrence
Transmission
from
Known
SNP
ASD
(n = 427)
CTRL
(n = 190)
2 c.203T>C L68P 1 0 Father No
6 c.643C>T R215C 0 1 Mother No
6 c.670G>A A224T 1 0 Not done Durand et
al.
6 c.734T>C I245T 270 124 Not done rs9616915
19 c.2161G>A A721T 28 10 Not done No
19 c.2265C +1delG S755Sfs × 1 1 0 de novo No
21 c.3893G>A R1298K 1 1 Mother No
21 c.3970G>A A1324T 0 1 Mother No
24 c.4960C>A P1654T 2 3 Not done Durand et
al.
a Exon numbering is according to Wilson et al. [2003].
b cDNA sequence as described by Moessner et al. [2007].
Page 2 sur 4
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The Leucine 68 residue is 100% conserved from vertebrates to sea urchin, C. elegans, and insects, and is also conserved between
SHANK3 and its close paralogs SHANK1 and SHANK2 (Fig. 1). Secondary structure modeling of this region of the SHANK3 protein using
nnPredict (http://www.cmpharm.ucsf.edu/ nomi/nnpredict.html) suggests that the Leu68 residue lies in an alpha-helical domain. Given
this high level of conservation, that a substitution of the leucine for a proline at this position would be predicted to disrupt an alpha-helical
domain, and that the missense is not found in normal control samples suggests that this missense may be a cause of ASD in this affected
individual. However, functional studies will be required to confirm that this mutation is indeed pathogenic.
The splicing variant was examined in more detail. It was found in a male patient diagnosed with autism disorder, but was absent from
blood DNA samples from either of the biological parents (verified by DNA fingerprinting using nine highly informative microsatellite
markers). The ASQ score for this patient was 23. The delivery was normal at birth. He sat at 6 months of age, walked at 15 months and
said his first words at 12-13 months of age. Both parents are non-affected by ASD and there is no known history of ASD in this family. This
therefore constitutes a de novo mutation in this affected individual. Deletion of a G residue from the highly conserved splice donor site
would be predicted to lead to aberrant splicing of the transcript (Fig. 1A). This was confirmed using RT-PCR (Fig. 2) of mRNA isolated
from a lymphoblastoid cell line derived from the affected individual. In addition to the expected wild-type 483 bp product, a 559 bp product
could be amplified from the mRNA of patient, but not from a control mRNA sample. DNA sequencing of the RT-PCR products showed the
expected 483 bp wild-type fragment, whereas the 559 bp product contained an additional 76 bp of sequence corresponding to the 5end of
intron 19. Thus, the 1 bp deletion leads to skipping of the appropriate splice donor site, and use of a cryptic splice donor site downstream
in intron 19. The aberrant transcript is predicted to encode a prematurely truncated SHANK3 peptide of 755 amino acid residues, and
lacking a large portion of the C-terminal domains.
Since the SHANK3 protein acts as a scaffolding protein, such a prematurely truncated peptide could act in a dominant negative fashion in
cells where it is expressed. Alternately, the aberrantly spliced transcript may be degraded, leading to reduced levels of SHANK3 protein.
This would be in agreement with Durand et al. [2007] who showed that abnormal SHANK3 gene dosage or premature truncation of the
peptide are associated with ASD [Durand et al., [2007]]. We have also detected rare non-synonymous variants both in ASD patients and
controls. The potential role of these remains to be determined.
In summary, we have identified novel mutations in the SHANK3 gene in ASD patients that underline the role of this gene in ASD. Together
with previous observations, these data support our de novo mutation hypothesis for ASD. Finally, these new mutations further support the
notion that ASD is caused by dysfunction of synaptic proteins.
Acknowledgements
We would like to thank the families who made this research possible by participating in our study. Thanks to the Synapse-to-Disease
teams for their work. This work was funded by Genome Canada and Genome Quebec.
Figure 1. A: Peptide sequence alignment of a portion of the SHANK3 protein from
different species (orthologous to the human residues 29-88) showing conservation of
the Leucine 68 residue (asterisk). Predicted secondary structure using nnPredict is
shown below the sequence from SHANK1 (H = helix; E = strand; - = no prediction). B:
Sequence of the splice donor and acceptor sites for intron 19 with flanking
sequences for wilt-type (WT) and mutant (c.2256C-1delG) alleles. Deletion of the first
base of the intron causes aberrant splicing, and premature truncation of the peptide
after codon 755. [Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
[Normal View 94K | Magnified View 189K]
Figure 2. mRNA expression of the human SHANK3 gene isolated from a
lymphoblastoid cell line derived from the affected individual carrying the splice site
deletion (Proband) and one control individual. The 483 bp band corresponds to the
correctly spliced normal allele in both the control and the proband, whereas the 559
bp band seen only in the proband contained an additional 76 bp of sequence
corresponding to the 5 end of intron 19.
[Normal View 23K | Magnified View 28K]
REFERENCES
Durand CM, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F, Nygren G, Rastam M, Gillberg IC, Anckarsater H, et al.
2007. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat
Genet 39(1): 25-27. Links
Gauthier J, Bonnel A, St-Onge J, Karemera L, Laurent S, Mottron L, Fombonne E, Joober R, Rouleau GA. 2005. NLGN3/NLGN4 gene
mutations are not responsible for autism in the Quebec population. Am J Med Genet B Neuropsychiatr Genet 132(1): 74-75. Links
Jamain S, Quach H, Betancur C, Rastam M, Colineaux C, Gillberg IC, Soderstrom H, Giros B, Leboyer M, Gillberg C, et al. 2003.
Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nat Genet 34(1): 27-29. Links
Kemper TL, Bauman M. 1998. Neuropathology of infantile autism. J Neuropathol Exp Neurol 57(7): 645-652. Links
Page 3 sur 4
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Moessner R, Marshall CR, Sutcliffe JS, Skaug J, Pinto D, Vincent J, Zwaigenbaum L, Fernandez B, Roberts W, Szatmari P, et al. 2007.
Contribution of SHANK3 Mutations to Autism Spectrum Disorder. Am J Hum Genet 81(6): 1289-1297. Links
Muhle R, Trentacoste SV, Rapin I. 2004. The genetics of autism. Pediatrics 113(5): 472-486. Links
Raymond GV, Bauman ML, Kemper TL. 1996. Hippocampus in autism: a Golgi analysis. Acta Neuropathol (Berl) 91(1): 117-119. Links
Wilson HL, Wong AC, Shaw SR, Tse WY, Stapleton GA, Phelan MC, Hu S, Marshall J, McDermid HE. 2003. Molecular characterisation
of the 22q13 deletion syndrome supports the role of haploinsufficiency of SHANK3/PROSAP2 in the major neurological symptoms. J Med
Genet 40(8): 575-584. Links
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Bon O.K, c'est le bordel en faisant le copier coller
mais si quelqu'un a donc BEAUCOUP de courage je peux l'envoyer par mail en format PDF... 
C'est moi l'ame charitable????