In addition to these core features, ASD are sometimes associated with specific endophenotypes such as macrocephaly and abnormal serotonin/melatonin blood levels. Although ASD are considered to be some of the most genetic of all psychiatric disorders, their basic causes remain largely unknown.
Our international research group gathers psychiatrists, psychologists, neuroscientists and geneticists to understand the causes of ASD. We previously identified the first synaptic pathway associated with ASD – the NLGN-NRXN-SHANK pathway. This pathway is known for playing a role in synapse formation and in the excitatory-inhibitory balance within the brain. In parallel, we identified the first mutations within the melatonin pathway, which probably contribute to the major sleep problems observed in patients with ASD. Our results highlight the genetic heterogeneity of ASD, but also point to common synaptic pathways that could constitute relevant targets for new treatments.
Our research project for the next years includes a thorough genomic and clinical profiling of 300-500 patients using high-throughput genotyping/sequencing and brain imaging. In parallel, we are focusing on a set of mutations that we identified within NLGN and SHANK genes by studying in depth their functional impacts at the mouse behaviour, neuronal and clinical levels. A special effort will be made to test if the observed deficits are reversible using mouse models and human induced pluripotent stem cells (iPSC). Our group is also developing new methods for analyzing whole genome and brain imaging data as well as new paradigms for characterizing mouse social and vocal behavior.
Overall, the aim of our research is to identify new genetic pathways involved in social interactions and communication in humans, and to offer an evidence-based approach to ASD that should help improve their diagnosis, care and integration.
AUTISM. FROM MICE TO MEN
1. Establishment of cohorts of patients with ASD
Our group is part of several international and national consortia that provide the specific multidisciplinary clinical (and basic science) in-depth expertise required to explore different facets of ASD.
For the past 25 years, the Paris Autism Research International Sib-pair (PARIS) Study, a collaborative project, coordinated by Professors Christopher Gillberg (Gothenbburg) and Marion Leboyer (Paris), joined the efforts of clinicians in Sweden and France, later also the Faroe Islands, with the purpose of gathering a large cohort of patients with ASD and their first degree relatives to perform genetic studies. Professor Thomas Bourgeron (Paris), who joined the project 15 years ago, is also the PI of the European High-Functioning autism Network (EU-HFAutism) that gathers groups in France, Germany and Spain to study the genetics of ASD patients without intellectual disability (HF-ASD, IQ>70). In France, our group has recently gathered clinical groups working on ASD. In each center, psychiatrists and geneticists are collaborating to include additional and well-characterized patients with ASD and their relatives. Additional efforts will be focused on the inclusion of large pedigrees with multiple affected subjects.
All the individuals with ASD included in the cohort are comprehensively clinically assessed by expert clinicians (psychiatrists, neuropediatricians and psychologists), who have particular skills in autism, and other neurodevelopmental disorders (ESSENCE). To date, within this collaborative project, we have investigated 808 families with ASD mostly from Sweden, France, and the Faroe Islands (616 trios, 118 families with two affected sibs, 18 families with three affected sibs and 4 multigenerational families). We have clinical data and DNA from most of these families including parents and unaffected sibllings. We are continuously recruiting new patients with ASD, and we expect to include 500 new patients by the end of 2015.
2. Genetics of ASD
In the last decade, many candidate susceptibility genes for ASD have been reported. However, in complex neurodevelopmental disorders, such as ASD, the identified variants may not fully segregate with the trait and are usually present only in a small subset of patients. We are therefore performing a detailed analysis of the genome of the patients, their relatives, and controls.
We are using complementary approaches to identify candidate susceptibility genes to ASD: high-throughput SNP genotyping, whole-exome and whole genome sequencing. The data generated are then analyzed using association and pathway analyses in order to detect significant genotype-phenotype correlations.
In 2003 our group was the first to report mutations of two X-linked neuroligins, NLGN3 and NLGN4X, in patients with autism or Asperger syndrome. Neuroligins are cell adhesion molecules with a crucial role in the formation of functional synapses. They are located at the postsynaptic side of the synapse and bind to neurexins located on the pre-synaptic side of the synapse. Following these results, we identified mutations in SHANK2 and SHANK3 that are scaffolding proteins of the postsynaptic density, which binds to NLGN, and are known to regulate the structural organization of dendritic spines.
Our current projects are focused on understanding the additive or episatic effect of more than one mutation affecting different genes in individuals with ASD (the multiple hit model). We are especially studying the interplay between synaptic and clock proteins in the susceptibility of ASD.
3. Intermediate phenotypes in ASD: Brain imaging and biochemistry
In addition to the clinical data, we are also studying brain imaging and biochemistry data as intermediate phenotypes in ASD.
Brain imaging: The differences in cranial perimeter among some ASD patients and controls have been suggested to result from an abnormally faster brain growth during infancy that levels out with age. These results have been interpreted in the framework of the influential central coherence theory which suggests that the lack of “high-level” and “low-level” coherence among individuals with ASD could be due to an abnormal profusion of local frontal lobe connections with the rest of the brain becoming virtually “disconnected”. We are currently exploring the link between the genetic and neuroimaging findings of ASD patients. We are developing various methods for the analysis of cortical folding (http://brainfolding.sourceforge.net and http://coactivationmap.sourceforge.net), the multivariate allometry of brain anatomy, and brain functional connectivity as well as developing mathematical models to describe the morphogenesis of cortical folding and its pattern in the human brain.
Biochemistry: Sleep difficulties are a major concern for families of ASD patients, but are often considered as an epiphenomenon, and therefore do not catch the attention of the medical scientific community. High blood serotonin and low blood melatonin levels have been repeatedly reported in patients with ASD, but the causes of these imbalances are still unknown. In 2008, we were first to report genetic and biochemical alterations of the melatonin pathway in patients with ASD. We identified deletions, partial duplications and loss of function mutations of ASMT, coding for the last enzyme of the melatonin synthesis pathway, in a subset of patients with ASD. Following these results, we developed new tools to study the ASMT protein by purifying the recombinant human protein, producing antibodies with high affinity and resolving its crystal structure. We are currently analyzing a large cohort of more than 800 individuals (>550 patients, >50 siblings; >200 controls) with clinical, biochemical and genetic data in order to understand the mechanisms accounting for the serotonin-melatonin pathway imbalance and the clinical consequences of this imbalance.
4. Cell and animal models of ASD
In order to characterize the mechanisms leading to ASD, we are studying several mouse models of ASD, and human neuronal cells derived from induced pluripotent stem cells (iPSC) of patients with ASD.
Mouse models of ASD: We are currently studying mice lacking SHANK2 or SHANK3 proteins. The aim is to identify the synaptic and microcircuit defects associated with ASD and to screen for molecules to reverse the defect. The methodologies include behaviorual analyses (with a focus on social communication), transcriptomic, biochemistry and proteomic as well as advanced optical tools to study microcircuit defects in cerebellum and cortex. Our previous results include the characterization of mouse models lacking NLGN4 (Jamain et al. PNAS 2008; Ey; Genes Brain Behav; 2012), SHANK2 and SHANK3 (Schmeisser et al. Nature 2012).
iPSC: We are currently studying neuronal cells derived from iPSC of patients with ASD carrying SHANK3 truncating mutations. The aim is to identify the synaptic defects associated with ASD and to screen for molecules to reverse the defects. The methodologies include: generation of neurons derived from iPSCs from patients with ASD, transcriptomic, advanced optical tools to study synaptic proteins (including STED microsopy) and physiology (neurotransmitter uncaging and fast confocal microscopy), neuronal biochemistry and proteomic, high-throughput molecule screening.
5. Major Results of the Laboratory
• The identification of genes associated with ASD (Jamain et al. Nature Genetics 2003; Durand et al. Nature Genetics, 2007; Szatsmari et al. Nature Genetics 2007; Pinto et al. Nature 2010 ; Anney et al. Hum. Mol. Genet. 2010 ; Leblond et al. PLoS Genetics 2012)
• The characterization of mouse models of ASD: NLGN4 KO : Jamain et al. PNAS 2008; Ey et al. Brain Behav. 2012; SHANK2 and SHANK3 KO (Schmeisser et al. Nature 2012).
• The identification of the first mutations in the melatonin pathway in humans (Melke et al Mol Psychiatry 2008; Chaste et al. PLoS One 2010; Chaste et al. J. Pineal Res. 2011 ; Pagan et al. BMC Medical Genetics 2011)
• New tools for studying ASMT, the last enzyme of the melatonin pathway (Ben-Abdallah Prot Expr and Purif (2011); Maronde et al. J. Pineal Res. 2011)
• Reviews on the genetics of ASD (Bourgeron Curr. Opin. Neurobiol. 2009; Toro et al. Trends in Genetics 2010; Ey et al., Autism Research 2011; Huguet et al. Annu. Rev. Genomics Hum. Genet. 2013; Delorme et al. Nature Medicine 2013).
6. General conclusion
The identification of new genes associated with ASD has consolidated our hypothesis that genetic mutations in synaptic proteins play an important role in the development of the disorder. These results highlight the genetic heterogeneity of ASD, but also point towards common synaptic pathways that could constitute a relevant target for new treatments. Our project now includes a better genomic and clinical profiling of the patients using high-throughput genotyping/sequencing, brain imaging and bochemistry. In parallel, we are focusing on a set of mutations that we identified within the NLGN and SHANK gene families by studying in depth their functional impacts at the animal, neuronal, and clinical levels. A special effort will be made to test whether the observed deficits are reversible using human induced pluripotent stem cells (iPSC) and animal models.
7. Main publications of our group
Delorme R, Ey E, Toro R, Leboyer M, Gillberg C, and Bourgeron T. (2013) Progress towards treatments for synaptic defects in autism. Nature Medicine 19:685-94.
Schmeisser MJ, Ey E, Kuebler A, Bockmann J, Wegener S, Stempel AV, Kuebler A, Janssen AL, Udvardi PT, Shiban E, Spilker C, Balschun D, Skryabin BV, tom Dieck S, Smalla KH, Montag D, Leblond CS, Faure P, Torquet N, Le Sourd AM, Toro R, Grabrucker AM, Shoichet SA, Schmitz D, Kreutz MR, Bourgeron T, Gundelfinger ED and Boeckers TM. (2012) Hyperactivity and autistic-like behaviours in mice lacking ProSAP1/Shank2. Nature 486 : 256-60.
Leblond CS, Heinrich J, Delorme R, Proepper C, Betancur C, Huguet G, Konyukh M, Chaste P, Ey E, Rastam M, Anckarsäter H, Nygren G, Gillberg IC, Melke J, Toro R, Regnault B, Fauchereau F, Mercati O, Lemière N, Skuse D, Poot M, Holt R, Monaco AP, Järvelä I, Kantojärvi K, Vanhala R, Curran S, Collier DA, Bolton P, Chiocchetti A, Klauck SM, Poustka F, Freitag CM, Waltes R, Kopp M, Duketis E, Bacchelli E, Minopoli F, Ruta L, Battaglia A, Mazzone L, Maestrini E, Sequeira AF, Oliveira B, Vicente A, Oliveira G, Pinto D, Scherer SW, Zelenika D, Delepine M, Lathrop M, Bonneau D, Guinchat V, Devillard F, Assouline B, Mouren MC, Leboyer M, Gillberg C, Boeckers TM, Bourgeron T. (2012) Genetic and functional analyses of SHANK2 mutations provide evidence for a multiple hit model of autism spectrum disorders. PLoS Genetics 8(2):e1002521.
Toro R, Konyukh M, Delorme R, Leblond C, Chaste P, Fauchereau F, Coleman M, Leboyer M, Gillberg C and Bourgeron T. (2010) Key role for gene dosage and synaptic homeostasis in autism spectrum disorders. Trends in Genetics 26:363-372.
Pinto D, Pagnamenta A, Klei L Merico D, Anney R , Merico D, Regan R, Conroy J, Magalhaes T, Correia C, Abrahams BS, Almeida J, Bacchelli E, Bader GD, Bailey AJ, Baird G, Battaglia A, Berney T, Bolshakova N, Bölte S, Bolton PF, Bourgeron T et al. (2010) Functional impact of global rare copy number variation in autism Nature 466 : 368-72.
Jamain S., Radyushkin K, Hammerschmidt K, Granon S, Boretius S, Varoqueaux F, Ramanantsoa N, Gallego J, Ronnenberg A, Winter D, Frahm J, Fischer J, Bourgeron T, Ehrenreich H, and Brose N. (2008) Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism. Proc Natl Acad Sci U S A. 105:1710-1715.
Melke J, Goubran-Botros H, Chaste P, Betancur C, Nygren G, AnckarsäterH, Rastam M, Ståhlberg O, Gillberg IC, Delorme R, Chabane N, Mouren-Simeoni MC, Fauchereau F, Durand CM, Chevalier F, Drouot X, Collet C, Launay JM, Leboyer M, Gillberg C, and Bourgeron T (2008) Abnormal Melatonin Synthesis in Autism Spectrum Disorders Molecular Psychiatry 13:90-98.
The Autism Genome Project Consortium; (2007) Mapping autism risk loci using genetic linkage and chromosomal rearrangements. Nature Genetics. 39:319-28.
Durand C, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F, Nygren G, Rastam M, Gillberg IC, Anckarsäter H, Sponheim E, Goubran-Botros H, Delorme R, Chabane N, Mouren-Simeoni MC, de Mas P, Bieth E, Rogé B, Héron D, Burglen L, Gillberg C, Leboyer M, Bourgeron T (2007) Mutations of the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nature Genetics 39:25-7.
Jamain S, Quach H, Betancur C, Råstam M, Colineaux C, Gillberg IC, Soderstrom H, Giros B, Leboyer M, Gillberg C, Bourgeron T and the Paris study (2003). Mutations of the X-linked neuroligins NLGN3 and NLGN4 are associated with autism Nature Genetics 34, 27-29.
Figure 1 Circos plot of de novo mutations in ASDAll coding-sequence variants and copy-number variants present in the two knowledgebases AutismKB and SFARI Gene are shown. A GeneMANIA network analysis (center) highlights proteins with synaptic function. Taken from Huguet et al. The genetic landscapes of autism spectrum disorders. Annual Review of Genomics and Human Genetics. 2013.