Sarah A. Shoichet

Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2

Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2−/− mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2−/− mutants with ProSAP2/Shank3αβ−/− mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.

A novel X-linked recessive mental retardation syndrome comprising macrocephaly and ciliary dysfunction is allelic to oral–facial–digital type I syndrome

We report on a large family in which a novel X-linked recessive mental retardation (XLMR) syndrome comprising macrocephaly and ciliary dysfunction co-segregates with a frameshift mutation in the OFD1 gene. Mutations of OFD1 have been associated with oral–facial–digital type 1 syndrome (OFD1S) that is characterized by X-chromosomal dominant inheritance and lethality in males. In contrast, the carrier females of our family were clinically inconspicuous, and the affected males suffered from severe mental retardation, recurrent respiratory tract infections and macrocephaly. All but one of the affected males died from respiratory problems in infancy; and impaired ciliary motility was confirmed in the index patient by high-speed video microscopy examination of nasal epithelium. This family broadens the phenotypic spectrum of OFD1 mutations in an unexpected way and sheds light on the complexity of the underlying disease mechanisms.

Mutations in the polyglutamine binding protein 1 gene cause X-linked mental retardation.

We found mutations in the gene PQBP1 in 5 of 29 families with nonsyndromic (MRX) and syndromic (MRXS) forms of X-linked mental retardation (XLMR). Clinical features in affected males include mental retardation, microcephaly, short stature, spastic paraplegia and midline defects. PQBP1 has previously been implicated in the pathogenesis of polyglutamine expansion diseases. Our findings link this gene to XLMR and shed more light on the pathogenesis of this common disorder.

Genome-wide copy number variation analysis in attention-deficit/hyperactivity disorder: association with neuropeptide Y gene dosage in an extended pedigree

Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental syndrome characterized by hyperactivity, inattention and increased impulsivity. To detect micro-deletions and micro-duplications that may have a role in the pathogenesis of ADHD, we carried out a genome-wide screen for copy number variations (CNVs) in a cohort of 99 children and adolescents with severe ADHD. Using high-resolution array comparative genomic hybridization (aCGH), a total of 17 potentially syndrome-associated CNVs were identified. The aberrations comprise 4 deletions and 13 duplications with approximate sizes ranging from 110 kb to 3 Mb. Two CNVs occurred de novo and nine were inherited from a parent with ADHD, whereas five are transmitted by an unaffected parent. Candidates include genes expressing acetylcholine-metabolizing butyrylcholinesterase (BCHE), contained in a de novo chromosome 3q26.1 deletion, and a brain-specific pleckstrin homology domain-containing protein (PLEKHB1), with an established function in primary sensory neurons, in two siblings carrying a 11q13.4 duplication inherited from their affected mother. Other genes potentially influencing ADHD-related psychopathology and involved in aberrations inherited from affected parents are the genes for the mitochondrial NADH dehydrogenase 1 α subcomplex assembly factor 2 (NDUFAF2), the brain-specific phosphodiesterase 4D isoform 6 (PDE4D6) and the neuronal glucose transporter 3 (SLC2A3). The gene encoding neuropeptide Y (NPY) was included in a ∼3 Mb duplication on chromosome 7p15.2-15.3, and investigation of additional family members showed a nominally significant association of this 7p15 duplication with increased NPY plasma concentrations (empirical family-based association test, P=0.023). Lower activation of the left ventral striatum and left posterior insula during anticipation of large rewards or losses elicited by functional magnetic resonance imaging links gene dose-dependent increases in NPY to reward and emotion processing in duplication carriers. These findings implicate CNVs of behaviour-related genes in the pathogenesis of ADHD and are consistent with the notion that both frequent and rare variants influence the development of this common multifactorial syndrome.

Mutations in Autism Susceptibility Candidate 2 (AUTS2) in patients with mental retardation

We report on three unrelated mentally disabled patients, each carrying a de novo balanced translocation that truncates the autism susceptibility candidate 2 (AUTS2) gene at 7q11.2. One of our patients shows relatively mild mental retardation; the other two display more profound disorders. One patient is also physically disabled, exhibiting urogenital and limb malformations in addition to severe mental retardation. The function of AUTS2 is presently unknown, but it has been shown to be disrupted in monozygotic twins with autism and mental retardation, both carrying a translocation t(7;20)(q11.2;p11.2) (de la Barra et al. in Rev Chil Pediatr 57:549–554, 1986; Sultana et al. in Genomics 80:129–134, 2002). Given the overlap of this autism/mental retardation (MR) phenotype and the MR-associated disorders in our patients, together with the fact that mapping of the additional autosomal breakpoints involved did not disclose obvious candidate disease genes, we ascertain with this study that AUTS2 mutations are clearly linked to autosomal dominant mental retardation.

Mutations in the ZNF41 gene are associated with cognitive deficits: identification of a new candidate for X-linked mental retardation

Nonsyndromic X-linked mental retardation (MRX) is defined by an X-linked inheritance pattern of low IQ, problems with adaptive behavior, and the absence of additional specific clinical features. The 13 MRX genes identified to date account for less than one-fifth of all MRX, suggesting that numerous gene defects cause the disorder in other families. In a female patient with severe nonsyndromic mental retardation and a de novo balanced translocation t(X;7)(p11.3;q11.21), we have cloned the DNA fragment that contains the X-chromosomal and the autosomal breakpoint. In silico sequence analysis provided no indication of a causative role for the chromosome 7 breakpoint in mental retardation (MR), whereas, on the X chromosome, a zinc-finger gene, ZNF41, was found to be disrupted. Expression studies indicated that ZNF41 transcripts are absent in the patient cell line, suggesting that the mental disorder in this patient results from loss of functional ZNF41. Moreover, screening of a panel of patients with MRX led to the identification of two other ZNF41 mutations that were not found in healthy control individuals. A proline-to-leucine amino acid exchange is present in affected members of one family with MRX. A second family carries an intronic splice-site mutation that results in loss of specific ZNF41 splice variants. Wild-type ZNF41 contains a highly conserved transcriptional repressor domain that is linked to mechanisms of chromatin remodeling, a process that is defective in various other forms of MR. Our results suggest that ZNF41 is critical for cognitive development; further studies aim to elucidate the specific mechanisms by which ZNF41 alterations lead to MR.

Truncation of the CNS-expressed JNK3 in a patient with a severe developmental epileptic encephalopathy

We have investigated the breakpoints in a male child with pharmacoresistant epileptic encephalopathy and a de novo balanced translocation t(Y;4)(q11.2;q21). By fluorescence in situ hybridisation, we have identified genomic clones from both chromosome 4 and chromosome Y that span the breakpoints. Precise mapping of the chromosome 4 breakpoint indicated that the c-Jun N-terminal kinase 3 (JNK3) gene is disrupted in the patient. This gene is predominantly expressed in the central nervous system, and it plays an established role in both neuronal differentiation and apoptosis. Expression studies in the patient lymphoblastoid cell line show that the truncated JNK3 protein is expressed, i.e. the disrupted transcript is not immediately subject to nonsense-mediated mRNA decay, as is often the case for truncated mRNAs or those harbouring premature termination codons. Over-expression studies with the mutant protein in various cell lines, including neural cells, indicate that both its solubility and cellular localisation differ from that of the wild-type JNK3. It is plausible, therefore, that the presence of the truncated JNK3 disrupts normal JNK3 signal transduction in neuronal cells. JNK3 is one of the downstream effectors of the GTPase-regulated MAP kinase cascade, several members of which have been implicated in cognitive function. In addition, two known JNK3-interacting proteins, β-arrestin 2 and JIP3, play established roles in neurite outgrowth and neurological development. These interactions are likely affected by a truncated JNK3 protein, and thereby provide an explanation for the link between alterations in MAP kinase signal transduction and brain disorders.

Characterisation of de novo MAPK10/JNK3 truncation mutations associated with cognitive disorders in two unrelated patients.

The c-Jun N-terminal kinases (JNKs) are stress-activated serine-threonine kinases that have recently been linked to various neurological disorders. We previously described a patient with intellectual disability (ID) and seizures (Patient 1), carrying a de novo chromosome translocation affecting the CNS-expressed MAPK10/JNK3 gene. Here, we describe a second ID patient (Patient 2) with a similar translocation that likewise truncates MAPK10/JNK3, highlighting a role for JNK3 in human brain development. We have pinpointed the breakpoint in Patient 2, which is just distal to that in Patient 1. In both patients, the rearrangement resulted in a predicted protein interrupted towards the C-terminal end of the kinase domain. We demonstrate that these truncated proteins, although capable of weak interaction with various known JNK scaffolds, are not capable of phosphorylating the classical JNK target c-Jun in vitro, which suggests that the patient phenotype potentially arises from partial loss of JNK3 function. We next investigated JNK3-binding partners to further explore potential disease mechanisms. We identified PSD-95, SAP102 and SHANK3 as novel interaction partners for JNK3, and we demonstrate that JNK3 and PSD-95 exhibit partially overlapping expression at synaptic sites in cultured hippocampal neurons. Moreover, JNK3, like JNK1, is capable of phosphorylating PSD-95 in vitro, whereas disease-associated mutant JNK3 proteins do not. We conclude that reduced JNK3 activity has potentially deleterious effects on neuronal function via altered regulation of a set of post-synaptic proteins.

Identification of candidate genes for sporadic amyotrophic lateral sclerosis by array comparative genomic hybridization.

Amyotrophic lateral sclerosis (ALS) is a devastating disorder of the central nervous system that leads to progressive loss of upper and lower motor neurons. Most cases are sporadic and of unknown aetiology. In this study, we screened 72 patients with sporadic ALS for the presence of DNA copy number variations, in order to identify novel candidate disease genes. We have used sub-megabase resolution BAC array comparative genomic hybridization to detect genomic imbalances in our ALS patient cohort. Aberrations with potential relevance for disease aetiology were verified by oligo array CGH. In 72 patients with sporadic ALS, we identified a total of six duplications and five deletions that scored above our threshold. Nine of these 11 variations were smaller than 1Mb, and five were observed exclusively in ALS patients. In conclusion, non-polymorphic sub-microscopic duplications and deletions observable by array CGH are frequent in patients with sporadic ALS. Analysis of such aberrations serves as a starting point in deciphering the aetiology of this complex disease, given that affected genes can be considered candidates for influencing disease susceptibility.

Synaptic MAGUK Multimer Formation Is Mediated by PDZ Domains and Promoted by Ligand Binding

To examine the scaffolding properties of PSD-95, we have taken advantage of established ligand/PDZ domain interactions and developed a cell-based assay for investigating protein complex formation. This assay enables quantitative analysis of PDZ domain-mediated protein clustering using bimolecular fluorescence complementation (BiFC). Two nonfluorescent halves of EYFP were fused to C-terminal PDZ ligand sequences to generate probes that sense for PDZ domain binding grooves of adjacent (interacting) molecules. When these probes are brought into proximity by the PDZ domains of a multiprotein scaffold, a functional fluorescent EYFP molecule can be detected. We have used this system to examine the properties of selected PSD-95 variants and thereby delineated regions of importance for PSD-95 complex formation. Further analysis led to the finding that PSD-95 multimerization is PDZ domain-mediated and promoted by ligand binding.