Unraveling molecular pathways shared by Kabuki and Kabuki-like syndromes.

Kabuki syndrome (KS) is a rare genetic syndrome characterized by a typical facial gestalt, variable degrees of intellectual disability, organ malformations, postnatal growth retardation and skeletal abnormalities. So far, KMT2D or KDM6A mutation has been identified as the main cause of KS, accounting for 56%-75% and 3%-8% of cases, respectively. Patients without mutations in 1 of the 2 causative KS genes are often referred to as affected by Kabuki-like syndrome. Overall, they represent approximately 30% of KS cases, pointing toward substantial genetic heterogeneity for this condition. Here, we review all currently available literature describing KS-like phenotypes (or phenocopies) associated with genetic variants located in loci different from KMT2D and KDM6A . We also report on a new KS phenocopy harboring a 5 Mb de novo deletion in chr10p11.22-11.21. An enrichment analysis aimed at identifying functional Gene Ontology classes shared by the 2 known KS causative genes and by new candidate genes currently associated with KS-like phenotypes primarily converges upon abnormal chromatin remodeling and transcriptional dysregulation as pivotal to the pathophysiology of KS phenotypic hallmarks. The identification of mutations in genes belonging to the same functional pathways of KMT2D and KDM6A can help design molecular screenings targeted to KS-like phenotypes.

Migraine genetics: current findings and future lines of research.

In the last two decades, migraine research has greatly advanced our current knowledge of the genetic contributions and the pathophysiology of this common and debilitating disorder. Nonetheless, this knowledge still needs to grow further and to translate into more effective treatments. To date, several genes involved in syndromic and monogenic forms of migraine have been identified, allowing the generation of animal models which have significantly contributed to current knowledge of the mechanisms underlying these rare forms of migraine. Common forms of migraine are instead posing a greater challenge, as they may most often stem from complex interactions between multiple common genetic variants, with environmental triggers. This paper reviews our current understanding of migraine genetics, moving from syndromic and monogenic forms to oligogenic/polygenic migraines most recently addressed with some success through genome-wide association studies. Methodological issues in study design and future perspectives opened by biomarker research will also be briefly addressed.

Anti-brain antibodies are associated with more severe cognitive and behavioral profiles in Italian children with Autism Spectrum Disorder.

Circulating 45 and 62kDa antibodies targeting the cerebellum were previously associated with Autism Spectrum Disorder (ASD), lower adaptive/cognitive function and aberrant behaviors. Moreover, 37, 39 and 73kDa maternal antibodies (mAb) targeting the fetal brain were previously correlated with broad autism spectrum, irritability, abnormal brain enlargement and impaired expressive language. The present study aims towards clinically characterizing individuals with brain-targeted IgG and/or exposed to maternal antibrain antibodies in a large sample of Italian autistic children (N=355), their unaffected siblings (N=142) and mothers (N=333). The presence of patient- and mother-produced anti-brain antibodies does not confer increased risk of autism within the same sibship. However, the 45 and 62kDa antibodies are correlated with autism severity: the 45kDa Ab is associated with cognitive impairment and lower scores at the Vineland Adaptive Behavior Scales, the 62kDa Ab with motor stereotypies, while both correlate with larger head circumference (all P<0.05). On the other hand, maternal 37, 39 and 73kDa antibrain antibodies, either alone or in combination, are correlated with impaired verbal and non-verbal language development, neurodevelopmental delay and sleep/wake cycle disturbances in their autistic children (P<0.05). Presence of the 62kDa autoAb in the child is significantly associated with presence of the 39 and/or 73kDa antibodies in his/her mother. Our results confirm and extend previous observations in an ethnically distinct sample, providing further evidence of a pathomorphic role for anti-brain antibodies in autism while demonstrating their familial clustering.

Altered calcium homeostasis in autism-spectrum disorders: evidence from biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier AGC1.

Autism is a severe developmental disorder, whose pathogenetic underpinnings are still largely unknown. Temporocortical gray matter from six matched patient-control pairs was used to perform post-mortem biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier (AGC), which participates in the aspartate/malate reduced nicotinamide adenine dinucleotide shuttle and is physiologically activated by calcium (Ca(2+)). AGC transport rates were significantly higher in tissue homogenates from all six patients, including those with no history of seizures and with normal electroencephalograms prior to death. This increase was consistently blunted by the Ca(2+) chelator ethylene glycol tetraacetic acid; neocortical Ca(2+) levels were significantly higher in all six patients; no difference in AGC transport rates was found in isolated mitochondria from patients and controls following removal of the Ca(2+)-containing postmitochondrial supernatant. Expression of AGC1, the predominant AGC isoform in brain, and cytochrome c oxidase activity were both increased in autistic patients, indicating an activation of mitochondrial metabolism. Furthermore, oxidized mitochondrial proteins were markedly increased in four of the six patients. Variants of the AGC1-encoding SLC25A12 gene were neither correlated with AGC activation nor associated with autism-spectrum disorders in 309 simplex and 17 multiplex families, whereas some unaffected siblings may carry a protective gene variant. Therefore, excessive Ca(2+) levels are responsible for boosting AGC activity, mitochondrial metabolism and, to a more variable degree, oxidative stress in autistic brains. AGC and altered Ca(2+) homeostasis play a key interactive role in the cascade of signaling events leading to autism: their modulation could provide new preventive and therapeutic strategies.

Preference for biological motion is reduced in ASD: implications for clinical trials and the search for biomarkers.

BACKGROUND: The neurocognitive mechanisms underlying autism spectrum disorder (ASD) remain unclear. Progress has been largely hampered by small sample sizes, variable age ranges and resulting inconsistent findings. There is a pressing need for large definitive studies to delineate the nature and extent of key case/control differences to direct research towards fruitful areas for future investigation. Here we focus on perception of biological motion, a promising index of social brain function which may be altered in ASD. In a large sample ranging from childhood to adulthood, we assess whether biological motion preference differs in ASD compared to neurotypical participants (NT), how differences are modulated by age and sex and whether they are associated with dimensional variation in concurrent or later symptomatology. METHODS: Eye-tracking data were collected from 486 6-to-30-year-old autistic (N = 282) and non-autistic control (N = 204) participants whilst they viewed 28 trials pairing biological (BM) and control (non-biological, CTRL) motion. Preference for the biological motion stimulus was calculated as (1) proportion looking time difference (BM-CTRL) and (2) peak look duration difference (BM-CTRL). RESULTS: The ASD group showed a present but weaker preference for biological motion than the NT group. The nature of the control stimulus modulated preference for biological motion in both groups. Biological motion preference did not vary with age, gender, or concurrent or prospective social communicative skill within the ASD group, although a lack of clear preference for either stimulus was associated with higher social-communicative symptoms at baseline. LIMITATIONS: The paired visual preference we used may underestimate preference for a stimulus in younger and lower IQ individuals. Our ASD group had a lower average IQ by approximately seven points. 18% of our sample was not analysed for various technical and behavioural reasons. CONCLUSIONS: Biological motion preference elicits small-to-medium-sized case-control effects, but individual differences do not strongly relate to core social autism associated symptomatology. We interpret this as an autistic difference (as opposed to a deficit) likely manifest in social brain regions. The extent to which this is an innate difference present from birth and central to the autistic phenotype, or the consequence of a life lived with ASD, is unclear.

Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression.

Protein kinase C enzymes play an important role in signal transduction, regulation of gene expression and control of cell division and differentiation. The fsI and betaII isoenzymes result from the alternative splicing of the PKCbeta gene (PRKCB1), previously found to be associated with autism. We performed a family-based association study in 229 simplex and 5 multiplex families, and a postmortem study of PRKCB1 gene expression in temporocortical gray matter (BA41/42) of 11 autistic patients and controls. PRKCB1 gene haplotypes are significantly associated with autism (P<0.05) and have the autistic endophenotype of enhanced oligopeptiduria (P<0.05). Temporocortical PRKCB1 gene expression was reduced on average by 35 and 31% for the PRKCB1-1 and PRKCB1-2 isoforms (P<0.01 and <0.05, respectively) according to qPCR. Protein amounts measured for the PKCbetaII isoform were similarly decreased by 35% (P=0.05). Decreased gene expression characterized patients carrying the 'normal' PRKCB1 alleles, whereas patients homozygous for the autism-associated alleles displayed mRNA levels comparable to those of controls. Whole genome expression analysis unveiled a partial disruption in the coordinated expression of PKCbeta-driven genes, including several cytokines. These results confirm the association between autism and PRKCB1 gene variants, point toward PKCbeta roles in altered epithelial permeability, demonstrate a significant downregulation of brain PRKCB1 gene expression in autism and suggest that it could represent a compensatory adjustment aimed at limiting an ongoing dysreactive immune process. Altogether, these data underscore potential PKCbeta roles in autism pathogenesis and spur interest in the identification and functional characterization of PRKCB1 gene variants conferring autism vulnerability.

Autistic phenotypes and genetic testing: state-of-the-art for the clinical geneticist.

Autism spectrum disorders represent a group of developmental disorders with strong genetic underpinnings. Several cytogenetic abnormalities or de novo mutations able to cause autism have recently been uncovered. In this study, the literature was reviewed to highlight genotype-phenotype correlations between causal gene mutations or cytogenetic abnormalities and behavioural or morphological phenotypes. Based on this information, a set of practical guidelines is proposed to help clinical geneticists pursue targeted genetic testing for patients with autism whose clinical phenotype is suggestive of a specific genetic or genomic aetiology.

Evaluating Sex and Age Differences in ADI-R and ADOS Scores in a Large European Multi-site Sample of Individuals with Autism Spectrum Disorder.

Research on sex-related differences in Autism Spectrum Disorder (ASD) has been impeded by small samples. We pooled 28 datasets from 18 sites across nine European countries to examine sex differences in the ASD phenotype on the ADI-R (376 females, 1763 males) and ADOS (233 females, 1187 males). On the ADI-R, early childhood restricted and repetitive behaviours were lower in females than males, alongside comparable levels of social interaction and communication difficulties in females and males. Current ADI-R and ADOS scores showed no sex differences for ASD severity. There were lower socio-communicative symptoms in older compared to younger individuals. This large European ASD sample adds to the literature on sex and age variations of ASD symptomatology.

Barrel pattern formation requires serotonin uptake by thalamocortical afferents, and not vesicular monoamine release.

Thalamocortical neurons innervating the barrel cortex in neonatal rodents transiently store serotonin (5-HT) in synaptic vesicles by expressing the plasma membrane serotonin transporter (5-HTT) and the vesicular monoamine transporter (VMAT2). 5-HTT knock-out (ko) mice reveal a nearly complete absence of 5-HT in the cerebral cortex by immunohistochemistry, and of barrels, both at P7 and adulthood. Quantitative electron microscopy reveals that 5-HTT ko affects neither the density of synapses nor the length of synaptic contacts in layer IV. VMAT2 ko mice, completely lacking activity-dependent vesicular release of monoamines including 5-HT, also show a complete lack of 5-HT in the cortex but display largely normal barrel fields, despite sometimes markedly reduced postnatal growth. Transient 5-HTT expression is thus required for barrel pattern formation, whereas activity-dependent vesicular 5-HT release is not.

Genetic vulnerability to drug abuse. The D2 dopamine receptor Taq I B1 restriction fragment length polymorphism appears more frequently in polysubstance abusers.

Alcoholics are more likely than nonalcoholics to display the Taq I A1 restriction fragment length polymorphism of the D2 dopamine receptor gene, according to four of six studies that examined alcoholics and controls. The current study examines whether the association observed in alcoholism might extend to other addictive substances by examining D2 dopamine receptor Taq I A and B restriction fragment length polymorphisms in polysubstance users and controls free of significant substance use. We hypothesized a stronger association for the B1 restriction fragment length polymorphism since it lies closer to dopamine receptor protein coding and 5' regulatory regions. Heavy polysubstance users and subjects with DSM-III-R psychoactive substance use diagnoses displayed significantly higher Taq I B1 frequencies than control subjects; Taq I A1 results for these comparisons were less robust. These results are consistent with a role for a D2 dopamine receptor gene variant marked by these restriction fragment length polymorphisms in enhanced substance abuse vulnerability.

D2 dopamine receptor gene TaqI A1 and B1 restriction fragment length polymorphisms: enhanced frequencies in psychostimulant-preferring polysubstance abusers.

Several lines of evidence suggest that presence of a D2 dopamine receptor (DRD2) gene variant marked by TaqI restriction fragment length polymorphisms (RFLPs) might contribute to vulnerability to substance abuse. Psychostimulants display the most robust enhancement of dopamine activity in mesolimbic/mesocortical circuits important for behavioral reward. The present study tests the hypothesis that a DRD2 gene variant might be more prominent in polysubstance users who preferentially use psychostimulants than in addicts with preferential opiate use or in those with no drug preference. Polysubstance users with histories of heavy daily preferential psychostimulant use more often displayed one or two copies of the TaqI A1 (27/62 = 43.5% vs 33/119 = 27.7% for controls), and B1 (20/62 = 32.3% vs 23/119 = 19.8% for controls) markers at the DRD2 locus. DRD2 gene marker distributions in abusers with more prominent opiate use, or those with no history of drug preference, were similar to control genotypes. Psychostimulant-preferring drug users also reported earlier onset of psychostimulant use. Our data are consistent with the hypothesis that DRD2 gene variants marked by these polymorphisms may work, probably in concert with other genetic and environmental factors, to enhance vulnerability to psychostimulant abuse.

Exclusion of close linkage of the dopamine transporter gene with schizophrenia spectrum disorders.

OBJECTIVE: Involvement of genetic factors in the pathogenesis of schizophrenia spectrum disorders has been indicated in twin, adoption, and familial aggregation studies; the pivotal role played by the dopamine transporter in dopaminergic neurotransmission makes it a candidate gene for these disorders. Detection of close linkage between a dopamine transporter marker and schizophrenia spectrum disorders would strongly support the existence of causal relationships between genetic mutations at the dopamine transporter locus and the disease phenotype. METHOD: The authors assessed the linkage between this gene and schizophrenia spectrum disorders by using polymorphic dopamine transporter gene markers in 156 subjects from 16 multiplex pedigrees with schizophrenia as well as schizophreniform, schizoaffective, and schizotypal disorders and mood-incongruent psychotic depression. RESULTS: Complete (theta = 0.0) linkage to the schizophrenia spectrum was excluded under both dominant and recessive models. CONCLUSIONS: These results indicate that allelic variants at the dopamine transporter locus do not provide major genetic contributions to the etiology of schizophrenia and related disorders in these pedigrees.

cDNA cloning of an orphan opiate receptor gene family member and its splice variant.

Radioligand binding and cDNA homology studies have suggested the existence of opiate receptors distinct from the recently-cloned mu, delta and kappa receptors. XOR1S, a rat brain cDNA whose predicted translation product displays 67-72% homology with those encoded by mu 1, delta 1 and kappa 1 opiate receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XOR1L variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XOR1 mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of opiate ligands nor reproducible opiate inhibition of forskolin-stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the opiate receptor gene family, including apparent differential splicing and expression in peripheral tissues.

Human mu opiate receptor. cDNA and genomic clones, pharmacologic characterization and chromosomal assignment.

A human mu opiate receptor cDNA has been identified from a cerebral cortical cDNA library using sequences from the rat mu opiate receptor cDNA. The human mu opiate receptor (h mu OR1) shares 95% amino acid identity with the rat sequence. The expressed mu OR1 recognized tested opiate drugs and opioid peptides in a sodium- and GTP-sensitive fashion with affinities virtually identical to those displayed by the rat mu opiate receptor. Effects on cyclic AMP are similar to those noted for the rat mu opiate receptor. An 18 kb genomic clone hybridizing with the h mu OR1 cDNA contains 63 and 489 bp exonic sequences flanked by splice donor/acceptor sequences. Analysis of hybridization to DNA prepared from human rodent hybrid cell lines and chromosomal in situ hybridization studies indicate localization to 6q24-25. An MspI polymorphism, producing a 3.7 kb band, may prove useful in assessing this gene's involvement in neuropsychiatric disorders involving opiatergic systems.

A human synaptic vesicle monoamine transporter cDNA predicts posttranslational modifications, reveals chromosome 10 gene localization and identifies TaqI RFLPs.

A human vesicular monoamine transporter cDNA has been identified by screening a human brainstem library using sequences from the rat brain synaptic vesicle monoamine transporter (SVMT) [(1992) Cell 70, 539-551; (1992) Proc. Natl. Acad. Sci. USA 89, 10993-10997]. The hSVMT shares 92% amino acid identity with the rat sequence, but displays one less consensus site for asparagine N-linked glycosylation and one more consensus site for phosphorylation by protein kinase C. The human SVMT gene maps to chromosome 10q25 using Southern blotting analysis of human/rodent hybrid cell lines and fluorescent in situ hybridization approaches. The cDNA, and a subclone, recognize TaqI polymorphisms that may prove useful to assess this gene's involvement in neuropsychiatric disorders involving monoaminergic brain systems.

Transcription factors: potential roles in drug-induced neuroplasticity.

Transcription factors act to regulate gene expression. Many transcription factor families have been discovered based on their roles in cell cycle events involved in development and oncogenesis. In post-mitotic neuronal cells, however, many transcription factor genes are "trans-synaptically" regulated: their patterns of expression can be dramatically altered by extracellular stimuli. Transcription factor proteins can then potently influence expression of other genes, whose products can directly alter neuronal function. The central nervous system (CNS) displays varying degrees of neuroplasticity in adult life. Flexible neurochemical pathways that link extracellular stimuli to long-term modifications in neuronal functions are likely to contribute substantially to this neuroplasticity. This review summarizes evidence supporting central roles for transcription factors in such neurochemical cascades. It furthermore illustrates how drugs of abuse can trigger and modulate neuroadaptive processes that could conceivably contribute to clinically relevant addiction phenomena such as craving, tolerance, sensitization, and withdrawal.

Dual effects of lephetamine on spontaneous and evoked neuronal firing in the somatosensory cortex of the rat.

Lephetamine is a central analgesic, recently shown to be abused by drug addicts and to induce dependence in humans. The drug was applied microiontophoretically on single neurones of the somatosensory cortex of the rat in vivo. Its activity on the spontaneous and evoked firing rate was recorded. Morphine and naloxone were employed to verify the hypothesis that a mu-opiate mechanism of action could be involved. The most frequent response evoked by lephetamine was a dose-dependent excitation non-reversible by naloxone. On the other hand, units inhibited or apparently unaffected by the drug, showed a selective anti-glutamate (and partly anti-acetylcholine) effect, which was reversed by either systemically- or iontophoretically-administered naloxone. Long-lasting (8-12 min) applications of lephetamine caused a progressive desensitization of cortical neurones to the inhibitory and anti-glutamate effect. The inhibitory activity of lephetamine and morphine was additive and an increased neuronal excitability was shown by a post-inhibitory rebound of glutamate-induced neuronal activity. The action exerted by lephetamine on glutamate-induced excitations and on postsynaptic excitability, its reversibility by naloxone and the occurrence of acute tolerance allow the conclusion that only the inhibitory effect of lephetamine is mediated by an opioid mechanism. The lephetamine-induced excitations, not reversed by naloxone, are difficult to interpret as opioid-mediated.

Medial prefrontal cortical injections of c-fos antisense oligonucleotides transiently lower c-Fos protein and mimic amphetamine withdrawal behaviours.

Prefrontal cerebral cortical areas display decreased expression of several transcription factor/immediate-early genes, including c-fos, during amphetamine withdrawal. Antisense strategies can help to test possible roles for this prefrontal c-fos down-regulation in the behavioural correlates of amphetamine withdrawal. Medial prefrontal cortical injections delivering 1.7 nmoles of anti c-fos oligonucleotides revealed an approximately 3 h half-life for phosphothioate and a 15 min half-life for phosphodiester oligonucleotides. Antisense phosphothioates complementary to the c-fos translational start site reduced levels of c-Fos protein, while exerting modest and variable effects on c-fos messenger RNA levels. Neither missense phosphorothioate nor antisense phosphodiester oligonucleotides significantly reduced levels of either c-fos messenger RNA or protein. Animals injected with anti c-fos phosphothioate oligonucleotides into the medial prefrontal cortex displayed marked reductions in linear locomotor activity and repetitive movements measured in a novel environment, effects not seen when missense oligonucleotides were used or when animals were accustomed to the activity monitor prior to antisense oligonucleotide injection. Behavioural changes produced by prefrontal cortical injections of c-fos antisense oligonucleotides closely mimic alterations recorded during amphetamine withdrawal. Prefrontal c-fos could thus conceivably play roles in the neurobiological underpinnings of psychostimulant withdrawal and of responses to stressors such as exposure to novel environments.

Genotypic association between dopamine transporter gene polymorphisms and schizophrenia.

Dopamine transporter (DAT) gene variants do not appear to provide widespread contributions to the etiology of schizophrenia spectrum disorders, according to linkage studies [Persico et al., 1995: Am J Psychiatry 152:134-136]. They may, however, produce modifying effects, more readily detectable in specific subpopulations of schizophrenics through associations analyses. We therefore compared polymorphic DAT gene variable number tandem repeat (VNTR) distributions in 84 controls and 147 patients, divided according to DSM-IIIR schizophrenia type criteria. No evidence of allelic association between DAT alleles and schizophrenia or any specific schizophrenia subtype was found. Interestingly, the DAT genotype distribution among schizophrenic patients did display a statistically significant departure from the genotype distribution found in controls. Such discrepancies may represent stigmata of assortative mating or may suggest a "modifying" contribution of homozygote DAT genotypes to pathogenetic processes underlying schizophrenia.

Lack of association between dopamine transporter gene polymorphisms and delusional disorder.

Potential contributions of dopamine transporter (DAT) gene variants to delusional disorder were investigated using association analysis. DAT gene VNTR polymorphisms were assessed in 61 delusional patients and 54 normal controls. No differences were found in either genotypic or allelic distributions. These findings do not support relevant contributions of DAT gene variants to the pathogenesis of delusional disorder.