Characterizing sleep disorders in an autism-specific collection of electronic health records.

OBJECTIVE/BACKGROUND: Sleep problems are common in people on the autism spectrum. This study reviews one detailed approach to querying the electronic health record (EHR) in a large tertiary care center. PATIENTS/METHODS: We developed methods for identifying people on the autism spectrum and defined their sleep problems using the key words, "sleep" or "melatonin", or International Classification of Diseases (ICD) codes. We examined treatment responses of these individuals to melatonin supplementation. RESULTS: Sleep problems were documented in 86% of patients with ages ranging from 6 to 30 years old. Our specific keyword search yielded more patients with sleep diagnoses than ICD codes alone. About two-thirds of patients who received melatonin supplementation reported benefit from its use. CONCLUSIONS: Our study provides a framework for using deidentified medical records to characterize sleep, a common co-occurring condition, in people on the autism spectrum. Using specific keywords could be helpful in future work that queries the EHR.

Brain and peripheral pharmacokinetics of levodopa in the cynomolgus monkey following administration of opicapone, a third generation nitrocatechol COMT inhibitor.

OBJECTIVE: The present study aimed at evaluating the effect of opicapone, a third generation nitrocatechol catechol-O-methyltransferase (COMT) inhibitor, on the systemic and central bioavailability of 3,4-dihydroxy-l-phenylalanine (levodopa) and related metabolites in the cynomolgus monkey. METHODS: Four monkeys, implanted with guiding cannulas for microdialysis probes, in the substantia nigra, dorsal striatum and prefrontal cortex, were randomized in two groups that received, in a crossover design, vehicle or 100 mg/kg opicapone for 14 days. Twenty-three hours after last administration of vehicle or opicapone, animals were challenged with levodopa/benserazide (12/3 mg/kg). Extracellular dialysate and blood samples were collected over 360 min (at 30 min intervals) for the assays of catecholamine and COMT activity. RESULTS: Opicapone increased levodopa systemic exposure by 2-fold not changing Cmax values and reduced both 3-O-methyldopa (3-OMD) exposure and Cmax values by 5-fold. These changes were accompanied by ∼76-84% reduction in erythrocyte COMT activity. In dorsal striatum and substantia nigra, opicapone increased levodopa exposure by 1.7- and 1.4-fold, respectively, reducing 3-OMD exposure by 5- and 7-fold respectively. DOPAC exposure was increased by 4-fold in the substantia nigra. In the prefrontal cortex, opicapone increased levodopa exposure and reduced 3-OMD levels by 2.3- and 2.4-fold, respectively. CONCLUSIONS: Opicapone behaved as long-acting COMT inhibitor that markedly increased systemic and central levodopa bioavailability. Opicapone is a strong candidate to fill the unmet need for COMT inhibitors that lead to more sustained levodopa levels in Parkinson's disease patients.

De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

Maternal transmission of a rare GABRB3 signal peptide variant is associated with autism.

Maternal 15q11-q13 duplication is the most common copy number variant in autism, accounting for ∼1-3% of cases. The 15q11-q13 region is subject to epigenetic regulation, and genomic copy number losses and gains cause genomic disorders in a parent-of-origin-specific manner. One 15q11-q13 locus encodes the GABA(A) receptor ß3 subunit gene (GABRB3), which has been implicated by several studies in both autism and absence epilepsy, and the co-morbidity of epilepsy in autism is well established. We report that maternal transmission of a GABRB3 signal peptide variant (P11S), previously implicated in childhood absence epilepsy, is associated with autism. An analysis of wild-type and mutant ß3 subunit-containing α1ß3γ2 or α3ß3γ2 GABA(A) receptors shows reduced whole-cell current and decreased ß3 subunit protein on the cell surface due to impaired intracellular ß3 subunit processing. We thus provide the first evidence of an association between a specific GABA(A) receptor defect and autism, direct evidence that this defect causes synaptic dysfunction that is autism relevant and the first maternal risk effect in the 15q11-q13 autism duplication region that is linked to a coding variant.

Oestradiol attenuates the cognitive deficit induced by acute phencyclidine treatment in mature female hooded-Lister rats.

Gender differences in psychiatric research are becoming more widely recognized, and changes in levels of the steroid hormone, oestrogen, over the menstrual or oestrous cycle are becoming increasingly implicated in alterations in cognitive strategies and capacities. The aim of this study is to investigate the interaction between oestrogen, NMDA receptor function and cognitive processing in rats. Forty-five mature female hooded-Lister rats received vehicle, 0.5, 5 or 10 microg/kg of oestradiol benzoate (EB, s.c. in olive oil) 24 h prior to an acute dose of 2 mg/kg phencyclidine (PCP, i.p. in 0.9% w/v saline), or vehicle (0.9% saline). After 30 min following PCP treatment, animals completed the novel object recognition task with a 1 min inter-trial interval to assess object recognition memory. Results show that 5 and 10 microg/kg of EB 24 h prior to 2 mg/kg PCP significantly (P < 0.01 and < 0.001, respectively) protected against the cognitive impairing effect of PCP in contrast to vehicle and 0.5 microg/kg EB plus PCP (not significant). EB may exert a neuromodulatory effect in this animal model leading to attenuation of the PCP-induced impairment in object recognition memory, suggesting an interaction between the gonadal steroids and NMDA receptor-mediated cognitive dysfunction, which is of potential relevance to schizophrenia.

Influence of gender on working and spatial memory in the novel object recognition task in the rat.

Gender differences in many behavioural tasks have been observed in both humans and laboratory animals. The novel object recognition (NOR) task is increasingly used to investigate drug effects on working memory processes, although, the influence of sexually dimorphic behaviours have not yet been evaluated. In addition, the role of natural fluctuations in the sex steroids during the oestrous cycle has received little attention during object recognition tasks. Therefore, the aim of the current study was to investigate the influence of gender and oestrous cycle phase on working and spatial memory using the NOR task. Animals were tested in the NOR task and the spatial NOR task. Male and female rats completed an acquisition trial followed by an inter-trial interval of a specified length, then a final retention trial. Vaginal cytology enabled the influence of oestrous cycle phase to be determined in both the NOR and spatial NOR, each animal was tested during one phase of their regular oestrous cycle only. It was found that female rats performed significantly better than male rats in the standard NOR paradigm (p<0.05 compared to no significance (NS) at 3h, respectively), while male rats showed improved memory in the spatial NOR paradigm compared with female rats (p<0.05 compared to NS at 3h, respectively). There was no influence of phase of oestrous cycle on the NOR task, however, during the spatial NOR there was a significant improvement in ability when oestrogen and progesterone levels have been shown to be at their lowest (i.e. p<0.05 during oestrous compared to NS at other stages). In conclusion, it is clear that gonadal hormones can influence components of memory and gender is an important consideration in experimental design.

Analysis of the RELN gene as a genetic risk factor for autism.

Several genome-wide screens have indicated the presence of an autism susceptibility locus within the distal long arm of chromosome 7 (7q). Mapping at 7q22 within this region is the candidate gene reelin (RELN). RELN encodes a signaling protein that plays a pivotal role in the migration of several neuronal cell types and in the development of neural connections. Given these neurodevelopmental functions, recent reports that RELN influences genetic risk for autism are of significant interest. The total data set consists of 218 Caucasian families collected by our group, 85 Caucasian families collected by AGRE, and 68 Caucasian families collected at Tufts University were tested for genetic association of RELN variants to autism. Markers included five single-nucleotide polymorphisms (SNPs) and a repeat in the 5'-untranslated region (5'-UTR). Tests for association in Duke and AGRE families were also performed on four additional SNPs in the genes PSMC2 and ORC5L, which flank RELN. Family-based association analyses (PDT, Geno-PDT, and FBAT) were used to test for association of single-locus markers and multilocus haplotypes with autism. The most significant association identified from this combined data set was for the 5'-UTR repeat (PDT P-value=0.002). These analyses show the potential of RELN as an important contributor to genetic risk in autism.

Linkage and association analysis at the serotonin transporter (SLC6A4) locus in a rigid-compulsive subset of autism.

Autism is a complex genetic neurodevelopmental disorder in which affected individuals display deficits in language, social relationships, and patterns of compulsive and stereotyped behaviors and rigidity. Linkage analysis in our dataset of 57 New England and 80 AGRE multiplex autism families reveals a multipoint heterogeneity LOD (HLOD) score of 2.74 at D17S1871 in 17q11.2. Analysis of phenotypic subsets shows an increased HLOD of 3.62 in families with compulsive behaviors and rigidity. The serotonin transporter locus (SLC6A4) maps nearby and is considered a functional candidate gene in autism and obsessive-compulsive disorder. We genotyped an insertion/deletion polymorphism in the promoter (5-HTTLPR), and seven single nucleotide polymorphisms (SNPs) across the 38-kb transcriptional unit. Transmission disequilibrium (TD) analysis reveals nominal association at a SNP in intron 5 (P = 0.02) as well as 5-HTTLPR (P = 0.01), corresponding to over-transmission of the short allele. TD analysis in the rigid-compulsive subset shows no evidence for association. Intermarker linkage disequilibrium was determined. All SNPs define a single haplotype block, while 5-HTTLPR lies 5' to this block. Three SNPs are sufficient to detect all common alleles (> or =5%) in this > 26-kb block. Analysis of haplotypes for these markers demonstrates no evidence for association to autism. These data indicate that a common allele within the coding region of SLC6A4 is not responsible for the observed linkage. However, the presence of heterogeneous disease variants within the block or the existence of a common disease-associated allele either upstream or downstream of this block is possible. In fact, such variants may well account for linkage to 17q11.2 in our families.

Dense linkage disequilibrium mapping in the 15q11-q13 maternal expression domain yields evidence for association in autism.

Autism [MIM 209850] is a neurodevelopmental disorder exhibiting a complex genetic etiology with clinical and locus heterogeneity. Chromosome 15q11-q13 has been proposed to harbor a gene for autism susceptibility based on (1) maternal-specific chromosomal duplications seen in autism and (2) positive evidence for linkage disequilibrium (LD) at 15q markers in chromosomally normal autism families. To investigate and localize a potential susceptibility variant, we developed a dense single nucleotide polymorphism (SNP) map of the maternal expression domain in proximal 15q. We analyzed 29 SNPs spanning the two known imprinted, maternally expressed genes in the interval (UBE3A and ATP10C) and putative imprinting control regions. With a marker coverage of 1/10 kb in coding regions and 1/15 kb in large 5' introns, this map was employed to thoroughly dissect LD in autism families. Two SNPs within ATP10C demonstrated evidence for preferential allelic transmission to affected offspring. The signal detected at these SNPs was stronger in singleton families, and an adjacent SNP demonstrated transmission distortion in this subset. All SNPs showing allelic association lie within islands of sequence homology between human and mouse genomes that may be part of an ancestral haplotype containing a functional susceptibility allele. The region was further explored for recombination hot spots and haplotype blocks to evaluate haplotype transmission. Five haplotype blocks were defined within this region. One haplotype within ATP10C displayed suggestive evidence for preferential transmission. Interpretation of these data will require replication across data sets, evaluation of potential functional effects of associated alleles, and a thorough assessment of haplotype transmission within ATP10C and neighboring genes. Nevertheless, these findings are consistent with the presence of an autism susceptibility locus in 15q11-q13.

Defining the autism minimum candidate gene region on chromosome 7.

Previous genetic and cytogenetic studies provide evidence that points to one or more autism susceptibility genes residing on chromosome 7q (AUTS1, 115-149 cM on the Marshfield map). However, further localization using linkage analysis has proven difficult. To overcome this problem, we examined the Collaborative Linkage Study of Autism (CLSA) data-set to identify only the families potentially linked to chromosome 7. Out of 94, 47 families were identified and 17 markers were used to generate chromosomal haplotypes. We performed recombination breakpoint analysis to determine if any portion of the chromosome was predominately shared across families. The most commonly shared region spanned a 6 cM interval between D7S501 and D7S2847. Additional markers at 1 cM intervals within this region were genotyped and association and recombination breakpoint analysis was again performed. Although no significant allelic association was found, the recombination breakpoint data points to a shared region between D7S496-D7S2418 (120-123 cM) encompassing about 4.5 Mb of genomic DNA containing over 50 genes.

Linkage disequilibrium at the Angelman syndrome gene UBE3A in autism families.

Autistic disorder is a neurodevelopmental disorder with a complex genetic etiology. Observations of maternal duplications affecting chromosome 15q11-q13 in patients with autism and evidence for linkage and linkage disequilibrium to markers in this region in chromosomally normal autism families indicate the existence of a susceptibility locus. We have screened the families of the Collaborative Linkage Study of Autism for several markers spanning a candidate region covering approximately 2 Mb and including the Angelman syndrome gene (UBE3A) and a cluster of gamma-aminobutyric acid (GABA(A)) receptor subunit genes (GABRB3, GABRA5, and GABRG3). We found significant evidence for linkage disequilibrium at marker D15S122, located at the 5' end of UBE3A. This is the first report, to our knowledge, of linkage disequilibrium at UBE3A in autism families. Characterization of null alleles detected at D15S822 in the course of genetic studies of this region showed a small (approximately 5-kb) genomic deletion, which was present at somewhat higher frequencies in autism families than in controls.

Possible dosage effect of maternally expressed genes on visual recognition memory in Prader-Willi syndrome.

Seventeen patients with Prader-Willi syndrome (7 with paternal deletion of chromosome 15q11-q13 and 10 with maternal uniparental disomy [UPD]), and 9 controls performed a computerized visual recognition task. A series of color digital photographs were presented; most were presented twice, but the remainder appeared only once. Photographs presented twice were separated in their presentation by either 0, 10, 30, 50 or 100 intervening photographs. Subjects indicated whether each photograph had been presented previously. This procedure was implemented twice, once using photographs of foods, and once using photographs of nonfood objects. As the number of intervening photographs between the first and second presentation increased, subjects were less likely to remember having seen the photograph before. Performance by UPD subjects was less affected by increasing the number of intervening photographs relative to the other two groups, suggesting they had superior visual recognition memory. This raises the possibility of a beneficial effect of having two copies maternally expressed genes on chromosome 15. UBE3A is suggested as a possible candidate for this effect.

Partial and generalized epilepsy with febrile seizures plus and a novel SCN1A mutation.

BACKGROUND: Generalized epilepsy with febrile seizures plus (GEFS+) is an autosomal dominant syndrome characterized by febrile seizures (FS) and a variety of afebrile generalized seizure types. GEFS+ has previously been linked to mutations in two genes encoding the voltage-gated sodium channel alpha-subunit (SCN1A) and beta1-subunit (SCN1B). We studied a large family with FS and partial as well as generalized seizure types. METHODS: All but two living affected family members were interviewed and examined. Information on deceased affected family members was sought. EEG for 11 affected family members and one unaffected family member were obtained. Genetic linkage analysis and mutation screening of SCN1A were performed on blood samples from 16 affected individuals and their first-degree relatives. RESULTS: There were 27 affected family members; 18 were alive at the time of the study. All affected family members had FS; seven had FS only, and 19 also had afebrile seizures. Eleven individuals continued to have FS beyond 6 years of age. FS were complex in 12 family members, usually with prolonged duration. The index patient had right temporal lobe epilepsy and hippocampal sclerosis. Four other patients had strong historical evidence of temporal lobe epilepsy, and three others had nonlocalizing evidence of partial epilepsy. Pedigree analysis indicated autosomal dominant transmission. All affected individuals who were tested and one asymptomatic individual had a sodium channel mutation of SCN1A, an A-->C transversion at nucleotide 3809 resulting in the substitution of lysine 1270 by threonine in the D3/S2 segment (designated as K1270T). CONCLUSIONS: Our findings indicate that partial epilepsy preceded by FS can be associated with sodium channel mutations and may represent a variant of GEFS+.

The spectrum of mutations in UBE3A causing Angelman syndrome.

Angelman syndrome (AS) is characterized by mental retardation, absence of speech, seizures and motor dysfunction. AS is caused by maternal deletions for chromosome 15q11-q13, paternal uniparental disomy (UPD), imprinting defects or loss-of-function mutations in the UBE3A locus which encodes E6-AP ubiquitin-protein ligase. The UBE3A gene is imprinted with paternal silencing in human brain and similar silencing of the Ube3a locus in Purkinje cells and hippocampal neurons in the mouse. We have sequenced the major coding exons for UBE3A in 56 index patients with a clinical diagnosis of AS and a normal DNA methylation pattern. The analysis identified disease-causing mutations in 17 of 56 patients (30%) including 13 truncating mutations, two missense mutations, one single amino acid deletion and one stop codon mutation predicting an elongated protein. Mutations were identified in six of eight families (75%) with more than one affected case, and in 11 of 47 isolated cases (23%); no mutation was found in one family with two siblings, one with a typical and one with an atypical phenotype. Mutations were de novo in nine of the 11 isolated cases. An amino acid polymorphism of threonine substituted for alanine at codon 178 was identified, and a 3 bp length polymorphism was found in the intron upstream of exon 8. In all informative cases, phenotypic expression was consistent with imprinting with a normal phenotype when a mutation was on the paternal chromosome and an AS phenotype when a mutation was on the maternal chromosome. Laboratory diagnosis and genetic counseling for AS are complex, and mutation analysis is valuable in clinically typical AS patients with a normal methylation analysis.

Integrated YAC contig map of the Prader-Willi/Angelman region on chromosome 15q11-q13 with average STS spacing of 35 kb.

Prader-Willi syndrome and Angelman syndrome are associated with parent-of-origin-specific abnormalities of chromosome 15q11-q13, most frequently a deletion of an approximately 4-Mb region. Because of genomic imprinting, paternal deficiency of this region leads to PWS and maternal deficiency to AS. Additionally, this region is frequently involved in other chromosomal rearrangements including duplications, triplications, or supernumerary marker formation. A detailed physical map of this region is important for elucidating the genes and mechanisms involved in genomic imprinting, as well as for understanding the mechanism of recurrent chromosomal rearrangments. An initial YAC contig extended from D15S18 to D15S12 and was comprised of 23 YACs and 21 STSs providing an average resolution of about one STS per 200 kb. To close two gaps in this contig, YAC screening was performed using two STSs that flank the gap between D15S18 and 254B5R and three STSs located distal to the GABRA5-149A9L gap. Additionally, we developed 11 new STSs, including seven polymorphic markers. Although several groups have developed whole-genome genetic and radiation hybrid maps, the depth of coverage for 15q11-q13 has been somewhat limited and discrepancies in marker order exist between the maps. To resolve the inconsistencies and to provide a more detailed map order of STSs in this region, we have constructed an integrated YAC STS-based physical map of chromosome 15q11-q13 containing 118 YACs and 118 STSs, including 38 STRs and 49 genes/ESTs. Using an estimate of 4 Mb for the size of this region, the map provides an average STS spacing of 35 kb. This map provides a valuable resource for identification of disease genes localized to this region as well as a framework for complete DNA sequencing.

Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.

Angelman syndrome (AS) is a human genetic disorder characterized by mental retardation, seizures, inappropriate laughter, abnormal galt, tremor and ataxia. There is strong genetic evidence that the disorder is associated with a maternally expressed, imprinted gene mapping to chromosome 15q11-13. Affected patients demonstrate varied molecular abnormalities, including large maternal deletions, uniparental paternal disomy (UPD). Imprinting mutations and loss of function mutations of E6-associated-protein (E6-AP) ubiquitin-protein ligase (UBE3A). All of these abnormalities are associated with loss of maternal expression of UBE3A. Although mutations in UBE3A cause AS, indicating that maternal-specific expression of UBE3A is essential for a normal phenotype, evidence for maternal-specific expression of UBE3A has been lacking. Using mice with partial paternal UPD encompassing Ube3a to differentiate maternal and paternal expression, we found by in situ hybridization that expression of Ube3a in Purkinje cells, hippocampal neurons and mitral cells of the olfactory bulb in UPD mice was markedly reduced compared to non-UPD littermates. In contrast, expression of Ube3a in other regions of the brain was only moderately or not at all reduced in UPD mice. The major phenotypic features of AS correlate with the loss of maternal-specific expression of Ube3a in hippocampus and cerebellum as revealed in the mouse model.

Sequencing and functional analysis of the SNRPN promoter: in vitro methylation abolishes promoter activity.

The gene encoding the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) maps to the Prader-Willi syndrome critical region on chromosome 15 and is expressed preferentially from the paternal allele. A CpG island encompassing the first exon of SNRPN is methylated on the inactive maternal allele. DNA sequence was determined for a cosmid containing the first three exons of SNRPN and extending 20 kb upstream and 15 kb downstream from the CpG island. This region is extremely rich in Alu elements and other repetitive sequences and contains a single CpG island, which includes numerous short direct repeat sequences. Functional analysis of the first exon revealed strong promoter activity for a 260-bp fragment extending 207 bp upstream from the exon. In vitro methylation of this 260-bp fragment abolished promoter activity completely, suggesting that the silencing of the maternal SNRPN allele may be a direct consequence of methylation of the promoter region.