[Show abstract][Hide abstract] ABSTRACT: Contactin-associated protein-like 2 gene (CNTNAP2), a member of the Neurexin gene superfamily, is one of the best-replicated risk genes for autism spectrum disorders (ASD). ASD are predominately genetically determined neurodevelopmental disorders characterized by impairments of language development, social interaction and communication, as well as stereotyped behavior and interests. Although CNTNAP2 expression levels were proposed to alter ASD risk, no study to date has focused on its 5' promoter. Here, we directly sequenced the CNTNAP2 5' promoter region of 236 German families with one child with ASD and detected four novel variants. Furthermore, we genotyped the three most frequent variants (rs150447075, rs34712024, rs71781329) in an additional sample of 356 families and found nominal association of rs34712024G with ASD and rs71781329GCG with language development. The four novel and the three known minor alleles of the identified variants were predicted to alter transcription factor binding sites (TFBS). At the functional level, the respective sequences spanning these seven variants were bound by nuclear factors. In a luciferase promoter assay, the respective minor alleles showed cell line-specific and differentiation stage-dependent effects at the level of promoter activation. The novel potential rare risk-variant M2, a G>A mutation -215 base pairs 5' of the transcriptional start site, significantly reduced promoter efficiency in HEK293T and in undifferentiated and differentiated neuroblastoid SH-SY5Y cells. This variant was transmitted to a patient with autistic disorder. The under-transmitted, protective minor G allele of the common variant rs34712024, in contrast, increased transcriptional activity. These results lead to the conclusion that the pathomechanism of CNTNAP2 promoter variants on ASD risk is mediated by their effect on TFBSs, and thus confirm the hypothesis that a reduced CNTNAP2 level during neuronal development increases liability for ASD.
[Show abstract][Hide abstract] ABSTRACT: Background
There is an urgent need for expanding and enhancing autism spectrum disorder (ASD) samples, in order to better understand causes of ASD.
In a unique public-private partnership, 13 sites with extensive experience in both the assessment and diagnosis of ASD embarked on an ambitious, 2-year program to collect samples for genetic and phenotypic research and begin analyses on these samples. The program was called The Autism Simplex Collection (TASC). TASC sample collection began in 2008 and was completed in 2010, and included nine sites from North America and four sites from Western Europe, as well as a centralized Data Coordinating Center.
Over 1,700 trios are part of this collection, with DNA from transformed cells now available through the National Institute of Mental Health (NIMH). Autism Diagnostic Interview-Revised (ADI-R) and Autism Diagnostic Observation Schedule-Generic (ADOS-G) measures are available for all probands, as are standardized IQ measures, Vineland Adaptive Behavioral Scales (VABS), the Social Responsiveness Scale (SRS), Peabody Picture Vocabulary Test (PPVT), and physical measures (height, weight, and head circumference). At almost every site, additional phenotypic measures were collected, including the Broad Autism Phenotype Questionnaire (BAPQ) and Repetitive Behavior Scale-Revised (RBS-R), as well as the non-word repetition scale, Communication Checklist (Children’s or Adult), and Aberrant Behavior Checklist (ABC). Moreover, for nearly 1,000 trios, the Autism Genome Project Consortium (AGP) has carried out Illumina 1 M SNP genotyping and called copy number variation (CNV) in the samples, with data being made available through the National Institutes of Health (NIH). Whole exome sequencing (WES) has been carried out in over 500 probands, together with ancestry matched controls, and this data is also available through the NIH. Additional WES is being carried out by the Autism Sequencing Consortium (ASC), where the focus is on sequencing complete trios. ASC sequencing for the first 1,000 samples (all from whole-blood DNA) is complete and data will be released in 2014. Data is being made available through NIH databases (database of Genotypes and Phenotypes (dbGaP) and National Database for Autism Research (NDAR)) with DNA released in Dist 11.0. Primary funding for the collection, genotyping, sequencing and distribution of TASC samples was provided by Autism Speaks and the NIH, including the National Institute of Mental Health (NIMH) and the National Human Genetics Research Institute (NHGRI).
TASC represents an important sample set that leverages expert sites. Similar approaches, leveraging expert sites and ongoing studies, represent an important path towards further enhancing available ASD samples.
[Show abstract][Hide abstract] ABSTRACT: Acute tryptophan depletion – converging evidence for decreasing central nervous serotonin synthesis in rodents and humans We read the comment provided by Simon N. Young (1) on the articles (2–5) in the special issue of Acta Psychiatrica Scandi-navica (6) dealing with the acute tryptophan depletion (ATD) methodology with great interest. ATD is a pharmacological method designed to lower central nervous system (CNS) syn-thesis of the neurotransmitter serotonin (5-HT) for a brief per-iod that can also be used in both adults and young people (7). As 5-HT plays an important role in behavioral inhibition (8– 10) and other important processes in the brain (11–14), ATD is a translational method to study the effects of changes in CNS 5-HT function that has particular value, as discussed at a recent symposium dedicated to the role of 5-HT in psychopa-thology (7–11, 15). The author of this particular comment expressed concerns that ATD might not always decrease CNS 5-HT synthesis and that the lack of the amino acid histidine (HIS) in the depletion mixtures used might influence the results due to the potential role of 5-HT–histamine interactions in any observed outcome. We appreciate the comments made and would like to address the issues raised, point by point. Young argues that 'there is no evidence that ATD does always decrease seroto-nin release (in humans)'. This is contradictory by decades of work in rodents and in humans demonstrating that ATD can decrease 5-HT synthesis and release in rodents and lower 5-HIAA in human CSF (16–19). In one of our laboratories, the acute tryptophan depletion (ATD) protocol termed 'Moja-De' has been shown to decrease 5-HT release in rodents (20, 21) and to lower tryptophan (TRP) comparably in humans (22), suggesting that this mixture successfully decreases 5-HT synthesis as postulated. While some experi-ments (23) fail to detect changes in central 5-HT function after ATD, this is the exception rather than the rule in published studies. The author of this comment was also concerned that there would be regional variations in the inhibition of serotonin function. This is logical and consistent with published data on the effects of Moja-De ATD in mice. Mouse studies indicated that depletion of TRP was comparable across different brain areas but that the extent of decrease in 5-HIAA varied by region (20, 21). Regional release of 5-HT is controlled by a combination of cell firing including regionally selective input, the concentration of 5-HT1b receptors on terminals, the amount of tryptophan hydroxylase, and many other factors (24). However, there is no evidence that 5-HT release happens only in selective regions, but we agree the magnitude of ATD effects on release is likely to vary between regions despite comparable depletion of TRP. As regards potential interactions between 5-HT and hista-mine, we agree that measurement of histidine after depletion of TRP or any other formula lacking HIS is of interest. Young has questioned the results of ATD experiments in which HIS was not included, stating that 'histidine is an essential amino acid'. However, the essentiality of this amino acid is not clearly established (25). It has been reported that HIS was not necessary for the maintenance of nitrogen balances in short-term (26, 27). Kriengsunyos et al. (28) observed after a long-term histidine depletion administered to healthy adults that there were no effects on the protein metabolism (urinary nitrogen excretion and nitrogen balance). They suggested that the essentiality of this amino acid in healthy adults is still unclear as there are some components that may serve as sources of HIS, although the data they reported indicate that this amount may not be enough for maintain the HIS pool. The other concern expressed by Young was that effects of ATD could reflect disruption of a histamine–serotonin inter-action, as ATD would cause a dramatic decrease in histamine synthesis. This is possible, as it is well established that the neurotransmitter histamine is formed from HIS (29), and his-tamine turnover seems to occur faster than other biogenic amines, such as norepinephrine or 5-HT (30). Therefore, in the absence of HIS, competition from the amino acid mix-tures could indeed lower histamine production. However, nei-ther the control nor the ATD mixture in most studies contains histidine, and so histamine would not be differen-tially affected by the ATD mixture, but should be compara-bly depleted in both control and ATD mixtures. Nevertheless, it is possible that some interaction between his-tamine depletion and 5-HT depletion could have behavioral effects. Unfortunately, no behavioral effects of histamine depletion have been clearly established in the literature. A study by Young and his collaborators of HIS depletion effects on sensory and motor behavior in healthy adults (31) showed that HIS in plasma decreased 20% and the ratio HIS/ΣLNAA decreased 59%, but there were no behavioral effects of this depletion. Finally, we disagree with the statement that 'the relevance of such animal studies to the far more complex human brain is uncertain'. It is well known that validation of trans-lational methods has allowed modeling many aspects of the neuropsychopathology with the use of appropriate animal models, the majority of them throughout the use of rodents (32, 33). Translation of behavioral findings is challenging, due to limits in extrapolating simple behavioral tasks in rodents to sophisticated behaviors in humans. However, bio-chemical studies of ATD effects in humans and rodents have shown considerable concordance. For example, our studies in humans (5, 22) have been validated in mice (20, 21), consistent with the field as described above (16–19). As Dr. Young points out, detailed anatomic studies of 5-HT synthesis in the human brain are technologically demanding and rarely conducted. However, the concordance between the dependent measures that can be collected in humans (CSF 5-HIAA for example) and comparable measures in
[Show abstract][Hide abstract] ABSTRACT: Autism spectrum disorders (ASD) are heterogeneous disorders with a high heritability and complex genetic architecture. Due to the central role of the fragile X mental retardation gene 1 protein (FMRP) pathway in ASD we investigated common functional variants of ASD risk genes regulating FMRP. We genotyped ten SNPs in two German patient sets (N = 192 and N = 254 families, respectively) and report association for rs7170637 (CYFIP1; set 1 and combined sets), rs6923492 (GRM1; combined sets), and rs25925 (CAMK4; combined sets). An additional risk score based on variants with an odds ratio (OR) >1.25 in set 1 and weighted by their respective log transmitted/untransmitted ratio revealed a significant effect (OR 1.30, 95 % CI 1.11-1.53; P = 0.0013) in the combined German sample. A subsequent meta-analysis including the two German samples, the "Strict/European" ASD subsample of the Autism Genome Project (1,466 families) and a French case/control (541/366) cohort showed again association of rs7170637-A (OR 0.85, 95 % CI 0.75-0.96; P = 0.007) and rs25925-G (OR 1.31, 95 % CI 1.04-1.64; P = 0.021) with ASD. Functional analyses revealed that these minor alleles predicted to alter splicing factor binding sites significantly increase levels of an alternative mRNA isoform of the respective gene while keeping the overall expression of the gene constant. These findings underpin the role of ASD candidate genes in postsynaptic FMRP regulation suggesting that an imbalance of specific isoforms of CYFIP1, an FMRP interaction partner, and CAMK4, a transcriptional regulator of the FMRP gene, modulates ASD risk. Both gene products are related to neuronal regulation of synaptic plasticity, a pathomechanism underlying ASD and may thus present future targets for pharmacological therapies in ASD.
Human Genetics 01/2014; 133(6). DOI:10.1007/s00439-013-1416-y · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 20 item observational measure of social functioning, the Impression of Interviewee rating scale, is one of three measures devised to assess the broader autism phenotype. The sample studied included families containing at least two individuals with autism spectrum disorder; observations were undertaken by the researcher who interviewed the subject. An exploratory factor analysis suggested a single factor was most appropriate (Cronbach's α of 0.78). There was a modest but significant retest correlation of 0.42. Correlations between live ratings and blind consensus ratings of vignettes were high (0.93). Correlations with the interview measures were moderate but statistically significant. In conclusion, the observational scale provides a promising start but further work is required before general use can be recommended.
Journal of Autism and Developmental Disorders 04/2013; 43(9). DOI:10.1007/s10803-013-1810-2 · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective:
Animal experiments and studies in adults have shown that the neurotransmitter serotonin (5-HT) plays an important role in learning and memory processes. However, data on this relationship in young persons are scarce, and neurodietary research in this age group is limited compared with the extensive literature on adults. Here, we aimed to explore the effects of a diminished central nervous 5-HT synthesis, which is achieved by acute tryptophan depletion (ATD) Moja-De , on memory function in young males with attention deficit hyperactivity disorder (ADHD).
Twenty-two male patients with ADHD (ages 9-15 years, mean 10.95 ± 1.17 years) received ATD, thus diminishing central nervous 5-HT synthesis, and a tryptophan-balanced amino acid load (BAL) in a randomized, double-blind, within-subject, crossover design study. Approximately 1.7 h after administration of ATD/BAL, verbal declarative memory was assessed using the 'Auditory Verbal-Learning-Test' (AVLT).
There were no significant effects of ATD administration on verbal declarative memory function.
In this study, changes in 5-HT neurotransmission were not associated with specific aspects of verbal declarative memory in young persons with ADHD. Future studies with healthy control groups that address effects of covarying attentional processes are warranted.
[Show abstract][Hide abstract] ABSTRACT: Taste is a hereditary trait and affects eating and dietary behavior. Nearly 70% of the population is sensitive for 6-n-propylthiouracil (PROP) which is typified as moderately-to-extremely bitter by this population group. It has been hypothesized that PROP-tasters are more sensitive to other bitter tastes, sweet taste and the texture of fats. Recently we could show that food preferences in healthy adolescents are associated with the number of the tongue's taste papillae and the sensitivity for PROP. The impact of PROP status in eating disorders is unknown.AimsThe aim of the present study was to evaluate the food consumption in PROP-tasters and nontasters of remitted female patients with anorexia nervosa (AN).Methods20 remitted female patients with AN were included and tested for PROP-sensitivity. 10 patients were PROP-nontaster (aged 22,9 ± 3,0 yrs., BMI 20,5 ± 2,0 kg/m2, remission period 6,7 ± 2,9 yrs.) and 10 patients with AN were PROP-taster (24,9 ± 2,9 yrs., BMI 22,3 ± 3,5 kg/m2, remission period 5,3 ± 2,2 yrs.). Taste perception was evaluated using taste strips (sweet, sour, salty, bitter). Food practice and choice were assessed by self-report. Several blood parameters including hormones and leptin were analyzed. For psychological evaluation SIAB-S, EDI-2 and ASR were used.ResultsRemitted PROP-taster with AN consume significantly more often ice cream, dumpling, lemonade, potato chips, candies and sweets.Conclusions
We hypothesize that PROP status may influence eating behaviour in eating disorders. The sensitivity for PROP possibly improves the course of AN.
European Psychiatry 12/2012; 27:1. DOI:10.1016/S0924-9338(12)74734-X · 3.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Autism spectrum disorders (ASD) present with a high genetic heterogeneity, and so far no single gene could be directly associated to ASD etiology. Genome wide association studies remained unsuccessful so far. Screening of candidate genes showed that 6-10% of ASD patients carry mutations in genes linked to neurogenesis and synaptic plasticity. However, the pathomechanisms underlying ASD remain unclear. Recently discovered mutations in the ribosomal protein L10 gene (RPL10) provided a new hint towards an altered translational capacity.
To further understand the contribution of an RPL10 mutation at protein level we used 2-dimensional differential-in-gel-electrophoresis (2D-DIGE) on lymphoblastoid cell lines (LCLs). Putative candidates were analyzed using tandem mass-spectrometry.
We compared cell lines carrying the RPL10 mutations with non-mutant allele carriers and a set of 10 ASD patients, not harbouring any RPL10 mutation, with 10 random controls. Validation of differentially regulated proteins was performed on mRNA and overall protein expression level applying RT-PCR and Western Blot methods.
Candidates differentially expressed due to a mutation in the RPL10 gene were associated to the same functional pathways deregulated in cell lines derived from ASD patients. These proteins were mainly related to oxidative phosphorylation and energy metabolism as well as control of mRNA and protein stability. Validation of these results showed that the alteration is mainly taking place at translational levels and suggested also alterations of yet uncharacterized post-translational modifications. Furthermore we observed a variant protein expression level in the ASD samples which in some candidates correlated with a variant mRNA expression level. Overall, the differential protein patterns identified in the LCLs from autistic patients in comparison to LCLs from controls may result from both quantitative (expression level) and qualitative (post-translational modifications) changes in translation.
The RPL10 mutations contribute to the molecular phenotype observed in the ASD derived cell lines by altering functionally related mechanisms. The identified candidates seem to be altered not only quantitatively, but also by a change in post-translational modifications. A further characterization of these modifications is needed. The association of the identified pathomechanisms to ASD is in agreement with already known candidate pathways. Therefore, we provide evidence that lymphoblastoid cell lines could serve as a tool to characterize the impact of a single mutation in a complex disorder.
2012 International Meeting for Autism Research; 05/2012
[Show abstract][Hide abstract] ABSTRACT: Evidence from animal studies suggests that leptin metabolism is associated with zinc (Zn) status. However, research investigating this relationship in adolescents and young adults with anorexia nervosa (AN) is scarce; the present study aims to fill that gap.
Serum concentrations of leptin, the soluble leptin receptor (sOB-R) and the free leptin index (FLI) were obtained in healthy control subjects (n=19), acutely ill individuals (n=14) and recovered patients with AN (n=15). Serum Zn concentrations noted in previous research data were also incorporated for all groups.
Leptin, FLI and Zn concentrations were higher in recovered subjects with AN when compared with acutely ill AN patients. Remitted patients showed higher sOB-R concentrations but no difference in FLI compared with the control group. Leptin and FLI were lower in the acutely ill patients compared with the control subjects, who showed no differences in Zn concentrations. Zn concentrations were not correlated with leptin, sOB-R or FLI concentrations in any of the three investigated subgroups.
The present investigation does not entirely support an association between Zn, Leptin and FLI concentrations in subjects with AN, possibly due to limited statistical power. Further research and replication of the present findings related to the interaction between leptin and Zn is warranted. However, with respect to serum leptin levels the data of the present investigation indicate that acutely ill and remitted patients with AN differ as regards serum leptin concentrations and FLI, which is in line with previous research.
[Show abstract][Hide abstract] ABSTRACT: Background:
Autism spectrum disorder (ASD) has a strong genetic background with a higher frequency of affected males suggesting involvement of X-linked genes and possibly also other factors causing the unbalanced sex ratio in the etiology of the disorder. Syndrome pathogenesis is associated with abnormal brain development and manifests in several specific brain regions, especially cerebellum, amygdala and hippocampus. Positive linkage findings in genome wide screens and association studies identified susceptibility regions and genes on the X chromosome.
The ribosomal protein L10 gene (RPL10) located in Xq28 was identified as a candidate susceptibility gene for ASD through RNA in situ hybridization experiments. We detected high expression of RPL10 in mouse hippocampus. A first screen in 317 cases with autistic disorder, 21 cases with Asperger syndrome, and 7 cases with PDD-NOS representing 296 families revealed two missense mutations, L206M and H213Q, in two independent male-male affected sib-pair families (Klauck et al. 2006, Mol Psychiatry 11, 1073-84). In the follow-up study presented here, further 175 patients (145 autistic disorder, 27 Asperger syndrome, 3 PDD-NOS) representing 169 independent families have been screened to enlarge the study group. To understand the involvement of RPL10 in the pathogenesis of ASD, the RPL10 mRNA level in patients with ASD and controls was quantified.
All seven exons of the RPL10 gene and the intronic snoRNA U70 have been screened by direct sequencing for mutations. RPL10 transcript levels were tested by quantitative RT-PCR on RNA extracted from lymphoblastoid cell lines (LCL) of different probands.
In the follow-up sample the H213Q mutation inherited from the carrier mother was identified in a male patient from a simplex family. The two different mutations, L206M and H213Q, are both located at the yet uncharacterized C-terminal end of the RPL10 protein, a constituent of the large ribosomal subunit. RPL10 itself is known to have impact on differential gene expression in yeast and man. Functional analyses in yeast revealed that both amino acid substitutions L206M and H213Q, respectively, confer hypomorphism with regard to the alteration of the translation process while keeping the basic translation functions intact. No statistical significant difference in RPL10 expression levels was found by testing 12 autistic patients including 2 patients from the two families carrying the H213Q mutation, 8 relatives of autistic patients and 7 controls. Moreover, the H213Q mutation has no effect on RPL10 expression in this cell system.
The mutations identified may have a modulating effect on translation processes of synaptogenesis in neuronal development with impact especially on those cognitive functions that are mediated through the limbic system. Alterations due to the inherited mutation at the transcript level may be too subtle to be identified in the LCL system. In future, the functional impact of both mutations in its hemizygous state on the X chromosome will be further analyzed by in vitro and in vivo modeling.
American Journal of Medical Genetics Part A 06/2011; 155A(6):1472-5. DOI:10.1002/ajmg.a.33977 · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Autism spectrum disorder (ASD) has a strong genetic background with a higher frequency of affected males suggesting involvement of X-linked genes. The complex genetic architecture points towards the contribution of rare genomic variants distributed over the complete genome targeting genes in pathways for synaptic signaling, cell adhesion, secretion or scaffolding. These are crucial processes in the establishment of synaptic plasticity (SP).
Objectives: We previously identified two rare missense mutations, L206M and H213Q, in the ribosomal protein L10 (RPL10) gene on Xq28 in three independent families. Functional analyses in yeast showed that both mutant alleles alter translation, a modulating mechanism of SP. Gene expression studies of RPL10 itself in lymphoblastoid cell lines (LCL) of male patients harboring the H213Q mutation showed no significant difference at the transcriptional level. Therefore, we aim to analyze the impact of RPL10 mutations on differential translation applying 2D gel/mass spectrometry (MS) methods using these mutant LCLs.
Methods: Protein extracts of two biological replicates for DIGE were prepared from LCLs established from the two male index patients of each family, their heterozygous carrier mothers, an unaffected brother and a heterozygous carrier sister, respectively, and a male and female control. Each of the samples was stained with Cy3 and compared versus the Cy5 stained internal control, which was an isogenic mixture of each of the samples. Data acquisition was performed on the Typhoon Scanner System and data were subsequently analyzed using DeCyder Software. Data were normalized against the internal standard and relative volumes of spots were calculated using normalized intensity values. Three different types of statistical analyses (two-tailed t-tests) were performed: patients versus mothers, patients versus non-patients and mutant allele carriers versus wild-type allele carriers.
Results: Following data analysis ten significantly up- or down-regulated spots in the samples of the mutation carriers were selected and picked from a preparative, Coomassie-stained 2D-PAGE. Proteins were digested with trypsin and analyzed by MALDI-MS. Interestingly three of the ten spots could be identified as posttranslational modifications of a single protein. This down-regulated protein is a central regulator in gluconeogenesis. One other up-regulated protein was identified as a peroxisomal member of the lipid metabolism. Validation via RT-PCR and Western Blot is in progress.
Conclusions: There is evidence from the literature that RPL10 is not only involved in the translation processes as a component of the large ribosomal subunit, but also in the establishment of the cytoskeleton, mRNA turnover and ageing. We will use information of differential protein expression caused by these autism specific RPL10 mutations to search for underlying pathways specifically relevant for synaptogenesis in neuronal development and therefore causative for the autism phenotype.
International Meeting for Autism Research 2011; 05/2011