Catarina Correia

Oxytocin receptor (OXTR) does not play a major role in the aetiology of autism: genetic and molecular studies

Oxytocin (OXT) has been hypothesized to play a role in aetiology of autism based on a demonstrated involvement in the regulation of social behaviours. It is postulated that OXT reduces activation of the amygdala, inhibiting social anxiety, indicating a neural mechanism for the effects of OXT in social cognition. Genetic variation at the oxytocin receptor gene (OXTR) has been reported to be associated with autism. We examined 18 SNPs at the OXTR gene for association in three independent autism samples from Ireland, Portugal and the United Kingdom. We investigated cis-acting genetic effects on OXTR expression in lymphocytes and amygdala region of the brain using an allelic expression imbalance (AEI) assay and by investigating the correlation between RNA levels and genotype in the amygdala region. No marker survived multiple correction for association with autism in any sample or in a combined sample (n=436). Results from the AEI assay performed in the lymphoblast cell lines highlighted two SNPs associated with relative allelic abundance in OXTR (rs237897 and rs237895). Two SNPs were found to be effecting cis-acting variation through AEI in the amygdala. One was weakly correlated with total gene expression (rs13316193) and the other was highlighted in the lymphoblast cell lines (rs237895). Data presented here does not support the role of common genetic variation in OXTR in the aetiology of autism spectrum disorders in Caucasian samples.

Increased BDNF levels and NTRK2 gene association suggest a disruption of BDNF/TrkB signaling in autism

The brain‐derived neurotrophic factor (BDNF), a neurotrophin fundamental for brain development and function, has previously been implicated in autism. In this study, the levels of BDNF in platelet‐rich plasma were compared between autistic and control children, and the role of two genetic factors that might regulate this neurotrophin and contribute to autism etiology, BDNF and NTRK2, was examined. We found that BDNF levels in autistic children (n = 146) were significantly higher (t = 6.82; P < 0.0001) than in control children (n = 50) and were positively correlated with platelet serotonin distribution (r = 0.22; P = 0.004). Heritability of BDNF was estimated at 30% and therefore candidate genes BDNF and NTRK2 were tested for association with BDNF level distribution in this sample, and with autism in 469 trio families. Genetic association analysis provided no evidence for BDNF or NTRK2 as major determinants of the abnormally increased BDNF levels in autistic children. A significant association with autism was uncovered for six single nucleotide polymorphisms (SNPs) [0.004 (Z(1df) = 2.85) < P < 0.039 (Z(1df) = 2.06)] and multiple haplotypes [5 × 10−4(χ(3df) = 17.77) < P < 0.042 (χ(9df) = 17.450)] in the NTRK2 gene. These results do not withstand correction for multiple comparisons, however, reflect a trend toward association that supports a role of NTRK2 as a susceptibility factor for the disorder. Genetic variation in the BDNF gene had no impact on autism risk. By substantiating the previously observed increase in BDNF levels in autistic children in a larger patient set, and suggesting a genetic association between NTRK2 and autism, this study integrates evidence from multiple levels supporting the hypothesis that alterations in BDNF/tyrosine kinase B (TrkB) signaling contribute to an increased vulnerability to autism.

Pharmacogenetics of risperidone therapy in autism: association analysis of eight candidate genes with drug efficacy and adverse drug reactions

Little has been reported on the factors, genetic or other, that underlie the variability in individual response, particularly for autism. In this study we simultaneously explored the effects of multiple candidate genes on clinical improvement and occurrence of adverse drug reactions, in 45 autistic patients who received monotherapy with risperidone up to 1 year. Candidate genes involved in the pharmacokinetics (CYP2D6 and ABCB1) and pharmacodynamics (HTR2A, HTR2C, DRD2, DRD3, HTR6) of the drug, and the brain-derived neurotrophic factor (BDNF) gene, were analysed. Using the generalized estimating equation method these genes were tested for association with drug efficacy, assessed with the Autism Treatment Evaluation Checklist, and with safety and tolerability measures, such as prolactin levels, body mass index (BMI), waist circumference and neurological adverse effects, including extrapyramidal movements. Our results confirm that risperidone therapy was very effective in reducing some autism symptoms and caused few serious adverse effects. After adjusting for confounding factors, the HTR2A c.-1438G>A, DRD3 Ser9Gly, HTR2C c.995G>A and ABCB1 1236C>T polymorphisms were predictors for clinical improvement with risperidone therapy. The HTR2A c.-1438G>A, HTR2C c.68G>C (p.C33S), HTR6 c.7154–2542C>T and BDNF c.196G>A (p.V66M) polymorphisms influenced prolactin elevation. HTR2C c.68G>C and CYP2D6 polymorphisms were associated with risperidone-induced increase in BMI or waist circumference. We thus identified for the first time several genes implicated in risperidone efficacy and safety in autism patients. Although association results require replication, given the small sample size, the study makes a preliminary contribution to the personalized therapy of risperidone in autism.

MECP2 coding sequence and 3'UTR variation in 172 unrelated autistic patients.

Mutations in the coding sequence of the methyl‐CpG‐binding protein 2 gene (MECP2), which cause Rett syndrome (RTT), have been found in male and female autistic subjects without, however, a causal relation having unequivocally been established. In this study, the MECP2 gene was scanned in a Portuguese autistic population, hypothesizing that the phenotypic spectrum of mutations extends beyond the traditional diagnosis of RTT and X‐linked mental retardation, leading to a non‐lethal phenotype in male autistic patients. The coding region, exon–intron boundaries, and the whole 3′UTR were scanned in 172 patients and 143 controls, by Detection of Virtually All Mutations‐SSCP (DOVAM‐S). Exon 1 was sequenced in 103 patients. We report 15 novel variants, not found in controls: one missense, two intronic, and 12 in the 3′UTR (seven in conserved nucleotides). The novel missense change, c.617G > C (p.G206A), was present in one autistic male with severe mental retardation and absence of language, and segregates in his maternal family. This change is located in a highly conserved residue within a region involved in an alternative transcriptional repression pathway, and likely alters the secondary structure of the MeCP2 protein. It is therefore plausible that it leads to a functional modification of MeCP2. MECP2 mRNA levels measured in four patients with 3′UTR conserved changes were below the control range, suggesting an alteration in the stability of the transcripts. Our results suggest that MECP2 can play a role in autism etiology, although very rarely, supporting the notion that MECP2 mutations underlie several neurodevelopmental disorders.

The impact of the metabotropic glutamate receptor and other gene family interaction networks on autism

Although multiple reports show that defective genetic networks underlie the aetiology of autism, few have translated into pharmacotherapeutic opportunities. Since drugs compete with endogenous small molecules for protein binding, many successful drugs target large gene families with multiple drug binding sites. Here we search for defective gene family interaction networks (GFINs) in 6,742 patients with the ASDs relative to 12,544 neurologically normal controls, to find potentially druggable genetic targets. We find significant enrichment of structural defects (P≤2.40E−09, 1.8-fold enrichment) in the metabotropic glutamate receptor (GRM) GFIN, previously observed to impact attention deficit hyperactivity disorder (ADHD) and schizophrenia. Also, the MXD-MYC-MAX network of genes, previously implicated in cancer, is significantly enriched (P≤3.83E−23, 2.5-fold enrichment), as is the calmodulin 1 (CALM1) gene interaction network (P≤4.16E−04, 14.4-fold enrichment), which regulates voltage-independent calcium-activated action potentials at the neuronal synapse. We find that multiple defective gene family interactions underlie autism, presenting new translational opportunities to explore for therapeutic interventions.

Characterization of pharmacogenetically relevant CYP2D6 and ABCB1 gene polymorphisms in a Portuguese population sample.

Differences in metabolism of drugs can lead to severe toxicity or therapeutic failure. In addition to cytochrome P450 2D6, which plays a critical role in drug metabolism, ABCB1 encoded P‐glycoprotein (PGP) is also an important determinant in drug bioavailability. The genes encoding these molecules are highly variable among populations and, given their clinical importance in drug therapy, determining CYP2D6 and ABCB1 allele frequencies in specific populations is very important for useful application in clinical settings. In this study the frequency of the pharmacologically relevant CYP2D6*3, *4, *5, *6 allelic variants and gene duplication, and ABCB1 C1236T and C3435T gene polymorphisms and their haplotypes was determined in a population sample of 100 Portuguese healthy subjects. CYP2D6 allele frequencies were 1.4% (*3), 13.3% (*4), 2.8% (*5), 1.8% (*6) and 6.1% (gene duplication), with 5% of the individuals classified as PM and 8.4% as UM. The frequencies obtained for the non‐functional alleles and for the CYP2D6 gene duplication are in agreement with other South European populations, and reinforce the previously suggested south/north gradient of CYP2D6 duplications. Allelic frequencies for the ABCB1 polymorphisms were 52% (3435C) and 54% (1236C) and the most common haplotype (1236C‐3435C) occurred with a frequency of 45.5%. Although allele and haplotype frequency data for ABCB1 in Southern Europe is limited, some discrepancies were found with other European populations, with possible therapeutic implications for PGP substrate drugs. Copyright

Association of the α4 integrin subunit gene (ITGA4) with autism

In the present work, we provide further evidence for the involvement of the integrin alpha‐4 precursor gene (ITGA4) in the etiology of autism, by replicating previous findings of a genetic association with autism in various independent populations. The ITGA4 gene maps to the autism linkage region on 2q31‐33 and is therefore a plausible positional candidate. We tested eight single nucleotide polymorphisms (SNPs) in the ITGA4 gene region for association with autism in a sample of 164 nuclear families. Evidence for association was found for the rs155100 marker (P = 0.019) and for a number of specific marker haplotypes containing this SNP (0.00053 < P < 0.022). α4 integrins are known to play a key role in neuroinflammatory processes, which are hypothesized to contribute to autism. In this study, an association was found between the ITGA4 rs1449263 marker and levels of a serum autoantibody directed to brain tissue, which was previously shown to be significantly more frequent in autistic patients than in age‐matched controls in our population. This result suggests that the ITGA4 gene could be involved in a neuroimmune process thought to occur in autistic patients and, together with previous findings, offers a new perspective on the role of integrins in the etiology of autism to which little attention has been paid so far.

Recurrent duplications of the annexin A1 gene (ANXA1) in autism spectrum disorders

BackgroundValidating the potential pathogenicity of copy number variants (CNVs) identified in genome-wide studies of autism spectrum disorders (ASD) requires detailed assessment of case/control frequencies, inheritance patterns, clinical correlations, and functional impact. Here, we characterize a small recurrent duplication in the annexin A1 (ANXA1) gene, identified by the Autism Genome Project (AGP) study.MethodsFrom the AGP CNV genomic screen in 2,147 ASD individuals, we selected for characterization an ANXA1 gene duplication that was absent in 4,964 population-based controls. We further screened the duplication in a follow-up sample including 1,496 patients and 410 controls, and evaluated clinical correlations and family segregation. Sequencing of exonic/downstream ANXA1 regions was performed in 490 ASD patients for identification of additional variants.ResultsThe ANXA1 duplication, overlapping the last four exons and 3’UTR region, had an overall prevalence of 11/3,643 (0.30%) in unrelated ASD patients but was not identified in 5,374 controls. Duplication carriers presented no distinctive clinical phenotype. Family analysis showed neuropsychiatric deficits and ASD traits in multiple relatives carrying the duplication, suggestive of a complex genetic inheritance. Sequencing of exonic regions and the 3’UTR identified 11 novel changes, but no obvious variants with clinical significance.ConclusionsWe provide multilevel evidence for a role of ANXA1 in ASD etiology. Given its important role as mediator of glucocorticoid function in a wide variety of brain processes, including neuroprotection, apoptosis, and control of the neuroendocrine system, the results add ANXA1 to the growing list of rare candidate genetic etiological factors for ASD.

Increased BDNF levels and association with the NTRK2 gene suggest a disruption of BDNF/TRKB signaling in autism

heterotopia, (non-TSC) focal dysplasia and polymicrogyria, were recruited in 15 years at our academic hospital. In an initial cohort of 113 patients, an etiological diagnosis was possible in 40% of the cases after systematic neuro-radiologic, clinical genetic, routine cytogenetic/FISH and molecular genetic tests (de Wit et al., Archives of Neurology, 2008). In DNA of additional 134 patients and parents, if available, analysis of copy number variations (CNV) was performed on Affymetrix 250K SNP arrays (CNAG program). CNVs were considered pathogenic if (1) not reported in the Toronto database (DGV) as polymorphic, (2) confirmed by independent techniques (FISH, Q-PCR), (3) included known microdeletion/duplication syndromes or (4) were de novo and included (candidate) genes, and (5) parents were available for testing. We identified pathogenic changes in about 13% of the patient samples, mostly from the polymicrogyria and heterotopia group. Additionally, a group of about 10 samples (7%) contained unclassified variants (UVs) including (1) inherited homozygous deletions/duplications from heterozygous parents, (2) deletions/duplications reported for rare and different clinical phenotypes. Analysis of candidate genes from these areas is ongoing in the patient cohort. Another approach to the identification of new genes for MCD using the same data set is represented by homozygosity mapping. In unrelated patients from consanguineous families we are pursuing analysis of overlapping genomic areas of homozygosity, in the assumption of autosomal recessive inheritance for rare MCD phenotypes. This approach gave us the opportunity to identify new syndromes associated with cortical brain malformations.

FGF/FGFR signaling pathway in autism: Identification of a novel mutation in FGF2 gene and association of FGF2, FGFR1, FGFR2 and FGFR3 genetic variants in a Portuguese population sample

Autism is a neurodevelopmental disorder of unclear etiology. Enlarged brain size is common in children with autism and recent studies suggest that an increased number of cortical excitatory neurons may underlie the large brain volume. Fibroblast Growth Factors (FGFs), a family of genes regulating cortical size and connectivity, may be involved in these developmental alterations. In this study we analyzed the involvement of FGF/FGFR signaling pathway genes in autism, through mutation screening and genetic association studies. Screening of mutations in exon 1 of the FGF2 gene was performed in 412 patients and 103 healthy controls. To further elucidate the involvement of the FGF genes in autism, we tested the FGF2, FGFR1, FGFR2 and FGFR3 genes for genetic association with autism in a sample of 238 nuclear families with one affected individual. For this purpose, 32 markers spanning the FGF2 gene, 44 SNPs in the FGFR1, 217 SNPs in FGFR2 gene and 35 SNPs in FGFR3 gene were analyzed using the Transmission Disequilibrium Test. We identified a novel p.T13 M mutation in exon 1 of FGF2 gene, in one male diagnosed with severe autism and macrocephaly. One marker in FGFR1 (p = 0.0312), four markers in introns 1, 2 and the 3’UTR region of the FGF2 gene (0.046 < p < 0.05) and one intergenic marker in downstream of the FGFR2 (p = 0.05) were nominally associated with autism. The genetic association analysis did not provide evidence for the FGF genes as common susceptibility factors for autism. However, the identified FGF2 mutation, together with a segmental duplication in the FGFR3 gene previously found in another autistic patient from our cohort, suggests that abnormal variation in these genes may represent a rare cause for autism. These findings further support the notion that rare sequence variation contributes to autism etiology.