Guiomar Oliveira

Genetic and Functional Analyses of SHANK2 Mutations Suggest a Multiple Hit Model of Autism Spectrum Disorders

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11–q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the “multiple hit model” for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.

Analysis of the neuroligin 3 and 4 genes in autism and other neuropsychiatric patients

SIR—Jamain et al reported a frameshift and a mis sense mutation in the X-linked neuroligin 4 (NLGN4, MIM# 300427) and neuroligin 3 (NLGN3, MIM# 300336) genes, respectively, in Swedish families with autism. A frameshift mutation in NLGN4 appeared de novo in the mother, cosegregated with an affected brother with Asperger syndrome and was absent in a normal brother. This frameshift mutation was not present in 600 unrelated control X-chromosomes. A missense mutation in NLGN3, R451C, was found in the mother and two sibs, one with autism and another with Asperger syndrome, but no other relatives were studied. It was not found in 300 unrelated control X-chromosomes. Laumonnier et al reported a large French family in which 10 males had nonspecific X-linked mental retardation, two had autism and one had pervasive developmental disorder. All affected patients were found to have the same frameshift mutation (1253delAG) in the NLGN4 gene. One obligate female carrier had mild mental retardation. The NLGN3 and NLGN4 genes map to Xq13 and Xp22.3, respectively. The NLGN3 gene spans 32 kb, and the NLGN4 gene spans 338 kb. NLGN3 has eight exons, encoding two alternatively spliced isoforms of 828 and 848 amino acids. The NLGN4 gene contains six exons and codes for a protein of 816 amino acids. All neuroligins contain an N-terminal hydrophobic sequence with the characteristics of a cleaved signal peptide followed by a large esterase homology domain, a highly conserved single transmembrane region, and a short cytoplasmic domain. To better understand the relationship between the NLGN4 gene and autism, the coding regions and associated splice junctions of the NLGN4 gene were scanned for mutations with DOVAM-S (Detection of Virtually All Mutations-SSCP) and direct sequencing in the following subjects: 148 unrelated patients with autism (76 Midwest US Caucasians and 72 Portuguese Caucasians; 122 males and 26 females), 48 patients without autism, including 24 Midwest US Caucasian patients with attention deficit hyperactivity disorder (ADHD) and 24 UK Caucasian patients with DSM-IV Bipolar I Disorder (BPD), as well as 48 Portuguese healthy control subjects. The Portuguese autistic patients were diagnosed using DSM-IV criteria, the Autism Diagnostic Interview-Revised (ADI-R) and the Childhood Autism Rating Scale (CARS). Idiopathic subjects were included in the study after clinical assessment and screening for known medical and genetic conditions associated with autism (fragile X, chromosomal disorders, neurocutaneous syndromes, metabolic disorders, infectious diseases). Neuropsychological evaluation was performed using the Ruth Griffiths Mental Developmental Scales or the Wechsler Intelligence Scale for Children (WISC), depending on the patient’s age. The Midwest autistic patients were diagnosed as described previously. Putative missense mutations were identified once each in the NLGN4 gene in four separate autistic patients (Table 1). G99S and K378R were found in unrelated Portuguese patients. V403M and R704C were found in unrelated Midwest patients. G99, K378 and V403 are located in the esterase domain and R704 is located in the cytoplasmic domain. Three of the structural changes, K378R, V403M and R704C, occur in asymptomatic mothers, while G99S occurs in a mother with learning disability. Comprehensive mutation scanning of 48 Portuguese healthy controls and sequencing of the appropriate exons in 288 healthy controls including 96 Portuguese and 192 Midwest US Caucasians (144 males and 192 females; 528 X-chromosomes total) did not reveal these four missense variants or any other structural changes (4/148 vs 0/336, P1⁄4 0.009 or 4/174 vs 0/528, P1⁄4 0.004, when the Fisher exact test is performed with patients or X-chromosome alleles, respectively). In addition, no structural variants were found in a pilot experiment performed on patients with ADHD and BPD (24 of each). Patient #1 has a younger brother with a diagnosed language disability and a global developmental quotient below the mean (Ruth Griffiths Mental Developmental Scales score of 89), who also carries the G99S variation. Their mother, who is heterozygous for the variation, had a documented learning disability. As also found by Laumonnier et al, sequence variation in NLGN4 may be segregating with autism and cognitive disability in this family. Patient #3 had an affected brother with V403M. He had three other unaffected sibs, including a sister without the variant and two brothers with V403M who had normal social function (making friends easily), normal school performance and no attentional problems, consistent with an absence of cosegregation between this variant and any phenotype. The three unaffected children had Social Communication Questionnaire (SCQ) (Lifetime) scores of 0. Proband #3 was specifically of Irish descent. In all, 50 normal female controls of Irish descent were sequenced, none of them had V403M. Molecular Psychiatry (2005) 10, 329–335 & 2005 Nature Publishing Group All rights reserved 1359-4184/05

Mitochondrial dysfunction in autism spectrum disorders: a population-based study

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Epidemiology of autism spectrum disorder in Portugal: prevalence, clinical characterization, and medical conditions

A minority of cases of autism has been associated with several different organic conditions, including bioenergetic metabolism deficiency. In a population-based study, we screened associated medical conditions in a group of 120 children with autism (current age range 11y 5mo to 14y 4mo, mean age 12y 11mo [SD 9.6mo], male:female ratio 2.9:1). Children were diagnosed using Diagnostic and Statistical Manual of Mental Disorders criteria, the Autism Diagnostic Interview--Revised, and the Childhood Autism Rating Scale; 76% were diagnosed with typical autism and 24% with atypical autism. Cognitive functional level was assessed with the Griffiths scale and the Wechsler Intelligence Scale for Children and was in the normal range in 17%. Epilepsy was present in 19 patients. Plasma lactate levels were measured in 69 patients, and in 14 we found hyperlactacidemia. Five of 11 patients studied were classified with definite mitochondrial respiratory chain disorder, suggesting that this might be one of the most common disorders associated with autism (5 of 69; 7.2%) and warranting further investigation.

Variants of the serotonin transporter gene ( SLC6A4 ) significantly contribute to hyperserotonemia in autism

The objective of this study was to estimate the prevalence of autistic spectrum disorder (ASD) and identify its clinical characterization, and medical conditions in a paediatric population in Portugal. A school survey was conducted in elementary schools, targeting 332 808 school‐aged children in the mainland and 10 910 in the Azores islands. Referred children were directly assessed using the Diagnostic and Statistical Manual of Mental Disorders (4th edn), the Autism Diagnostic Interview–Revised, and the Childhood Autism Rating Scale. Clinical history and a laboratory investigation was performed. In parallel, a systematic multi‐source search of children known to have autism was carried out in a restricted region. The global prevalence of ASD per 10 000 was 9.2 in mainland, and 15.6 in the Azores, with intriguing regional differences. A diversity of associated medical conditions was documented in 20%, with an unexpectedly high rate of mitochondrial respiratory chain disorders.

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

The role of the serotonin system in the etiology and pathogenesis of autism spectrum disorders (ASD) is not clearly defined. High levels of platelet serotonin (5-HT) have been consistently found in a proportion of patients, and it is known that specific 5-HT transporter gene (SLC6A4) variants modulate transporter reuptake function, therefore possibly influencing the occurrence of hyperserotonemia in a subset of autistic patients. We have examined the association of platelet serotonin levels with two SLC6A4 polymorphisms, 5-HTT gene-linked polymorphic region (HTTLPR) in the promoter and intron 2 variable number of tandem repeats (VNTR), in a sample of 105 ASD patients, their parents, and 52 control children. Quantitative transmission disequilibrium test (QTDT) results showed a significant effect on 5-HT levels of each SLC6A4 marker (P=0.017 for HTTLPR; P=0.047 for intron 2 VNTR) and of haplotypes of the two markers (P=0.017), with a major contribution of the L.Stin2.10 haplotype (P=0.0013). A 5-HT mean value in the range of hyperserotonemia was associated with the homozygous L.Stin2.10 haplotype (H (1,N=97)=7.76, P=0.0054), which occurred in 33% of hyperserotonemic patients against 6% of patients with normal 5-HT levels (Fishers exact test: P=0.013, OR=8). Allele interaction at the HTTLPR locus was found, with a significant dominance variance effect on 5-HT levels. We found no transmission disequilibrium of any of the SLC6A4 variants in ASD. Our results show that the SLC6A4 gene is a significant factor in the determination of 5-HT levels, and that specific SLC6A4 variants are associated with an increased risk for hyperserotonemia in our sample of autistic patients. The biological mechanism, however, is unlikely to involve the SLC6A4 gene solely. The associated SLC6A4 alleles likely interact with other genes or environmental factors to produce the abnormally high 5-HT levels observed in this subset of autistic patients, who possibly represent a separate etiological group.

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

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.

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

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.

Stereotypies in Rett syndrome Analysis of 83 patients with and without detected MECP2 mutations

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.

Identification of novel genetic causes of Rett syndrome-like phenotypes

Background: Hand stereotypies are considered a hallmark of Rett syndrome (RTT) and are usually described as symmetric movements at the midline. However, related pathologies may show the same type of involuntary movement. Furthermore, patients with RTT also have stereotypies with other localizations that are less well characterized. Methods: We analyzed stereotypies in 83 patients with RTT, 53 with and 30 without a mutation detected in the MECP2 gene. Patients were observed and videotaped always by the same pediatric neurologist. Stereotypies were classified, and data were submitted to statistical analysis for comparison of mutation-positive and -negative patients and analysis of their evolution with the disease. Results: All the patients showed hand stereotypies that coincided with or preceded the loss of purposeful hand movements in 62% of the patients with MECP2 mutations.The hair pulling stereotypy was more frequent in the group with detected mutations, whereas hand washing was not. Hand gaze was absent in all RTT patients with MECP2 mutations. Patients with MECP2 mutations also had more varied stereotypies, and the number of stereotypies displayed by each patient decreased significantly with age in this group. In all patients, stereotypies other than manual tended to disappear with the evolution of the disease. Conclusions: Although symmetric midline hand stereotypies were not specific to patients with an MECP2 mutation, some of the other stereotypies seemed to be more characteristic of this group. In patients younger than 10 years and meeting the necessary diagnostic criteria of Rett syndrome, the association of hand stereotypies without hand gaze, bruxism, and two or more of the other stereotypies seemed to be highly indicative of the presence of an MECP2 mutation.