The genetic architecture of autism spectrum disorders(ASDs) and the potential importance of common regulatory genetic variants

Currently, there is great interest in identifying genetic variants that contribute to the risk of developing autism spectrum disorders(ASDs), due in part to recent increases in the frequency of diagnosis of these disorders worldwide. While there is nearly universal agreement that ASDs are complex diseases, with multiple genetic and environmental contributing factors, there is less agreement concerning the relative importance of common vs rare genetic variants in ASD liability. Recent observations that rare mutations and copy number variants(CNVs) are frequently associated with ASDs, combined with reduced fecundity of individuals with these disorders, has led to the hypothesis that ASDs are caused primarily by de novo or rare genetic mutations. Based on this model, large-scale whole-genome DNA sequencing has been proposed as the most appropriate method for discovering ASD liability genes. While this approach will undoubtedly identify many novel candidate genes and produce important new insights concerning the genetic causes of these disorders, a full accounting of the genetics of ASDs will be incomplete absent an understanding of the contributions of common regulatory variants, which are likely to influence ASD liability by modifying the effects of rare variants or, by assuming unfavorable combinations, directly produce these disorders. Because it is not yet possible to identify regulatory genetic variants by examination of DNA sequences alone, their identification will require experimentation. In this essay, I discuss these issues and describe the advantages of measurements of allelic expression imbalance(AEI) of m RNA expression for identifying cis-acting regulatory variants that contribute to ASDs.     

Mutation screening of melatonin-related genes in patients with autism spectrum disorders

Background

One consistent finding in autism spectrum disorders (ASD) is a decreased level of the pineal gland hormone melatonin and it has recently been demonstrated that this decrease to a large extent is due to low activity of the acetylserotonin O-methyltransferase (ASMT), the last enzyme in the melatonin synthesis pathway. Moreover, mutations in the ASMT gene have been identified, including a splice site mutation, that were associated with low ASMT activity and melatonin secretion, suggesting that the low ASMT activity observed in autism is, at least partly, due to variation within the ASMT gene.

Methods

In the present study, we have investigated all the genes involved in the melatonin pathway by mutation screening of AA-NAT (arylalkylamine N-acetyltransferase), ASMT, MTNR1A, MTNR1B (melatonin receptor 1A and 1B) and GPR50 (G protein-coupled receptor 50), encoding both synthesis enzymes and the three main receptors of melatonin, in 109 patients with autism spectrum disorders (ASD). A cohort of 188 subjects from the general population was used as a comparison group and was genotyped for the variants identified in the patient sample.

Results

Several rare variants were identified in patients with ASD, including the previously reported splice site mutation in ASMT (IVS5+2T>C). Of the variants affecting protein sequence, only the V124I in the MTNR1B gene was absent in our comparison group. However, mutations were found in upstream regulatory regions in three of the genes investigated, ASMT, MTNR1A, and MTNR1B.

Conclusions

Our report of another ASD patient carrying the splice site mutation IVS5+2T>C, in ASMT further supports an involvement of this gene in autism. Moreover, our results also suggest that other melatonin related genes might be interesting candidates for further investigation in the search for genes involved in autism spectrum disorders and related neurobehavioral phenotypes. However, further studies of the novel variants identified in this study are warranted to shed light on their potential role in the pathophysiology of these disorders.     

Iodine in autism spectrum disorders

ObjectiveThe aim of our study was to assess the iodine status of Polish boys with severe autism compared to their healthy peers and evaluate the relationship between urinary iodine, thyroid hormones, body mass index and Autism Spectrum Disorder (ASD) symptomatology.Subjects and methodsTests were performed in 40 boys with ASD and 40 healthy boys, aged 2–17 from the same geographic region in Poland. Urinary iodine (UI), free triiodothyronine (fT3), free thyroxine (fT4), thyroid stimulating hormone (TSH), BMI, and individual symptoms measured by the Childhood Autism Rating Scale (CARS) were correlated.Validated ion chromatography method with pulsed amperometric detection was applied for the determination of urinary iodine after optimized alkaline digestion in a closed system assisted with microwaves.Results19 out of 40 children with ASD had mild to moderate iodine deficiency. Statistically significant lower levels of UI, fT3 and fT4 and higher levels of TSH were found in the autistic group when compared with the control group. Concentration of iodine in urine was negatively associated with clinician’s general impression for children between 11 and 17 years. Emotional response, adaptation to environmental change, near receptor responsiveness, verbal communication, activity level, and intellectual functioning are more associated with UI than other symptoms listed in CARS.ConclusionThe severity of certain symptoms in autism is associated with iodine status in maturing boys. Thyroid hormones were within normal reference ranges in both groups while urinary iodine was significantly lower in autistic boys suggesting that further studies into the nonhormonal role of iodine in autism are required.     

High-throughput sequencing of mGluR signaling pathway genes reveals enrichment of rare variants in autism

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism.     

SHANK1 and autism spectrum disorders

Autism spectrum disorders(ASD) are highly heterogeneous pediatric developmental disorders with estimated heritability more than 70%. Although the genetic factors in ASD are mainly unknown, a large number of gene mutations have been found, especially in genes involved in neurogenesis. The Neurexin-Neuroligin-Shank(NRXN-NLGN-SHANK) pathway plays a key role in the formation, maturation and maintenance of synapses, consistent with the hypothesis of neurodevelopmental abnormality in ASD. Presynaptic NRXNs interact with postsynaptic NLGNs in excitatory glutamatergic synapses. SHANK proteins function as core components of the postsynaptic density(PSD) by interacting with multiple proteins. Recently, deletions and point mutations of the SHANK1 gene have been detected in ASD individuals, indicating the involvement of SHANK1 in ASD. This review focuses on the function of SHANK1 protein, Shank1 mouse models, and the molecular genetics of the SHANK1 gene in human ASD.     

Scan-statistic approach identifies clusters of rare disease variants in LRP2, a gene linked and associated with autism spectrum disorders, in three datasets

Cluster-detection approaches, commonly used in epidemiology and astronomy, can be applied in the context of genetic sequence data for the identification of genetic regions significantly enriched with rare disease-risk variants (DRVs). Unlike existing association tests for sequence data, the goal of cluster-detection methods is to localize significant disease mutation clusters within a gene or region of interest. Here, we focus on a chromosome 2q replicated linkage region that is associated with autism spectrum disorder (ASD) and that has been sequenced in three independent datasets. We found that variants in one gene, LRP2, residing on 2q are associated with ASD in two datasets (the combined variable-threshold-test p value is 1.2 × 10(-5)). Using a cluster-detection method, we show that in the discovery and replication datasets, variants associated with ASD cluster preponderantly in 25 kb windows (adjusted p values are p(1) = 0.003 and p(2) = 0.002), and the two windows are highly overlapping. Furthermore, for the third dataset, a 25 kb region similar to those in the other two datasets shows significant evidence of enrichment of rare DRVs. The region implicated by all three studies is involved in ligand binding, suggesting that subtle alterations in either LRP2 expression or LRP2 primary sequence modulate the uptake of LRP2 ligands. BMP4 is a ligand of particular interest given its role in forebrain development, and modest changes in BMP4 binding, which binds to LRP2 near the mutation cluster, might subtly affect development and could lead to autism-associated phenotypes.     

The complex genetics in autism spectrum disorders

Autism spectrum disorders(ASD) are a pervasive neurodevelopmental disease characterized by deficits in social interaction and nonverbal communication, as well as restricted interests and stereotypical behavior. Genetic changes/heritability is one of the major contributing factors, and hundreds to thousands of causative and susceptible genes, copy number variants(CNVs), linkage regions, and micro RNAs have been associated with ASD which clearly indicates that ASD is a complex genetic disorder. Here, we will briefly summarize some of the high-confidence genetic changes in ASD and their possible roles in their pathogenesis.     

Discovery of allelic variants of HOXA1 and HOXB1: genetic susceptibility to autism spectrum disorders

BACKGROUND: Family studies have demonstrated that the autism spectrum disorders (ASDs) have a major genetic etiologic component, but expression and penetrance of the phenotype are variable. Mice with null mutations of Hoxa1 or Hoxb1, two genes critical to hindbrain development, have phenotypic features frequently observed in autism, but no naturally occurring variants of either gene have been identified in mammals. METHODS: By sequencing regions of genomic DNA of patients with autism spectrum disorders, we detected a substitution variant at HOXA1 and an insertion variant at HOXB1, both in coding regions of the genes. Fifty-seven individuals ascertained for a diagnosis of an ASD, along with 166 of their relatives, were typed for these variants. Two non-ASD populations were typed, and the frequency of the newly identified alleles was determined in all groups. The genotypes of the ASD families were tested for conformation to Hardy-Weinberg proportions and Mendelian expectations for gene transmission. RESULTS: The frequency of the variants was 10-25% in persons of European or African origin. In the ASD families, there was a significant deviation from the HOXA1 genotype ratios expected from Hardy-Weinberg proportions (P = 0.005). Among affected offspring, a significant deviation from Mendelian expectation in gene transmission (P = 0.011) was observed. No statistically significant effects were detected when the same analyses were applied to the HOXB1 locus, but there was evidence of an interaction between HOXA1, HOXB1, and gender in susceptibility to ASDs. CONCLUSIONS: The results support a role for HOXA1 in susceptibility to autism, and add to the existing body of evidence implicating early brain stem injury in the etiology of ASDs.     

Paraoxonase 1 activities and polymorphisms in autism spectrum disorders

Autism spectrum disorders (ASD) comprise a complex and heterogeneous group of conditions of unknown aetiology, characterized by significant disturbances in social, communicative and behavioural functioning. Recent studies suggested a possible implication of the high-density lipoprotein associated esterase/lactonase paraoxonase 1 (PON1) in ASD. In the present study, we aimed at investigating the PON1 status in a group of 50 children with ASD as compared to healthy age and sex matched control participants. We evaluated PON1 bioavailability (i.e. arylesterase activity) and catalytic activity (i.e. paraoxonase activity) in plasma using spectrophotometric methods and the two common polymorphisms in the PON1 coding region (Q192R, L55M) by employing Light Cycler real-time PCR. We found that both PON1 arylesterase and PON1 paraoxonase activities were decreased in autistic patients (respectively, P < 0.001, P < 0.05), but no association with less active variants of the PON1 gene was found. The PON1 phenotype, inferred from the two-dimensional enzyme analysis, had a similar distribution in the ASD group and the control group. In conclusion, both the bioavailability and the catalytic activity of PON1 are impaired in ASD, despite no association with the Q192R and L55M polymorphisms in the PON1 gene and a normal distribution of the PON1 phenotype.     

Genetically meaningful phenotypic subgroups in autism spectrum disorders

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with strong evidence for genetic susceptibility. However, the effect sizes for implicated chromosomal loci are small, hard to replicate and current evidence does not explain the majority of the estimated heritability. Phenotypic heterogeneity could be one phenomenon complicating identification of genetic factors. We used data from the Autism Diagnostic Interview‐Revised, Autism Diagnostic Observation Schedule, Vineland Adaptive Behavior Scales, head circumferences, and ages at exams as classifying variables to identify more clinically similar subgroups of individuals with ASD. We identified two distinct subgroups of cases within the Autism Genetic Resource Exchange dataset, primarily defined by the overall severity of evaluated traits. In addition, there was significant familial clustering within subgroups (odds ratio, OR ≈ 1.38–1.42, P < 0.00001), and genotypes were more similar within subgroups compared to the unsubgrouped dataset (Fst = 0.17 ± 0.0.0009). These results suggest that the subgroups recapitulate genetic etiology. Using the same approach in an independent dataset from the Autism Genome Project, we similarly identified two distinct subgroups of cases and confirmed this severity‐based dichotomy. We also observed evidence for genetic contributions to subgroups identified in the replication dataset. Our results provide more effective methods of phenotype definition that should increase power to detect genetic factors influencing risk for ASD .     

CACNA1H mutations in autism spectrum disorders

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type Ca(V)1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type Ca(V)3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p = 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce Ca(V)3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype.     

Enhanced visual temporal resolution in autism spectrum disorders

Cognitive functions that rely on accurate sequencing of events, such as action planning and execution, verbal and nonverbal communication, and social interaction rely on well-tuned coding of temporal event-structure. Visual temporal event-structure coding was tested in 17 high-functioning adolescents and adults with autism spectrum disorder (ASD) and mental- and chronological-age matched typically-developing (TD) individuals using a perceptual simultaneity paradigm. Visual simultaneity thresholds were lower in individuals with ASD compared to TD individuals, suggesting that autism may be characterised by increased parsing of temporal event-structure, with a decreased capability for integration over time. Lower perceptual simultaneity thresholds in ASD were also related to increased developmental communication difficulties. These results are linked to detail-focussed and local processing bias.     

Network- and attribute-based classifiers can prioritize genes and pathways for autism spectrum disorders and intellectual disability

Autism spectrum disorders (ASD) are a group of related neurodevelopmental disorders with significant combined prevalence (～1%) and high heritability. Dozens of individually rare genes and loci associated with high-risk for ASD have been identified, which overlap extensively with genes for intellectual disability (ID). However, studies indicate that there may be hundreds of genes that remain to be identified. The advent of inexpensive massively parallel nucleotide sequencing can reveal the genetic underpinnings of heritable complex diseases, including ASD and ID. However, whole exome sequencing (WES) and whole genome sequencing (WGS) provides an embarrassment of riches, where many candidate variants emerge. It has been argued that genetic variation for ASD and ID will cluster in genes involved in distinct pathways and protein complexes. For this reason, computational methods that prioritize candidate genes based on additional functional information such as protein-protein interactions or association with specific canonical or empirical pathways, or other attributes, can be useful. In this study we applied several supervised learning approaches to prioritize ASD or ID disease gene candidates based on curated lists of known ASD and ID disease genes. We implemented two network-based classifiers and one attribute-based classifier to show that we can rank and classify known, and predict new, genes for these neurodevelopmental disorders. We also show that ID and ASD share common pathways that perturb an overlapping synaptic regulatory subnetwork. We also show that features relating to neuronal phenotypes in mouse knockouts can help in classifying neurodevelopmental genes. Our methods can be applied broadly to other diseases helping in prioritizing newly identified genetic variation that emerge from disease gene discovery based on WES and WGS.