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Analysis of the effects of rare variants on splicing identifies alterations in GABAA receptor genes in autism spectrum disorder individuals.

ABSTRACT: A large-scale sequencing screen of X-linked synaptic genes in individuals with autism spectrum disorder (ASD) or schizophrenia (SCZ), two common neurodevelopmental disorders, identified many variants most of which have no easily predictable effect on gene function. In this report, we evaluated the impact of these rare missense and silent variants on gene splicing. For this purpose, we used complementary in silico analyses, in vitro minigene-based assays and RNA prepared from lymphoblastoid cells derived from patients with these mutations. Our goal was to identify the variants which might either create or disrupt an acceptor splice site, a donor splice site or an exonic splicing enhancer, thus leading to aberrant splicing that could be involved in the pathogenesis of ASD or SCZ. We identified truncating mutations in distinct X-linked gamma-aminobutyric acid A (GABAA) receptor subunit-encoding genes, GABRQ and GABRA3, in two different families. Furthermore, missense and silent variants in nuclear RNA export factor 5 and histone deacetylase 6 were shown to partially disrupt the protein. While genes from the GABAergic pathway have previously been thought to be involved in the pathophysiology of ASD, this is the first report of ASD patients with truncating mutations in GABA receptors genes.

SUBMITTER: Piton A

PROVIDER: S-EPMC3722945 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Analysis of the effects of rare variants on splicing identifies alterations in GABAA receptor genes in autism spectrum disorder individuals.

Piton Amélie A Jouan Loubna L Rochefort Daniel D Dobrzeniecka Sylvia S Lachapelle Karine K Dion Patrick A PA Gauthier Julie J Rouleau Guy A GA

European journal of human genetics : EJHG 20121121 7

A large-scale sequencing screen of X-linked synaptic genes in individuals with autism spectrum disorder (ASD) or schizophrenia (SCZ), two common neurodevelopmental disorders, identified many variants most of which have no easily predictable effect on gene function. In this report, we evaluated the impact of these rare missense and silent variants on gene splicing. For this purpose, we used complementary in silico analyses, in vitro minigene-based assays and RNA prepared from lymphoblastoid cells ...[more]

PMID: 23169495

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Project description:BackgroundThe Autism Sequencing Consortium identified 102 high-confidence autism spectrum disorder (ASD) genes, showing that individuals with ASD and with potentially damaging single nucleotide variation (pdSNV) in these genes had lower cognitive levels and delayed age at walking, when compared to ASD participants without pdSNV. Here, we made use of a Swedish sample of individuals with ASD (called PAGES, for Population-Based Autism Genetics & Environment Study) to evaluate the frequency of pdSNV and their impact on medical and psychiatric phenotypes, using an epidemiological frame and universal health reporting. We then combine findings with those for potentially damaging copy number variation (pdCNV).MethodsSNV and CNV calls were generated from whole-exome sequencing and chromosome microarray data, respectively. Birth and medical register data were used to collect phenotypes.ResultsOf 808 individuals assessed by sequencing, 69 (9%) had pdSNV in the 102 ASC genes, and 144 (18%) had pdSNV in the 102 ASC genes or in a larger set of curated neurodevelopmental genes (from the Deciphering Developmental Disorders study, the gene2phenotype database, and the Radboud University gene lists). Three or more individuals had pdSNV in GRIN2B, POGZ, SATB1, DYNC1H1, SCN8A, or CREBBP. In comparison, out of the 996 individuals from whom CNV were called, 105 (11%) carried one or more pdCNV, including four or more individuals with CNV in the recurrent 15q11q13, 22q11.2, and 16p11.2 loci. Carriers of pdSNV were more likely to have intellectual disability (ID) and epilepsy, while carriers of pdCNV showed increased rates of congenital anomalies and scholastic skill disorders. Carriers of either pdSNV or pdCNV were more likely to have ID, scholastic skill disorders, and epilepsy.LimitationsThe cohort only included individuals with autistic disorder, the more severe form of ASD, and phenotypes are defined from medical registers. Not all genes studied are definitively ASD genes, and we did not have de novo information to aid in classification.ConclusionsIn this epidemiological sample, rare pdSNV were more common than pdCNV and the combined yield of potentially damaging variation was substantial at 27%. The results provide compelling rationale for the use of high-throughout sequencing as part of routine clinical workup for ASD and support the development of precision medicine in ASD.

Project description:Although the neurobiological basis of autism spectrum disorder (ASD) is not fully understood, recent studies have indicated the potential role of GABAA receptors in the pathophysiology of ASD. GABAA receptors play a crucial role in various neurodevelopmental processes and adult neuroplasticity. However, the mechanism(s) of regulation of GABAA receptors in ASD remains poorly understood.Postmortem middle frontal gyrus tissues (13 ASD and 13 control subjects) were used. In vitro studies were performed in primary cortical neurons at days in vitro (DIV) 14. The protein levels were examined by western blotting. Immunofluorescence studies were employed for cellular localization. The gene expression was determined by RT-PCR array and qRT-PCR.A significant decrease in GABAA?1 protein, but not mRNA levels was found in the middle frontal gyrus of ASD subjects indicating a post-translational regulation of GABAA receptors in ASD. At the cellular level, treatment with proteasomal inhibitor, MG132, or lactacystin significantly increased GABAA?1 protein levels and Lys48-linked polyubiquitination of GABAA?1, but reduced proteasome activity in mouse primary cortical neurons (DIV 14 from E16 embryos). Moreover, treatment with betulinic acid, a proteasome activator significantly decreased GABAA?1 protein levels in cortical neurons indicating the role of polyubiquitination of GABAA?1 proteins with their subsequent proteasomal degradation in cortical neurons. Ubiquitination specific RT-PCR array followed by western blot analysis revealed a significant increase in SYVN1, an endoplasmic reticulum (ER)-associated degradation (ERAD) E3 ubiquitin ligase in the middle frontal gyrus of ASD subjects. In addition, the inhibition of proteasomal activity by MG132 increased the expression of GABAA?1 in the ER. The siRNA knockdown of SYVN1 significantly increased GABAA?1 protein levels in cortical neurons. Moreover, reduced association between SYVN1 and GABAA?1 was found in the middle frontal gyrus of ASD subjects.SYVN1 plays a critical role as an E3 ligase in the ubiquitin proteasome system (UPS)-mediated GABAA?1 degradation. Thus, inhibition of the ubiquitin-proteasome-mediated GABAA?1 degradation may be an important mechanism for preventing GABAA?1 turnover to maintain GABAA?1 levels and GABA signaling in ASD.

Project description:In addition to core deficits in social-communication abilities and repetitive behaviors and interests, many patients with autism spectrum disorder (ASD) experience developmental comorbidities, including sensorimotor issues. Sensorimotor issues are common in ASD and associated with more severe clinical symptoms. Importantly, sensorimotor behaviors are precisely quantifiable and highly translational, offering promising targets for neurophysiological studies of ASD. We used functional MRI to identify brain regions associated with sensorimotor behavior using a visually guided precision gripping task in individuals with ASD (n = 20) and age-, IQ-, and handedness-matched controls (n = 18). During visuomotor behavior, individuals with ASD showed greater force variability than controls. The blood oxygen level-dependent signal for multiple cortical and subcortical regions was associated with force variability, including motor and premotor cortex, posterior parietal cortex, extrastriate cortex, putamen, and cerebellum. Activation in the right premotor cortex scaled with sensorimotor variability in controls but not in ASD. Individuals with ASD showed greater activation than controls in left putamen and left cerebellar lobule VIIb, and activation in these regions was associated with more severe clinically rated symptoms of ASD. Together, these results suggest that greater sensorimotor variability in ASD is associated with altered cortical-striatal processes supporting action selection and cortical-cerebellar circuits involved in feedback-guided reactive adjustments of motor output. Our findings also indicate that atypical organization of visuomotor cortical circuits may result in heightened reliance on subcortical circuits typically dedicated to motor skill acquisition. Overall, these results provide new evidence that sensorimotor alterations in ASD involve aberrant cortical and subcortical organization that may contribute to key clinical issues in patients.NEW & NOTEWORTHY This is the first known study to examine functional brain activation during precision visuomotor behavior in autism spectrum disorder (ASD). We replicate previous findings of elevated force variability in ASD and find these deficits are associated with atypical function of ventral premotor cortex, putamen, and posterolateral cerebellum, indicating cortical-striatal processes supporting action selection and cortical-cerebellar circuits involved in feedback-guided reactive adjustments of motor output may be key targets for understanding the neurobiology of ASD.

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Analysis of the effects of rare variants on splicing identifies alterations in GABAA receptor genes in autism spectrum disorder individuals.

Publications

Analysis of the effects of rare variants on splicing identifies alterations in GABAA receptor genes in autism spectrum disorder individuals.

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