Autism and Nonsyndromic Mental Retardation Associated with a De Novo Mutation in the NLGN4X Gene Promoter Causing an Increased Expression Level

doi:10.1016/j.biopsych.2009.05.008

Biological Psychiatry

Volume 66, Issue 10, 15 November 2009, Pages 906-910

https://doi.org/10.1016/j.biopsych.2009.05.008 Get rights and content

Background

Pathogenic mutations in the X-linked Neuroligin 4 gene (NLGN4X) in autism spectrum disorders (ASDs) and/or mental retardation (MR) are rare. However, nothing is known regarding a possible altered expression level of NLGN4X that would be caused by mutations in regulatory sequences. We investigated this issue by analyzing these regions in patients with ASDs and no mutation in the NLGN4X coding sequence.

Methods

We studied 96 patients who met all DSM-IV criteria for autism. The entire coding sequence and the regulatory sequences of the NLGN4X gene were analyzed by polymerase chain reaction and direct sequencing.

Results

We identified a de novo 1 base pair (−335G>A) substitution located in the promoter region in a patient with autism and nonsyndromic profound MR. Interestingly, this variation is associated with an increased level of the NLGN4X transcript in the patient compared with male control subjects as well as his father. Further in vitro luciferase reporter and electrophoretic mobility shift assays confirmed, respectively, that this mutation increases gene expression and is probably caused by altered binding of transcription factors in the mutated promoter sequence.

Conclusions

This result brings further insight about the phenotypic spectrum of NLGN4X mutations and suggests that the analysis of the expression level of NLGN4X might detect new cases.

Section snippets

Subjects

Autistic patients (80 male subjects and 16 female subjects) were recruited through the Child Psychiatry Center at the University Hospital of Tours (France) by a multidisciplinary team. All patients were Caucasians. Written informed consent was obtained from parents. Exclusion criteria included: severe sensory problems (e.g., visual impairment or hearing loss); significant motor impairments (e.g., failure to sit by 12 months or walk by 24 months); and identified metabolic, genetic, or

Results

We did not detect any mutation or polymorphism in the NLGN4X coding sequence of all patients. However, we identified a 1-bp substitution G>A located in the promoter region sequence 335 bp upstream from the transcription initiation site in a boy with autism and profound MR (Figure 1). The sequence variation has been confirmed in a second independent PCR with genomic DNA extracted from peripheral blood. This variation occurred de novo, and a paternity test using different independent and

Discussion

We report here the first description of a de novo substitution in the promoter region of the NLGN4X gene in a patient with autism and nonsyndromic profound MR. This variation is associated with an increased level of the NLGN4X transcript in the LCLs of the patient compared with that of male control subjects as well as that of his father. However, we acknowledge that the presence of only one causative variation in our population of 96 autistic patients—the use of the nonneuronal cell line HEK

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Autism Spectrum Disorders (ASD) are a group of neurodevelopmental disorders (NDDs) characterized by difficulties in social interaction and communication, repetitive behavior, and restricted interests. While ASD have been proven to have a strong genetic component, current research largely focuses on coding regions of the genome. However, non-coding DNA, which makes up for ∼99% of the human genome, has recently been recognized as an important contributor to the high heritability of ASD, and novel sequencing technologies have been a milestone in opening up new directions for the study of the gene regulatory networks embedded within the non-coding regions. Here, we summarize current progress on the contribution of non-coding alterations to the pathogenesis of ASD and provide an overview of existing methods allowing for the study of their functional relevance, discussing potential ways of unraveling ASD's “missing heritability”.
Novel frameshift mutation in Indian autistic population causes neuroligin and neurexin binding defect
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Whole genome studies identify NLGN4X as the candidate gene for association study for autism (Yu et al., 2013; Yuen et al., 2017). Several denovo variations of NGN4X are associated with autism and contribute to the etiology of autism (Zhang et al., 2009; Daoud et al., 2009; Wang et al., 2018; Martínez et al., 2017). Subsequently, few studies in different autism population failed to find the variants to prove the involvement of the NLGN4X gene in autism so maybe NLGN4X variants account the only a small proportion of autism (Wermter et al., 2008; Vincent et al., 2004; Gauthier et al., 2005; Blasi et al., 2006) But Neuroligin is dynamically regulated through post translational modification.
Autism is a complex neurodevelopmental condition which is clinically, etiologically and genetically heterogeneous. The predisposition of genes and genetic loci has been under study over the last few decades. Autism is increasing dramatically over the last few years in India. Mutations in two x-linked neuroligin genes viz. NLGN3 and NLGN4X have been recently implicated in autism pathogenesis. In the current study, for the first time in India, we screened the genetic variations in transmembrane neuroligin 4× gene in the Indian autistic population using sequencing technology. 1828 mentally ill children were screened around the North Karnataka region of India and 136 autistic children were identified, out of which 108 autistic children who were fulfilling the inclusion criteria were taken for the study. DNA was isolated from the clinical samples and exonic regions were amplified. Sanger sequencing was carried to screen the genetic variations and expression profiling was performed using Real time PCR. Sequencing results revealed 25 different variants (eight 5′UTR variants, four missense variants, four synonymous variants, one frameshift variant and eight 3′ UTR variants). One novel frameshift variation (p.Gly214GlyfsTer2) was recorded in 19 (17.5%) autistic individuals. This novel frameshift mutation leads to a premature stop codon. Relative expression of the NLGN4X gene in autistic children with novel frameshift mutation was largely reduced compared to the control group [0.052 and 1.217 respectively, p-value 0.00001]. The present study shows novel frameshift mutation causes abnormal NLGN4× protein that leads to loss of binding to presynaptic neurexin during synapses. Therefore neuroligin 4× is the potential target gene in the Indian autistic population for future functional analysis.
Comparative mapping of selected structural determinants on the extracellular domains of cholinesterase-like cell-adhesion molecules
2021, Neuropharmacology
Citation Excerpt :
The resulting substitution, localized in the extracellular domain of NLGN3 (Fig. 1), was shown to alter trafficking of the mutant protein through the intracellular secretory pathway (Comoletti et al., 2004; Chih et al., 2004; De Jaco et al., 2006). Other rare variants were also found for the other NLGNs, of which most were linked to autism and related neurodevelopmental disorders (Laumonnier et al., 2004; Yan et al., 2005; Talebizadeh et al., 2006; Lawson-Yuen et al., 2008; Daoud et al., 2009; Zhang et al., 2009; Pampanos et al., 2009; Sun et al., 2011; Xu et al., 2014; Landini et al., 2016; Nakanishi et al., 2017; Quartier et al., 2019; Shillington et al., 2020; and others). NRT, a 846-residue transmembrane protein with a type-II topology and whose extracellular ChE-like domain comprises residues 347–846 (Fig. 1; Table 1), is found only in insects (de la Escalera et al., 1990).
Cell adhesion generally involves formation of homophilic or heterophilic protein complexes between two cells to form transcellular junctions. Neural cell-adhesion members of the α/β-hydrolase fold superfamily of proteins use their extracellular or soluble cholinesterase-like domain to bind cognate partners across cell membranes, as illustrated by the neuroligins. These cell-adhesion molecules currently comprise the synaptic organizers neuroligins found in all animal phyla, along with three proteins found only in invertebrates: the guidance molecule neurotactin, the glia-specific gliotactin, and the basement membrane protein glutactin. Although these proteins share a cholinesterase-like fold, they lack one or more residues composing the catalytic triad responsible for the enzymatic activity of the cholinesterases. Conversely, they are found in various subcellular localisations and display specific disulfide bonding and N-glycosylation patterns, along with individual surface determinants possibly associated with recognition and binding of protein partners. Formation of non-covalent dimers typical of the cholinesterases is documented for mammalian neuroligins, yet whether invertebrate neuroligins and their neurotactin, gliotactin and glutactin relatives also form dimers in physiological conditions is unknown. Here we provide a brief overview of the localization, function, evolution, and conserved versus individual structural determinants of these cholinesterase-like cell-adhesion proteins.
This article is part of the special issue entitled ‘Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield’.
Neuroligins and neurexins
2020, Synapse Development and Maturation: Comprehensive Developmental Neuroscience
The differentiation and functional specification of synapses are critical for the assembly and function of neuronal networks. Adhesion molecules that bridge the synaptic cleft have emerged as important regulators of the establishment, maintenance, and functional characteristics of synaptic connections. This chapter focuses on one pair of such trans-synaptic interaction partners, the neurexins (NRXs) and neuroligins (NLs), which form a heterophilic adhesion complex. We will first briefly discuss the discovery of these synaptic proteins and then summarize conclusions from structural and cell biological studies on the synaptic function of NRX and NL proteins. In the last section of the chapter, we will then highlight the involvement of NRX and NL functions in neuropsychiatric and neurodevelopmental disorders.
A strategic plan to identify key neurophysiological mechanisms and brain circuits in autism
2018, Journal of Chemical Neuroanatomy
Citation Excerpt :
These molecules are involved in synaptic development and conservation, where they play an important role in the balance between excitatory and inhibitory transmission. Other studies have pointed to genes encoding proteins localized at the glutamatergic synapse such as the SHANK3 protein, which binds with the neuroligins, enabling membrane proteins to bind to the cytoskeleton of the synapse, and also to the KCNMA1 gene involved in a postsynaptic channel regulating neuronal excitability (Daoud et al., 2009). The hypothesis of a deficit in synaptic development and functioning is now at the frontline in pathophysiological research on autism.
Autism and Autism Spectrum Disorder (ASD) cover a large variety of clinical profiles which share two main dimensions: social and communication impairment and repetitive behaviors or restricted interests, which are present during childhood. There is now no doubt that genetic factors are a major component in the etiology of autism but precise physiopathological pathways are still being investigated. Furthermore, developmental trajectories combined with compensatory mechanisms will lead to various clinical and neurophysiological profiles which together constitute this Autism Spectrum Disorder. To better understand the pathophysiology of autism, comprehension of key neurophysiological mechanisms and brain circuits underlying the different bioclinical profiles is thus crucial. To achieve this goal we propose a strategy which investigates different levels of information processing from sensory perception to complex cognitive processing, taking into account the complexity of the stimulus and whether it is social or non-social in nature. In order to identify different developmental trajectories and to take into account compensatory mechanisms, we further propose that such protocols should be carried out in individuals from childhood to adulthood representing a wide variety of clinical forms.
Etiologies underlying sex differences in Autism Spectrum Disorders
2014, Frontiers in Neuroendocrinology
Citation Excerpt :
For example, as discussed above, deletions or duplications including Xp22.3 have been reported in individuals diagnosed with ASD. One of the genes of interest on Xp22.3, besides the STS gene discussed above, is neuroligin 4 (NLGN4X) because it has been implicated with autism in many studies (Carrel et al., 2006; Daoud et al., 2009; Glessner et al., 2009; Laumonnier et al., 2007; Marshall et al., 2008; Pampanos et al., 2009; but see Gauthier et al., 2005; Vincent et al., 2004 for two screening studies using samples of 196 and 96 autistic probands that found no association between NLGN4X and ASD indicating that this gene is only a causative factor underlying the development of ASD in a fraction of the cases). NLGN4X encodes cell adhesion molecules involved in the formation of functional synapses.
The male predominance of Autism Spectrum Disorders (ASD) is one of the best-known, and at the same time, one of the least understood characteristics of these disorders. In this paper we review genetic, epigenetic, hormonal, and environmental mechanisms underlying this male preponderance. Sex-specific effects of Y-linked genes (including SRY expression leading to testicular development), balanced and skewed X-inactivation, genes that escape X-inactivation, parent-of-origin allelic imprinting, and the hypothetical heterochromatin sink are reviewed. These mechanisms likely contribute to etiology, instead of being simply causative to ASD. Environments, both internal and external, also play important roles in ASD’s etiology. Early exposure to androgenic hormones and early maternal immune activation comprise environmental factors affecting sex-specific susceptibility to ASD. The gene–environment interactions underlying ASD, suggested here, implicate early prenatal stress as being especially detrimental to boys with a vulnerable genotype.

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Research ReportAutism and Nonsyndromic Mental Retardation Associated with a De Novo Mutation in the NLGN4X Gene Promoter Causing an Increased Expression Level

Background

Methods

Results

Conclusions

Section snippets

Subjects

Results

Discussion

Am J Hum Genet

Am J Hum Genet

Methods

Neuron

Cell

Neuron

Am J Hum Genet

The genetics of autistic disorders and its clinical relevance: A review of the literature

Eur J Hum Genet

Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism

Nat Genet

Mutation screening of X-chromosomal neuroligin genes: No mutations in 196 autism probands

Am J Med Genet B Neuropsychiatr Genet

NLGN3/NLGN4 gene mutations are not responsible for autism in the Quebec population

Am J Med Genet B Neuropsychiatr Genet

Research Report
Autism and Nonsyndromic Mental Retardation Associated with a De Novo Mutation in the NLGN4X Gene Promoter Causing an Increased Expression Level