X Chromosome Inactivation and Alternative Splicing In Autism: An Integrated Approach to Cross Link Gene Regulatory Processes

Background: Numerous susceptibility genes and chromosomal abnormalities have been associated with autism spectrum disorders (ASD), but most discoveries either fail to be replicated or account for a small effect. Inconclusive results could in part be a reflection of heterogeneous phenotype and indicate the need to employ strategies that identify more homogeneous groups of ASD subjects. An increased prevalence of autism in males suggests a role for the X chromosome. Evaluating X chromosome inactivation (XCI) in autistic females has been recommended by us and other groups. Despite some interesting hypotheses (e.g., imprinting and epigenetic mechanisms), it is not yet clearly understood if and how XCI might be important to understanding the mechanism of autism. One way to tackle this important topic is to assess if autistic females with X-inactivation skewness (XIS) have a unique genomic profile. Exploring such a question first requires knowing how this gene regulatory process is connected with other genomic processes. X chromosome inactivation is a DNA methylation process. Previously we have shown the trend of a higher degree of XIS in autistic females compared to non-autistic females. We also reported on the potential role of aberrant alternative splicing in X-linked neuroligin genes in autism. More recent findings have shown a cross link between DNA methylation and alternative splicing. These lines of evidence prompted us to systematically evaluate alternative splicing of NLGN3 in relation to autistic females’ XCI status. 

Objectives: To assess the relationship between XCI patterns and alternative splicing, we evaluated the expression level of multiple alternative splicing transcripts for NLGN3, an X-linked autism gene, in autistic females with and without XIS.

Methods:  Subjects were ascertained from the Autism Genetics Resource Exchange (AGRE). XCI status was determined using the Androgen Receptor gene assay on blood-derived genomic DNA. TaqMan gene expression experiments for multiple NLGN3 splice variants were conducted using RNA from AGRE lymphoblastoid cell lines. Exon boundaries/compositions of amplified products were confirmed by DNA sequencing. 

Results: In addition to the two known NLGN3 variants, we identified three novel splice variants for this gene. A distinct, statistically significant pattern was detected in the expression level of examined alternatively spliced NLGN3 isoforms for autistic females with XIS compared to those without XIS and controls.

Conclusions:  This is the first study to evaluate the expression level of NLGN3 at the alternative splicing level taking into consideration the X-inactivation status of autistic females.  Our study indicates that NLGN3 undergoes complex splicing process resulting in multiple splice variants. Furthermore, our expression data demonstrates that autistic females with XIS may represent a more homogeneous subset of this highly heterogeneous population. Applying such a stratification method may provide a way to better understand underlying genetic and epigenetic mechanism in autism.  Our study introduces a novel and effective method to connect different lines of genomic data in unraveling the etiology of this complex neurodevelopmental disorder.