In a preprint uploaded to the Research Square* server, researchers used a mix of in vivo mice models and next-generation sequencing techniques to elucidate the pathomechanistic connection between autism and myotonic muscular dystrophy type 1.

Their results suggest that CTG expansion within the DMPK gene and muscleblind-like factor inhibition induces developmental mis-splicing, which in turn cause myotonic muscular dystrophy type 1 and autistic social deficits.

Study: Autistic traits in myotonic dystrophy type 1 as a result of MBNL inhibition and RNA mis-splicing. Image Credit: SewCreamStudio/Shutterstock.com

*Vital notice: Research Square publishes preliminary scientific reports that are usually not peer-reviewed and, subsequently, shouldn’t be considered conclusive, guide clinical practice/health-related behavior, or treated as established information.

What are ASD and DM1?

Autism spectrum disorder (ASD) is a neurodevelopmental disability affecting the brain. It’s characterised by social communication or interaction problems, and restricted or repetitive behaviors or interests.

The condition affects one in 36 children, just about all of whom (95%) suffer from physical or mental comorbidities. ASD is a clinically heterogeneous condition, and despite a whole bunch of genes having been related to the incapacity, the molecular mechanisms underlying ASD remain unknown.

Previous whole-genome sequencing studies have identified tandem repeat mutations as related to ASD, contributing ~4% to ASD risk. Tandem repeats are sequences of two or more DNA bases repeated quite a few times in a head-to-tail manner on a chromosome.

They are frequently present within the non-coding region of DNA, but when these mutations arise in coding genes, they could have severe consequences, including myotonic muscular dystrophy type 1 (DM1).

The myotonic dystrophy protein kinase (DMPK) gene is certainly one of many liable for proper muscle development and performance. Research has shown that mutations on this gene correlate with autism.

One of the common mutations affecting one in every 2,100 newborns is a CTG expansion (CTGexp) within the gene’s 3′ untranslated region (3’UTR). These DMPK 3’UTR CTGexp mutations cause DM1, a variable genetic condition characterised by muscle weakness and wasting, myotonia, cataract, and sometimes cardiac conduction abnormalities.

When present, DMPK 3’UTR CTGexp  RNAs present in DM1 neurons and muscles lead to the inhibition of muscleblind-like (MBNL) RNA-binding proteins through the formation of biomolecular structures called RNA foci.

Under normal conditions, MBNL proteins act as trans-acting aspects, regulating alternative splicing (AS) during fetal development. Their inhibition is usually recommended to lead to ‘mis-splicing’ correlating with specific DM1 clinical profiles, especially myotonia.

DM1 and ASD have been identified as comorbidities, with the latter correlating with the age of onset of the previous. Like in DM1, developmental mis-splicing occurs in neuronal microexons (miEs), identified in ~30% of idiopathic ASD brains.

These microexons control protein-protein interaction networks and encode post-translational modification sites, that are crucial in normal nervous system development. Mutations in miEs have been shown to trigger ASD-like symptoms in murine models, including social avoidance.

While the processes and mechanisms underlying DM1 mis-splicing have been well documented, ASD mis-splicing stays poorly understood. Further, molecular mechanisms linking ASD and DM1 have hitherto been unstudied.

Concerning the study

In the current preprint, researchers employed integrative genomic- and transcriptomic techniques (next-generation sequencing) together with in vivo studies using multiple genetically modified DM1 murine models to elucidate the molecular mechanisms underpinning mis-splicing in DM1-associated ASD.

Their analyses focused on neuronal miEs mis-splicing in ASD-risk genes modulated by MBNL proteins, specifically MBNL1 and MBNL2, during murine and human brain development.

Researchers first analyzed human prefrontal cortex RNA-seq data from DM1-positive (study) and DM1-negative (control) cohorts. They compared the change of percent spliced (DPSI) in 38 ASD-relevant gene sets retrieved from previous studies and located that 76% of their dataset showed significant enrichment of mis-spliced events in study cohorts.

They further identified mis-splicing within the Duchenne muscular dystrophy (DMD) gene, a comorbidity of ASD. Their evaluation revealed that CTGexp size was significantly correlated with the variety of mis-spliced events in these genes, verifying that DMPK 3’UTR CTGexp prefrontal cortex mutations inhibit AS in ASD-relevant genes.

To elucidate the involvement of MBNL protein regulation within the prefrontal cortex, genetically modified mice models’ (Mbnl cDKO) frontal cortex samples were in comparison with those from wild-type (WT) mice.

Results showed that, just like humans, 61% of ASD-relevant gene sets were mis-splice-enriched in Mbnl cDKO murine frontal cortexes. Of those, 55 ASD-risk genes overlapped between humans and mice with ASD.

Transcriptomic analyses of neuronal miEs were employed to guage the extent of mis-splicing in these microexons. Comparisons between WT and Mbnl cDKO miEs revealed that the previous had significantly fewer (4%) mis-spliced events than the latter (10%).

When these analyses focused on ASD-risk genes, these contrasts were as high as 35%. Comparative in silico modeling of miE-encoded protein structure was employed to evaluate if mis-splices in miEs can impact peptide structures.

Modeling results suggest that internal or C-terminal protein structures is perhaps altered if miEs are mis-spliced.

Gene expression data from five mammalian brains (including humans) was analyzed to infer the role of MBNL proteins and their AS in ASD-risk genes during organismal development.

“Our evaluation showed an evolutionarily conserved increase of MBNL2 expression during neonate/P0 to middle childhood/P14 brain development. Although MBNL1 expression increases concurrently, its expression within the developed brain is roughly 3-fold lower than MBNL2.”

Reverse transcription-polymerase chain response (RT-PCR) splicing evaluation of MBNL2 in WT and Mbnl cDKO mice revealed that the loss or alteration of MBNL2 caused severe alterations in AS of ASD-risk genes in numerous parts of Mbnl cDKO brains, especially the hippocampus.

Finally, social interaction in vivo tests were conducted on WT, Mbnl2 knockout, and Dmpk 3’UTR CTGexp knockin genetically modified mice.

Three-chamber tests which included familiar mice, stranger mice, and novel inanimate objects, were employed to check the sociability of those animals. WT mice spent significantly more time with strange mice than their genetically modified counterparts.

There was no difference in time spent with novel inanimate objects, highlighting that sociability, and never curiosity, was impacted by alterations in MBNL2 and DM1.

Conclusions

In the current preprint, researchers utilized next-generation sequencing technologies to elucidate the pathomechanistic relevance of mis-splicing in DM1-associated ASD.

Their results revealed that tandem CTG repeats within the DMPK gene significantly increase the occurrence of each DM1 and ASD via inhibition of MBNL proteins, especially MBNL2.

The length and variety of tandem repeats were positively correlated with the clinical severity of DM1 and ASD. These results suggest that MBNL protein inhibitions alter neuronal development, leading to these diseases.

In vivo comparisons between genetically modified murine models (altered to reflect these ASD-relevant genotypes) and their regular counterparts to guage sociability confirmed the transcriptomic and genomic analyses.

They revealed that alternations or mis-splicing of CTG tandem repeats and MBNL proteins lead to significantly reduced sociability in mice.

“Our results provide insights into the molecular mechanism underlying DM1-associated ASD where developmental mis-splicing of ASD-linked genes arises by lack of MBNL activity as a result of CUG repeat expansions. Understanding this pathomechanistic connection provides a possibility for greater in-depth investigations of mechanistic threads in autism.”

*Vital notice: Research Square publishes preliminary scientific reports that are usually not peer-reviewed and, subsequently, shouldn’t be considered conclusive, guide clinical practice/health-related behavior, or treated as established information.