The genetic profiling of the Autism Spectrum Disorder
PART I: Autism spectrum disorder (ASD)
What is ASD?
Autism spectrum disorder (ASD) is an umbrella term for a group of complex neurodevelopmental disorders, which links up to 150 rare diseases, including PTEN Hamartoma Tumour Syndrome (PHTS), Fragile X, RETT Syndrome, Phelan McDermid Syndrome, and more.1
Understanding ASD deeper at a genetic level
The clinical diagnosis of ASD has been challenging because of the highly variable observed somatic abnormality representation seen on patients.1,2 With little molecular understanding of ASD in the early days, the traditional clinical diagnosis of ASD was mostly syndrome-based. These symptom-based diagnostic tests could be time-costly with multi-disciplinary interview-based assessments involving speech and language therapists, pediatricians, child development centre psychiatrists and psychologists.1,2,3 Many ASD patients presenting complex ASD symptoms do not get an accurate diagnosis until much older in life, which costs them the opportunity to get proper treatments. As recent advancements in genetic sequencing techniques continue to provide precise data of genetic variants found in ASD patients, genetic analysis is leading us to a deeper understanding of the etiology of ASD. Studies have revealed that ASD susceptibility is the consequence of hundreds of gene variants combined, which can be detected through genetic sequencing as a cost-effective approach. Thus, shifting from a clinical syndrome-based ASD diagnosis to a genetic etiology-based ASD diagnosis may provide more promising results in patient diagnosis.
Genetic sequencing & Exploring genetic etiology of ASD
Genetic sequencing is the method of mapping out the components that made up genes. Through obtaining a blueprint of individual gene sequences, gaining knowledge of the etiology of ASD at a genetic and molecular basis becomes much more accessible. Progressing genetic sequencing techniques has led us to the era of High-throughput sequencing (HTS). The advent of HTS techniques has enabled the sequencing of millions of DNA fragments simultaneously, thus, acts as a tool that is highly sensitive and accurate in detecting genetic variants and gene expression variation in ASD patients. With the real-time sequencing power and the increasingly abundant sequence database, genetic sequencing advancements have led to the identifications of many genetic markers associated with ASD. Therefore, the technique is shining a light on the molecular mechanisms that underlie ASD. With the greater characterization of the genetics of ASD, high-throughput genetic sequencing techniques can hopefully facilitate the diagnosis of ASD on a molecular basis and identify potential therapeutic strategies.2,3,4,5,6,7
PART II: Genetic components of the ASD aetiology
Genetic variants have been found to play a significant role in the development of ASD given that ASD-associated genes have as high as ~50% chance of being passed from parents to the next generation.3 Meanwhile, the identification of causal genetic variants remains limited to a small subset of ASD patients with a strong family history or patients with ASD associated monogenic developmental disorders. Overall, the complexity of genetic variation combined with clinical symptoms continues to cause difficulties in effective diagnosis and therapeutic development.7
About 75% reported ASD cases to have unknown causes, which are categorized as non-syndromic idiopathic ASD in scientific articles. While the cause of ASD remains largely unknown and undetectable, the path to unravelling this secret is hopeful. In the past decade, the strongest breakthrough in understanding the biochemical mechanism and the clinical feature of ASD comes from a genetic analysis in studies focusing on families. About 25% of the ASD cases are detected by cytogenetics or sequence-based analysis to involve ASD-causing genetic variations. Examples include Chromosome 15q duplications, Fragile X syndrome, Rett syndrome.
Variants associated with ASD have been found in genes that are essential in brain development, neuronal signalling and transcription regulation, especially within the three major cellular pathways for synaptic function, translation and WNT signalling.7 Synapses between neurons are the key for signal transduction in the nervous system and WNT signalling is a molecular pathway that is in charge of regulating neural development. Transcription and translation are essential for creating the cellular machinery that functions to sustain life. Therefore, they require tight monitoring and regulation to avoid errors that may cause disease. Variants of ASD-associated genes that function in these three cellular pathways cause defects in synaptogenesis, synaptic functions, WNT signalling during brain maturation, and neuronal activity.7 These genes are often transcription factors, splicing factors or chromatin regulators, which often result in widespread gene dysregulation since they regulate the processing of many RNA at many steps such as upstream transcription regulation, pre-mRNA splicing and post-transcriptional RNA processing.7
In the search for ASD-susceptibility genes, the SFARI database presented 913 identified genes, including genes with an excess of rare de novo protein-truncating variants (PTVs) or copy-number variants (CNVs). Husson et al. (2020) presented a more finely categorized collection of susceptibility genes to aid in ASD diagnosis and a guideline for rare susceptibility variants categorization, which could contribute to the foundation of genetic etiology-based ASD diagnosis.
PART III: ASD – associated biology & Genetic sequencing technique
ASD-associated biology revealed by genetic sequencing techniques
Various techniques such as transcriptomic analysis, microarray analysis, PCR and RNA-Seq are used to evaluate gene expression in a high-throughput manner to understand the molecular mechanisms underlying ASD. Transcriptomic analysis, the analysis of gene expression through measuring RNA, has allowed us to identify changes in gene expression (the physiological representation of a gene within our bodies) associated with ASD in specific brain regions, such as the prefrontal cortex, the superior temporal cortex, and the cerebellum.3 Through transcriptomic analysis, another study found that genes involved in immune response showed reduced expression in the temporal cortex of ASD patients, which suggested that inflammation in the brain might be contributing to ASD-associated neurological defects.3 Transcriptome analysis focusing on gene regulatory programmes and feedback loops revealed downregulation of specific genes and the disruption of splicing of highly conserved neuronal microexons (pieces of genes that are less than 27 nucleotides in length) as mechanisms underlying ASD.5 Overall, the transcriptomic analysis uses sequencing data to convey a meaningful understanding of biological processes in the event of alternative splicing of the microexon to reveal the abnormalities in molecular mechanisms in ASD.
Sequence analysis techniques have also contributed to deepening the understanding of ASD etiology at a molecular level. One essential molecule in ASD research is the neurotransmitter, glutamate. It functions in modulating memory and learning, which are usually impaired in ASD. Using microarray analysis, the technique to measure the expression of a large number of genes at multiple regions of the genome, research has revealed that the cerebellum of ASD patients showed an increase in the expression of the glutamate transporter, excitatory amino acid transporter 1 (EAAT1) and an increase in the AMPA receptor component, glutamate receptor 1 (GRIA1), which are essential to glutamate signalling.3 Moreover, another sequencing analysis technique called reverse transcription (RT)-PCR has verified the misregulation of neuronal mRNA expression of EEAT1 and GRIA1, which are two functionally ASD-linked genes.3 The gene expression analysis technique, chromosomal microarray analysis, has a 7-9% diagnostic yield capacity with some limitations in the sensitivity towards single nucleotide variants (the alteration of a single unit of DNA sequences) and small indels.5 In comparison, the High-throughput genetic sequencing with a higher sensitivity revealed a significant decrease in the expression of relevant genes between the frontal and the temporal cortex in ASD.3 Consequently, a list of genes was identified with ASD-specific regional expression loss in glutamatergic neurons (SLC17A6, CPLX2, MET and SLC6A7) and GABAergic neurons (PVALB and SYT2).3 Moreover, research with RNA-seq gene profiling performed on pluripotent (the potential of differentiating into various tissue types in an organism) stem cell-derived brain organoids (a simplified version of 3D organ created in a laboratory-controlled environment) from ASD patients also identified the highest number of differentially expressed protein-coding genes (2,433 genes) compared to the number of genes (1,097 genes) identified from post-mortem brain tissues of ASD patients.3
As an increasing amount of gene sequences of ASD-associated patients is collected as a database, the accumulated knowledge of gene variants contributes to an increasingly precise understanding of ASD etiology at a genetic and molecular level, which could possibly provide guidelines of ASD diagnosis through genetic sequencing.
Future directions of next-generation sequencing in understanding ASD pathophysiology
Current genomic studies revealed genetic profiles and molecular characteristics of ASD, including disrupted expression of RNA. Though an increasing number of genes are identified to be associated with ASD, there remains much to be learned about the genetic underpinnings of ASD. The advancements in HST sequencing provide a cost-effective and powerful technique that is an excellent tool for identifying genes related to ASD diagnosis.5 Moreover, genome-wide experiments employing CRISPR-Cas9 have recently enabled high-throughput screening of alternative splicing regulators (AS) related to ASD-associated cellular and developmental pathology.5
Overall, the fast-progressing genome analyzing techniques such as transcriptome analysis, RNA-seq analysis, sequencing platforms, computational analysis tools, single-cell sequencing and genome editing provide researchers powerful tools to advance understanding of ASD pathology, therefore, advance the current diagnostic methods of ASD.
Zheng Song
Works Cited:
Autism spectrum disorder. Genetic and Rare Diseases Information Center. https://rarediseases.info.nih.gov/diseases/10248/autism-spectrum-disorder. Published December 16, 2016. Accessed October 16, 2020.
Fernandez, B. A. & Scherer, S. W. Syndromic autism spectrum disorders: moving from a clinically defined to a molecularly defined approach. Dialogues Clin. Neurosci. 19, 353–371 (2017).
Gaugler, T. et al. Most genetic risk for autism resides with common variation. Nat. Genet. 46, 881–885 (2014).
Gonatopoulos-Pournatzis T, Blencowe BJ. Microexons: at the nexus of nervous system development, behaviour and autism spectrum disorder. Current Opinion in Genetics & Development. 65,22-33 (2020).
Husson T. et al. Rare genetic susceptibility variants assessment in autism spectrum disorder: detection rate and practical use. Translational Psychiatry. 10, 77 (2020).
Sandin, S. et al. The familial risk of autism. JAMA 311,1770–1777 (2014).
Quesnel-Vallières, M., Weatheritt, R.J., Cordes, S.P. et al. Autism spectrum disorder: insights into convergent mechanisms from transcriptomics. Nat Rev Genet. 20, 51–63 (2019).
Amaral, David. G. “Advancing Research Through the Gift of Brain Donation.” Autism BrainNet, 2021 Simonsfoundation, 2021, www.autismbrainnet.org/.
Cite This Article:
Song Z., Lee K., Chowdhury F., Lombo L., Chharawala, V. The Genetic Profiling of the Autism Spectrum Disorder. Illustrated by F. Choudhary. Rare Disease Review. March 2021. DOI:10.13140/RG.2.2.34408.19205.
