Analysis of hybrid RNA/DNA profiles in blood from autistic patients reveals differences in mRNA and ncRNA regions

Autism spectrum disorder (ASD) is a set of genetically heterogenous neurodevelopmental disorders characterized by core symptoms including impaired social interaction, communication deficits, and restricted or stereotyped behaviors. While a significant number of cases are not explained by Mendelian inheritance, there is growing evidence in implication of non-coding RNAs (ncRNAs) in development and inheritance of ASD. Transcriptional studies face challenges from patient-specific variations in gene expression and technical differences in preserving RNA integrity. We propose that isolating RNA from DNA/RNA hybrids provides a robust method to capture transcriptional information. We performed a whole transcriptome analysis on blood samples from ASD patients and healthy controls to investigate transcripts associated with DNA/RNA hybrids. We identified 278,300 novel transcripts across 68,487 DNA/RNA hybrid loci, with significant enrichment in exonic and intronic regions. The novel long non-coding RNAs (lncRNAs) we found showed higher expression levels compared to known transcripts. Differential expression analysis revealed 301 significantly upregulated and 401 downregulated transcripts in ASD samples. Through qRT-PCR validation, we confirmed the significant upregulation of RN7SK and SMARCC2 associated with DNA/RNA hybrids in ASD patients. Pathway and enrichment analyses highlighted mitochondrial dysfunction and energy metabolism. Our results suggest that ncRNAs can form DNA/RNA hybrids that influence gene expression, providing preliminary insights into the mechanisms of transcriptional dysregulation in ASD. Overall design: DNA-associated RNA molecules were purified form 12 blood samples (6 autism patients, 6 Controls). 10-100 ng of RNA after Dnase digestion were recovered and analyzed by high throughput sequencing on Illumina.