[Molecular diagnosis of genetic polymorphisms related to autism spectrum disorder in children based on multi-PCR...

Establish a multiplex PCR targeted sequencing technology to detect the single nucleotide polymorphism (SNP) sites of disease-related genes in children with autism spectrum disorder, and explore its application value as a diagnostic biomarker for autism spectrum disorder. Through a literature review, 357 candidate risk genes and their 603 mutation sites highly associated with the occurrence of autism spectrum disorder (ASD) were selected. The MFEprimer software was used to design a multiplex PCR primer library. Using this primer library and targeted gene sequencing technology, SNP polymorphisms in ASD-related risk genes were screened in 105 children with ASD and 71 healthy controls from the outpatient department of Rehabilitation Medicine in Huangshi Maternity and Children's Health Hospital from January to August 2024. The selected polymorphic SNP sites were then validated by Sanger sequencing, in order to assess the clinical application value of the multiplex PCR-based targeted sequencing technology established in this study. The results showed that analysis of the multiplex PCR-based targeted sequencing results showed that, among the 105 children with ASD, 42 individuals carried the RELN (rs12666897) mutation site, with a mutation frequency of 40.00%; 22 individuals carried the AUTS2 (rs3735260) mutation site, with a mutation frequency of 20.95%. In contrast, among the 71 healthy controls, the mutation frequencies of RELN (rs12666897) and AUTS2 (rs3735260) were 18.31%(13 cases) and 5.63%(4 cases), respectively. The differences in mutation frequencies of these two SNPs between the ASD patients and healthy controls were statistically significant (respectively 0.002 7, 0.004 7, P<0.01). Sanger sequencing validation of the genotypes at these two sites showed complete concordance with the multiplex PCR-based targeted sequencing results, suggesting that these two SNPs may be associated with the risk of ASD onset. In conclusion, the multiplex PCR targeted high-throughput sequencing technology that established can screen for differentially expressed genes in children with ASD. Candidate gene polymorphism sites are closely associated with ASD in children, and have the potential to become new diagnostic biomarkers for ASD identification.