m⁶A-mRNA Reader YTHDF2 Identified as a Potential Risk Gene in Autism With Disproportionate Megalencephaly

Among autistic individuals, a subphenotype of disproportionate megalencephaly (ASD-DM) seen at three years of age is associated with co-occurring intellectual disability and poorer prognoses later in life. However, many of the genes contributing to ASD-DM have yet to be delineated. In this study, we identified additional ASD-DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD-DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection, totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD-DM genes harboring de novo protein-impacting variants. Our findings include 14 high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM, including CHD8 and PTEN. By performing functional network analysis, we expanded to additional candidate genes, including one previously implicated in ASD-DM (PIK3CA) as well as 184 additional genes connected with ASD or DM alone. Using zebrafish, we modeled a de novo tandem duplication impacting YTHDF2, encoding an N6-methyladenosine (m⁶A)-mRNA reader, in an ASD-DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size, while overexpressing YTHDF2 resulted in increased head/brain size matching that of the proband. Single-cell transcriptomes of YTHDF2 gain-of-function larvae point to reduced expression of Fragile-X-syndrome-associated FMRP-target genes globally and in the developing brain, providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m⁶A reader, YTHDC1, in our ASD-DM cohort. Though we highlight only two cases to date, our study provides support for the m⁶A-RNA modification pathway as potentially contributing to this severe form of autism.