Autism Spectrum Disorder (ASD) displays a notable male bias in prevalence. Research into rare (<0.1) genetic variants on the X chromosome has implicated over 20 genes in ASD pathogenesis, such as MECP2, DDX3X, and DMD. The "female protective effect" in ASD suggests that females may require a higher genetic burden to manifest similar symptoms as males, yet the mechanisms remain unclear. Despite technological advances in genomics, the complexity of the biological nature of sex chromosomes leave them underrepresented in genome-wide studies. Here, we conducted an X chromosome-wide association study (XWAS) using whole-genome sequencing data from 6,873 individuals with ASD (82% males) across Autism Speaks MSSNG, Simons Simplex Cohort SSC, and Simons Foundation Powering Autism Research SPARK, alongside 8,981 population controls (43% males). We analyzed 418,652 X-chromosome variants, identifying 59 associated with ASD (p-values 7.9x10-6 to 1.51x10-5), surpassing Bonferroni-corrected thresholds. Key findings include significant regions on chrXp22.2 (lead SNP=rs12687599, p=3.57x10-7) harboring ASB9/ASB11, and another encompassing DDX53/PTCHD1-AS long non-coding RNA (lead SNP=rs5926125, p=9.47x10-6). When mapping genes within 10kb of the most significantly associated SNPs, 91 genes were found, 17 of which yielded association with ASD (GRPR, AP1S2, DDX53, HDAC8, PCDH19, PTCHD1, PCDH11X, PTCHD1-AS, DMD, SYAP1, CNKSR2, GLRA2, 56 OFD1, CDKL5, GPRASP2, NXF5, SH3KBP1). FGF13 emerged as a novel X-linked ASD candidate gene, highlighted by sex-specific differences in minor allele frequencies. These results reveal significant new insights into X chromosome biology in ASD, confirming and nominating genes and pathways for further investigation.

At the time of this study and its publication, S.W.S. served on the Scientific Advisory Committee of Population Bio. Intellectual property from aspects of his research held at The Hospital for Sick Children are licensed to Athena Diagnostics and Population Bio. These relationships did not influence data interpretation or presentation during this study but are disclosed for potential future considerations.

This study was funded by CGEn HostSeq/CIHR fellowship (CGE 185054),SickKids Restracomp Fellowship, the Intramural Research Program of the NIH and the National Institute on Aging (NIA), Canadian Institutes of Health Research Sex and Gender Science Chair (GSB 171373) and the Northbridge Chair in Pediatric Research at The Hospital for Sick Children and the University of Toronto.

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The Research Ethics Board (Ethics committee) of The Hospital for Sick Children gave ethical approval for this work.

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