Chromosome X-Wide Common Variant Association Study (XWAS) in Autism Spectrum Disorder

Abstract

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 7.9×10^-6 to 1.51×10^-5), surpassing Bonferroni-corrected thresholds. Key findings include significant regions on chrXp22.2 (lead SNP=rs12687599, p=3.57×10^-7) harboring ASB9/ASB11, and another encompassing DDX53/PTCHD1-AS long non-coding RNA (lead SNP=rs5926125, p=9.47×10^-6). When mapping genes within 10kb of the 59 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, 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.

Figure 1:. XWAS workflow.

A) Outline of the XWAS pipeline detailing data sources including MSSNG (Autism Speaks), SSC (Simons Simplex Cohort), SPARK (Simons Foundation Powering Autism Research), 1KGP (1000 Genome Project), HostSeq (The Host Genome Sequencing Initiative), and MGRB (Medical Genome Reference Bank). The significance threshold was determined using Bonferroni correction, individually calculated for the Male-XWAS, Female-XWAS, and Both-XWAS approaches. For Meta-XWAS, we used the threshold inferred from the Both-XWAS result. B) Replication and robustness studies conducted.

Figure 2.. ASD-XWAS manhattan and qq plots.

Each panel shows a Manhattan plot on the left part and qqPlot on the right part. The graphs result from XWAS testing using 6,873 ASD individuals (5,639 males and 1,234 females) and 8,981 controls (3,911 males and 5,070 females) with a total of 418,652 X chromosomal variants originated from WGS data (46 variants in PAR regions) for (A) Male-XWAS, B) Female-XWAS, C) Both-XWAS, and D) the Meta-XWAS, a meta-analysis from the sex stratified approaches implemented on GWAMA.

Figure 3.. Annotation details for the genomic risk Locus 1_Male-XWAS and 1_Both XWAS.

A) Details for the genomic risk locus 1_Male-XWAS. The upper panel shows the LocusZoom plot for the correspondent region with the lead SNP rs12687599 highlighted in purple. The used LD reference panel was Europeans from 1000G data for both sexes together. Following the LocusZoom plot, on the left, we provide annotation results displaying CADD and RegulomeDB scores. On the right, the Manhattan plot illustrates the gene-based test computed by MAGMA in FUMA. The SNPs were mapped to 704 protein-coding genes, hence the genome-wide significance threshold (indicated by the red dashed line in the plot) was conservatively set at P = 0.05/704 = 7.10×10⁻⁵. B) LocusZoom plot of the genomic locus 1_Both-XWAS, followed by the CADD and Regulome profiles of the same region.

Figure 4.. sdMaf Results.

A) Left, the Manhattan plot illustrates the sdMAF p-values obtained from ASD datasets exclusively. Right, the Manhattan plot represents the sdMAF p-values obtained from control datasets only. B) The LocusZoom plot displays the region identified in the sdMAF-cases results, highlighting the gene FGF13. The LD reference panel used was Europeans from 1000G data for both sexes together.

Figure 5.. Rare Variant Frequency Analysis.

The figure compares the frequencies of rare variants among different groups: ASD-Probands (red bars), ASD-Unaffected Siblings (green bars), and ASD-Parents (gray bars). The left panel shows the frequency of rare predicted damaging SNVs (<0.1% frequency in general population) across 11 genes (ASB9, ASB11, TXLNG, PDHA1, PTCHD1-AS, DMD, HDAC8, PCDH11X, PCDH19, HTR2C, ENOX2, FGF13) detected through XWAS common variant data analysis (Table 2). The right panel illustrates the frequency of rare CNV deletions overlapping exons (< 1% frequency in general population), found in four XWAS-genes (PTCHD1-AS, DMD, ENOX2, FGF13). In each graph, the corresponding p-value from a conditional logistic regression is shown at the bottom, conducted separately for males, females, and both sexes combined (using “sex” as covariate).

Figure 6.. Gene Expression by Brain Regions in different development times.

Brain map showing the gene expression levels in different parts of the brain in five developmental stages (Early Fetal, Late fetal, Early childhood, Childhood/Teenage and Adulthood). Left to right shows the gene expression levels from all 12 ASD-candidate genes with available expression data (ASB11, ASB9, DMD, ENOX2, FGF13, HDAC8, HTR2C, PABPC1L2A, PCDH11X, PCDH19, PDHA1, TXLNG), followed by three genes from Male-XWAS (ASB11, ASB9, PCDH19), three genes from Female-XWAS (TXLNG, HTR2C, ENOX2) and the correspondent control comparison with all the ~800 X chromosome genes in both sexes and also in male brains only and female brains only. The color scales go from blue (downregulated) to red (upregulated).