Chromosome X-wide common variant association study 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 symptoms similar to those in males, yet the mechanisms remain unclear. Despite technological advances in genomics, the complexity of the biological nature of sex chromosomes leaves 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 Collection (SSC), and Simons 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 Xp22.2 (lead SNP rs12687599, p = 3.57 × 10^-7) harboring ASB9/ASB11 and another encompassing DDX53 and the PTCHD1-AS long non-coding RNA (lead SNP rs5926125, p = 9.47 × 10^-6). When mapping genes within 10 kb 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, and SH3KBP1). FGF13 emerged as an X-linked ASD candidate gene, highlighted by sex-specific differences in minor allele frequencies. These results reveal significant insights into X chromosome biology in ASD, confirming and nominating genes and pathways for further investigation.

Keywords: ASB9/ASB11; DMD; PTCHD1-AS/DDX53; X chromosome; XWAS; autism; common-rare variant association.

Graphical abstract

Figure 1

XWAS workflow (A) Outline of the XWAS pipeline detailing data sources including MSSNG (Autism Speaks), SSC (Simons Simplex Collection), SPARK (Simons Powering Autism Research), 1KGP (1000 Genomes Project), HostSeq (the CanCOGeN 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) Consistency analysis and robustness studies conducted.

Figure 2

ASD-XWAS Manhattan and qqPlots Manhattan plots are shown on the left and qqPlots on the right. The graphs resulted 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 chromosome variants originating from WGS data (46 variants in PARs) 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. The green horizontal line in the Manhattan plots indicates the threshold for significance. The y axis displays the −log10 of the p value for each tested variant, derived from logistic regression analysis.

Figure 3

Annotation details for the genomic risk loci 1_Male-XWAS and 1_Both XWAS (A) Details for the genomic risk locus 1_Male-XWAS. (i) The LocusZoom plot for the correspondent region (chromosome X 15–15.5 Mb) with the lead SNP rs12687599 highlighted in purple (genome build hg38). The significance threshold was defined by an X-chromosome-specific Bonferroni correction. The y axis displays the −log10 of the p value for each tested variant, derived from logistic regression analysis. The LD reference panel used was Europeans from the 1000G data for both sexes together. Following the LocusZoom plot, in (ii), we provide annotation results displaying CADD and RegulomeDB scores (both in hg37 given the genome build availability on FUMA at the time). In (iii), 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 (hg38) of the genomic locus 1_Both-XWAS, followed by the CADD and Regulome profiles (hg37) of the same region.

Figure 4

sdMAF results capture signal at FGF13 (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 FGF13. The LD reference panel used was Europeans from the 1000G data for both sexes together.

Figure 5

Rare variant frequency analysis The frequencies of rare variants are compared among different groups: ASD probands (red bars), ASD-unaffected siblings (purple bars), and ASD parents (gray bars). The left shows the frequency of rare predicted damaging SNVs (<0.1% frequency in the general population) across 11 genes (ASB9, ASB11, TXLNG, PDHA1, DMD, HDAC8, PCDH11X, PCDH19, HTR2C, ENOX2, and FGF13) detected through XWAS common variant data analysis (Table 2). The right illustrates the frequency of rare CNV deletions overlapping exons (<1% frequency in general population) found in four XWAS genes (PTCHD1-AS, DMD, ENOX2, and FGF13). In each graph, the conditional logistic regression coefficients are next to each bar in the panel plot, and significant p values (p < 0.05) are highlighted with a star at the bottom of the plots. This analysis was conducted separately for males, females, and both sexes combined (using “sex” as covariate).

Figure 6

Gene expression by brain regions at different development times Brain map showing the gene expression levels in different parts of the brain at 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, and TXLNG), followed by three genes from the male-XWAS (ASB11, ASB9, and PCDH19), three genes from the female-XWAS (TXLNG, HTR2C, and 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).