In phase 1, we had >80% power to detect a genotype relative risk (GRR) of 1.3 for any single SNP, with a type 1 error rate of 0.05 and an allele frequency of 0.10. The small number of individuals with African ancestry in our dataset significantly decreases the power for this subset (12% power for GRR of 1.3, α=0.05, MAF=0.10). Our stratified CMH test identified six nominally significant variants (p≤ 0.05) that do not survive Bonferroni correction (Table S1
). Using a continuous variable to control for stratification in a logistic regression framework, we identified six SNPs with nominal p-values (p ≤ 0.05) through our single marker tests of association (; ; Table S3
). These nominal associations do not survive either Bonferroni correction or our less conservative assessment via permutation testing using RVASSOC in the homogeneous Caucasian non-Hispanic subset of phase 1 (observed max χ2
=4.81, p=0.93). Of note, four of these six nominally significant variants overlap the stratified and logistic regression approaches; out of these four overlapping variants, two (mtDNA positions 9899 and 10589) tag subgroups of haplogroup L.
A manhattan plot of the phase 1 logistic regresstion results.
In phase 2, we examined both the major European haplogroups and European haplogroup defining SNPs for association to ASD susceptibility. Our haplogroup analysis yielded no significant difference in the frequency of cases versus controls for any particular haplogroup (). There are too few SNPs in this phase to control for population substructure using the principal components approach we used in phase 1. Instead, we used the haplogroups as covariates in a logistic regression analysis. We see no significant difference between our cases and controls for any of these single haplogroup defining SNPs (). Interestingly, individuals with self-reported African ancestry belong to a number of haplogroups other than haplogroup L.
Given that the samples in phase 3 are almost completely overlapping with those genotyped in phase 1 and 2, we chose not to perform single marker association tests of the 139 SNPs previously examined in those phases within this dataset. The advantage of this phase rests in capturing rare variants which cannot be powerfully tested with single marker tests of association. We confirmed the homogeneity of the phase 3 dataset which was based on self-report using the mtGIF calculated with χ2 test statistics from all 941 polymorphic positions. Subsequently, we performed a joint test of these variants, many of which have MAF ≤ 0.01 and are spread across the mitochondrial genome; this returned no significant results (observed max χ2 =6.34, p=0.70).
Furthermore, we examined whether we detected more rare variation in total among cases when compared to controls specifically in the coding regions of the 13 protein subunits of the mitochondrial electron transport chain. We found no significant difference between our cases and controls for either the number of synonymous or non-synonymous changes in these genes ().
We specifically analyzed our phase 3 dataset to determine if any of our samples contained any of these rare variations as reported and organized by the MitoMap project (http://www.mitomap.org
). We identified 25 variants which were not previously examined in phases 1 or 2 that were in the MitoMap tables of ‘mtDNA Mutations with Reports of Disease-Associations’ from http://www.mitomap.org
(). These tables contain any mtDNA variant that is reported in the literature of disease associations, but not necessarily replicated in subsequent investigations. A single variant (A13637G), previously associated to Lieber’s Hereditary Optic Neuropathy (LHON), demonstrated a nominally significant p-value before multiple testing correction (p=0.02) using the Fisher’s Exact test. This rare allele was found in eight ASD cases and a single control.
Finally, we examined our phase 3 dataset for evidence of heteroplasmy and mutational burden differences between cases and controls. We examined the mutational burden in a specific subset of the variants detected, however failed to find any significant difference between our ASD case and control datasets (). No significant difference was found between cases and controls in the heteroplasmy analysis using individual raw intensity allele data (data not shown).