Genetic Control over mtDNA and Its Relationship to Major Depressive Disorder

Abstract

Control over the number of mtDNA molecules per cell appears to be tightly regulated, but the mechanisms involved are largely unknown. Reversible alterations in the amount of mtDNA occur in response to stress suggesting that control over the amount of mtDNA is involved in stress-related diseases including major depressive disorder (MDD). Using low-coverage sequence data from 10,442 Chinese women to compute the normalized numbers of reads mapping to the mitochondrial genome as a proxy for the amount of mtDNA, we identified two loci that contribute to mtDNA levels: one within the TFAM gene on chromosome 10 (rs11006126, p value = 8.73 × 10(-28), variance explained = 1.90%) and one over the CDK6 gene on chromosome 7 (rs445, p value = 6.03 × 10(-16), variance explained = 0.50%). Both loci replicated in an independent cohort. CDK6 is thus a new molecule involved in the control of mtDNA. We identify increased rates of heteroplasmy in women with MDD, and show from an experimental paradigm using mice that the increase is likely due to stress. Furthermore, at least one heteroplasmic variant is significantly associated with changes in the amount of mtDNA (position 513, p value = 3.27 × 10(-9), variance explained = 0.48%) suggesting site-specific heteroplasmy as a possible link between stress and increase in amount of mtDNA. These findings indicate the involvement of mitochondrial genome copy number and sequence in an organism's response to stress.

Figure 1

Two Loci Associated with mtDNA Manhattan plot of genome-wide association for amount of mtDNA (A). Detailed views of two loci associated with amount of mtDNA over the TFAM region on chromosome 10 at 60.1 Mb (B) and the CDK6 gene on chromosome 7 at position 92.4 Mb (C) are shown. The −log10 p values of imputed SNPs associated with amount of mtDNA are shown on the left y axis. The horizontal axis gives chromosomal position in megabases (Mb). Genes within the regions are shown in the bottom panels. Linkage disequilibrium of each SNP with top SNP, shown in large purple diamond, is indicated by its color. The plots were drawn using LocusZoom [9]. See also Figure S1.

Figure 2

Accessibility of mtDNA for Variant Calling Mean per site read depth across 10,442 samples for all sites on the mtDNA. Reads included in the per site read count have passed the following quality control criteria: (1) mapping quality >59, (2) both ends of paired-end read map uniquely to the mtDNA reference NC_012920.1, (3) they do not contain mismatches to the mtDNA reference with total length of 5 bp or above, and (4) base at site in question has base quality of higher than 20. Sites with more than 10% samples with read depth less than 10 would not be used for calling homoplasmic variants, and sites with more than 10% samples with read depth less than 50 would not be used for calling heteroplasmic variants. See also Experimental Procedures.

Figure 3

Heteroplasmy Counts in Stressed and Control Mice The figures shows (A) boxplot of the amount of mtDNA quantified from high-coverage sequencing of long-range PCR of mtDNA from liver samples (controlled for starting mass of genomic DNA extracted from mice livers then quantile normalized among all 12 mice) of six female mid-age C56BL/6J mice exposed to a 4-week chronic stress protocol (shown in red), compared to that from the liver samples of six female, mid-age C56BL/6J control mice (shown in blue). Normalized amount of mtDNA from liver samples of six mice was significantly higher than that in non-stressed control mice mean (fold increase = 2.16, p value = 0.0040), and (B) the number of heteroplasmic variant sites found in high-coverage sequencing of long-range PCR of mtDNA (down-sampled to equal coverage of 500 reads per site) from liver samples of six female mid-age C56BL/6J mice exposed to a 4-week chronic stress protocol, compared to that from the liver samples of six female, mid-age C56BL/6J control mice, where each point represents one mouse; stressed mice had significantly higher number of heteroplasmic variants in mtDNA per sample (mean fold increase = 1.46, p value = 0.029).