Mitochondrial DNA sequence variation is largely conserved at birth with rare de novo mutations in neonates

Abstract

Objective: Mitochondrial DNA (mtDNA) encodes the proteins of the electron transfer chain to produce adenosine triphosphate through oxidative phosphorylation, and is essential to sustain life. mtDNA is unique from the nuclear genome in so much as it is solely maternally inherited (non-mendelian patterning), and shows a relatively high rate of mutation due to the absence of error checking capacity. While it is generally assumed that most new mutations accumulate through the process of heteroplasmy, it is unknown whether mutations initiated in the mother are inherited, occur in utero, or occur and accumulate early in life. The purpose of this study is to examine the maternally heritable and de novo mutation rate in the fetal mtDNA through high-fidelity sequencing from a large population-based cohort.

Study design: Samples were obtained from 90 matched maternal (blood) and fetal (placental) pairs. In addition, a smaller cohort (n = 5) of maternal (blood), fetal (placental), and neonatal (cord blood) trios were subjected to DNA extraction and shotgun sequencing. The whole genome was sequenced on the Illumina HiSeq platform (Illumina Inc., San Diego, CA), and haplogroups and mtDNA variants were identified through mapping to reference mitochondrial genomes (NC_012920).

Results: We observed 665 single nucleotide polymorphisms and 82 insertions-deletions variants identified in the cohort at large. We achieved high sequencing depth of the mtDNA to an average depth of 65X (range, 20-171X) coverage. The proportions of haplogroups identified in the cohort are consistent with the patient's self-identified ethnicity (>90% Hispanic), and all maternal-fetal pairs mapped to the identical haplogroup. Only variants from samples with average depth >20X and allele frequency >1% were included for further analysis. While the majority of the maternal-fetal pairs (>90%) demonstrated identical variants at the single nucleotide level, we observed rare mitochondrial single nucleotide polymorphism discordance between maternal and fetal mitochondrial genomes.

Conclusion: In this first in-depth sequencing analysis of mtDNA from maternal-fetal pairs at the time of birth, a low rate of de novo mutations appears in the fetal mitochondrial genome. This implies that these mutations likely arise from the maternal heteroplasmic pool (eg, in the oocyte), and accumulate later in the offspring's life. These findings have key implications for both the occurrence and screening for mitochondrial disorders.

Keywords: maternal mitochondrial transmission; mitochondrial DNA mutations; mitochondrial DNA variation; mitochondrial DNA heritability; mitochondrial heteroplasmy.

Figure 1. Number of SNPs discovered in samples is independent of sequencing depth

The X axis shows the sequencing depth from low to high (min 20, max 171). The Y axis depicts the number of SNPs discovered per subject sample.

Figure 2. Cladogram projection depicting the relationship and proportion of subjects for each haplogroup

No variation in the mtDNA haplogroup was observed to be discordant or variant between any maternal-fetal pair tested. As anticipated by our subjects self-identified race and ethnicity, the majority of subjects are in haplogroup A, B, C, D which are the dominate haplogroups in the Hispanic population.

Figure 3. Comparisons among five sets of trio subject samples

The outer most circle represents the 16.569 Kb circular mitochondrial reference genome. Trio subject samples include specimens from paired cord blood (red), maternal blood (yellow) and fetal placental tissue (green). mtSNPs were identified for each sample and are depicted as non-variant or variant from the reference mitochondrial genome. Each set of trio subject samples clustered together, and are thus shown in parallel.

Figure 4. Detailed analysis of maternal-fetal mitochondrial genomes

The outer most circle represents 16.569 Kb circular mitochondrial reference genome, with inner circles representing each maternal-fetal pair in parallel sequence. The black dots depicts identical mtSNPs as detected in both subjects comprising the maternal-fetal pair. The red dots show mtSNPs which were observed in the mom but not the fetus, and the blue dots represent mtSNPs that only appear in the fetus (and conversely not the mom). The color of the inner circle is based on the haplogroup assignment as previously depicted in Figure 2, and enables visualization of non-haplogroup defining mtSNPs.