Background In humans, vitamin B-12 (cobalamin) transport involves 3 paralogous proteins: transcobalamin, haptocorrin, and intrinsic factor. Zebrafish (Danio rerio) express 3 genes that encode proteins homologous to known B-12 carrier proteins: tcn2 (a transcobalamin ortholog) and 2 atypical β-domain-only homologs, tcnba and tcnbb. Objectives Given the orthologous relation between zebrafish Tcn2 and human transcobalamin, we hypothesized that zebrafish carrying null mutations of tcn2 would exhibit phenotypes consistent with vitamin B-12 deficiency. Methods First-generation and second-generation tcn2–/– zebrafish were characterized using phenotypic assessments, metabolic analyses, viability studies, and transcriptomics. Results Homozygous tcn2–/– fish produced from a heterozygous cross are viable and fertile but exhibit reduced growth, which persists into adulthood. When first-generation female tcn2–/– fish are bred, their offspring exhibit gross developmental and metabolic defects. These phenotypes are observed in all offspring from a tcn2–/– female regardless of the genotype of the male mating partner, suggesting a maternal effect, and can be rescued with vitamin B-12 supplementation. Transcriptome analyses indicate that offspring from a tcn2–/– female exhibit expression profiles distinct from those of offspring from a tcn2+/+ female, which demonstrate dysregulation of visual perception, fatty acid metabolism, and neurotransmitter signaling pathways. Conclusions Our findings suggest that the deposition of vitamin B-12 in the yolk by tcn2–/– females may be insufficient to support the early development of their offspring. These data present a compelling model to study the effects of vitamin B-12 deficiency on early development, with a particular emphasis on transgenerational effects and gene–environment interactions.

The analysis of somatic variation in the mitochondrial genome requires deep sequencing of mitochondrial DNA. This is ordinarily achieved by selective enrichment methods, such as PCR amplification or probe hybridization. These methods can introduce bias and are prone to contamination by nuclear-mitochondrial sequences (NUMTs), elements that can introduce artefacts into heteroplasmy analysis. We isolated intact mitochondria using differential centrifugation and alkaline lysis and subjected purified mitochondrial DNA to a sequence-independent and PCR-free method to obtain ultra-deep (>80,000X) sequencing coverage of the mitochondrial genome. This methodology avoids false-heteroplasmy calls that occur when long-range PCR amplification is used for mitochondrial DNA enrichment. Previously published methods employing mitochondrial DNA purification did not measure mitochondrial DNA enrichment or utilise high coverage short-read sequencing. Here, we describe a protocol that yields mitochondrial DNA and have quantified the increased level of mitochondrial DNA post-enrichment in 7 different mouse tissues. This method will enable researchers to identify changes in low frequency heteroplasmy without introducing PCR biases or NUMT contamination that are incorrectly identified as heteroplasmy when long-range PCR is used.

One-carbon metabolism is a complex network of metabolic reactions that are essential for cellular function including DNA synthesis. Vitamin B12 and folate are micronutrients that are utilized in this pathway and their deficiency can result in the perturbation of one-carbon metabolism and subsequent perturbations in DNA replication and repair. This effect has been well characterized in nuclear DNA but to date, mitochondrial DNA (mtDNA) has not been investigated extensively. Mitochondrial variants have been associated with several inherited and age-related disease states; therefore, the study of factors that impact heteroplasmy are important for advancing our understanding of the mitochondrial genome's impact on human health. Heteroplasmy studies require robust and efficient mitochondrial DNA enrichment to carry out in-depth mtDNA sequencing. Many of the current methods for mtDNA enrichment can introduce biases and false positive results. Here we use a method that overcomes these limitations and have applied it to assess mitochondrial heteroplasmy in mouse models of altered one-carbon metabolism. Vitamin B12 deficiency was found to cause increased levels of mitochondrial DNA heteroplasmy across all tissues that were investigated. Folic acid supplementation also contributed to elevated mitochondrial DNA heteroplasmy across all mouse tissues investigated. Heteroplasmy analysis of human data from the Framingham Heart Study suggested a potential sex-specific effect of folate and vitamin B12 status on mitochondrial heteroplasmy. This is a novel relationship that may have broader consequences for our understanding of one-carbon metabolism, mitochondrial related disease and the influence of nutrients on DNA mutation rates.