The underlying molecular diagnosis of patients with mitochondrial diseases can be due to nuclear or mitochondrial DNA (mtDNA) pathogenic variants. In humans, mtDNA is generally inherited from the mother. Thus, the cascade diagnostic investigations, genetic counseling, and reproductive choices for patients with pathogenic mtDNA variants are currently based on that premise.1

In a recent study, Luo et al.2 reported evidence of paternal transmission of mtDNA to offspring in three families. Some of the issues emerging from these findings relate specifically to mitochondrial disease diagnosis and genetic counseling and could prompt concerns about current practice.

The possibility of paternal inheritance of mtDNA has been reported previously in mice, including across multiple generations.3,4 Tissue-specific inheritance of paternal mtDNA in humans has also been reported in a single individual with mitochondrial myopathy whose skeletal muscle had a de novo homoplasmic 2-bp deletion in the MT-ND2 gene on the paternal mtDNA haplotype, while only maternal mtDNA was detected in other tissues tested.5 The recent finding by Luo et al.2 prompts reconsideration of whether paternal inheritance may be more common in humans than previously considered. To our knowledge, there have not been any other systematic investigations in human trios.


All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients included in the study. This project was approved by the Human Research Ethics Committee of the Sydney Children’s Hospitals Network (ID number 10/CHW/114).

We studied genome sequencing (GS) data of DNA extracted from blood samples of 41 pediatric patients recruited in Australia with suspected mitochondrial disease and their parents, as previously reported.6 Thirty-seven of the patients had trio GS, while in four patients DNA from the father was not available.

Variants were filtered using Seave7 and reported relative to the Revised Cambridge Reference Sequence NC_012920.1. Variants with heteroplasmy levels >5% were included in the analysis and if discordant with the mother, lower levels of heteroplasmy were further investigated using the bioinformatic pipeline mity (Puttick et al., unpublished). Variants in positions where common sequencing errors occur due to a homopolymer stretch (MT:302–315 and MT:3105–3109) were excluded.


Using GS we observed a median of medians coverage of 4586× (range 1179–22,965×) in the mitochondrial genome. We identified a median of 31 mtDNA variants per proband (Table 1). In all studied families, homoplasmic variants detected in the mother were also found in the offspring.

Table 1 Mitochondrial DNA (mtDNA) variant segregation

Only five probands had any heteroplasmic variants. In two families, heteroplasmic variants detected in the mother presented lower heteroplasmy levels in the probands (families 8 and 30); in one family (family 15) the m.16150C>T heteroplasmic variant (mutant load 25%) detected in the mother was not present in the proband. These different heteroplasmy levels can be explained by the mitochondrial bottleneck during oogenesis.

In one family (family 32), two de novo heteroplasmic variants (m.15617G>A; mutant load 12% and m.153A>G; mutant load 96%) were detected in the proband that were not detected in the mother or father. In family 3, a de novo heteroplasmic variant (m.7925G>A; mutant load 70%) was present in the proband that was not identified in the mother’s blood; no DNA was available from the father, however, all 37 variants present in the mother were also identified in the proband and they both shared the same haplotype (T2a1b1a).

We did not find any homoplasmic or heteroplasmic variants in the cohort that segregated only with the father. In addition, none of these families presented with high numbers of heteroplasmic variants or other suggestive patterns of biparental inheritance of their mtDNA. However, because only blood samples were analyzed, tissue-specific heteroplasmy cannot be ruled out.


Previous studies reporting paternal mtDNA transmission have been controversial, with most cases later regarded as sample cross-contamination.8 The recent study by Luo et al.2 has reactivated the concern about the possibility of paternal mtDNA transmission; they described three multigeneration families with a large number of mtDNA variants with high levels of heteroplasmy, corresponding to mixed haplogroups in a pattern apparently corresponding to autosomal dominant inheritance. They attempted to exclude sample contamination by conducting the mtDNA sequencing in different laboratories, however, their results have also been challenged. Lutz-Bonengel9 and Vissing10 speculated that the apparent biparental mtDNA inheritance pattern in the families could be attributed to amplification of nuclear mtDNA segments (NuMTs).9,10 Luo et al.11 contested that claim by arguing that it is unlikely that the NuMT would amplify with the different sequencing methods used. In addition, they argued that there would be a low probability of all offspring studied inheriting the NuMT variants. Nonetheless, they plan to conduct further single-cell sequencing studies with additional families to rule out that possibility. Even if paternal mtDNA transmission is confirmed, the mechanism of this event is still not clear, as well as the potential consequences of this phenomenon, given that no actual causal relationship with mitochondrial disease was established in the reported families.

Our results further support the prevailing view that paternal inheritance of mtDNA appears to be a rare event in humans, and suggest that the scenario described by Luo et al.2 is uncommon. We suggest that genetic counseling and genetic investigations for families with suspected mitochondrial disease do not need to change. Nonetheless, it may be worth considering sequencing paternal mtDNA where a patient has an apparent de novo mtDNA variant.

We believe that this is the largest cohort of patients with suspected mitochondrial disease undergoing trio GS reported to date. As access to GS technology and genomic data sharing become more widespread in clinical practice,12 the analysis of nuclear and mtDNA sequencing data in larger cohorts could aid in better understanding the frequency and consequences of biparental mtDNA inheritance events, and could lead to the identification of variants in nuclear genes involved in paternal mtDNA elimination that may be contributing to this mechanism.2,13