**3. Conclusions**

This review has discussed 20 of the 30 supernumerary subunits of human complex I. Missense and various other point mutations have been described for 17 of the subunits. One subunit, NDUFV3, has several known amino acids substitutions, but there is no evidence that they are causative for disease. All of the mutations, the diseases they are associated with, and their effects on assembly of complex I are summarized in Table 2. The gene of one subunit NDUFA3 was completely deleted, along with several neighboring genes. This provided evidence for haplosufficiency, since no mitochondrial disease characteristics occurred in those individuals. The gene for another subunit NDUFB6 had a mutation in the promoter, resulting in a change in the expression level.


**Table 2.** Summary of mutations with associated diseases and effects on assembly.


**Table 2.** *Cont.*


**Table 2.** *Cont.*

<sup>1</sup> Assembly: ••• normal or near normal; •• intermediate level; • little or no assembly. NT: not tested. When 2 mutations are listed, they are two alleles of the same gene, or if indicated, one is a second gene. See the text for more details. <sup>2</sup> p.Asn76Metfs\*4 is a frameshift mutation at codon 76 that converts Asn (AAT) to Met (ATG) with a stop codon appearing 4 codons downstream. p.Tyr97\* is a nonsense mutation at Tyr76 to TAG.

Among the 17 subunits that were found to have missense or other point mutations, nearly all were homozygous or compound heterozygous and therefore lacked a normal allele. This supports the likely general trend of haplosufficiency. One exception might be in NDUFB11, which is found on the X chromosome. We suggest that mosaicism in female patients due to X-inactivation might explain variable outcomes from heterozygous null mutations. To resolve such questions, it is important to extensively sequence the genome to identify other possible mutations related to the disease state. Another discrepancy was found to occur in NDUFB3, in which one girl with one homozygous mutation died in infancy [78]; meanwhile, several boys with the same mutation, in the hemizygous form, survived into adolescence and adulthood [80]. Deep sequencing might be the answer, again to try to identify other mutations.

Among the missense mutations, many are found at subunit interfaces. Such mutations are predicted to negatively impact assembly, at least local assembly. Others are found at interior sites of the proteins, and it can be predicted that protein folding will be disrupted and lead to a nonfunctional state, an effect similar to that of a gene knockout. The results of the knockouts of each of the supernumerary subunits in cell culture [11] have demonstrated that most are essential for complex I assembly. The same general trend can be seen with missense mutations in the supernumerary subunits. Essential subunits might include those that serve to stabilize the structure of complex I or that might act as chaperones in the assembly process. In recent years, analyses of complex I assembly have increased, but there have not been a great number of mutants analyzed at sufficient depth. The network of interactions among

supernumerary subunits, as well as with core subunits, suggests that the instability of complex I might reflect a cooperative assembly and binding of the subunits. The loss of one supernumerary subunit could impact multiple other subunits such that assembly is not completed. Even if complex I is able to assemble, its stability might be marginal, eventually leading to a loss of function.

In conclusion, the increasingly high-resolution structures of complex I from human and other species have helped to interpret the possible roles of the supernumerary subunits and the loss of function due to mutation. Unbiased DNA sequencing will be necessary to find all of the mutations that might exist in various disease states, as illustrated by the mutations discovered in NDUFB11 to be associated with congenital sideroblastic anemia [77]. In this way, other rare diseases might become better understood and treated.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2075-1729/10/11/296/s1, Table S1: Interactions of Human Complex I Subunits, Video S1: Structure of Human Complex I.

**Author Contributions:** Conceptualization, S.B.V.; Investigation, Q.-C.L.D., D.H.P., A.N.J., M.P., H.A.A., F.Z. and S.B.V.; Writing—Original Draft Preparation, Q.-C.L.D., D.H.P. and S.B.V.; Writing—Review & Editing, Q.-C.L.D., D.H.P., A.N.J., M.P., H.A.A., F.Z. and S.B.V.; Supervision, S.B.V.; Funding Acquisition, S.B.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Institutes of Health, USA, grant number 1R15GM126507, and the American Heart Association, grant number 17AIREA33661165.

**Acknowledgments:** Q.-C.L.D., D.H.P., A.N.J. and M.P. acknowledge support of the Engaged Learning and Hamilton Scholars programs at Southern Methodist University.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
