Rare primary mitochondrial DNA mutations and probable synergistic variants in Leber's hereditary optic neuropathy

Abstract

Background: Leber's hereditary optic neuropathy (LHON) is a maternally inherited blinding disorder, which in over 90% of cases is due to one of three primary mitochondrial DNA (mtDNA) point mutations (m.11778G>A, m.3460G>A and m.14484T>C, respectively in MT-ND4, MT-ND1 and MT-ND6 genes). However, the spectrum of mtDNA mutations causing the remaining 10% of cases is only partially and often poorly defined.

Methodology/principal findings: In order to improve such a list of pathological variants, we completely sequenced the mitochondrial genomes of suspected LHON patients from Italy, France and Germany, lacking the three primary common mutations. Phylogenetic and conservation analyses were performed. Sixteen mitochondrial genomes were found to harbor at least one of the following nine rare LHON pathogenic mutations in genes MT-ND1 (m.3700G>A/p.A132T, m.3733G>A-C/p.E143K-Q, m.4171C>A/p.L289M), MT-ND4L (m.10663T>C/p.V65A) and MT-ND6 (m.14459G>A/p.A72V, m.14495A>G/p.M64I, m.14482C>A/p.L60S, and m.14568C>T/p.G36S). Phylogenetic analyses revealed that these substitutions were due to independent events on different haplogroups, whereas interspecies comparisons showed that they affected conserved amino acid residues or domains in the ND subunit genes of complex I.

Conclusions/significance: Our findings indicate that these nine substitutions are all primary LHON mutations. Therefore, despite their relative low frequency, they should be routinely tested for in all LHON patients lacking the three common mutations. Moreover, our sequence analysis confirms the major role of haplogroups J1c and J2b (over 35% in our probands versus 6% in the general population of Western Europe) and other putative synergistic mtDNA variants in LHON expression.

Figure 1. Phylogenetic tree of 16 complete mtDNA sequences from LHON patients.

Rare LHON mutations are shown in bold. The position of rCRS is indicated for reading off sequence motifs. Mutations are shown on the branches; they are transitions unless a base is explicitly indicated. The prefix @ designates reversions, while suffixes indicate: transversions (to A, G, C, or T), indels (+, d), gene locus (∼t, tRNA; ∼r, rRNA), synonymous or non-synonymous changes (s or ns) and heteroplasmies (h). Recurrent mutations are underlined. One mtDNA sequence (I; black circle) from a control subject (GenBank accession number FJ190383) was also included to illustrate that sequences 11 and 12 acquired independently the LHON mutation m.14568C>T. The haplogroup affiliation of each haplotype is based on mutational motifs and follows the most updated human phylogeny .

Figure 2. Sequence alignment and conservation analysis of ND1, ND4L and ND6 protein sequences.

Alignments of protein sequences from eukaryotes and mammals, including Homo sapiens, are reported. The upper lines represent the human sequence with the amino acid changes induced by the corresponding LHON mutation. Mutated positions are in bold and indicated by an asterisk. Different shading corresponds to increasing conservation levels: amino acid conservation between 70% and 90% are highlighted in light grey, amino acid conservation between 90% and 99% are highlighted in dark grey, and invariant positions (100% conservation) are highlighted in black. Alignment gaps are indicated by a hyphen (-).

Figure 3. RFLP evaluation of heteroplasmy.

Electrophoresis on Metaphor gel of the restriction fragments is shown. Wild type and mutant fragments are indicated with the corresponding size, expressed as base pairs (bps). Non affected negative control individual is indicated as C. DNA samples were extracted from whole blood, except when indicated: whole blood is indicated as B, urinary epithelium is indicated as U, platelet fraction is indicated as P, hairs are indicated as H and skeletal muscle is indicated as M. Molecular weight marker is indicated as MWM.