Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees

Abstract

The mitochondrial DNA (mtDNA) of 87 index cases with Leber hereditary optic neuropathy (LHON) sequentially diagnosed in Italy, including an extremely large Brazilian family of Italian maternal ancestry, was evaluated in detail. Only seven pairs and three triplets of identical haplotypes were observed, attesting that the large majority of the LHON mutations were due to independent mutational events. Assignment of the mutational events into haplogroups confirmed that J1 and J2 play a role in LHON expression but narrowed the association to the subclades J1c and J2b, thus suggesting that two specific combinations of amino acid changes in the cytochrome b are the cause of the mtDNA background effect and that this may occur at the level of the supercomplex formed by respiratory-chain complexes I and III. The families with identical haplotypes were genealogically reinvestigated, which led to the reconnection into extended pedigrees of three pairs of families, including the Brazilian family with its Italian counterpart. The sequencing of entire mtDNA samples from the reconnected families confirmed the genealogical reconstruction but showed that the Brazilian family was heteroplasmic at two control-region positions. The survey of the two sites in 12 of the Brazilian subjects revealed triplasmy in most cases, but there was no evidence of the tetraplasmy that would be expected in the case of mtDNA recombination.

Figure 1

Two of the extended pedigrees, after the reconnection of apparently unrelated familial groups. A, The relationship between the BL04 (Carelli et al. ; Lodi et al. 2002) and LB68 pedigrees. These pedigrees are from the same Italian region (Emilia). Both probands, indicated by the dotted arrows, suffered typical LHON, with severe visual loss in their twenties; their mtDNAs were homoplasmic for the 3460/ND1 mutation on an identical H haplotype (table 1). An excess of affected females (4:2) is apparent, and all available affected subjects (4 of 6) harbored only homoplasmic mutant mtDNA. In contrast, the survey of available unaffected maternally related individuals showed that the 3460 mutation was heteroplasmic in multiple subjects from both families. Arrows indicate the subjects available for DNA testing of the 3460 mutation; an asterisk (*) indicates heteroplasmy, whereas the absence of the asterisk indicates homoplasmy. The results of the DNA testing suggest that the risk of developing LHON increases consistently only when the mutant mtDNA shifts to homoplasmy and partially explains the unusual prevalence of affected females in the BL04/LB68 extended family. B, The reconstructed 6-generation pedigree generated from the families LB105 (Vergani et al. ; Carelli et al. ; Lodi et al. 2000) and BL02, both also originating from the same geographical area (Abruzzo). All available subjects along the maternal line in both families were homoplasmic for the 11778/ND4 mutation. The male:female ratio (9:3) in the extended family matches the reported values for the 11778/ND4 mutation (Carelli et al. 2004).

Figure 2

The reconstructed pedigree generated from families BSL07 and LB10. The BSL07 family originates from the state of Espirito Santo in Brazil and is a nuclear subset of the large Brazilian SOA-BR pedigree, which has been completely reconstructed and investigated from a genetic, epidemiological, and clinical point of view (Sadun et al. , ; Salomao et al. 2004). Briefly, the SOA-BR family numbers ∼360 family members, 128 of whom are maternally related. Of the latter, 35 are affected (23 are alive, 2 of whom are recent cases and became affected in the past 2 years). All 122 individuals investigated are homoplasmic for the 11778/ND4 mutation. The woman—daughter of the most recent common female ancestor of the entire pedigree, who migrated to Brazil and gave rise to the Brazilian branch—was born in 1861 in the Veneto region of northern Italy, the same geographical area from which the LB10 family originates.

Figure 3

Tree encompassing the complete mtDNA sequences (GenBank) observed in the three pairs of reconnected LHON-affected families—BL04 and LB68 (haplogroup H12), LB105 and BL02 (haplogroup J1c), BSL07 and LB10 (haplogroup J1d)—together with those of mtDNAs that are representative of all other subclades of J. Sequences 2 (J2a) and 3 (J2b) are new and from Italian control individuals, whereas sequence 1 (J1b) corresponds to B30 (Palanichamy et al. 2004). The tree is rooted using the reference sequence rCRS (Andrews et al. 1999)—a member of haplogroup H2b—as an outgroup. Mutations are shown on the branches; they are transitions unless the base change is explicitly indicated. Insertions are suffixed with a plus sign (+) and the inserted nucleotide, and deletions have a “d” prefix. Heteroplasmic positions (boxed) in BSL07 are indicated by an “h” following the nucleotide position. In LB10, 16092C was homoplasmic and 16129A was absent. Underlining indicates recurrent mutations; “s” indicates synonymous mutations, whereas “ns” indicates nonsynonymous mutations. The arrows indicate the nonsynonymous J polymorphisms that might have a role in modulation of LHON expression. LHON mutations are shown in italics and were added to the tree after phylogeny construction.