Figure 3. Homoplasmy of the m.15992A>T MT-TP mutation.

(A) Strategy of the mispairing PCR for restriction analysis of the m.15992A>T mutation proportion. Italic characters indicate the primers used to amplify the 200-bp-long mtDNA fragment from position 15820 to 16019; DdeI restriction site is highlighted in cyan blue; the nucleotide position 15992 in underlined bold blue character; the mispairing position in the backward primer is in underlined bold red. (B) Restriction fragments of mtDNA fragments cut with DdeI. P1, P2, P3, P4, P5, and P6 = patients 1, 2, 3, 4, 5, and 6 respectively; C = wild-type controls; M = DNA from muscle, L = DNA from blood leukocytes DNA, U = DNA from urinary sediment; B = DNA from buccal cells; F = DNA from cultured skin fibroblasts. The need for mispairing is shown in the upper panel with the control sample (C) amplified with and without mispairing and then cut with DdeI. Without mispairing, the WT restriction fragment run only slightly above the mutated fragments, in agreement with their length being only 5 bp longer than the mutant fragments. With mispairing abolishing the DdeI site at position 15,996, all samples amplified from patients' tissues run homogeneously at a longer distance than wild-type control mtDNA fragment in agreement with their 28 bp shorter length. The upper panel also shows that the mutation appeared homoplasmic in every tissue tested in several members of family 1. The rest of the panels show that the mutation appeared homoplasmic in every member analyzed in the 2 families where DNA from blood leukocytes was available (figure 1). It also appeared homoplasmic in cultured fibroblasts derived from a skin biopsy of patients 1, 2, 3, and 4 and in the cybrid clones obtained from either patient 2 or patient 4 fibroblasts. mtDNA = mitochondrial DNA; WT = wild type.