Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication - PubMed (original) (raw)
Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication
Y Wang et al. Proc Natl Acad Sci U S A. 2001.
Abstract
The recently discovered aging-dependent large accumulation of point mutations in the human fibroblast mtDNA control region raised the question of their occurrence in postmitotic tissues. In the present work, analysis of biopsied or autopsied human skeletal muscle revealed the absence or only minimal presence of those mutations. By contrast, surprisingly, most of 26 individuals 53 to 92 years old, without a known history of neuromuscular disease, exhibited at mtDNA replication control sites in muscle an accumulation of two new point mutations, i.e., A189G and T408A, which were absent or marginally present in 19 individuals younger than 34 years. These two mutations were not found in fibroblasts from 22 subjects 64 to 101 years of age (T408A), or were present only in three subjects in very low amounts (A189G). Furthermore, in several older individuals exhibiting an accumulation in muscle of one or both of these mutations, they were nearly absent in other tissues, whereas the most frequent fibroblast-specific mutation (T414G) was present in skin, but not in muscle. Among eight additional individuals exhibiting partial denervation of their biopsied muscle, four subjects >80 years old had accumulated the two muscle-specific point mutations, which were, conversely, present at only very low levels in four subjects < or =40 years old. The striking tissue specificity of the muscle mtDNA mutations detected here and their mapping at critical sites for mtDNA replication strongly point to the involvement of a specific mutagenic machinery and to the functional relevance of these mutations.
Figures
Figure 1
(A) Shown are the portion of the main control region of human mtDNA containing the initiation sites for rRNA-encoding DNA (rDNA; H1) and whole H-strand transcription (H2) and for L-strand transcription and synthesis of the primer of H-strand synthesis (L; ref. 6) and the primary origin for H-strand DNA synthesis (OH1; ref. 3), as well as the map positions of the CSB1, CSB2, and CSB3 (conserved sequence blocks 1, 2, and 3; ref. 7), of the DLP4 and DLP6 segments chosen for DGGE analysis in this work, of the overlapping DLP3, DLP7 (1), and TRNA1 segments (8), and of the large fragment, encompassing all of the above segments (Init-Tra-Rep), used for cloning. (B) Scheme of the approaches followed to carry out a preliminary screening of the mtDNA samples for the presence of mutations in the DLP4 and/or DLP6 segment by first-round DGGE; then, to identify and quantify the mutations by cloning of the whole Init-Tra-Rep fragment; followed by second-round DGGE and sequencing (1); and, finally, to carry out a large-scale screening of mtDNA samples for the identified mutations by allele-specific termination of primer extension (1). Phe, tRNAPhe gene.
Figure 2
Autoradiograms showing the electrophoretic patterns obtained in the analysis of different mutations by allele-specific termination of primer extension. (A) Detection of the A189G mutation in DLP4 segments PCR-amplified from muscle mtDNA of individuals without any history of neuromuscular disease (group A, see Materials and Methods), comparing 19 individuals aged 19 to 34 years and 23 individuals aged 53 to 92 years old. (B) Detection of the T408A mutation in DLP6 segments amplified from muscle mtDNA of the individuals indicated in A. A section of the gel has been cut out for space considerations. The extended primer, which terminated at the site of the mutation, corresponds to the lower band of each doublet (indicated by M arrow), whereas the upper band is a spurious product visible also in the lane for the wild-type DLP6 (W). (C) Detection of the T414G mutation in DLP6 fragments amplified from muscle mtDNA of the individuals indicated in_A_. (D) Detection of the A189G mutation in DLP4 segments amplified from fibroblast mtDNA of 32 individuals from 20-week fetal (FW) to 101 years old. In A and_D_, some samples exhibited consistently in repeated runs a relatively minor band migrating slower than the extended primer terminated on the wild-type template (W), which, most likely, resulted from a conformational change in the template associated with a polymorphism(s) in DLP4; this hypothesis is supported by the sequencing data for individuals 22 y-1 and 25 y-2 (data not shown), for 79 y (Table 1), and for 48 y (1), and by the presence of the same band in multiple tissues from 79 y and 90 y (see below, Fig. 3). In A, C, and D, the weak abnormal bands migrating faster than the mutant or wild-type band, but which were present also in the PE lane and/or W lane (see below), were due to spurious products. In the lanes for 19 y, 21 y, 60 y-2, 75 y, 77 y-1, 77 y-2, and 78 y of A and in the lanes for FW and 75 y-1 of D, a band [migrating approximately mid-way between the extension product corresponding to the wild-type sequence (terminating at position 187) and that corresponding to the mutant sequence (terminating at position 189)] represents the extended primer prematurely terminated on the wild-type template, due presumably to an A to G transition at position 188. The number above each lane represents the age of the individual analyzed. P, primer; PE, primer + Sequenase; W and M, primer extension products obtained on plasmid DNA carrying a cloned DLP4 or DLP6 fragment with wild-type and, respectively, mutant sequence.
Figure 3
Primer extension data for the detection of the A189G, T408A, and T414G mutations in DLP4 and DLP6 segments amplified from mtDNA of different tissues of 79 y, 84 y-1, and 90 y. Mi, intercostal muscle; Mp, psoas muscle; Md, deltoid muscle; H, heart; Li, liver; Sp, spleen; Ly, lymph node; and Sk, skin. Symbols W and M are explained in Fig. 2 legend.
Figure 4
(A) Diagram summarizing the age distribution and frequency of the A189G, T408A, and T414G mutations in mtDNA from skeletal muscles of individuals of a wide range of ages of group A (see_Materials and Methods_). The number below each tick on the abscissae axes indicates the age of the individuals analyzed (in years). Solid bars represent the primer extension data, the striped bars, the data from the DGGE-cloning-sequencing analysis. (B) Scheme of a portion of the mtDNA main control region showing the positions of the two muscle-specific mtDNA mutations detected in the present work (thick arrows) and of the fibroblast-specific mtDNA T414G mutation (thin arrow). The positions of binding of the mitochondrial transcription factor A (the densely hatched rectangle indicates a position of high affinity binding), and the site of the promoter for L-strand transcription (LSP) are shown. Other symbols are explained in Fig. 1_A_ legend.
Figure 5
(A) Primer extension data for the detection of the A189G, T408A, and T414G mutations in mtDNA from biopsied quadriceps muscle of eight individuals exhibiting partial muscle denervation (group B in Materials and Methods). (B) Diagram summarizing the frequency of the A189G, T408A, and T414G mutations in the eight individuals. Bars are as in Fig. 4.
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