Progressive damage to mitochondrial DNA (mtDNA) during life is thought to contribute to aging processes. However, this idea has been difficult to reconcile with the small fraction of mtDNA so far found to be altered. Here, examination of mtDNA revealed high copy point mutations at specific positions in the control region for replication of human fibroblast mtDNA from normal old, but not young, individuals. Furthermore, in longitudinal studies, one or more mutations appeared in an individual only at an advanced age. Some mutations appeared in more than one individual. Most strikingly, a T414G transversion was found, in a generally high proportion (up to 50 percent) of mtDNA molecules, in 8 of 14 individuals above 65 years of age (57 percent) but was absent in 13 younger individuals.
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 <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. R ecently, the discovery of an aging-dependent large accumulation of point mutations in the control region for mtDNA replication of human skin fibroblasts has been reported (1). Particularly striking was the demonstration, in a generally high proportion of molecules (up to 50%), of a T to G transversion at position 414 in the original Cambridge sequence (2), within the promoter for the synthesis of the RNA primer of mtDNA heavy (H)-strand synthesis (3) and for light (L)-strand transcription (4). This mutation was present in more than 50% of the individuals above 65 years of age and absent in younger individuals. In the present work, to investigate the occurrence of these aging-dependent mutations in other cell types, in particular, in postmitotic cells, a screening was carried out for the detection in human muscle of aging-related specific point mutations in the DLP4 and DLP6 segments of the main mtDNA control region, which were the segments previously found to carry the fibroblast mtDNA mutations (1). These two mtDNA segments correspond to one of the hypervariable portions of the main control region (5), and were chosen, for the purpose of analysis, as containing each a uniform melting domain (Y.M., unpublished data). They carry critical sequences for mtDNA replication (Fig. 1A). In particular, DLP4 contains the primary origin of H-strand mtDNA synthesis (O H1 ) (3), whereas DLP6 contains the promoter and start site for H-strand replication RNA...
The presence of a genetic component in longevity is well known. Here, the association of a mtDNA mutation with a prolonged life span in humans was investigated. Large-scale screening of the mtDNA main control region in leukocytes from subjects of an Italian population revealed a homoplasmic C150T transition near an origin of heavy mtDNA-strand synthesis in Ϸ17% of 52 subjects 99 -106 years old, but, in contrast, in only 3.4% of 117 younger individuals (P ؍ 0.0035). Evidence was obtained for the contribution of somatic events, under probable nuclear genetic control, to the striking selective accumulation of the mutation in centenarians. In another study, among leukocyte mtDNA samples from 20 monozygotic and 18 dizygotic twins, 60 -75 years old, 30% (P ؍ 0.0007) and 22% (P ؍ 0.011), respectively, of the individuals involved exhibited the homoplasmic C150T mutation. In a different system, i.e., in five human fibroblast longitudinal studies, convincing evidence for the agingrelated somatic expansion of the C150T mutation, up to homoplasmy, was obtained. Most significantly, 5 end analysis of nascent heavy mtDNA strands consistently revealed a new replication origin at position 149, substituting for that at 151, only in C150T mutationcarrying samples of fibroblasts or immortalized lymphocytes. Considering the aging-related health risks that the centenarians have survived and the developmental risks of twin gestations, it is proposed that selection for a remodeled replication origin, inherited or somatically acquired, provides a survival advantage and underlies the observed high incidence of the C150T mutation in centenarians and twins. R ecently we reported a large aging-dependent accumulation of tissue-specific point mutations at critical control sites for mtDNA replication in human skin fibroblasts and skeletal muscle (1-3). The T414G transversion within the promoter for light (L)-strand transcription and for synthesis of the RNA primer of heavy (H)-strand synthesis (4, 5) ( Fig. 1) was found in a generally high proportion (up to 50%) of mtDNA molecules in skin fibroblast cultures from 8 of 14 normal individuals above 65 years of age, but was absent in fibroblast cultures from 13 younger individuals (1). The age distribution and the results of two longitudinal studies indicated clearly that the T414G mutation was not inherited (1). A search for possible point mutations in the main mtDNA control region of skeletal muscle revealed, surprisingly, the presence of two mutations that had not been observed in fibroblast mtDNA (2). In particular, an A189G transition, very close to the main origin of H-strand synthesis (position 191), in 11-64% of the mtDNA and a T408A transversion, within the promoter for the RNA primer of H-strand synthesis (Fig. 1), in 2-16% of the mtDNA were found in the muscle from the majority of 27 normal individuals above 53 years old, while being absent or marginally present in the muscle from 19 individuals younger than 34 years. Both the fibroblast T414G mtDNA mutation and the muscle A189G a...
The number of new cervical adenocarcinoma (AD) cases has risen slowly, however, its histological similarity to other tumor types and the difficulty of identifying the site of the original tumor makes the diagnosis of cervical AD particularly challenging. We investigated a novel molecular biomarker for cervical AD through the integration of multiple methods of genomic analysis. Tumor samples in discovery set were obtained from 87 patients who underwent radiotherapy, including 31 cervical AD. Microarray analysis and quantitative polymerase chain reaction analysis were performed to screen a candidate diagnostic molecule for cervical AD, and its clinical significance was investigated by immunohistochemical analysis (IHC). We found a difference between biopsy samples of AD and squamous cell carcinoma (SCC) in the expression and genomic copy number of Villin1 (VIL1), which maps to 2q35. IHC revealed 14 VIL1-positive tumors; 13 cervical AD and one small cell carcinoma of cervix, while none of SCC or endometrial AD was VIL1-positive. Kaplan-Meier survival curves revealed worse disease-free survival in VIL1-positive tumors. The marker was validated by newly enrolled 65 patients, and VIL1 positive staining showed 52% of sensitivity and 100% of selectivity for cervical AD. In conclusion, we have identified VIL1 as a novel biomarker of cervical AD. VIL1, a major structural component of the brush border cyto skeleton, which was recently found to be an epithelial cellspecific anti-apoptotic protein. Our study suggests the existence of a subtype of cervical tumors which are VIL1 positive with poor radioresponse.
Lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC) is known to associate with a significant decrease of 5-year survival. Genetic factors related to the difference of the LNM status in the OSCC have been not fully elucidated. Array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) were conducted using primary tumor materials resected from 54 OSCC patients with (n=22) or without (n=32) cervical LNM. Frequent gain was observed at the 11q13 region exclusively in patients with cervical LNM, which was confirmed by real-time QPCR experiments using 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. It was revealed that two distinct amplification cores existed, which were separated by a breakpoint between MYEOV and CCND1 in the 11q13 region. The combination of copy number amplification at CTTN (core 2) and/or TPCN2/MYEOV (core 1), selected from each core, was most significantly associated with cervical LNM (P=0.0035). Two amplification cores at the 11q13 region may have biological impacts on OSCC cells to spread from the primary site to local lymph nodes. Further study of a larger patient series should be conducted to validate these results.
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