Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondrial patients
Introduction
The human mitochondrial genome is a circular double-stranded molecule of 16569 base pairs (bp); it encodes 2 ribosomal RNAs, 22 transfer RNAs for mitochondrial protein synthesis and 13 structural subunits of the mitochondrial respiratory chain. The only noncoding region is the 1.1-kb mitochondrial DNA (mtDNA) control region (the Displacement loop (D-loop) region), which contains crucial elements for mtDNA replication and transcription. MtDNA is replicated at much higher rate than nuclear DNA and has less efficient proof-reading DNA repair activity mechanisms. Thus, mtDNA is more vulnerable to attacks by reactive oxygen species and free radicals that are generated by electron leak of the mitochondrial respiratory chain [1]. Mitochondrial functions are especially critical for tissues (i.e. muscle and brain) that are highly dependent on aerobic metabolism. Mitochondrial disorders are human genetic diseases with extremely variable clinic and genetic features [2], [3], [4]. Pathogenic mtDNA defects can be divided into two groups: large-scale rearrangements (from a few hundred base pairs to >10 kb) and point mutations affecting both protein-encoding genes and RNA genes. Diseases associated with mtDNA rearrangements include sporadically occurring mtDNA deletions, maternally inherited mtDNA duplications and Mendelian-inherited mtDNA multiple deletions. Diseases associated with point mutations are almost exclusively maternally inherited. In all mitochondrial disorders, the levels of mutated mtDNA is high (from 50% to 90% of total mtDNA) and can be easily detected by Southern blot analysis (for large-scale rearrangements) or by PCR/RFLP analysis (usually for point mutations).
It has been established that mtDNA abnormalities occur also in nonpathological conditions at extremely low levels (detectable only with more sensitive procedures, i.e. radio-labelled PCR, two-step PCR amplification or other PCR-based techniques). The accumulation of deleterious rearrangements in the mitochondrial genome has been proposed as a potential biomarker of ageing [5]. Several large-scale deletions have been observed to increase in an age-dependent manner in different post-mitotic tissues including skeletal muscle, heart and central nervous system [6], [7]; mtDNA with multiple deletions was observed at very low levels in post-mitotic cells of healthy subjects [8]; several reports have shown that pathogenic mtDNA tRNALeu A3243G and tRNALys A8344G mutations accumulate with age also in healthy individuals [9], [10], [11], [12].
On the other hand, high heteroplasmic levels of ageing-related point mutations (up to 65%) localised in the D-loop region of skin fibroblasts and skeletal muscles have been recently observed only in aged normal individuals [13], [14]. In particular, two somatic ageing-related point mutations, i.e. A189G and T408A, show a striking skeletal muscle specificity and are characterised by a wide heterogeneous distribution at single-fiber level [15]. At present, both the role and the phenotypic effects of these specific mutations are unknown, and whether these mtDNA alterations are of primary or secondary consequence in ageing process is yet to be determined.
To better characterise the ageing-related accumulation of these specific mtDNA point mutations in the D-loop region, we evaluated their presence and frequencies in 91 muscle biopsies from both normal and mitochondrial patients of various ages.
Section snippets
Patients
We analysed 91 muscle biopsies from unrelated subjects (aged 6 to 82 years at the time when diagnosis was sought) referred to our Department of Neurology. Twenty one subjects were free of any neuromuscular disorder and considered as healthy controls; 70 patients were diagnosed to be affected by mitochondrial disorders as reported [16]. Molecular genetic studies (see Genetic analysis) performed on muscle DNA from these patients allowed us to classify them according to mtDNA molecular defects in
Results
We evaluated the presence of the mtDNA D-loop A189G and T408A point mutations in muscle biopsies from 91 unrelated individuals of different ages; 21 subjects, without any known clinical history of neuromuscular disease, were referred as healthy controls. As shown in Table 1, a high proportion of the subjects (59.3%) carries at least one of the two point mutations.
Fig. 1 shows the distribution over age of the two point mutations across the different categories in all 91 individuals.
To better
Discussion
One important feature of the ageing process is the progressive decline in bioenergy production at tissue/organ level with advancing of years. A potential explanation is the progressive and gradual accumulation of various mtDNA mutational events [24]. Although it is known that mtDNA alterations accumulate in somatic tissues during ageing, there has been no evidence so far regarding the simultaneous accumulation of specific ageing-related D-loop point mutations in genetically defined
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2014, MitochondrionCitation Excerpt :Studies indicate that dysfunctional ETS proteins could increase the escape of free electrons from the ETS, resulting in more oxidative damage (Harman, 1956). In concordance with these theories, studies have revealed that somatic mutations and large deletions in mtDNA accumulate with age, especially in high oxygen consumer tissues (Arnheim and Cortopassi, 1992), such as the brain (Bandy and Davison, 1990; Corral-Debrinski et al., 1992a; Soong et al., 1992), skeletal muscle (Cao et al., 2001; Del Bo et al., 2002b) and heart (Corral-Debrinski et al., 1992b; Hattori et al., 1991). These genetic alterations eventually give rise to age-associated phenotypes and reduced lifespan, as demonstrated in mtDNA mutator mice (Trifunovic et al., 2004).