Volume 5, Issue 1, Pages 1-6 (March 2003)

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Mitochondrial mutagenesis in the brain in forensic and pathological research

Received 27 November 2002; received in revised form 5 December 2002; accepted 18 January 2003.

Abstract

Accumulation of alterations to the mitochondrial DNA (mtDNA) would be expected to significantly impair the bioenergetic function of mitochondria in the affected host cells. Many of these changes have been associated with several specific diseases and the process of aging. These mutations may be the result of mitochondrial oxidative stress, which is increased with age of individuals and specific degenerative diseases.

Our aim with this review is to summarize the recent literature on the occurrence of mtDNA alterations and its possible relation to age-depending degenerative processes with special regards to the brain. Additionally, we show how these alterations could be used in fields of pathology and forensic medicine.

Keywords: Mitochondrial DNA, DNA degeneration, Forensic neuropathology, Aging

Article Outline

• Abstract

• 1. Introduction

• 2. Point mutations of mtDNA seem to accumulate randomly

• 3. The structure of mtDNA alterations in different organ systems is multiple

• 4. mtDNA alteration are possibly associated with neurodegenerative diseases

• 5. The 4977 bp deletion is called ‘the common deletion’ and might be a marker molecule of aging

• 6. The amount of 4977 bp dmtDNA increases region-specific in the human brain

• 7. mtDNA copy number may also increase with advanced age

• 8. Conclusions

• References

• Copyright

1. Introduction

Mitchondrial DNA (mtDNA) evolves at a faster rate than nuclear DNA. In 1979, it was estimated that nucleotide substitutions are present at a ten-fold higher frequency in mtDNA than in nuclear DNA. The rate of mtDNA evolution is faster than that of nuclear DNA [1], [2], which may reflect an increased susceptibility to mutations [3]. There appear to be several characteristics of mtDNA which would favor high mutagenesis, including relatively high turnover both in mitotic and postmitotic cells that increases the chances of mutations, and the susceptibility of mitochondrial structures to oxidative stress which is supported by several reports detecting lipid peroxidation [4], protein oxidation [5], and mtDNA mutations [6]. The analysis of human mtDNA is becoming increasingly important in forensic sciences. Especially in the identification of poorly preserved skeletons or other human remains, sequence analysis of the human D-Loop region is widely employed [7], [8]. In this work, we summarize the recent results and investigations in mitochondrial genetics and their application possibilities for pathological and forensic medicine.

2. Point mutations of mtDNA seem to accumulate randomly

There are several reports showing contradictory results concerning the increase of point mutations with age. In 1998, Liu et al. [9] showed a correlation between three different mtDNA mutations and age, including the probably most frequent 3243 A to G point mutation with a correlation coefficient of r=0.57 [9] and confirmed earlier results [10], [11]. By using a quantitative PCR method, a different occurrence of point mutations in mtDNA of human muscle was presented [12]. Point mutations were detectable at a variety of positions at the mitochondrial genome of both young and old individuals indicating random occurrences at the level of base substitutions. They seemed to be primarily spontaneous in origin and arise either from DNA replication errors or reactions of DNA with endogenous metabolites [13]. The finding, that the original tissue samples displayed a spectrum similar to that observed in human cell culture, suggest a common pathway and seems to disprove the hypothesis that environmental mutagens are important contributors to mitochondrial point mutagenesis. Pallotti et al. [14] used a modified polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), a so-called ‘double PCR and digestion (DPD)’ method, in which a minority of mutated mtDNA sequences can be enriched. They detected levels of point mutations between 0.002 and 0.040% without any correlation to the age of the subject [14]. The discrepancy of these reports concerning the age-dependence may be due to several factors including the specific mutations, the authors investigated, or the method of detection, the tissue or the age of the sample itself.

The question arises why the mtDNA is affected by so many induced sequence alterations but can still be investigated for identification purposes. One explanation could be that no point mutation exceeds the amount which can be detected by sequencing analysis, i.e. about 5–10%. Nevertheless, it should be stressed that reports exist on heteroplasmy of mtDNA sequences in the D-loop within one individual [15] even though other investigations did not detect point mutations in the control region in normal aging and neurodegenerative human brains, suggesting that mutations in the D-loop region did not contribute to the aging and degenerative process in vivo [16] and thus obviously would not be a disturbing factor for forensic analysis. Other reports of an age-related accumulation of sequence alterations in the D-loop have been contradictory since both possibilities, accumulation [9] and no accumulation [14] have been reported.

3. The structure of mtDNA alterations in different organ systems is multiple

In addition to sequence analysis of the mitochondrial D-loop region for identification purposes, mtDNA has been investigated for rearrangements in recent years. Alterations to the structure of mtDNA are identified in a variety of tissues in rats [17], [18], mice [19], rhesus monkeys [20], nematodes [21], Drosophila melanogaster [22], and also in almost all human tissues, be it postmitotic differentiated tissues such as skeletal muscle [23], heart muscle [24], [25] and brain [26], or in highly replicative tissues such as skin [27], [28] or blood [29]. Even in hair follicles [30], oocytes [31] or sperm cells [32], mutated mtDNA was found. Alterations to mtDNA include large deletions [33], point mutations [10], insertions, and short duplications [34], [35]. By using a special PCR covering the whole mitochondrial genome, fragmentation of mtDNA into more than 100 types of different sized deleted molecules could be observed [24].

Apparently, mutations occur spontaneously, possibly triggered by oxidative stress, and accumulate in various tissues with age. The employment of the highly specific and sensitive PCR led to the detection of more than 50 different alterations of human mtDNA that are associated with, or responsible for specific human diseases. Many of these deletions have been demonstrated to also be associated with advanced age [12], [35].

4. mtDNA alteration are possibly associated with neurodegenerative diseases

The discovery that mtDNA mutations are of pathological importance [36], [37], and that mitochondria plays an important role in the mechanisms of aging [3], [25], [35], [38] and cell death [39], [40] shows the importance of mitochondria in pathology research. The spectrum of phenotypes has expanded from rare myopathies to multiple diseases representing virtually all branches of medicine. The possibility that some of the most common and devastating degenerative diseases seem to involve mitochondria implicates the importance of investigations of mitochondrial genetics and biochemical changes in these organelles. Although mitochondrial mutations are present at only relatively low levels (usually <2%) in the whole tissue, it could be possible that mutations clonally expand within one cell and exceed a defined threshold which could possibly cause defects of mitochondrial oxidative metabolism and may lead to cell death.

There are many reports on mitochondria and mtDNA and a possible involvement with degenerative diseases, e.g. Parkinson's disease or Alzheimer's disease [41], [42], [43], [44], [45], [46]. Still, investigations are contradictory or not yet confirmed. Screening for a specific substitution did not reveal any differences between brains from normal elderly persons or patients with Alzheimer's or Parkinson's disease [45], [47].

5. The 4977 bp deletion is called ‘the common deletion’ and might be a marker molecule of aging

The most common deletion of mtDNA is the 4977bp deletion, which has been observed in biopsy [48] and autopsy material from individuals aged 20 years and older [49], [50]. It occurs in the highest percentage and with the best correlation to age in well-differentiated tissues such as brain and muscle tissue [23]. The reason for this relatively high frequency of the common deletion is probably the structure of the DNA flanking their breakpoints. Hou et al. [51] could show a retarded and increased mobility in this special region and suggest that these frequencies are rendered to assume a more distorted structure than B-DNA by the two flanking best-inducing DNA sequences in organelles and thereby render this region to be more vulnerable to attacks by reactive oxygen species and free radicals [51]. Using a kinetic PCR, we detected levels starting from 0.00049 to 0.14% for the 4977bp deleted mtDNA in skeletal muscle of aged individuals older than 20 years [50]. The findings summarized here confirm many other investigation regarding the occurrence of the common deletion in skeletal muscle and its accumulation with age up to a physiological amount of less than 1% deleted mtDNA [9], [12], [48], [52], [53].

Thus, the common 4977bp deletion with its strong correlation to the age of an individual might be a possible tool for the estimation of the age of an unknown individual based only on soft tissue. However, the confidence interval is rather broad [49], [50], [54]. Therefore the method is not as reliable as an age estimation based on the racemization of aspartic acid in bones or teeth [55], [56].

6. The amount of 4977 bp dmtDNA increases region-specific in the human brain

Older individuals show an accumulation of a heterogeneous array of mtDNA rearrangements as well as heterogeneity of mutations between different brain regions within the same individual [57], which we could confirm detecting the common deletion in five different brain regions. The amount of 4977bp dmtDNA increased with advancing age in four investigated regions: caudate nucleus, substantia nigra, putamen, and parietal cortex, but not in cerebellar tissue. The strongest correlation to age and the highest amount of deleted mtDNA was found in caudate nucleus, followed by putamen, substantia nigra, and parietal cortex, while in cerebellar tissue the deletion could only be detected in very low amounts in six out of 26 persons. Subsequent histological investigations of these cases revealed signs of prolonged ischemic events in five patients [58]. Thus, it might be hypothesized, that the 4977bp deletion is an indicator for oxidative stress and that its accumulation correlates with oxidative damage sustained some weeks or more ago.

7. mtDNA copy number may also increase with advanced age

In addition to the qualitative changes of mtDNA mentioned above, the copy number of mtDNA in animal tissue has also been shown to change with age. Petruzella et al. detected using southern hybridization a significant decrease of the D-loop number in rat brain tissues [59]. The mtDNA is apparently affected by multiple deletions which also occur in an age-depending correlation [9] as it does the increase of the copy number of the total mitochondrial genome in human tissue [60]. This phenomenon, e.g. the amount of total mtDNA in human dentin was already investigated under the aspect of age dependency for a forensic approach [61].

8. Conclusions

In this review, we have summarized some of the latest papers on mitochondrial mutagenesis in human tissues and shown how alterations of mtDNA seem to be involved in degenerative processes during normal aging and pathological events.

Improved detection methods, especially modifications of the PCR, led to better detection thresholds in recent years. They allow the detection of deletions in tissues, that usually contain only a very low amount of mutated DNA, e.g. skin [27], blood [29] or bone [62], or enable the detection of the common 4977bp deletion in individuals younger than 20 years of age or from less template DNA [63]. Using real time PCR, even a single molecule can reproducibly be detected and quantified [54]. The development of single cell PCR can be useful to study mtDNA alterations and their direct influence on biochemical function in the mitochondria such as COX activity [64], [65]. These improved detection methods may contribute to more insight to the occurrence of certain deletions or the content of total mtDNA and their relation to degenerative and harmful processes in the organism. The possible role of mtDNA alterations in carcinogenesis processes [66], [67] or other phenomenons, such as sudden infant death syndrome (SIDS) [68] makes the investigation of human mitochondrial genetics even more important.

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a Institute of Legal Medicine, Christian Albrecht University of Kiel, 24105 Kiel, Germany

b Institute of Legal Medicine, Medical University of Lübeck, 23562 Lübeck, Germany

Corresponding author. Tel.: +49-431-597-3600; fax: +49-431-597-3612

PII: S1344-6223(03)00003-8

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