H. Pfeiffer

Expanding the forensic German mitochondrial DNA control region database: genetic diversity as a function of sample size and microgeography.

Abstract Mitochondrial DNA control region sequences were determined in 109 unrelated German Caucasoid individuals from north west Germany for both hypervariable regions 1 (HV1) and 2 (HV2) and 100 polymorphic nucleotide positions (nps) were found, 63 in HV1 and 37 in HV2. A total of 100 different mtDNA lineages was revealed, of which 7 were shared by 2 individuals and 1 by 3 individuals. The probability of drawing a HV1 sequence match within the north west Germans or within published sets of south Germans and west Austrians is similar (within a factor of 2) to drawing a sequence match between any two of these three population samples. Furthermore, HV1 sequences of 700 male inhabitants of one village in Lower Saxony were generated and these showed a nearly linear increase of the number of different haplotypes with increasing number of individuals, demonstrating that the commonly used haplotype diversity measure (Nei 1987) for population samples tends to underestimate mtDNA diversity in the actual population.

A comparative study on the immunohistochemical detection of early myocardial damage.

Abstract The study was undertaken to evaluate the kinetics and distribution patterns of several immunohistochemical markers in ischemically and hypoxically damaged myocardium. The myocardium of 8 cases of acute myocardial infarction (AMI), 8 cases of diagnosed acute cardiac death (ACD) and 12 cases of acute exogenic hypoxia (AEH) due to CO poisoning or hanging were analysed for depletion of the cardiac antigens FABP, troponin C and T, desmin and myoglobin, loss of CD59 and deposition of the plasma antigens fibrinogen, fibronectin and the terminal complement complex C5b-9. The visualisation of the terminal complement complex was positive as early as 30 min after onset of symptoms of AMI. Depletion of cellular antigens started earlier than the deposition of plasma antigens. The deposition of fibronectin and fibrinogen began earlier than the detection of C5b-9 but later than the depletion of the cellular antigens. Our findings indicate that for the immunohistochemical detection of very early myocardial damage, the depletion of myoglobin is at least of the same rank or better than depletion of FABP and troponin.

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.

Mitochondrial DNA extraction and typing from isolated dentin-experimental evaluation in a Korean population

Abstract This study reports mtDNA polymorphisms in both hypervariable segments HV1 and HV2 of the non coding D-loop region from 60 unrelated Koreans. In contrast to two previous Korean data base studies, mtDNA was extracted separately from pulp tissue and root dentin of teeth obtained from dentists. Dentin turned out to be a reliable source of mitochondrial DNA. This can be of practical importance in forensic identification case work after a long post-mortem interval since pulp decomposes rapidly. The extraction method is explained in detail. The mtDNA polymorphisms obtained from 60 teeth of unrelated Koreans were compared with the already existing Korean data base.

Influence of soil storage and exposure period on DNA recovery from teeth.

Abstract A study was performed to determine the influence of garden soil on the deoxyribonucleic acid (DNA) recovery from teeth depending on the duration of storage. In the first series 24 teeth supplied by dentists were exposed to garden soil storage for a maximum of 18 weeks. Selected samples were excavated for DNA extraction at time intervals of 6,12 and 18 weeks. For the second series 20 teeth were stored for one year in garden soil. Following phenol/chloroform extraction with decalcification (first series) and without decalcification prior to extraction (second series) DNA was quantified, amplified using the polymerase chain reaction (PCR) for the tandem repeat loci D1S80, tyrosine hydroxylase, intron 1 (TH01) and Von Willebrand factor, intron A (VWA) (first series), human alpha fibrinogen (FGA) (second series) and sequenced in the hypervariable regions 1 and 2 (HV1, HV2) of the mitochondrial DNA (second series). The DNA concentration of the extracts after the first 6 weeks in soil was reduced by more than 90%. Amplification and direct sequencing of HV1 and HV2 of the mitochondrial genome was the most successful DNA technique.

The results of an mtDNA study of 1200 inhabitants of a German village in comparison to other Caucasian databases and its relevance for forensic casework

Abstract Mitochondrial DNA control region sequences were determined in 1200 male volunteers from one village area of Lower Saxony for the hypervariable region 1 (HV1). The 154 variable positions found resulted in 460 different haplotypes with a haplotype diversity value of 0.98165. The number of different haplotypes showed a nearly linear increase with the number of individuals typed. The haplotype diversity approached saturation level at a value of approximately 0.981 after typing 400 individuals. Furthermore, the number of different haplotypes and the haplotype diversity were calculated for four short amplicons of HV1 in order to establish the most variable section with a high efficiency for forensic casework.

Mitochondrial DNA typing from human axillary, pubic and head hair shafts – success rates and sequence comparisons

Abstract The analysis of mitochondrial DNA (mtDNA) from shed hairs has gained high importance in forensic casework since telogen hairs are one of the most common types of evidence left at the crime scene. In this systematic study of hair shafts from 20 individuals, the correlation of mtDNA recovery with hair morphology (length, diameter, volume, colour), with sex, and with body localisation (head, armpit, pubis) was investigated. The highest average success rate of hypervariable region 1 (HV 1) sequencing was found in head hair shafts (75%) followed by pubic (66%) and axillary hair shafts (52%). No statistically significant correlation between morphological parameters or sex and the success rate of sequencing was found. MtDNA sequences of buccal cells, head, pubic and axillary hair shafts did not show intraindividual differences. Heteroplasmic base positions were observed neither in the hair shafts nor in control samples of buccal cells.

Mitochondrial DNA in human hair shafts--existence of intra-individual differences?

Abstract The sequences of the hypervariable region 1 (HV1) of the mitochondrial DNA control region from multiple hair shafts from 10 unrelated individuals were compared to determine the frequency of differences in hairs from one individual. The extraction method described herein showed an average success rate of 67% for all 150 hair shafts tested in HV1. The mtDNA sequences from the hair shafts matched the sequences from the corresponding blood and saliva samples taken from the same donor and no evidence of heteroplasmy was found. The results emphasize the reliability of DNA extraction and mtDNA typing from human hair shafts for forensic purposes.

Demonstration and semi-quantification of mtDNA from human dentine and its relation to age.

Abstract In order to study if mitochondrial DNA (mtDNA) could be retrieved from isolated human dentine, small pieces of dentine were cut out from the central part of the apical half of wisdom teeth from 21 individuals aged 15 to 85 years. The dental pulp was used as a control. After extraction, amplification and agarose gel electrophoresis the amount of mtDNA was semi-quantified from the intensity of the stained bands in the gel. Mitochondrial DNA was retrieved from all samples and the sequences were identical in pulp and dentine from each individual. There was a clear age-dependent decrease in the amount of amplified mtDNA. Since the odontoblastic processes in the apical dentine undergo degeneration with age and the dentinal tubules subsequently become occluded with calcium phosphate crystals, the conclusion was drawn that even after dissolution of the odontoblastic processes, at least remains of the mtDNA are trapped in the dentine. This well protected mtDNA could thus be regarded a good source of DNA in identification cases with severe degradation.

A rapid mtDNA assay of 22 SNPs in one multiplex reaction increases the power of forensic testing in European Caucasians

We have developed a multiplex mitochondrial (mtDNA) assay of 21 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable region 1 (HVR1) and hypervariable region 2 (HVR2) that can be amplified in a single reverse touchdown polymerase chain reaction. Single base extension using the SNaPshot technique is also carried out as one multiplex. Besides the nine major European haplogroups (i.e. H, I, J, K, T, U, V, W, and X), 16 additional subclades (i.e. N1, X2, X2b, U2′-4/7′-9′, J/T, J1, J1c, HV, H1, H1a1, H1c, H3, H4, H6a, H7a H10) can be detected and classified into a phylogenetic mtDNA tree. By analyzing 130 Caucasoid samples from Germany, 36 different haplotypes were found resulting in a power of discrimination of 93.2%. Although 49% of all samples belonged to superhaplogroup H, the most common haplotype, i.e., haplogroup-specific SNPs plus haplogroup unspecific SNPs, had a frequency of only 18%. This assay is applicable for high-throughput mtDNA analysis and forensic mass screening. It will give additional information to the common control region sequencing of HVR1 and HVR2.