Walter Bär

DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing

Sequence analysis of human mitochondrial DNA (mtDNA) is being used widely to characterize forensic biological specimens, particularly when there is insufficient nuclear DNA in samples for typing. Hair shafts, bones, teeth and other samples that are severely decomposed may be subjected to mtDNA analysis, e.g. [1–5]. Although many of the quality assurance, quality control and interpretational guidelines used for PCR-based nuclear DNA analyses apply to mtDNA analysis, there are some features of mtDNA that warrant specific consideration: (1) mtDNA is maternally inherited; (2) heteroplasmy; and (3) the greater sensitivity of detection of mtDNA typing. It is imperative that guidelines consider the features of mtDNA and that practices do not exceed the state-of-knowledge on mtDNA. In a effort to refine previously published guidelines [6] and to assist those currently using mtDNA protocols and those considering implementing mtDNA analysis, the DNA Commission of the ISFG met on 16th August 1999 in San Francisco to develop current guidelines. The following are the recommendations by the DNA Commission on the use of mtDNA analysis.

Fulminant Liver Failure in Association with the Emetic Toxin of Bacillus cereus

BACKGROUND A 17-year-old boy and his father had acute gastroenteritis after eating spaghetti and pesto that had been prepared four days earlier. Within two days, fulminant liver failure and rhabdomyolysis developed in the boy and he died. The father had hyperbilirubinemia and rhabdomyolysis but recovered. We investigated the cause of these illnesses. METHODS Bacteria were isolated and characterized by conventional methods, and bacterial toxins were quantified by immunoassays and cell-culture techniques. The effect of the isolated toxin on the rates of oxidation of various substrates was analyzed in rat-liver mitochondria. RESULTS Autopsy of the boys liver revealed diffuse microvesicular steatosis and midzonal necrosis that suggested impaired beta-oxidation of liver mitochondria due to a mitochondrial toxin. There was no evidence of ingestion of heavy metals, halogenated compounds, hepatotoxic drugs, or staphylococcal enterotoxin. However, high concentrations of Bacillus cereus emetic toxin were found in both the residue from the pan used to reheat the food and the boys liver and bile. B. cereus was cultured from the intestinal contents and the pan residue. The emetic toxin isolated from the B. cereus cultures was found to be a mitochondrial toxin. CONCLUSIONS Fulminant liver failure developed after the ingestion of food contaminated with the B. cereus emetic toxin. The toxin inhibits hepatic mitochondrial fatty-acid oxidation, indicating that it caused liver failure in this patient.

mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR

mRNA profiling is a promising new method for the identification of body fluids from biological stains. Major advantages of mRNA profiling are the possibility of detecting several body fluids in one multiplex reaction and of simultaneously isolating DNA without loss of material. A reverse transcription endpoint polymerase chain reaction (PCR) method and a realtime PCR assay were established for the identification of blood, saliva, semen, vaginal secretions and menstrual blood, and were compared to conventional enzymatic and immunologic tests. The results for specificity, sensitivity and suitability to biological stains were satisfying and mRNA stability was demonstrated for up to 2-year-old stains. Two novel multiplex assays were created with the endpoint PCR primers: multiplex 1 amplifies two markers for each of the above mentioned body fluids and is suited for screening; multiplex 2 was designed for the detection of blood, vaginal secretions and menstrual blood. The results demonstrate that both endpoint PCR and realtime PCR are suitable for the identification of body fluids in forensic stains and represent an effective alternative to conventional enzymatic and immunologic tests.

Postmortem stability of DNA

High-molecular-weight DNA was recovered postmortem in sufficient quantities from various human organ tissues as well as from blood, although not all organs were equally well suitable. Good DNA stability was found in brain cortex, lymph nodes and psoas muscle over a period of three weeks postmortem. Spleen and kidney showed good DNA stability up to five days postmortem but after longer periods, rapid degradation was observed. Yields of DNA from blood were not consistent because of the non homogeneity of samples. Blood clots were rich with DNA. Generally, the amount of degraded DNA correlated directly with the duration of the postmortem period. However in some cases, DNA degradation was already prominent after a short period. However in some cases, DNA degradation was already prominent after a short period. Case histories showed that high environmental temperature at the site of death and/or infectious diseases prior to death were the main factors for rapid autolysis. Gradual disappearance to complete loss of the long fragments (15-23 kb) was observed in DNA fingerprinting using the minisatellite probe 33.15. No extra-bands were noted, thus excluding erroneous conclusions. However, evidentiary value of older samples was lower.

DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing.

Sequence analysis of human mitochondrial DNA (mtDNA) is being used widely to characterize forensic biological specimens, particularly when there is insufficient nuclear DNA in samples for typing. Hair shafts, bones, teeth and other samples that are severely decomposed may be subjected to mtDNA analysis, e.g. [1–5]. Although many of the quality assurance, quality control and interpretational guidelines used for PCR-based nuclear DNA analyses apply to mtDNA analysis, there are some features of mtDNA that warrant specific consideration: (1) mtDNA is maternally inherited; (2) heteroplasmy; and (3) the greater sensitivity of detection of mtDNA typing. It is imperative that guidelines consider the features of mtDNA and that practices do not exceed the state-of-knowledge on mtDNA. In a effort to refine previously published guidelines [6] and to assist those currently using mtDNA protocols and those considering implementing mtDNA analysis, the DNA Commission of the ISFG met on 16th August 1999 in San Francisco to develop current guidelines. The following are the recommendations by the DNA Commission on the use of mtDNA analysis.

Report of the European DNA profiling group (EDNAP) — towards standardisation of short tandem repeat (STR) loci

This paper describes a collaborative exercise intended to demonstrate whether uniformity of DNA profiling results could be achieved between European laboratories using short tandem repeat (STR) loci. Two different STRs were chosen--HUMTH01 and the AT-rich HUMACTBP2 (SE33). The former locus has only five common alleles, whereas the latter is complex and has at least 30 alleles. Laboratories were asked to test seven blood stains and to report the results to the coordinating laboratory. The exercise demonstrated that the simple STR systems such as HUMTH01 are more amenable to adoption as standard loci than complex AT-rich systems.

RNA/DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.

mRNA profiling for the identification of blood—Results of a collaborative EDNAP exercise

A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests.

Report of a European collaborative exercise comparing DNA typing results using a single locus VNTR probe

A collaborative exercise was carried out in 1989 among 12 European forensic laboratories using the single locus VNTR probe pYNH24, the restriction enzyme HinfI, the same set of human genomic DNA samples, and a standardized DNA size marker. The objectives of the exercise were: (1) to study the degree of variation within and between laboratories, (2) to obtain information on requirements for technical standardization allowing the exchange of typing results and (3) to compare different approaches for the identification of allelic DNA fragments of unknown size. Each laboratory carried out up to 10 independent typing experiments using the same DNA samples. The results were analysed independently by two laboratories using three different methods. The results of the exercise demonstrate the correlation of typing that can be achieved within and between laboratories under conditions of minimal standardization.

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

In the present work, we have evaluated eight reportedly blood-specific mRNA markers (HBB, HBA, ALAS2, CD3G, ANK1, PBGD, SPTB, AQP9) in an attempt to determine the most suitable ones for use in forensic applications based on their sensitivities, specificities and performance with casework samples. While varying levels of expression were observed, all markers were relatively sensitive requiring as little as 1ng of RNA input into the reverse transcription (RT) reaction. In singleplex reactions, seven of the eight analyzed blood markers (all except AQP9) demonstrated a high degree of specificity for blood. In multiplex reactions, non-reproducible cross-reactivity was observed for several of the mRNA markers, which was reduced and, in most cases, eliminated when less input total RNA was used. Additionally, some cross-reactivity was observed with tissue and animal samples. Despite differences in the observed sensitivity and specificity of the blood markers examined in this study, a number of the candidates appear to be suitable for inclusion in appropriately validated multiplex mRNA-based body fluid identification systems.