Niels Morling

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.

DNA Commission of the International Society of Forensic Genetics: recommendations on forensic analysis using Y- chromosome STRs

During the past few years the DNA commission of the International Society of Forensic Genetics has published a series of documents providing guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. This latest report addresses a relatively new area, namely Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). This report addresses nomenclature, use of allelic ladders, population genetics and reporting methods.

HLA-D and -DR antigens in genetic analysis of insulin dependent diabetes mellitus

SummaryThree groups of patients with insulin-dependent diabetes mellitus, ascertained by different procedures, were investigated for HLA-A, B, C and D antigens (n=164), and a subset (n=93) for HLA-DR. Both HLA-D/DR3 and D/DR4 were strongly positively associated and D/DR2 was negatively associated with insulin-dependent diabetes. HLA-DR4 was found to be a better marker for insulin-dependent diabetes than Dw4. The HLA-B associations (B8, B15 and B18) were clearly secondary to the increases of HLA-D/DR3 and D/DR 4. The HLA associations did not differ between familial and isolated cases indicating that these two groups may well have a common genetic background. Based on analysis of HLA-haplotype sharing in affected sibling pairs, a simple dominant model of inheritance could be ruled out, and a simple recessive model was found unlikely. The relative risks for the HLA-Dw3,4 and HLA-DR3,4 phenotype were 21.2 and 44.4 respectively and exceeded those of both the HLA-Dw3 and HLA-DR3 (5.6 and 4.3) as well as the HLA-Dw4 and DR4 (10.1 and 10.5) phenotypes. This argues against an intermediate genetic model but further studies are needed to clarify whether there is more than one susceptibility gene for insulin-dependent diabetes mellitus within the HLA-system.

ISFG: Recommendations on biostatistics in paternity testing

The Paternity Testing Commission (PTC) of the International Society for Forensic Genetics has taken up the task of establishing the biostatistical recommendations in accordance with the ISO 17025 standards and a previous set of ISFG recommendations specific to the genetic investigations in paternity cases. In the initial set, the PTC recommended that biostatistical evaluations of paternity are based on a likelihood ratio principle - yielding the paternity index, PI. Here, we have made five supplementary biostatistical recommendations. The first recommendation clarifies and defines basic concepts of genetic hypotheses and calculation concerns needed to produce valid PIs. The second and third recommendations address issues associated with population genetics (allele probabilities, Y-chromosome markers, mtDNA, and population substructuring) and special circumstances (deficiency/reconstruction and immigration cases), respectively. The fourth recommendation considers strategies regarding genetic evidence against paternity. The fifth recommendation covers necessary documentation, reporting details and assumptions underlying calculations. The PTC strongly suggests that these recommendations should be adopted by all laboratories involved in paternity testing as the basis for their biostatistical analysis.

Association between HLA-DR2 and Production of Tumour Necrosis Factor α and Interleukin 1 by Mononuclear Cells Activated by Lipopolysaccharide

The production of tumour necrosis factor (TNF) and interleukin 1 (IL‐1) by lipopolysaccharide‐activated mononuclear cells from 39 healthy donors was studied in vitro by bioassay and ELISA. The donors were typed for HLA‐A, ‐B, ‐C, ‐DR, and ‐DP antigens. There was no detectable production of TNFβ (lymphotoxin). The intracellular levels of bioactive TNFα were minimal or undetectable in all cases. Cells from HLA‐DR2+ individuals secreted significantly lower amounts of TNFα than cells from HLA‐DR2− donors [2 ng/ml (1.5–4.4) and 7.5 ng/ml (3.9–8.3) respectively (medians 25–75%); P<0.01]. The difference disappeared if the cells were preactivated for 2 days with 1000 U/ml of recombinant gamma interferon (rIFN‐γ). In some individuals, the TNFα response increased considerably after IFN‐γ priming, in particular in those possessing the HLA‐DR2 antigen. In contrast, there was no detectable difference in the production of IL‐1β between the donors, and the IL‐1β response decreased significantly after rIFN‐γ priming in HLA‐DR2+ individuals [2.3 ng/ml (1.1–8.4) versus 7.2 ng/ml (5–7.9); P<0.05] and in HLA‐DR2− individuals [3 ng/ml (1.1–5.3) versus 5.7 ng/ml (3.9–7.5); P<0.01]. There was no correlation between the TNFα and IL‐1 responses and any of the other HLA‐DR, ‐DP, or ‐B antigens. There was a significant positive correlation between the levels of TNFα measured by ELISA and by cytotoxicity assay. However, the TNFα‐containing supernatants from 9 out of 37 individuals appeared to contain inhibitor(s) of the biological activity of TNFα. The presence of inhibitor(s) was not associated with any HLA antigens.

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.

Next generation sequencing and its applications in forensic genetics

It has been almost a decade since the first next generation sequencing (NGS) technologies emerged and quickly changed the way genetic research is conducted. Today, full genomes are mapped and published almost weekly and with ever increasing speed and decreasing costs. NGS methods and platforms have matured during the last 10 years, and the quality of the sequences has reached a level where NGS is used in clinical diagnostics of humans. Forensic genetic laboratories have also explored NGS technologies and especially in the last year, there has been a small explosion in the number of scientific articles and presentations at conferences with forensic aspects of NGS. These contributions have demonstrated that NGS offers new possibilities for forensic genetic case work. More information may be obtained from unique samples in a single experiment by analyzing combinations of markers (STRs, SNPs, insertion/deletions, mRNA) that cannot be analyzed simultaneously with the standard PCR-CE methods used today. The true variation in core forensic STR loci has been uncovered, and previously unknown STR alleles have been discovered. The detailed sequence information may aid mixture interpretation and will increase the statistical weight of the evidence. In this review, we will give an introduction to NGS and single-molecule sequencing, and we will discuss the possible applications of NGS in forensic genetics.

DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. A previous recommendation published in 2001 has already addressed Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). Since then, the use of Y-STRs has become very popular, and numerous new loci have been introduced. The current recommendations address important aspects to clarify problems regarding the nomenclature, the definition of loci and alleles, population genetics and reporting methods.

Multiplex PCR and minisequencing of SNPs— a model with 35 Y chromosome SNPs

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.

ISFG: Recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations

The use of non-human DNA typing in forensic science investigations, and specifically that from animal DNA, is ever increasing. The term animal DNA in this document refers to animal species encountered in a forensic science examination but does not include human DNA. Non-human DNA may either be: the trade and possession of a species, or products derived from a species, which is contrary to legislation; as evidence where the crime is against a person or property; instances of animal cruelty; or where the animal is the offender. The first instance is addressed by determining the species present, and the other scenarios can often be addressed by assigning a DNA sample to a particular individual organism. Currently there is little standardization of methodologies used in the forensic analysis of animal DNA or in reporting styles. The recommendations in this document relate specifically to animal DNA that is integral to a forensic science investigation and are not relevant to the breeding of animals for commercial purposes. This DNA commission was formed out of discussions at the International Society for Forensic Genetics 23rd Congress in Buenos Aires to outline recommendations on the use of non-human DNA in a forensic science investigation. Due to the scope of non-human DNA typing that is possible, the remit of this commission is confined to animal DNA typing only.