Sex determination of forensic samples: co-amplification and simultaneous detection of a Y-specific and an X-specific DNA sequence

Summary The detection of restriction fragment length polymorphisms (RFLP) (1) in DNA extracted from forensic samples remains impossible in a significant number of cases due to deterioration and contamination of the biological material and the extremely low quantities of DNA isolated. The polymerase chain reaction (PCR) is a recent and particularly convenient method for analysing and typing very small amounts (10–20ng) of degraded human DNA (2, 3). DNA analysis at the level of a few cells present in forensic samples such as bloodstains, semen stains, vaginal swabs and head hair bulbs now appears possible using DNA amplification. A PCR protocol [4, 5] was adapted to simultaneously amplifiy a Y-specific DNA repeat sequence from the DYZ1 locus [6] and an X-specific DNA repeat sequence from the DXS424 locus [7]. The co-amplified Y-specific DNA fragment (102 bp) and X-specific DNA fragments (181–199 bp) were visualized on an ethidium bromide-stained 4% agarose gel. The male or female type of the amplified DNA extracted from blood samples, bloodstains, semen stains, vaginal swabs, brain tissue and 1, 2, 5, or 10 head hair bulbs was determined.     

Post-mortem redistribution of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") in the rabbit. Part II: post-mortem infusion in trachea or stomach

Drug concentrations in autopsy samples can also be influenced by post-mortem gastric diffusion when the stomach contains a substantial amount of the drug or by diffusion from the trachea when agonal aspiration or post-mortem regurgitation of vomit occurs. This was studied in a rabbit animal model in which MDMA solutions were infused post mortem either in the trachea or in the stomach. At 24, 48 or 72 h post mortem, samples including cardiac blood, vitreous humour, urine, bile, gastric content and several tissues were taken for toxicological analysis. After post-mortem tracheal infusion, MDMA can easily diffuse not only into the lungs but also in great quantities into the cardiac blood and, to a lesser extent, into the cardiac muscle. MDMA was also found in the closely adjacent diaphragm and in the upper abdominal organs, including the liver and the stomach. Following post-mortem infusion into the stomach, considerable MDMA levels were found in cardiac blood and muscle, both lungs, diaphragm and liver tissue when the solution was concentrated nearby the lower oesophageal sphincter. However, when the MDMA solution was present deeper in the stomach, MDMA levels were high in the spleen and the liver and relatively low in cardiac blood and muscle. In both experiments, MDA levels in most tissues were low or below the limit of quantitation, but were substantial in cardiac blood and muscle, lungs and diaphragm, indicating that MDMA can be metabolised to MDA after death. These results in the rabbit model indicate that the diffusion of MDMA out of the stomach content, or due to aspirated vomit and gastro-oesophageal reflux can lead to considerable post-mortem redistribution and thus should be taken into account in current forensic practice in order to draw the right conclusions when a peripheral blood sample is not available.     

Analytical confirmation of lethal heroin overdose by the use of liquid chromatography methods

BACKGROUND/AIM: Heroin is diacetylated morphine. Its ability to induce euphoria has led to its frequent abuse, giving rise to psychological and physical dependence. It has a short half-life, of approximately 2-6 min. In the brain, heroin undergoes deacetylation to 6-monoacetylmorphine (6-MAM) and morphine. Detection of 6-acetylmorphine in the urine is indicative of heroin use. The aim of this study was to compare sensitivity and reliability of two analytical methods, a multicolumn liquid chromatography system with UV scanning detector (HPLC-UV) and liquid chromatography-mass spectrometry detection (LC-MS) in opiate determining in post mortem material. METHODS: Post mortem samples (blood, urine and vitreous humor) were analyzed by liquid chromatography with UV and MS detection. The samples were prepared by liquid-liquid extraction with mixture chloroform-isopropanol (9:1). Separation was performed on C8 column with mobile phase composed of 55% acetonitrile-glacial acetic acid (99:1) and 45% 20 mM ammonium acetate. RESULTS: The analysis of blood samples, urine, and eye liquid by the use of multicolumn HPLC-UV method confirmed the presence of morphine in the samples of blood and urine, codeine only in urine, and 6-MAM in the samples of urine and eye liquid. Using LC-MS method morphine was confirmed in all of the samples, while codeine was confirmed in urine and in the sample of eye liquid. In the samples of eye liquid and urine 6-MAM was confirmed. CONCLUSION: For determination of opiates in post mortem material LC-MS technique is more sensitive and reliable as compared to multicolumn liquid chromatography.     

Development of a mRNA profiling multiplex for the inference of organ tissues

Forensic characterisation of organ tissue generally occurs through histological and immunological assays of limited sensitivity. Here, we explore an alternative approach and examine a total of 41 candidate mRNA markers for their ability to differentiate between brain, lung, liver, skeletal muscle, heart, kidney and skin. Various selection rounds are applied involving 85 organ tissues (36 excised autopsy specimens and 49 frozen tissue sections, with at least ten specimens for each organ type), 20 commercially available RNAs from different human tissues and at least two specimens of blood, saliva, semen, vaginal mucosa, menstrual secretion or touch samples. Finally, 14 markers are regarded tissue-specific and included in an endpoint RT-PCR multiplex together with one general muscle, one blood and one housekeeping marker. This 17-plex is successfully used to analyse a blind test set of 20 specimens including mixtures, and samples derived from stabbing of organ tissues. With the blind test set samples, it is shown that an earlier described interpretation strategy for RNA cell typing results [1] is also effective for tissue inference. As organ-typing is embedded in a procedure of combined DNA/RNA extraction and analysis, both donor and organ type information is derived from the same sample. Some autopsy specimens presented DNA profiles characteristic for degraded DNA. Nevertheless, the organ-typing multiplex could generate full RNA profiles, which is probably due to small sizes of the amplicons. This assay provides a novel tool for analysis of samples from violent crimes.     

Criminal anticipation of DNA investigations resulting in mutilation of a corpse

A corpse of a female was found in her apartment in a state of advanced putrefaction. Both hands were amputated, the external genitals were excised and the body parts had been removed from the scene. The subsequent investigations proved that the body parts had been severed post mortem. The cause of death was determined to be manual strangulation. A 33-year-old man later confessed that he had strangled the victim 9 days prior to discovery of the body and that he had had sexual intercourse post mortem. According to the confession, the rational motive for the subsequent mutilation was to eliminate biological stains (e.g. semen inside the vagina, epithelial cells below the fingernails from scratching) suitable for forensic DNA analysis. This constitutes a new type of defensive mutilation intended to prevent the identification of the perpetrator. An increase in the occurrence would be detrimental to the elucidation rate of homicides: in a total of 171 homicides investigated at this institute, DNA analysis of biological stains gave reliable evidence in 45 cases. Received: 26 February 1999 / Received in revised form: 15 July 1999     

Heroin body packer's death in Haryana; India: A case report

We report a case of death due to heroin leakage in a body packer, attempting to smuggle the drug by concealing it in his gastro-intestinal tract. The body was recovered 3–5 days of incidence that was confirmed by autopsy. Fifty pellets (packages) were recovered from the body, 42 identical oval shaped “egg” packages were found in the stomach out of which two were damaged, 6 in small intestine, 2 in large intestine. The total weight of the powder was 267 g. Toxicological analysis of the powder samples from the damaged package and other 48 packages was performed and was found positive for heroin, caffeine and codeine. The main pathological findings at autopsy were pulmonary and cerebral edema. This case illustrates the challenges in postmortem evaluation of narcotic fatalities and the need to consider factors such as ante-mortem history, thorough post mortem examination, toxicology results and photography in forensic diagnosis. This case is unique in the sense that cause of death was intoxication caused by leakage of heroin from damaged packages detected at autopsy and demonstrates that body packing is an existing problem in India.     

Impact of antiseizure medication level on the incidence of sudden unexplained death from epilepsy in patients from an inner city emergency department

Sudden unexplained death from seizures (SUDS) accounts for death in approximately 10% of the epileptic population. SUDS usually occurs in young males with a history of seizure disorders who are in otherwise good health. No definitive anatomical lesions are found at autopsy that explain death. There is however, a correlation between SUDS and subtherapeutic levels of antiseizure medications. The purpose of this study was to retrospectively review and compare drug levels from a seizure patient that presented to an inner city emergency department to those from the medical examiners office. This study was prompted by a wrongful death claim for substandard care in a known seizure disorder patient. The claim alleged that the death was directly attributed to subtherapeutic seizure medication levels. We report the results from 150 seizure patients that presented to the emergency department with a chief complaint of a recent seizure and of 163 patients that were examined post mortem. 58% of the emergency department patients who were taking phenytoin, 79% taking carbamazapine and 82% of the patients taking phenobarbital had subtherapeutic levels. No patient in this population died and only 13% required hospital admission. These levels were comparable to the post mortem population.     

Comparison of DNA extraction and amplification from ancient human bone and mummified soft tissue

South american precolumbian male mummies were employed as source material for a comparative investigation of bone and soft tissues by DNA analysis. The suitability of the DNA extracts from both sources was tested and evaluated by their effectiveness as target DNA in PCR amplifications. The results suggest that skeletal material should be given preference over soft tissues for PCR analysis if the material is severely degraded. This seems to be independent of the specific anatomical origin of the samples.     

Investigation of single nucleotide polymorphism loci susceptible to degradation by ultraviolet light

DNA in biological fluids is often degraded by environmental factors. Given that single nucleotide polymorphism (SNP) analyses require shorter amplicons than short tandem repeat (STR) analyses do, their use in human identification using degraded samples has recently attracted attention. Although various SNP loci are used to analyze degraded samples, it is unclear which ones are more appropriate. To characterize and identify SNP loci that are susceptible or resistant to degradation, we artificially degraded DNA, obtained from buccal swabs from 11 volunteers, by exposure to ultraviolet (UV) light for different durations (254 nm for 5, 15, 30, 60, or 120 min) and analyzed the resulting SNP loci. DNA degradation was assessed using gel electrophoresis, STR, and SNP profiling. DNA fragmentation occurred within 5 min of UV irradiation, and successful STR and SNP profiling decreased with increasing duration. However, 73% of SNP loci were still detected correctly in DNA samples irradiated for 120 min, a dose that rendered STR loci undetectable. The unsuccessful SNP typing and the base call failure of nucleotides neighboring the SNPs were traced to rs1031825, and we found that this SNP was susceptible to UV light. When comparing the detection efficiencies of STR and SNP loci, SNP typing was more successful than STR typing, making it effective when using degraded DNA. However, it is important to use rs1031825 with caution when interpreting SNP analyses of degraded DNA.     

Performance evaluation of a mitogenome capture and Illumina sequencing protocol using non-probative,case-type skeletal samples: Implications for the use of a positive control in a next-generation sequencing procedure

Next-generation ancient DNA technologies have the potential to assist in the analysis of degraded DNA extracted from forensic specimens. Mitochondrial genome (mitogenome) sequencing, specifically, may be of benefit to samples that fail to yield forensically relevant genetic information using conventional PCR-based techniques. This report summarizes the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory’s (AFMES-AFDIL) performance evaluation of a Next-Generation Sequencing protocol for degraded and chemically treated past accounting samples. The procedure involves hybridization capture for targeted enrichment of mitochondrial DNA, massively parallel sequencing using Illumina chemistry, and an automated bioinformatic pipeline for forensic mtDNA profile generation. A total of 22 non-probative samples and associated controls were processed in the present study, spanning a range of DNA quantity and quality. Data were generated from over 100 DNA libraries by ten DNA analysts over the course of five months.The results show that the mitogenome sequencing procedure is reliable and robust, sensitive to low template (one ng control DNA) as well as degraded DNA, and specific to the analysis of the human mitogenome. Haplotypes were overall concordant between NGS replicates and with previously generated Sanger control region data. Due to the inherent risk for contamination when working with low-template, degraded DNA, a contamination assessment was performed. The consumables were shown to be void of human DNA contaminants and suitable for forensic use. Reagent blanks and negative controls were analyzed to determine the background signal of the procedure. This background signal was then used to set analytical and reporting thresholds, which were designated at 4.0X (limit of detection) and 10.0X (limit of quantiation) average coverage across the mitogenome, respectively. Nearly all human samples exceeded the reporting threshold, although coverage was reduced in chemically treated samples resulting in a ∼58% passing rate for these poor-quality samples. A concordance assessment demonstrated the reliability of the NGS data when compared to known Sanger profiles. One case sample was shown to be mixed with a co-processed sample and two reagent blanks indicated the presence of DNA above the analytical threshold. This contamination was attributed to sequencing crosstalk from simultaneously sequenced high-quality samples to include the positive control. Overall this study demonstrated that hybridization capture and Illumina sequencing provide a viable method for mitogenome sequencing of degraded and chemically treated skeletal DNA samples, yet may require alternative measures of quality control.     

Autosomal SNP typing of forensic samples with the GenPlex™ HID System: Results of a collaborative study

The GenPlex™ HID System (Applied Biosystems – AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250–500 pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500 pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500 pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500 pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power.     

Comparison of DNA extraction methods for identification of human remains

After mass disasters, where the bodies of victims have been degraded, soft tissues are often completely lost, with the result that bones and teeth are all that remain for identification. The aim of this study was to investigate three DNA extraction methods that are currently used with degraded bones in forensic contexts: a silica-based extraction protocol used by the International Commission for Missing Persons (ICMP), a total demineralisation method used by the US Armed Forces DNA Identification Laboratory (AFDIL) and a standard organic DNA extraction method used by the Australian Federal Police (AFP). The methods were compared by real-time PCR quantitation and STR profiling. The silica and organic methods were not significantly different for either of these measures for bone but were both significantly more successful than the demineralisation method. Bovine serum albumin (BSA) was found to significantly improve the DNA yields from real-time PCR and the number of reportable alleles from STR typing, especially when using bone samples.     

Applicability of the parentally imprinted allele (PIA) typing of a VNTR upstream the H19 gene to forensic samples of different tissues

The parentally imprinted allele (PIA) typing that we have recently developed determines parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene. The usefulness of this typing was demonstrated by its application to blood samples in paternity cases. However, its applicability to other tissue DNA remains to be tested. DNA samples from fifteen different postmortem tissues such as cerebrum, skeletal muscle and skin were examined, all of which were obtained from three autopsy cases 2-11h after death. DNA was digested with a methylation-sensitive HhaI enzyme and diluted solutions of the digests were subjected to the first PCR amplification, providing amplification of only the paternal H19 methylated allele. Subsequent VNTR typing was carried out for the amplified products to determine which allele was of paternal origin. No tissue-dependent difference was observed and all the samples examined, though degraded, were successfully used for determining the paternal allele. These results substantiate the usefulness of PIA typing in forensic examinations. Its application to two identity cases, a burned male body and a male body with adipocere formation, was also shown.     

Post-mortem redistribution of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") in the rabbit. Part I: experimental approach after in vivo intravenous infusion

Post-mortem redistribution is known to influence blood and tissue levels of various drugs. An animal model was used in an attempt to elucidate this problem for the amphetamine analogue, 3,4-methylenedioxymethamphetamine (MDMA). Rabbits received 1 mg/kg MDMA intravenously (iv) and were killed 2 h later in order to simulate the state of complete distribution in the body. MDMA and 3,4-methylenedioxyamphetamine (MDA) concentrations were determined in blood, urine, bile, vitreous humour, and various tissues (eye globe walls, brain, cardiac muscle, lungs, liver, kidneys, iliopsoas muscle and adipose tissue) using a high pressure liquid chromatographic (HPLC) procedure with fluorescence detection. In the first group (control group, sampling immediately post mortem) considerable MDMA concentrations were found in the brain and both lungs. In addition, our data indicate the elimination of MDMA by hepatic biotransformation and excretion via the bile. When the animals were preserved either 24 or 72 h post mortem (second group), an increase of MDMA and MDA levels in the liver and the eye globe walls was noticed. In the lungs, on the other hand, they tended to decline as a function of increasing post-mortem interval. MDMA levels in cardiac and iliopsoas muscle were fairly comparable and remained stable up to 72 h after death. In the third group, ligation of the large vessels around the heart took place immediately post mortem, but significant differences in blood and tissue MDMA concentrations between rabbits of group 2 and 3 could not be demonstrated. We therefore conclude that post-mortem redistribution of MDMA at the cellular level (viz. by pure diffusion gradient from higher to lower concentrations) is more important than its redistribution via the vascular pathway. Finally, MDA levels were relatively low in all samples, thus indicating that this is not a major metabolite in the rabbit, at least within the first 2 h after administration.     

HLA typing of aortic tissues from unidentified bodies using hot start polymerase chain reaction-sequence specific primers

This study investigated the identification of unidentified bodies through HLA DNA typing of aortic tissues. Eight aortas were collected at autopsy from six bodies found in water, one burnt body, and one mummified body, respectively. DNA was extracted from 10-20 mg of aortic tissue with a PUREGENE DNA Isolation Kit and phenol/chloroform. HLA (A, B, DR or DQ locus) alleles were typed using the hot start polymerase chain reaction-sequence specific primers (PCR-SSP) method with a Dynal AllSet(+) SSP "low resolution" kit for each locus. The aorta was still retained in degraded samples, those from the drowned, the burnt, and the mummified bodies. In each case, approximately 0.04-3.84 microg of DNA was recovered from 1 mg of fixed tissue. We typed 8/8 for the DR, 4/4 for the DQ, 3/3 for the A, and 3/3 for the B locus from the samples. Based on these results and the finding that DNA extraction is easier from the aorta than from other samples such as bone or tooth, we considered this method to be useful for forensic samples.     

Detection of the age-dependent 4977 bp deletion of mitochondrial DNA

In recent years a number of mitochondrial DNA (mtDNA) deletions have been detected in various tissues from individuals over 20 years of age. It has been postulated that these deletions are associated with natural aging. In order to determine whether a correlation exists between age and the amount of deleted 4977 bp mtDNA, we used two PCR reactions to study total DNA (nuclear and mitochondrial DNA) extracted from skeletal muscle (m. iliopsoas) obtained at autopsy from 93 individuals representing a wide age spectrum (range: 3 months–97 years). The primer pair L15/H15 was used to amplify a 533 bp fragment of intact mtDNA to determine the percentage of total DNA. A second PCR with the primer pair L35/H35 was then employed to amplify a 667 bp fragment of the deleted mtDNA. The amount of template DNA necessary to amplify the specific fragments of deleted mtDNA was found to decrease with age. Whereas no 4977 bp deletion could be detected in subjects under 20 years of age even with 1000 ng of total DNA, in individuals aged 21 to 30 years 1000 ng total DNA were sufficient. Only 1 ng total DNA was needed in all individuals over 70. Our results show that the 4977 bp deletion can be a useful marker of natural aging in human subjects. Received: 24 September 1996 / Received in revised form: 24 April 1997     

Screening for single nucleotide polymorphisms in highly degraded DNA by using the amplified fragment length polymorphism technique

Short tandem repeat (STR) analysis is generally used for human identification of forensic samples; however, standard STR analysis sometimes fails to generate full profiles since DNA is frequently degraded by various environmental factors. Recently, single nucleotide polymorphism (SNP) analysis has attracted attention for human identification since the shorter amplicons are better suited for degraded samples. Though various SNP loci are used for analysis of degraded samples, it is unclear which ones are more appropriate. To identify SNPs that were resistant to degradation, we artificially degraded DNA obtained from the buccal swabs of six volunteers and the K562 cell line by heat treatment. Subsequently, the amplified fragment length polymorphism (AFLP) technique was used for SNP screening. We focused on the AFLP bands detected in both the heat-treated and untreated samples, and DNA extracted from these bands was directly sequenced. DNA degradation increased as the duration of heat treatment increased, and no STR profiles could be generated after 6 h of heat treatment. When the AFLP band patterns were compared between 6 h heat-treated and untreated samples, eight common bands were detected. The sequences of the DNA fragments of these common bands had higher adenine-thymine (A-T) content and included 17 SNPs. The SNPs detected in the heat-treated and untreated samples were considered to be resistant to degradation. Although there was a little information available in databases regarding the nine SNPs identified in this study, this study shows that some of these SNPs might be useful for human identification of extremely degraded DNA.     

Real-time PCR assay for the detection of picoplankton DNA distribution in the tissues of drowned rabbits

The detection of plankton DNA is one of the important methods for the diagnosis of drowning from postmortem tissues. This study investigated the quantities of picoplankton (Cyanobacteria) DNA in the lung, liver, kidney tissues and blood of drowned and non-drowned rabbits, and the sensitivity of detection of picoplankton DNA by polymerase chain reaction (PCR) detect for the diagnosis of death from drowning. For this purpose, the DNA of the 16S ribosomal RNA gene of picoplankton was quantitatively assayed from the tissues of drowned and non-drowned rabbits immersed in water after death. Each of the liver, kidney and lung tissues and blood were obtained from drowned and non-drowned rabbits. Picoplankton DNA in the tissues was extracted using the DNeasy® Blood & Tissue kit to determine the yield of picoplankton DNA from each tissue. TaqMan real-time PCR was performed for quantitative analysis of picoplankton DNA. Target DNA was detected in the liver, kidney and lung samples obtained from the drowned rabbits, while no picoplankton DNA was detected in the non-drowned rabbit tissues (except in lung samples). The results verified that direct PCR for the detection of picoplankton DNA is useful for the diagnosis of drowning. Although we observed seasonal changes in the quantity of picoplankton in river water, we were able to detect DNA from various organs of drowned bodies during the season when picoplankton were not the most abundant.