Current perspectives in the interpretation of gunshot residues in forensic science: A review.

The traces produced when a firearm is discharged can provide important information in cases when questions regarding a possible association of the firearm with a person of interest (POI), time since discharge or shooting distance are raised. With advances in technology, the forensic challenges presented by these traces, known as gunshot residues (GSR), are moving from the analytical domain to the interpretation of the analytical results. Different interpretation frameworks are currently competing. Formal classification of particles, using standards such as that produced by ASTM, focusses only on evaluation of evidence at the sub-source level. Another approach, based on the application of Bayesian reasoning - namely the case-by-case approach - has been proposed that allows evaluation of evidence in regards to activity-related questions. This alternative approach allows an evaluation of the evidence that is more closely aligned to judicial and investigative aims. This paper critically presents the state of the art in regards to GSR interpretation in a holistic manner.

Molecular genetic analysis of 1053 Danish individuals with clinical signs of familial hypercholesterolemia.

The lipid disorder familial hypercholesterolemia (FH) predisposes to cardiovascular disease. With a prevalence of approximately one in 500 in the general Caucasian population, FH is one of the most frequent single-gene disorders. As the mutational spectra vary between populations, it is crucial to identify the mutations in a given population in order to implement a molecular genetic screening strategy. A total of 1053 referred individuals with clinical signs of FH were investigated, and mutations were identified in 425 individuals. Fifty-four different mutations were identified, of which 13 are novel. The five most frequent mutations accounted for 56.3% of all disease-causing mutations. The majority of the remaining mutations were of a private nature only encountered in single families. In this study, a reliable molecular genetic screening protocol was established, and the relevance of performing presymptomatic genetic analysis as part of a preventive strategy was documented. We have acquired knowledge of the mutational spectra in the Danish population and thus will be able to trace mutations in their relatives through our index cases.

Expression and characterization of six clinically relevant uroporphyrinogen decarboxylase gene mutations.

The functional consequence of six uroporphyrinogen decarboxylase (UROD) gene mutations found in Danish patients with familial porphyria cutanea tarda was investigated. Wild-type UROD and the 6 mutants (3 missense, 1 nonsense and 2 frameshift mutants) were cloned and expressed using the prokaryotic gGEX-6P system, in which the protein is produced in fusion with glutathione S-transferase (GST). Enzymatic activity of the purified recombinant mutant fusion proteins ranged from undetectable to less than 12% of the recombinant wild-type protein. Mutant proteins cleaved from the GST part did not retain any catalytic activity. These observations can be ascribed to the structure/function relationships of the enzyme, and the fact that the enzyme is a dimer in its active form. Although the clinical manifestation of familial porphyria cutanea tarda is complex, the findings support the notion that different mutations may affect individuals differently.

Characterization of two isoalleles and three mutations in both isoforms of purified recombinant human porphobilinogen deaminase.

Defects in the enzyme porphobilinogen deaminase (PBG-D) are associated with acute intermittent porphyria (AIP). Human PBG-D is transcribed into a housekeeping or an erythroid form as a result of differential promoter usage and splicing. In addition, three pairs of isoallelic forms have been described. However, whether the enzymatic properties of housekeeping and erythroid forms differ is unknown. In this study the two isoallelic forms, K210 and E210, were cloned and expressed in Escherichia coli together with three mutations associated with a clinical AIP phenotype. The mutations were introduced in the K210 isoallelic background and expressed as both the housekeeping and the erythroid form. The proteins were expressed as GST fusions and purified to homogeneity. Initial experiments revealed that the GST-PBG-D fusions and the purified PBG-D obtained by proteolytic removal of the GST moiety had enzymatic properties that were indistinguishable. Consequently, all analyses with mutant PBG-D were performed on the GST-fusion proteins. Comparison of the wild-type proteins revealed a significant difference in Km between isoalleles with a Km of 9 microM for K210 and 7 microM for E210, whereas no significant difference in activity or kinetics between the housekeeping and the erythroid isoforms was observed. The mutant proteins showed 0.3-1.0% wild-type activity, depending on mutation. There was a clear correlation between yield of recombinant protein and CRIM status of patients. Furthermore, co-expression of the mutant proteins with the bacterial chaperone GroESL did not affect protein yield or function to any significant extent, supporting the view that the investigated mutations primarily influence structure and function and not folding of the proteins.

Mutations in endoglin and in activin receptor-like kinase 1 among Danish patients with hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is a rare disorder with one per 6000-10,000 affected individuals in the general Caucasian population. HHT is genetically heterogeneous, involving at least two loci HHT1 mapping to chromosome 9q34.1 and HHT2 mapping to chromosome 12q31. The loci have been identified as endoglin (ENG) and activin receptor-like kinase 1 (ALK1). In order to gain knowledge of the genotype distribution and prevalence in the Danish population and to establish a reproducible and sensitive molecular genetic test method, we developed a denaturating gradient gel electrophoresis protocol for mutation scanning of the two loci. Twenty-five Danish HHT families were tested. A total of eight new as well as seven previously reported mutations were identified. A founder mutation was characterized present in seven families and possibly introduced around 350 years ago. In one individual, a presumed spontaneous mutation was characterized. The method developed proved to be very sensitive for mutation detection in both ENG and ALK1. Genetic screening in HHT families facilitates an early treatment strategy for silent HHT manifestations in first degree relatives.

[Randomized controlled trial of structured, individualized treatment of type 2 diabetes mellitus. The project of Diabetes Care in General Practice]. / Randomiseret kontrolleret undersøgelse af struktureret, individualiseret behandling af type 2-diabetes mellitus. Projektet Diabetesomsorg i almen praksis.

INTRODUCTION: We assessed the effect of a multifaceted intervention directed at general practitioners to improve type 2 diabetes care. MATERIALS AND METHODS: Three hundred and eleven Danish practices with 474 general practitioners were randomised to structured personal care (intervention group) or routine care (comparison group). Of 970 surviving patients (aged 40+ years) diagnosed with diabetes in 1989-1991, 874 (90.1%) were assessed after 6 years. Intervention comprised regular follow-up and individualized goal-setting, supported by reminders to doctors, clinical guidelines, feed-back, and continuing medical education. RESULTS: Predefined non-fatal outcomes and mortality were the same in both groups. The following risk factor levels were lower in the intervention patients than in the comparison patients: fasting plasma glucose (7.9 vs 8.7 mmol/l, medians, P = 0.0007), haemoglobin A1c (8.5 vs 9.0%, P < 0.0001, normal range 5.4-7.4%), systolic blood pressure (145 vs 150 mmHg, P = 0.0004), and cholesterols (6.0 vs 6.1 mmol/l, P = 0.029, baseline-adjusted). Both groups had sustained a weight loss since diagnosis (2.6 vs 2.0 kg). Metformin was the only drug used more frequently in the intervention group (24 vs 15%). Intervention doctors arranged more follow-up consultations, referred fewer patients to diabetes clinics, and were more optimistic in their goal-setting. DISCUSSION: In primary care, individualized goal-setting with educational and surveillance support may for at least six years bring risk factors of patients with type 2 diabetes to a level that in other trials has been shown to reduce diabetic complications, but without adverse weight gain.

Randomised controlled trial of structured personal care of type 2 diabetes mellitus.

OBJECTIVE: To assess the effect of a multifaceted intervention directed at general practitioners on six year mortality, morbidity, and risk factors of patients with newly diagnosed type 2 diabetes. DESIGN: Pragmatic, open, controlled trial with randomisation of practices to structured personal care or routine care; analysis after 6 years. SETTING: 311 Danish practices with 474 general practitioners (243 in intervention group and 231 in comparison group). PARTICIPANTS: 874 (90.1%) of 970 patients aged >/=40 years who had diabetes diagnosed in 1989-91 and survived until six year follow up. INTERVENTION: Regular follow up and individualised goal setting supported by prompting of doctors, clinical guidelines, feedback, and continuing medical education. MAIN OUTCOME MEASURES: Predefined clinical non-fatal outcomes, overall mortality, risk factors, and weight. RESULTS: Predefined non-fatal outcomes and mortality were the same in both groups. The following risk factor levels were lower for intervention patients than for comparison patients (median values): fasting plasma glucose concentration (7.9 v 8.7 mmol/l, P=0.0007), glycated haemoglobin (8.5% v 9.0%, P<0.0001; reference range 5.4-7.4%), systolic blood pressure (145 v 150 mm Hg, P=0.0004), and cholesterol concentration (6.0 v 6.1 mmol/l, P=0.029, adjusted for baseline concentration). Both groups had lost weight since diagnosis (2.6 v 2.0 kg). Metformin was the only drug used more frequently in the intervention group (24% (110/459) v 15% (61/415)). Intervention doctors arranged more follow up consultations, referred fewer patients to diabetes clinics, and set more optimistic goals. CONCLUSIONS: In primary care, individualised goals with educational and surveillance support may for at least six years bring risk factors of patients with type 2 diabetes to a level that has been shown to reduce diabetic complications but without weight gain.

Certification of the mass concentration of creatine kinase isoenzyme 2 (CK-MB) in the reference material BCR 608.

We describe the certification of a mass concentration value in the already prepared creatine kinase-2 reference material (BCR 608). Creatine kinase-2 was purified from human heart. The purified enzyme was diluted in order to measure its protein concentration by the Doetsch method. A protein concentration value of 124.30+/-13.17 mg/l was assigned to the stock solution of purified creatine kinase-2. This stock solution was diluted in 25 mmol/l piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES) pH 7.2, containing 2 mmol/l ADP, 5 mmol/l 2-mercaptoethanol, 154 mmol/l sodium chloride and 50 g/l human albumin to obtain a stable liquid standard of known creatine kinase-2 mass concentration (80.36 microg/l). This standard was then used to recalculate the creatine kinase-2 mass concentration measured in the BCR 608 material by immunoassay. The mass concentration of creatine kinase-2 in samples of reconstituted BCR 608 was certified to be 93.30+/-9.65 microg/l.

[Questions as evident key to knowledge: teaching medical students evidence-based medicine]. / Spørgsmålet som (e)vidensnøgle: undervisning af medicinstuderende i evidensbaseret medicin.

INTRODUCTION: In Denmark, as in other countries, there is an increasing focus on evidence-based medicine (EBM) as a necessary tool for using modern sources of information, but until now EBM training has not been incorporated in our undergraduate curriculum. MATERIAL AND METHODS: This course is given in the ninth semester (out of 13) and the subject matter is clinical biochemistry. The course consists of seven (one-hour) lectures over three weeks. First, the EBM method is introduced, then the students split up into small groups, choose their own diagnostic problem, and carry out a structured search. The search leads to the choice of a scientific article, which is subsequently presented to and discussed by all the students. We asked about their opinion of the course by questionnaire. RESULTS: The answers to the questionnaire confirmed that the students have improved their ability to read and assess scientific articles and to seek information, and it has stimulated them to understand concepts instead of memorising details. DISCUSSION: Although the assessment of this course was positive, it can no doubt be improved and further developed. In our opinion the use of EBM should not be confined to one course. The medical students should be introduced to EBM at an early stage, thereby abling them to practice it throughout their training.

Calibration, specificity and trueness of a postheparin plasma lipoprotein lipase assay.

Measurement of lipoprotein lipase activity in postheparin plasma is generally accompanied by moderate within-run variation CV(W-R) (<10%) and higher between-run variation CV(B-R) (5-25%). A calibration system was introduced in order to improve the reproducibility of measurements and to compare lipoprotein lipase activities from different days. Every day a calibration curve for lipoprotein lipase activity was constructed. Fifteen calibration curves designed over 2 years, show linearity over the whole biological spectrum and a considerable reduction of between-run variation in lipoprotein lipase activity, from 42% to 5.3% as estimated from two control postheparin plasma samples. The lipoprotein lipase calibration system is an easy and very cheap arrangement, which makes it possible to compare lipoprotein lipase activities achieved over years. When the lipoprotein lipase control values are compared with reference lipoprotein lipase samples determined in other lipase laboratories, the calibration-control system becomes an important tool for reducing analytical bias. The article reviews the original analytical criteria of catalytic measurement of lipoprotein lipase activity and describes the implementation of the calibration-control system. We describe a model for reduction of the analytical variability in the measurement of lipoprotein lipase activity. Other standardization efforts need to be made in the future, especially to define the reference material for calibration.

Production and certification of an enzyme reference material for adenosine deaminase 1 (BCR 647).

BACKGROUND: We describe the preparation of a lyophilised reference material containing purified human adenosine deaminase 1 and the certification of its catalytic concentration. METHODS: The enzyme was purified from human erythrocytes. RESULTS: The enzyme was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts (<0.4%) of alanine aminotransferase, aspartate aminotransferase and L-lactate dehydrogenase were detected in the purified fraction. The purified adenosine deaminase had a molar mass of 41600 g/mol and an isoelectric pH at 4.7, 4.85 and 5.0. The material was prepared by diluting the purified adenosine deaminase in a matrix containing 50 mmol/l Tris-HCl buffer pH 7.4 and 30 g/l human serum albumin; dispensing in vials and freeze-drying. The batch was homogeneous and the predicted loss of adenosine deaminase activity per year on the basis of accelerated degradation studies was 0.006% at -20 degrees C and 0.04% at 4 degrees C. The certified value for adenosine deaminase catalytic concentration in the reconstituted reference material is (2.55+/-0.09) microkat/l when measured by the method that uses adenosine as substrate and glutamate dehydrogenase as auxiliary enzyme at 37 degrees C. CONCLUSIONS: The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the adenosine deaminase catalytic concentration measurements.

[Medical graduates from the Odense University 1972-1998. Recruitment and employment]. / Laegevidenskabelige kandidater fra Odense Universitet 1972-1998. Rekruttering og beskaeftigelse.

The aim was to create and maintain a registration of all medical graduates from Odense University from the first graduates in 1972 and to describe the graduates' background and subsequent professional employment. All graduates from 1972 to 1998 are included. About 55% of the graduates came from secondary schools in the country og Funen, 20% from southern Jutland and 15% from Copenhagen and Jutland north of Vejle. About 45% became general practitioners after 5-22 years (median 11 years), a third of them in the county of Funen. Twenty-five percent became consultants after 11-17 years (median 14 years), twenty-five percent of these in the county of Funen. The cumulated death rate was 5% for the ages 30-55 years. At 17 years after graduation 83% had become specialists, at ten years the number was 55%. In conclusion, Odense University recruits 75% of its medical students from southern Denmark. Of the graduates that become general practitioners, about two-thirds are employed in southern Denmark, for consultants the corresponding figure is 50%.

Association between CYP1A2 polymorphism and susceptibility to porphyria cutanea tarda.

Individuals with the most common form of the porphyrias, porphyria cutanea tarda (PCT), are believed to be genetically predisposed to development of clinically overt disease through mutations and polymorphisms in genes associated with known precipitating factors. In this study, we have examined a group of Danish patients with PCT for the presence of the C/A polymorphism in intron 1 of CYP1A2. The results demonstrate that the frequency of the highly inducible A/A genotype is increased in both familial and sporadic PCT. This suggests that inheritance of this genotype is a susceptibility factor in development of PCT.

Biochemical markers of ischaemia for the early identification of acute myocardial infarction without St segment elevation.

Blood was collected on admission and after 1-2 h in 130 consecutive patients admitted with typical chest pain in order to assess the capacity of myoglobin, fatty-acid-binding protein (FABP), CK-MB mass, and troponin I (TnI) in the early identification of acute myocardial infarction (AMI) without ST elevation. Using the maximum value within 6 h of onset of symptoms, AMI was detected with a 90-95% sensitivity and a 81-94% specificity by FABP at a cut-off level 8-12 midrog/l, or 81-86% and 89-93%, respectively, by myoglobin at a cut-off level 70-90 microg/l. CK-MB mass and TnI had low sensitivity, albeit very high specificity. As almost all AMI patients were identified within 6 h, serial measurements of FABP or myoglobin ruled out AMI with a very high degree of certainty. Due to the low prevalence of AMI (16%), the positive predictive values were modest (47-73%), yet increasing the probability of AMI by a factor 3-4. Myoglobin and FABP are very useful markers in the early triage of chest pain patients.

Uroporphyrinogen decarboxylase gene mutations in Danish patients with porphyria cutanea tarda.

Decreased uroporphyrinogen decarboxylase (UROD) activity is a characteristic feature of the most common of the porphyrias, porphyria cutanea tarda (PCT). A subgroup of the clinically overt PCT cases is associated with mutations in the gene encoding UROD and inherited as an autosomal-dominant trait. In this study, DNAs from 53 Danish PCT patients were subjected to genetic analysis for UROD mutations using denaturing gradient gel electrophoresis. Eleven genetic variations, seven of which are possible disease causing, were identified. All but one of these mutations were previously unknown, lending further support to the assumption that PCT is a heteroallelic disease. Only 11% of the examined patients were previously recognized as familial PCT cases. However, possible disease-related UROD mutations were identified in 24% of the examined patients, indicating that genetic analysis of PCT patients may improve differentiation between familial and sporadic PCT cases.

Phenotypes and genotypes for CYP2D6 and CYP2C19 in a black Tanzanian population.

AIMS: CYP2D6 and CYP2C19 are polymorphically expressed enzymes that show marked interindividual and interethnic variation. The aim of this study was to determine the frequency of the defective alleles in CYP2D6 and CYP2C19 in Africans and to test whether the genotype for CYP2C19 is better correlated with the proguanil/cylcoguanil ratio than the mephenytoin S/R ratio. METHODS: Two hundred and sixteen black Tanzanians were phenotyped for CYP2D6 with the use of sparteine, and for CYP2C19 with the use of mephenytoin and proguanil. Of these 196 subjects were also genotyped for CYP2D6 (including the CYP2D6*1, CYP2D6*3 and CYP2D6*4 alleles) and 195 were genotyped for CYP2C19 (including the CYP2C19*1, CYP2C19*2 and the CYP2C19*3 alleles). Furthermore 100 subjects were examined for the allele duplication in CYP2D6, leading to ultrarapid metabolism, with long PCR. RESULTS: The sparteine metabolic ratio (MR) was statistically significantly higher in the Tanzanian group of homozygous, extensive metabolizers compared to a historical control group of white Danish extensive metabolizers. Only one poor metabolizer for CYP2D6 (MR=124 and genotype CYP2D6*1/CYP2D6*4 ) was found. The gene frequencies were 0.96 for the CYP2D6*1 allele and 0.04 for the CYP2D6*4 allele. No CYP2D6*3 alleles were found. Nine subjects had an allele duplication in CYP2D6 (9%). For CYP2C19 there were seven subjects (3. 6%) who were phenotyped as poor metabolizers, but only three subjects (1.5%) had a genotype (CYP2C19*2/CYP2C19*2 ) indicative of poor metabolism. The gene frequencies were 0.90 for the CYP2C19*1 allele and 0.10 for the CYP2C19*2 allele. No CYP2C19*3 alleles were found. The mephenytoin S/R ratios were not bimodally distributed. CONCLUSIONS: Both the genotyping and phenotyping results show that there is a substantial difference between an African black population and a Caucasian population in the capacity to metabolize drugs via CYP2D6 and CYP2C19.

Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis. Identification and characterization of six novel mutations associated with familial PCT.

The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able to detect the underlying UROD mutation in 10 previously characterized DNA samples as well as a new mutation in each of six previously unexamined PCT patients. The six novel UROD mutations comprise three missense mutations (M01T, F229L, and M324T), two splice mutations (IVS3-2A-->T and IVS5-2A-->G) leading to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual catalytic activity, whereas the two missense mutants retained some residual activity. Furthermore, the missense mutants exhibited a considerable increase in thermolability. The six new mutations bring to a total of 29 the number of disease-related mutations in the UROD gene. The DGGE assay presented greatly improves the genetic diagnosis of fPCT and HEP, thereby facilitating the detection of familial UROD deficient patients as well as the discrimination between familial and sporadic PCT cases.