Estimation of time since death by vitreous humor hypoxanthine, potassium, and ambient temperature.

Measurement of vitreous humor potassium (K(+)) has since the 1960s been recognized as an adjunct for estimation of time since death. In 1991 we introduced hypoxanthine (Hx) as a new marker. Furthermore we demonstrated that time since death estimation was more accurate when ambient temperature was included in the calculations, both for K(+) and for Hx. In this paper we present a refined method. The subjects consist of 132 cases with known time of death and ambient temperature. One sample from each subject was used in the calculations. Vitreous humor Hx levels were available in all subjects, while K(+) was measured in 106 of the subjects, due to insufficient volume of vitreous humor. Linear regression analysis was applied to model the correlation between vitreous humor Hx and K(+), taking the interactions with temperature into consideration. The diagrams published in 1991, which also included ambient temperature, estimated median time since death with range between the 10th and 90th percentile, whereas the linear regression analysis presented in this paper estimates mean time since death with a corresponding 95% interval of confidence. We conclude that time since death may be estimated with relatively high precision applying vitreous humor Hx and K(+) concentrations combined with ambient temperature.

SPG20, a novel biomarker for early detection of colorectal cancer, encodes a regulator of cytokinesis.

Colorectal cancer is a common disease with high mortality. Suitable biomarkers for detection of tumors at an early curable stage would significantly improve patient survival. Here, we show that the SPG20 (spastic paraplegia-20) promoter, encoding the multifunctional Spartin protein, is hypermethylated in 89% of colorectal carcinomas, 78% of adenomas and only 1% of normal mucosa samples. SPG20 methylation was also present in a pilot series of stool samples and corresponding tumors from colorectal cancer patients. SPG20 promoter hypermethylation resulted in loss of mRNA expression in various cancer types and subsequent depletion of Spartin. We further showed that Spartin downregulation in cancer cells resulted in cytokinesis arrest, which was reversed when SPG20 methylation was inhibited. The present study identifies SPG20 promoter hypermethylation as a biomarker suitable for non-invasive detection of colorectal cancer, and a possible mechanism for cytokinesis arrest in colorectal tumorigenesis.

The brain-derived neutrophic factor val66met polymorphism and sudden unexpected infant death.

AIM: Findings of hypoxia prior to death and involvement of a dysregulation of the serotonergic network in sudden infant death syndrome (SIDS) may indicate that brain-derived neutrophic factor (BDNF) also is of importance with regard to sudden unexpected infant death. Based on this, the purpose of this study was to investigate the BDNF val66met polymorphism in SIDS cases, cases of infectious death and controls. METHODS: The polymorphism was investigated in 163 SIDS cases, 34 cases of infectious death and 121 controls, using real-time PCR and fluorescence melting curve analysis. RESULTS: There were no differences in val66met genotype distribution between neither the SIDS cases nor the cases of infectious death and controls (p = 0.95 and p = 0.52, respectively). CONCLUSION: The study indicates that the val66met polymorphism is not important for sudden unexpected infant death. However, several other SNPs in the BDNF gene, as well as in other genes involved in this pathway, including G-protein, have to be investigated to fully exclude any involvement of BDNF in SIDS.

Bacteriological investigation--significance of time lapse after death.

In cases of sudden unexpected death in infants and children (SUDI), microbiological investigation has been an important part of the autopsy protocol at the University of Oslo for the last 15 years. The purpose of this study was to compare the microbiological findings in samples taken at hospital admittance shortly after death and at autopsy. Blood cultures and cerebrospinal fluid (CSF) were collected both at the hospital and at autopsy; organ samples were additionally collected at autopsy. Hospital samples were collected at a median of 4.5 h (95% confidence interval [CI] 3.25-5) and autopsy samples at a median of 24.25 h (95% CI 22-25.5) after death. The proportion of positive cultures was stable over time; the post mortal time had no influence on bacterial growth. As long as the autopsy is performed within 48 h after death, prior microbiological examination is unnecessary. Blood culture, CSF and lung specimens are the best predictors in our study.

Determination of cotinine in pericardial fluid and whole blood by liquid chromatography-tandem mass spectrometry.

Cotinine is the main metabolite of nicotine and is used as an indicator of exposure to tobacco smoke. A method has been developed for quantification of cotinine in pericardial fluid and whole blood collected from autopsy casework involving cases of infant death. Sample clean-up was achieved by solid-phase extraction with a mixed-mode column. Cotinine was quantified by liquid chromatography-tandem mass spectrometry. Positive ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analyte and one for the internal standard, cotinine-d(3). The calibration range was 0.9-176 ng/mL for cotinine in both matrixes. The recovery of the analyte ranged from 86 to 92%, and the between-assay precisions ranged from 4 to 6% relative standard deviation. Whole blood and pericardial fluid samples from 95 infant deaths obtained during autopsy were analyzed. A strong correlation (R(2) = 0.97) was found between the cotinine concentrations in pericardial fluid and blood. The correlation was not affected by the postmortem time interval. This study demonstrates that pericardial fluid may be an alternative specimen to blood for quantification of cotinine in forensic autopsies.

Molecular genetic analysis of long QT syndrome in Norway indicating a high prevalence of heterozygous mutation carriers.

Mutations in the KCNQ1, HERG, SCN5A, minK and MiRP1 genes cause long QT syndrome (LQTS), of which there are two forms: the Romano Ward syndrome and the Jervell and Lange-Nielsen syndrome. We have performed DNA sequencing of the LQTS-associated genes in 169 unrelated patients referred for genetic testing with respect to Romano Ward syndrome and in 13 unrelated patients referred for genetic testing with respect to Jervell and Lange-Nielsen syndrome. A total of 37 different mutations in the 5 genes, of which 20 were novel, were identified. Among patients with the most stringent clinical criteria of Romano Ward syndrome, a mutation was identified in 71%. Twelve of the 13 unrelated patients referred for genetic testing with respect to Jervell and Lange-Nielsen syndrome were provided with a molecular genetic diagnosis. Cascade genetic screening of 505 relatives of index patients with molecularly defined LQTS identified 251 mutation carriers. The observed penetrance was 41%. Although caution must be exerted, the prevalence of heterozygotes for mutations in the LQTS-associated genes in Norway could be in the range 1/100-1/300, based on the prevalence of patients with Jervell and Lange-Nielsen syndrome.

Frequency of acute asymptomatic myocardial infarction and an estimate of infarct age in cases of abrupt sudden death observed in a forensic autopsy material.

The aim of this study is to determine the frequency of acute infarcts at autopsy in cases of unexpected abrupt deaths in persons with coronary heart disease. In addition, we want to estimate the time between onset of infarct and death based on evolving tissue changes in the infarct known to occur during the first hours. Thirty cases of unexpected, abrupt deaths were selected from a forensic autopsy material. Half of them had a preliminary diagnosis of coronary heart disease, the other half a preliminary diagnosis not involving the heart or chest area. Complete autopsies were performed. The myocardium and the coronary arteries were sampled and examined without knowledge of the gross findings or to which group the case belonged. Myocardial infarcts and acute coronary changes were found in both groups, less frequently in the non-coronary group. The age of the myocardial and coronary lesions was estimated by observing morphological characteristics changing with time, e.g. increasing polymorphonuclear leucocytes in the infarcted myocardium, and increasing amount of fibrin in thrombi. The majority of cases in the coronary group died with an extensive asymptomatic myocardial infarction, which probably had lasted 5-6 hrs or less. Acute changes in the right coronary artery and its area of supply prevailed. Acute myocardial infarcts were observed also in a minority of the non-coronary group, but myocardial infarction was not the cause of death in any of them. Abrupt coronary death is most often preceded by an extensive asymptomatic myocardial infarction within the last 5-6 hrs.

Genetic and protein markers related to in situ growth and multiplicity in small sporadic colorectal adenomas.

BACKGROUND: Some early genetic events in the development of colorectal adenomas are known, but their relationship to in vivo growth characteristics is uncertain. This study compared in situ size changes and other clinicopathological variables with selected genetic and protein markers. METHODS: 56 adenomas (< or = 10 mm) from 39 patients were analysed for APC, CTNNB1 and K-ras mutations, allelic imbalance on 1p and 18q, microsatellite instability and immunohistochemical expression of HLA-DR, BAX, BCL-2 and Ki-67. For 42 of the adenomas, in situ growth was measured over 3 years. The total number of polyps in each patient was recorded. RESULTS: K-ras was mutated in 8/56 adenomas. None of the regressing adenomas revealed such mutations, compared to 20% in those that maintained or increased their size. Multivariate linear regression analysis showed that tumour growth was higher in females compared to males, and was even higher in the presence of a K-ras mutation. APC mutations were found in 37/56 adenomas. CTNNB1 mutations were found in 2/19 adenomas without APC mutation. Deletions of 1p were found in 12/56 adenomas and, seemingly, most frequent in patients with few tumours. The most frequently expressed protein was BAX (33/41), but neither this nor the other proteins showed associations with an in situ growth pattern. CONCLUSION: The multivariate linear regression model showed that patient gender and the presence of K-ras mutation had significant effects on tumour growth. The lack of the proliferative stimulus resulting from a K-ras mutation may contribute to the process of adenoma regression.

APC and CTNNB1 mutations in a large series of sporadic colorectal carcinomas stratified by the microsatellite instability status.

BACKGROUND: Adenomatous polyposis coli (APC) and beta-catenin (encoded by CTNNB1) are important components in the WNT signalling pathway, a pathway altered in nearly all colorectal tumours. Conflicting results are reported on whether APC mutations are less common in tumours with a high degree of microsatellite instability (MSI-H) than in microsatellite stable (MSS) ones, and whether mutations in the regulatory domain of CTNNB1 substitute for APC mutations in the MSI-H tumours. METHODS: A consecutive series of 218 primary colorectal carcinomas, stratified by MSI status, were analysed for mutations in the APC gene (by the protein truncation test) and in the CTNNB1 gene (by single-strand conformation polymorphism). RESULTS: APC mutations detected in 66% of the patients were significantly more frequent in the MSS and MSI-L (low) tumours than in the MSI-H tumour group (P < 0.001). The MSI-H tumours tended to have more frameshift mutations than the MSS/MSI-L tumours. The majority of the APC mutations were located in the mutation cluster region (MCR). Patients that had lost all beta-catenin binding sites of the APC gene showed a shorter survival time than patients who retained some or all of these binding sites (P = 0.045). Two mutations were found in the CTNNB1 gene, but neither of them was located in the regulatory domain in exon 3. CONCLUSION: This study confirms that APC mutations are less frequent in MSI-H tumours than in MSS and MSI-L tumours. However, CTNNB1 mutations do not substitute for APC mutations in MSI-H tumours in these Norwegian patients.

Are substitutions in the first hypervariable region of the mitochondrial DNA displacement-loop in sudden infant death syndrome due to maternal inheritance?

AIM: To investigate whether all substitutions in the first hypervariable region (HVR1) in sudden infant death syndrome (SIDS) can be recovered along the maternal line of the family (inherited), or whether SIDS victims have new substitutions compared to maternal relatives (somatic mutations) that may be related to environmental factors. METHODS: Seventy-one SIDS/mother pairs, including 11 families with SIDS, mother and mother's relatives and/or SIDS siblings, were studied. The HVR1 sequence was recorded in the base-pair range 16056-16400. The recorded HVR1 sequence was compared with the Cambridge sequence, and differences were recorded as substitutions. The substitution pattern in the SIDS victims was compared with the pattern found in family members along the maternal line. RESULTS: All the substitutions found in SIDS victims could be traced in the maternal line of the family; in 5 cases this was observed through three generations, and in 3 cases through four generations. DISCUSSION: In patients with known mitochondrial (mt) DNA disease, a large number of sequence variants have been found in the D-loop region. Substitutions in the D-loop may be part of a haplotype with mutations elsewhere in the mtDNA. CONCLUSION: HVR1 substitutions in SIDS victims are hereditary and not due to somatic mutations.

Strong HLA-DR expression in microsatellite stable carcinomas of the large bowel is associated with good prognosis.

Progression of colorectal cancer may follow either of two main genetic routes: the chromosome- or microsatellite-instability pathways. Association between the patients' prognosis and microsatellite instability has been questioned. Improved survival has previously been found in patients with expression of HLA-DR antigens on their tumour cells. In this study, the expression of HLA-DR antigen was investigated by immunohistochemistry in 357 large bowel carcinomas stratified by microsatellite instability status. Sixteen per cent of the tumours showed strong HLA-DR expression and 35% had weak DR expression. We confirmed that patients with strong positive HLA-DR staining had improved survival (P<0.001) compared to patients with no HLA-DR expression. Strong epithelial HLA-DR staining was significantly associated with high level of microsatellite instability (P<0.001). In the subgroup of tumours with characteristics typical of the chromosomal instability phenotype, i.e. in microsatellite-stable tumours, the patients positive for the HLA-DR determinants showed better survival than those without HLA-DR expression. The protective effect of HLA-DR expression on survival was confirmed by multivariate analysis, both in the whole patient group and in the microsatellite-stable/microsatellite instability-low group. This might be explained by enhanced T-cell mediated anti-tumour immune responses against tumour cells in the HLA-DR positive tumours. The finding of better patient survival in the subgroup of strong HLA-DR positive microsatellite-stable tumours may have clinical implications for these patients.

Comparative epidemiology of sudden infant death syndrome and sudden intrauterine unexplained death.

BACKGROUND: Unexplained antepartum stillbirth and sudden infant death syndrome (SIDS) are major contributors to perinatal and infant mortality in the western world. A relation between them has been suggested. As an equivalent of SIDS, only cases validated by post mortem examination are diagnosed as sudden intrauterine unexplained death (SIUD). OBJECTIVE: To test the hypothesis that SIDS and SIUD have common risk factors. METHODS: Registration comprised all stillbirths in Oslo and all infant deaths in Oslo and the neighbouring county, Akershus, Norway during 1986-1995. Seventy six cases of SIUD and 78 of SIDS were found, along with 582 random controls surviving infancy, all singletons. Odds ratios were obtained by multiple logistic regression analysis. RESULTS: Whereas SIUD was associated with high maternal age, overweight/obesity, smoking, and low education, SIDS was associated with low maternal age, smoking, male sex, multiparity, proteinuria during pregnancy, and fundal height exceeding +2 SD. Thus the effects of maternal age were opposite in SIUD and SIDS (adjusted odds ratio 1.39 (95% confidence interval 1.17 to 1.66) per year, p < 0.0005). Heavy smoking, male sex, and a multiparous mother was less likely in SIUD than in SIDS (0.22 (0.06 to 0.83), 0.22 (0.07 to 0.78), and 0.03 (<0.01 to 0.17) respectively). Overweight/obesity and low fundal height were more common in SIUD than in SIDS (7.45 (1.49 to 37.3) and 13.8 (1.56 to 122) respectively). CONCLUSIONS: The differences in risk factors do not support the hypothesis that SIDS and SIUD have similar determinants in maternal or fetal characteristics detectable by basic antenatal care.

WNT1 inducible signaling pathway protein 3, WISP-3, a novel target gene in colorectal carcinomas with microsatellite instability.

BACKGROUND & AIMS: Microsatellite instability (MSI) is the phenotype of colorectal carcinomas with defect mismatch repair. Genes with repetitive sequences within their coding regions are targets for mutations in these tumors. We have evaluated 2 novel candidate genes for potential involvement in development of MSI colorectal carcinomas and compared them with alterations in known target genes. METHODS: The MSI status was determined by multiplex polymerase chain reactions (PCRs) of 5-17 markers in a Norwegian series of 275 colorectal carcinomas. All MSI tumors were analyzed for gene mutations using fluorescence PCR followed by capillary electrophoresis. Two novel candidate genes, WNT1-inducible signaling pathway protein 3 (WISP-3) and caspase-1, and 9 known target genes were analyzed. RESULTS: Thirteen percent of the tumors were MSI-high (H) and 12% were MSI-low (L). Thirty-three of 37 MSI-H vs. 1 of 34 MSI-L tumors showed mutations in the target genes (P < 0.001). WISP-3 was mutated in 31% of the MSI-H tumors. The frequencies of frameshift mutations in the known target genes were comparable with other studies. CONCLUSIONS: The relative high frequency of mutation, higher than those seen for other known target genes, the predicted truncation of the protein product, and the homology with WISP-1 and WISP-2, 2 proteins induced downstream of WNT1 signaling, strongly suggest WISP-3 as a novel target in development of MSI-H colorectal carcinomas.

[Therapeutic cloning--future medicine or an ethical dead end?]. / Terapeutisk kloning--fremtidens medisin eller en etisk blindgate?

BACKGROUND: Treatment with stem cells has given promising results in animal experiments and may be relevant in a variety of diseases, including heart disease, cancer, diabetes, Parkinson's disease and Alzheimer's disease. However, the use of pluripotent stem cells grown from blastocysts available after in vitro fertilization or from fetuses raises difficult issues in medical ethics. The same goes for therapeutic cloning. RESULTS: Attitudes to this mode of treatment differ from country to country. In Britain, Parliament has accepted therapeutic cloning (February 2001), while the German Bundestag has banned the procedure. The EU Parliament has recommended European countries not to allow therapeutic cloning, and President George W. Bush takes a more critical stand than did the Clinton administration. DISCUSSION: The debate over embryonal cells and therapeutic cloning involves both scientists and politicians. In Norway, the Biotechnology Advisory Board wants to open up for research on fertilized eggs and for therapeutic cloning. The Storting, Norway's legislature, will have to reach a decision on these issues when the Biotechnology Act come up for revision, probably in 2002. In the debate in Norway, it has been claimed that accepting therapeutic cloning would be a violation of a traditional western norm: Man should always be an end onto himself, never be used as an instrument to other ends. Furthermore, some scientists think that one should use adult stem cells from adults--this also because the procedure seems less risky. Bone marrow transplantation with autologous bone marrow stem cells has been used for 30 years, and recent research has disclosed that such stem cells may be reprogrammed, for instance from bone marrow stem cells to nerve cells.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Changes in the epidemiological pattern of sudden infant death syndrome in southeast Norway, 1984-1998: implications for future prevention and research.

AIM: To look for changes in risk factors for sudden infant death syndrome (SIDS) after decrease and stabilisation of the SIDS rate. METHODS: Questionnaires were distributed to parents of 174 SIDS infants, dying between 1984 and 1998, and 375 age and sex matched controls in southeast Norway. RESULTS: The proportion of infants sleeping prone has decreased, along with the decrease in SIDS rate for the region during the periods studied, but over half of the SIDS victims are still found in the prone position. As the number of SIDS cases has decreased, additional risk factors have become more significant. Thus, after 1993, a significantly increased risk of SIDS is seen when the mother smokes during pregnancy. After 1993, young maternal age carries an increased risk. Maternal smoking and young maternal age are associated with each other. For SIDS victims, an increase in the number of infants found dead while co-sleeping is seen, and the age peak between 2 and 4 months and the winter peak have become less pronounced. CONCLUSION: Changes in risk factor profile following the decrease in SIDS rate in the early 1990s, as well as consistency of other factors, provides further clues to SIDS prevention and to the direction of further studies of death mechanisms.

A strategy combining flow sorting and comparative genomic hybridization for studying genetic aberrations at different stages of colorectal tumorigenesis in ulcerative colitis.

BACKGROUND: DNA aneuploidy has been shown to increase the risk of developing dysplasia in ulcerative colitis (UC) and is related to tumorigenesis in the colorectum. Therefore, it is of particular interest to study genetic aberrations behind DNA aneuploidization during colorectal carcinogenesis. We wanted to elucidate further the relationship between mucosal morphology and DNA aberrations in UC. METHODS: DNA flow cytometry was applied to multiple lesions including regenerative, dysplastic, and carcinomatous mucosa from the colectomy specimen of a male patient with long-standing UC. The lesions harbored multiple DNA aneuploid stemlines that were subjected to flow sorting. We analyzed gene alterations by degenerate oligonucleotide primer (DOP; universal primers) polymerase chain reaction (PCR)-based comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) in diploid and aneuploid sorted cells. RESULTS: DOP-PCR-based CGH shows gains and losses that can be verified by FISH. We show that with this approach one can study genetic evolution of distinct DNA diploid and aberrant subpopulations through defined stages of colorectal tumorigenesis. This includes getting information related to tumor heterogeneity that cannot be obtained by CGH with DNA extracted from nonsorted cell populations. Genetic imbalance was also detected in diploid nondysplastic flow-sorted mucosal cells from the same bowel. CONCLUSIONS: Similar gains and losses were found in aneuploid dysplasias and carcinomas at widely separated locations in the same bowel, indicating a common selection pressure in different areas of the same bowel. The common aberrations may be of importance for progression from dysplasia to carcinoma.

[Intracytoplasmic sperm injection--risk of abnormalities]. / Intracytoplasmatisk spermieinjeksjon--risiko for misdannelser.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) is performed in a large number of fertility clinics; since 1991, several thousand children have been conceived by this method. Intracytoplasmic sperm injection is different than traditional in vitro-fertilization (IVF) in that a single spermatozoon is mechanically transferred into a mature oocyte using a glass pipette. Concern has been raised as to whether intracytoplasmic sperm injection damages the chromosomes and/or the spindle apparatus in the oocyte, leading to an increased risk of congenital abnormalities. MATERIAL AND METHODS: We have reviewed the literature on congenital abnormalities in children conceived by intracytoplasmatic sperm injection. RESULTS AND INTERPRETATION: Although the reports are methodologically heterogeneous, most articles conclude that intracytoplasmatic sperm injection does not cause a statistically significant increased risk of congenital abnormalities compared to traditional in vitro fertilization or normally conceived children. However, some studies do report an increased risk. The issue cannot be finally settled until even larger studies on children conceived by intracytoplasmatic sperm injection are published.

Mitochondrial DNA point mutations detected in four cases of sudden infant death syndrome.

The aim of this study was to investigate the tRNA(Leu(UUR)) gene and the first part of the ND1 gene in mitochondrial DNA (mtDNA) in cases of sudden infant death syndrome (SIDS). A total of 158 cases of SIDS and 97 controls were included in the study, and the base pairs in the range 3230-3330 were investigated using polymerase chain reaction (PCR) and temporal temperature gradient electrophoresis (TTGE). If a band shift was detected by TTGE, the area investigated and the D-loop was sequenced. Three different point mutations (T3290C, T3308C and T3308G) were detected in four of the SIDS cases, while none of the controls were mutated. We also found a high D-loop substitution rate in these four cases. The findings indicate that mtDNA mutations may play a role in some cases of SIDS.