Elevated germline mutation rate in teenage fathers.

Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural 'cell-cycle counter'. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77-196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as 'A-dark spermatogonia'.

FREM1 mutations cause bifid nose, renal agenesis, and anorectal malformations syndrome.

An autosomal-recessive syndrome of bifid nose and anorectal and renal anomalies (BNAR) was previously reported in a consanguineous Egyptian sibship. Here, we report the results of linkage analysis, on this family and on two other families with a similar phenotype, which identified a shared region of homozygosity on chromosome 9p22.2-p23. Candidate-gene analysis revealed homozygous frameshift and missense mutations in FREM1, which encodes an extracellular matrix component of basement membranes. In situ hybridization experiments demonstrated gene expression of Frem1 in the midline of E11.5 mouse embryos, in agreement with the observed cleft nose phenotype of our patients. FREM1 is part of a ternary complex that includes FRAS1 and FREM2, and mutations of the latter two genes have been reported to cause Fraser syndrome in mice and humans. The phenotypic variability previously reported for different Frem1 mouse mutants suggests that the apparently distinct phenotype of BNAR in humans may represent a previously unrecognized variant of Fraser syndrome.

Cytokines and sudden infant death.

BACKGROUND: It has been hypothesised that inflammatory reactions could play an important role in the pathway(s) leading to sudden and unexpected death in infancy. On a molecular level, these reactions are regulated by various cytokines. METHODS: To characterise the role of IL-1ß, IL-6 and TNFα more precisely, the concentrations of these cytokines were determined quantitatively using specific ELISA techniques in serum and cerebrospinal fluid (CSF) in 119 cases of sudden infant death. The infants were grouped into four categories (SIDS, SIDS with infection, natural death due to infection and unnatural death). RESULTS: A good correlation was found between CSF and serum for IL-6 (Spearman correlation coefficients (SCC), 0.73) and also for TNFα (SCC, 0.57), although the CSF concentrations were lower than that from the serum. There were no significant differences between the categories of death for any of the serum or CSF cytokines. Compared with normal values, increased serum concentrations of IL-1ß, IL-6 and TNFα were found in 70%, 69% and 38% of the cases respectively, indicating possible agonal or post-mortem changes of cytokine concentrations. In three cases very high cytokine concentrations were found (mainly for IL-6). This may have contributed to the mechanism of death (cytokine storm) in two of the cases. CONCLUSIONS: In a small group of patients, very high cytokine concentrations are a possible explanation for the cause of death ("cytokine storm").

Haemorrhages into the back and auxiliary breathing muscles after death by hanging.

We describe and discuss haemorrhages discovered in the back and auxiliary breathing muscles of a population of cases of suicidal death by hanging. Intramuscular haemorrhages were present in approximately 30% of the cases. Pre-existing illnesses with an increased tendency to bleed or an anticoagulant medication did not exist; corresponding skin and subcutaneous fatty tissue structures were intact in each case. In cases of death by hanging, the occurrence of muscle haemorrhages of this type may be explained pathophysiologically by the occurrence of increased respiratory exertions and/or seizures during the hanging process. Although the results of our study do not indicate an obligatory autopsy finding, evidence of internal haemorrhaging into the back and auxiliary breathing muscles may be called upon following consideration of differential diagnostic aspects as a further diagnostic indication of vital hanging.

Bowel wall hemorrhage after death by hanging.

We describe and discuss autopsy findings of bowel wall hemorrhage in a study population comprising cases of suicidal death by hanging. Intramural hemorrhages were seen in approximately 12% of the cases examined; no preexisting bowel diseases were found. In hanging deaths with a longer agonal phase, we opine that abdominal congestion during the hanging process provides a viable pathophysiological explanation for bowel wall hemorrhage. Though we are not dealing here with obligatory autopsy findings, the detection of bowel wall hemorrhage might be used as another sign of vital hanging after considering differential diagnostic aspects.

Petechial bleedings in sudden infant death.

The autopsy reports of 484 cases of deceased infants (201 females, 283 males) were analysed retrospectively for the existence of external and internal petechial bleedings (PET). The cases were divided into five groups on the basis of the cause of death (sudden infant death syndrome, sepsis, airway infections, asphyxia and trauma). Internal PET (pleural, pericardial, epicardial, thymic and peritoneal) were observed in each group with a lower prevalence in cases of trauma. The highest prevalence of external (cutaneous and conjunctival) PET was detected in cases of asphyxia (38% and 31%, respectively). However, even if with low prevalence, such bleedings were detected in every group. Factors like sex, age, cardiopulmonary resuscitation and its duration did not influence the presence of PET. The detection of external PET at autopsy is a suspicious finding that suggests asphyxia. Because of the possible natural origin of these bleedings, the medicolegal investigation has to be as complete as possible and has to include histology as mandatory.

Haplotype-assisted characterization of germline mutations at short tandem repeat loci.

In this study, 98 families with 101 mutations were analyzed in depth in which a mutation had been observed at one of the four loci D3S1358, FGA, ACTBP2, and VWA. To determine the origin (male/female) of the mutation, five to seven polymorphic flanking markers were selected for each locus concerned and used to construct family-specific haplotypes. Additionally, all alleles of the STR system concerned were sequenced. With this duplicate approach, it was possible to identify the mutated structure and/or mutation event in the vast majority of cases. The ratio of one-step to two-step mutations was 100:1. The ratio of paternal to maternal mutations was 76:8. The ratio of gains to losses was 47:50. Also, the mutation rates in two systems, ACTBP2 and VWA, were clearly higher than those given in the literature.

Reaction patterns of pulmonary macrophages in protracted asphyxiation.

Do long periods of asphyxiation trigger the proliferation of pulmonary macrophages and the formation of giant cells? Three groups have been defined: six autopsy cases with time periods of suffocation >25 min (long protracted asphyxiation), eight cases with estimated time periods of suffocation 10-25 min (short protracted asphyxiation) and nine cases where death had occurred immediately (very severe trauma). The stain used was haematoxylin and eosin (H&E), and the immunohistochemical stainings were performed using antibodies CD 68, MRP 8, MRP 14 and NP 57. The intraalveolar macrophages and giant cells were counted in H&E sections. For the immunohistochemical stainings, a scoring was used in order to compare the groups. In protracted asphyxiation, the number of intraalveolar macrophages was definitely elevated. A significant increase of giant cells was observed in the cases of long protracted asphyxiation. CD 68 showed clearly elevated numbers in both asphyxiation groups. Early-stage macrophages are significantly increased in protracted asphyxiation. With increasing time periods of asphyxiation, the results become more significant. The results show that the length of the agony period stimulates the proliferation of pulmonary macrophages and the formation of giant cells.

Developing equine mtDNA profiling for forensic application.

Horse mtDNA profiling can be useful in forensic work investigating degraded samples, hair shafts or highly dilute samples. Degraded DNA often does not allow sequencing of fragments longer than 200 nucleotides. In this study we therefore search for the most discriminatory sections within the hypervariable horse mtDNA control region. Among a random sample of 39 horses, 32 different sequences were identified in a stretch of 921 nucleotides. The sequences were assigned to the published mtDNA types A-G, and to a newly labelled minor type H. The random match probability within the analysed samples is 3.61%, and the average pairwise sequence difference is 15 nucleotides. In a "sliding window" analysis of 200-nucleotide sections of the mtDNA control region, we find that the known repetitive central motif divides the mtDNA control region into a highly diverse segment and a markedly less discriminatory segment.

Evaluating length heteroplasmy in the human mitochondrial DNA control region.

We present allelic data for three known and one new C-tract in the human mitochondrial DNA (mtDNA) control region, and we measure intergenerational mutation rates at such C-tracts. In detail, in a sample of 1,172 mtDNA sequences, we demonstrate the existence of an instability threshold of eight consecutive cytosines, at and above which the phenomenon of length heteroplasmy arises. To determine mutation rates, we draw on mtDNA sequences in up to four generations of 248 pedigrees for families living in high or low-radiation environmental conditions. The high-radiation sample gives the most conservative (fastest) mutation rate likely to be encountered in any forensic context. We find that the C-tract mutation rate is up to 6% per generation, and we observe an excess of cytosine gains over losses. Case studies and guidelines for evaluating mtDNA heteroplasmy are provided.

Leptomeningeal neurons are a common finding in infants and are increased in sudden infant death syndrome.

Developmental abnormalities of the brain, in particular, the brainstem potentially affecting centers for breathing, circulation and sleep regulation, are thought to be involved in the etiology of sudden infant death syndrome (SIDS). In order to investigate whether leptomeningeal neurons could serve as morphological indicators for a developmental failure or retardation in cerebral maturation, we evaluated the density of isolated leptomeningeal neurons (without associated glia) in 15 brain regions of 24 SIDS and 8 control cases, representing part of the German Study on sudden infant death. Leptomeningeal neurons were encountered in 79% of SIDS and 68% of control cases. More leptomeningeal neurons in SIDS versus control cases were found in lower pons (p = 0.002), upper pons (p = 0.016), cerebellar hemispheres (p = 0.012), lower medulla oblongata (p = 0.039), and temporal lobe (p = 0.041). Summarizing the data according to gross anatomical region of origin (i.e., brainstem, cerebellum or cerebrum), higher numbers of leptomeningeal neurons in SIDS cases were only found in the brainstem (p = 0.006 vs. 0.13 and 0.19, respectively). Our data show that single leptomeningeal neurons are present in most normal infantile brains. The age-dependent increase of leptomeningeal neurons among SIDS cases may either (a) represent a delayed maturation or retardation, i.e., a later or slower reduction of neurons or a delayed peak in occurrence (shift toward an older age), or (b) may be interpreted as a generally increased occurrence of leptomeningeal neurons among SIDS cases as a result of a diffuse developmental abnormality during central nervous system maturation.

Meiosis study in a population sample from Nigeria: allele frequencies and mutation rates of 16 STR loci.

Allele frequencies for the 16 short tandem repeat (STR) loci D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, ACTBP2, CSF1PO, FGA, TH01, TPOX and VWA were determined for 337 immigrants from Nigeria. All loci were in Hardy-Weinberg equilibrium. More than 6,000 meiotic transfers were investigated and ten mutations were observed. Single mutations were observed in the STR systems D2S1338, D3S1358, D7S820, D8S1179, D16S539 and FGA, whereas two mutations were observed in the systems D21S11 and VWA.

Ultrasound-accelerated formalin fixation improves the preservation of nucleic acids extraction in histological sections.

The aim of the present study was to examine an ultrasound-accelerated fixation technique that reduces the exposure time of the tissue to formaldehyde with respect to the analysis of nucleic acids. We extracted and analysed DNA and RNA from three series of autopsy specimens from five routine cases. Two series were shortly fixed in 4% buffered formalin (15 and 30 min, respectively) whilst being irradiated with high-frequency, high-intensity ultrasound. The last series (control) was routinely fixed in 4% buffered formalin for 24-48 h without irradiation. Although sufficient amounts of DNA of good quality could be extracted and amplified from all three series, the peak heights obtained from conventional fixation were smaller and allele dropout occurred more often, especially for the longer amplicons. RNA yield depended on the fixation procedure, i.e. the shortest fixation time led to the highest RNA yield and quality. No differences were observed with regard to the quality of the histological slides both with conventional and immunohistochemical staining methods. Keeping in mind the increasing need for molecular diagnosis, this fixation technique can be useful to ensure stable quality of nucleic acids in archived autopsy specimens.

NHE3 in the human brainstem: implication for the pathogenesis of the sudden infant death syndrome (SIDS)?

Previous studies have demonstrated an inverse correlation between the degree of respiratory drive and NHE3 mRNA expression in the brainstem of awake rabbits. Here we show that the levels of NHE3 mRNA extractable from kryo-conserved tissue are highly variable also in the human brainstem. As an insufficient drive to breath may be a final event causing sudden infant death, we compared the expression of NHE3 mRNA in a collective of children who died from non-natural causes to an equal number of SIDS victims. Evaluation of signals from NHE3 RT-PCR showed higher values for the SIDS collective than for the control group. We suggest that the level of NHE3 expression in brainstem tissue may contribute to the vulnerability of infants for SIDS.

Fatal leaflet escape in an Edwards TEKNA aortic prosthesis.

The case is reported of a 26-year-old male patient who died eight years after the replacement of an aortic valve with a bileaflet mechanical valve (TEKNA; Edwards, USA). Following prosthesis implantation, the patient had been in a good state of health, and his death occurred unexpectedly. Forensic autopsy revealed a leaflet escape, with two fragments of the leaflet being found bilaterally in the common iliac arteries. Death occurred due to an acute cardiac insufficiency. Immunohistochemical investigations revealed fresh myocardial fiber necroses. Stereomicroscopic and scanning electron microscopic investigations demonstrated surface erosions of the leaflet. Although the valve was withdrawn from the market in June 2000, it had previously been implanted in over 18,000 patients. Thus, from a clinical viewpoint, the question of using a prophylactic replacement in affected patients must be discussed.

Incidence of alcohol dependence among drunken drivers.

To discriminate 'alcoholics' and 'non-alcoholics', individual Alc-Indices (determined by methanol, acetone, 2-propanol, gamma-GT and CDT-concentrations) were calculated in a collective of 327 alcohol-impaired drivers with regard to the blood alcohol concentration, the time of the event and the age of the drivers. Applying this new defined Alc-Index, 48% of the drivers investigated could be characterised as alcohol dependent. The prevalence of alcoholics among individuals with blood alcohol concentrations (BAC) higher than 1.9per thousand was more than 80%. The diagnostic value of alcohol concentrations for the recognition of 'alcoholics', considering the legal limit in Germany (1.1per thousand) as well as statistically calculated limits, were compared to the Alc-Index.

Synthesis of a psilocin hapten and a protein-hapten conjugate.

Derivatives of psilocin with omega-functionalized alkyl spacers in position 1 of the indole ring were synthesized as haptens for use in a radioimmunoassay. Whereas the psilocin analogues with a 3-aminopropyl and a 4-aminobutyl moiety at the indole nitrogen decomposed during synthesis, the analogous 3-carboxypropyl psilocin derivative proved to be stable. This compound was coupled to bovine serum albumin (BSA) using the N-hydroxysuccinimide ester-mediated conjugation. The protein-hapten conjugate was characterized by matrix-assisted laser desorption ionization mass spectrometry. The mass spectrometry data indicated an average incorporation ratio of 4-5 molecules of psilocin hapten per molecule of BSA.

The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives.

This paper presents an overview of the organisation and the results of the collaborative exercises (CE) of the European DNA Profiling (EDNAP) Group's mitochondrial DNA population database project (EMPOP). The aim of the collaborative exercises was to determine whether uniformity of mtDNA sequencing results could be achieved among different laboratories. These were asked to sequence either the complete mtDNA control region or the two hypervariable regions HVI (16024-16365) and HVII (73-340) from DNA extracts, buccal swabs or bloodstains, proceeding in accordance with the protocol and strategies used in each individual laboratory. The results of the collaborative exercises were employed to identify possible sources of errors that could arise during the analysis and interpretation of mtDNA profiles. These findings were taken as a basis to tentatively make suitable arrangements for the construction of a high quality mtDNA database. One hundred fifty mtDNA profiles were submitted to the evaluating laboratory, and disaccording profiles were classified into four groups corresponding to the source of error: clerical errors, sample mix-ups, contaminations and discrepancies with respect to the mtDNA nomenclature. Overall, 14 disaccording haplotypes (16 individual errors) were observed. The errors included 10 clerical errors, 3 interpretation problems, 2 cases of sample mix-up and 1 case of point heteroplasmic mixture, where the 2 sequencing reactions brought inconsistent base calls. This corresponds to an error rate of 10.7% in a virtual mtDNA database consisting of the collaborative exercise results. However, this estimate is still conservative compared to conclusions drawn by authors of meanwhile numerous publications critically reviewing published mtDNA population databases. Our results and earlier published concerns strongly emphasize the need for appropriate safety regulations when mtDNA profiles are compiled for database purposes in order to accomplish the high standard required for mtDNA databases that are used in the forensic context.