Gene variants associated with obstructive sleep apnea (OSA) in relation to sudden infant death syndrome (SIDS).

BACKGROUND: Both obstructive sleep apnea (OSA) and (at least a fraction of) sudden infant death syndrome (SIDS) are associated with impaired respiration. For OSA, an association with several gene variants was identified. Therefore, our hypothesis is that these polymorphisms might be of relevance in SIDS as well. METHODS: Twenty-four single nucleotide polymorphisms (SNPs) in 21 candidate genes connected to OSA, were genotyped in a total of 282 SIDS cases and 374 controls. Additionally, subgroups based on factors codetermining the SIDS risk (age, sex, season, and prone position) were established and compared as well. RESULTS: Two of the analyzed SNPs showed nominally significant differences between SIDS and control groups: rs1042714 in ADRB2 (adrenoceptor beta 2) and rs1800541 in EDN1 (endothelin 1). In the subgroup analyses, 10 further SNPs gave significant results. Nevertheless, these associations did not survive adjustment for multiple testing. CONCLUSIONS: Our results suggest that there might be a link between SIDS and OSA and its resulting respiratory and cardiovascular problems, albeit this predisposition might be dependent on the combination with other, hitherto unknown gene variants. These findings may encourage replication studies to get a better understanding of this connection.

Transcriptomic signatures of neuronal differentiation and their association with risk genes for autism spectrum and related neuropsychiatric disorders.

Genes for autism spectrum disorders (ASDs) are also implicated in fragile X syndrome (FXS), intellectual disabilities (ID) or schizophrenia (SCZ), and converge on neuronal function and differentiation. The SH-SY5Y neuroblastoma cell line, the most widely used system to study neurodevelopment, is currently discussed for its applicability to model cortical development. We implemented an optimal neuronal differentiation protocol of this system and evaluated neurodevelopment at the transcriptomic level using the CoNTeXT framework, a machine-learning algorithm based on human post-mortem brain data estimating developmental stage and regional identity of transcriptomic signatures. Our improved model in contrast to currently used SH-SY5Y models does capture early neurodevelopmental processes with high fidelity. We applied regression modelling, dynamic time warping analysis, parallel independent component analysis and weighted gene co-expression network analysis to identify activated gene sets and networks. Finally, we tested and compared these sets for enrichment of risk genes for neuropsychiatric disorders. We confirm a significant overlap of genes implicated in ASD with FXS, ID and SCZ. However, counterintuitive to this observation, we report that risk genes affect pathways specific for each disorder during early neurodevelopment. Genes implicated in ASD, ID, FXS and SCZ were enriched among the positive regulators, but only ID-implicated genes were also negative regulators of neuronal differentiation. ASD and ID genes were involved in dendritic branching modules, but only ASD risk genes were implicated in histone modification or axonal guidance. Only ID genes were over-represented among cell cycle modules. We conclude that the underlying signatures are disorder-specific and that the shared genetic architecture results in overlaps across disorders such as ID in ASD. Thus, adding developmental network context to genetic analyses will aid differentiating the pathophysiology of neuropsychiatric disorders.

Molecular characterization of disseminated pilocytic astrocytomas.

AIM: Pilocytic astrocytomas represent the most common paediatric tumours of the central nervous system. Dissemination through the ventricular system occurs rarely in patients with pilocytic astrocytomas; however, it is more common in infants with diencephalic tumours, and is associated with a poor outcome. Despite histological similarities with classic pilocytic astrocytomas, it is still unclear whether disseminated pilocytic astrocytomas may have specific molecular features. METHODS: Seventeen disseminated pilocytic astrocytomas were investigated using the molecular inversion probe array and screened for the presence of gene fusions (KIAA1549-BRAF) and mutations (BRAF, RAS and FGFR1). RESULTS: Along with evidence of a constitutive MAPK activation in all cases, the molecular inversion probe array, fluorescence in situ hybridization analysis and mutational study revealed KIAA1549-BRAF fusions in 66% and BRAF(V600E) mutations in 5% of cases. No KRAS, HRAS, NRAS or FGFR1 mutations were found. CONCLUSIONS: disseminated pilocytic astrocytomas showed genetic features similar to classic pilocytic astrocytoma, including a similar incidence of KIAA1549-BRAF fusions, BRAF mutations and a stable genetic profile. Given common activation of the MAPK pathway, the use of specific inhibitors can be hypothesized for the treatment of disseminated pilocytic astrocytomas, along with standard chemo- and/or radiotherapy.

Mitochondrial deoxyribonucleic acid may play a role in a subset of sudden infant death syndrome cases.

AIM: It has been suggested that progressive adenosine triphosphate (ATP) depletion could play a key role in sudden infant death syndrome (SIDS). Because mitochondrial deoxyribonucleic acid (mtDNA) codes for a subset of essential genes for oxidative phosphorylation, we investigated 22 mtDNA polymorphisms in a large sample of Caucasian SIDS cases. METHODS: A total of 774 samples were analysed, 365 from infant SIDS cases (mean age 131 days) and 409 from controls. These were investigated for the presence of 22 haplogroup-specific single nucleotide polymorphisms (SNPs), using a SNaPshot assay, a mini-sequencing assay that combines polymerase chain reaction (PCR) and sequencing. RESULTS: No significant differences in assigned haplogroups could be detected between the groups. With regard to gender and age, we found significant correlations for SNP positions 3010, 8251, 13 708, 14 470, 15 904 and 16 519. The most prominent result was the A allele in SNP 14 470 in male SIDS cases (p = 0.01). CONCLUSION: This is the largest study on mtDNA polymorphisms in SIDS to date, and our results indicate that mtDNA may play a role in a subset of SIDS cases. In order to complement these significant results, it is important to consider nuclear gene coding for mitochondrial proteins in future studies.

The German Stain Commission: recommendations for the interpretation of mixed stains.

In the course of forensic DNA analysis, the interpretation of DNA profiles of mixed stains, i.e. cell material from more than a single donor, has become increasingly more important. The German Stain Commission, a joint commission of Institutes of Forensic Science and Legal Medicine, has therefore developed guidelines aiming to harmonize the evaluation of mixed stains in German criminal cases.

Characterization of human myocardial fibroblasts immortalized by HPV16 E6--E7 genes.

Human myocardial fibroblasts (HMF) have proved to be useful as a species specific cell culture system in various studies on myocarditis and cardiac remodelling. However, their use is limited, since they are hard to obtain and lifespan is short due to replicative senescence. To overcome these disadvantages, we transfected primary HMF with the E6 and E7 genes of the oncogenic human papillomavirus (HPV) 16. Successful transfection was demonstrated in 3 of 12 experiments by detection of E6-E7 gene transcription with nucleic acid sequence based amplification (NASBA). No significant change of phenotype was noted in the emerging cell lines (HMF(1226D), HMF(1321D), HMF(1226K)), but their in vitro lifespan was increased by 20 to 30 population doublings until cells entered crisis. A single subclone of HMF(1226K) had a transformed phenotype and continued to proliferate indefinitely. This subclone (HMF(1226K/I)) was considered to be immortalized and telomerase activity was detected. Despite the increased risk of mutations due to abrogation of p53 function, HMF(1226K/I) and the HMF lines with an increased lifespan retained the properties of primary HMF cells, as they expressed fibroblast markers (prolyl-4-hydroxylase, vimentin), cytokines (interleukin 1 alpha, 6, 8), and angiotensin II receptors and still were permissive for coxsackievirus B3 infection.

Vaginorectal impalement injury in a 2-year-old child--caused by sexual abuse or an accident?

Anogenital impalement injuries are rarely encountered in clinical or forensic practice. If seen in children and if incurred under suspect circumstances, sexual abuse must be considered in the differential diagnosis. Here we describe the case of a 2-year-old girl admitted to hospital with a vaginorectal impalement injury. According to the girl's parents, she had slipped in the bathroom and fallen onto the handle of an upright toilet bowl brush. Since a second slight anal injury was present, the parents' account appeared inconsistent. Therefore, physicians from the Institute of Legal Medicine were consulted to investigate the possibility of underlying sexual abuse. Because they could not rule out that the injuries could have been caused by sexual abuse, they recommended having the police immediately examine the site of the purported accident for evidence. The police and forensic investigations, however, verified the parents' account of an accidental injury. Thus, in this case, the initiation of a police inquiry, which is not compulsory in Germany even when sexual abuse is strongly suspected, led to the exoneration of the father.

Reliability of SE33 typing by capillary electrophoresis.

A ladder of 24 ACTBP2 (SE33) alleles was separated 175 times by denaturing capillary electrophoresis on an ABI Prism 310 Genetic Analyzer using polymer POP-4. The mean standard deviation of fragment size determination was 0.083 bp. Fragments in the whole allelic range of ACTBP2 could be typed with high precision and reproducibility if adjacent fragments differed by at least two nucleotides. The capacity of resolving 1 bp differences was tested by repeatedly running a ACTBP2*14.2/14.3/31.2/31.3 allelic mixture. The 14.2/14.3 fragment pair could be separated in 98%, the 31.2/31.3 fragment pair only in 65% of all runs. Reliable separation of this difficult fragment mixture could exclusively achieved by using POP-6.

Laser-assisted microdissection and short tandem repeat PCR for the investigation of graft chimerism after solid organ transplantation.

The detection of donor-derived cells in the blood and tissues of graft recipients after solid organ transplantation is a readily observed phenomenon called microchimerism. Yet very little is known about the persistence and integration of recipient-derived cells in the transplanted organ, indicating a form of intragraft chimerism. To further study this phenomenon and its possible influence on graft acceptance or rejection, we developed the following novel approach. Immunohistochemically labeled cells were isolated by means of laser-based microdissection and subsequent laser pressure catapulting from paraffine-embedded posttransplantation biopsies. The following use of a highly sensitive PCR assay analyzing one polymorphic short tandem repeat (STR) marker enabled us to clearly identify the genotypes in samples containing as little as 10 isolated cells. The combination of laser-based microdissection and STR-PCR thus provides a powerful tool for the genotyping of even very few cells isolated from routinely processed biopsies after solid organ transplantation.

Evaluation of serological screening of cadaveric sera for donor selection for cornea transplantation.

Human corneas are explanted for grafting as late as 72 h after death, for example, from medical examiner cases. Currently, infection of the donor with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) is excluded in most cornea banks by serological testing of the cadaveric serum only. The reliability of this strategy was investigated by testing paired cadaveric and premortem sera of 33 potential donors. Results were discordant in 17 of 33 donors by at least one assay. Most frequently, HBsAg enzyme-linked immunosorbent assay (ELISA) yielded false-positive results with the cadaveric serum (16 of 33 serum pairs). Virus safety of the graft was affected in a single case, which was HCV antibody negative in the cadaveric serum, but positive in the premortem serum (confirmed by HCV-RIBA strip immunoassay). Forensic DNA profiling by polymerase chain reaction (PCR) of both serum samples confirmed that these were derived from the same individual. In conclusion, the results indicate that serological testing of cadaveric sera is not a reliable method for screening of potential cornea donors, and may not be sufficient for the virus safety of cornea grafts. Therefore, other screening strategies such as detection of viral nucleic acids by PCR should be evaluated.

Body and head position, covering of the head by bedding and risk of sudden infant death (SID).

We examined the position of the body and head, and the covering of the head by bedding on discovery in cases of sudden infant death (SID) in Lower Saxony. Between 1.1.1986 and 31.12.1992 structured, questionnaire-based interviews were carried out with parents of 140 SID victims. Control data were taken from a population-based cross-sectional study on infant sleeping position performed by the German Health Office (BGA) in autumn 1991. Of the SID cases 86.4% were discovered in the prone position [odds ratio (OR) = 7.4, 95% confidence interval (CI) = 4.3, 12.7] and 41.4% were found with the head covered. These infants were significantly older than those where the head was uncovered (p < 0.001) and covering of the head showed a significant association with a risk of SID (OR = 20.8; 95% CI = 11.5-37.6). Of the SID cases 27.9% were discovered in the face-down position. These infants were significantly younger than the infants who were discovered with the head in a side or supine position (p < 0.001). This study confirms the increased risk of SID associated with the prone position and suggests that this association could be related to the development of hypoxaemia or hypercapnia. Together with other factors such as heat stress or an infection, hypoxaemia or hypercapnia could culminate in SID if the arousal from sleep and auto-resuscitation apparently fails.

Menin mutations in patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN-1) is a familial cancer syndrome with parathyroid, pituitary and enteropancreatic tumors. The disease phenotype segregates with markers on chromosome 11q13. Very recently a new gene was cloned from this region and was found to carry mutations in 14 of 15 unrelated MEN-1 patients. The gene was termed menin and is predicted to code for a tumor suppressor protein of 610 amino acids, but its precise function is totally unknown. To confirm this finding we used PCR from genomic DNA and direct sequencing to analyze exons 2 through 10 of the menin gene in eight patients from four pedigrees with MEN-1 syndrome or an affected relative. We identified four different heterozygous mutations, three of them are novel: one nonsense mutation, one large deletion of 32 bp and two insertions, all of them located in exon 2. Our results confirm that patients with MEN-1 carry mutations in the menin gene.

Hyperthermia in sudden infant death.

To determine whether preterminal hyperthermia is significantly associated with sudden infant death (SID), 140 structured interviews with parents of SID victims were compared with questionnaires filled in by a control group of parents living in the same area. All SID autopsies were performed between 1986 and 1992 at the Institute of Legal Medicine of Hannover Medical School according to the same protocol. Signs of profuse sweating (i.e. moist head, damp clothing or bedding) were present at the scene of death in 35.7% of cases. SID victims with signs of profuse sweating were more frequently found under their bedding (p < 0.001), were older (178 vs. 130 days) and the time period between when they were last seen alive and when they were found dead was longer (6.5 vs. 4.5 hours p < 0.01) compared to cases without sweating. Sweat on the head [odds ratio (OR) = 1.9; 95% confidence interval (CI) = 1.0, 3.6], and sweaty clothing and bedding (OR = 17.9; 95% CI = 8.7; 37.1) showed a significant association with the risk for SID. The pathophysiological basis for hyperthermia is SID remains to be determined. Hyperthermia could result from infection, overinsulation from excessive clothing with high environmental temperatures, covering of the infant's head or immature central thermoregulatory centres. The influence on the fatal outcome and the role in the pathogenesis of these deaths requires further research.

[Subendocardial hemorrhage from the forensic medicine viewpoint]. / Subendokardiale Hämorrhagien aus forensisch-medizinischer Sicht.

The histological examination of cardial tissue samples concerning routine autopsy casework showed microscopic discrepancies in case of macroscopically found "subendocardial haemorrhages". In an attempt to analyse this phenomenon we compared the macroscopic and microscopic findings in 175 samples of endocardium from tissues presenting such haemorrhages of varying intensity: Only 8.6% showed a histological correlate and therefore could be verified as true haemorrhage. 53.7% presented mere hyperaemia and the remaining 37.7% even showed no microscopic finding at all. A satisfactory reason for this phenomenon could not be given by this investigation: The histological findings, however, do not depend on their cardial localization nor do they differ in relation to the cause of death or due to resuscitative actions. A possible cause of the described discrepancies may be a loss of haemoglobin of the erythrocytes. The results of this investigation point out that it might be necessary to reconsider the use of the term "subendocardial haemorrhage".

Haptoglobin subtyping by isoelectric focusing in ultrathin-layer polyacrylamide gels. Population genetic data for Hanover and Lower Saxony.

This report describes a method for subtyping haptoglobin by means of isoelectric focusing in 0.2-mm ultrathin-layer polyacrylamide gels. Haptoglobin (Hp) is purified by ion-exchange chromatography and reduced. The well-known advantages of ultrathin-layer gels combine high isoelectrophoretic resolution of the Hp subtypes with less demands for time and material and make sequential visualization by fixation and protein staining possible. The distribution of the Hp subtypes in 1500 unrelated adults from Hanover and Lower Saxony is presented. Allelic frequencies are calculated to be: Hp*2FF = 0.0030; *2FS = 0.5620; *2SS = 0.0290; *1F = 0.1537; *1S = 0.2523. Segregation analysis for 68 matings shows an autosomal codominant mode of transmission in all cases. For the population investigated the chance of isolated paternity exclusion with the subtyped Hp system amounts to 33.91%.