A de Novo BRCA1 Pathogenic Variant in a 29-Year-Old Woman with Triple-Negative Breast Cancer.

Introduction: Germline pathogenic variants in the BRCA1 and BRCA2 genes lead to a highly increased lifetime risk for breast and ovarian cancer. These variants are usually inherited and reports of de novo occurrences are a very rare phenomenon. Case Presentation: We report on a breast cancer patient with a de novo BRCA1 variant c.121C>T (p.His41Tyr). The pathogenic variant was detected in leukocyte DNA of a patient with negative family history who had developed early onset, triple-negative breast cancer. The variant was not found in any of the maternal and paternal tissues tested, but it was detected in multiple samples representing all three germ layers of the affected carrier, which renders somatic mosaicism unlikely. Conclusion: This case highlights the importance of including early onset of disease and triple negativity of the tumor as criteria for genetic testing, even in patients without family history. Considering the availability of effective breast cancer treatments in patients with pathogenic variants in the BRCA genes, this finding underscores the importance of genetic testing in breast cancer patients.

Enteric-Coated Cysteamine Bitartrate in Cystinosis Patients.

Cystinosis is a severe inherited metabolic storage disease caused by the lysosomal accumulation of cystine. Lifelong therapy with the drug cysteamine bitartrate is necessary. Cysteamine cleaves intralysosomal cystine, and thereafter, it can exit from the organelle. The need for frequent dosing every 6 h and the high prevalence of gastrointestinal side effects lead to poor therapy adherence. The purpose of our study was to improve cysteamine treatment by comparing the efficacy of two cysteamine formulas. This is highly relevant for the long-term outcome of cystinosis patients. The cystine and cysteamine levels of 17 patients taking immediate-release cysteamine (IR-cysteamine/Cystagon®) and 6 patients taking encapsulated delayed-release cysteamine (EC-cysteamine) were analyzed. The EC-cysteamine levels showed a near-ideal pharmacokinetic profile indicative of delayed release (longer Tmax and Tmin), and the corresponding cystine levels showed few fluctuations. In addition, the Cmax of IR-cysteamine was greater, which was responsible for unbearable side effects (e.g., nausea, vomiting, halitosis, lethargy). Treatment with EC-cysteamine improves the quality of life of cystinosis patients because the frequency of intake can be reduced to 2-3 times daily and it has a more favorable pharmacokinetic profile than IR-cysteamine. In particular, cystinosis patients with no access to the only approved delayed-release cysteamine Procysbi® could benefit from a cost-effective alternative.

Effects of solvent-based adhesive removal on the subsequent dual analysis of fingerprint and DNA.

The combined approach of classical fingerprinting and DNA profiling is a powerful tool in forensic investigations of latent "touch" traces. However, little attention has been paid to the organic solvents frequently used in dactyloscopic laboratories to facilitate the separation of adhesive evidence prior to fingerprint development and downstream effects on subsequent DNA profiling. In the present study, we tested a selection of adhesive removers (n = 9) and assessed their potential impact on DNA recovery and amplification by PCR. Thereby, we identified and characterized novel PCR inhibitors. All investigated chemicals contain volatile organic compounds that evaporate under normal indoor atmospheric conditions. Exposure to certain solvents resulted in increased DNA degradation, but only if evaporation was prevented. A series of adhesive-removal experiments were conducted with prepared mock evidence (self-adhesive postage stamps affixed to paper envelope) to investigate the impact of treatment time and the location of applied traces on DNA recovery and dactyloscopy, respectively. Due to the early onset of print decomposition, we found that only a short treatment time was compatible with the development of fingerprints on the adhesive side of a stamp. Solvents also removed DNA from the adhesive surface, thus resulting in a marked shift in the substrate distribution of recovered DNA from the stamp to the envelope, but not in the reverse direction. Furthermore, we observed that treatment with conventional fingerprint reagents lead to a significant reduction in the amounts of DNA recovered from stamps, while the additional use of adhesive removers did not significantly enhance this effect.

A comparison of immediate release and delayed release cysteamine in 17 patients with nephropathic cystinosis.

BACKGROUND: Nephropathic cystinosis is a rare and severe metabolic disease leading to an accumulation of cystine in lysosomes which especially harms kidney function. A lifelong therapy with the aminothiol cysteamine can delay the development of end-stage renal disease and the necessity of kidney transplantation. The purpose of our study was to compare the effectiveness of immediate-release and delayed-release cysteamine on cystine and cysteamine levels as well as assessing the onset of adverse effects. METHODS: We retrospectively analysed cystine and cysteamine levels of 17 patients after a single dose of immediate-release cysteamine (Cystagon®, Mylan Pharmaceuticals, Canonsburg, PA and Recordati Pharma GmbH) as well as a single dose of delayed-release cysteamine (Procysbi®; Horizon Pharma USA and Chiesi Farmaceutici S.p.A., Parma, Italy) respectively. Data were collected during a period of three years in the context of optimizing the individual treatment regimens. The dose of DR-cysteamine was reduced to 70% of the equivalent dose of IR-cysteamine. The efficacy of both formulas in depleting white blood cells' cystine levels and their side effects were compared. RESULTS: Immediate (IR)- and delayed-release (DR) cysteamine effectively decreased intracellular cystine levels under the target value of 0.5 nmol cystine/mg protein, while fewer side effects occurred under DR-cysteamine. Mean maximum levels of cysteamine were reached after 60 min with IR-cysteamine and after 180 min with DR-cysteamine. CONCLUSION: A therapy with DR-cysteamine is as effective as IR-cysteamine while less side effects were reported. Our data show that DR-cysteamine should be dosed higher than 70% of the equivalent dose of IR-cysteamine in order to decrease cystine levels over an extended period of time. Moreover, our data suggest increasing the dosing scheme of Procysbi® to three times daily, to prevent a rapid decrease and achieve a steadier decline in cystine levels. Due to the more convenient dosing scheme, DR-cysteamine might ameliorate therapy adherence and improve patients' quality of life.

First genetic evidence of leprosy in early medieval Austria.

Leprosy used to be a widespread, dreaded disease in Europe during the middle ages, and it still remains an important health problem in some parts of the world today. Herein, we present data on the earliest 'Austrian' (an adult female from the early medieval period) proven to have suffered from leprosy. Manifestations of the disease were first identified during a systematic screening of pathological changes in skeletons recovered from an archaeological site in Pottenbrunn (Lower Austria). In the present study, DNA extracts from selected cranial and postcranial bone samples were investigated using polymerase chain reaction primers specific to the Mycobacterium leprae (M. leprae) repetitive element (RLEP). M. leprae traces were detected in extracts from nasal and palatine bones. Sequence analysis of informative polymorphic sites supports previous reports indicating that European M. leprae strains fall into single nucleotide polymorphism group 3. In summary, these findings put Austria on the map of confirmed leprosy cases in ancient Europe.

Rapid genetic detection of ingested Amanita phalloides.

Mushrooms are often poorly digested by humans. Thus, their remains (tissues, spores) may persist in the gastrointestinal tract and can be detected in feces several days after mushroom consumption. In this report, we present protocols for the rapid PCR-based detection of fungal traces in a variety of complex samples. Novel primers were designed to amplify portions of ribosomal DNA from deadly poisonous European members of the genus Amanita, namely the death cap (A. phalloides), the destroying angel (A. virosa) and the fool's mushroom (A. verna), respectively. Assay sensitivity was sufficient to discover diluted DNA traces in amounts below the genomic content of a single target mushroom cell. Specificity testing was performed with DNA extracts from a variety of mushroom species. Template amplification was exclusively observed with intended targets and it was not compromised by a vast excess of non-target DNA (i.e. DNA from human and human fecal origin, respectively). A series of experiments was conducted with prepared specimens in order to follow the course of mushroom food processing and digestion. Amplification by direct PCR was successful with raw, fried and digested mixed mushrooms. To improve assay performance with fecal samples, a rapid protocol for sample pre-processing (including water-ether sedimentation and bead beating) and a modified PCR reaction mix were applied. Thereby, it was possible to detect the presence of A. phalloides DNA in spiked feces as well as in clinical samples (vomit, stool) from two independent cases of suspected mushroom poisoning.

Molecular identification of traces from the White-tailed Sea Eagle.

PURPOSE: Over the preceeding decades, after periods of dramatic decline and extinction in many parts of Europe, the White-tailed Sea Eagle (Haliaeetus albicilla) has re-colonized traditional breeding areas. However, this large apex predator remains threatened, not only by the bioaccumulation of environmental pollutants, but also by targeted poisoning and poaching. In connection with a forensic case, a novel PCR assay was developed for the sensitive and specific detection of sea eagle DNA traces in questioned samples of unknown origin. METHODS: The assay amplifies a fragment of the popular phylogenetic marker gene cytochrome b. Primers were designed to bind sites with relatively high variability between homologous sequences from H. albicilla and other related European birds of prey. RESULTS: Assay sensitivity was sufficient for single cell analysis. Specificity was tested in vitro and the primers did not cross-detect DNA from humans, chicken and the following raptors: Common Buzzard (Buteo buteo), Northern Goshawk (Accipiter gentilis), Red Kite (Milvus milvus) and Black Kite (Milvus migrans). Applicability for the analysis of poor quality samples was demonstrated with extracts from field-collected small molted down feathers that did not contain detectable amounts of sea eagle nuclear DNA. Amplicons of the expected size were generated, purified and sequenced. Sequence data were subjected to Basic Local Alignment Search Tool analysis and affiliated with cytochrome b from H. albicilla. CONCLUSIONS: The novel PCR primers allowed for the correct assignment of traces from H. albicilla, even in mixed samples and in cases with limited and degraded biological material.

Application of direct PCR in a forensic case of yew poisoning.

Intoxications with yew (Taxus spp.) pose a challenge to forensic toxicology because a variety of Taxus ingredients have been associated with its toxic effects. To provide preliminary evidence in cases where plant material is available, we introduce a novel direct PCR assay for the detection of DNA traces from Taxus spp. This assay has been successfully applied to a forensic case of suicidal poisoning via ingestion of Taxus leaves. PCR primers were designed to target a sequence located in the internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA, which is well conserved among species of the genus Taxus and can, therefore, be exploited to discriminate between Taxus and other conifers. Because ITS1 exists as a multicopy sequence within the plant genome, the assay provides enough sensitivity to work with trace amounts that are below the DNA content of a single cell. Specificity of the assay was tested with DNA extracts from Taxaceae and selected representatives from other related plant families (Cephalotaxaceae, Cupressaceae and Pinaceae). When combined with the commercial Phire® Plant Direct PCR Kit (Finnzymes), the primers allowed application of a two-step cycling protocol (without the annealing step), and because direct PCR requires only little sample pre-treatment, results from PCR could be obtained within 1.5 h after analysis had begun. Direct PCR was performed with diluted gastric content from the forensic case. Amplification products of the expected size were purified and sequenced. Sequence data were subjected to Basic Local Alignment Search Tool analysis and affiliated with ITS1 from Taxus spp.

Evaluation of a novel tagging and tissue preservation system for potential use in forensic sample collection.

The authors describe a new, easy-to-use barcode-based tissue collection, preservation and body tracking system, which might prove instrumental in the containment of mass fatalities such as aircraft accidents, war related accidents, environmental disasters (e.g. earthquakes, hurricanes, and floods) terrorist bombings or mass murders.