576 Genetik und Evolution
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- DNA typing (2)
- Short tandem repeat (STR) (2)
- Whole genome amplification (2)
- apoptosis (2)
- 5-Oxoprolinase (1)
- 5-oxoprolinuria (1)
- ACacylcarnitines (1)
- AMAtypical myopathy (1)
- ASPA (1)
- Area under the curve (1)
Background: Atypical myopathy (AM), an acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in horses, induce changes in mitochondrial metabolism. Only few veterinary laboratories offer diagnostic testing for this disease. Inborn and acquired MADD exist in humans, therefore determination of organic acids (OA) in urine and acylcarnitines (AC) in blood by assays available in medical laboratories can serve as AM diagnostics. The evolution of OA and AC profiles in surviving horses is unreported.
Methods: AC profiles using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and OA in urine using gas chromatography mass spectrometry (GC–MS) were determined in dried blot spots (DBS, n = 7) and urine samples (n = 5) of horses with AM (n = 7) at disease presentation and in longitudinal samples from 3/4 survivors and compared to DBS (n = 16) and urine samples (n = 7) from control horses using the Wilcoxon test.
Results: All short- (C2-C5) and medium-chain (C6-C12) AC in blood differed significantly (p < 0.008) between horses with AM and controls, except for C5:1 (p = 0.45) and C5OH + C4DC (p = 0.06). In AM survivors the AC concentrations decreased over time but were still partially elevated after 7 days. 14/62 (23%) of OA differed significantly between horses with AM and control horses. Concentrations of ethylmalonic acid, 2-hydroxyglutaric acid and the acylglycines (butyryl-, valeryl-, and hexanoylglycine) were highly elevated in the urine of all horses with AM at the day of disease presentation. In AM survivors, concentrations of those metabolites were initially lower and decreased during remission to approach normalization after 7 days.
Conclusion: OA and AC profiling by specialized human medical laboratories was used to diagnose AM in horses. Elevation of specific metabolites were still evident several days after disease presentation, allowing diagnosis via analysis of samples from convalescent animals.
Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analysing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.
Polymerase Chain Reaction
(2021)
DNA Sequencing
(2021)
Isolation of DNA and RNA
(2021)
In forensic DNA profiling, the occurrence of complex mixed profiles is currently a common issue. Cases involving intimate swabs or skin flake tape liftings are prone to mixed profiles, because of more than one donor contributing to a DNA sample. By DNA profiling of single spermatozoa and skin flakes, problems associated with mixed profile could ideally be overcome. However, PCR is not a sensitive enough method to generate DNA profiles by STRs on single cells. Moreover, high quality intact DNA is required, but is not always available in skin flakes due to degradation. Additionally, single skin flakes are difficult to discriminate from other similar looking particles on tape liftings used to secure DNA samples from evidence. The main purpose of this study was to develop a method that enables DNA profiling of single sperm cells and skin flakes. After studying multiple whole genome amplification (WGA) protocols, REPLI-g Single Cell WGA was selected due to its suitability in the pre-amplification step of template DNA. Micromanipulation was used to isolate single spermatozoa. Furthermore, micromanipulation in combination with REPLI-g Single Cell WGA resulted in successful DNA profiling of single spermatozoa by using autosomal STRs as well as X- and Y-chromosomal STRs. The single spermatozoa DNA profiling method described in this thesis was successfully used to identify male contributors from mock intimate swabs with a mixture of semen from multiple male contributors. Different dyes were analysed to develop a staining method to discriminate skin flakes from other particles including particles such as those from hair cosmetic products. From all dyes tested, Orange G was the only dye which successfully discriminated skin flakes from hair product particles. Also, an alkaline based lysis protocol was developed that allowed PCR to be carried out directly on the lysates of single skin flakes. Furthermore, REPLI-g Single Cell WGA was tested on single skin flakes. In contrast to the single spermatozoa, REPLI-g Single Cell WGA was not successful in DNA profiling of single skin flakes. The single skin flake DNA profiling method described in this thesis was successfully used in correctly identifying contributors from mock mixed DNA evidence. Additionally, a small amplicon-based NGS method was tested on single skin flakes. Compared to the PCR and CE approach, the small amplicon-based NGS method improved DNA profiling of single skin flakes, giving a significant increase in allele recovery. In conclusion, this study shows circumventing mixtures is possible by DNA profiling of single spermatozoa, using micromanipulation and WGA. Furthermore, DNA profiling of single skin flakes has been improved by the staining of tape liftings methodology with Orange G, alkaline lysis, direct-PCR and a small amplicon-based NGS approach. Nonetheless, future work is required to assess the performance of the single spermatozoa method on mock swabs with more diluted semen. Also, commercially available NGS kits should be tested with single skin flakes and compared with the in-house NGS method.
Survival of patients with pediatric acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-SCT) is mainly compromised by leukemia relapse, carrying dismal prognosis. As novel individualized therapeutic approaches are urgently needed, we performed whole-exome sequencing of leukemic blasts of 10 children with post–allo-SCT relapses with the aim of thoroughly characterizing the mutational landscape and identifying druggable mutations. We found that post–allo-SCT ALL relapses display highly diverse and mostly patient-individual genetic lesions. Moreover, mutational cluster analysis showed substantial clonal dynamics during leukemia progression from initial diagnosis to relapse after allo-SCT. Only very few alterations stayed constant over time. This dynamic clonality was exemplified by the detection of thiopurine resistance-mediating mutations in the nucleotidase NT5C2 in 3 patients’ first relapses, which disappeared in the post–allo-SCT relapses on relief of selective pressure of maintenance chemotherapy. Moreover, we identified TP53 mutations in 4 of 10 patients after allo-SCT, reflecting acquired chemoresistance associated with selective pressure of prior antineoplastic treatment. Finally, in 9 of 10 children’s post–allo-SCT relapse, we found alterations in genes for which targeted therapies with novel agents are readily available. We could show efficient targeting of leukemic blasts by APR-246 in 2 patients carrying TP53 mutations. Our findings shed light on the genetic basis of post–allo-SCT relapse and may pave the way for unraveling novel therapeutic strategies in this challenging situation.
Die Diskussion über Nutzen und Risiken der Biound Gentechnik beherrschte die Jahre von 1998 bis 2001. Die „grüne Gentechnik“ kämpfte auch 1997 gegen wachsende Widerstände. Erst als sich im Jahr 2001 die Medien mit der „roten“ Gentechnik befassten, sank der Anteil kritischer Vorbehalte und stieg die Akzeptanz gegenüber gentechnisch herstellten Medikamenten in der Bevölkerung. Die Entschlüsselung der menschlichen Erbanlage und die Diskussion um das Klonen von Menschen sowie die Forschung mit embryonalen Stammzellen führte neue Aspekte in die öffentliche Diskussion ein. Die Debatte findet seither auf allen politischen Ebenen statt. Im Zusammenhang mit der Genforschung werden in den Medien immer häufiger Moral und Ökonomie gegenübergestellt und gegeneinander abgewogen. In diesem Wirrwarr von unterschiedlichen und häufig auch widerstreitenden Interessen ist es nicht einfach, sich zurechtzufinden und schließlich zu einer eigenen Position zu gelangen. Umso wichtiger ist daher eine nüchterne Darstellung der Sachverhalte.
Distinct types of relay neurons in the hindbrain process somatosensory or viscerosensory information. How neurons choose between these two fates is unclear. We show here that the homeobox gene Lbx1 is essential for imposing a somatosensory fate on relay neurons in the hindbrain. In Lbx1 mutant mice, viscerosensory relay neurons are specified at the expense of somatosensory relay neurons. Thus Lbx1 expression distinguishes between the somatosensory or viscerosensory fate of relay neurons.
Mutations in SELENBP1, encoding a novel human methanethiol oxidase, cause extraoral halitosis
(2017)
Selenium-binding protein 1 (SELENBP1) has been associated with several cancers, although its exact role is unknown. We show that SELENBP1 is a methanethiol oxidase (MTO), related to the MTO in methylotrophic bacteria, that converts methanethiol to H2O2, formaldehyde, and H2S, an activity not previously known to exist in humans. We identified mutations in SELENBP1 in five patients with cabbage-like breath odor. The malodor was attributable to high levels of methanethiol and dimethylsulfide, the main odorous compounds in their breath. Elevated urinary excretion of dimethylsulfoxide was associated with MTO deficiency. Patient fibroblasts had low SELENBP1 protein levels and were deficient in MTO enzymatic activity; these effects were reversed by lentivirus-mediated expression of wild-type SELENBP1. Selenbp1-knockout mice showed biochemical characteristics similar to those in humans. Our data reveal a potentially frequent inborn error of metabolism that results from MTO deficiency and leads to a malodor syndrome.
Nutrigenomics and nutrigenetics are receiving growing attention from a diverse range of stakeholders including health care professionals, citizens, governments, insurers and industry. Currently there is special focus on research on how our food influences us and might cause discomfort or even symptoms of disease, but also the fact that several food intolerances are caused by genetic alterations. The strong interest expressed by certain segments of the general population for predictive and preventive diagnostic testing in relation to diet and ways in which this can improve overall health led to a fast growing market of nutrigenetic based tests. This puts pressures and challenges on governments and insurers for how best to reimburse new genetic tests. These discussions are best informed by a sound understanding of nutrigenetics science and technology, its promises and challenges, which are addressed in this paper. For example, some of the most common food intolerances caused by genetic variations are lactose intolerance, inherited fructose intolerance, congenital sucrase isomaltase deficiency (sucrose intolerance), celiac disease, glucose-6-phosphate deficiency (favism), alcohol intolerance and hemochromatosis. The increasing understanding of molecular mechanisms associated with these conditions is stimulating the development of a broad range of diagnostics allowing any person with adequate resources to have their genetic predispositions determined. However, many of the currently available tests cover only one of the above mentioned diseases or a small set of responsible mutations, which is in strong contrast to the evolution of medicine towards a more holistic approach as, for example, P4 medicine. Additionally, available tests are often not based on evidence or other guidelines for genetic test development as recommended by the ACCE evidentiary framework. In this paper we discuss the most common nutrigenetic diseases and their potential and demonstrated impacts on public health, as well as ways to devise personalized diet informed by human genomics variation in the future.
