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Establishing nucleic acid-based assays for genetic newborn screening (NBS) provides the possibility to screen for genetically encoded diseases like spinal muscular atrophy (SMA), best before the onset of symptoms. Such assays should be easily scalable to 384-well reactions that make the screening of up to 2000 samples per day possible. We developed a test procedure based on a cleanup protocol for dried blood spots and a quantitative (q)PCR to screen for a homozygous deletion of exon 7 of the survival of motor neuron 1 gene (SMN1) that is responsible for >95% of SMA patients. Performance of this setup is evaluated in detail and tested on routine samples. Our cleanup method for nucleic acids from dried blood spots yields enough DNA for diverse subsequent qPCR applications. To date, we have applied this approach to test 213,279 samples within 18 months. Thirty patients were identified and confirmed, implying an incidence of 1:7109 for the homozygous deletion. Using our cleanup method, a rapid workflow could be established to prepare nucleic acids from dried blood spot cards. Targeting the exon 7 deletion, no invalid, false-positive, or false-negative results were reported to date. This allows timely identification of the disease and grants access to the recently introduced treatment options, in most cases before the onset of symptoms. Carriers are not identified, thus, there are no concerns of whether to report them.

Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgSTI-3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Δtgst2, the modest impairment in Δtggt1 and Δtggt1/Δtgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Δtggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Δtggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Δtggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disorder, which causes loss of renal proximal tubular function and progressive loss of glomerular function, finally leading to end stage renal failure at school age. In the course of the disease most patients will need kidney transplantation if treatment has not been started before clinical manifestation. With an effective treatment available, a newborn screening assay is highly demanded. Since newborns with cystinosis usually do not show symptoms within the first months of life and no biochemical markers are easily detectable, a DNA-based method seems to be an obvious tool for early diagnosis. Screening was performed using high-throughput nucleic acid extraction followed by 384-well qPCR and melting analysis for the three most frequent variants (57 kb deletion NC_000017.11:g.3600934_3658165del (GRCh38); c.18_21del GACT; c.926dupG) responsible for the defective lysosomal membrane protein cystinosin (CTNS). To increase sensitivity, all heterozygous samples identified in qPCR assay were verified and screened for additional variants by applying next generation sequencing. From January 2018 to July 2019 nearly 292,000 newborns were successfully screened. We identified two newborns with a homozygous 57 kb deletion and a second one with heterozygous 57 kb deletion and a G>C substitution at position c.-512 on the second allele. Cystinosis is an example for diseases caused by a limited number of high prevalence and a high number of low prevalence variants. We have shown that qPCR combined with NGS can be used as a high throughput, cost effective tool in newborn screening for such diseases.

Toxoplasma gondii is an obligate intracellular parasite and which is able to infect and replicate within nearly any eukaryotic, nucleated cell. In the intermediate host the parasite occurs in two different stages, during the acute infection as the fast replicating tachyzoite and after the immune response, during the chronic phase of the infection, as a nearly dormant bradyzoite. These bradyzoites produce a cyst wall which can not be detected by the immune system. As a bradyzoite, the parasite persists lifelong in his intermediate host.The two known isoforms of enolase and lactate-dehydrogenase are differently expressed during stage conversion and leads to the hypothesis that the parasite has a different need of its C-metabolism. It is also questionable, whether the parasite’s mitochondrion harbour a complete TCA-cycle.The aim of this thesis was to examine the compartmentation of the C-metabolic pathways in the protozoan parasite Toxoplasma gondii, with special focus on the localization of the different isoenzymes in the mitochondria, cytosol and apicoplast.The localization of the identified glycolytic isoenzymes, all enzymes needed for a complete TCA-cycle as well as several enzymes of associated pathways was performed using immunofluorescence staining. The parasite was stably transfected with a plasmid containing the complete or partial open reading frame of the corresponding gene followed by a myc-tag, which allowed to detect the produced fusion proteins with myc-tag specific antibodies. As an alternative approach polyclonal antisera were produced to confirm the localization of some proteins.Only one PDH-complex is encoded in the genome and all subunits could be recognized in the apicoplast exclusively by indirect immunofluorescence staining and was confirmed with the polyclonal antisera. The presence of the PDH-complex inside the apicoplast explains how the acetyl-CoA for the type II fatty acid biosynthesis in the apicoplast is provided. In addition, isoenzymes of the pyruvate-kinase, triosephosphate-isomerase and phosphoglycerate-kinase could be localized to the apicoplast via indirect immunofluorescence staining. A triosephosphate-translocator was also found to be localized to the apicoplast. All these enzymes provide essential substrates for the biosynthesis of isoprenoids and phospholipids. Together with the previously described plastidic isoform of the glyceraldehyd-3-phosphate-dehydrogenase a new metabolic model for parts of the apicoplast metabolism could be developed. These plastidic isoforms of the glycolytic pathway are connected and catalyze the reaction during which reduction power and energy is produced. After the import of dihydroxyaceton-phosphate (DHAP) by the triosephosphate-translocator DHAP is reduced to 3-phosphoglycerate in 3 steps in which NAD(P)H and ATP are produced and at the end 3-phosphoglycerate is exported back into the cytosol to be used for further reactions in the glycolytic pathway.Due to the absence of the PDH-complex in the mitochondria and and thus the loss of the natural acetyl-CoA source for the TCA-cycle, it was unclear whether there was a functional TCA-cycle in the mitochondria or not. The genome of Toxoplasma gondii encodes all enzymes needed for a complete TCA-cycle and they could all be localized to the mitochondria by immunofluorescence. The presence of all these enzymes and a complete respiratory chain leads to assume a complete cycle.A functional characterisation of the TCA-cycle was done by the construction of a conditional knock-out mutant of the succinyl-CoA-synthetase alpha subunit. After the integration of an additional copy under the control of a tetracycline inducible promoter, the endogenous locus of this gene was destroyed by homologous integration of a knock-out plasmid. The disruption of the TCA-cycle does not lead to a lethal phenotype. Thus it seems that a complete cycle is not essential for the survival of the parasite. Further analysis of the knock out mutant showed a reduced growth rate in comparison to the wild type. The addition of external succinate to the media led to a complementation of the phenotype.However, for a functional TCA-cycle the presence of acetyl-CoA is absolutely essential. Acetyl-CoA might be provided by the degradation of branched amino acid, which is supported by the fact that an essential enzyme, the branched-chain oxoglutarate dehydrogenase, could also be identified and localized to the mitochondria.