High-throughput genetic newborn screening for spinal muscular atrophy by rapid nucleic acid extraction from dried blood spots and 384-well qPCR.

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.

Next generation sequencing as second-tier test in high-throughput newborn screening for nephropathic cystinosis.

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.

Molecular based newborn screening in Germany: Follow-up for cystinosis.

BACKGROUND: Newborn screening (NBS) programs for treatable metabolic disorders have been enormously successful, but molecular-based screening has not been broadly implemented so far. METHODS: This prospective pilot study was performed within the German NBS framework. DNA, extracted from dried blood cards was collected as part of the regular NBS program. As cystinosis has a prevalence of only 1:100,000-1:200,000, a molecular genetic assay for detection of the SMN1 gene mutation with a higher prevalence was also included in the screening process, a genetic defect that leads to spinal muscular atrophy (SMA). First tier multiplex PCR was employed for both diseases. The cystinosis screening employed assays for the three most common CTNS mutations covering 75% of German patients; in case of heterozygosity for one of these mutations, samples were screened by next generation sequencing (NGS) of the CTNS exons for 101 CTNS mutations. A detection rate of 98.5% is predicted using this approach. RESULTS: Between January 15, 2018 and May 31, 2019, 257,734 newborns were screened in Germany for cystinosis. One neonate was diagnosed with cystinosis, consistent with the known incidence of the disease. No false positive or false negatives were detected so far. Screening, communication of findings to parents, and confirmation of diagnosis were accomplished in a multi-disciplinary setting. This program was accomplished with the cooperation of hospitals, physicians, and parents. In the neonate diagnosed with cystinosis, oral cysteamine treatment began on day 18. After 16 months of treatment the child has no clinical signs of renal tubular Fanconi syndrome. CONCLUSIONS: This pilot study demonstrates the efficacy of a molecular-based neonatal screening program for cystinosis using an existing national screening framework.

Apicoplast: keep it or leave it.

Most Apicomplexans possess a relic plastid named apicoplast, originating from secondary endosymbiosis of a red algae. This non-photosynthetic organelle fulfils important metabolic functions and confers sensitivity to antibiotics. The tasks of this organelle is compared across the phylum of Apicomplexa, highlighting its role in metabolic adaptation to different intracellular niches.

Host-derived glucose and its transporter in the obligate intracellular pathogen Toxoplasma gondii are dispensable by glutaminolysis.

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 (TgST1-3), 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 Deltatgst2, the modest impairment in Deltatggt1 and Deltatggt1/Deltatgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Deltatggt1 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 Deltatggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Deltatggt1 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.

Targeting the transcriptional and translational machinery of the endosymbiotic organelle in apicomplexans.

Apicomplexans are obligate intracellular parasites causing devastating disease in both humans and livestock. Nearly all apicomplexans, with the exception of Cryptosporidium, contain two endosymbiontic organelles carrying their own DNA; the mitochondrion and the plastid-like organelle called the apicoplast. The apicoplast is an attractive drug target as it harbors not only metabolic pathways not found in the host cell, but it is also dependent on its ancient transcriptional and translational machinery. These parasites rely on the plastid, and inhibition of its function or loss of this organelle leads to immediate or delayed death. Replication of plastidic DNA shows differences between the members of this phylum. In Plasmodium parasites, two forms of replication are observed--unidirectional single-stranded replication and a rolling circle mechanism--whereas in Toxoplasma gondii only the rolling circle is found. Targeting enzymes involved in DNA-replication leads to a delayed death of the parasite. Most of the genes in the apicoplast genome encode elements of their own transcriptional and translational machinery, and they are highly similar to those found in bacteria. Several anti-bacterials which target this machinery are also active against apicomplexan parasites and inhibition leads mostly to the delayed death phenomenon.

Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii.

The apicomplexan parasite Toxoplasma gondii displays some unusual localisations of carbohydrate converting enzymes, which is due to the presence of a vestigial, non-photosynthetic plastid, referred to as the apicoplast. It was recently demonstrated that the single pyruvate dehydrogenase complex (PDH) in T. gondii is exclusively localised inside the apicoplast but absent in the mitochondrion. This raises the question about expression, localisation and function of enzymes for the tricarboxylic acid (TCA)-cycle, which normally depends on PDH generated acetyl-CoA. Based on the expression and localisation of epitope-tagged fusion proteins, we show that all analysed TCA cycle enzymes are localised in the mitochondrion, including both isoforms of malate dehydrogenase. The absence of a cytosolic malate dehydrogenase suggests that a typical malate-aspartate shuttle for transfer of reduction equivalents is missing in T. gondii. We also localised various enzymes which catalyse the irreversible steps in gluconeogenesis to a cellular compartment and examined mRNA expression levels for gluconeogenesis and TCA cycle genes between tachyzoites and in vitro bradyzoites. In order to get functional information on the TCA cycle for the parasite energy metabolism, we created a conditional knock-out mutant for the succinyl-CoA synthetase. Disruption of the sixth step in the TCA cycle should leave the biosynthetic parts of the cycle intact, but prevent FADH2 production. The succinyl-CoA synthetase depletion mutant displayed a 30% reduction in growth rate, which could be restored by supplementation with 2 microM succinate in the tissue culture medium. The mitochondrial membrane potential in these parasites was found to be unaltered. The lack of a more severe phenotype suggests that a functional TCA cycle is not essential for T. gondii replication and for maintenance of the mitochondrial membrane potential.

Identification of novel bradyzoite-specific Toxoplasma gondii genes with domains for protein-protein interactions by suppression subtractive hybridization.

By using suppression subtractive hybridization we identified five so far uncharacterized stage specific genes in Toxoplasma gondii, which are induced during tachyzoite-to-bradyzoite differentiation. The mRNA level of a putative zinc-finger protein was increased 23-fold in bradyzoites, while the remaining four genes displayed induction levels >100-fold. Two of these genes predict proteins with domains for protein-protein interactions. One protein (ANK1) contains both, a TPR-domain and an ankyrin motif, which consists of seven repeats. ANK1 was shown by epitope tagging experiments to be localized in the cytosol. In a fraction of parasites, the myc-tagged fusion protein was additionally localized in the nucleus, which is in agreement with the presence of a bipartite nuclear targeting sequence in ANK1. The identification of bradyzoite-specific proteins with TPR- and ankyrin-domains supports the concept that during stage conversion a variety of proteins which are involved in protein-protein interactions are induced, thereby assisting the rebuilding of the proteome.

Carbohydrate metabolism in the Toxoplasma gondii apicoplast: localization of three glycolytic isoenzymes, the single pyruvate dehydrogenase complex, and a plastid phosphate translocator.

Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites' viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.