Mapping the functional landscape of frequent phenylalanine hydroxylase (PAH) genotypes promotes personalised medicine in phenylketonuria

Background: In phenylketonuria, genetic heterogeneity, frequent compound heterozygosity, and the lack of functional data for phenylalanine hydroxylase genotypes hamper reliable phenotype prediction and individualised treatment. Methods: A literature search revealed 690 different phenylalanine hydroxylase genotypes in 3066 phenylketonuria patients from Europe and the Middle East. We determined phenylalanine hydroxylase function of 30 frequent homozygous and compound heterozygous genotypes covering 55% of the study population, generated activity landscapes, and assessed the phenylalanine hydroxylase working range in the metabolic (phenylalanine) and therapeutic (tetrahydrobiopterin) space. Results: Shared patterns in genotype-specific functional landscapes were linked to biochemical and pharmacological phenotypes, where (1) residual activity below 3.5% was associated with classical phenylketonuria unresponsive to pharmacological treatment; (2) lack of defined peak activity induced loss of response to tetrahydrobiopterin; (3) a higher cofactor need was linked to inconsistent clinical phenotypes and low rates of tetrahydrobiopterin response; and (4) residual activity above 5%, a defined peak of activity, and a normal cofactor need were associated with pharmacologically treatable mild phenotypes. In addition, we provide a web application for retrieving country-specific information on genotypes and genotype-specific phenylalanine hydroxylase function that warrants continuous extension, updates, and research on demand. Conclusions: The combination of genotype-specific functional analyses with biochemical, clinical, and therapeutic data of individual patients may serve as a powerful tool to enable phenotype prediction and to establish personalised medicine strategies for dietary regimens and pharmacological treatment in phenylketonuria

Mapping the functional landscape of frequent phenylalanine hydroxylase (PAH) genotypes promotes personalised medicine in phenylketonuria

Background: In phenylketonuria, genetic heterogeneity, frequent compound heterozygosity, and the lack of functional data for phenylalanine hydroxylase genotypes hamper reliable phenotype prediction and individualised treatment. Methods: A literature search revealed 690 different phenylalanine hydroxylase genotypes in 3066 phenylketonuria patients from Europe and the Middle East. We determined phenylalanine hydroxylase function of 30 frequent homozygous and compound heterozygous genotypes covering 55% of the study population, generated activity landscapes, and assessed the phenylalanine hydroxylase working range in the metabolic (phenylalanine) and therapeutic (tetrahydrobiopterin) space. Results: Shared patterns in genotype-specific functional landscapes were linked to biochemical and pharmacological phenotypes, where (1) residual activity below 3.5% was associated with classical phenylketonuria unresponsive to pharmacological treatment; (2) lack of defined peak activity induced loss of response to tetrahydrobiopterin; (3) a higher cofactor need was linked to inconsistent clinical phenotypes and low rates of tetrahydrobiopterin response; and (4) residual activity above 5%, a defined peak of activity, and a normal cofactor need were associated with pharmacologically treatable mild phenotypes. In addition, we provide a web application for retrieving country-specific information on genotypes and genotype-specific phenylalanine hydroxylase function that warrants continuous extension, updates, and research on demand. Conclusions: The combination of genotype-specific functional analyses with biochemical, clinical, and therapeutic data of individual patients may serve as a powerful tool to enable phenotype prediction and to establish personalised medicine strategies for dietary regimens and pharmacological treatment in phenylketonuria

Edgetic Perturbations Contribute to Phenotypic Variability in PEX26 Deficiency

Peroxisomes share metabolic pathways with other organelles and peroxisomes are embedded into key cellular processes. However, the specific function of many peroxisomal proteins remains unclear and restricted knowledge of the peroxisomal protein interaction network limits a precise mapping of this network into the cellular metabolism. Inborn peroxisomal disorders are autosomal or X-linked recessive diseases that affect peroxisomal biogenesis (PBD) and/or peroxisomal metabolism. Pathogenic variants in the PEX26 gene lead to peroxisomal disorders of the full Zellweger spectrum continuum. To investigate the phenotypic complexity of PEX26 deficiency, we performed a combined organelle protein interaction screen and network medicine approach and 1) analyzed whether PEX26 establishes interactions with other peroxisomal proteins, 2) deciphered the PEX26 interaction network, 3) determined how PEX26 is involved in further processes of peroxisomal biogenesis and metabolism, and 4) showed how variant-specific disruption of protein-protein interactions (edgetic perturbations) may contribute to phenotypic variability in PEX26 deficient patients. The discovery of 14 novel protein-protein interactions for PEX26 revealed a hub position of PEX26 inside the peroxisomal interactome. Analysis of edgetic perturbations of PEX26 variants revealed a strong correlation between the number of affected protein-protein interactions and the molecular phenotype of matrix protein import. The role of PEX26 in peroxisomal biogenesis was expanded encompassing matrix protein import, division and proliferation, and membrane assembly. Moreover, the PEX26 interaction network intersects with cellular lipid metabolism at different steps. The results of this study expand the knowledge about the function of PEX26 and refine genotype-phenotype correlations, which may contribute to our understanding of the underlying disease mechanism of PEX26 deficiency

The interplay between genotype, metabolic state and cofactor treatment governs phenylalanine hydroxylase function and drug response

The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH4 cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH4 concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH4 loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH4 ratio. Finally, evaluation of data from BH4 loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH4-responsive PAH deficienc

Varicella Zoster Virus ORF25 Gene Product: An Essential Hub Protein Linking Encapsidation Proteins and the Nuclear Egress Complex

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.Fil: Vizoso Pinto, María Guadalupe. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Pothineni, Venkata R.. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Haase, Rudolf. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Woidy, Mathias. Ludwig Maximilians Universitat; AlemaniaFil: Lotz Havla, Amelie. Ludwig Maximilians Universitat; AlemaniaFil: Gersting, Soren W.. Ludwig Maximilians Universitat; AlemaniaFil: Muntau, Ania C.. Ludwig Maximilians Universitat; AlemaniaFil: Haas, Jurgen. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Sommer, Marvin. University of Stanford; Estados UnidosFil: Arvin, Ann M.. University of Stanford; Estados UnidosFil: Baiker, Armin. Bavarian Health and Food Safety Authority; Alemani

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9–mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.publishedVersio

Biallelic MADD variants cause a phenotypic spectrum ranging from developmental delay to a multisystem disorder

In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands