Seroconversion to HCoV-NL63 in Rhesus Macaques

HCoV-NL63 is a recently identified respiratory virus. Its pathogenesis has not been fully unraveled because an animal model is currently lacking. Here we examined whether rhesus macaques encounter HCoV-NL63 infections during life, by examining the levels of antibodies to HCoV-NL63 in time. The animals were followed for 7 up till 19 years, and in three animals we observed a steep rise in antibodies during follow up, indicative of a natural infection with HCoV-NL63

Terminal osseous dysplasia with pigmentary defects and cardiomyopathy caused by a novel FLNA variant

Terminal osseous dysplasia with pigmentary defects (TODPD), also known as digitocutaneous dysplasia, is one of the X‐linked filaminopathies caused by a variety of FLNA‐variants. TODPD is characterized by skeletal defects, skin fibromata and dysmorphic facial features. So far, only a single recurrent variant (c.5217G>A;p.Val1724_Thr1739del) in FLNA has found to be responsible for TODPD. We identified a novel c.5217+5G>C variant in FLNA in a female proband with skeletal defects, skin fibromata, interstitial lung disease, epilepsy, and restrictive cardiomyopathy. This variant causes mis‐splicing of exon 31 predicting the production of a FLNA‐protein with an in‐frame‐deletion of 16 residues identical to the miss‐splicing‐effect of the recurrent TODPD c.5217G>A variant. This mis‐spliced transcript was explicitly detected in heart tissue, but was absent from blood, skin, and lung. X‐inactivation analyses showed extreme skewing with almost complete inactivation of the mutated allele (>90%) in these tissues, except for heart. The mother of the proband, who also has fibromata and skeletal abnormalities, is also carrier of the FLNA‐variant and was diagnosed with noncompaction cardiomyopathy after cardiac screening. No other relevant variants in cardiomyopathy‐related genes were found. Here we describe a novel variant in FLNA (c.5217+5G>C) as the second pathogenic variant responsible for TODPD. Cardiomyopathy has not been described as a phenotypic feature of TODPD before

A second case of glutaminase hyperactivity: Expanding the phenotype with epilepsy

Glutaminase (GLS) hyperactivity was first described in 2019 in a patient with profound developmental delay and infantile cataract. Here, we describe a 4-year-old boy with GLS hyperactivity due to a de novo heterozygous missense variant in GLS, detected by trio whole exome sequencing. This boy also exhibits developmental delay without dysmorphic features, but does not have cataract. Additionally, he suffers from epilepsy with tonic clonic seizures. In line with the findings in the previously described patient with GLS hyperactivity, in vivo 3 T magnetic resonance spectroscopy (MRS) of the brain revealed an increased glutamate/glutamine ratio. This increased ratio was also found in urine with UPLC-MS/MS, however, inconsistently. This case indicates that the phenotypic spectrum evoked by GLS hyperactivity may include epilepsy. Clarifying this phenotypic spectrum is of importance for the prognosis and identification of these patients. The combination of phenotyping, genetic testing, and metabolic diagnostics with brain MRS and in urine is essential to identify new patients with GLS hyperactivity and to further extend the phenotypic spectrum of this disease

The detection of a strong episignature for Chung–Jansen syndrome, partially overlapping with Börjeson–Forssman–Lehmann and White–Kernohan syndromes

Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the PHIP gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that PHIP haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic PHIP variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung–Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung–Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White–Kernohan syndrome (caused by variants in DDB1 gene) and Börjeson–Forssman–Lehmann syndrome (caused by variants in PHF6 gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung–Jansen, Börjeson–Forssman–Lehmann and White–Kernohan syndromes.</p

The detection of a strong episignature for Chung-Jansen syndrome, partially overlapping with Börjeson-Forssman-Lehmann and White-Kernohan syndromes

Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the PHIP gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that PHIP haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic PHIP variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung-Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung-Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White-Kernohan syndrome (caused by variants in DDB1 gene) and Börjeson-Forssman-Lehmann syndrome (caused by variants in PHF6 gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung-Jansen, Börjeson-Forssman-Lehmann and White-Kernohan syndromes

Glutamate metabolism revisited : Clarifying the pathophysiology of GLS mutations

Inborn Errors of Metabolism (IEMs) are a class of inherited genetic disorders caused by variants in genes coding for proteins that function in metabolism. The research presented in this thesis describes the identification and disease mechanisms of two novel IEMs caused by mutations in the GLS gene. This gene encodes the protein glutaminase, which converts the amino acid glutamine into glutamate. Glutamine is a source for many proteins and is important for ammonia detoxification. Glutamate is an important neurotransmitter and plays a pivotal role in energy metabolism and redox homeostasis. Excess of these amino acids however can be harmful. A balance between these amino acids is therefore essential. In chapter 1, the required steps for the identification of new IEMs are elaborated. Furthermore, it is exemplified how the development of genetic and analytical techniques improved our ability to discover the genetic cause of IEMs. In chapter 2, we describe a new IEM in 4 children of 2 unrelated families, caused by bi-allelic loss-of-function mutations in GLS. This leads to neonatal, respiratory insufficiency, epilepsy and death. This was achieved through a “genome first approach”, in which Whole Exome Sequencing identified the variants in the GLS gene, which co-segregated within the family, and targeted metabolic analyses confirmed loss-of-function. In chapter 3, we describe another novel IEM caused by a GLS mutation. This mutation caused GLS hyperactivity, resulting in low glutamine and high glutamate levels. This patient had early onset cataract diagnosed at age 3 months, dermatological abnormalities, axial hypotonia and profound developmental delay. In a cell model in which the GLS hyperactivity mutation was expressed, we showed that metabolic compensatory mechanisms were activated: glutaminase protein expression was decreased, while glutamine synthetase (the reciprocal enzyme) protein expression was increased. We furthermore demonstrated that redox capacity buffer was decreased. We demonstrated the causality of GLS hyperactivity for cataract formation, as zebrafish with GLS hyperactivity developed cataract, which could be alleviated with GLS activity inhibition. To visualize the lens of the zebrafish embryo (smaller than 0.1mm) and lens-opacities, we developed a fluorescence microscopic method based on UV-Illumination (FUVI) in chapter 6. In chapter 4 the extensive downstream metabolic consequences of GLS hyperactivity were examined by untargeted metabolomics. This method provides an extensive “metabolic-fingerprint”. It is less specific than targeted metabolic techniques, but enables the detection of thousands of metabolites with one test. We demonstrated that GLS hyperactivity not only affected glutamine and glutamate levels, but that it has far-reaching downstream metabolic consequences. GLS loss-of-function and GLS hyperactivity are not the only inborn errors of glutamate metabolism. In chapter 5 we review all reported inborn errors of enzymes of glutamate metabolism and provide an overview of their clinical and biochemical characteristics. By drawing phenotypic and biochemical parallels, we attempt to create insight into the clinical effect of disturbed glutamate metabolism. By drawing parallels between phenotypic features of these patients, we showed that neurological, ophthalmological and dermatological features were often exhibited. This provides us with knowledge about the importance of regulated glutamate homeostasis for these tissues

Identification of a Loss-of-Function Mutation in the Context of Glutaminase Deficiency and Neonatal Epileptic Encephalopathy

Importance The identification and understanding of the monogenic causes of neurodevelopmental disorders are of high importance for personalized treatment and genetic counseling. Objective To identify and characterize novel genes for a specific neurodevelopmental disorder characterized by refractory seizures, respiratory failure, brain abnormalities, and death in the neonatal period; describe the outcome of glutaminase deficiency in humans; and understand the underlying pathological mechanisms. Design, Setting, and Participants We performed exome sequencing of cases of neurodevelopmental disorders without a clear genetic diagnosis, followed by genetic and bioinformatic evaluation of candidate variants and genes. Establishing pathogenicity of the variants was achieved by measuring metabolites in dried blood spots by a hydrophilic interaction liquid chromatography method coupled with tandem mass spectrometry. The participants are 2 families with a total of 4 children who each had lethal, therapy-refractory early neonatal seizures with status epilepticus and suppression bursts, respiratory insufficiency, simplified gyral structures, diffuse volume loss of the brain, and cerebral edema. Data analysis occurred from October 2017 to June 2018. Main Outcomes and Measures Early neonatal epileptic encephalopathy with glutaminase deficiency and lethal outcome. Results A total of 4 infants from 2 unrelated families, each of whom died less than 40 days after birth, were included. We identified a homozygous frameshift variant p.(Asp232Glufs*2) in GLS in the first family, as well as compound heterozygous variants p.(Gln81*) and p.(Arg272Lys) in GLS in the second family. The GLS gene encodes glutaminase (Enzyme Commission 3.5.1.2), which plays a major role in the conversion of glutamine into glutamate, the main excitatory neurotransmitter of the central nervous system. All 3 variants probably lead to a loss of function and thus glutaminase deficiency. Indeed, glutamine was increased in affected children (available z scores, 3.2 and 11.7). We theorize that the potential reduction of glutamate and the excess of glutamine were a probable cause of the described physiological and structural abnormalities of the central nervous system. Conclusions and Relevance We identified a novel autosomal recessive neurometabolic disorder of loss of function of glutaminase that leads to lethal early neonatal encephalopathy. This inborn error of metabolism underlines the importance of GLS for appropriate glutamine homeostasis and respiratory regulation, signal transduction, and survival

Metabolic fingerprinting reveals extensive consequences of GLS hyperactivity

Background: High glutaminase (GLS;EC3.5.1.2) activity is an important pathophysiological phenomenon in tumorigenesis and metabolic disease. Insight into the metabolic consequences of high GLS activity contributes to the understanding of the pathophysiology of both oncogenic pathways and inborn errors of glutamate metabolism. Glutaminase catalyzes the conversion of glutamine into glutamate, thereby interconnecting many metabolic pathways. Methods: We developed a HEK293-based cell-model that enables tuning of GLS activity by combining the expression of a hypermorphic GLS variant with incremental GLS inhibition. The metabolic consequences of increasing GLS activity were studied by metabolic profiling using Direct-Infusion High-Resolution Mass-Spectrometry (DI-HRMS). Results and conclusions: Of 12,437 detected features [m/z], 109 features corresponding to endogenously relevant metabolites were significantly affected by high GLS activity. As expected, these included strongly decreased glutamine and increased glutamate levels. Additionally, increased levels of tricarboxylic acid (TCA) intermediates with a truncation of the TCA cycle at the level of citrate were detected as well as increased metabolites of transamination reactions, proline and ornithine synthesis and GABA metabolism. Levels of asparagine and nucleotide metabolites showed the same dependence on GLS activity as glutamine. Of the nucleotides, especially metabolites of the pyrimidine thymine metabolism were negatively impacted by high GLS activity, which is remarkable since their synthesis depend both on aspartate (product of glutamate) and glutamine levels. Metabolites of the glutathione synthesizing γ-glutamyl-cycle were either decreased or unaffected. General significance: By providing a metabolic fingerprint of increasing GLS activity, this study shows the large impact of high glutaminase activity on the cellular metabolome