Beyond nephronophthisis: Retinal dystrophy in the absence of kidney dysfunction in childhood expands the clinical spectrum of CEP83 deficiency.

The CEP83 protein is an essential part in the first steps of ciliogenesis, causing a ciliopathy if deficient. As a core component of the distal appendages of the centriole, CEP83 is located in almost all cell types and is involved in the primary cilium assembly. Previously reported CEP83 deficient patients all presented with nephronophthisis and kidney dysfunction. Despite retinal degeneration being a common feature in ciliopathies, only one patient also had retinitis. Here, we present two unrelated patients, who both presented with retinitis pigmentosa, without nephronophthisis or any form of kidney dysfunction. Both patients harbor bi-allelic variants in CEP83. This report expands the current clinical spectrum of CEP83 deficiency. For timely diagnosis of CEP83 deficiency, we advocate that CEP83 should be included in gene panels for inherited retinal diseases.

Cross-Omics: Integrating Genomics with Metabolomics in Clinical Diagnostics.

Next-generation sequencing and next-generation metabolic screening are, independently, increasingly applied in clinical diagnostics of inborn errors of metabolism (IEM). Integrated into a single bioinformatic method, these two -omics technologies can potentially further improve the diagnostic yield for IEM. Here, we present cross-omics: a method that uses untargeted metabolomics results of patient's dried blood spots (DBSs), indicated by Z-scores and mapped onto human metabolic pathways, to prioritize potentially affected genes. We demonstrate the optimization of three parameters: (1) maximum distance to the primary reaction of the affected protein, (2) an extension stringency threshold reflecting in how many reactions a metabolite can participate, to be able to extend the metabolite set associated with a certain gene, and (3) a biochemical stringency threshold reflecting paired Z-score thresholds for untargeted metabolomics results. Patients with known IEMs were included. We performed untargeted metabolomics on 168 DBSs of 97 patients with 46 different disease-causing genes, and we simulated their whole-exome sequencing results in silico. We showed that for accurate prioritization of disease-causing genes in IEM, it is essential to take into account not only the primary reaction of the affected protein but a larger network of potentially affected metabolites, multiple steps away from the primary reaction.

Untargeted Metabolomics for Metabolic Diagnostic Screening with Automated Data Interpretation Using a Knowledge-Based Algorithm.

Untargeted metabolomics may become a standard approach to address diagnostic requests, but, at present, data interpretation is very labor-intensive. To facilitate its implementation in metabolic diagnostic screening, we developed a method for automated data interpretation that preselects the most likely inborn errors of metabolism (IEM). The input parameters of the knowledge-based algorithm were (1) weight scores assigned to 268 unique metabolites for 119 different IEM based on literature and expert opinion, and (2) metabolite Z-scores and ranks based on direct-infusion high resolution mass spectrometry. The output was a ranked list of differential diagnoses (DD) per sample. The algorithm was first optimized using a training set of 110 dried blood spots (DBS) comprising 23 different IEM and 86 plasma samples comprising 21 different IEM. Further optimization was performed using a set of 96 DBS consisting of 53 different IEM. The diagnostic value was validated in a set of 115 plasma samples, which included 58 different IEM and resulted in the correct diagnosis being included in the DD of 72% of the samples, comprising 44 different IEM. The median length of the DD was 10 IEM, and the correct diagnosis ranked first in 37% of the samples. Here, we demonstrate the accuracy of the diagnostic algorithm in preselecting the most likely IEM, based on the untargeted metabolomics of a single sample. We show, as a proof of principle, that automated data interpretation has the potential to facilitate the implementation of untargeted metabolomics for metabolic diagnostic screening, and we provide suggestions for further optimization of the algorithm to improve diagnostic accuracy.

Hypothesis: determining phenotypic specificity facilitates understanding of pathophysiology in rare genetic disorders.

In the rapidly growing group of rare genetic disorders, data scarcity demands an intelligible use of available data, in order to improve understanding of underlying pathophysiology. We hypothesize, based on the principle that clinical similarities may be indicative of shared pathophysiology, that determining phenotypic specificity could provide unsuspected insights in pathophysiology of rare genetic disorders. We explored our hypothesis by studying subunit deficiencies of the conserved oligomeric Golgi (COG) complex, a subgroup of congenital disorders of glycosylation (CDG). In this systematic data assessment, all 45 reported patients with COG-CDG were included. The vocabulary of the Human Phenotype Ontology was used to annotate all phenotypic features and to assess occurrence in other genetic disorders. Gene occurrence ratios were calculated by dividing the frequency in the patient cohort over the number of associated genes, according to the Human Phenotype Ontology. Prioritisation based on phenotypic specificity was highly informative and captured phenotypic features commonly associated with glycosylation disorders. Moreover, it captured features not seen in any other glycosylation disorder, among which episodic fever, likely reflecting underappreciated other cellular functions of the COG complex. Interestingly, the COG complex was recently implicated in the autophagy pathway, as are more than half of the genes underlying disorders that present with episodic fever. This suggests that whereas many phenotypic features in these patients are caused by disrupted glycosylation, episodic fever might be caused by disrupted autophagy. Thus, we here demonstrate support for our hypothesis that determining phenotypic specificity could facilitate understanding of pathophysiology in rare genetic disorders.

Retrospective evaluation of the Dutch pre-newborn screening cohort for propionic acidemia and isolated methylmalonic acidemia: What to aim, expect, and evaluate from newborn screening?

Evidence for effectiveness of newborn screening (NBS) for propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) is scarce. Prior to implementation in the Netherlands, we aim to estimate the expected health gain of NBS for PA and MMA. In this national retrospective cohort study, the clinical course of 76/83 Dutch PA and MMA patients, diagnosed between January 1979 and July 2019, was evaluated. Five clinical outcome parameters were defined: adverse outcome of the first symptomatic phase, frequency of acute metabolic decompensations (AMD), cognitive function, mitochondrial complications, and treatment-related complications. Outcomes of patients identified by family testing were compared with the outcomes of their index siblings. An adverse outcome due to the first symptomatic phase was recorded in 46% of the clinically diagnosed patients. Outcome of the first symptomatic phase was similar in 5/9 sibling pairs and better in 4/9 pairs. Based on the day of diagnosis of the clinically diagnosed patients and sibling pair analysis, a preliminary estimated reduction of adverse outcome due to the first symptomatic phase from 46% to 36%-38% was calculated. Among the sibling pairs, AMD frequency, cognitive function, mitochondrial, and treatment-related complications were comparable. These results suggest that the health gain of NBS for PA and MMA in overall outcome may be limited, as only a modest decrease of adverse outcomes due to the first symptomatic phase is expected. With current clinical practice, no reduced AMD frequency, improved cognitive function, or reduced frequency of mitochondrial or treatment-related complications can be expected.

Assessing the Pre-Analytical Stability of Small-Molecule Metabolites in Cerebrospinal Fluid Using Direct-Infusion Metabolomics.

Metabolomics studies aiming to find biomarkers frequently make use of historical or multicenter cohorts. These samples often have different pre-analytical conditions that potentially affect metabolite concentrations. We studied the effect of different storage conditions on the stability of small-molecule metabolites in cerebrospinal fluid to aid a reliable interpretation of metabolomics data. Three cerebrospinal fluid pools were prepared from surplus samples from the Amsterdam Dementia Cohort biobank. Aliquoted pools were exposed to different storage conditions to assess the temperature and freeze/thaw stability before final storage at -80 °C: storage up to four months at -20 °C and up to one week at either 5-8 °C or 18-22 °C and exposure to up to seven freeze/thaw cycles. Direct-infusion high-resolution mass spectrometry was performed, resulting in the identification of 1852 m/z peaks. To test the storage stability, principal component analyses, repeated measures analysis of variance, Kruskal‒Wallis tests, and fold change analyses were performed, all demonstrating that small-molecule metabolites in the cerebrospinal fluid (CSF) are relatively unaffected by 1‒3 freeze/thaw cycles, by storage at -20 °C up to two months, by storage at 5-8 °C for up to 72 h, or by storage at 18-22 °C for up to 8 h. This suggests that these differences do not affect the interpretation of potential small-molecule biomarkers in multicenter or historical cohorts and implies that these cohorts are suitable for biomarker studies.

Aspartylglycosamine is a biomarker for NGLY1-CDDG, a congenital disorder of deglycosylation.

BACKGROUND: NGLY1-CDDG is a congenital disorder of deglycosylation caused by a defective peptide:N-glycanase (PNG). To date, all but one of the reported patients have been diagnosed through whole-exome or whole-genome sequencing, as no biochemical marker was available to identify this disease in patients. Recently, a potential urinary biomarker was reported, but the data presented suggest that this marker may be excreted intermittently. METHODS: In this study, we performed untargeted direct-infusion high-resolution mass spectrometry metabolomics in seven dried blood spots (DBS) from four recently diagnosed NGLY1-CDDG patients, to test for small-molecule biomarkers, in order to identify a potential diagnostic marker. Results were compared to 125 DBS of healthy controls and to 238 DBS of patients with other diseases. RESULTS: We identified aspartylglycosamine as the only significantly increased compound with a median Z-score of 4.8 (range: 3.8-8.5) in DBS of NGLY1-CDDG patients, compared to a median Z-score of -0.1 (range: -2.1-4.0) in DBS of healthy controls and patients with other diseases. DISCUSSION: The increase of aspartylglycosamine can be explained by lack of function of PNG. PNG catalyzes the cleavage of the proximal N-acetylglucosamine residue of an N-glycan from the asparagine residue of a protein, a step in the degradation of misfolded glycoproteins. PNG deficiency results in a single N-acetylglucosamine residue left attached to the asparagine residue which results in free aspartylglycosamine when the glycoprotein is degraded. Thus, we here identified aspartylglycosamine as the first potential small-molecule biomarker in DBS for NGLY1-CDDG, making a biochemical diagnosis for NGLY1-CDDG potentially feasible.

De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

Pathophysiology of propionic and methylmalonic acidemias. Part 2: Treatment strategies.

Despite realizing increased survival rates for propionic acidemia (PA) and methylmalonic acidemia (MMA) patients, the current therapeutic regimen is inadequate for preventing or treating the devastating complications that still can occur. The elucidation of pathophysiology of these complications allows us to evaluate and rethink treatment strategies. In this review we display and discuss potential therapy targets and we give a systematic overview on current, experimental and unexplored treatment strategies in order to provide insight in what we have to offer PA and MMA patients, now and in the future. Evidence on the effectiveness of treatment strategies is often scarce, since none were tested in randomized clinical trials. This raises concerns, since even the current consensus on best practice treatment for PA and MMA is not without controversy. To attain substantial improvements in overall outcome, gene, mRNA or enzyme replacement therapy is most promising since permanent reduction of toxic metabolites allows for a less strict therapeutic regime. Hereby, both mitochondrial-associated and therapy induced complications can theoretically be prevented. However, the road from bench to bedside is long, as it is challenging to design a drug that is delivered to the mitochondria of all tissues that require enzymatic activity, including the brain, without inducing any off-target effects. To improve survival rate and quality of life of PA and MMA patients, there is a need for systematic (re-)evaluation of accepted and potential treatment strategies, so that we can better determine who will benefit when and how from which treatment strategy.

Pathophysiology of propionic and methylmalonic acidemias. Part 1: Complications.

Over the last decades, advances in clinical care for patients suffering from propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) have resulted in improved survival. These advances were possible thanks to new pathophysiological insights. However, patients may still suffer from devastating complications which largely determine the unsatisfying overall outcome. To optimize our treatment strategies, better insight in the pathophysiology of complications is needed. Here, we perform a systematic data-analysis of cohort studies and case-reports on PA and MMA. For each of the prevalent and rare complications, we summarize the current hypotheses and evidence for the underlying pathophysiology of that complication. A common hypothesis on pathophysiology of many of these complications is that mitochondrial impairment plays a major role. Assuming that complications in which mitochondrial impairment may play a role are overrepresented in monogenic mitochondrial diseases and, conversely, that complications in which mitochondrial impairment does not play a role are underrepresented in mitochondrial disease, we studied the occurrence of the complications in PA and MMA in mitochondrial and other monogenic diseases, using data provided by the Human Phenotype Ontology. Lastly, we combined this with evidence from literature to draw conclusions on the possible role of mitochondrial impairment in each complication. Altogether, this review provides a comprehensive overview on what we, to date, do and do not understand about pathophysiology of complications occurring in PA and MMA and about the role of mitochondrial impairment herein.

Direct-infusion based metabolomics unveils biochemical profiles of inborn errors of metabolism in cerebrospinal fluid.

BACKGROUND: For inborn errors of metabolism (IEM), metabolomics is performed for three main purposes: 1) development of next generation metabolic screening platforms, 2) identification of new biomarkers in predefined patient cohorts and 3) for identification of new IEM. To date, plasma, urine and dried blood spots are used. We anticipate that cerebrospinal fluid (CSF) holds additional - valuable - information, especially for IEM with neurological involvement. To expand metabolomics to CSF, we here tested whether direct-infusion high-resolution mass spectrometry (DI-HRMS) based non-quantitative metabolomics could correctly capture the biochemical profile of patients with an IEM in CSF. METHODS: Eleven patient samples, harboring eight different IEM, and thirty control samples were analyzed using DI-HRMS. First we assessed whether the biochemical profile of the control samples represented the expected profile in CSF. Next, each patient sample was assigned a 'most probable diagnosis' by an investigator blinded for the known diagnoses of the patients. RESULTS: the biochemical profile identified using DI-HRMS in CSF samples resembled the known profile, with - among others - the highest median intensities for mass peaks annotated with glucose, lactic acid, citric acid and glutamine. Subsequent analysis of patient CSF profiles resulted in correct 'most probable diagnoses' for all eleven patients, including non-ketotic hyperglycinaemia, propionic aciduria, purine nucleoside phosphorylase deficiency, argininosuccinic aciduria, tyrosinaemia type I, hyperphenylalaninemia and hypermethioninaemia. CONCLUSION: We here demonstrate that DI-HRMS based non-quantitative metabolomics accurately captures the biochemical profile of this set of patients in CSF, opening new ways for using metabolomics in CSF in the metabolic diagnostic laboratory.

Direct Infusion Based Metabolomics Identifies Metabolic Disease in Patients' Dried Blood Spots and Plasma.

In metabolic diagnostics, there is an emerging need for a comprehensive test to acquire a complete view of metabolite status. Here, we describe a non-quantitative direct-infusion high-resolution mass spectrometry (DI-HRMS) based metabolomics method and evaluate the method for both dried blood spots (DBS) and plasma. 110 DBS of 42 patients harboring 23 different inborn errors of metabolism (IEM) and 86 plasma samples of 38 patients harboring 21 different IEM were analyzed using DI-HRMS. A peak calling pipeline developed in R programming language provided Z-scores for ~1875 mass peaks corresponding to ~3835 metabolite annotations (including isomers) per sample. Based on metabolite Z-scores, patients were assigned a 'most probable diagnosis' by an investigator blinded for the known diagnoses of the patients. Based on DBS sample analysis, 37/42 of the patients, corresponding to 22/23 IEM, could be correctly assigned a 'most probable diagnosis'. Plasma sample analysis, resulted in a correct 'most probable diagnosis' in 32/38 of the patients, corresponding to 19/21 IEM. The added clinical value of the method was illustrated by a case wherein DI-HRMS metabolomics aided interpretation of a variant of unknown significance (VUS) identified by whole-exome sequencing. In summary, non-quantitative DI-HRMS metabolomics in DBS and plasma is a very consistent, high-throughput and nonselective method for investigating the metabolome in genetic disease.

Hypothesis: lobe A (COG1-4)-CDG causes a more severe phenotype than lobe B (COG5-8)-CDG.

The conserved oligomeric Golgi (COG) complex consists of eight subunits organized in two lobes: lobe A (COG1-4) and lobe B (COG5-8). The different functional roles of COG lobe A and lobe B might result in distinct clinical phenotypes in patients with COG-CDG (congenital disorders of glycosylation). This hypothesis is supported by three observations. First, knock-down of COG lobe A components affects Golgi morphology more severely than knock-down of COG lobe B components. Second, nearly all of the 27 patients with lobe B COG-CDG had bi-allelic truncating mutations, as compared with only one of the six patients with lobe A COG-CDG. This represents a frequency gap which suggests that bi-allelic truncating mutations in COG lobe A genes might be non-viable. Third, in support, large-scale exome data of healthy adults (Exome Aggregation Consortium (ExAC)) underline that COG lobe A genes are less tolerant to genetic variation than COG lobe B genes. Thus, comparable molecular defects are more detrimental in lobe A COG-CDG than in lobe B COG-CDG. In a larger perspective, clinical phenotypic severity corresponded nicely with tolerance to genetic variation. Therefore, genomic epidemiology can potentially be used as a photographic negative for mutational severity.

Participatory methods in pediatric participatory research: a systematic review.

Meaningful child participation in medical research is seen as important. In order to facilitate further development of participatory research, we performed a systematic literature study to describe and assess the available knowledge on participatory methods in pediatric research. A search was executed in five databases: PubMed, CINAHL, PsycINFO, Scopus, and Cochrane. After careful screening of relevant papers, finally 24 documents were included in our analysis. Literature on participatory methods in pediatric research appears generally to be descriptive, whereby high-quality evidence is lacking. Overall, five groups of participatory methods for children could be distinguished: observational, verbal, written, visual, and active methods. The choice for one of these methods should be based on the child's age, on social and demographic characteristics, and on the research objectives. To date, these methods are still solely used for obtaining data, yet they are suitable for conducting meaningful participation. This may result in a successful partnership between children and researchers. Researchers conducting participatory research with children can use this systematic review in order to weigh the current knowledge about the participatory methods presented.

Key features and clinical variability of COG6-CDG.

The conserved oligomeric Golgi (COG) complex consists of eight subunits and plays a crucial role in Golgi trafficking and positioning of glycosylation enzymes. Mutations in all COG subunits, except subunit 3, have been detected in patients with congenital disorders of glycosylation (CDG) of variable severity. So far, 3 families with a total of 10 individuals with biallelic COG6 mutations have been described, showing a broad clinical spectrum. Here we present 7 additional patients with 4 novel COG6 mutations. In spite of clinical variability, we delineate the core features of COG6-CDG i.e. liver involvement (9/10), microcephaly (8/10), developmental disability (8/10), recurrent infections (7/10), early lethality (6/10), and hypohidrosis predisposing for hyperthermia (6/10) and hyperkeratosis (4/10) as ectodermal signs. Regarding all COG6-related disorders a genotype-phenotype correlation can be discerned ranging from deep intronic mutations found in Shaheen syndrome as the mildest form to loss-of-function mutations leading to early lethal CDG phenotypes. A comparison with other COG deficiencies suggests ectodermal changes to be a hallmark of COG6-related disorders. Our findings aid clinical differentiation of this complex group of disorders and imply subtle functional differences between the COG complex subunits.