Vascular Ehlers-Danlos Syndrome: A Comprehensive Natural History Study in a Dutch National Cohort of 142 Patients.

BACKGROUND: Vascular Ehlers-Danlos syndrome (vEDS) is a rare connective tissue disorder with a high risk for arterial, bowel, and uterine rupture, caused by heterozygous pathogenic variants in COL3A1. The aim of this cohort study is to provide further insights into the natural history of vEDS and describe genotype-phenotype correlations in a Dutch multicenter cohort to optimize patient care and increase awareness of the disease. METHODS: Individuals with vEDS throughout the Netherlands were included. The phenotype was charted by retrospective analysis of molecular and clinical data, combined with a one-time physical examination. RESULTS: A total of 142 individuals (50% female) participated the study, including 46 index patients (32%). The overall median age at genetic diagnosis was 41.0 years. More than half of the index patients (54.3%) and relatives (53.1%) had a physical appearance highly suggestive of vEDS. In these individuals, major events were not more frequent (P=0.90), but occurred at a younger age (P=0.01). A major event occurred more often and at a younger age in men compared with women (P<0.001 and P=0.004, respectively). Aortic aneurysms (P=0.003) and pneumothoraces (P=0.029) were more frequent in men. Aortic dissection was more frequent in individuals with a COL3A1 variant in the first quarter of the collagen helical domain (P=0.03). CONCLUSIONS: Male sex, type and location of the COL3A1 variant, and physical appearance highly suggestive of vEDS are risk factors for the occurrence and/or early age of onset of major events. This national multicenter cohort study of Dutch individuals with vEDS provides a valuable basis for improving guidelines for the diagnosing, follow-up, and treatment of individuals with vEDS.

Impressive clinical improvement and disappearance of neuropathic pain in an adult patient with hypophosphatasia treated with asfotase alfa.

Hypophosphatasia (HPP) is a rare disorder, resulting from loss-of-function variants of the ALPL gene encoding non-tissue specific alkaline phosphatase (TNSALP). Presentation varies largely, with increased severity usually occurring with earlier disease onset. Here we describe the clinical improvement of a 57-year-old woman with childhood onset HPP, after initiating treatment with asfotase alfa (Strensiq®). This was started because of the rapid and progressive radiological deterioration of bone structure after placement of nails in both upper legs for spontaneous atypical femur fracture (AFF) - like fractures. Initiation of treatment, not only resulted in stabilization of bone structure on X-rays, but within a few weeks there was a dramatic reduction of burning pain sensations in the lower legs, attributed in retrospect to neuropathic pain, and also almost complete disappearance of headaches. Additionally, unhealed metatarsal fractures finally healed after almost 10 years. Drug efficacy was further evaluated through -quality of life questionnaires and multiple tests conducted by the physiotherapist, and showed clear improvements. Within 3 months after starting asfotase alfa, the patient was able to carry out her daily tasks indoors without relying on a walker and even started electric bike rides for 20 km/day. In conclusion, treatment with asfotase alfa, halted rapid radiological bone deterioration after bilateral intramedullary femoral pen placement and strongly increased quality of life, marked by rapid disappearance of neuropathic pain, reduction in headaches and musculoskeletal pains, and enhanced muscle strength and mobility. The quick and almost complete disappearance of neuropathic pain and headache suggests a relation with disturbed levels of metabolites in HPP.

Osteosclerotic Metaphyseal Dysplasia Due to a Likely Pathogenic LRRK1 Variant as a Cause of Recurrent Long Bone Fractures.

Osteosclerotic metaphyseal dysplasia (OSMD) is a very rare autosomal-recessive disease caused by mutations in the leucine-rich repeat kinase 1 (LRRK1) gene. It is a sclerosing skeletal dysplasia characterized by osteosclerosis of the long bones, predominantly at the metaphyses and vertebrae. Phenotypic features can be short stature, pathological fractures, delayed development, and hypotonia, but they are not uniformly present, and relatively few cases are known from the literature. A 40-year-old man was seen at our bone center because of nonspontaneous multiple peripheral low-energy trauma fractures since puberty. He had no other complaints and his family history was negative. Except for a relatively short stature (167 cm; -1.5 SD), there were no abnormalities on examination, including laboratory tests. Initially, a suspicion was raised of osteogenesis imperfecta, but bone mineral density was high and X-rays of the whole skeleton showed osteosclerosis of the metaphyses of long bones and vertebrae. Whole-exome sequencing showed a homozygous, likely pathogenic, variant (American College of Medical Genetics and Genomics criteria class 4) in the LRRK1 gene, fitting the diagnosis of OSMD. In conclusion, we described a 40-year-old patient with osteosclerotic metaphyseal dysplasia caused by a homozygous variant in the LRRK1 gene, resulting in multiple fractures of the long bones without other features of the disease, adding to the phenotypic variation of OSMD. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Early-Onset Pectus Excavatum Is More Likely to Be Part of a Genetic Variation.

BACKGROUND: Potential underlying genetic variations of pectus excavatum (PE) are quite rare. Only one-fifth of PE cases are identified in the first decade of life and thus are of congenital origin. The objective of this study is to test if early-onset PE is more likely to be part of genetic variations than PE that becomes apparent during puberty or adolescence. MATERIALS AND METHODS: Children younger than 11 years who presented with PE to the outpatient clinic of the Department of Pediatric Surgery at our center between 2014 and 2020 were screened by two clinical geneticists separately. Molecular analysis was performed based on the differential diagnosis. Data of all young PE patients who already had been referred for genetic counseling were analyzed retrospectively. RESULTS: Pathogenic genetic variations were found in 8 of the 18 participants (44%): 3 syndromic disorders (Catel-Manzke syndrome and two Noonan syndromes), 3 chromosomal disorders (16p13.11 microduplication syndrome, 22q11.21 microduplication syndrome, and genetic gain at 1q44), 1 connective tissue disease (Loeys-Dietz syndrome), and 1 neuromuscular disorder (pathogenic variation in BICD2 gene). CONCLUSION: Early-onset PE is more likely to be part of genetic variations than PE that becomes apparent during puberty or adolescence. Referral for genetic counseling should therefore be considered. TRIAL REGISTRATION: NCT05443113.

Prevalence of Monogenic Bone Disorders in a Dutch Cohort of Atypical Femur Fracture Patients.

Atypical femur fractures (AFFs), considered rare associations of bisphosphonates, have also been reported in patients with monogenic bone disorders without bisphosphonate use. The exact association between AFFs and monogenic bone disorders remains unknown. Our aim was to determine the prevalence of monogenic bone disorders in a Dutch AFF cohort. AFF patients were recruited from two specialist bone centers in the Netherlands. Medical records of the AFF patients were reviewed for clinical features of monogenic bone disorders. Genetic variants identified by whole-exome sequencing in 37 candidate genes involved in monogenic bone disorders were classified based on the American College of Medical Genetics and Genomics (ACMG) classification guidelines. Copy number variations overlapping the candidate genes were also evaluated using DNA array genotyping data. The cohort comprises 60 AFF patients (including a pair of siblings), with 95% having received bisphosphonates. Fifteen AFF patients (25%) had clinical features of monogenic bone disorders. Eight of them (54%), including the pair of siblings, had a (likely) pathogenic variant in either PLS3, COL1A2, LRP5, or ALPL. One patient carried a likely pathogenic variant in TCIRG1 among patients not suspected of monogenic bone disorders (2%). In total, nine patients in this AFF cohort (15%) had a (likely) pathogenic variant. In one patient, we identified a 12.7 Mb deletion in chromosome 6, encompassing TENT5A. The findings indicate a strong relationship between AFFs and monogenic bone disorders, particularly osteogenesis imperfecta and hypophosphatasia, but mainly in individuals with symptoms of these disorders. The high yield of (likely) pathogenic variants in AFF patients with a clinical suspicion of these disorders stresses the importance of careful clinical evaluation of AFF patients. Although the relevance of bisphosphonate use in this relationship is currently unclear, clinicians should consider these findings in medical management of these patients. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Deleterious, protein-altering variants in the transcriptional coregulator ZMYM3 in 27 individuals with a neurodevelopmental delay phenotype.

Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator ZMYM3, located on the X chromosome. Most (n = 24) individuals were males, 17 of which have a maternally inherited variant; six individuals (4 male, 2 female) harbor de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n = 26) are missense, including six that recurrently affect two residues. Four unrelated probands were identified with inherited variation affecting Arg441, a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation resulting in p.Arg1294Cys (c.3880C>T). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one variant, p.Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to definitively support a causative role for variation in ZMYM3, the totality of the evidence, including 27 affected individuals, recurrent variation at two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally confirmed functional effects strongly support ZMYM3 as an NDD-associated gene.

DNA methylation episignature for Witteveen-Kolk syndrome due to SIN3A haploinsufficiency.

PURPOSE: Witteveen-Kolk syndrome (WITKOS) is a rare, autosomal dominant neurodevelopmental disorder caused by heterozygous loss-of-function alterations in the SIN3A gene. WITKOS has variable expressivity that commonly overlaps with other neurodevelopmental disorders. In this study, we characterized a distinct DNA methylation epigenetic signature (episignature) distinguishing WITKOS from unaffected individuals as well as individuals with other neurodevelopmental disorders with episignatures and described 9 previously unpublished individuals with SIN3A haploinsufficiency. METHODS: We studied the phenotypic characteristics and the genome-wide DNA methylation in the peripheral blood samples of 20 individuals with heterozygous alterations in SIN3A. A total of 14 samples were used for the identification of the episignature and building of a predictive diagnostic biomarker, whereas the diagnostic model was used to investigate the methylation pattern of the remaining 6 samples. RESULTS: A predominantly hypomethylated DNA methylation profile specific to WITKOS was identified, and the classifier model was able to diagnose a previously unresolved test case. The episignature was sensitive enough to detect individuals with varying degrees of phenotypic severity carrying SIN3A haploinsufficient variants. CONCLUSION: We identified a novel, robust episignature in WITKOS due to SIN3A haploinsufficiency. This episignature has the potential to aid identification and diagnosis of individuals with WITKOS.

Integration of metabolomics with genomics: Metabolic gene prioritization using metabolomics data and genomic variant (CADD) scores.

The integration of metabolomics data with sequencing data is a key step towards improving the diagnostic process for finding the disease-causing genetic variant(s) in patients suspected of having an inborn error of metabolism (IEM). The measured metabolite levels could provide additional phenotypical evidence to elucidate the degree of pathogenicity for variants found in genes associated with metabolic processes. We present a computational approach, called Reafect, that calculates for each reaction in a metabolic pathway a score indicating whether that reaction is deficient or not. When calculating this score, Reafect takes multiple factors into account: the magnitude and sign of alterations in the metabolite levels, the reaction distances between metabolites and reactions in the pathway, and the biochemical directionality of the reactions. We applied Reafect to untargeted metabolomics data of 72 patient samples with a known IEM and found that in 81% of the cases the correct deficient enzyme was ranked within the top 5% of all considered enzyme deficiencies. Next, we integrated Reafect with Combined Annotation Dependent Depletion (CADD) scores (a measure for gene variant deleteriousness) and ranked the metabolic genes of 27 IEM patients. We observed that this integrated approach significantly improved the prioritization of the genes containing the disease-causing variant when compared with the two approaches individually. For 15/27 IEM patients the correct affected gene was ranked within the top 0.25% of the set of potentially affected genes. Together, our findings suggest that metabolomics data improves the identification of affected genes in patients suffering from IEM.

The MAP3K7 gene: Further delineation of clinical characteristics and genotype/phenotype correlations.

Mitogen-activated protein 3 kinase 7 (MAP3K7) encodes the ubiquitously expressed transforming growth factor ß-activated kinase 1, which plays a crucial role in many cellular processes. Mutationsin the MAP3K7 gene have been linked to two distinct disorders: frontometaphyseal dysplasia type 2 (FMD2) and cardiospondylocarpofacial syndrome (CSCF). The fact that different mutations can induce two distinct phenotypes suggests a phenotype/genotype correlation, but no side-by-side comparison has been done thus far to confirm this. Here, we significantly expand the cohort and the description of clinical phenotypes for patients with CSCF and FMD2 who carry mutations in MAP3K7. Our findings support that in contrast to FMD2-causing mutations, CSCF-causing mutations in MAP3K7 have a loss-of-function effect. Additionally, patients with pathogenic mutations in MAP3K7 are at risk for (severe) cardiac disease, have symptoms associated with connective tissue disease, and we show overlap in clinical phenotypes of CSCF with Noonan syndrome (NS). Together, we confirm a molecular fingerprint of FMD2- versus CSCF-causing MAP3K7 mutations and conclude that mutations in MAP3K7 should be considered in the differential diagnosis of patients with syndromic congenital cardiac defects and/or cardiomyopathy, syndromic connective tissue disorders, and in the differential diagnosis of NS.

Specific delivering of RNAi using Spike's aptamer-functionalized lipid nanoparticles for targeting SARS-CoV-2: A strong anti-Covid drug in a clinical case study.

Coronavirus (SARS-CoV-2) as a global pandemic has attracted the attention of many scientific centers to find the right treatment. We expressed and purified the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and specific RBD aptamers were designed using SELEX method. RNAi targeting nucleocapsid phosphoprotein was synthesized and human lung cells were inoculated with aptamer-functionalized lipid nanoparticles (LNPs) containing RNAi. The results demonstrated that RBD aptamer having KD values of 0.290 nm possessed good affinity. Based on molecular docking and efficacy prediction analysis, siRNA molecule was showed the best action. LNPs were appropriately functionalized by aptamer and contained RNAi molecules. Antiviral assay using q-PCR and ELISA demonstrated that LNP functionalized with 35 µm Apt and containing 30 nm RNAi/ml of cell culture had the best antiviral activity compared to other concentrations. Applied aptamer in the nanocarrier has two important functions. First, it can deliver the drug (RNAi) to the surface of epithelial cells. Second, by binding to the SARS-CoV-2 spike protein, it inhibits the virus entrance into cells. Our data reveal an interaction between the aptamer and the virus, and RNAi targeted the virus RNA. CT scan and the clinical laboratory tests in a clinical case study, a 36-year old man who presented with severe SARS-CoV-2, demonstrated that inhalation of 10 mg Apt-LNPs-RNAi nebulized/day for six days resulted in an improvement in consolidation and ground-glass opacity in lungs on the sixth day of treatment. Our findings suggest the treatment of SARS-CoV-2 infection through inhalation of Aptamer-LNPs-RNAi.

Expanding the phenotype of ASXL3-related syndrome: A comprehensive description of 45 unpublished individuals with inherited and de novo pathogenic variants in ASXL3.

The study aimed at widening the clinical and genetic spectrum of ASXL3-related syndrome, a neurodevelopmental disorder, caused by truncating variants in the ASXL3 gene. In this international collaborative study, we have undertaken a detailed clinical and molecular analysis of 45 previously unpublished individuals with ASXL3-related syndrome, as well as a review of all previously published individuals. We have reviewed the rather limited functional characterization of pathogenic variants in ASXL3 and discuss current understanding of the consequences of the different ASXL3 variants. In this comprehensive analysis of ASXL3-related syndrome, we define its natural history and clinical evolution occurring with age. We report familial ASXL3 pathogenic variants, characterize the phenotype in mildly affected individuals and discuss nonpenetrance. We also discuss the role of missense variants in ASXL3. We delineate a variable but consistent phenotype. The most characteristic features are neurodevelopmental delay with consistently limited speech, significant neuro-behavioral issues, hypotonia, and feeding difficulties. Distinctive features include downslanting palpebral fissures, hypertelorism, tubular nose with a prominent nasal bridge, and low-hanging columella. The presented data will inform clinical management of individuals with ASXL3-related syndrome and improve interpretation of new ASXL3 sequence variants.

Mucolipidosis type II and type III: a systematic review of 843 published cases.

PURPOSE: Mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma are rare autosomal recessive lysosomal storage disorders. Data on the natural course of the diseases are scarce. These data are important for counseling, therapies development, and improvement of outcome. The aim of this study is to gain knowledge on the natural history of ML by obtaining data on survival, symptom onset, presenting symptoms, diagnosis, and pathogenic variants associated with the MLII or MLIII phenotype. METHODS: A systematic review on all published MLII and MLIII cases between 1968 and August 2019 was performed. RESULTS: Three hundred one articles provided data on 843 patients. Median age at diagnosis: 0.7 for MLII and 9.0 years for MLIII. Median survival: 5.0 for MLII and 62.0 years for MLIIIII. Median age of death: 1.8 for MLII and 33.0 years for MLIII. Most frequent causes of death in all ML were pulmonary and/or cardiac complications. Pathogenic variants were described in 388 patients (GNPTAB: 571, GNPTG 179). CONCLUSION: This review provides unique insights into the natural history of MLII and MLIII, with a clear genotype-phenotype correlation with the most frequent pathogenic variant c.3503_3504del in MLII and in MLIII alpha/beta c.22A>G for GNPTAB. All pathogenic GNPTG variants resulted in MLIII gamma.

Bi-allelic variants in the ER quality-control mannosidase gene EDEM3 cause a congenital disorder of glycosylation.

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

Association between pectus excavatum and congenital genetic disorders: A systematic review and practical guide for the treating physician.

BACKGROUND: Pectus excavatum (PE) could be part of a genetic disorder, which then has implications regarding comorbidity, the surgical correction of PE, and reproductive choices. However, referral of a patient presenting with PE for genetic analysis is often delayed because additional crucial clinical signs may be subtle or even missed in syndromic patients. We reviewed the literature to inventory known genetic disorders associated with PE and create a standardized protocol for clinical evaluation. METHODS: A systematic literature search was performed in electronic databases. Genetic disorders were considered associated with PE if studies reported at least five cases with PE. Characteristics of each genetic disorder were extracted from the literature and the OMIM database in order to create a practical guide for the clinician. RESULTS: After removal of duplicates from the initial search, 1632 citations remained. Eventually, we included 119 full text articles, representing 20 different genetic disorders. Relevant characteristics and important clinical signs of each genetic disorder were summarized providing a standardized protocol in the form of a scoring list. The most important clinical sign was a positive family history for PE and/or congenital heart defect. CONCLUSIONS: Twenty unique genetic disorders have been found associated with PE. We have created a scoring list for the clinician that systematically evaluates crucial clinical signs, thereby facilitating decision making for referral to a clinical geneticist.

Comprehensive study of 28 individuals with SIN3A-related disorder underscoring the associated mild cognitive and distinctive facial phenotype.

Witteveen-Kolk syndrome (OMIM 613406) is a recently defined neurodevelopmental syndrome caused by heterozygous loss-of-function variants in SIN3A. We define the clinical and neurodevelopmental phenotypes related to SIN3A-haploinsufficiency in 28 unreported patients. Patients with SIN3A variants adversely affecting protein function have mild intellectual disability, growth and feeding difficulties. Involvement of a multidisciplinary team including a geneticist, paediatrician and neurologist should be considered in managing these patients. Patients described here were identified through a combination of clinical evaluation and gene matching strategies (GeneMatcher and Decipher). All patients consented to participate in this study. Mean age of this cohort was 8.2 years (17 males, 11 females). Out of 16 patients ≥ 8 years old assessed, eight (50%) had mild intellectual disability (ID), four had moderate ID (22%), and one had severe ID (6%). Four (25%) did not have any cognitive impairment. Other neurological symptoms such as seizures (4/28) and hypotonia (12/28) were common. Behaviour problems were reported in a minority. In patients ≥2 years, three were diagnosed with Autism Spectrum Disorder (ASD) and four with Attention Deficit Hyperactivity Disorder (ADHD). We report 27 novel variants and one previously reported variant. 24 were truncating variants; three were missense variants and one large in-frame gain including exons 10-12.

Using Out-of-Batch Reference Populations to Improve Untargeted Metabolomics for Screening Inborn Errors of Metabolism.

Untargeted metabolomics is an emerging technology in the laboratory diagnosis of inborn errors of metabolism (IEM). Analysis of a large number of reference samples is crucial for correcting variations in metabolite concentrations that result from factors, such as diet, age, and gender in order to judge whether metabolite levels are abnormal. However, a large number of reference samples requires the use of out-of-batch samples, which is hampered by the semi-quantitative nature of untargeted metabolomics data, i.e., technical variations between batches. Methods to merge and accurately normalize data from multiple batches are urgently needed. Based on six metrics, we compared the existing normalization methods on their ability to reduce the batch effects from nine independently processed batches. Many of those showed marginal performances, which motivated us to develop Metchalizer, a normalization method that uses 10 stable isotope-labeled internal standards and a mixed effect model. In addition, we propose a regression model with age and sex as covariates fitted on reference samples that were obtained from all nine batches. Metchalizer applied on log-transformed data showed the most promising performance on batch effect removal, as well as in the detection of 195 known biomarkers across 49 IEM patient samples and performed at least similar to an approach utilizing 15 within-batch reference samples. Furthermore, our regression model indicates that 6.5-37% of the considered features showed significant age-dependent variations. Our comprehensive comparison of normalization methods showed that our Log-Metchalizer approach enables the use out-of-batch reference samples to establish clinically-relevant reference values for metabolite concentrations. These findings open the possibilities to use large scale out-of-batch reference samples in a clinical setting, increasing the throughput and detection accuracy.

Untargeted Metabolomics-Based Screening Method for Inborn Errors of Metabolism using Semi-Automatic Sample Preparation with an UHPLC- Orbitrap-MS Platform.

Routine diagnostic screening of inborn errors of metabolism (IEM) is currently performed by different targeted analyses of known biomarkers. This approach is time-consuming, targets a limited number of biomarkers and will not identify new biomarkers. Untargeted metabolomics generates a global metabolic phenotype and has the potential to overcome these issues. We describe a novel, single platform, untargeted metabolomics method for screening IEM, combining semi-automatic sample preparation with pentafluorophenylpropyl phase (PFPP)-based UHPLC- Orbitrap-MS. We evaluated analytical performance and diagnostic capability of the method by analysing plasma samples of 260 controls and 53 patients with 33 distinct IEM. Analytical reproducibility was excellent, with peak area variation coefficients below 20% for the majority of the metabolites. We illustrate that PFPP-based chromatography enhances identification of isomeric compounds. Ranked z-score plots of metabolites annotated in IEM samples were reviewed by two laboratory specialists experienced in biochemical genetics, resulting in the correct diagnosis in 90% of cases. Thus, our untargeted metabolomics platform is robust and differentiates metabolite patterns of different IEMs from those of controls. We envision that the current approach to diagnose IEM, using numerous tests, will eventually be replaced by untargeted metabolomics methods, which also have the potential to discover novel biomarkers and assist in interpretation of genetic data.