Early diagnostics and treatment of vitamin B12 deficiency (B12D) in infants, mainly maternally conditioned, is crucial in preventing possible developmental delay and neurological deficits. Currently, B12D is rarely listed in regular newborn screening panels and mostly regarded as an incidental finding. The aim of this study was to evaluate a targeted newborn screening strategy for detection of suspected B12D. A decision strategy based on the primary parameters propionylcarnitine and methionine for selection of samples to be analyzed for total homocysteine by mass spectrometry was established. Therefore, 93,116 newborns were initially screened. Concentrations of vitamin B12 and holotranscobalamin in serum were obtained from clinical follow-up analyses of recalled newborns. Moreover, an extremely sensitive mass spectrometric method to quantify methylmalonic acid from the dried blood spots was developed. Overall, 0.15% of newborns were screened positive for suspected B12D, of which 64% had vitamin B12 concentrations below 148 pM. We also determined a cutoff value for methylmalonic acid in dried blood spots indicative for B12D in infants. Overall, we calculated a prevalence of 92/100,000 for suspected B12D in the Austrian newborns. In conclusion, we present a screening algorithm including second-tier measurement of total homocysteine that allows detection of low B12 serum concentrations with a high detection rate and low false-positive rate.

No abstract available

Early-onset epileptic encephalopathy (EE) and combined developmental and epileptic encephalopathies (DEE) are clinically and genetically heterogeneous severely devastating conditions. Recent studies emphasized de novo variants as major underlying cause suggesting a generally low-recurrence risk. In order to better understand the full genetic landscape of EE and DEE, we performed high-resolution chromosomal microarray analysis in combination with whole-exome sequencing in 63 deeply phenotyped independent patients. After bioinformatic filtering for rare variants, diagnostic yield was improved for recessive disorders by manual data curation as well as molecular modeling of missense variants and untargeted plasma-metabolomics in selected patients. In total, we yielded a diagnosis in ∼42% of cases with causative copy number variants in 6 patients (∼10%) and causative sequence variants in 16 established disease genes in 20 patients (∼32%), including compound heterozygosity for causative sequence and copy number variants in one patient. In total, 38% of diagnosed cases were caused by recessive genes, of which two cases escaped automatic calling due to one allele occurring de novo. Notably, we found the recessive gene SPATA5 causative in as much as 3% of our cohort, indicating that it may have been underdiagnosed in previous studies. We further support candidacy for neurodevelopmental disorders of four previously described genes (PIK3AP1, GTF3C3, UFC1, and WRAP53), three of which also followed a recessive inheritance pattern. Our results therefore confirm the importance of de novo causative gene variants in EE/DEE, but additionally illustrate the major role of mostly compound heterozygous or hemizygous recessive inheritance and consequently high-recurrence risk.

Genes implicated in the Golgi and endosomal trafficking machinery are crucial for brain development, and mutations in them are particularly associated with postnatal microcephaly (POM).

Rett syndrome (RTT) is defined as a rare disease caused by mutations of the methyl-CpG binding protein 2 (MECP2). It is one of the most common causes of genetic mental retardation in girls, characterized by normal early psychomotor development, followed by severe neurologic regression. Hitherto, RTT lacks a specific biomarker, but altered lipid homeostasis has been found in RTT model mice as well as in RTT patients. We performed LC-MS/MS lipidomics analysis to investigate the cerebrospinal fluid (CSF) and plasma composition of patients with RTT for biochemical variations compared to healthy controls. In all seven RTT patients, we found decreased CSF cholesterol levels compared to age-matched controls (n = 13), whereas plasma cholesterol levels were within the normal range in all 13 RTT patients compared to 18 controls. Levels of phospholipid (PL) and sphingomyelin (SM) species were decreased in CSF of RTT patients, whereas the lipidomics profile of plasma samples was unaltered in RTT patients compared to healthy controls. This study shows that the CSF lipidomics profile is altered in RTT, which is the basis for future (functional) studies to validate selected lipid species as CSF biomarkers for RTT.

Deficiency of antiquitin (α-aminoadipic semialdehyde dehydrogenase), an enzyme involved in lysine degradation and encoded by ALDH7A1, is the major cause of vitamin B6 -dependent epilepsy (PDE-ALDH7A1). Despite seizure control with high dose pyridoxine (PN), developmental delay still occurs in approximately 70% of patients. We aimed to investigate metabolic perturbations due to possible previously unidentified roles of antiquitin, which may contribute to developmental delay, as well as metabolic effects of high dose pyridoxine supplementation reflecting the high doses used for seizure control in patients with PDE-ALDH7A1. Untargeted metabolomics by high resolution mass spectrometry (HRMS) was used to analyze plasma of patients with PDE-ALDH7A1 and two independently generated lines of cultured ReNcell CX human neuronal progenitor cells (NPCs) with CRISPR/Cas mediated antiquitin deficiency. Accumulation of lysine pathway metabolites in antiquitin-deficient NPCs and western-blot analysis confirmed knockdown of ALDH7A1. Metabolomics analysis of antiquitin-deficient NPCs in conditions of lysine restriction and PN supplementation identified changes in metabolites related to the transmethylation and transsulfuration pathways and osmolytes, indicating a possible unrecognized role of antiquitin outside the lysine degradation pathway. Analysis of plasma samples of PN treated patients with PDE-ALDH7A1 and antiquitin-deficient NPCs cultured in conditions comparable to the patient plasma samples demonstrated perturbation of metabolites of the gamma-glutamyl cycle, suggesting potential oxidative stress-related effects in PN-treated patients with PDE-ALDH7A1. We postulate that a model of human NPCs with CRISPR/Cas mediated antiquitin deficiency is well suited to characterize previously unreported roles of antiquitin, relevant to this most prevalent form of pyridoxine-dependent epilepsy.

Antiquitin (ATQ) deficiency is the main cause of pyridoxine dependent epilepsy characterized by early onset epileptic encephalopathy responsive to large dosages of pyridoxine. Despite seizure control most patients have intellectual disability. Folinic acid responsive seizures (FARS) are genetically identical to ATQ deficiency. ATQ functions as an aldehyde dehydrogenase (ALDH7A1) in the lysine degradation pathway. Its deficiency results in accumulation of α-aminoadipic semialdehyde (AASA), piperideine-6-carboxylate (P6C) and pipecolic acid, which serve as diagnostic markers in urine, plasma, and CSF. To interrupt seizures a dose of 100 mg of pyridoxine-HCl is given intravenously, or orally/enterally with 30 mg/kg/day. First administration may result in respiratory arrest in responders, and thus treatment should be performed with support of respiratory management. To make sure that late and masked response is not missed, treatment with oral/enteral pyridoxine should be continued until ATQ deficiency is excluded by negative biochemical or genetic testing. Long-term treatment dosages vary between 15 and 30 mg/kg/day in infants or up to 200 mg/day in neonates, and 500 mg/day in adults. Oral or enteral pyridoxal phosphate (PLP), up to 30 mg/kg/day can be given alternatively. Prenatal treatment with maternal pyridoxine supplementation possibly improves outcome. PDE is an organic aciduria caused by a deficiency in the catabolic breakdown of lysine. A lysine restricted diet might address the potential toxicity of accumulating αAASA, P6C and pipecolic acid. A multicenter study on long term outcomes is needed to document potential benefits of this additional treatment. The differential diagnosis of pyridoxine or PLP responsive seizure disorders includes PLP-responsive epileptic encephalopathy due to PNPO deficiency, neonatal/infantile hypophosphatasia (TNSALP deficiency), familial hyperphosphatasia (PIGV deficiency), as well as yet unidentified conditions and nutritional vitamin B6 deficiency. Commencing treatment with PLP will not delay treatment in patients with pyridox(am)ine phosphate oxidase (PNPO) deficiency who are responsive to PLP only.

Mitochondrial respiratory chain dysfunction has been identified in a number of neurodegenerative disorders. Infantile cerebellar-retinal degeneration associated with mutations in the mitochondrial aconitase 2 gene (ACO2) has been recently described as a neurodegenerative disease of autosomal recessive inheritance. To date there is no biomarker for ACO2 deficiency and diagnosis relies on genetic analysis. Here we report global metabolic profiling in eight patients with ACO2 deficiency. Using an LC-MS-based metabolomics platform we have identified several metabolites with affected plasma concentrations including the tricarboxylic acid cycle metabolites cis-aconitate, isocitrate and alpha-ketoglutarate, as well as phosphoenolpyruvate and hydroxybutyrate. Taken together we report a diagnostic metabolic fingerprint for mitochondrial aconitase 2 deficiency.

4H or POLR3-related leukodystrophy is an autosomal recessive disorder typically characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism, caused by biallelic pathogenic variants in POLR3A, POLR3B, POLR1C, and POLR3K. The endocrine and growth abnormalities associated with this disorder have not been thoroughly investigated to date.

No abstract available

Considerable non-allelic heterogeneity for autosomal recessively inherited Charcot-Marie-Tooth (ARCMT) disease has challenged molecular testing and often requires a large amount of work in terms of DNA sequencing and data interpretation or remains unpractical. This study tested the value of SNP array-based whole-genome homozygosity mapping as a first step in the molecular genetic diagnosis of sporadic or ARCMT in patients from inbred families or outbred populations with the ancestors originating from the same geographic area. Using 10 K 2.0 and 250 K Nsp Affymetrix SNP arrays, 15 (63%) of 24 CMT patients received an accurate genetic diagnosis. We used our Java-based script eHoPASA CMT-easy Homozygosity Profiling of SNP arrays for CMT patients to display the location of homozygous regions and their extent of marker count and base-pairs throughout the whole genome. CMT4C was the most common genetic subtype with mutations detected in SH3TC2, one (p.E632Kfs13X) appearing to be a novel founder mutation. A sporadic patient with severe CMT was homozygous for the c.250G > C (p.G84R) HSPB1 mutation which has previously been reported to cause autosomal dominant dHMN. Two distantly related CMT1 patients with early disease onset were found to carry a novel homozygous mutation in MFN2 (p.N131S). We conclude that SNP array-based homozygosity mapping is a fast, powerful, and economic tool to guide molecular genetic testing in ARCMT and in selected sporadic CMT patients.

Defects of mitochondrial oxidative phosphorylation (OXPHOS) are associated with a wide range of clinical phenotypes and time courses. Combined OXPHOS deficiencies are mainly caused by mutations of nuclear genes that are involved in mitochondrial protein translation. Due to their genetic heterogeneity it is almost impossible to diagnose OXPHOS patients on clinical grounds alone. Hence next generation sequencing (NGS) provides a distinct advantage over candidate gene sequencing to discover the underlying genetic defect in a timely manner. One recent example is the identification of mutations in MTFMT that impair mitochondrial protein translation through decreased formylation of Met-tRNA(Met). Here we report the results of a combined exome sequencing and candidate gene screening study. We identified nine additional MTFMT patients from eight families who were affected with Leigh encephalopathy or white matter disease, microcephaly, mental retardation, ataxia, and muscular hypotonia. In four patients, the causal mutations were identified by exome sequencing followed by stringent bioinformatic filtering. In one index case, exome sequencing identified a single heterozygous mutation leading to Sanger sequencing which identified a second mutation in the non-covered first exon. High-resolution melting curve-based MTFMT screening in 350 OXPHPOS patients identified pathogenic mutations in another three index cases. Mutations in one of them were not covered by previous exome sequencing. All novel mutations predict a loss-of-function or result in a severe decrease in MTFMT protein in patients' fibroblasts accompanied by reduced steady-state levels of complex I and IV subunits. Being present in 11 out of 13 index cases the c.626C>T mutation is one of the most frequent disease alleles underlying OXPHOS disorders. We provide detailed clinical descriptions on eleven MTFMT patients and review five previously reported cases.

Despite abundant evidence for pathogenicity of large copy number variants (CNVs) in neurodevelopmental disorders (NDDs), the individual significance of genome-wide rare CNVs <500 kb has not been well elucidated in a clinical context.

Mucopolysaccharidoses (MPS) are monogenic metabolic disorders that significantly affect the skeleton. Eleven enzyme defects in the lysosomal degradation of glycosaminoglycans (GAGs) have been assigned to the known MPS subtypes (I-IX). Arylsulfatase K (ARSK) is a recently characterised lysosomal hydrolase involved in GAG degradation that removes the 2-O-sulfate group from 2-sulfoglucuronate. Knockout of Arsk in mice was consistent with mild storage pathology, but no human phenotype has yet been described.

Microcephaly is a sign of many genetic conditions but has been rarely systematically evaluated. We therefore comprehensively studied the clinical and genetic landscape of an unselected cohort of patients with microcephaly.

Disorders of the mitochondrial energy metabolism are clinically and genetically heterogeneous. An increasingly recognized subgroup is caused by defective mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, with defects in 13 genes being linked to human disease to date. Mutations in three of them, NFU1, BOLA3, and IBA57, affect the assembly of mitochondrial [4Fe-4S] proteins leading to an impairment of diverse mitochondrial metabolic pathways and ATP production. Patients with defects in these three genes present with lactic acidosis, hyperglycinemia, and reduced activities of respiratory chain complexes I and II, the four lipoic acid-dependent 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). To date, five different NFU1 pathogenic variants have been reported in 15 patients from 12 families. We report on seven new patients from five families carrying compound heterozygous or homozygous pathogenic NFU1 mutations identified by candidate gene screening and exome sequencing. Six out of eight different disease alleles were novel and functional studies were performed to support the pathogenicity of five of them. Characteristic clinical features included fatal infantile encephalopathy and pulmonary hypertension leading to death within the first 6 months of life in six out of seven patients. Laboratory investigations revealed combined defects of pyruvate dehydrogenase complex (five out of five) and respiratory chain complexes I and II+III (four out of five) in skeletal muscle and/or cultured skin fibroblasts as well as increased lactate (five out of six) and glycine concentration (seven out of seven). Our study contributes to a better definition of the phenotypic spectrum associated with NFU1 mutations and to the diagnostic workup of future patients.

To identify the gene defect in patients with hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) who are negative for TUBB4A mutations.

Neurodevelopmental disorders (NDD) caused by protein phosphatase 2A (PP2A) dysfunction have mainly been associated with de novo variants in PPP2R5D and PPP2CA, and more rarely in PPP2R1A. Here, we aimed to better understand the latter by characterizing 30 individuals with de novo and often recurrent variants in this PP2A scaffolding Aα subunit.

In the highly dynamic field of metabolomics, we have developed a method for the analysis of hydrophilic metabolites in various biological samples. Therefore, we used hydrophilic interaction chromatography (HILIC) for separation, combined with a high-resolution mass spectrometer (MS) with the aim of separating and analyzing a wide range of compounds. We used 41 reference standards with different chemical properties to develop an optimal chromatographic separation. MS analysis was performed with a set of pooled biological samples human cerebrospinal fluid (CSF), and human plasma. The raw data was processed in a first step with Compound Discoverer 3.1 (CD), a software tool for untargeted metabolomics with the aim to create a list of unknown compounds. In a second step, we combined the results obtained with our internally analyzed reference standard list to process the data along with the Lipid Data Analyzer 2.6 (LDA), a software tool for a targeted approach. In order to demonstrate the advantages of this combined target-list based and untargeted approach, we not only compared the relative standard deviation (%RSD) of the technical replicas of pooled plasma samples (n = 5) and pooled CSF samples (n = 3) with the results from CD, but also with XCMS Online, a well-known software tool for untargeted metabolomics studies. As a result of this study we could demonstrate with our HILIC-MS method that all standards could be either separated by chromatography, including isobaric leucine and isoleucine or with MS by different mass. We also showed that this combined approach benefits from improved precision compared to well-known metabolomics software tools such as CD and XCMS online. Within the pooled plasma samples processed by LDA 68% of the detected compounds had a %RSD of less than 25%, compared to CD and XCMS online (57% and 55%). The improvements of precision in the pooled CSF samples were even more pronounced, 83% had a %RSD of less than 25% compared to CD and XCMS online (28% and 8% compounds detected). Particularly for low concentration samples, this method showed a more precise peak area integration with its 3D algorithm and with the benefits of the LDAs graphical user interface for fast and easy manual curation of peak integration. The here-described method has the advantage that manual curation for larger batch measurements remains minimal due to the target list containing the information obtained by an untargeted approach.

The metabolic defect in glycogen storage disease type I (GSDI) results in fasting hypoglycemia and typical secondary metabolic abnormalities (eg, hypertriglyceridemia, hyperlactatemia, hyperuricemia). The aim of this study was to assess further perturbations of the metabolic network in GSDI patients under ongoing treatment.