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Newborn screening (NBS) is one of the most effective measures of secondary prevention. While the benefit of NBS on the clinical long-term outcomes of children with inherited metabolic diseases (IMD) has been demonstrated, the potential burden of families living with an early diagnosed and treated child with an IMD has not been thoroughly investigated. The aim of this longitudinal questionnaire-based study on 369 families living with a child with an IMD was to investigate the psychosocial and financial burden following a true-positive NBS. The reported psychosocial burden differed between children and their parents, and was associated with the child's age, diagnosis, and treatment. At younger ages, parent-reported burden was higher for the parents than for the individual child, while it increased for children and decreased for parents as the child grew older. Furthermore, psychosocial burden increased if the child required a strict dietary treatment and was at risk of metabolic decompensation. Regardless of diagnosis and treatment, the developmental delay of their child independently increased the parental psychosocial burden. Financial burden was reported by 24% of all families, and was higher in low-income families and in families whose children required dietary treatment. In conclusion, a substantial psychosocial and financial burden was revealed for children and their families after true-positive NBS. Since this burden is likely to have a negative impact on the long-term individual health benefits of NBS, this study underlines the importance of regularly assessing the psychosocial and financial needs of these families., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The current German newborn screening (NBS) panel includes 13 inherited metabolic diseases (IMDs). In addition, a NBS pilot study in Southwest Germany identifies individuals with propionic acidemia (PA), methylmalonic acidemia (MMA), combined and isolated remethylation disorders (e.g., cobalamin [cbl] C and methylenetetrahydrofolate reductase [MTHFR] deficiency), cystathionine β-synthase (CBS) deficiency, and neonatal cbl deficiency through one multiple-tier algorithm. The long-term health benefits of screened individuals are evaluated in a multicenter observational study. Twenty seven screened individuals with IMDs (PA [N = 13], MMA [N = 6], cblC deficiency [N = 5], MTHFR deficiency [N = 2] and CBS deficiency [N = 1]), and 42 with neonatal cbl deficiency were followed for a median of 3.6 years. Seventeen screened IMD patients (63%) experienced at least one metabolic decompensation, 14 of them neonatally and six even before the NBS report (PA, cbl-nonresponsive MMA). Three PA patients died despite NBS and immediate treatment. Fifteen individuals (79%) with PA or MMA and all with cblC deficiency developed permanent, mostly neurological symptoms, while individuals with MTHFR, CBS, and neonatal cbl deficiency had a favorable clinical outcome. Utilizing a combined multiple-tier algorithm, we demonstrate that NBS and specialized metabolic care result in substantial benefits for individuals with MTHFR deficiency, CBS deficiency, neonatal cbl deficiency, and to some extent, cbl-responsive MMA and cblC deficiency. However, its advantage is less evident for individuals with PA and cbl-nonresponsive MMA. SYNOPSIS: Early detection through newborn screening and subsequent specialized metabolic care improve clinical outcomes and survival in individuals with MTHFR deficiency and cystathionine-β-synthase deficiency, and to some extent in cobalamin-responsive methylmalonic acidemia (MMA) and cblC deficiency while the benefit for individuals with propionic acidemia and cobalamin-nonresponsive MMA is less evident due to the high (neonatal) decompensation rate, mortality, and long-term complications., (© 2024 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Background and Aims: Pediatric acute liver failure (PALF) is a life-threatening condition. In Europe, the main causes are viral infections (12%-16%) and inherited metabolic diseases (14%-28%). Yet, in up to 50% of cases the underlying etiology remains elusive, challenging clinical management, including liver transplantation. We systematically studied indeterminate PALF cases referred for genetic evaluation by whole-exome sequencing (WES), and analyzed phenotypic and biochemical markers, and the diagnostic yield of WES in this condition., Approach and Results: With this international, multicenter observational study, patients (0-18 y) with indeterminate PALF were analyzed by WES. Data on the clinical and biochemical phenotype were retrieved and systematically analyzed., Results: In total, 260 indeterminate PALF patients from 19 countries were recruited between 2011 and 2022, of whom 59 had recurrent PALF. WES established a genetic diagnosis in 37% of cases (97/260). Diagnostic yield was highest in children with PALF in the first year of life (41%), and in children with recurrent acute liver failure (64%). Thirty-six distinct disease genes were identified. Defects in NBAS (n=20), MPV17 (n=8), and DGUOK (n=7) were the most frequent findings. When categorizing, the most frequent were mitochondrial diseases (45%), disorders of vesicular trafficking (28%), and cytosolic aminoacyl-tRNA synthetase deficiencies (10%). One-third of patients had a fatal outcome. Fifty-six patients received liver transplantation., Conclusions: This study elucidates a large contribution of genetic causes in PALF of indeterminate origin with an increasing spectrum of disease entities. The high proportion of diagnosed cases and potential treatment implications argue for exome or in future rapid genome sequencing in PALF diagnostics., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Despite numerous studies in human patients and animal models for phenylketonuria (PKU; OMIM#261600), the pathophysiology of PKU and the underlying causes of brain dysfunction and cognitive problems in PKU patients are not well understood. In this study, lumbar cerebral spinal fluid (CSF) was obtained immediately after blood sampling from early-treated adult PKU patients who had fasted overnight. Metabolite and amino acid concentrations in the CSF of PKU patients were compared with those of non-PKU controls. The CSF concentrations and CSF/plasma ratios for glucose and lactate were found to be below normal, similar to what has been reported for glucose transporter1 (GLUT1) deficiency patients who exhibit many of the same clinical symptoms as untreated PKU patients. CSF glucose and lactate levels were negatively correlated with CSF phenylalanine (Phe), while CSF glutamine and glutamate levels were positively correlated with CSF Phe levels. Plasma glucose levels were negatively correlated with plasma Phe concentrations in PKU subjects, which partly explains the reduced CSF glucose concentrations. Although brain glucose concentrations are unlikely to be low enough to impair brain glucose utilization, it is possible that the metabolism of Phe in the brain to produce phenyllactate, which can be transported across the blood-brain barrier to the blood, may consume glucose and/or lactate to generate the carbon backbone for glutamate. This glutamate is then converted to glutamine and carries the Phe-derived ammonia from the brain to the blood. While this mechanism remains to be tested, it may explain the correlations of CSF glutamine, glucose, and lactate concentrations with CSF Phe., Competing Interests: Declaration of competing interest The authors declare no competing or conflicts of interest. C.C. is employe of Bruker Biospin GmbH, Ettlingen, Germany. N.B. is Deputy Editor-in-Chief of Molecular Genetics and Metabolism., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: This study aims to elucidate the long-term benefit of newborn screening (NBS) for individuals with long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiency, inherited metabolic diseases included in NBS programs worldwide., Methods: German national multicenter study of individuals with confirmed LCHAD/MTP deficiency identified by NBS between 1999 and 2020 or selective metabolic screening. Analyses focused on NBS results, confirmatory diagnostics, and long-term clinical outcomes., Results: Sixty-seven individuals with LCHAD/MTP deficiency were included in the study, thereof 54 identified by NBS. All screened individuals with LCHAD deficiency survived, but four with MTP deficiency (14.8%) died during the study period. Despite NBS and early treatment neonatal decompensations (28%), symptomatic disease course (94%), later metabolic decompensations (80%), cardiomyopathy (28%), myopathy (82%), hepatopathy (32%), retinopathy (17%), and/or neuropathy (22%) occurred. Hospitalization rates were high (up to a mean of 2.4 times/year). Disease courses in screened individuals with LCHAD and MTP deficiency were similar except for neuropathy, occurring earlier in individuals with MTP deficiency (median 3.9 vs. 11.4 years; p = 0.0447). Achievement of dietary goals decreased with age, from 75% in the first year of life to 12% at age 10, and consensus group recommendations on dietary management were often not achieved., Interpretation: While NBS and early treatment result in improved (neonatal) survival, they cannot reliably prevent long-term morbidity in screened individuals with LCHAD/MTP deficiency, highlighting the urgent need of better therapeutic strategies and the development of disease course-altering treatment., (© 2024 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Biallelic pathogenic variants in neuroblastoma-amplified sequence (NBAS) cause a pleiotropic multisystem disorder. Three clinical subgroups have been defined correlating with the localisation of pathogenic variants in the NBAS gene: variants affecting the C-terminal region of NBAS result in SOPH syndrome (short stature, optic atrophy, Pelger-Huët anomaly), variants affecting the Sec 39 domain are associated with infantile liver failure syndrome type 2 (ILFS2) and variants affecting the ß-propeller domain give rise to a combined phenotype. However, there is still unexplained phenotypic diversity across the three subgroups, challenging the current concept of genotype-phenotype correlations in NBAS-associated disease. Therefore, besides examining the genetic influence, we aim to elucidate the potential impact of pre-symptomatic diagnosis, emergency management and other modifying variables on the clinical phenotype. We investigated genotype-phenotype correlations in individuals sharing the same genotypes (n = 30 individuals), and in those sharing the same missense variants with a loss-of-function variant in trans (n = 38 individuals). Effects of a pre-symptomatic diagnosis and emergency management on the severity of acute liver failure (ALF) episodes also were analysed, comparing liver function tests (ALAT, ASAT, INR) and mortality. A strong genotype-phenotype correlation was demonstrated in individuals sharing the same genotype; this was especially true for the ILFS2 subgroup. Genotype-phenotype correlation in patients sharing only one missense variant was still high, though at a lower level. Pre-symptomatic diagnosis in combination with an emergency management protocol leads to a trend of reduced severity of ALF. High genetic impact on clinical phenotype in NBAS-associated disease facilitates monitoring and management of affected patients sharing the same genotype. Pre-symptomatic diagnosis and an emergency management protocol do not prevent ALF but may reduce its clinical severity., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Analytical and therapeutic innovations led to a continuous but variable extension of newborn screening (NBS) programmes worldwide. Every extension requires a careful evaluation of feasibility, diagnostic (process) quality and possible health benefits to balance benefits and limitations. The aim of this study was to evaluate the suitability of 18 candidate diseases for inclusion in NBS programmes. Utilising tandem mass spectrometry as well as establishing specific diagnostic pathways with second-tier analyses, three German NBS centres designed and conducted an evaluation study for 18 candidate diseases, all of them inherited metabolic diseases. In total, 1 777 264 NBS samples were analysed. Overall, 441 positive NBS results were reported resulting in 68 confirmed diagnoses, 373 false-positive cases and an estimated cumulative prevalence of approximately 1 in 26 000 newborns. The positive predictive value ranged from 0.07 (carnitine transporter defect) to 0.67 (HMG-CoA lyase deficiency). Three individuals were missed and 14 individuals (21%) developed symptoms before the positive NBS results were reported. The majority of tested candidate diseases were found to be suitable for inclusion in NBS programmes, while multiple acyl-CoA dehydrogenase deficiency, isolated methylmalonic acidurias, propionic acidemia and malonyl-CoA decarboxylase deficiency showed some and carnitine transporter defect significant limitations. Evaluation studies are an important tool to assess the potential benefits and limitations of expanding NBS programmes to new diseases., (© 2023 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Newborn screening (NBS) allows early identification of individuals with rare disease, such as isovaleric aciduria (IVA). Reliable early prediction of disease severity of positively screened individuals with IVA is needed to guide therapeutic decision, prevent life-threatening neonatal disease manifestation in classic IVA and over-medicalization in attenuated IVA that may remain asymptomatic. We analyzed 84 individuals (median age at last study visit 8.5 years) with confirmed IVA identified by NBS between 1998 and 2018 who participated in the national, observational, multicenter study. Screening results, additional metabolic parameters, genotypes, and clinical phenotypic data were included. Individuals with metabolic decompensation showed a higher median isovalerylcarnitine (C5) concentration in the first NBS sample (10.6 vs. 2.7 μmol/L; p < 0.0001) and initial urinary isovalerylglycine concentration (1750 vs. 180 mmol/mol creatinine; p = 0.0003) than those who remained asymptomatic. C5 was in trend inversely correlated with full IQ (R = -0.255; slope = -0.869; p = 0.0870) and was lower for the "attenuated" variants compared to classic genotypes [median (IQR; range): 2.6 μmol/L (2.1-4.0; 0.7-6.4) versus 10.3 μmol/L (7.4-13.1; 4.3-21.7); N = 73]. In-silico prediction scores (M-CAP, MetaSVM, and MetaLR) correlated highly with isovalerylglycine and ratios of C5 to free carnitine and acetylcarnitine, but not sufficiently with clinical endpoints. The results of the first NBS sample and biochemical confirmatory testing are reliable early predictors of the clinical course of IVA, facilitating case definition (attenuated versus classic IVA). Prediction of attenuated IVA is supported by the genotype. On this basis, a reasonable algorithm has been established for neonates with a positive NBS result for IVA, with the aim of providing the necessary treatment immediately, but whenever possible, adjusting the treatment to the individual severity of the disease., (© 2023 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Phenylketonuria (PKU) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase gene. Depending on the severity of the genetic mutation, medical treatment, and patient dietary management, elevated phenylalanine (Phe) may occur in blood and brain tissues. Research has recently shown that high Phe not only impacts the central nervous system, but also other organ systems (e.g., heart and microbiome). This study used ex vivo proton nuclear magnetic resonance ( 1 H-NMR) analysis of urine samples from PKU patients (mean 14.9 ± 9.2 years, n = 51) to identify the impact of elevated blood Phe and PKU treatment on metabolic profiles. Our results found that 24 out of 98 urinary metabolites showed a significant difference ( p < 0.05) for PKU patients compared to age-matched healthy controls ( n = 51) based on an analysis of urinary metabolome. These altered urinary metabolites were related to Phe metabolism, dysbiosis, creatine synthesis or intake, the tricarboxylic acid (TCA) cycle, end products of nicotinamide-adenine dinucleotide degradation, and metabolites associated with a low Phe diet. There was an excellent correlation between the metabolome and genotype of PKU patients and healthy controls of 96.7% in a confusion matrix model. Metabolomic investigations may contribute to a better understanding of PKU pathophysiology.

There are about 1500 genetic metabolic diseases. A small number of treatable diseases are diagnosed by newborn screening programs, which are continually being developed. However, most diseases can only be diagnosed based on clinical symptoms or metabolic findings. The main biological fluids used are urine, plasma and, in special situations, cerebrospinal fluid. In contrast to commonly used methods such as gas chromatography and high performance liquid chromatography mass spectrometry, ex vivo proton spectroscopy ( 1 H-NMR) is not yet used in routine clinical practice, although it has been recommended for more than 30 years. Automatic analysis and improved NMR technology have also expanded the applications used for the diagnosis of inborn errors of metabolism. We provide a mini-overview of typical applications, especially in urine but also in plasma, used to diagnose common but also rare genetic metabolic diseases with 1 H-NMR. The use of computer-assisted diagnostic suggestions can facilitate interpretation of the profiles. In a proof of principle, to date, 182 reports of 59 different diseases and 500 reports of healthy children are stored. The percentage of correct automatic diagnoses was 74%. Using the same 1 H-NMR profile-targeted analysis, it is possible to apply an untargeted approach that distinguishes profile differences from healthy individuals. Thus, additional conditions such as lysosomal storage diseases or drug interferences are detectable. Furthermore, because 1 H-NMR is highly reproducible and can detect a variety of different substance categories, the metabolomic approach is suitable for monitoring patient treatment and revealing additional factors such as nutrition and microbiome metabolism. Besides the progress in analytical techniques, a multiomics approach is most effective to combine metabolomics with, for example, whole exome sequencing, to also diagnose patients with nondetectable metabolic abnormalities in biological fluids. In this mini review we also provide our own data to demonstrate the role of NMR in a multiomics platform in the field of inborn errors of metabolism., (© 2022 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Newborn screening (NBS) for inherited metabolic diseases (IMDs) substantially shortens a patient's journey. It enables the early start of metabolic treatment which might prevent potentially lethal neonatal disease manifestations, while promoting favorable development and long-term clinical outcomes. This study aims to assess growth in screened individuals with IMDs under different dietary regimes. Anthropometric data (3585 prospective measures) of 350 screened individuals with IMDs born between 1999 and 2018 and participating in a German prospective multicenter observational study were evaluated. Overall, birth measures were within the reference ranges, suggesting unaffected prenatal growth, except for phenylketonuria (weight) and glutaric aciduria Type 1 (head circumference). After birth, longitudinal analysis of anthropometric measures revealed a loss of height standard deviation score (SDS; -0.5 SDS; p < 0.0001), head circumference SDS (-0.2 SDS; p = 0.0028), but not for weight SDS (0.1 SDS; p = 0.5097) until the age of 18 years, while BMI SDS increased (0.4 SDS; p < 0.0001). The significant interaction with age and diet groups was pronounced for the linear growth in individuals receiving diets being low in protein, long-chain triglycerides, and galactose (p < 0.001). Identification by NBS and subsequent early (dietary) treatment cannot completely protect against alterations in growths. Disease-specific (e.g., metabolic impairments, neurotoxins) and dietary-specific (e.g., diets reduced in protein) factors may have an amplified impact on longitudinal growth. Therefore, alongside other important follow-ups, the continuous observation of the anthropometric development of screened individuals with IMDs needs special attention to early identify and support individuals at risk., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

To prevent maternal phenylketonuria (PKU) syndrome low phenylalanine concentrations (target range, 120-360 μmol/L) during pregnancy are recommended for women with PKU. We evaluated the feasibility and effectiveness of current recommendations and identified factors influencing maternal metabolic control and children's outcome. Retrospective study of first successfully completed pregnancies of 85 women with PKU from 12 German centers using historical data and interviews with the women. Children's outcome was evaluated by standardized IQ tests and parental rating of child behavior. Seventy-four percent (63/85) of women started treatment before conception, 64% (54/85) reached the phenylalanine target range before conception. Pregnancy planning resulted in earlier achievement of the phenylalanine target (18 weeks before conception planned vs. 11 weeks of gestation unplanned, p < 0.001) and lower plasma phenylalanine concentrations during pregnancy, particularly in the first trimester (0-7 weeks of gestation: 247 μmol/L planned vs. 467 μmol/L unplanned, p < 0.0001; 8-12 weeks of gestation: 235 μmol/L planned vs. 414 μmol/L unplanned, p < 0.001). Preconceptual dietary training increased the success rate of achieving the phenylalanine target before conception compared to women without training (19 weeks before conception vs. 9 weeks of gestation, p < 0.001). The majority (93%) of children had normal IQ (mean 103, median age 7.3 years); however, IQ decreased with increasing phenylalanine concentration during pregnancy. Good metabolic control during pregnancy is the prerequisite to prevent maternal PKU syndrome in the offspring. This can be achieved by timely provision of detailed information, preconceptual dietary training, and careful planning of pregnancy., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The mitochondrial malate aspartate shuttle system (MAS) maintains the cytosolic NAD+/NADH redox balance, thereby sustaining cytosolic redox-dependent pathways, such as glycolysis and serine biosynthesis. Human disease has been associated with defects in four MAS-proteins (encoded by MDH1 , MDH2 , GOT2 , SLC25A12 ) sharing a neurological/epileptic phenotype, as well as citrin deficiency ( SLC25A13 ) with a complex hepatopathic-neuropsychiatric phenotype. Ketogenic diets (KD) are high-fat/low-carbohydrate diets, which decrease glycolysis thus bypassing the mentioned defects. The same holds for mitochondrial pyruvate carrier (MPC) 1 deficiency, which also presents neurological deficits. We here describe 40 (18 previously unreported) subjects with MAS-/MPC1-defects (32 neurological phenotypes, eight citrin deficiency), describe and discuss their phenotypes and genotypes (presenting 12 novel variants), and the efficacy of KD. Of 13 MAS/MPC1-individuals with a neurological phenotype treated with KD, 11 experienced benefits-mainly a striking effect against seizures. Two individuals with citrin deficiency deceased before the correct diagnosis was established, presumably due to high-carbohydrate treatment. Six citrin-deficient individuals received a carbohydrate-restricted/fat-enriched diet and showed normalisation of laboratory values/hepatopathy as well as age-adequate thriving. We conclude that patients with MAS-/MPC1-defects are amenable to dietary intervention and that early (genetic) diagnosis is key for initiation of proper treatment and can even be lifesaving.

The recent description of biallelic DNAJC30 variants in Leber hereditary optic neuropathy (LHON) and Leigh syndrome challenged the longstanding assumption for LHON to be exclusively maternally inherited and broadened the genetic spectrum of Leigh syndrome, the most frequent paediatric mitochondrial disease. Herein, we characterize 28 so far unreported individuals from 26 families carrying a homozygous DNAJC30 p.Tyr51Cys founder variant, 24 manifesting with LHON, two manifesting with Leigh syndrome, and two remaining asymptomatic. This collection of unreported variant carriers confirms sex-dependent incomplete penetrance of the homozygous variant given a significant male predominance of disease and the report of asymptomatic homozygous variant carriers. The autosomal recessive LHON patients demonstrate an earlier age of disease onset and a higher rate of idebenone-treated and spontaneous recovery of vision in comparison to reported figures for maternally inherited disease. Moreover, the report of two additional patients with childhood- or adult-onset Leigh syndrome further evidences the association of DNAJC30 with Leigh syndrome, previously only reported in a single childhood-onset case., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background: Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics., Methods: We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage., Results: We detected on average 12,500 genes per sample including around 60% of all disease genes-a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions., Conclusion: Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics., (© 2022. The Author(s).)

Objectives: Fibroblast growth factor 23 (FGF23) controls the production and degradation of biologically active vitamin D, 1,25(OH) 2 D 3 , and phosphate reabsorption in the kidney as a hormone synthesized by bone cells. Additional paracrine effects in other organs exist as well. As a biomarker, the FGF23 plasma concentration increases in renal and cardiovascular diseases, and is correlated with outcome. The regulation of FGF23 is incompletely understood and dependent on several factors, including oxidative stress. L-homocysteine is an amino acid produced in methionine metabolism, and can be converted into further metabolites depending on the availability of vitamin B. Hyperhomocysteinemia is a potential cardiovascular risk factor. Our study aimed to explore whether homocysteine impacts FGF23 synthesis., Methods: Experiments were performed in UMR106 osteoblast-like cells. Fgf23 gene expression and FGF23 protein concentration were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Oxidative stress was determined by 2',7'-dichlorofluorescein diacetate fluorescence., Results: Homocysteine dose-dependently upregulated Fgf23 gene expression and protein synthesis. Moreover, homocysteine imposed oxidative stress on UMR106 cells. The effect of homocysteine on Fgf23 was abrogated by antioxidant ascorbic acid., Conclusions: Homocysteine is a potent stimulator of FGF23 production, an effect at least in part mediated by oxidative stress. The homocysteine-dependent upregulation of FGF23 presumably contributes to its role as a cardiovascular risk factor., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: ATP synthase (ATPase) is responsible for the majority of ATP production. Nevertheless, disease phenotypes associated with mutations in ATPase subunits are extremely rare. We aimed at expanding the spectrum of ATPase-related diseases., Methods: Whole-exome sequencing in cohorts with 2,962 patients diagnosed with mitochondrial disease and/or dystonia and international collaboration were used to identify deleterious variants in ATPase-encoding genes. Findings were complemented by transcriptional and proteomic profiling of patient fibroblasts. ATPase integrity and activity were assayed using cells and tissues from 5 patients., Results: We present 10 total individuals with biallelic or de novo monoallelic variants in nuclear ATPase subunit genes. Three unrelated patients showed the same homozygous missense ATP5F1E mutation (including one published case). An intronic splice-disrupting alteration in compound heterozygosity with a nonsense variant in ATP5PO was found in one patient. Three patients had de novo heterozygous missense variants in ATP5F1A, whereas another 3 were heterozygous for ATP5MC3 de novo missense changes. Bioinformatics methods and populational data supported the variants' pathogenicity. Immunohistochemistry, proteomics, and/or immunoblotting revealed significantly reduced ATPase amounts in association to ATP5F1E and ATP5PO mutations. Diminished activity and/or defective assembly of ATPase was demonstrated by enzymatic assays and/or immunoblotting in patient samples bearing ATP5F1A-p.Arg207His, ATP5MC3-p.Gly79Val, and ATP5MC3-p.Asn106Lys. The associated clinical profiles were heterogeneous, ranging from hypotonia with spontaneous resolution (1/10) to epilepsy with early death (1/10) or variable persistent abnormalities, including movement disorders, developmental delay, intellectual disability, hyperlactatemia, and other neurologic and systemic features. Although potentially reflecting an ascertainment bias, dystonia was common (7/10)., Interpretation: Our results establish evidence for a previously unrecognized role of ATPase nuclear-gene defects in phenotypes characterized by neurodevelopmental and neurodegenerative features. ANN NEUROL 2022;91:225-237., (© 2021 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Biallelic pathogenic variants in CLPP , encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.

Subdural hematoma (SDH) was initially reported in 20% to 30% of patients with glutaric aciduria type 1 (GA1). A recent retrospective study found SDH in 4% of patients, but not in patients identified by newborn screening (NBS). 168 MRIs of 69 patients with GA1 (age at MRI 9 days - 73.8 years, median 3.2 years) were systematically reviewed for presence of SDH, additional MR and clinical findings in order to investigate the frequency of SDH and potential risk factors. SDH was observed in eight high-excreting patients imaged between 5.8 and 24.4 months, namely space-occupying SDH in two patients after minor accidental trauma and SDH as an incidental finding in six patients without trauma. In patients without trauma imaged at 3 to 30 months (n = 36, 25 NBS, 27/9 high/low excreters), incidence of SDH was 16.7% (16% in NBS). SDH was more common after acute (33.3%) than insidious onset of dystonia (14.3%) or in asymptomatic patients (5.9%). It was only seen in patients with wide frontoparietal CSF spaces and frontotemporal hypoplasia. High excreters were over-represented among patients with SDH (6/27 vs 0/9 low excreters), acute onset (10/12), and wide frontoparietal CSF spaces (16/19). Incidental SDH occurs despite NBS and early treatment in approximately one in six patients with GA1 imaged during late infancy and early childhood. Greater risk of high excreters is morphologically associated with more frequent enlargement of external CSF spaces including frontotemporal hypoplasia, and may be furthered aggravated by more pronounced alterations of cerebral blood volume and venous pressure., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The aim of the study was a systematic evaluation of cognitive development in individuals with glutaric aciduria type 1 (GA1), a rare neurometabolic disorder, identified by newborn screening in Germany. This national, prospective, observational, multi-centre study includes 107 individuals with confirmed GA1 identified by newborn screening between 1999 and 2020 in Germany. Clinical status, development, and IQ were assessed using standardized tests. Impact of interventional and non-interventional parameters on cognitive outcome was evaluated. The majority of tested individuals (n = 72) showed stable IQ values with age (n = 56 with IQ test; median test age 11 years) but a significantly lower performance (median [IQR] IQ 87 [78-98]) than in general population, particularly in individuals with a biochemical high excreter phenotype (84 [75-96]) compared to the low excreter group (98 [92-105]; p = 0.0164). For all patients, IQ results were homogenous on subscale levels. Sex, clinical motor phenotype and quality of metabolic treatment had no impact on cognitive functions. Long-term neurologic outcome in GA1 involves both motor and cognitive functions. The biochemical high excreter phenotype is the major risk factor for cognitive impairment while cognitive functions do not appear to be impacted by current therapy and striatal damage. These findings implicate the necessity of new treatment concepts., (© 2021. The Author(s).)

Background: During the initial 26-week SPARK (Safety Paediatric efficAcy phaRmacokinetic with Kuvan®) study, addition of sapropterin dihydrochloride (Kuvan®; a synthetic formulation of the natural cofactor for phenylalanine hydroxylase, tetrahydrobiopterin; BH 4 ), to a phenylalanine (Phe)-restricted diet, led to a significant improvement in Phe tolerance versus a Phe-restricted diet alone in patients aged 0-4 years with BH 4 -responsive phenylketonuria (PKU) or mild hyperphenylalaninaemia (HPA). Based on these results, the approved indication for sapropterin in Europe was expanded to include patients < 4 years of age. Herein, we present results of the SPARK extension study (NCT01376908), evaluating the long-term safety, dietary Phe tolerance, blood Phe concentrations and neurodevelopmental outcomes in patients < 4 years of age at randomisation, over an additional 36 months of treatment with sapropterin., Results: All 51 patients who completed the 26-week SPARK study period entered the extension period. Patients who were previously treated with a Phe-restricted diet only ('sapropterin extension' group; n = 26), were initiated on sapropterin at 10 mg/kg/day, which could be increased up to 20 mg/kg/day. Patients previously treated with sapropterin plus Phe-restricted diet, remained on this regimen in the extension period ('sapropterin continuous' group; n = 25). Dietary Phe tolerance increased significantly at the end of the study versus baseline (week 0), by 38.7 mg/kg/day in the 'sapropterin continuous' group (95% CI 28.9, 48.6; p < 0.0001). In the 'sapropterin extension' group, a less pronounced effect was observed, with significant differences versus baseline (week 27) only observed between months 9 and 21; dietary Phe tolerance at the end of study increased by 5.5 mg/kg/day versus baseline (95% CI - 2.8, 13.8; p = 0.1929). Patients in both groups had normal neuromotor development and growth parameters., Conclusions: Long-term treatment with sapropterin plus a Phe-restricted diet in patients who initiated sapropterin at < 4 years of age with BH 4 -responsive PKU or mild HPA maintained improvements in dietary Phe tolerance over 3.5 years. These results continue to support the favourable risk/benefit profile for sapropterin in paediatric patients (< 4 years of age) with BH 4 -responsive PKU. Frequent monitoring of blood Phe levels and careful titration of dietary Phe intake to ensure adequate levels of protein intake is necessary to optimise the benefits of sapropterin treatment. Trial registration ClinicalTrials.gov, NCT01376908. Registered 17 June 2011, https://clinicaltrials.gov/ct2/show/NCT01376908 ., (© 2021. The Author(s).)

Introduction: Phenylketonuria (PKU) is a rare, treatable inborn error of metabolism with frequent neurological and neuropsychiatric complications, especially in undiagnosed or insufficiently treated individuals. Given the wide range of clinical presentations and the importance of treatment implications, we here delineate the neurological and neuropsychiatric symptom spectrum in a large cohort of previously unreported adults with late-treated PKU., Methods: We consecutively evaluated late-treated PKU cases and pooled clinical and paraclinical data, including video-material, from three centers with expertise in complex movement disorders, inborn errors of metabolism and pediatrics., Results: 26 individuals were included (10 females, median age 52 years). Developmental delay and intellectual disability were omnipresent with severe impairment of expressive communication noted in 50% of cases. Movement disorders were prevalent (77%), including tremor (38%, mostly postural), stereotypies (38%), and tics (19%). One case had neurodegenerative levodopa-responsive parkinsonism. Mild ataxia was noted in 54% of cases and 31% had a history of seizures. Neuropsychiatric characteristics included obsessive-compulsive (35%) and self-injurious behaviors (31%), anxiety (27%), depression (19%) and features compatible with those observed in individuals with autism spectrum disorder (19%). Neuroimaging revealed mild white matter changes. Adherence to dietary treatment was inconsistent in the majority of cases, particularly throughout adolescence., Conclusion: A history of movement disorders, particularly tremor, stereotypies and tics, in the presence of developmental delay, intellectual disability and neuropsychiatric features, such as obsessive-compulsive and self-injurious behaviors in adults should prompt the diagnostic consideration of PKU. Initiation and adherence to (dietary) treatment can ameliorate the severity of these symptoms., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Isovaleric aciduria (IVA), a metabolic disease with severe (classic IVA) or attenuated phenotype (mild IVA), is included in newborn screening (NBS) programs worldwide. The long-term clinical benefit of screened individuals, however, is still rarely investigated. A national, prospective, observational, multi-center study of individuals with confirmed IVA identified by NBS between 1998 and 2018 was conducted. Long-term clinical outcomes of 94 individuals with IVA were evaluated, representing 73.4% (for classic IVA: 92.3%) of the German NBS cohort. In classic IVA (N = 24), NBS prevented untimely death except in one individual with lethal neonatal sepsis (3.8%) but did not completely prevent single (N = 10) or recurrent (N = 7) metabolic decompensations, 13 of them occurring already neonatally. IQ (mean ± SD, 90.7 ± 10.1) was mostly normal but below the reference population (P = .0022) and was even lower in individuals with severe neonatal decompensations (IQ 78.8 ± 7.1) compared to those without crises (IQ 94.7 ± 7.5; P = .01). Similar results were obtained for school placement. In contrast, individuals with mild IVA had excellent neurocognitive outcomes (IQ 105.5 ± 15.8; normal school placement) and a benign disease course (no metabolic decompensation, normal hospitalization rate), which did not appear to be impacted by metabolic maintenance therapy. In conclusion, NBS reduces mortality in classic IVA, but does not reliably protect against severe neonatal metabolic decompensations, crucial for favorable neurocognitive outcome. In contrast, individuals with mild IVA had excellent clinical outcomes regardless of metabolic maintenance therapy, questioning their benefit from NBS. Harmonized stratified therapeutic concepts are urgently needed., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Glutaric aciduria type 1 (GA1) is a rare neurometabolic disorder, caused by inherited deficiency of glutaryl-CoA dehydrogenase, mostly affecting the brain. Early identification by newborn screening (NBS) significantly improves neurologic outcome. It has remained unclear whether recommended therapy, particular low lysine diet, is safe or negatively affects anthropometric long-term outcome. This national prospective, observational, multi-centre study included 79 patients identified by NBS and investigated effects of interventional and non-interventional parameters on body weight, body length, body mass index (BMI) and head circumference as well as neurological parameters. Adherence to recommended maintenance and emergency treatment (ET) had a positive impact on neurologic outcome and allowed normal anthropometric development until adulthood. In contrast, non-adherence to ET, resulting in increased risk of dystonia, had a negative impact on body weight (mean SDS -1.07; P = .023) and body length (mean SDS -1.34; P = -.016). Consistently, longitudinal analysis showed a negative influence of severe dystonia on weight and length development over time (P < .001). Macrocephaly was more often found in female (mean SDS 0.56) than in male patients (mean SDS -0.20; P = .049), and also in individuals with high excreter phenotype (mean SDS 0.44) compared to low excreter patients (mean SDS -0.68; P = .016). In GA1, recommended long-term treatment is effective and allows for normal anthropometric long-term development up to adolescence, with gender- and excreter type-specific variations. Delayed ET and severe movement disorder result in poor anthropometric outcome., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Leber's hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in mitochondrial DNA (mtDNA). A molecular diagnosis is achieved in up to 95% of cases, the vast majority of which are accounted for by 3 mutations within mitochondrial complex I subunit-encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON were recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knockout cellular model, we measured reduced turnover of specific complex I N-module subunits and a resultant impairment of complex I function. These results demonstrate that DNAJC30 is a chaperone protein needed for the efficient exchange of complex I subunits exposed to reactive oxygen species and integral to a mitochondrial complex I repair mechanism, thereby providing the first example to our knowledge of a disease resulting from impaired exchange of assembled respiratory chain subunits.

Objective: To evaluate the relationship between circulating phenylalanine and brain function as well as neuropsychiatric symptoms in adults with phenylketonuria., Methods: In this prospective cross-sectional study, early-treated patients with phenylketonuria older than 30 years and age- and sex-matched controls were included. Extensive neurologic evaluation, neuropsychological and behavioral testing, sensory and motor evoked potentials, and MRI were performed. CSF concentrations of neurodegenerative markers were evaluated in addition in a subset of 10 patients., Results: Nineteen patients with phenylketonuria (median age 41 years) with different phenylalanine levels (median 873 μmol/L) entered the study. They showed higher prevalence of neurologic symptoms, cognitive and behavioral abnormalities, autonomic dysfunction, alterations in neurophysiologic measures, and atrophy in putamen and right thalamus compared to controls. In CSF, patients with phenylketonuria exhibited higher β-amyloid 1-42 ( p = 0.003), total tau ( p < 0.001), and phosphorylated tau ( p = 0.032) levels compared to controls. Plasma phenylalanine levels highly correlated with the number of failed neuropsychological tests ( r = 0.64, p = 0.003), neuropsychiatric symptoms ( r = 0.73, p < 001), motor evoked potential latency ( r = 0.48, p = 0.030), and parietal lobe atrophy., Conclusions: Our study provides strong evidence for a correlation between phenylalanine levels and clinical, neuropsychological, neurophysiologic, biochemical, and imaging alterations in adult patients with phenylketonuria., (© 2020 American Academy of Neurology.)

Background : Tyrosinaemia type 1 is a rare inherited metabolic disease caused by an enzyme defect in the tyrosine degradation pathway. It is treated using nitisinone and a low-protein diet. In a workshop in 2013, a group of nutritional specialists from Germany, Switzerland and Austria agreed to advocate a simplified low-protein diet and to allow more natural protein intake in patients with tyrosinaemia type 1. This retrospective study evaluates the recommendations made at different treatment centers and their impact on clinical symptoms and metabolic control. Methods : For this multicenter study, questionnaires were sent to nine participating treatment centers to collect data on the general therapeutic approach and data of 47 individual patients treated by those centers. Results : Dietary simplification allocating food to 3 categories led to increased tyrosine and phenylalanine blood concentrations without weighing food. Phenylalanine levels were significantly higher in comparison to a strict dietary regimen whereas tyrosine levels in plasma did not change. Non-inferiority was shown for the simplification and liberalization of the diet. Compliance with dietary recommendations was higher using the simplified diet in comparison to the stricter approach. Age correlates negatively with compliance. Conclusions : Simplification of the diet with increased natural protein intake based on three categories of food may be implemented in the diet of patients with tyrosinaemia type 1 without significantly altering metabolic control. Patient compliance is strongly influencing tyrosine blood concentrations. A subsequent prospective study with a larger sample size is necessary to get a better insight into the effect of dietary recommendations on metabolic control.

Background: Neonatal intrahepatic cholestasis caused by citrin deficiency (CD) is a rare inborn error of metabolism due to variants in the SLC25A13 gene encoding the calcium-binding protein citrin. Citrin is an aspartate-glutamate carrier located within the inner mitochondrial membrane., Case Presentation: We report on two siblings of Romanian-Vietnamese ancestry with citrin deficiency. Patient 1 is a female who presented at age 8 weeks with cholestasis, elevated lactate levels and recurrent severe hypoglycemia. Diagnosis was made by whole exome sequencing and revealed compound heterozygosity for the frameshift variant c.852&#95;855del, p.Met285Profs*2 and a novel deletion c.(69 + 1&#95;70-1)&#95;(212 + 1&#95;231-1)del in SLC25A13. The girl responded well to dietary treatment with a lactose-free, MCT-enriched formula. Her younger brother (Patient 2) was born 1 year later and also found to be carrying the same gene variants. Dietary treatment from birth was able to completely prevent clinical manifestation until his current age of 4.5 months., Conclusions: As CD is a well-treatable disorder it should be ruled out early in the differential diagnosis of neonatal cholestasis. Due to the combination of hepatopathy, lactic acidosis and recurrent hypoglycemia the clinical presentation of CD may resemble hepatic mitochondrial depletion syndrome.

Background: ECHS1 encodes the mitochondrial short chain enoyl CoA hydratase 1 (SCEH). Biallelic ECHS1 variants have been associated with Leigh-like presentations and milder phenotypes with paroxysmal exercise-induced dystonia., Patients/methods: We used exome sequencing to investigate molecular bases of paroxysmal and non-paroxysmal dystonia in three patients and performed functional studies in fibroblasts. Disease presentation and response upon dietary interventions were documented., Results: We identified compound heterozygous ECHS1 missense variants in all individuals; all of them harbouring an c.518C > T (p.Ala173Val) variant. SCEH activity was impaired in patients' fibroblasts, respiratory chain-, and pyruvate-dehydrogenase-complex activities were normal in one individual. Patient 1 presented from the age of 2.5 years on with paroxysmal opisthotonic posturing. Patient 2 had a first metabolic crisis at the age 20 months developing recurrent exercise-induced dystonic episodes. Disease history of patient 3 was unremarkable for neurological findings until he first presented at the age of 20 years with persistent dystonia. Ketogenic diet had beneficial effects in patient 1. Neither ketogenic nor low protein diets led to milder symptoms in patient 2. Patient 3 benefits from low protein diet with improvement of his torticollis., Conclusions: In line with literature, our findings corroborate that the pathogenic ECHS1 variant c.518C > T (p.Ala173Val) is associated with milder phenotypes characterized by paroxysmal and non-paroxysmal dystonia. Because of the potentially treatable defect, especially in milder affected patients, it is important to consider SCEH deficiency not only in patients with Leigh-like syndrome but also in patients with paroxysmal dystonia and normal neurological findings between episodes., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: Although extended newborn screening (NBS) programs have been introduced more than 20 years ago, their impact on the long-term clinical outcome of individuals with inherited metabolic diseases (IMDs) is still rarely investigated., Methods: We studied the clinical outcomes of individuals with IMDs identified by NBS between 1999 and 2016 in a prospective multicenter observational study., Results: In total, 306 screened individuals with IMDs (115 with phenylketonuria and 191 with other IMDs with a lifelong risk for metabolic decompensation) were followed for a median time of 6.2 years. Although the risk for metabolic decompensation was disease-specific and NBS could not prevent decompensations in every individual at risk ( n = 49), the majority did not develop permanent disease-specific signs (75.9%), showed normal development (95.6%) and normal cognitive outcome (87.7%; mean IQ: 100.4), and mostly attended regular kindergarten (95.2%) and primary school (95.2%). This demonstrates that not only individuals with phenylketonuria, serving as a benchmark, but also those with lifelong risk for metabolic decompensation had a favorable long-term outcome. High NBS process quality is the prerequisite of this favorable outcome. This is supported by 28 individuals presenting with first symptoms at a median age of 3.5 days before NBS results were available, by the absence of neonatal decompensations after the report of NBS results, and by the challenge of keeping relevant process parameters at a constantly high level., Conclusions: NBS for IMDs, although not completely preventing clinical presentations in all individuals, can be considered a highly successful program of secondary prevention., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2020 by the American Academy of Pediatrics.)

Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Acute hyperammonemia without signs of common causes in the elderly might be challenging to identify. We report the oldest case known to date of a female patient with late onset ornithine carbamyltransferase deficiency (OTC), which was unmasked after a protein overload due to nutritional supplements. Our case illustrates how environmental factors (protein overload) in previously unknown OTC in the elderly leads to hyperammonemic encephalopathy and highlights that early treatment prevents persisting neurological deficits and should be considered in absence of common causes of hyperammonemic encephalopathy., Case Presentation: A 68-year-old woman presented with acute confusion, which progressed into a deep coma (Glasgow-Coma-Scale score 3) within a few hours. The only remarkable finding was a plasma ammonia (NH3) concentration of 697 μmmol/l (range 12-47 μmmol/). Third party history revealed that the patient disliked meat for most of her life (meat = protein, which needs to be metabolized) and had taken nutritional supplements (since supplements often have a high protein-ratio) 2 days before the symptoms started. Protein catabolism results in NH3, which is metabolized via the urea cycle. Consequently, the acute hyperammonemia in our patient was thought to be related to an inherited metabolic disorder, which only unmasked itself as a result of an overload of the corresponding metabolite (in this case protein). Since ornithine carbamyltransferase deficiency (OTC) is the most common inherited urea cycle disorder, this diagnosis became likely and was confirmed later via genetic and metabolic testing (amino acids, orotic acid, etc.). After 2 weeks of treatment (dialysis, low-protein-diet, nitrogen-lowering medication) the patient was discharged in a healthy condition without any neurological deficits., Conclusion: OTC is a x-chromosomal linked disorder, that usually manifests in newborn infants and children, but also rarely in adults and even rarer in the elderly (50- till 60-years-old), where it is probably underdiagnosed. In case of hyperammonemic encephalopathy - regardless of the underlying cause -, treatment should be started early to prevent persisting neurological deficits. OTC should be considered in absence of common causes of hyperammonemic encephalopathy.

Background: Phenylketonuria (PKU; OMIM#261600) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene resulting in high phenylalanine (Phe) in blood and brain. If not treated early this results in intellectual disability, behavioral and psychiatric problems, microcephaly, motor deficits, eczematous rash, autism, seizures, and developmental problems. There is a controversial discussion of whether patients with PKU have an additional risk for atherosclerosis due to interference of Phe with cholesterol synthesis and LDL-cholesterol regulation. Since cholesterol also plays a role in membrane structure and myelination, better insight into the clinical significance of the impact of Phe on lipoprotein metabolism is desirable. In 22 treated PKU patients (mean age 38.7 years) and 14 healthy controls (mean age 35.2 years), we investigated plasma with NMR spectroscopy and quantified 105 lipoprotein parameters (including lipoprotein subclasses) and 24 low molecular weight parameters. Analysis was performed on a 600 MHz Bruker AVANCE IVDr spectrometer as previously described., Results: Concurrent plasma Phe in PKU patients showed a wide range with a mean of 899 μmol/L (50-1318 μmol/L). Total cholesterol and LDL-cholesterol were significantly lower in PKU patients versus controls: 179.4 versus 200.9 mg/dL (p < 0.02) and 79.5 versus 104.1 mg/dL (p < 0.0038), respectively. PKU patients also had lower levels of 22 LDL subclasses with the greatest differences in LDL2 Apo-B, LDL2 Particle Number, LDL2-phospholipids, and LDL2-cholesterol (p < 0.0001). There was a slight negative correlation of total cholesterol and LDL-cholesterol with concurrent Phe level. VLDL5-free cholesterol, VLDL5-cholesterol, VLDL5-phospholipids, and VLDL4-free cholesterol showed a significant (p < 0.05) negative correlation with concurrent Phe level. There was no difference in HDL and their subclasses between PKU patients and controls. Tyrosine, glutamine, and creatinine were significantly lower in PKU patients compared to controls, while citric and glutamic acids were significantly higher., Conclusions: Using NMR spectroscopy, a unique lipoprotein profile in PKU patients can be demonstrated which mimics a non-atherogenic profile as seen in patients treated by statins.

Objective: To delineate the phenotypic and genotypic spectrum in carriers of mitochondrial MT-ATP6 mutations in a large international cohort., Methods: We analyzed in detail the clinical, genetical, and neuroimaging data from 132 mutation carriers from national registries and local databases from Europe, USA, Japan, and China., Results: We identified 113 clinically affected and 19 asymptomatic individuals with a known pathogenic MT-ATP6 mutation. The most frequent mutations were m.8993 T > G (53/132, 40%), m.8993 T > C (30/132, 23%), m.9176 T > C (30/132, 23%), and m.9185 T > C (12/132, 9%). The degree of heteroplasmy was high both in affected (mean 95%, range 20%-100%) and unaffected individuals (mean 73%, range 20%-100%). Age at onset ranged from prenatal to the age of 75 years, but almost half of the patients (49/103, 48%) became symptomatic before their first birthday. In 28 deceased patients, the median age of death was 14 months. The most frequent symptoms were ataxia (81%), cognitive dysfunction (49%), neuropathy (48%), seizures (37%), and retinopathy (14%). A diagnosis of Leigh syndrome was made in 55% of patients, whereas the classic syndrome of neuropathy, ataxia, and retinitis pigmentosa (NARP) was rare (8%)., Conclusions: In this currently largest series of patients with mitochondrial MT-ATP6 mutations, the phenotypic spectrum ranged from asymptomatic to early onset multisystemic neurodegeneration. The degree of mutation heteroplasmy did not reliably predict disease severity. Leigh syndrome was found in more than half of the patients, whereas classic NARP syndrome was rare. Oligosymptomatic presentations were rather frequent in adult-onset patients, indicating the need to include MT-ATP6 mutations in the differential diagnosis of both ataxias and neuropathies., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Purpose: Skeletal muscle growth and regeneration rely on muscle stem cells, called satellite cells. Specific transcription factors, particularly PAX7, are key regulators of the function of these cells. Knockout of this factor in mice leads to poor postnatal survival; however, the consequences of a lack of PAX7 in humans have not been established., Methods: Here, we study five individuals with myopathy of variable severity from four unrelated consanguineous couples. Exome sequencing identified pathogenic variants in the PAX7 gene. Clinical examination, laboratory tests, and muscle biopsies were performed to characterize the disease., Results: The disease was characterized by hypotonia, ptosis, muscular atrophy, scoliosis, and mildly dysmorphic facial features. The disease spectrum ranged from mild to severe and appears to be progressive. Muscle biopsies showed the presence of atrophic fibers and fibroadipose tissue replacement, with the absence of myofiber necrosis. A lack of PAX7 expression was associated with satellite cell pool exhaustion; however, the presence of residual myoblasts together with regenerating myofibers suggest that a population of PAX7-independent myogenic cells partially contributes to muscle regeneration., Conclusion: These findings show that biallelic variants in the master transcription factor PAX7 cause a new type of myopathy that specifically affects satellite cell survival.

Pyruvate carboxylase deficiency (PCD) is caused by biallelic mutations of the PC gene. The reported clinical spectrum includes a neonatal form with early death (type B), an infantile fatal form (type A), and a late-onset form with isolated mild intellectual delay (type C). Apart from homozygous stop-codon mutations leading to type B PCD, a genotype-phenotype correlation has not otherwise been discernible. Indeed, patients harboring biallelic heterozygous variants leading to PC activity near zero can present either with a fatal infantile type A or with a benign late onset type C form. In this study, we analyzed six novel patients with type A (three) and type C (three) PCD, and compared them with previously reported cases. First, we observed that type C PCD is not associated to homozygous variants in PC. In silico modeling was used to map former and novel variants associated to type A and C PCD, and to predict their potential effects on the enzyme structure and function. We found that variants lead to type A or type C phenotype based on the destabilization between the two major enzyme conformers. In general, our study on novel and previously reported patients improves the overall understanding on type A and C PCD., (© 2019 Wiley Periodicals, Inc.)

Mitochondrial dynamics such as fission and fusion play a vital role in normal brain development and neuronal activity. DNM1L encodes a dynamin-related protein 1 (Drp1), which is a GTPase essential for proper mitochondrial fission. The clinical phenotype of DNM1L mutations depends on the degree of mitochondrial fission deficiency, ranging from severe encephalopathy and death shortly after birth to initially normal development and then sudden onset of refractory status epilepticus with very poor neurologic outcome. We describe a case of a previously healthy 3-year-old boy with a mild delay in speech development until the acute onset of a refractory status epilepticus with subsequent epileptic encephalopathy and very poor neurologic outcome. The de novo missense mutation in DNM1L (c.1207C > T, p.R403C), which we identified in this case, seems to determine a unique clinical course, strikingly similar to four previously described patients in literature with the identical de novo heterozygous missense mutation in DNM1L ., Competing Interests: None., (Georg Thieme Verlag KG Stuttgart · New York.)

Biogenic amines synthesis in phenylketonuria (PKU) patients with high phenylalanine (Phe) concentration is thought to be impaired due to inhibition of tyrosine and tryptophan hydroxylases and competition with amino acids at the blood-brain barrier. Dopamine and serotonin deficits might explain brain damage and progressive neuropsychiatric impairment in adult PKU patients. Ten early treated adult PKU patients (mean age 38.2 years) and 15 age-matched controls entered the study. Plasma and cerebrospinal fluid (CSF) Phe, 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP), 3,4-dihydroxy-l-phenylalanine (l-DOPA) and homovanillic acid (HVA) were analyzed. Voxel-based morphometry statistical nonparametric mapping was used to test the age-corrected correlation between gray matter atrophy and CSF biogenic amines levels. 5-HIAA and 5-HTP were significantly reduced in PKU patients compared to controls. Significant negative correlations were found between CSF 5-HIAA, HVA, and 5-HTP and Phe levels. A decrease in 5-HIAA and 5-HTP concentrations correlated with precuneus and frontal atrophy, respectively. Lower HVA levels correlated with occipital atrophy. Biogenic amines deficits correlate with specific brain atrophy patterns in adult PKU patients, in line with serotonin and dopamine projections. These findings may support a more rigorous Phe control in adult PKU to prevent neurotransmitter depletion and accelerated brain damage due to aging., (© 2019 SSIEM.)

Background: Primary microcephaly and profound global developmental delay have been considered the core clinical phenotype in patients with bi-allelic PRUNE1 mutations., Methods: Linkage analysis and whole-exome sequencing (WES) in a multiplex family and extraction of further cases from a WES repository containing 571 children with severe developmental disabilities and neurologic symptoms., Results: We identified bi-allelic PRUNE1 mutations in twelve children from six unrelated families. All patients who survived beyond the first 6 months of life had early-onset global developmental delay, bilateral spastic paresis, dysphagia and difficult-to-treat seizures, while congenital or later-evolving microcephaly was not a consistent finding. Brain MRI showed variable anomalies with progressive cerebral and cerebellar atrophies and T2-hyperintense brain stem lesions. Peripheral neuropathy was documented in five cases. Disease course was progressive in all patients and eight children died in the first or early second decade of life. In addition to the previously reported missense mutation p.(Asp106Asn), we observed a novel homozygous missense variant p.(Leu172Pro) and a homozygous contiguous gene deletion encompassing most of the PRUNE1 gene and part of the neighboring BNIPL gene., Conclusions: PRUNE1 deficiency causes severe early-onset disease affecting the central and peripheral nervous systems. Microcephaly is probably not a universal feature., Competing Interests: All authors state that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy., Results: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers., Conclusions: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

Objective: Untreated individuals with glutaric aciduria type 1 (GA1) commonly present with a complex, predominantly dystonic movement disorder (MD) following acute or insidious onset striatal damage. Implementation of GA1 into newborn screening (NBS) programs has improved the short-term outcome. It remains unclear, however, whether NBS changes the long-term outcome and which variables are predictive., Methods: This prospective, observational, multicenter study includes 87 patients identified by NBS, 4 patients missed by NBS, and 3 women with GA1 identified by positive NBS results of their unaffected children., Results: The study population comprises 98.3% of individuals with GA1 identified by NBS in Germany during 1999-2016. Overall, cumulative sensitivity of NBS is 95.6%, but it is lower (84%) for patients with low excreter phenotype. The neurologic outcome of patients missed by NBS is as poor as in the pre-NBS era, and the clinical phenotype of diagnosed patients depends on the quality of therapeutic interventions rather than noninterventional variables. Presymptomatic start of treatment according to current guideline recommendations clearly improves the neurologic outcome (MD: 7% of patients), whereas delayed emergency treatment results in acute onset MD (100%), and deviations from maintenance treatment increase the risk of insidious onset MD (50%). Independent of the neurologic phenotype, kidney function tends to decline with age, a nonneurologic manifestation not predicted by any variable included in this study., Interpretation: NBS is a beneficial, disease-changing intervention for GA1. However, improved neurologic outcome critically depends on adherence to recommended therapy, whereas kidney dysfunction does not appear to be impacted by recommended therapy. Ann Neurol 2018;83:970-979., (© 2018 American Neurological Association.)

Background: Mitochondrial diseases, a group of multi-systemic disorders often characterized by tissue-specific phenotypes, are usually progressive and fatal disorders resulting from defects in oxidative phosphorylation. MTO1 (Mitochondrial tRNA Translation Optimization 1), an evolutionarily conserved protein expressed in high-energy demand tissues has been linked to human early-onset combined oxidative phosphorylation deficiency associated with hypertrophic cardiomyopathy, often referred to as combined oxidative phosphorylation deficiency-10 (COXPD10)., Material and Methods: Thirty five cases of MTO1 deficiency were identified and reviewed through international collaboration. The cases of two female siblings, who presented at 1 and 2years of life with seizures, global developmental delay, hypotonia, elevated lactate and complex I and IV deficiency on muscle biopsy but without cardiomyopathy, are presented in detail., Results: For the description of phenotypic features, the denominator varies as the literature was insufficient to allow for complete ascertainment of all data for the 35 cases. An extensive review of all known MTO1 deficiency cases revealed the most common features at presentation to be lactic acidosis (LA) (21/34; 62% cases) and hypertrophic cardiomyopathy (15/34; 44% cases). Eventually lactic acidosis and hypertrophic cardiomyopathy are described in 35/35 (100%) and 27/34 (79%) of patients with MTO1 deficiency, respectively; with global developmental delay/intellectual disability present in 28/29 (97%), feeding difficulties in 17/35 (49%), failure to thrive in 12/35 (34%), seizures in 12/35 (34%), optic atrophy in 11/21 (52%) and ataxia in 7/34 (21%). There are 19 different pathogenic MTO1 variants identified in these 35 cases: one splice-site, 3 frameshift and 15 missense variants. None have bi-allelic variants that completely inactivate MTO1; however, patients where one variant is truncating (i.e. frameshift) while the second one is a missense appear to have a more severe, even fatal, phenotype. These data suggest that complete loss of MTO1 is not viable. A ketogenic diet may have exerted a favourable effect on seizures in 2/5 patients., Conclusion: MTO1 deficiency is lethal in some but not all cases, and a genotype-phenotype relation is suggested. Aside from lactic acidosis and cardiomyopathy, developmental delay and other phenotypic features affecting multiple organ systems are often present in these patients, suggesting a broader spectrum than hitherto reported. The diagnosis should be suspected on clinical features and the presence of markers of mitochondrial dysfunction in body fluids, especially low residual complex I, III and IV activity in muscle. Molecular confirmation is required and targeted genomic testing may be the most efficient approach. Although subjective clinical improvement was observed in a small number of patients on therapies such as ketogenic diet and dichloroacetate, no evidence-based effective therapy exists., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The biogenesis of the multi-subunit vacuolar-type H + -ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that ATP6AP2 deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into Drosophila led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both ATP6AP2 mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in ATP6AP2 lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy., (© 2017 Rujano et al.)

Mitochondrial protein synthesis involves an intricate interplay between mitochondrial DNA encoded RNAs and nuclear DNA encoded proteins, such as ribosomal proteins and aminoacyl-tRNA synthases. Eukaryotic cells contain 17 mitochondria-specific aminoacyl-tRNA synthases. WARS2 encodes mitochondrial tryptophanyl-tRNA synthase (mtTrpRS), a homodimeric class Ic enzyme (mitochondrial tryptophan-tRNA ligase; EC 6.1.1.2). Here, we report six individuals from five families presenting with either severe neonatal onset lactic acidosis, encephalomyopathy and early death or a later onset, more attenuated course of disease with predominating intellectual disability. Respiratory chain enzymes were usually normal in muscle and fibroblasts, while a severe combined respiratory chain deficiency was found in the liver of a severely affected individual. Exome sequencing revealed rare biallelic variants in WARS2 in all affected individuals. An increase of uncharged mitochondrial tRNA Trp and a decrease of mtTrpRS protein content were found in fibroblasts of affected individuals. We hereby define the clinical, neuroradiological, and metabolic phenotype of WARS2 defects. This confidently implicates that mutations in WARS2 cause mitochondrial disease with a broad spectrum of clinical presentation., (© 2017 Wiley Periodicals, Inc.)

Objective: 3-Methylglutaconic aciduria, dystonia-deafness, hepatopathy, encephalopathy, Leigh-like syndrome (MEGDHEL) syndrome is caused by biallelic variants in SERAC1., Methods: This multicenter study addressed the course of disease for each organ system. Metabolic, neuroradiological, and genetic findings are reported., Results: Sixty-seven individuals (39 previously unreported) from 59 families were included (age range = 5 days-33.4 years, median age = 9 years). A total of 41 different SERAC1 variants were identified, including 20 that have not been reported before. With the exception of 2 families with a milder phenotype, all affected individuals showed a strikingly homogeneous phenotype and time course. Severe, reversible neonatal liver dysfunction and hypoglycemia were seen in >40% of all cases. Starting at a median age of 6 months, muscular hypotonia (91%) was seen, followed by progressive spasticity (82%, median onset = 15 months) and dystonia (82%, 18 months). The majority of affected individuals never learned to walk (68%). Seventy-nine percent suffered hearing loss, 58% never learned to speak, and nearly all had significant intellectual disability (88%). Magnetic resonance imaging features were accordingly homogenous, with bilateral basal ganglia involvement (98%); the characteristic "putaminal eye" was seen in 53%. The urinary marker 3-methylglutaconic aciduria was present in virtually all patients (98%). Supportive treatment focused on spasticity and drooling, and was effective in the individuals treated; hearing aids or cochlear implants did not improve communication skills., Interpretation: MEGDHEL syndrome is a progressive deafness-dystonia syndrome with frequent and reversible neonatal liver involvement and a strikingly homogenous course of disease. Ann Neurol 2017;82:1004-1015., (© 2017 American Neurological Association.)

Across a variety of Mendelian disorders, ∼50-75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.