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Objective: Ornithine transcarbamylase deficiency (OTC-D) is an X-linked metabolic disease and the most common urea cycle disorder. Due to high phenotypic heterogeneity, ranging from lethal neonatal hyperammonemic events to moderate symptoms and even asymptomatic individuals, the prediction of the disease course at an early disease stage is very important to individually adjust therapies such as medical treatment or liver transplantation. In this translational study, we developed a severity-adjusted classification system based on in vitro residual enzymatic OTC activity. Methods: Applying a cell-based expression system, residual enzymatic OTC activities of 71 pathogenic OTC variants were spectrophotometrically determined and subsequently correlated with clinical and biochemical outcome parameters of 119 male individuals with OTC-D (mOTC-D) as reported in the UCDC and E-IMD registries. Results: Integration of multiple data sources enabled the establishment of a robust disease prediction model for mOTC-D. Residual enzymatic OTC activity not only correlates with age at first symptoms, initial peak plasma ammonium concentration and frequency of metabolic decompensations but also predicts mortality. The critical threshold of 4.3% residual enzymatic activity distinguishes a severe from an attenuated phenotype. Interpretation: Residual enzymatic OTC activity reliably predicts the disease severity in mOTC-D and could thus serve as a tool for severity- adjusted evaluation of therapeutic strategies and counselling patients and parents.

Additional file 2: Fig. S1. Overview of the study. Fig. S2. Quality control. Fig. S3. DNA-RNA sample matching. Fig. S4. Aberrant events per sample. Fig. S5. Rare variants among expression outliers. Fig. S6. Power analysis of overexpression outliers. Fig. S7. Power analysis of underexpression outliers with respect to biological coefficient of variation. Fig. S8. Cases with many mtDNA expression outliers. Fig. S9. Rare variants among splicing outliers. Fig. S10. Splicing prediction algorithms evaluation. Fig. S11. Complex pattern of aberrant splicing. Fig. S12. Analysis of variants called by RNA-seq. Fig. S13. Rare variants leading to outliers. Fig. S14. Diagnostic rate across cohorts.

Additional file 2: Table S2. Blood Phe levels in patients with treatment-emergent hypophenylalaninaemia.

Additional file 1: Table S1. Status at the end of the extension period—ITTE population.

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. ispartof: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research vol:1867 issue:7 ispartof: location:Netherlands status: Published online

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. publishersversion published

ObjectiveTo delineate the phenotypic and genotypic spectrum in carriers of mitochondrial MT-ATP6 mutations in a large international cohort.MethodsWe 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.ResultsWe 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%).ConclusionsIn 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.

Additional file 1: Table S1. Terminology of 117 lipoprotein parameters measured with the lipoprotein distribution (LPD) prediction method. The 105 measured parameters are presented in the first table, while the 12 parameters, which are calculated from the original ones, are presented at the end of the table (From https://pubs.acs.org/doi/abs/10.1021/acs.analchem.8b02412 with changes). Table S2. Results of low molecular weight metabolites in PKU patients and controls: mean, standard deviation and p significance (t-test) are presented in the first table; lipoprotein subclasses (explanation in Table S1) are presented in the second table. Figure S1. Correlation between 2 years mean blood phenylalanine (Phe) level and concurrent Phe level in 7 PKU patients (historical blood Phe levels measured in dried blood by tandem mass spectrometry).

TMEM165 is involved in a rare genetic human disease named TMEM165‐CDG (congenital disorders of glycosylation). It is Golgi localized, highly conserved through evolution and belongs to the uncharacterized protein family 0016 (UPF0016). The use of isogenic TMEM165 KO HEK cells was crucial in deciphering the function of TMEM165 in Golgi manganese homeostasis. Manganese is a major cofactor of many glycosylation enzymes. Severe Golgi glycosylation defects are observed in TMEM165 Knock Out Human Embryonic Kidney (KO HEK) cells and are rescued by exogenous manganese supplementation. Intriguingly, we demonstrate in this study that the observed Golgi glycosylation defect mainly depends on fetal bovine serum, particularly its manganese level. Our results also demonstrate that iron and/or galactose can modulate the observed glycosylation defects in TMEM165 KO HEK cells. While isogenic cultured cells are widely used to study the impact of gene defects on proteins' glycosylation patterns, these results emphasize the importance of the use of validated fetal bovine serum in glycomics studies. 43;2

ollaborators: Bloxam S, Brody L, Caspi L, Elsbecker S, Fierro L, Lynn A, Mullins M, Mütze U, Papaleo C, Payan I, Seminara J, Simpson K, Singer R, Wallis K, Alber FD, Babikian T, Bender H, Boys C, Breiger D, Buerger C, Caudle SE, NguyenDriver M, Kerr E, Mamak E, Sanz JH, Tangen R, Wilkening G, Cederbaum S, Feigenbaum A, Kerr DS, LichterKonecki U, Seashore MR, Berry SA, Burrage L, Coughlin C, Diaz GA, Gallagher RC, Gropman A, Harding CO, Lee B, Le Mons C, Lawrence Merritt J 2nd, Nagamani SCS, Schulze A, Stricker T, Tuchman M, Waisbren S, WeisfeldAdams J, Wong D, Yudkoff M, Arnoux J, Bari Cacute I, Bosch AM, Chabrol B, Chakrapani A, CortèsSaladefont E, Couce ML, Eyskens F, de Laet C, de Meirleir L, Freisinger P, Gleich F, Grünewald S, Häberle J, Hwu W, Jalan A, Karall D, Lindner M, Lund AM, Martinelli D, Murphy E, Mühlhausen C, Olivieri G, Ottolenghi C, Rodrigues E, Rubert L, Sarajlija A, Schiff M, Sokal E, SykutCegielska J, Walter JH, Williams M, Zeman J. BACKGROUND: To improve our understanding of urea cycle disorders (UCDs) prospectively followed by two North American (NA) and European (EU) patient cohorts. AIMS: Description of the NA and EU patient samples and investigation of the prospects of combined and comparative analyses for individuals with UCDs. METHODS: Retrieval and comparison of the data from 1095 individuals (NA: 620, EU: 475) from two electronic databases. RESULTS: The proportion of females with ornithine transcarbamylase deficiency (fOTC-D), particularly those being asymptomatic (asfOTC-D), was higher in the NA than in the EU sample. Exclusion of asfOTC-D resulted in similar distributions in both samples. The mean age at first symptoms was higher in NA than in EU patients with late onset (LO), but similar for those with early (≤ 28 days) onset (EO) of symptoms. Also, the mean age at diagnosis and diagnostic delay for EO and LO patients were similar in the NA and EU cohorts. In most patients (including fOTC-D), diagnosis was made after the onset of symptoms (59.9%) or by high-risk family screening (24.7%), and less often by newborn screening (8.9%) and prenatal testing (3.7%). Analysis of clinical phenotypes revealed that EO patients presented with more symptoms than LO individuals, but that numbers of symptoms correlated with plasma ammonium concentrations in EO patients only. Liver transplantation was reported for 90 NA and 25 EU patients. CONCLUSIONS: Combined analysis of databases drawn from distinct populations opens the possibility to increase sample sizes for natural history questions, while comparative analysis utilizing differences in approach to treatment can evaluate therapeutic options and enhance long-term outcome studies.

OBJECTIVE Individuals with urea cycle disorders (UCDs) often present with intellectual and developmental disabilities. The major aim of this study was to evaluate the impact of diagnostic and therapeutic interventions on cognitive outcomes in UCDs. METHODS This prospective, observational, multicenter study includes data from 503 individuals with UCDs who had comprehensive neurocognitive testing with a cumulative follow-up of 702 patient-years. RESULTS The mean cognitive standard deviation score (cSDS) was lower in symptomatic than in asymptomatic (p < 0.001, t test) individuals with UCDs. Intellectual disability (intellectual quotient < 70, cSDS < -2.0) was associated with the respective subtype of UCD and early disease onset, whereas height of the initial peak plasma ammonium concentration was inversely associated with neurocognitive outcomes in mitochondrial (proximal) rather than cytosolic (distal) UCDs. In ornithine transcarbamylase and argininosuccinate synthetase 1 deficiencies, we did not find evidence that monoscavenger therapy with sodium or glycerol phenylbutyrate was superior to sodium benzoate in providing cognitive protection. Early liver transplantation appears to be beneficial for UCDs. It is noteworthy that individuals with argininosuccinate synthetase 1 and argininosuccinate lyase deficiencies identified by newborn screening had better neurocognitive outcomes than those diagnosed after the manifestation of first symptoms. INTERPRETATION Cognitive function is related to interventional and non-interventional variables. Early detection by newborn screening and early liver transplantation appear to offer greater cognitive protection, but none of the currently used nitrogen scavengers was superior with regard to long-term neurocognitive outcome. Further confirmation could determine these variables as important clinical indicators of neuroprotection for individuals with UCDs. ANN NEUROL 2019.

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).

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 2 years 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., Highlights • The phenotypic and genotypic spectrum of MTO1 deficiency is more variable than previously reported • Hallmark features: cardiomyopathy, lactic acidosis, developmental delay, failure to thrive, seizures, optic atrophy, ataxia • 19 pathogenic MTO1 mutations (splice-site, frameshift, missense) were identified with a geno-phenotype relation • Suspect MTO1 deficiency based on clinical features, mitochondrial markers in body fluids, low complex I III IV activity • Although ketogenic diet exerted subjective improvement in some cases, there is exists no evidence-based effective therapy

Due to an unfortunate error during the typesetting process, the collaborators were presented incorrectly.

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. Electronic supplementary material The online version of this article (10.1186/s13023-018-0784-8) contains supplementary material, which is available to authorized users.

WOS: 000418389700017, PubMed ID: 29205472, Objective3-Methylglutaconic aciduria, dystonia-deafness, hepatopathy, encephalopathy, Leigh-like syndrome (MEGDHEL) syndrome is caused by biallelic variants in SERAC1. MethodsThis multicenter study addressed the course of disease for each organ system. Metabolic, neuroradiological, and genetic findings are reported. ResultsSixty-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. InterpretationMEGDHEL 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, Else Kroner-Fresenius Stiftung; German Bundesministerium fur Bildung und Forschung (BMBF)Federal Ministry of Education & Research (BMBF); Horizon2020 through the E-Rare project GENOMIT [01GM1603, 01GM1207, FWF I 2741-B26]; Vereinigung zur Forderung Padiatrischer Forschung und Fortbildung Salzburg; Wellcome Centre for Mitochondrial Research [203105/Z/16/Z]; Medical Research Council (MRC) Centre for Translational Research in Neuromuscular Disease, Mitochondrial Disease Patient Cohort (UK)Medical Research Council UK (MRC) [G0800674]; Lily Foundation; UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children, M.S. was supported by the Else Kroner-Fresenius Stiftung" This study was supported by the German Bundesministerium fur Bildung und Forschung (BMBF) and Horizon2020 through the E-Rare project GENOMIT (01GM1603 and 01GM1207 for HP and FWF I 2741-B26 for J.A.M. J.A.M., S.B.W., W.S. were supported by the Vereinigung zur Forderung Padiatrischer Forschung und Fortbildung Salzburg. R.W.T. was supported by the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z), the Medical Research Council (MRC) Centre for Translational Research in Neuromuscular Disease, Mitochondrial Disease Patient Cohort (UK) (G0800674), the Lily Foundation and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children.

Summary of neuromotor developmental milestones – ITT population. Data show (a) the proportion of patients with normal development in the area of assessment at baseline, (b) Week 12, and (c) Week 26 treated with sapropterin plus the Phe-restricted diet, or Phe-restricted diet alone. P-values show comparison of results between treatment groups at Week 26 using the chi-squared test. Table S1. PAH genotypes of sapropterin responders (n=37). (DOCX 180 kb)

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.

Acute liver failure (ALF) in infancy and childhood is a life-threatening emergency. Few conditions are known to cause recurrent acute liver failure (RALF), and in about 50% of cases, the underlying molecular cause remains unresolved. Exome sequencing in five unrelated individuals with fever-dependent RALF revealed biallelic mutations in NBAS. Subsequent Sanger sequencing of NBAS in 15 additional unrelated individuals with RALF or ALF identified compound heterozygous mutations in an additional six individuals from five families. Immunoblot analysis of mutant fibroblasts showed reduced protein levels of NBAS and its proposed interaction partner p31, both involved in retrograde transport between endoplasmic reticulum and Golgi. We recommend NBAS analysis in individuals with acute infantile liver failure, especially if triggered by fever.

Table S2. Calculation of European incidence of ACAD9 deficiency (DOCX 36Â kb)

Table S1. Compound heterozygous and homozygous ACAD9 variants identified in 67 patients present in this study (DOCX 75Â kb)

Additional data. (DOCX 38Â kb)

Figure S1. Representative picture of Complex I assembly in fibroblasts of individual 1 (A, upper panels) Two-dimensional BN/SDS-PAGE separation and quantification of fluorescent-labelled mitochondrial complexes from 10 mg patient (left) and control fibroblasts (right) are shown. (A, lower panels) show silver stained 2 D gels. (B) Quantified Supercomplexes in 2D gels from control and patient fibroblast with and without bezafibrate treatment for 72 h. (C) Panoramaplots of 2D gels with assignment of signals used for quantification of complexes. Assignment of complexes: O, OGDC, oxoglutarate dehydrogenase complex; V, complex V or ATP synthase; III, complex III or cytochrome c reductase; IV, complex IV or cytochrome c oxidase; S, supercomplexes composed of respiratory chain complexes I, III, and IV. 2-D gels were scanned side by side for direct comparison and are shown as pseudocolors. (pdf). (PDF 1109 kb)

Table S3. Clinical characteristics of the 67 patients present in this study (DOCX 72Â kb)

Figure S1. Representative picture of Complex I assembly in fibroblasts of individual 1 (A, upper panels) Two-dimensional BN/SDS-PAGE separation and quantification of fluorescent-labelled mitochondrial complexes from 10 mg patient (left) and control fibroblasts (right) are shown. (A, lower panels) show silver stained 2 D gels. (B) Quantified Supercomplexes in 2D gels from control and patient fibroblast with and without bezafibrate treatment for 72 h. (C) Panoramaplots of 2D gels with assignment of signals used for quantification of complexes. Assignment of complexes: O, OGDC, oxoglutarate dehydrogenase complex; V, complex V or ATP synthase; III, complex III or cytochrome c reductase; IV, complex IV or cytochrome c oxidase; S, supercomplexes composed of respiratory chain complexes I, III, and IV. 2-D gels were scanned side by side for direct comparison and are shown as pseudocolors. (pdf). (PDF 1109 kb)

Table S2. Calculation of European incidence of ACAD9 deficiency (DOCX 36Â kb)

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.

Table S3. Clinical characteristics of the 67 patients present in this study (DOCX 72Â kb)

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.

Additional data. (DOCX 38Â kb)

Table S1. Compound heterozygous and homozygous ACAD9 variants identified in 67 patients present in this study (DOCX 75Â kb)

Mitochondrial diseases are a genetically and phenotypically diverse collection of diseases due to defects in over 400 genes with roles converging on aerobic energy metabolism. Their heterogeneity leads to significant challenges in making an accurate diagnosis. This thesis analyses the diagnostic power of whole exome sequencing, validates variants in known and novel disease genes, and applies an integrative multi-omic approach encompassing genetic, phenotypic, RNA sequencing, and quantitative proteomic data, to discover and simultaneously validate diagnoses in patients. Mitochondriale Erkrankungen sind eine genetisch und phänotypisch vielfältige Gruppe von Erkrankungen, die auf Defekte in 400 Genen zurückzuführen sind und deren Funktion im Energiestoffwechsel konvergiert. Ihre Heterogenität führt zu enormen Herausforderungen bei der Diagnosestellung. Diese Arbeit analysiert das diagnostische Potential der Exomsequenzierung, validiert Varianten in bekannten und neuen Krankheitsgenen und wendet einen integrativen Ansatz an, der genetische, Phänotyp-, RNA- und proteomische Daten umfasst, um Diagnosen zu entdecken und zu validieren.

A cohort of 150 patients with isolated respiratory chain complex I (RCC I) deficiency was screened for pathogenic mutations in five new candidate genes. No pathogenic mutations were identified explaining the RCC I defect. The molecular-genetic results of a previously performed mutation screen were matched with the patients’ phenotypes, which showed a rather heterogeneous spectrum of clinical signs. The results of the genotype-phenotype correlation studies including other published cases can be helpful for choosing nuclear encoded candidate genes and thus improve molecular-genetic diagnostics. 150 Patienten mit isoliertem Defekt des Komplex I der Atmungskette wurden auf pathogenetische Mutationen innerhalb fünf neuer Kandidatengene gescreent. Es wurden keine pathogenetischen Mutationen gefunden, welche den Defekt erklären. Die molekulargenetischen Ergebnisse wurden mit den Phänotypen verglichen, wobei ein heterogenes Spektrum an klinischen Symptomen beobachtet wurde. Die Ergebnisse der Genotyp-Phänotyp-Korrelationsstudie, welche publizierte Fälle einbezieht, können bei der Auswahl nukleär kodierter Kandidatengene hilfreich sein und so die molekulargenetische Diagnostik verbessern.

Item does not contain fulltext 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.

Die Ursachen für hepatozerebrale Syndrome im Kindesalter sind vielfältig und u.a. durch Störungen im mitochondrialen Stoffwechsel verursacht. Deren genetische Ursachen wiederum sind sehr heterogen. Wir untersuchten 44 Kinder mit hepatozerebralem Syndrom und mitochondrialer Dysfunktion nach Mutationen im POLG1- und DGUOK- Gen, die bei der Synthese der mitochondrialen DNA eine zentrale Rolle spielen. Dabei konnten wir in 21 Patienten pathogene Mutationen nachweisen. Das phänotypische Spektrum reicht von neonatal beginnender Leberinsuffizienz mit variabler neurologischer und muskulä We studied 44 patients with hepatocerebral syndrome and mitochondrial dysfunction, and detected pathogenic mutations in POLG 1 and DGUOK in 21 patients. The phenotypical spectrum ranges from early onset liver failure with various neurological and muscular involvement to Alpers syndrome a disorder with intractable seizures and liver dysfunction. Thus mutations of POLG1 and DGUOK are relatively common causes of hepatocerebral syndrome in childhood.

Zur seltenen Gruppe der Mukolipidosen gehören die Sialidose, Mukolipidose II, III und IV. In der vorliegenden retrospektiven Studie wurden die Krankheitsverläufe von dreizehn Mukolipidose II-Patienten mittels Fragebogen hinsichtlich Schwangerschaft, Geburtsverlauf, physischer und psychomotorischer Entwicklung, Erstmanifestation der Symptome, Behandlung und ärztlicher Betreuung erfasst: Dysostosis multiplex, Gelenksteifigkeit und Gesichtsdysmorphie können als Leitsymptome angesehen werden. Die physische und psychomotorische Retardierung ist, ebenso wie die Organbeteiligung variabel, im Allgemeinen aber schwer. Empfehlungen zur Diagnostik und Betreuung werden gegeben. To the rare group of mucolipidosis belong sialidosis, mucolipidosis II, III and IV, In this retrospective study was covered the clinical course of 13 mucolipidosis II-patients with the help of questionnaire regarding pregnancy, birth, physical and psychomotoric development, onset, treatment and medical care. Dysostosis multiplex, joint stiffness and facial dysmorphia are guiding symptoms. Physical and psychomotor retardation are variable, in general serious, also organic involvement. Recommendation for diagnostics and medical care are given.

Diese Arbeit untersucht die Korrelationen zwischen Ultraschall-Knochendichtemessung (BUA: Broadband-Ultrasound-Attenuation; MHz/dB und SOS: Speed of sound; m/s) des Kalkaneus und Knochenstoffwechselparametern (unspez. Alk. Phosphatase: AP; Prokollagen-I-Propeptid: PICP; Parathormon: PTH) bei 38 chron. niereninsuffizienten (NIOD; N=7), dialysepflichtigen (NIMD; N=7) und nierentransplantierten (NITRAN; N=24) Kindern über 12 Monate. Es zeigen sich Korrelationen von BUA mit SOS bei NITRAN (0.497, p

Das Marfan-Syndrom (MFS) ist eine seltene, vererbte Bindegewebserkrankung mit variabler Expressivität, die sich klinisch an zahlreichen Organsystemen manifestieren kann. Die Diagnosestellung erfolgt nach den Kriterien der Genter Nosologie. In einer Spezialambulanz wurden in Zusammenarbeit mit dem Deutschen Herzzentrum München 32 Patienten mit Verdacht auf MFS untersucht und betreut. Je nach Ausprägung der Symptome wurden die Patienten in drei Gruppen aufgeteilt. Bei ca. einem Drittel wurde die Diagnose sicher gestellt, bei einem weiteren Drittel konnte lediglich der dringende Verdacht geäußert werden. Die restlichen Patienten wiesen nur einzelne Merkmale auf. Bei der Betreuung der Patienten mit MFS bedürfen Patienten mit dringendem Verdacht auf MFS engmaschiger Kontrollen, um Komplikationen frühzeitig zu erfassen und adäquat zu behandeln. The Marfan syndrome (MFS) is a rare hereditary disease of the connective tissue with variable expressivity. Numerous organs can be affected. The diagnosis is made according to the nosology of Gent. 32 patients that were suspected to be affected by the Marfan syndrome were examined ambulatory in cooperation with the Deutschen Herzzentrum München. Depending on the grade of manifestations of the symptoms patients were divided into three groups. In approximately one third of the cases the diagnosis MFS could be confirmed, in one third of the patients it was only possible to speak of a strong suspicion. The remaining patients only showed single characteristics of the syndrome. In long-term follow-up of patients with Marfan syndrome close-meshed control examinations are needed especially for patients stronlgy suspected to be affected by the MFS to detect complications in time and to treat them appropriately.

Primary coenzyme Q10 (CoQ10) deficiencies are rare, clinically heterogeneous disorders caused by mutations in several genes encoding proteins involved in CoQ10 biosynthesis. CoQ10 is an essential component of the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. By whole-exome sequencing, we identified five individuals carrying biallelic mutations inCOQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex that contains most of the CoQ10 biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency, and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory-distress syndrome, and epileptic encephalopathy. The fifth subject had an early-onset but slowly progressive clinical course dominated by neurological deterioration with hardly any involvement of other organs. All available specimens from affected subjects showed reduced amounts of CoQ10 and often displayed a decrease in CoQ10-dependent ETC complex activities. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model; oxidative growth, strongly impaired in strains lacking COQ4, was corrected by expression of human wild-type COQ4 cDNA but failed to be corrected by expression of COQ4 cDNAs with any of the mutations identified in affected subjects. COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations and associated with CoQ10 deficiency.