Mitochondrial diseases are severe, inherited metabolic disorders that affect the paediatric population. They affect the functioning of mitochondrial oxidative phosphorylation (OXPHOS) apparatus either directly or indirectly. Since mutations in mtDNA are responsible for only 25 % of paediatric cases and next-generation sequencing does not always provide a conclusive diagnosis, the biochemical approach still represents a valuable tool in diagnostics. Mitochondrial defects can be identified in tissue biopsies (muscle or skin). However, they also often manifest in peripheral blood cells. We developed a protocol for isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) from 5 ml of children's blood using Ficoll centrifugation which can be utilised for subsequent functional measurements on thawed samples. Furthermore, we evaluated the diagnostic utility of the optimised high-resolution oxygraphy protocol using digitonin-permeabilized cryopreserved PBMCs on 47 samples from patients with confirmed or suspected mitochondrial disease. Overall, the diagnosis was confirmed in 72 % of cases, while the analysis of cryopreserved PBMCs provided a false negative outcome in 13 % of cases. Our study demonstrates a sensitive, fast, and non-invasive approach for the diagnostics of various types of mitochondrial disorders, especially those of nuclear genetic origin manifesting in paediatric patients.

• Klíčová slova
• Cryopreservation, Diagnostics, Glycolysis, Mitochondrial diseases, OXPHOS, Oxidative phosphorylation, PBMC, Peripheral blood mononuclear cells, Respirometry,
• MeSH
• buněčné dýchání MeSH
• dítě MeSH
• kojenec MeSH
• kryoprezervace * MeSH
• leukocyty mononukleární * metabolismus   MeSH
• lidé MeSH
• mitochondriální nemoci * diagnóza   genetika   metabolismus   MeSH
• mitochondrie * metabolismus   MeSH
• mladiství MeSH
• oxidativní fosforylace MeSH
• předškolní dítě MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Cardiac involvement (CI) in phosphomannomutase 2-congenital disorders of glycosylation (PMM2-CDG) is part of the multisystemic presentation contributing to high mortality rates. The most common cardiac manifestations are pericardial effusion, cardiomyopathy, and structural heart defects. A genotype-phenotype correlation with organ involvement has not yet been described. We analyzed clinical, biochemical, and molecular genetic data of 222 patients from eight European centers and characterized the natural course of patients with CI. Fifty-seven patients (45 children) presented with CI, of whom 24 died (median age 21 months, standard deviation 49.8). Pericardial effusion was the most frequent manifestation (55.4%), occurring mostly within the first 6 months of life. The most common pathogenic variants in patients with CI were p.(Arg141His) in 74%, followed by p.(Val231Met) in 36%, which is 3.5 times higher than in PMM2-CDG patients without CI (p < 0.0001). Twenty-one out of 36 patients with p.(Val231Met) had CI; among them, 15 died, compared to 33 out of 166 patients without p.(Val231Met) who had CI (p < 0.0001). Nine out of 33 patients died (p = 0.0015), indicating greater clinical severity. Furthermore, the p.(Val231Met) variant is predominant in Eastern Europe, suggesting a founder effect. Cardiac complications in PMM2-CDG patients are common and serious. The variant p.(Val231Met) profoundly influences the extent of CI and mortality rates. Therefore, we recommend cardiac surveillance be included in the follow-up protocols for PMM2-CDG.

• Klíčová slova
• CDG, PMM2, cardiac involvement, p.(Val231Met) genotype–phenotype correlations, pericardial effusion,
• MeSH
• dítě MeSH
• fenotyp * MeSH
• fosfotransferasy (fosfomutasy) * genetika   nedostatek   MeSH
• genetické asociační studie MeSH
• kardiomyopatie genetika   MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mutace MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• stupeň závažnosti nemoci MeSH
• vrozené poruchy glykosylace * genetika   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH
• Názvy látek
• fosfotransferasy (fosfomutasy) * MeSH
• phosphomannomutase 2, human MeSH Prohlížeč

BACKGROUND: Transport protein particle (TRAPP) is a multiprotein complex that functions in localising proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in diseases affecting muscle, brain, eye and to some extent liver. We present three patients who are compound heterozygotes for a missense variant and a structural variant in the TRAPPC11 gene. TRAPPC11 structural variants have not yet been described in association with a disease. In order to reveal the estimated genesis of identified structural variants, we performed sequencing of individual breakpoint junctions and analysed the extent of homology and the presence of repetitive elements in and around the breakpoints. METHODS: Biochemical methods including isoelectric focusing on serum transferrin and apolipoprotein C-III, as well as mitochondrial respiratory chain complex activity measurements, were used. Muscle biopsy samples underwent histochemical analysis. Next-generation sequencing was employed for identifying sequence variants associated with neuromuscular disorders, and Sanger sequencing was used to confirm findings. RESULTS: We suppose that non-homologous end joining is a possible mechanism of deletion origin in two patients and non-allelic homologous recombination in one patient. Analyses of mitochondrial function performed in patients' skeletal muscles revealed an imbalance of mitochondrial metabolism, which worsens with age and disease progression. CONCLUSION: Our results contribute to further knowledge in the field of neuromuscular diseases and mutational mechanisms. This knowledge is important for understanding the molecular nature of human diseases and allows us to improve strategies for identifying disease-causing mutations.

• Klíčová slova
• Neuromuscular Diseases,
• MeSH
• delece genu MeSH
• dítě MeSH
• dospělí MeSH
• kosterní svaly patologie   metabolismus   MeSH
• lidé MeSH
• missense mutace genetika   MeSH
• svalové dystrofie * genetika   patologie   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• TRAPPC11 protein, human MeSH Prohlížeč

• Klíčová slova
• Case report, Dystonia, Genetic disorder, Smith-Magenis syndrome,
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

Carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamylase (OTC) deficiencies are rare urea cycle disorders, which can lead to life-threatening hyperammonemia. Liver transplantation (LT) provides a cure and offers an alternative to medical treatment and life-long dietary restrictions with permanent impending risk of hyperammonemia. Nevertheless, in most patients, metabolic aberrations persist after LT, especially low plasma citrulline levels, with questionable clinical impact. So far, little is known about these alterations and there is no consensus, whether l-citrulline substitution after LT improves patients' symptoms and outcomes. In this multicentre, retrospective, observational study of 24 patients who underwent LT for CPS1 (n = 11) or OTC (n = 13) deficiency, 25% did not receive l-citrulline or arginine substitution. Correlation analysis revealed no correlation between substitution dosage and citrulline levels (CPS1, p = 0.8 and OTC, p = 1). Arginine levels after liver transplantation were normal after LT independent of citrulline substitution. Native liver survival had no impact on mental impairment (p = 0.67). Regression analysis showed no correlation between l-citrulline substitution and failure to thrive (p = 0.611) or neurological outcome (p = 0.701). Peak ammonia had a significant effect on mental impairment (p = 0.017). Peak plasma ammonia levels correlate with mental impairment after LT in CPS1 and OTC deficiency. Growth and intellectual impairment after LT are not significantly associated with l-citrulline substitution.

• Klíčová slova
• carbamoyl phosphate synthetase 1, citrulline, liver transplantation, ornithine transcarbamylase, substitution, urea cycle disorders,
• MeSH
• amoniak metabolismus   MeSH
• arginin terapeutické užití   MeSH
• citrulin MeSH
• hyperamonemie * farmakoterapie   MeSH
• karbamoylfosfát metabolismus   terapeutické užití   MeSH
• karbamoylfosfátsynthasa (amoniak) metabolismus   MeSH
• lidé MeSH
• nemoc z nedostatku ornithinkarbamoyltransferázy * chirurgie   MeSH
• ornithinkarbamoyltransferasa MeSH
• retrospektivní studie MeSH
• transplantace jater * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• pozorovací studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amoniak MeSH
• arginin MeSH
• citrulin MeSH
• karbamoylfosfát MeSH
• karbamoylfosfátsynthasa (amoniak) MeSH
• ornithinkarbamoyltransferasa MeSH

Limb girdle muscular dystrophies (LGMD) are a genetically heterogeneous group of muscular dystrophies. The study presents an overview of molecular characteristics of a large cohort of LGMD patients who are representative of the Czech LGMD population. We present 226 LGMD probands in which 433 mutant alleles carrying 157 different variants with a supposed pathogenic effect were identified. Fifty-four variants have been described only in the Czech LGMD population so far. LGMD R1 caplain3-related is the most frequent subtype of LGMD involving 53.1% of patients with genetically confirmed LGMD, followed by LGMD R9 FKRP-related (11.1%), and LGMD R12 anoctamin5-related (7.1%). If we consider identified variants, then all but five were small-scale variants. One large gene deletion was identified in the LAMA2 gene and two deletions in each of CAPN3 and SGCG. We performed comparison our result with other published studies. The results obtained in the Czech LGMD population clearly differ from the outcome of other LGMD populations in two aspects-we have a more significant proportion of patients with LGMD R1 calpain3-related and a smaller proportion of LGMD R2 dysferlin-related.

• Klíčová slova
• LGMD, genetic diagnostics, limb girdle muscular dystrophy, next generation sequencing,
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• tisková chyba MeSH

PMM2-CDG is the most common defect among the congenital disorders of glycosylation. In order to investigate the effect of hypoglycosylation on important cellular pathways, we performed extensive biochemical studies on skin fibroblasts of PMM2-CDG patients. Among others, acylcarnitines, amino acids, lysosomal proteins, organic acids and lipids were measured, which all revealed significant abnormalities. There was an increased expression of acylcarnitines and amino acids associated with increased amounts of calnexin, calreticulin and protein-disulfid-isomerase in combination with intensified amounts of ubiquitinylated proteins. Lysosomal enzyme activities were widely decreased as well as citrate and pyruvate levels indicating mitochondrial dysfunction. Main lipid classes such as phosphatidylethanolamine, cholesterol or alkyl-phosphatidylcholine, as well as minor lipid species like hexosylceramide, lysophosphatidylcholines or phosphatidylglycerol, were abnormal. Biotinidase and catalase activities were severely reduced. In this study we discuss the impact of metabolite abnormalities on the phenotype of PMM2-CDG. In addition, based on our data we propose new and easy-to-implement therapeutic approaches for PMM2-CDG patients.

• Klíčová slova
• CDG-Ia, Congenital disorders of glycosylation, Fibroblasts, OMICS, PMM2-CDG, Therapeutic ideas,
• MeSH
• aminokyseliny metabolismus   MeSH
• fosfotransferasy (fosfomutasy) * genetika   nedostatek   MeSH
• glykosylace MeSH
• lidé MeSH
• lipidy MeSH
• vrozené poruchy glykosylace * genetika   terapie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• fosfotransferasy (fosfomutasy) * MeSH
• lipidy MeSH

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic.

• Klíčová slova
• ACMG, Dopamine, Neurotransmitter disorder, Serotonin, Variant effect prediction,
• MeSH
• aminokyseliny genetika   MeSH
• dekarboxylasy aromatických L-aminokyselin * nedostatek   MeSH
• dopamin metabolismus   MeSH
• genotyp MeSH
• lidé MeSH
• prevalence MeSH
• vrozené poruchy metabolismu aminokyselin * epidemiologie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• DDC protein, human MeSH Prohlížeč
• dekarboxylasy aromatických L-aminokyselin * MeSH
• dopamin MeSH

Leber hereditary optic neuropathy is a primary mitochondrial disease characterized by acute visual loss due to the degeneration of retinal ganglion cells. In this study, we describe a patient carrying a rare missense heteroplasmic variant in MT-ND1, NC_012920.1:m.4135T>C (p.Tyr277His) manifesting with a typical bilateral painless decrease of the visual function, triggered by physical exercise or higher ambient temperature. Functional studies in muscle and fibroblasts show that amino acid substitution Tyr277 with His leads to only a negligibly decreased level of respiratory chain complex I (CI), but the formation of supercomplexes and the activity of the enzyme are disturbed noticeably. Our data indicate that although CI is successfully assembled in the patient's mitochondria, its function is hampered by the m.4135T>C variant, probably by stabilizing CI in its inactive form. We conclude that the m.4135T>C variant together with a combination of external factors is necessary to manifest the phenotype.

• Klíčová slova
• complex I, mitochondria, mtDNA, optic neuropathy, supercomplexes,
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH