Development of a human mitochondrial oligonucleotide microarray (h-MitoArray) and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

<p>Abstract</p> <p>Background</p> <p>To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray) allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F₁F_oATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes.</p> <p>Results</p> <p>Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known <it>ATP6/ATP8 </it>defect in patients from the M group and indicated several candidate disease causing genes for nuclear defects.</p> <p>Conclusion</p> <p>Our analysis showed that deficiency in the ATP synthase protein complex amount is generally accompanied by only minor changes in expression of ATP synthase related genes. It also suggested that the site (mtDNA vs nuclear DNA) and the severity (ATP synthase content) of the underlying defect have diverse effects on cellular gene expression phenotypes, which warrants further investigation of cell cycle regulatory and signal transduction pathways in other OXPHOS disorders and related pharmacological models.</p

Mutation of Nogo-B Receptor, a Subunit of cis-Prenyltransferase, Causes a Congenital Disorder of Glycosylation

SummaryDolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice, and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provide a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation

POLR3B-associated leukodystrophy: clinical, neuroimaging and molecular-genetic analyses in four patients: clinical heterogeneity and novel mutations in POLR3B gene

Introduction and aim of the study. White matter disorders represent a spectrum of neurological diseases frequently associated with an unfavourable prognosis and a delay in diagnostics. We report the broad phenotypic spectrum of a rare hypomyelinating leukodystrophy and three novel mutations. Further, we aim to explore the role of the combined clinical and neuroimaging diagnostic approach in the era of whole exome sequencing. Materials and methods. We present a clinical, neuroimaging and molecular-genetic characterisation of four patients from three families suffering from a rare genetic leukoencephalopathy. Two severely affected siblings (P1, P2) manifested a profound developmental delay, cerebellar symptomatology, microcephaly, failure to thrive, short stature and delayed teeth eruption with oligodontia. The other two patients (P3, P4), on the contrary, suffer from substantially less serious impairment with mild to moderate developmental delay and cerebellar symptomatology, delayed teeth eruption, or well-manageable epilepsy. In all four patients, magnetic resonance revealed cerebellar atrophy and supratentorial hypomyelination with T2-weight hypointensities in the areas of the ventrolateral thalamic nuclei, corticospinal tract and the dentate nuclei. Results. Using whole-exome sequencing in P1, P2 and P3, and targeted sequencing in P4, pathogenic variants were disclosed in POLR3B, a gene encoding one of 17 subunits of DNA-dependent RNA polymerase III — all patients were compound heterozygotes for point mutations. Three novel mutations c.727A&gt;G (p.Met243Val) and c.2669G&gt;A (p.Arg890His) (P1, P2), and c.1495G&gt;A (p.Met499Val) (P3) were found. Magnetic resonance revealed the characteristic radiological pattern of POLR3-leukodystrophies in our patients. Conclusion and clinical implications. The diagnosis of POLR3-associated leukodystrophies can be significantly accelerated using the combined clinical and neuroradiological recognition pattern. Therefore, it is of crucial importance to raise the awareness of this rare disorder among clinicians. Molecular-genetic analyses are indispensable for a swift diagnosis confirmation in cases of clear clinical suspicion, and for diagnostic search in patients with less pronounced symptomatology. They represent an invaluable tool for unravelling the complex genetic background of heritable white matter disorders

A Novel MTTK Gene Variant m.8315A&gt;C as a Cause of MERRF Syndrome

In this study, we report on a novel heteroplasmic pathogenic variant in mitochondrial DNA (mtDNA). The studied patient had myoclonus, epilepsy, muscle weakness, and hearing impairment and harbored a heteroplasmic m.8315A&gt;C variant in the MTTK gene with a mutation load ranging from 71% to &gt;96% in tested tissues. In muscle mitochondria, markedly decreased activities of respiratory chain complex I + III and complex IV were observed together with mildly reduced amounts of complex I and complex V (with the detection of V*- and free F1-subcomplexes) and a diminished level of complex IV holoenzyme. This pattern was previously seen in other MTTK pathogenic variants. The novel variant was not present in internal and publicly available control databases. Our report further expands the spectrum of MTTK variants associated with mitochondrial encephalopathies in adults

Isoelectric Focusing of Serum Apolipoprotein C-III as a Sensitive Screening Method for the Detection of O-glycosylation Disturbances

Apolipoprotein C-III (ApoC-III) is a glycoprotein carrying the most common O-linked glycan structure and is abundantly present in serum, what renders it a suitable marker for analysis of O-glycosylation abnormalities. Isoelectric focusing followed by a Western blot of ApoC-III, using PhastSystem™ Electrophoresis System (GE Healthcare), was introduced as a rather simple and rapid method for screening of certain subtypes of inherited glycosylation disorders. The study’s aim was to establish this method in our laboratory, what included performing the analysis in a group of 170 healthy individuals to set the reference range of detected relative amounts of sialylated ApoC-III isoforms and to evaluate the gender- and age-dependent differences. A significant relative increase of asialo-ApoC-III with growing age was found. Secondly, we examined serum from patients with selected metabolic disorders and detected minor O-glycosylation changes in diseases such as Prader-Willi syndrome, PGM1 (phosphoglucomutase 1) or MAN1B (class 1B alpha-1,2-mannosidase) deficiency. Our results show that this method allows for a sensitive detection of ApoC-III O-glycosylation status, however this might be modulated by several factors (i.e. nutrition, medication) whose exact role remains to be determined

Noninvasive diagnostics of mitochondrial disorders in isolated lymphocytes with high resolution respirometry

Background: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts. Methods: To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue Native electrophoreses. Results: Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations. Conclusion: Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, IV and V manifesting in peripheral blood lymphocytes. General significance: The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients

Case report: A rare variant m.4135T>C in the MT-ND1 gene leads to Leber hereditary optic neuropathy and altered respiratory chain supercomplexes

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&gt;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&gt;C variant, probably by stabilizing CI in its inactive form. We conclude that the m.4135T&gt;C variant together with a combination of external factors is necessary to manifest the phenotype

Generation of two human iPSC lines from dermal fibroblasts of adult- and juvenile-onset Huntington's disease patients and two healthy donors

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a mutation in the HTT gene. To generate human-induced pluripotent stem cells (hiPSCs), we used dermal fibroblasts from 1 healthy adult control (K-Pic2), 1 HD manifest patient (M-T2), 1 healthy juvenile control (jK-N1), and 1 juvenile HD patient (jHD-V1). HD stage of patients was assessed by neurological tests and donors were without comorbidities and were non-smokers. Characterization showed that the obtained hiPSCs have the same number of CAG repeats as the parental fibroblast lines, express pluripotency markers and have the ability to differentiate into all 3 germ layers

Deterioration of mitochondrial bioenergetics and ultrastructure impairment in skeletal muscle of a transgenic minipig model in the early stages of Huntington's disease

Skeletal muscle wasting and atrophy is one of the more severe clinical impairments resulting from the progression of Huntington's disease (HD). Mitochondrial dysfunction may play a significant role in the etiology of HD, but the specific condition of mitochondria in muscle has not been widely studied during the development of HD. To determine the role of mitochondria in skeletal muscle during the early stages of HD, we analyzed quadriceps femoris muscle from 24-, 36-, 48- and 66-month-old transgenic minipigs that expressed the N-terminal portion of mutated human huntingtin protein (TgHD) and age-matched wild-type (WT) siblings. We found altered ultrastructure of TgHD muscle tissue and mitochondria. There was also significant reduction of activity of citrate synthase and respiratory chain complexes (RCCs) I, II and IV, decreased quantity of oligomycin-sensitivity conferring protein (OSCP) and the E2 subunit of pyruvate dehydrogenase (PDHE2), and differential expression of optic atrophy 1 protein (OPA1) and dynamin-related protein 1 (DRP1) in the skeletal muscle of TgHD minipigs. Statistical analysis identified several parameters that were dependent only on HD status and could therefore be used as potential biomarkers of disease progression. In particular, the reduction of biomarker RCCII subunit SDH30 quantity suggests that similar pathogenic mechanisms underlie disease progression in TgHD minipigs and HD patients. The perturbed biochemical phenotype was detectable in TgHD minipigs prior to the development of ultrastructural changes and locomotor impairment, which become evident at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression in skeletal muscle in HD, which is in concordance with the mobility problems observed in this model