Regionalized Pathology Correlates with Augmentation of mtDNA Copy Numbers in a Patient with Myoclonic Epilepsy with Ragged-Red Fibers (MERRF-Syndrome)

Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89–100%. However, mtDNA copy numbers were increased 3–7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that “futile” stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome

Monogenic variants in dystonia: an exome-wide sequencing study

Background Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia. Methods For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow. Findings We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222;excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism. Interpretation In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations

Entwicklung eines transgenen Tiermodells für die Messung von intrazellulärem ATP

Mitochondriopathies are a group of clinically heterogeneous, mostly hereditary multisystemic disorders based on a deficient mitochondrial supply of ATP. Tissues with high energy demand, such as the brain or muscles, are predominantly affected. Symptoms include epileptic seizures, ataxia or muscle weakness. The underlying pathomechanisms are still not completely understood. In this context, the measurement of compartment-specific ATP levels represents an interesting approach. My doctoral thesis deals with the question whether the detection and quantification of intra-mitochondrial ATP in situ by means of the luciferase reaction would be possible. To explore this, I designed a gene construct containing the luciferase gene plus a mitochondrial targeting sequence and the antigenic fusion peptide FLAG™-tag. Additionally, I added the Shine-Dalgarno sequence, necessary for translation in prokaryotes, and the Kozak consensus sequence including the eukaryotic start codon. The gene sequence of interest was inserted into the plasmid pENTR1A_DS. This vector is part of the Gateway® Recombination System, a cloning tool based on the site- specific recombination pathway of the bacteriophage lambda being independent of appropriate restriction enzyme sites. This considerably facilitates cloning of fragments into different vector systems. I generated a vector for mammalian cell lines, (pcDNA3.2/V5_cox8a-flag-luc) and a targeting vector designed to insert the construct into the ROSA26 locus of the mouse (pROSA26_cox8a-flag- luc). I verified that transfection of HEK293 cells with the pcDNA3.2/V5_cox8a- flag-luc vector resulted in protein translation of a protein of about 70 kDa. By means of immunofluorescence, I identified the localization of the protein as intra-mitochondrial. I was able to confirm the functionality of the luciferase protein in transfected HEK293 and COS1 cells by means of a luciferin-luciferase assay. A luminometer detects the light emission in relative light units. Increasing amounts of the substrate luciferin led to a rise in light intensity. The second vector (pROSA26_cox8a-flag-luc) was constructed for the generation of a knock-in animal model with constitutive intra-mitochondrial luciferase expression. The transgenic mouse model will serve in future studies to bring forward the understanding of the pathomechanisms of mitochondriopathies.Mitochondriopathien sind eine Gruppe klinisch heterogener, meist genetisch bedingter Multi-System-Erkrankungen, die auf einer eingeschränkten mitochondrialen Adenosintriphosphat (ATP)-Bereitstellung beruhen. Gewebe mit hohem Energiebedarf wie das Gehirn oder die Skelettmuskulatur sind vorrangig betroffen, Symptome wie epileptische Anfälle, Ataxie und Muskelschwäche sind häufig. Die zu Grunde liegenden Pathomechanismen sind bei weitem nicht vollständig verstanden. In diesem Kontext wäre die Messung kompartiment- spezifischer ATP-Spiegel ein interessanter Ansatz. Die vorliegende Dissertation behandelt die Fragestellung, ob die Erfassung und Quantifizierung des intramitochondrialen ATPs in situ mittels der Luciferase-Reaktion möglich ist. Dafür habe ich ein Genkonstrukt entworfen, welches aus dem Luciferase-Gen mit einer mitochondrialen Ziel-Sequenz besteht. Zusätzlich enthält das Konstrukt die kodierende Sequenz für das FLAG™-tag Peptid, welches den Nachweis des Konstruktes mittels eines spezifischen Antikörpers verbessert. Ausserdem fügte ich die Shine-Dalgarno-Sequenz, die für die optimale Translation in Prokaryoten erforderlich ist, und die Kozak-Sequenz, die das eukaryotische Startcodon beinhaltet, ein. Die Gensequenz wurde in das Plasmid pENTR1A_DS eingebracht. Dieser Vektor ist Teil des Gateway® Recombination Systems, einer Klonierungs-Methode, die sich des Mechanismus der ortsspezifischen Rekombination des Bakteriophagen lambda bedient. Dadurch ist die Klonierung des interessierenden Gens in verschiedene Vektor-Systeme deutlich einfacher. Ich erstellte sowohl einen Vektor zur Transfektion von Säugetier-Zellkulturen (pcDNA3.2/V5_cox8a-flag-luc), als auch einen Vektor zur Insertion des Konstruktes in den ROSA26-Locus muriner embryonaler Stammzellen (pROSA26_cox8a-flag-luc). Die Ergebnisse zeigen, dass die Transfektion von HEK293-Zellen mit pcDNA3.2/V5_cox8a-flag-luc zu einer Genexpression eines Proteins der Größe 70 kDa führt. Mittels Immunfluoreszenz konnte ich das Protein intra-mitochondrial lokalisieren. Die Funktionsfähigkeit des Proteins, welches als das Enzym Luciferase unter Anwesenheit von Luciferin und ATP Licht emittiert, konnte ich in transfizierten HEK293- und COS1-Zellen mittels des Luciferase-Assays nachweisen. Ein Luminometer erfasste die Licht-Emission in relativen Licht-Einheiten. Die Zufuhr steigender Mengen an Substrat (Luciferin) führte zu einer Erhöhung der Licht-Intensität. Ich entwickelte den zweiten Vektor, pROSA26_cox8a-flag-luc, für die Generierung eines knock-in Tiermodells mit konstitutiver, intra-mitochondrialer Luciferase-Expression. Das transgene Maus-Modell kann zum besseren Verständnis der Pathomechanismen von Mitochondriopathien beitragen

Cystic fibrosis in disguise – the wolf in sheep’s clothing, a case report

Background!#!Childhood hypoglycemia in combination with hepatomegaly is suspicious for inborn errors of metabolism. Cystic fibrosis typically presents with failure to thrive, pulmonary and gastrointestinal symptoms. Hepatic involvement and hypoglycemia can occur in a significant number of patients, although hepatomegaly is uncommon.!##!Case presentation!#!A 28 months old boy was presented with recurrent upper airways infections, progressive lethargy and weight loss. Clinically hepatomegaly was the main presenting feature and hypoglycemia (minimum 1.4 mmol/l) was noted as were elevated transaminases. The patient did not produce enough sweat to analyze it. Infectious causes for hepatitis were excluded and a broad metabolic work-up initiated. A therapy with starch was initiated to control hypoglycemia. In further course loose stools were reported and pancreatic elastase was found to be reduced. A further sweat test yielded pathological chloride concentration and genetic testing confirmed the diagnosis of cystic fibrosis.!##!Conclusions!#!Cystic fibrosis is a systemic disease and less common presentations need to be considered. Even in the age of CF-newborn screening in many countries CF needs to be ruled out in typical and atypical clinical presentations and diagnostics need to be repeated if inconclusive

Variants in Mitochondrial ATP Synthase Cause Variable Neurologic Phenotypes

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.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

PORTEC-4a: international randomized trial of molecular profile-based adjuvant treatment for women with high-intermediate risk endometrial cancer

Background Vaginal brachytherapy is currently recommended as adjuvant treatment in patients with high-intermediate risk endometrial cancer to maximize local control and has only mild side effects and no or limited impact on quality of life. However, there is still considerable overtreatment and also some undertreatment, which may be reduced by tailoring adjuvant treatment to the patients' risk of recurrence based on molecular tumor characteristics. Primary objectives To compare the rates of vaginal recurrence in women with high-intermediate risk endometrial cancer, treated after surgery with molecular-integrated risk profile-based recommendations for either observation, vaginal brachytherapy or external pelvic beam radiotherapy or with standard adjuvant vaginal brachytherapy Study hypothesis Adjuvant treatment based on a molecular-integrated risk profile provides similar local control and recurrence-free survival as current standard adjuvant brachytherapy in patients with high-intermediate risk endometrial cancer, while sparing many patients the morbidity of adjuvant treatment and reducing healthcare costs. Trial design A multicenter, international phase III randomized trial (2:1) of molecular-integrated risk profile-based adjuvant treatment (experimental arm) or adjuvant vaginal brachytherapy (standard arm). Major inclusion/exclusion criteria Women aged 18 years and over with a histological diagnosis of high-intermediate risk endometrioid endometrial cancer after total abdominal or laparoscopic hysterectomy and bilateral salpingo-oophorectomy. High-intermediate risk factors are defined as: (i) International Federation of Gynecology and Obstetrics stage IA (with invasion) and grade 3; (ii) stage IB grade 1 or 2 with age >= 60 and/or lymph-vascular space invasion; (iii) stage IB, grade 3 without lymph-vascular space invasion; or (iv) stage II (microscopic and grade 1). Endpoints The primary endpoint is vaginal recurrence. Secondary endpoints are recurrence-free and overall survival; pelvic and distant recurrence; 5-year vaginal control (including treatment for relapse); adverse events and patient-reported symptoms and quality of life; and endometrial cancer-related healthcare costs. Sample size 500 eligible and evaluable patients. Estimated dates for completing accrual and presenting results Estimated date for completing accrual will be late 2021. Estimated date for presentation of (first) results is expected in 2023

PORTEC-4a: international randomized trial of molecular profile-based adjuvant treatment for women with high-intermediate risk endometrial cancer

BACKGROUND: Vaginal brachytherapy is currently recommended as adjuvant treatment in patients with high-intermediate risk endometrial cancer to maximize local control and has only mild side effects and no or limited impact on quality of life. However, there is still considerable overtreatment and also some undertreatment, which may be reduced by tailoring adjuvant treatment to the patients' risk of recurrence based on molecular tumor characteristics. PRIMARY OBJECTIVES: To compare the rates of vaginal recurrence in women with high-intermediate risk endometrial cancer, treated after surgery with molecular-integrated risk profile-based recommendations for either observation, vaginal brachytherapy or external pelvic beam radiotherapy or with standard adjuvant vaginal brachytherapy STUDY HYPOTHESIS: Adjuvant treatment based on a molecular-integrated risk profile provides similar local control and recurrence-free survival as current standard adjuvant brachytherapy in patients with high-intermediate risk endometrial cancer, while sparing many patients the morbidity of adjuvant treatment and reducing healthcare costs. TRIAL DESIGN: A multicenter, international phase III randomized trial (2:1) of molecular-integrated risk profile-based adjuvant treatment (experimental arm) or adjuvant vaginal brachytherapy (standard arm). MAJOR INCLUSION/EXCLUSION CRITERIA: Women aged 18 years and over with a histological diagnosis of high-intermediate risk endometrioid endometrial cancer after total abdominal or laparoscopic hysterectomy and bilateral salpingo-oophorectomy. High-intermediate risk factors are defined as: (i) International Federation of Gynecology and Obstetrics stage IA (with invasion) and grade 3; (ii) stage IB grade 1 or 2 with age ≥60 and/or lymph-vascular space invasion; (iii) stage IB, grade 3 without lymph-vascular space invasion; or (iv) stage II (microscopic and grade 1). ENDPOINTS: The primary endpoint is vaginal recurrence. Secondary endpoints are recurrence-free and overall survival; pelvic and distant recurrence; 5-year vaginal control (including treatment for relapse); adverse events and patient-reported symptoms and quality of life; and endometrial cancer-related healthcare costs. SAMPLE SIZE: 500 eligible and evaluable patients. ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: Estimated date for completing accrual will be late 2021. Estimated date for presentation of (first) results is expected in 2023. TRIAL REGISTRATION: The trial is registered at clinicaltrials.gov (NCT03469674) and ISRCTN (11659025)

Monogenic variants in dystonia: an exome-wide sequencing study

Background Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia. Methods For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow. Findings We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222; excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism. Interpretation In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations