Rhabdomyolysis: a genetic perspective

Rhabdomyolysis (RM) is a clinical emergency characterized by fulminant skeletal muscle damage and release of intracellular muscle components into the blood stream leading to myoglobinuria and, in severe cases, acute renal failure. Apart from trauma, a wide range of causes have been reported including drug abuse and infections. Underlying genetic disorders are also a cause of RM and can often pose a diagnostic challenge, considering their marked heterogeneity and comparative rarity. In this paper we review the range of rare genetic defects known to be associated with RM. Each gene has been reviewed for the following: clinical phenotype, typical triggers for RM and recommended diagnostic approach. The purpose of this review is to highlight the most important features associated with specific genetic defects in order to aid the diagnosis of patients presenting with hereditary causes of recurrent RM.</p

Rhabdomyolysis: a genetic perspective

Rhabdomyolysis (RM) is a clinical emergency characterized by fulminant skeletal muscle damage and release of intracellular muscle components into the blood stream leading to myoglobinuria and, in severe cases, acute renal failure. Apart from trauma, a wide range of causes have been reported including drug abuse and infections. Underlying genetic disorders are also a cause of RM and can often pose a diagnostic challenge, considering their marked heterogeneity and comparative rarity.In this paper we review the range of rare genetic defects known to be associated with RM. Each gene has been reviewed for the following: clinical phenotype, typical triggers for RM and recommended diagnostic approach. The purpose of this review is to highlight the most important features associated with specific genetic defects in order to aid the diagnosis of patients presenting with hereditary causes of recurrent RM

Genetic defects are common in myopathies with tubular aggregates

Objective: A group of genes have been reported to be associated with myopathies with tubular aggregates (TAs). Many cases with TAs still lack of genetic clarification. This study aims to explore the genetic background of cases with TAs in order to improve our knowledge of the pathogenesis of these rare pathological structures. Methods: Thirty-three patients including two family members with biopsy confirmed TAs were collected. Whole-exome sequencing was performed on 31 unrelated index patients and a candidate gene search strategy was conducted. The identified variants were confirmed by Sanger sequencing. The wild-type and the mutant p.Ala11Thr of ALG14 were transfected into human embryonic kidney 293 cells (HEK293), and western blot analysis was performed to quantify protein expression levels. Results: Eleven index cases (33%) were found to have pathogenic variant or likely pathogenic variants in STIM1, ORAI1, PGAM2, SCN4A, CASQ1 and ALG14. Among them, the c.764A>T (p.Glu255Val) in STIM1 and the c.1333G>C (p.Val445Leu) in SCN4A were novel. Western blot analysis showed that the expression of ALG14 protein was severely reduced in the mutant ALG14 HEK293 cells (p.Ala11Thr) compared with wild type. The ALG14 variants might be associated with TAs in patients with complex multisystem disorders. Interpretation: This study expands the phenotypic and genotypic spectrums of myopathies with TAs. Our findings further confirm previous hypothesis that genes related with calcium signalling pathway and N-linked glycosylation pathway are the main genetic causes of myopathies with TAs

Genetic Investigations of Sporadic Inclusion Body Myositis and Myopathies with Structural Abnormalities and Protein Aggregates in Muscle

The application of whole-exome sequencing (WES) has not only dramatically accelerated the discovery of pathogenic genes of Mendelian diseases, but has also shown promising findings in complex diseases. This thesis focuses on exploring genetic risk factors for a large series of sporadic inclusion body myositis (sIBM) cases, and identifying disease-causing genes for several groups of patients with abnormal structure and/or protein aggregates in muscle. Both conventional and advanced techniques were applied. Based on the International IBM Genetics Consortium (IIBMGC), the largest sIBM cohort of blood and muscle tissue for DNA analysis was collected as the initial part of this thesis. Candidate gene studies were carried out and revealed a disease modifying effect of an intronic polymorphism in TOMM40, enhanced by the APOE ε3/ε3 genotype. Rare variants in SQSTM1 and VCP genes were identified in seven of 181 patients, indicating a mutational overlap with neurodegenerative diseases. Subsequently, a first whole-exome association study was performed on 181 sIBM patients and 510 controls. This reported statistical significance of several common variants located on chromosome 6p21, a region encompassing genes related to inflammation/infection. WES was performed on a group of 35 cases with tubular aggregates/cylindrical spirals, and detected rare variants in known/candidate genes. Disease-causing genes were identified in four families with protein aggregates in muscle also by WES. In one family identified with a novel homozygous deletion in SBF1 with a rare autosomal-recessive neuromuscular condition, functional analysis was carried out indicating a loss-of-function mechanism underlying the pathogenesis of the disease. The collection of a large series of sIBM patients through the IIBMGC has been shown here to reveal important genetic findings and will be a valuable resource for the future. WES proved to be important in sIBM and also to be an efficient method to investigate the genetics basis of rare complex muscle disorders

Application of Proteomics to Human Hypertrophic Cardiomyopathy

This work describes a preliminary study to evaluate the use of proteomics in the study of human hypertrophic cardiomyopathy. Both gel and mass spectrometry techniques were used for the identification and analysis of myocardial proteins in whole tissue lysate. Qualitative and quantitative proteomic methods were used to understand disease mechanisms and identify and validate novel biomarkers. Disease caused by mutations in beta myosin heavy chain, MYH7 and myosin binding protein-C, MYBPC3 were studied alongside patients where no genetic variant could be identified. Left ventricular septal myectomy samples showed changes in protein expression compared with control tissue. Novel mutations in MYH7 were confirmed by identification of mutant peptide sequences. Disease mechanisms were investigated by studying interactions between up- and down-regulated proteins involved in various pathways. Enriched protein groups included those involved in cytoskeletal protein binding and energy production. Novel findings included the identification of carbonic anhydrase III in cardiomyocytes. A targeted and multiplexed MRM-MS assay was developed to validate potential biomarkers in tissue and correlated with clinical phenotype. The assay was further applied to screen these biomarkers in urine. Novel findings included increased expression of lumican, a small leucine-rich proteoglycan that controls the assembly of collagen fibres in the extracellular matrix. Lumican concentration was highest in a sub-group of patients with evidence of scarring on cardiac magnetic resonance imaging, making it a potential marker of progressive disease. This is the first comprehensive global proteomic study of human hypertrophic cardiomyopathy. It has identified differences in the expression of several proteins in the myocardium not described previously, but highly relevant to pathophysiology of this disease. A targeted proteomic translational assay, capable of quantitating 35 peptides in less than 10 minutes has been developed

Διερεύνηση με Αλληλούχηση Επόμενης Γενιάς ασθενών με επαναλαμβανόμενα επεισόδια ραβδομυόλυσης

Η Ραβδομυόλυση αποτελεί μια σοβαρή διαταραχή που οφείλεται σε άμεσο ή έμμεσο τραυματισμό των μυών με αποτέλεσμα την καταστροφή των μυϊκών κυττάρων και την απελευθέρωση ενδοκυτταρικών πρωτεϊνών, όπως η μυοσφαιρίνη, αλλά και ηλεκτρολυτών στην κυκλοφορία. Οι εκφάνσεις της διαταραχής αυτής μπορεί να κυμαίνονται από ήπιες, όπως μυϊκό άλγος και κράμπες έως και επικίνδυνες για την ζωή όπως μυοσφαιρινουρία, οξεία νεφρική ανεπάρκεια (AKI), σοβαρές ηλεκτρολυτικές διαταραχές, καρδιολογικά προβλήματα ακόμη και θάνατος. Την χαρακτηριστική τριάδα των συμπτωμάτων της ραβδομυόλυσης αποτελούν, τα υψηλά επίπεδα CK, τα σκουρόχρωμα ούρα και η μυαλγία/μυϊκή αδυναμία. Οι παράγοντες που είναι υπεύθυνοι για την πρόκληση ραβδομυόλυσης μπορεί να είναι γενετικοί, για παράδειγμα παραλλαγές σε γονίδια όπως η κοινή παθογόνος παραλλαγή p.Ser113Leu του γονιδίου CPT2, είτε να σχετίζονται με τραυματισμούς, κατάχρηση αλκοόλ, λήψη φαρμάκων και παράνομων ουσιών, λοιμώξεις, τοξίνες και έντονη μυϊκή δραστηριότητα. Η θεραπευτική αντιμετώπιση της ραβδομυόλυσης περιλαμβάνει τρεις προσεγγίσεις: την ενδοφλέβια αποκατάσταση των υγρών (IV Fluid Therapy), την χορήγηση διουρητικών ή/και διττανθρακικού και την αντιμετώπιση των ηλεκτρολυτικών ανισορροπιών. Στην παρούσα διπλωματική εργασία αναλύθηκαν με τη βοήθεια των βιοπληροφορικών εργαλείων Varsome Clinical, Franklin by Genoox και VarAFT τα αποτελέσματα Whole Exome Sequencing (WES) από 8 ασθενείς με συμπτώματα ραβδομυόλυσης. Επίσης, διερευνήθηκαν με αλληλούχηση Sanger 40 ασθενείς ομόζυγοι για την κοινή παραλλαγή p.Ser113Leu του γονιδίου CPT2 προκειμένου να εντοπιστεί η πιθανή παρουσία της παραλλαγής p.Glu43Lys του γονιδίου ACADM και να μελετηθεί η επίπτωσή της στο φαινότυπο συνδυαστικά με την παρουσία παραλλαγών στο CPT2 ως τροποποιητικός γενετικός παράγοντας. Στους περισσότερους ασθενείς, από την μελέτη των αιτιών παραπομπής και των γενετικών αποτελεσμάτων δεν προέκυψε σαφές συμπέρασμα που να αιτιολογεί την κλινική τους εικόνα καθώς οι παραλλαγές που βρέθηκαν στα γονίδια δεν ήταν ιδιαίτερα πληροφοριακές. Εξαίρεση αποτελεί ο ασθενής P1, ο οποίος βρέθηκε ομόζυγος για την παθογόνο παραλλαγή c.338C&gt;T του γονιδίου CPT2 και ο οποίος διαγνώστηκε με CPTII ανεπάρκεια. Τα αποτελέσματα της αλληλούχησης Sanger στους 40 ασθενείς, έδειξαν ότι κανένας από τους ασθενείς δεν έφερε την παραλλαγή c.127G&gt;A του γονιδίου ACADM που αναζητούσαμε. Συνεπώς, δεν μπορέσαμε να καταλήξουμε σε σαφή συμπεράσματα σχετικά με το ποια είναι η πιθανή αιτία που ευθύνεται για την κλινική εικόνα των ασθενών με τα επαναλαμβανόμενα επεισόδια ραβδομυόλυσης αλλά ούτε σχετικά με το ποια είναι η πιθανή επίπτωση της παραλλαγής c. 127G&gt;A του γονιδίου ACADM στο φαινότυπο ασθενών με ραβδομυόλυση ομόζυγων για την παθογόνο παραλλαγή στο CPT2.Rhabdomyolysis is a serious disorder due to direct or indirect muscle injury resulting in the destruction of muscle cells and the release of intracellular proteins, such as myoglobin, and electrolytes into the circulation. The manifestations of this disorder can range from mild, such as muscle pain and cramps, to life-threatening such as myoglobinuria, acute kidney injury (AKI), severe electrolyte imbalances, cardiac problems, and even death. The typical triad of symptoms of rhabdomyolysis is high CK levels, dark-colored urine, and myalgia/muscle weakness. The factors responsible for causing rhabdomyolysis may be genetic, for example, variants in genes such as the common pathogenic variant p. Ser113Leu of the CPT2 gene, or related to injury, alcohol abuse, drug and substance abuse, infections, toxins, and intense muscle activity. The therapeutic management of rhabdomyolysis involves three approaches: intravenous fluid restoration (IV Fluid Therapy), administration of diuretics and/or bicarbonate, and management of electrolyte imbalances. In this thesis, Whole Exome Sequencing (WES) results from 8 patients with symptoms of rhabdomyolysis were analyzed using the bioinformatics tools Varsome Clinical, Franklin by Genoox, and VarAFT. In addition, 40 homozygous patients were investigated by Sanger sequencing for the common p. Ser113Leu variant of the CPT2 gene in order to identify the possible presence of the p. Glu43Lys variant of the ACADM gene and to study its impact on the phenotype in combination with the presence of CPT2 variants as a modifying genetic factor. In most patients, the study of the referral causes and genetic results did not yield a clear conclusion to explain their clinical picture as the variants found in the genes were not very informative. An exception was patient P1, who was found homozygous for the pathogenic c.338C&gt;T variant of the CPT2 gene and who was diagnosed with CPTII deficiency. The results of Sanger sequencing in the 40 patients showed that none of the patients carried the c.127G&gt;A variant of the ACADM gene that we were looking for. Therefore, we could not come to clear conclusions about the possible cause of the clinical picture of patients with recurrent episodes of rhabdomyolysis, nor about the possible impact of the c. 127G&gt;A variant of the ACADM gene on the phenotype of patients with rhabdomyolysis homozygous for the pathogenic variant in CPT2

Energy and carbon metabolism during the awakening from metabolic dormancy in cyanobacteria

Bacterial dormancy plays a crucial role in the survival and spread of bacterial populations. The capability of resuming growth after a dormant period allows bacterial cells to survive in an ever-changing environment. Cyanobacteria represent a diverse group of prokaryotes with an exceptional ability to adapt to different environmental conditions. One of the most common challenges cyanobacteria face in nature is nitrogen limitation. When the unicellular cyanobacterium Synechocystis sp. PCC 6803 lacks a source of combined nitrogen, cells undergo a metabolic adaptation that leads to a dormant state that allows them to survive these conditions for a prolonged period of time. This adaptation follows a genetically determined program and involves the degradation of most of the thylakoid membranes and the synthesis of glycogen stores. In the quiescent state, proper control of energy homeostasis and glycogen metabolism are essential for survival. In the present study, the regulation of the energy and carbon metabolism during nitrogen starvation was investigated. Dormant cells were shown to rely on a different mechanism of ATP synthesis than vegetative cells. During vegetative growth most of the cellular ATP is produced by the ATP synthases in a reaction that requires an electrochemical proton gradient across the thylakoid membrane, which is generated by photosynthetic or respiratory electron transport. In nitrogen-starved cells, the number of thylakoid membranes is very reduced, which implies a reduced capacity of thylakoidal ATP synthesis. Under these circumstances, cells rely on the ATP synthases located in the cytoplasmic membrane and on an extracellular electrochemical sodium gradient for ATP synthesis. This study unraveled the transient utilization of a sodium-motive force for energy generation as a survival strategy in response to adverse environmental conditions. Addition of a nitrogen source to dormant cells initiates the resuscitation program. Nitrogen assimilation triggers glycogen degradation, which provides the necessary energy and metabolic intermediates to regenerate the degraded cellular components. This work revealed that glycogen catabolism is induced by dephosphorylation and activation of phosphoglucomutase 1 (Pgm1), which acts as a metabolic valve to avoid premature usage of the glycogen stores before a nitrogen source is available. Remarkably, this regulatory mechanism seems to be evolutionary conserved. Only a specific glycogen mobilization strategy was shown to enable successful resuscitation, which involves the glycogen phosphorylase GlgP2, the oxidative pentose phosphate (OPP) pathway and the Enter- Doudoroff (ED) pathway. Furthermore, OPP cycle protein (OpcA) and glucose-6- phosphate dehydrogenase (G6PDH), the proteins involved in the first reaction of the OPP and ED pathways, were shown to interact with Pgm1 during recovery. These interactions might result in the formation of a metabolon that directs the carbon flux into the OPP and ED pathways to ensure effective awakening from dormancy