Molecular basis of myotonic disorders and new diagnostic techniques

Myotoniset sairaudet ovat lihastauteja, joihin liittyy myotoniaa. Myotonia on tila, jossa lihaksen tahdonalaisen supistuksen jälkeinen rentoutuminen viivästyy. Tyypin 1 (DM1) ja tyypin 2 (DM2) myotoniset dystrofiat ovat monielinsairauksia, jotka johtuvat DNA-mutaatioista geeneissä DMPK ja ZNF9. Aikuisiällä alkavalla DM1-taudin muodolla ja DM2-taudilla on kuitenkin selviä eroavaisuuksia kuten taudin vaikeusasteessa sekä eri lihaksien ja lihassyytyyppien vaurioitumisessa. Suomessa. DM2-taudin kliininen taudinmääritys on erittäin vaikeaa, koska oireet ovat hyvin vaihtelevat, eikä niiden raja-alueita tarkkaan tunneta. Taudin molekyylipatogeneesiin liittyen, ZNF9-proteiini on ajateltu olevan vailla merkitystä taudissa. Väitöskirjatyössä todistamme, että ZNF9-ilmentymä DM2-potilaissa on poikkeava monella eri tasolla ja voi ainakin osittain selittää joitakin tautien välisiä eroja. Diagnostiikan tarkkuuden parantaminen on tärkeää, jotta oireellisten potilaiden oikea lopullinen diagnoosi ja asianmukainen hoito toteutuisivat. Myosiinien eri entsyymikudoskemiallisia ATPaasi-ominaisuuksia on käytetty lihassyytyyppien erotteluun ja sitä on hyödynnetty lihasnäytteiden rutiinidiagnostiikassa yli neljän vuosikymmenen ajan. ATPaasi-värjäysmenetelmä on jokseenkin työläs ja värjäysmenetelmässä on monia haittapuolia. Lisäksi, joidenkin tyypin 2A/IIA lihassyiden surkastuminen on ominainen piirre DM2-taudille ja jäävät pääsääntöisesti tunnistamatta ATPaasi-värjäysmenetelmällä. Tässä väitöskirjatutkimuksessa kehitettiin uusi luotettava immunokudoskemiallinen myosiinien raskasketjujen kaksoisvärjäysmenetelmä eri syytyyppien erotteluun sekä kyseessä olevien hyvin surkastuneiden IIA lihassyiden tunnistamiseen. DM2-diagnostiikan lisäksi pystyimme tällä uudella menetelmällä tunnistamaan täysin uuden taudin, jossa nopeat tyypin IIA lihassyyt puuttuivat potilaan lihasnäytteestä kokonaan. Kongenitaalinen myotonia on ei-dystrofinen myotonia -sairaus, jonka aiheuttaa mutaatiot kloridikanavageenissä (CLCN1). Nykyään oireellisten potilaiden lopullinen diagnoosi saavutetaan usein molekyyligeneettisellä DNA-testauksella. Geenitestien lisääntyneen käytön seurauksena esiintyy myös monia tapauksia, joissa geenitulokset eivät tarjoa täyttä selvyyttä kliiniselle taudille. Tässä väitöskirjatutkimuksessa kehitetty immunokudoskemiallinen värjäysmenetelmä kloridikanava-proteiinin (ClC-1) tunnistamiseen tarjosi hyödyllisen menetelmän proteiinin arviointiin lihasleikkeistä ja tämän uuden menetelmän avulla löytyi myös uusia mutaatioita. Tässä väitöskirjatutkimuksessa kehitetyt uudet menetelmät yhdistettynä geenitestaukseen ovat tehokas tapa saavuttaa lopullinen diagnoosi myotoniaa sairastaville potilaille. Lisäksi diagnostiset mahdollisuudet ovat tarkentuneet myös muiden lihastautien osalta. Kattava ymmärrys geneettisten sairauksien molekyylitason patomekanismeista ja näiden osoittaminen uusilla menetelmillä on paitsi diagnostiikan kannalta tärkeä, mutta myös perusedellytys mahdollisten tulevaisuuden terapiavaihtoehtojen kehittämiselle.Myotonic disorders are primary diseases of the muscle characterized by myotonia, the delayed relaxation of skeletal muscles after voluntary contraction. Myotonic disorders include the myotonic dystrophies (DMs) and non-dystrophic myotonias. Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are multisystemic disorders caused by tri- (CTG)n or tetranucleotide (CCTG)n repeat expansion mutations in transcribed but not translated regions of the genes DMPK and ZNF9, respectively. Adult onset DM1 and DM2 share some features in the clinical presentation as well as in the molecular genetics and pathomechanisms. However, they also show distinct differences, including disease severity and involvement of muscles and muscle fiber types. So far, over 300 DM2 patients have been identified in Finland, which is in contrast to the prevalence estimate of 1/105 (corresponding to 50 patients) reported previously. DM2 is of particular interest, because it shows a wide range of clinical manifestations and therefore a makes clinical diagnosis extremely difficult. Regarding molecular pathogenesis it has been suggested that ZNF9 is of no significance for the disease pathogenesis, and that the disease is caused solely by RNA toxicity as a result of the underlying repeat expansion mutation. Such an exclusive explanation does not however explain the higher amount of toxic RNA in DM2 than DM1 muscle still resulting in milder clinical manifestations in DM2 compared to DM1. In this thesis work, we were able to show that ZNF9 expression in DM2 patients is in fact altered at multiple levels. While toxic RNA effects likely explain overlapping phenotypic manifestations between DM1 and DM2, abnormal ZNF9 levels in DM2 may account for at least some of the differences. It is important to improve diagnostic accuracy in order to efficiently identify symptomatic patients for correct final diagnosis and appropriate medical attention and management. The different enzyme histochemical ATPase properties of myosins to separate the muscle fiber types have been utilized in diagnostic muscle biopsy routine for more than four decades. The ATPase staining method is rather laborious and has several disadvantages, such as weakening of staining over time and non-specific staining of capillaries, making the distinction of extremely atrophic muscle fibers difficult. Extremely small atrophic type 2/IIA fibers are characteristic for DM2 and usually remain undetected using the ATPase staining method. A reliable and advanced immunohistochemical myosin double staining method for the identification of fiber types, including these highly atrophic type IIA fibers in routine diagnostics was developed in this thesis work. With this double staining method, it is easily possible to distinguish all different fiber types using a one slide technique. In addition to the obvious usefulness in DM2 diagnostics we were able to identify a completely new disease with this technique, because of the absence of fast type IIA fibers in patients muscle biopsies. The disease is caused by disruptive recessive mutations in the MYH2 gene resulting in total absence of MyHC IIA protein and correspondingly total lack of fast type IIA muscle fibers. Myotonia congenita is a non-dystrophic myotonia disease caused by mutations in the chloride channel gene (CLCN1). Currently, final diagnosis of patients with symptoms is frequently obtained by molecular genetic DNA testing. However, the increased use of genetic testing also results in many cases where the genetic results do not provide full clarification of the clinical disease. In this thesis work, the developed immunohistochemical staining method for chloride channel protein (ClC-1) in muscle fibers proved to be a robust method for the assessment of sarcolemmal ClC-1 protein on muscle sections. This method provided means to identify new mutations, to reclassify the W118G CLCN1 change as a moderately pathogenic mutation, and to clarify the final diagnosis in myotonia patients in whom only one recessive mutation had been identified by genetic testing. The methods developed in this thesis work combined with genetic testing are powerful approaches to achieving final diagnosis in patients with myotonic disorders. Comprehensive understanding of the molecular pathomechanisims of genetic diseases is also one of the pre-requisites for the future development of therapeutical options

Molecular basis of myotonic disorders and new diagnostic techniques

Myotoniset sairaudet ovat lihastauteja, joihin liittyy myotoniaa. Myotonia on tila, jossa lihaksen tahdonalaisen supistuksen jälkeinen rentoutuminen viivästyy. Tyypin 1 (DM1) ja tyypin 2 (DM2) myotoniset dystrofiat ovat monielinsairauksia, jotka johtuvat DNA-mutaatioista geeneissä DMPK ja ZNF9. Aikuisiällä alkavalla DM1-taudin muodolla ja DM2-taudilla on kuitenkin selviä eroavaisuuksia kuten taudin vaikeusasteessa sekä eri lihaksien ja lihassyytyyppien vaurioitumisessa. Suomessa. DM2-taudin kliininen taudinmääritys on erittäin vaikeaa, koska oireet ovat hyvin vaihtelevat, eikä niiden raja-alueita tarkkaan tunneta. Taudin molekyylipatogeneesiin liittyen, ZNF9-proteiini on ajateltu olevan vailla merkitystä taudissa. Väitöskirjatyössä todistamme, että ZNF9-ilmentymä DM2-potilaissa on poikkeava monella eri tasolla ja voi ainakin osittain selittää joitakin tautien välisiä eroja. Diagnostiikan tarkkuuden parantaminen on tärkeää, jotta oireellisten potilaiden oikea lopullinen diagnoosi ja asianmukainen hoito toteutuisivat. Myosiinien eri entsyymikudoskemiallisia ATPaasi-ominaisuuksia on käytetty lihassyytyyppien erotteluun ja sitä on hyödynnetty lihasnäytteiden rutiinidiagnostiikassa yli neljän vuosikymmenen ajan. ATPaasi-värjäysmenetelmä on jokseenkin työläs ja värjäysmenetelmässä on monia haittapuolia. Lisäksi, joidenkin tyypin 2A/IIA lihassyiden surkastuminen on ominainen piirre DM2-taudille ja jäävät pääsääntöisesti tunnistamatta ATPaasi-värjäysmenetelmällä. Tässä väitöskirjatutkimuksessa kehitettiin uusi luotettava immunokudoskemiallinen myosiinien raskasketjujen kaksoisvärjäysmenetelmä eri syytyyppien erotteluun sekä kyseessä olevien hyvin surkastuneiden IIA lihassyiden tunnistamiseen. DM2-diagnostiikan lisäksi pystyimme tällä uudella menetelmällä tunnistamaan täysin uuden taudin, jossa nopeat tyypin IIA lihassyyt puuttuivat potilaan lihasnäytteestä kokonaan. Kongenitaalinen myotonia on ei-dystrofinen myotonia -sairaus, jonka aiheuttaa mutaatiot kloridikanavageenissä (CLCN1). Nykyään oireellisten potilaiden lopullinen diagnoosi saavutetaan usein molekyyligeneettisellä DNA-testauksella. Geenitestien lisääntyneen käytön seurauksena esiintyy myös monia tapauksia, joissa geenitulokset eivät tarjoa täyttä selvyyttä kliiniselle taudille. Tässä väitöskirjatutkimuksessa kehitetty immunokudoskemiallinen värjäysmenetelmä kloridikanava-proteiinin (ClC-1) tunnistamiseen tarjosi hyödyllisen menetelmän proteiinin arviointiin lihasleikkeistä ja tämän uuden menetelmän avulla löytyi myös uusia mutaatioita. Tässä väitöskirjatutkimuksessa kehitetyt uudet menetelmät yhdistettynä geenitestaukseen ovat tehokas tapa saavuttaa lopullinen diagnoosi myotoniaa sairastaville potilaille. Lisäksi diagnostiset mahdollisuudet ovat tarkentuneet myös muiden lihastautien osalta. Kattava ymmärrys geneettisten sairauksien molekyylitason patomekanismeista ja näiden osoittaminen uusilla menetelmillä on paitsi diagnostiikan kannalta tärkeä, mutta myös perusedellytys mahdollisten tulevaisuuden terapiavaihtoehtojen kehittämiselle.Myotonic disorders are primary diseases of the muscle characterized by myotonia, the delayed relaxation of skeletal muscles after voluntary contraction. Myotonic disorders include the myotonic dystrophies (DMs) and non-dystrophic myotonias. Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are multisystemic disorders caused by tri- (CTG)n or tetranucleotide (CCTG)n repeat expansion mutations in transcribed but not translated regions of the genes DMPK and ZNF9, respectively. Adult onset DM1 and DM2 share some features in the clinical presentation as well as in the molecular genetics and pathomechanisms. However, they also show distinct differences, including disease severity and involvement of muscles and muscle fiber types. So far, over 300 DM2 patients have been identified in Finland, which is in contrast to the prevalence estimate of 1/105 (corresponding to 50 patients) reported previously. DM2 is of particular interest, because it shows a wide range of clinical manifestations and therefore a makes clinical diagnosis extremely difficult. Regarding molecular pathogenesis it has been suggested that ZNF9 is of no significance for the disease pathogenesis, and that the disease is caused solely by RNA toxicity as a result of the underlying repeat expansion mutation. Such an exclusive explanation does not however explain the higher amount of toxic RNA in DM2 than DM1 muscle still resulting in milder clinical manifestations in DM2 compared to DM1. In this thesis work, we were able to show that ZNF9 expression in DM2 patients is in fact altered at multiple levels. While toxic RNA effects likely explain overlapping phenotypic manifestations between DM1 and DM2, abnormal ZNF9 levels in DM2 may account for at least some of the differences. It is important to improve diagnostic accuracy in order to efficiently identify symptomatic patients for correct final diagnosis and appropriate medical attention and management. The different enzyme histochemical ATPase properties of myosins to separate the muscle fiber types have been utilized in diagnostic muscle biopsy routine for more than four decades. The ATPase staining method is rather laborious and has several disadvantages, such as weakening of staining over time and non-specific staining of capillaries, making the distinction of extremely atrophic muscle fibers difficult. Extremely small atrophic type 2/IIA fibers are characteristic for DM2 and usually remain undetected using the ATPase staining method. A reliable and advanced immunohistochemical myosin double staining method for the identification of fiber types, including these highly atrophic type IIA fibers in routine diagnostics was developed in this thesis work. With this double staining method, it is easily possible to distinguish all different fiber types using a one slide technique. In addition to the obvious usefulness in DM2 diagnostics we were able to identify a completely new disease with this technique, because of the absence of fast type IIA fibers in patients muscle biopsies. The disease is caused by disruptive recessive mutations in the MYH2 gene resulting in total absence of MyHC IIA protein and correspondingly total lack of fast type IIA muscle fibers. Myotonia congenita is a non-dystrophic myotonia disease caused by mutations in the chloride channel gene (CLCN1). Currently, final diagnosis of patients with symptoms is frequently obtained by molecular genetic DNA testing. However, the increased use of genetic testing also results in many cases where the genetic results do not provide full clarification of the clinical disease. In this thesis work, the developed immunohistochemical staining method for chloride channel protein (ClC-1) in muscle fibers proved to be a robust method for the assessment of sarcolemmal ClC-1 protein on muscle sections. This method provided means to identify new mutations, to reclassify the W118G CLCN1 change as a moderately pathogenic mutation, and to clarify the final diagnosis in myotonia patients in whom only one recessive mutation had been identified by genetic testing. The methods developed in this thesis work combined with genetic testing are powerful approaches to achieving final diagnosis in patients with myotonic disorders. Comprehensive understanding of the molecular pathomechanisims of genetic diseases is also one of the pre-requisites for the future development of therapeutical options

Diagnostically important muscle pathology in DNAJB6 mutated LGMD1D

Introduction: Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. LGMD1D is caused by mutated DNAJB6 and the molecular pathogenesis is mediated by defective chaperonal function leading to impaired handling of misfolded proteins which normally would be degraded. Here we aim to clarify muscle pathology of LGMD1D in order to facilitate diagnostic accuracy. After following six Finnish LGMD1D families, we analysed 21 muscle biopsies obtained from 15 patients at different time points after the onset of symptoms. All biopsies were obtained from the lower limb muscles and processed for routine histochemistry, extensive immunohistochemistry and electron microscopy. Results: Histopathological findings were myopathic or dystrophic combined with rimmed vacuolar pathology, and small myofibrillar aggregates. These myofibrillar inclusions contained abnormal accumulation of a number of proteins such as myotilin, aB-crystallin and desmin on immunohistochemistry, and showed extensive myofibrillar disorganization with excess of Z-disk material on ultrastructure. Later in the disease process the rimmed vacuolar pathology dominated with rare cases of pronounced larger pleomorphic myofibrillar aggregates. The rimmed vacuoles were reactive for several markers of defect autophagy such as ubiquitin, TDP-43, p62 and SMI-31. Conclusions: Since DNAJB6 is known to interact with members of the chaperone assisted selective autophagy complex (CASA), including BAG3 - a known myofibrillar myopathy causing gene, the molecular muscle pathology is apparently mediated through impaired functions of CASA and possibly other complexes needed for the maintenance of the Z-disk and sarcomeric structures. The corresponding findings on histopathology offer clues for the diagnosis.Peer reviewe

Abnormal Splicing of NEDD4 in Myotonic Dystrophy Type 2 Possible Link to Statin Adverse Reactions

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2

Distinct Muscle Biopsy Findings in Genetically Defined Adult-Onset Motor Neuron Disorders

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Gene Expression Profiling in Tibial Muscular Dystrophy Reveals Unfolded Protein Response and Altered Autophagy

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Combined Synergistic Effects of Aqueous Extracts of Parquetina nigrescens, Camellia sinensis and Telfaria occidentalis on Bone Marrow Haemopoietic Multipotent Stem Cells Proliferation in Irradiated Guinea Pigs

Cancer which is one of the most threatening human diseases is most commonly treated by chemotherapy and radiotherapy. However, these therapies are not tumor-specific. Normal tissues, particularly the bone marrow (BM), are extremely vulnerable to cytotoxicity caused by these therapies. How rapidly patients recover from these treatment modalities greatly depends on the percentage of resting stem cells remaining after such treatment. Antidotes are required for the untoward side effects of these therapies. As a means to protect stem cells or help damaged stem cells to recover, the use of biological response modifiers (BRMs) has received attention. The use of fruits or vegetables has the benefits of providing a cocktail of many different phytochemicals with multiple actions including antioxidant and anti-inflammatory effects. Certain whole-food extracts, such as blueberry, dietary fatty acids, particularly oleic acid and linoleic acid have been reported recently to actively promote the proliferation of haemopoietic stem cells [1]

Lamin A/C mutation affecting primarily the right side of the heart

LMNA mutations are amongst the most important causes of familial dilated cardiomyopathy. The most important cause of arrhythmogenic right ventricular cardiomyopathy (ARVC) is desmosomal pathology. The aim of the study was to elucidate the role of LMNA mutations among Finnish cardiomyopathy patients. We screened 135 unrelated cardiomyopathy patients for LMNA mutations. Because of unusual phenotype, two patients were screened for the known Finnish ARVC-related mutations of desmosomal genes, and their Plakophilin-2b gene was sequenced. Myocardial samples from two patients were examined by immunohistochemical plakoglobin staining and in one case by electron microscopy. We found a new LMNA mutation Phe237Ser in a family of five affected members with a cardiomyopathy affecting primarily the right side of the heart. The phenotype resembles ARVC but does not fulfill the Task Force Criteria. The main clinical manifestations of the mutation were severe tricuspid insufficiency, right ventricular enlargement and failure. Three of the affected patients died of the heart disease, and the two living patients received heart transplants at ages 44 and 47. Electron microscopy showed nuclear blebbing compatible with laminopathy. Immunohisto - chemical analysis did not suggest desmosomal pathology. No desmosomal mutations were found. The Phe237Ser LMNA mutation causes a phenotype different from traditional cardiolaminopathy. Our findings suggest that cardiomyopathy affecting primarily the right side of the heart is not always caused by desmosomal pathology. Our observations highlight the challenges in classifying cardiomyopathies, as there often is significant overlap between the traditional categories.Peer reviewe

The global burden of cancer attributable to risk factors, 2010-19 : a systematic analysis for the Global Burden of Disease Study 2019

Background Understanding the magnitude of cancer burden attributable to potentially modifiable risk factors is crucial for development of effective prevention and mitigation strategies. We analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 to inform cancer control planning efforts globally. Methods The GBD 2019 comparative risk assessment framework was used to estimate cancer burden attributable to behavioural, environmental and occupational, and metabolic risk factors. A total of 82 risk-outcome pairs were included on the basis of the World Cancer Research Fund criteria. Estimated cancer deaths and disability-adjusted life-years (DALYs) in 2019 and change in these measures between 2010 and 2019 are presented. Findings Globally, in 2019, the risk factors included in this analysis accounted for 4.45 million (95% uncertainty interval 4.01-4.94) deaths and 105 million (95.0-116) DALYs for both sexes combined, representing 44.4% (41.3-48.4) of all cancer deaths and 42.0% (39.1-45.6) of all DALYs. There were 2.88 million (2.60-3.18) risk-attributable cancer deaths in males (50.6% [47.8-54.1] of all male cancer deaths) and 1.58 million (1.36-1.84) risk-attributable cancer deaths in females (36.3% [32.5-41.3] of all female cancer deaths). The leading risk factors at the most detailed level globally for risk-attributable cancer deaths and DALYs in 2019 for both sexes combined were smoking, followed by alcohol use and high BMI. Risk-attributable cancer burden varied by world region and Socio-demographic Index (SDI), with smoking, unsafe sex, and alcohol use being the three leading risk factors for risk-attributable cancer DALYs in low SDI locations in 2019, whereas DALYs in high SDI locations mirrored the top three global risk factor rankings. From 2010 to 2019, global risk-attributable cancer deaths increased by 20.4% (12.6-28.4) and DALYs by 16.8% (8.8-25.0), with the greatest percentage increase in metabolic risks (34.7% [27.9-42.8] and 33.3% [25.8-42.0]). Interpretation The leading risk factors contributing to global cancer burden in 2019 were behavioural, whereas metabolic risk factors saw the largest increases between 2010 and 2019. Reducing exposure to these modifiable risk factors would decrease cancer mortality and DALY rates worldwide, and policies should be tailored appropriately to local cancer risk factor burden. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.Peer reviewe