TIA1-muutosten vaikutus stressigranuloiden muodostumiseen

Welander Distal Myopathy (WDM) is caused by the p.E384K mutation in the TIA1 gene. The mutation supposedly causes the disease by a gain-of-function mechanism related to the formation of stress granules (Hackman et al. 2013). Also environmental factors have been proposed to affect the development of the disease: an increased number of stress granules has been observed in cells treated with cold shock compared to cells kept in 37 °C (Hofmann et al. 2012). When patients with WDM-like symptoms have been screened for changes in TIA1, an p.N357S-change has been found enriched in these patients. The p.N357S-change has earlier been reported as a polymorphism. The change in question is located in the same prion-like domain in exon 5, in which the p.E384K-mutation also lies. Therefore, the p.N357S-change could affect the predisposition to aggregation. The pro gradu project is divided into two parts: • The effect of the p.N357S polymorphism on stress granule formation in arsenite and possibly other stress treated cells • The effect of cold shock on stress granule formation on wild type and p.E384K TIA1 The results indicate, that the p.N357S change in TIA1 causes a change in the translated protein’s behavior. Similarly to the p.E384K change, the p.N357S change also induces an increased amount of stress granules in arsenite treated cells. However, the results also show that the stress granules recover faster in fluorescence recovery after photobleaching (FRAP) studies p.N357S transfected cells as compared to TIA1 p.E384K and wild type transfected cells. The cold shock experiment indicates that there is a difference in the stress granule formation between cells transfected with p.E384K and wild type TIA1. This supports previously published results of the effect of the p.E384K change on the stress response and stress granule formation, and also the use of cold shock as a stress inducing treatment. Used methods: PCR, transformation, DNA-extraction, cell culture, transfection, induction of stress granule formation by arsenite treatment and cold shock. The cells are cultivated on well plates, imaged and the data is analyzed with an automatized high content image analysis method (the CellInsight-platform). p.N357S cells were also analyzed with FRAP.Welanders distala myopati (WDM) orsakas av mutationen p.E384K i genen TIA1. Mutationen antas vara sjukdomsalstrande på grund av en ökad produktion av protein, som relaterats till formationen av stressgranuler (Hackman et al. 2013). Även omgivningsfaktorer har föreslagits verka i sjukdomens utveckling: en ökad mängd stressgranuler har observerats i celler som behandlats med köldshock jämfört med celler som förvarats i 37°C (Hofmann et al. 2012). I patienter med WDM-liknande symptom som undersökts för förändringar i TIA1 har en p.N357S-förändring noterats förrikad. Denna förändring har tidigare anmälts som en polymorfism. Förändringen i fråga ligger i samma prionliknande domän i exon 5 som WDM-orsakande förändringen p.E384K. Därmed kunde p.N357S-förändringen öka predispositionen till aggregering. Pro gradu –arbetet är uppdelat i två delar: • p.N357S-polymorfismens effekt på stressgranulsbildningen i arsenitbehandle celler • Köldshockens effekt på stressgranulsbildningen Resultaten påvisar, att förändringen p.N357S i TIA1 orsakar en förändring i det translaterade proteinets beteende. I likhet med p.E384K-förändringen orsakar även p.N357S en ökad mängd stressgranuler i arsenitbehandlade celler. Däremot tyder resultaten på att stressgranulerna återbildas snabbare i fluorescence recovery after photobleaching-studier (FRAP) I p.N357S-transfekterade celler än i celler som transfekterats med TIA1 p.E384K och vildtyp. Köldshocksexperimenten tyder på att det finns en viss skillnad mellan bildningen av stressgranuler i celler transfekterade med p.E384K och vildtyps-TIA1. Detta stöder tidigare publicerade resultat om p.E384K-förändringens påverkan på stressresponsen och stressgranulsbildningen, och även köldshock som stressinducerande behandling. Använda metoder: PCR, transformation, DNA-ekstraktion, cellkultur, transfektion, induktion av stressgranulsformation med arsenitbehandling och köldshock. Cellerna kultiveras på brunnsplattor, fotograferas och datat analyseras med en automatiserad High Content biildanalysmetod (CellInsight-platform). p.N357S-celler analyserades även med FRAP

Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses

Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses

Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses.Peer reviewe

A custom ddPCR method for the detection of copy number variations in the nebulin triplicate region

The human genome contains repetitive regions, such as segmental duplications, known to be prone to copy number variation. Segmental duplications are highly identical and homologous sequences, posing a specific challenge for most mutation detection methods. The giant nebulin gene is expressed in skeletal muscle. It harbors a large segmental duplication region composed of eight exons repeated three times, the so-called triplicate region. Mutations in nebulin are known to cause nemaline myopathy and other congenital myopathies. Using our custom targeted Comparative Genomic Hybridization arrays, we have previously shown that copy number variations in the nebulin triplicate region are pathogenic when the copy number of the segmental duplication block deviates two or more copies from the normal number, which is three per allele. To complement our Comparative Genomic Hybridization arrays, we have established a custom Droplet Digital PCR method for the detection of copy number variations within the nebulin triplicate region. The custom Droplet Digital PCR assays allow sensitive, rapid, high-throughput, and cost-effective detection of copy number variations within this region and is ready for implementation a screening method for disease-causing copy number variations of the nebulin triplicate region. We suggest that Droplet Digital PCR may also be used in the study and diagnostics of other segmental duplication regions of the genome.Peer reviewe

Congenital asymmetric distal myopathy with hemifacial weakness caused by a heterozygous large de novo mosaic deletion in nebulin

We report the first mosaic mutation, a deletion of exons 11-107, identified in the nebulin gene in a Finnish patient presenting with a predominantly distal congenital myopathy and asymmetric muscle weakness. The female patient is ambulant and currently 26 years old. Muscle biopsies showed myopathic features with type 1 fibre predominance, strikingly hypotrophic type 2 fibres and central nuclei, but no nemaline bodies. The deletion was detected in a copy number variation analysis based on next-generation sequencing data. The parents of the patient did not carry the deletion. Mosaicism was detected using a custom, targeted comparative genomic hybridisation array. Expression of the truncated allele, less than half the size of full-length nebulin, was confirmed by Western blotting. The clinical and histological picture resembled that of a family with a slightly smaller deletion, and that in patients with recessively inherited distal forms of nebulin-caused myopathy. Asymmetry, however, was a novel feature. (c) 2021 Elsevier B.V. All rights reserved.Peer reviewe

Copy number variation analysis increases the diagnostic yield in muscle diseases

Objective: Copy number variants (CNVs) were analyzed from next-generation sequencing data, with the aim of improving diagnostic yield in skeletal muscle disorder cases.& para;& para;Methods: Four publicly available bioinformatic analytic tools were used to analyze CNVs from sequencing data from patients with muscle diseases. The patients were previously analyzed with a targeted gene panel for single nucleotide variants and small insertions and deletions, without achieving final diagnosis. Variants detected by multiple CNV analysis tools were verified with either array comparative genomic hybridization or PCR. The clinical significance of the verified CNVs was interpreted, considering previously identified variants, segregation studies, and clinical information of the patient cases.& para;& para;Results: Combining analysis of all different mutation types enabled integration of results and identified the final cause of the disease in 9 myopathy cases. Complex effects like compound heterozygosity of different mutation types and compound disease arising from variants of different genes were unraveled. We identified the first large intragenic deletion of the titin (TTN) gene implicated in the pathogenesis of a severe form of myopathy. Our work also revealed a "double-trouble" effect in a patient carrying a single heterozygous insertion/deletion mutation in the TTN gene and a Becker muscular dystrophy causing deletion in the dystrophin gene.& para;& para;Conclusions: Causative CNVs were identified proving that analysis of CNVs is essential for increasing the diagnostic yield in muscle diseases. Complex severe muscular dystrophy phenotypes can be the result of different mutation types but also of the compound effect of 2 different genetic diseases.Peer reviewe

Dominantly inherited distal nemaline/cap myopathy caused by a large deletion in the nebulin gene

We report the first family with a dominantly inherited mutation of the nebulin gene (NEB). This 100kb in-frame deletion encompasses NEB exons 14-89, causing distal nemaline/cap myopathy in a three-generation family. It is the largest deletion characterized in NEB hitherto. The mutated allele was shown to be expressed at the mRNA level and furthermore, for the first time, a deletion was shown to cause the production of a smaller mutant nebulin protein. Thus, we suggest that this novel mutant nebulin protein has a dominant-negative effect, explaining the first documented dominant inheritance of nebulin-caused myopathy. The index patient, a young man, was more severely affected than his mother and grandmother. His first symptom was foot drop at the age of three, followed by distal muscle atrophy, slight hypomimia, high-arched palate, and weakness of the neck and elbow flexors, hands, tibialis anterior and toe extensors. Muscle biopsies showed myopathic features with type 1 fibre predominance in the index patient and nemaline bodies and cap-like structures in biopsies from his mother and grandmother. The muscle biopsy findings constitute a further example of nemaline bodies and cap-like structures being part of the same spectrum of pathological changes. (C) 2019 Elsevier B.V. All rights reserved.Peer reviewe

Copy number variants in genes causing neuromuscular disorders

The aim of this thesis project was to improve the methods of molecular genetic mutation analysis in neuromuscular disorders, mainly focusing on challenging repetitive intragenic regions and making these methods more accessible. The large skeletal muscle genes nebulin and titin contain large repetitive, i.e., segmental duplication regions. These regions cause hurdles for standard copy number variation detection methods and are therefore often omitted from analysis. The segmental duplication region of nebulin has been previously shown to harbor pathogenic copy number variants; when the number of duplicated blocks deviates by two or more blocks per allele from the normal number, the alteration is considered a pathogenic recessive mutation. We developed a custom comparative genomic hybridization array for the detection of copy number variants in neuromuscular disorder-causing genes. The array covers all 11 known nemaline myopathy genes and 176 additional known and potential neuromuscular disorder genes. The genes are divided by coverage into three groups, all of which allow copy number variant detection at the exon level. Using the array, we detected the largest nebulin deletion (loss of genetic material) hitherto in a patient presenting with an asymmetric distal myopathy. We were also able to determine that the deletion was present in the patient in a mosaic state, which may contribute to the asymmetric phenotype of the patient. The deletion is the first causative dominant de novo deletion described in nebulin. We have previously used the comparative genomic hybridization array for routine screening of samples from patients in whom a copy number variant in the nebulin segmental duplication region is suspected to be a causative mutation. Moreover, the array detected previously unrecorded recurrent copy number variation in the segmental duplication region of titin. The array, however, is both relatively costly and laborious to run and yields far more data than necessary. In addition, the repetitive nature of the titin segmental duplication region does not allow for exact copy number determination by the comparative genomic hybridization array alone. We, therefore, set out to develop custom droplet digital PCR assays for routine screening of the nebulin segmental duplication region and assays to verify the copy number variation of the titin segmental duplication region. Using the droplet digital PCR assays targeting the nebulin segmental duplication region, we were able to determine the copy number of the region adequately. The droplet digital PCR assays targeting the titin segmental duplication region confirmed that the region is subject to copy number variation, revealing a novel putative pathogenic or modifying mechanism in the gene.Målet med detta doktorsavhandlingsarbete var att förbättra de molekylärgenetiska metoderna för mutationsanalys vid neuromuskulära sjukdomar med speciellt fokus på repetitiva regioner inom gener, och att göra dessa metoder lättare tillgängliga. De stora skelettmuskelgenerna nebulin och titin innehåller stora segmentella duplikationsregioner. Dessa regioner utmanar standardmetoderna inom analysen av förändringar i antalet segmentkopior, och lämnas därför ofta bort vid analyser. Den segmentala duplikationsregionen i nebulin har tidigare visats vara föremål för sjukdomsorsakande varianter i antalet segmentkopior; när antalet duplicerade block avviker med två eller fler stycken per allel från det normala antalet, anses förändringen vara en recessiv sjukdomsorsakande mutation. Vi utvecklade en komparativ genomisk hybridisations-mikromatris för att påvisa förändringar i antalet kopior av gener för neuromuskulära sjukdomar. Mikromatrisen täcker alla 11 kända nemalinmyopatiorsakande gener, tillsammans med 176 andra kända eller möjliga gener för neuromuskulära sjukdomar, populärt kallade muskelsjukdomar. Generna är indelade i tre grupper på basis av täckning, varav alla möjliggör bestämning av förändringar i antalet kopior på exonnivå. Med hjälp av mikromatrisen kunde vi hitta den största nebulindeletionen (det största bortfallet av genetiskt material) hittills i en patient med en asymmetrisk distal myopati. Vi kunde också se att deletionen var närvarande i mosaiktillstånd, vilket kan bidra till den asymmetriska muskelsvagheten hos patienten. Deletionen uppstod som en nymutation och är den första sjukdomsorsakande dominanta de novo deletion som beskrivits i nebulingenen. Vi har tidigare använt mikromatrisen för rutinanalys av prover från patienter hos vilka en förändring i antalet nebulinsegmentkopior har misstänkts vara sjukdomsorsakande. Ytterligare upptäckte vi med hjälp av mikromatrisen tidigare okänd kopieantalsvariation i titinets segmentala duplikationsområdet. Mikromatrisen är dock både såväl förhållandevis dyrt som arbetsdrygt, och producerar mycket mer data än vad som är nödvändigt. Dessutom tillåter inte den repetitiva naturen av titinets segmentala duplikationsregion inte exakt bestämning av antalet kopior med hjälp av endast mikromatrisen. Därmed satsade vi på att utveckla målspecifika droplet-digital-PCR-primers och hydrolysprober för rutinsållning och kopieantalsbestämning av nebulinets segmentala duplikationsregion, och primers och hydrolysprober i ett försök att verifiera variationen i antalet segmentkopior i titinets segmentala duplikationsregion. Droplet-digital-PCR primer-hydrolysprobparen för nebulinets segmentala duplikationsområde kunde adekvat bestämma antalet kopior i regionen. Droplet-digital-PCR-primer-hydrolysprobparen för titinets segmentala duplikationsområdet bekräftade förekomsten av förändringar i antalet kopior i området, och avslöjade därmed en möjlig ny sjukdomsorsakande eller modifierande mekanism hos genen

Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses