Novel genetic defects in titinopathies and other muscular dystrophies

Muscular dystrophies are a clinically and genetically heterogeneous group of inherited disorders that cause progressive weakness and atrophy of muscles. They are divided into several subgroups based on the clinical and genetic findings. Mutations in dozens of different genes are known to cause the disorders, but unsolved cases exist. Accurate molecular diagnosis is important for genetic counseling and management of the diseases. The aims of this doctoral study were to identify still unknown genetic defects in muscular dystrophy patients using Sanger sequencing and next-generation sequencing (NGS) methods and to develop a targeted NGS gene panel for molecular diagnostics. In the course of this study more than 30 muscular dystrophy families or sporadic patients obtained a molecular diagnosis. A majority of the identified mutations were located in TTN. The gene encodes titin, which is a giant muscle protein having a central role in aspects of development, structure and function of the striated muscle. Due to the huge size of the gene it was not routinely sequenced before the NGS era. Muscle disorders caused by mutations in TTN are called titinopathies. In the first study, missense mutations in TTN exon 343 were identified in 12 families from seven different countries having hereditary myopathy with early respiratory failure (HMERF). The findings considerable expanded the mutational spectrum of HMERF and highlighted the geographically wide occurrence of the disease as well as the role of TTN exon 343 as a mutational hotspot region. In the second study, additional mutations in patients having previously reported tibial muscular dystrophy (TMD) mutation in TTN exon 362 or 363, but atypical phenotypes, were identified. The patients had a frameshift mutation in the other TTN allele causing a more severe TMD phenotype or limb-girdle muscular dystrophy (LGMD). In the last study, recessive distal myopathy patients were identified to have novel mutations in TTN exon 362 or 363 in either homozygous state or in compound heterozygosity with a nonsense or frameshift mutation. The patients had similar more severe TMD phenotype as the patients in the previous study. The findings expanded the list of distal myopathies with a new category: juvenile or early adult onset recessive distal titinopathy. In addition, a few patients having other novel recessive TTN mutations and novel phenotypes were identified in this doctoral study. The targeted NGS gene panel, MyoCap, was designed to capture and sequence 180 myopathy related genes simultaneously. Altogether 61 myopathy patients, negative for previous candidate gene approaches, were screened using the assay and 19 (31%) of them obtained a molecular diagnosis. MyoCap has high sensitivity and specificity and it is easily updated when new discoveries of myopathy causing genes are made. It provides fast and reliable detection of mutations in myopathy patients and it has been implemented in the molecular diagnostics of myopathy patients in Finland. It is now the first method of choice in diagnostics if no clear candidate gene can be determined by conventional clinical examinations.Lihasdystrofiat ovat sekä kliinisesti että geneettisesti heterogeeninen joukko perinnöllisiä sairauksia, jotka aiheuttavat etenevää lihasten heikentymistä ja surkastumista. Taudit on jaettu useisiin alaryhmiin kliinisten ja geneettisten löydösten perusteella. Lihasdystrofioita aiheuttavia geenivirheitä tunnetaan kymmenissä eri geeneissä, mutta selvittämättömiä tapauksia on myös vielä paljon jäljellä. Geenivirheen löytäminen on tärkeää neuvonnan ja taudin hoitamisen kannalta. Tämän väitöskirjatutkimuksen tarkoituksena oli selvittää uusia geenivirheitä lihasdystrofiapotilailta käyttäen sekä perinteistä Sanger- että uuden sukupolven sekvensointimenetelmiä ja lisäksi kehittää geenipaneeli lihastautidiagnostiikkaan. Tutkimuksen aikana yli 30 perhettä tai yksittäistä potilasta saatiin diagnosoitua. Suuri osa löydetyistä uusista mutaatioista sijaitsi TTN-geenissä. Geeni koodaa titiiniä, jättimäistä lihasproteiinia, jolla on keskeinen rooli poikkijuovaisen lihaksen kehityksessä, rakenteessa ja toiminnassa. TTN-geenin suuresta koosta johtuen sitä ei ole aikaisemmin laajasti sekvensoitu. TTN-geenin mutaatioiden aiheuttamia lihastauteja kutsutaan titinopatioiksi. Ensimmäisessä osatutkimuksessa uusia mutaatioita löytyi perinnöllistä HMERF-tautia sairastavilta perheiltä TTN-eksonista 343. Löydökset laajensivat HMERF-mutaatioiden kirjoa ja korostivat eksonin 343 roolia HMERF-mutaatioalueena. Toisessa osatutkimuksessa potilailta, joilla oli tibiaalista lihasdystrofiaa (TMD) aiheuttava mutaatio TTN-eksonissa 362 tai 363, mutta tavallisesta TMD:stä poikkeava fenotyyppi, löytyi toisesta TTN-alleelista frameshift-mutaatio. Tämä johti vakavampaan TMD-tautiin ja osalla potilaista hartia-lantiotyypin lihasrappeumaan (LGMD). Viimeisessä osatutkimuksessa distaalista myopatiaa sairastavilta perheiltä löytyi uusia mutaatioita TTN-eksoneista 362 tai 363 ja lisäksi toisesta TTN-alleelista framshift- tai nonsense-mutaatio. Potilailla oli samanlainen aikaisemmin alkava ja vakavampi TMD-tauti kuin edellisen tutkimuksen potilailla. Nämä löydökset lisäsivät distaalimyopatioihin uuden kategorian: nuoruudessa alkava resessiivinen distaalinen titinopatia. Näiden lisäksi väitöskirjatutkimuksessa löydettiin uusia resessiivisiä TTN-mutaatioita myös muutamalta muulta potilailta, joilla oli uusi aiemmin kuvaamaton fenotyyppi. MyoCap-geenipaneeli suunniteltiin kattamaan 180 lihastautigeenin sekvensointi samanaikaisesti. Tutkimuksessa sitä käytettiin yhteensä 61 potilaan sekvensoimiseen, joilta ei oltu löydetty geenivirhettä aiemmissa tutkimuksissa. Potilaista 19 (31%) saatiin diagnosoitua. MyoCap on tarkka menetelmä ja siihen on helppo lisätä uusia tautigeenejä, kun niitä löydetään. Se tarjoaa nopean ja tehokkaan tavan paikantaa mutaatioita ja se on nykyisin käytössä lihastautipotilaiden diagnostiikassa. Sitä käytetään varsinkin potilailla, joiden tautigeeniä ei pystytä suoraan päättelemään kliinisissä tutkimuksissa

Re-evaluation of the phenotype caused by the common MATR3 p.Ser85Cys mutation in a new family

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

Novel mutations in DNAJB6 gene cause a very severe early-onset limb-girdle muscular dystrophy 1D disease

DNAJB6 is the causative gene for limb-girdle muscular dystrophy 1D (LGMD1D). Four different coding missense mutations, p.F89I, p.F93I, p.F93L, and p.P96R, have been reported in families from Europe, North America and Asia. The previously known mutations cause mainly adult-onset proximal muscle weakness with moderate progression and without respiratory involvement. A Finnish family and a British patient have been studied extensively due to a severe muscular dystrophy. The patients had childhood-onset LGMD, loss of ambulation in early adulthood and respiratory involvement; one patient died of respiratory failure aged 32. Two novel mutations, c.271T > A (p.F91I) and c.271T > C (p.F91L), in DNAJB6 were identified by whole exome sequencing as a cause of this severe form of LGMD1D. The results were confirmed by Sanger sequencing. The anti-aggregation effect of the mutant DNAJB6 was investigated in a filter-trap based system using transient transfection of mammalian cell lines and polyQ-huntingtin as a model for an aggregation-prone protein. Both novel mutant proteins show a significant loss of ability to prevent aggregation. (C) 2015 Elsevier B.V. All rights reserved.Peer reviewe

A 'second truncation' in TTN causes early onset recessive muscular dystrophy

Mutations in the gene encoding the giant skeletal muscle protein titin are associated with a variety of muscle disorders, including recessive congenital myopathies cardiomyopathy, limb girdle muscular dystrophy (LGMD) and late onset dominant distal myopathy. Heterozygous truncating mutations have also been linked to dilated cardiomyopathy. The phenotypic spectrum of titinopathies is emerging and expanding, as next generation sequencing techniques make this large gene amenable to sequencing. We undertook whole exome sequencing in four individuals with LGMD. An essential splice site mutation, previously reported in dilated cardiomyopathy, was identified in all families in combination with a second truncating mutation. Affected individuals presented with childhood onset proximal weakness associated with joint contractures and elevated CK. Cardiac dysfunction was present in two individuals. Muscle biopsy showed increased internal nuclei and immunoblotting identified reduction or absence of calpain-3 and demonstrated a marked reduction of C-terminal titin fragments. We confirm the co-occurrence of cardiac and skeletal myopathies associated with recessive truncating titin mutations. Compound heterozygosity of a truncating mutation previously associated with dilated cardiomyopathy and a 'second truncation' in TTN was identified as causative in our skeletal myopathy patients. These findings add to the complexity of interpretation and genetic counselling for titin mutations. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

Titin mutation segregates with hereditary myopathy with early respiratory failure

In 2001, we described an autosomal dominant myopathy characterized by neuromuscular ventilatory failure in ambulant patients. Here we describe the underlying genetic basis for the disorder, and we define the neuromuscular, respiratory and radiological phenotype in a study of 31 mutation carriers followed for up to 31 years. A combination of genome-wide linkage and whole exome sequencing revealed the likely causal genetic variant in the titin (TTN) gene (g.274375T>C; p.Cys30071Arg) within a shared haplotype of 2.93 Mbp on chromosome 2. This segregated with the phenotype in 21 individuals from the original family, nine subjects in a second family with the same highly selective pattern of muscle involvement on magnetic resonance imaging and a third familial case with a similar phenotype. Comparing the mutation carriers revealed novel features not apparent in our original report. The clinical presentation included predominant distal, proximal or respiratory muscle weakness. The age of onset was highly variable, from early adulthood, and including a mild phenotype in advanced age. Muscle weakness was earlier onset and more severe in the lower extremities in nearly all patients. Seven patients also had axial muscle weakness. Respiratory function studies demonstrated a gradual deterioration over time, reflecting the progressive nature of this condition. Cardiomyopathy was not present in any of our patients despite up to 31 years of follow-up. Magnetic resonance muscle imaging was performed in 21 affected patients and revealed characteristic abnormalities with semitendinosus involvement in 20 of 21 patients studied, including 3 patients who were presymptomatic. Diagnostic muscle histopathology most frequently revealed eosinophilic inclusions (inclusion bodies) and rimmed vacuoles, but was non-specific in a minority of patients. These findings have important clinical implications. This disease should be considered in patients with adult-onset proximal or distal myopathy and early respiratory failure, even in the presence of non-specific muscle pathology. Muscle magnetic resonance imaging findings are characteristic and should be considered as an initial investigation, and if positive should prompt screening for mutations in TTN. With 363 exons, screening TTN presented a major challenge until recently. However, whole exome sequencing provides a reliable cost-effective approach, providing the gene of interest is adequately captured

Structure and analysis of human obscurin Ig59.

<p><b>A</b>. Cartoon of the Ig59 crystal structure, showing the typical Ig-like fold. <b>B</b>. Comparison between the lowest RMSD Ig59 NMR structure and the X-ray structure. <b>C</b>. CD plot of WT obscurin Ig58-59 (black squares) and p.Arg4444Trp (open circles). <b>D</b>. MD simulated average models of WT (blue) and p.Arg4444Trp (red). Ca position. The side chains for Arg4444/Trp4444 are shown. E. RMSD vs residue number comparison of the mutant model to the wild-type model. The Arg4444Trp site is colored in red.</p