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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.

Importance Mutations in the titin gene (TTN) cause a wide spectrum of genetic diseases. The interpretation of the numerous rare variants identified inTTNis a difficult challenge given its large size. Objective To identify genetic variants in titin in a cohort of patients with muscle disorders. Design, Setting, and Participants In this case series, 9 patients with titinopathy and 4 other patients with possibly disease-causing variants inTTNwere identified. Titin mutations were detected through targeted resequencing performed on DNA from 504 patients with muscular dystrophy, congenital myopathy, or other skeletal muscle disorders. Patients were enrolled from 10 clinical centers in April 2012 to December 2013. All of them had not received a diagnosis after undergoing an extensive investigation, including Sanger sequencing of candidate genes. The data analysis was performed between September 2013 and January 2017. Sequencing data were analyzed using an internal custom bioinformatics pipeline. Main Outcomes and Measures The identification of novel mutations in theTTNgene and novel patients with titinopathy. We performed an evaluation of putative causative variants in theTTNgene, combining genetic, clinical, and imaging data with messenger RNA and/or protein studies. Results Of the 9 novel patients with titinopathy, 5 (55.5%) were men and the mean (SD) age at onset was 25 (15.8) years (range, 0-46 years). Of the 4 other patients (3 men and 1 woman) with possibly disease-causingTTNvariants, 2 (50%) had a congenital myopathy and 2 (50%) had a slowly progressive distal myopathy with onset in the second decade. Most of the identified mutations were previously unreported. However, all the variants, even the already described mutations, require careful clinical and molecular evaluation of probands and relatives. Heterozygous truncating variants or unique missense changes are not sufficient to make a diagnosis of titinopathy. Conclusions and Relevance The interpretation ofTTNvariants often requires further analyses, including a comprehensive evaluation of the clinical phenotype (deep phenotyping) as well as messenger RNA and protein studies. We propose a specific workflow for the clinical interpretation of genetic findings in titin.

Centronuclear myopathies (CNMs) are a group of clinically and genetically heterogeneous muscle disorders. To date, mutation in 7 different genes has been reported to cause CNMs but 30 % of cases still remain genetically undefined. Genetic investigations are often expensive and time consuming. Clinical and morphological clues are needed to facilitate genetic tests and to choose the best approach for genetic screening. We aimed to describe genotype–phenotype correlation in an Italian cohort of patients affected by CNMs, to define the relative frequencies of its defined genetic forms and to draw a diagnostic algorithm to address genetic investigations. We recruited patients with CNMs from all the Italian tertiary neuromuscular centers following clinical and histological criteria. All selected patients were screened for the four ‘canonical’ genes related to CNMs: MTM1, DNM2, RYR1 and BIN1. Pathogenetic mutations were found in 38 of the 54 screened patients (70 %), mostly in patients with congenital onset (25 of 30 patients, 83 %): 15 in MTM1, 6 in DNM2, 3 in RYR1 and one in TTN. Among the 13 patients with a childhood–adolescence onset, mutations were found in 6 patients (46 %), all in DNM2. In the group of the 11 patients with adult onset, mutations were identified in 7 patients (63 %), again in DNM2, confirming that variants in this gene are relatively more common in late-onset phenotypes. The present study provides the relative molecular frequency of centronuclear myopathy and of its genetically defined forms in Italy and also proposes a diagnostic algorithm to be used in clinical practice to address genetic investigations.