Molecular Genetics of Inclusion Body Myositis and Late-Onset Rimmed-Vacuolar Distal Myopathy

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Supervisors

Professor Bjarne Udd, MD, PhD

Folkhälsan Research Center, Helsinki, Finland

Department of Medical and Clinical Genetics, Medicum, University of Helsinki, Helsinki, Finland

Neuromuscular Research Center, University of Tampere, and Tampere University Hospital, Tampere, Finland Department of Neurology, Vaasa Central Hospital, Vaasa, Finland

Docent Peter Hackman, PhD

Folkhälsan Research Center, Helsinki, Finland

Department of Medical Genetics, Medicum, University of Helsinki, Helsinki, Finland

Thesis Advisory Committee

Docent Mikaela Grönholm, PhD

Drug Research Program Helsinki, Faculty of Pharmacy, TRIMM, Translational Immunology Research Program, Faculty of Medicine, University of Helsinki, Finland Docent Kati Donner, PhD

Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland

Reviewers

Professor Merilee Needham, MD, PhD

Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Western Australia, Australia

Hannele Koillinen, MD, PhD

Department of Clinical Genetics, Turku University Hospital, Turku, Finland

Opponent

Professor Kevin P. Campbell, PhD

Department of Molecular Physiology and Biophysics, Department of Neurology, Howard Hughes Medical Institute, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa city, Iowa, United States of America

Custos

Professor Juha Partanen, PhD

Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland

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Cover concept: Mridul Johari

This concept describes ‘perspective’ and reimagines the jharokha of Hawa Mahal, an iconic landmark in Jaipur (India), the birth city of the doctoral candidate Mridul Johari. Here the jharokha of Hawa Mahal gives the impression that we are looking into a muscle and provide an interesting perspective on the molecular pathology of muscle diseases. Each jharokha shows DNA, RNA, and Proteins, and individually they can only tell a part of the story. The broken jharokha to the right represent muscle fiber degeneration due to rimmed vacuoles or protein aggregates.

Our perspective or ‘view’ is often limited by what we want to focus on and for a better comprehension we must ‘zoom out’ and look at the whole picture and in turn the whole molecular pathology.

Cover artwork: Monika Jasnauskaite (Twitter: Mjasnauskaite, Instagram: artbymonikajasnauskaite)

ISBN 978-951-51-7696-7 (paperback) ISBN 978-951-51-7697-4 (PDF)

The Faculty of Biological and Environmental Sciences uses the Urkund system (plagiarism recognition) to examine all doctoral dissertations.

Press: Unigrafia Oy Year: 2021

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“If you can meet with Triumph and Disaster and treat those two imposters just the same”

- Rudyard Kipling

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To my Amma

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List of Original Publications

This thesis is based on the following articles, referred to in the text by their Roman numerals. Additionally, some unpublished (U) and non-peer-reviewed works are also presented.

I Johari M, Arumilli M, Palmio J, Savarese M, Tasca G, Mirabella M, Sandholm N, Lohi H, Hackman P, Udd B.

Association study reveals novel risk loci for sporadic inclusion body myositis. Eur J Neurol. 2017 Apr;24(4):572-577. doi:

10.1111/ene.13244. Epub 2017 Feb 24. PMID: 28233382.

II Johari, M., Vihola, A., Palmio, J., Jokela, M., Jonson, P. H., Sarparanta, J., Huovinen, S., Savarese, M., Hackman, P.,

& Udd, B. (2021). Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis. bioRxiv, 2021.2006.2030.450477. (pre-print/submitted manuscript)

III Johari M, Sarparanta J, Vihola A, Jonson PH, Savarese M, Jokela M, Torella A, Piluso G, Said E, Vella N, Cauchi M, Magot A, Magri F, Mauri E, Kornblum C, Reimann J, Stojkovic T, Romero NB, Luque H, Huovinen S, Lahermo P, Donner K, Comi GP, Nigro V, Hackman P, Udd B. Missense mutations in small muscle protein X-linked (SMPX) cause distal myopathy with protein inclusions. Acta Neuropathol. 2021 Aug;142(2):375-393. doi: 10.1007/s00401-021-02319-x. Epub 2021 May 11. PMID: 33974137.

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The following publications are relevant in the context of rimmed vacuolar distal myopathies but are not included in the thesis:

A. Jonson PH, Palmio J, Johari M, Penttilä S, Evilä A, Nelson I, Bonne G, Wiart N, Meyer V, Boland A, Deleuze JF, Masson C, Stojkovic T, Chapon F, Romero NB, Solé G, Ferrer X, Ferreiro A, Hackman P, Richard I, Udd B. Novel mutations in DNAJB6 cause LGMD1D and distal myopathy in French families. Eur J Neurol. 2018 May;25(5):790-794. doi:

10.1111/ene.13598. Epub 2018 Mar 30. PMID: 29437287.

B. Berardo A, Lornage X, Johari M, Evangelista T, Cejas C, Barroso F, Dubrovsky A, Bui MT, Brochier G, Saccoliti M, Bohm J, Udd B, Laporte J, Romero NB, Taratuto AL.

HNRNPDL-related muscular dystrophy: expanding the clinical, morphological and MRI phenotypes. J Neurol. 2019 Oct;266(10):2524-2534. doi: 10.1007/s00415-019-09437-3.

Epub 2019 Jul 2. PMID: 31267206.

C. Savarese M, Johari M, Johnson K, Arumilli M, Torella A, Töpf A, Rubegni A, Kuhn M, Giugliano T, Gläser D, Fattori F, Thompson R, Penttilä S, Lehtinen S, Gibertini S, Ruggieri A, Mora M, Maver A, Peterlin B, Mankodi A, Lochmüller H, Santorelli FM, Schoser B, Fajkusová L, Straub V, Nigro V, Hackman P, Udd B. Improved Criteria for the Classification of Titin Variants in Inherited Skeletal Myopathies. J Neuromuscul Dis. 2020;7(2):153-166. doi: 10.3233/JND- 190423. PMID: 32039858.

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Author Contributions

I

Conceptualization of the study MJ, MA, MS, PH, BU Recruitment, evaluation of patients, and

data collection

JP, GT, MM, BU

Bioinformatic analysis MA

Genetic data analysis, interpretation, and curation

MJ

Validation MJ

Statistical analysis MJ, MA

Writing the original draft MJ, MA, JP

II

Conceptualization of the study MJ, PHJ, MS, PH, BU Recruitment, evaluation of patients, and

data collection JP, MEJ, BU

Histopathological studies AV, SH

Bioinformatic analysis MJ

Genetic data analysis and interpretation MJ

Statistical analysis MJ

Visualization MJ

Writing the original draft MJ

III

Conceptualization of the study MJ, JS, AV, PHJ, MS, MEJ, VN, PH, BU Recruitment, evaluation of patients, and

data collection

MEJ, ES, NV, MC, AM, FM, EM, CK, JR, TS, NR, GC, BU

Histopathological studies AV, SH, NR

Bioinformatic analysis MJ

Genetic data analysis and interpretation MJ, MS

Validation MJ, AV, JS, PHJ

Statistical analysis MJ, JS, PHJ

Visualization MJ, JS, AV, PHJ

Writing the original draft MJ, JS, AV, PHJ, MEJ

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Abbreviations

ACMG American College of Medical Genetics and Genomics

BAM binary alignment/map

bp base pair

BWA Burrows-Wheeler Aligner

CK creatine kinase

cM centimorgan

CNV copy number variation

COX cytochrome c oxidase or Complex IV

EM electron microscopy

EMG electromyography

ES exome sequencing

ExAC Exome Aggregation Consortium EWAS exome-wide association analysis FDR false discovery rate

fIBM/f-IBM familial inclusion body myositis GATK Genome Analysis Toolkit

GNE UDP-N-Acetylglucosamine-2-

Epimerase/N-Acetylmannosamine Kinase

GS genome sequencing

GWAS genome-wide association analysis gnomAD genome Aggregation Database HLA human leukocyte antigen

HNRNPA1 heterogenous nuclear ribonucleoprotein A1

HNRNPA2B1 heterogenous nuclear ribonucleoprotein A2/B1

HTS High Throughput Sequencing

hIBM hereditary inclusion body myopathy HMERF hereditary myopathy with early

respiratory failure IBM inclusion body myositis

IBMPFD inclusion body myopathy with Paget disease with or without frontotemporal dementia

IGV Integrative Genomics Viewer

IM-VAMP inflammatory myopathy with vacuoles, aggregates and mitochondrial pathology

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IIM idiopathic inflammatory myopathies IPA Ingenuity Pathway Analysis

LC3 microtubule-associated proteins 1A/1B light chain 3B

LGMD limb-girdle muscular dystrophy

LOF loss-of-function

MAF minor allele frequency

MHC-I major histocompatibility complex I MHC-II major histocompatibility complex II MPS massively parallel sequencing MRI magnetic resonance imaging MSP multisystem proteinopathy

mtDNA mitochondrial DNA

OMIM Online Mendelian Inheritance in Man OPDM oculopharyngodistal myopathy

OPMD oculopharyngeal muscular dystropy PCA principal component analysis sIBM/s-IBM sporadic inclusion body myositis SDH succinate dehydrogenase

SISu Sequencing Initiative Suomi SMPX small muscle protein X-linked SNP single nucleotide polymorphism

SQSTM1 sequestosome-1

SV structural variation

TARDBP TAR DNA binding protein

TIA1 cytotoxic granule-associated RNA binding protein 1

TMD tibial muscular dystrophy VCP valosin-containing protein VUS variant of unknown or uncertain

significance

WDM Welander distal myopathy

XLMTM X-linked myotubular myopathy

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Abstract

Molecular genetics of late-onset neuromuscular diseases is often challenging to study due to limited patient and family material. This thesis aimed to unravel the genetics of late-onset myopathy patients with inclusion body myositis (IBM) and yet unknown inherited distal myopathy phenotype.

IBM is an inflammatory myopathy of idiopathic nature showing characteristic quadriceps and finger flexor muscle weakness and rimmed vacuolar pathology. The molecular etiology of IBM is of key interest due to co-existing cytotoxic T- cell activity. However, the precedence of degenerative pathology and autoimmune features remain unclear, hampering the eventual development of appropriate therapeutic options. Using DNA and RNA sequencing-based data analysis, we aimed at understanding the different components of the molecular pathomechanisms in IBM. Using a limited case-control study, we identified the association of HLA-DQB1 in Finnish IBM patients. Additionally, by performing a comprehensive transcriptomics analysis we observed differential expression and splicing patterns in genes associated with maintaining calcium homeostasis, particularly during different T-cell activity and regulation stages. Disturbed antigen-driven T-cell hyperactivity and eventual loss of T-cell apoptosis could be one of the mechanisms behind the refractoriness of immune therapies in IBM patients.

Patients with late-onset rare diseases often experience a long diagnostic journey in multiple and often invasive diagnostic procedures and ineffective symptomatic treatments.

Due to limited study material, it is often difficult to ascertain the molecular diagnosis of previously unknown inherited myopathies. Identifying new rare disease-causing genes requires collaborations from different diagnostic centers, sharing deep phenotypic and genomic data. We identified a novel type of slowly progressing late-onset distal myopathy

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with rimmed vacuoles caused by mutations in the small muscle protein X-linked (SMPX) gene in patients from five different countries. Our genetic analysis revealed four different missense mutations, including two different founder mutations in Europe, indicating that the prevalence of this disease may be higher, and therefore SMPX should be considered in all unsolved male patients with late-onset rimmed vacuolar myopathy.

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Referat

Molekylgenetik vid sent debuterande neuromuskulära sjukdomar är ofta utmanande att studera på grund av begränsat patient- och familjematerial. Syftet med denna avhandling var att klarlägga genetiska faktorer hos sent debuterande myopatipatienter med inklusionskroppsmyosit (IBM) och en hittills okänd genetisk distal myopati.

IBM är en inflammatorisk myopati av idiopatisk natur som uppvisar karakteristisk muskelsvaghet i anteriora lårmuskler och fingerflexorer samt rimmad vakuolär muskelpatologi.

IBMs molekylära etiologi är av avgörande intresse på grund av den coexisterande inflammatoriska cytotoxiska T- cellaktiviteten. Men vad som är primärt, den degenerativa patologin eller de autoimmuna egenskaperna, är fortfarande oklart och hindrar förståelsen av etiologin och eventuell utveckling av lämpliga terapeutiska alternativ. Med hjälp av DNA- och RNA-sekvensbaserad dataanalys syftade vi till att förstå de olika komponenterna i de molekylära patomekanismerna i IBM. Med hjälp av en begränsad fallkontrollstudie identifierade vi en association men HLA- DQB1 hos finska IBM-patienter. Dessutom, via en omfattande transkriptomikanalys, observerade vi differentierad expression och splitsningsmönster hos gener associerade med att upprätthålla kalciumhomeostasen, särskilt under olika stadier och regleringen av T-cellaktivitet. Störd antigen-pådriven T- cell hyperaktivitet och eventuell förlust av T-cell apoptos kan vara en av mekanismerna bakom bristande effekt av immunterapier för IBM-patienter.

Patienter med sällsynta muskelsjukdomar genomgår ofta en långvarig diagnostisk precedur omfattande multipla och ofta invasiva diagnostiska undersökningat och ineffektiva symptomatiska behandlingar. På grund av begränsade studiematerial är det ofta svårt att fastställa den molekylära diagnosen hos sent debuteande ärftliga myopatier.

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Identifiering av nya generna som orsakar sällsynta sjukdomar kräver samarbete mellan olika diagnostiska centra innefattande detaljerade fenotypuppgifter och genetiska data.

Vi har identifierat en ny typ av sent debuterande distal myopati med rimmade vakuolär patologi förorsakad av mutationer i small muscle protein X-linked (SMPX) genen hos patienter från fem olika länder. Vår genetiska analys avslöjade fyra olika missense -mutationer, inkluderande två olika grundarmutationer i Europa, vilket indikerar att förekomsten av denna sjukdom torde vara högre, och SMPX bör screenas för mutationer i andra ouppklarade sent debuterande vakuolära myopatier hos män.

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1 Introduction

Myopathies are inherited or acquired diseases that result from direct abnormalities of the muscle fibers, ion channels, or metabolism manifesting in muscle weakness, fatigue, cramps, contractures, myalgias, muscle stiffness, or myoglobinuria (Barohn et al., 2014; Dimachkie, 2014). Major myopathic disorders present with proximal muscle weakness in the pelvic and shoulder girdle (Hilton-Jones & Turner, 2014). Less often, weakness in the distal muscles of hands and/or feet is more prominent.

Myopathies, however, are clinically quite heterogeneous, and the initial symptoms often overlap with other neuromuscular disorders, e.g., selective weakness and atrophy in distal muscles are more commonly seen in neuropathies (Barohn et al., 2014). Thorough clinical examinations determining the onset and distribution of weakness and the rate of progression and variability are critical in establishing a differential diagnosis. Additionally, measurements of Creatine Kinase (CK) levels, electrodiagnostic studies, muscle imaging, histopathology, and molecular genetics are needed to establish the final myopathy diagnosis. The age of onset is significant in the differential diagnosis. In congenital myopathies, the symptoms appear at birth or during the first year of life. Many myopathies manifest later, in childhood, juvenile or adult age (18 years or older), or very late in life. Muscle magnetic resonance imaging (MRI) studies can determine the preferential involvement of different muscles, which in some diseases may be pathognomonic and lead to a more specific diagnosis (Straub et al., 2012; Bugiardini et al., 2018).

Histopathological examination of muscle biopsies may identify well-known pathological markers for different myopathies:

fiber type abnormalities, myofibrillar pathology, inflammation, rimmed vacuoles, and cytoplasmic inclusions (Dubowitz et al., 2013). In addition to these examinations, family history and a well-planned molecular genetic analysis are essential in

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differentiating between different overlapping presentations and providing a more comprehensive clinical and molecular diagnosis to the patient (Hilton-Jones & Turner, 2014).

Inherited myopathies often have a positive family history indicating a Mendelian inheritance and genetic basis of the disease. On the contrary, most acquired myopathies are sporadic but may have genetic risk factors that predispose an individual to the disease. In addition to these, ultra-rare myopathies or myopathies of yet unknown genetic causes, X- linked disorders, and myopathies caused by frequent de novo mutations may often present as sporadic cases, thus complicating the molecular diagnosis and prolonging the diagnostic journey of patients.

The studies presented in this doctoral thesis (Fig 1) aimed at unraveling possible molecular genetic factors involved in Inclusion body myositis (IBM), a more common, late-onset acquired myopathy, usually observed sporadically.

Additionally, this thesis aimed to find the molecular diagnosis of other yet unsolved myopathies not caused by any of the known neuromuscular disease-causing genes.

Fig 1: A graphical representation of the different phenotypes studied, and different methodological techniques used in this

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thesis. The group of muscles indicated by red color represent quadriceps, wrist flexion, finger flexion, and ankle dorsiflexion muscles. Image created with BioRender.com

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2 Review of the Literature

2.1 Distal myopathies

Distal myopathies are inherited primary muscle diseases causing predominant weakness first in hands or feet with varying ages of onset, clinical presentations, and histopathological findings (Savarese et al., 2020). Historically, Gowers first described two young patients with distal muscle weakness in the hand, foot, and facial muscles (Gowers, 1902) as cases with a distal myopathy phenotype. In 1951 the neurologist Lisa Welander described a large Swedish cohort of 249 patients from 72 pedigrees with autosomal dominant inheritance and a late-onset distal myopathy (Welander, 1951), later termed Welander Distal Myopathy (WDM). Since then, many forms of distal myopathies with onset ranging from infantile to late adulthood and different modes of inheritance have been reported.

Preferentially, finger or wrist extensors/flexors or ankle dorsiflexor/plantar flexor muscles are first affected in the distal myopathies. However, a late-onset and severe atrophy of distal muscles can also mirror distal hereditary motor neuropathies (Udd, 2014; Milone & Liewluck, 2019). Biopsy from the affected muscle of patients with distal myopathies shows a wide variety of myopathic structural changes such as the presence of rimmed vacuoles, myofibrillar pathology, cytoplasmic proteinaceous inclusions, or just nonspecific myopathic changes: fiber size variation, increase of internal nuclei, or rarely necrotic fibers.

In recent years, molecular genetic studies have become essential in confirming the final differential diagnosis of distal myopathies besides extensive clinical examinations, electromyography (EMG), MRI, and histopathological analysis (Udd, 2014). More than thirty different genes have been identified to date, causing different types of distal myopathies

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(Table 1). A few of these genes are also associated with other clinical disease phenotypes. For example, a heterozygous rearrangement of 11 bp in the last exon 364 of the titin gene (TTN) is the cause of Tibial muscular dystrophy, TMD (Hackman et al., 2002), an autosomal dominant, adult-onset distal myopathy. However, when observed in homozygosity, this Finnish founder FINmaj mutation causes early-onset LGMD R10 (Udd et al., 1992; Hackman et al., 2002).

Additionally, several nonsense or frameshift mutations in the last or the second last exon 363 may cause an early onset recessive distal titinopathy (Evila et al., 2017). In an increasingly complex picture of TTN related phenotypes, heterozygous missense variants in exon 344 of TTN result in yet another phenotype known as hereditary myopathy with early respiratory failure (HMERF) (Palmio et al., 2014) while truncating mutations in TTN are associated with dilated cardiomyopathies (Itoh-Satoh et al., 2002; Herman et al., 2012) and biallelic variants associated with various forms of recessive congenital titinopathies (Oates et al., 2018).

Table 1: Different genes and associated distal myopathy phenotypes, indicated by their OMIM numbers and specific pathological markers. AD = Autosomal dominant, AR = Autosomal recessive, RV = Rimmed vacuoles.

Causative Gene

Inheritance pattern

OMIM number of associated

distal myopathies

Reporting publications

Pathology

ACTN2 AD 618655 (Savarese et

al., 2019)

Myofibrillar + RV

CAV3 AD 614321 (Tateyama et

al., 2002)

Nonspecific myopathic

changes CRYAB AD 608810 (Vicart et al.,

1998)

Myofibrillar +

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cytoplasmic inclusions

DNAJB6 AD 603511

(Ruggieri et al., 2015;

Jonson et al., 2018)

Myofibrillar + RV

FLNC AD 614065 (Duff et al.,

2011)

changes

HNRNPA1 AD - (Beijer et al.,

2021)

RV + cytoplasmic

inclusions

HSPB8 AD (Ghaoui et

al., 2016)

Myofibrillar + RV

LDB3 AD 609452 (Griggs et al.,

2007)

Myofibrillar + RV

MATR3 AD -

(Senderek et al., 2009;

Palmio et al., 2016)

RV

MB AD - (Olivé et al.,

2019)

cytoplasmic inclusions

MYH7 AD 160500 (Meredith et

al., 2004)

Type 1 fiber hypotrophy

MYOT AD -

(Pénisson- Besnier et al.,

2006)

Myofibrillar

± RV

PLIN4 AD - (Ruggieri et

al., 2020) RV

TIA1 AD 604454 (Hackman et

al., 2013) RV

VCP AD -

(Haubenberg er et al., 2005; Palmio

et al., 2011)

RV

NOTCH2NLC AD - (Sone et al.,

2019) RV

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LRP12 AD - (Saito et al.,

2020) RV

GIPC1 AD - (Deng et al.,

2020) RV

SQSTM1 +

TIA1 digenic - (Lee et al.,

2018)

RV + Cytoplasmic

inclusions

ADSS1 AR 617030 (Park et al.,

2016) RV

ANO5 AR - (Bolduc et

al., 2010)

changes

DYSF AR 606768 (Liu et al.,

1998)

changes

GNE AR 605820 (Eisenberg et

al., 2001)

Myofibrillar + RV

DES AD/AR 601419 (Sjöberg et

al., 1999)

Myofibrillar + RV

NEB AD/AR 256030

(Wallgren- Pettersson et

al., 2007;

Kiiski et al., 2019)

Nemaline rods

RYR1 AD/AR -

(Laughlin et al., 2017;

Jokela et al., 2019)

Core myopathy

TTN AD/AR 600334 (AD)

/ - (AR)

(Hackman et al., 2002;

Palmio et al., 2014; Evila et al., 2017)

RV

SMPX X-linked - (Johari et al., 2021)

Myofibrillar + RV + cytoplasmic

inclusions

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2.1.1 Rimmed-vacuolar distal myopathies

Rimmed vacuoles are not specific to distal myopathies but are also part of the pathology in other inherited neuromuscular diseases and inclusion body myositis. On electron microscopy (EM), the rimmed vacuoles are focal disorganized autophagic areas and consist of autophagosomal components, accumulated debris material, myeloid membrane figures, and misfolded protein aggregates (Ii et al., 1986). They may also contain nuclear proteins (Greenberg et al., 2006) suggesting that more than one formation mechanism of rimmed vacuoles may occur.

Rimmed vacuoles are also observed in the multisystemic proteinopathies, caused by mutations in HNRNPA1, HNRNPA2B1, VCP, and the digenic SQSTM1 + TIA1. Due to the presence of rimmed vacuoles and cytoplasmic inclusions, these MSPs were referred to as hereditary inclusion body myopathies in earlier literature.

2.2 Molecular genetics of distal myopathies

The nuclear genome consists of approximately 3 billion base pairs (bp), divided into 22 pairs of autosomes and the sex chromosomes X and Y. Additionally, a circular DNA molecule of 16,569 nucleotides is present in the mitochondria known as the mitochondrial genome. Approximately 1-2% of the nuclear genome comprises protein-coding regions or genes. The rest of the non-coding DNA includes regulatory DNA, sequential repeats, introns, pseudogenes, transposons, and non-coding RNAs (Brown, 2002).

Every individual has two copies of an autosomal gene, referred to as the alleles they inherit from each biological parent. Genetic variations, which differ from a major allele, are responsible for differences between individual genomes. These genetic variations (single nucleotide variations, copy number

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variations, repeats, or expansions) can be harmless or pathogenic, resulting in different Mendelian and non- Mendelian genetic disorders.

2.2.1 Inheritance patterns and family history

Commonly studied distal myopathies show monogenic inheritance, including Mendelian inheritance (Table 1). When inherited from one affected parent, a disease has an autosomal dominant (AD) inheritance, and the affected individual has a 50% risk of transmitting it to the next generation. In AD families, a proper collection of the history of muscle weakness and other symptoms in the family can be highly informative.

Exceptionally, in some cases, non-paternity issues can be confounding.

In some cases, the pathogenic mutation is a ‘new mutation’

or a de novo, arisen during meiosis, resulting in an affected child who may then transmit the disease to the next generation in an autosomal dominant manner. De novo mutations can be due to germ cell mosaicism or occur in the fertilized egg itself.

These mutations usually are the most deleterious form of rare genetic variation as they have been subjected to less stringent evolutionary bias (Veltman & Brunner, 2012). Sporadic cases when biological parents are asymptomatic, and family history is negative is a perfect instance of rare disease cases caused by de novo variants. A good example is de novo mutations in MYH7, which cause approximately 30% of all Laing distal myopathy cases (Lamont et al., 2014). Identification of de novo mutations requires the availability of family material. In late- onset, sporadic cases, the diagnosis of a de novo mutation is thus quite challenging.

Not every member carrying the disease mutation(s) may have a similar onset or show equal severity of symptoms in a myopathy family. In such cases modifying variants in cis or trans or reduced penetrance of alleles can explain the highly variable onset of symptoms. The mutated allele may also have

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variable expressivity resulting in heterogenous clinical presentation in the same family. An example of reduced penetrance and variable expressivity is seen in distal myopathy caused by mutations in MYH7 (Feinstein-Linial et al., 2016).

In the case of autosomal recessive inheritance, the affected individuals have two mutated alleles inherited from the parents. In such cases, both parents must be carriers of at least one mutated allele, resulting in either homozygosity of the same variant or compound heterozygosity of two different variants in the same gene. In compound heterozygosity, an exception can be when one of the mutated alleles is de novo. In some cases, individuals with autosomal recessive inheritance may present as sporadic cases due to negative family history.

In inbred families, a comprehensive family and pedigree analysis can often show affected individuals in a horizontal line, as seen, e.g., in the Indian subcontinent leading to a higher rate of homozygous mutations in DYSF and GNE (Chakravorty et al., 2020).

In certain muscle diseases, the inheritance deviates from classical Mendelian description of autosomal dominant and recessive phenotypes if affected males are seen in an overwhelmingly high proportion, suggesting an X-linked inheritance of the disease. The classical understanding of X- linked diseases is that all hemizygous males are affected, and females are often asymptomatic carriers. However, skewed inactivation of the X chromosome in females can result in the later onset of symptoms or a milder manifestation of the disease. This is seen in X-linked myotubular myopathy (XLMTM) caused by mutations in MTM1 (Pierson et al., 2005), and in Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) caused by mutations in DMD (Viggiano et al., 2016). In X-linked families, most affected individuals are reported with no family history, generally due to the absence of another affected male sibling or maternal uncles. A comprehensive family history analysis for several generations can help in understanding the transmission of the

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disease. X-linked disorders are neither dominant nor recessive and, due to variable penetrance and expressivity, are referred to only as X-linked in modern concepts of Mendelian genetics (Dobyns et al., 2004). To date, only one X-linked distal myopathy phenotype has been described (Johari et al., 2021).

Abnormalities in mtDNA, such as large deletions or pathogenic point mutations resulting in abnormal mitochondrial proteins, are uncommon in distal myopathies but mutations in POLG with secondary multiple mtDNA deletions may cause a distal phenotype (Castiglioni et al., 2018). Multiple mitochondrial DNA deletions are also common in IBM (Oldfors et al., 1993).

2.2.2 Genetic and phenotypic variability

Most inherited myopathies are monogenic and are caused by mutations within a single gene. However, the molecular understanding of the variability in different distal myopathy phenotypes is difficult to dissect as they show genetic and phenotypic variability. This heterogeneity can also be intrafamilial when patients in the same family show different levels of disease severity. Allelic heterogeneity is an example of this genetic variability, quite commonly observed in distal myopathy genes where different variants in the same gene can cause the same or similar phenotypes. Another instance of genetic variability is locus heterogeneity, where mutations in different genes can result in a similar phenotype. For example, mutations in HNRNPA1, HNRNPA2B1, VCP, SQSTM1, and TIA1, can cause multisystemic proteinopathies.

Phenotypic heterogeneity is a less precise term referring to different mutations in one gene responsible for different phenotypes (Hilton-Jones & Turner, 2014). For example, missense and protein-truncating or splice variants in TTN can cause distal myopathies, congenital myopathies, HMERF, LGMD, and cardiomyopathies. Similarly, mutations in FLNC and MYH7 can cause both distal myopathies, proximal

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phenotypes, and cardiomyopathies. However, they can also cause disorders in entirely different organ systems, e.g., mutations in GNE can cause recessive distal myopathy or a dominant rare metabolic disorder called Sialuria (Seppala et al., 1999).

2.2.3 Molecular diagnostic strategies

A correct molecular diagnosis is essential for patients with genetic myopathies as identification of the causative mutation(s) facilitates informative genetic counseling, proper patient care, and management, as well as the administration of appropriate therapeutic interventions. Several diagnostic strategies exist for identifying pathogenic or putative pathogenic variants in patients with inherited myopathies.

These involve knowing the mode of inheritance in the pedigree, availability of appropriate muscle tissue, and family DNA material.

2.2.3.1 DNA sequencing

In an unsolved myopathy family, DNA is collected from patients and available and informative family members. The causative genes can be identified in cohorts with established genetic diagnostic methodologies for most of the currently known myopathies. However, most patients with myopathies referred for genetic testing remain without a final diagnosis due to yet unknown molecular mechanisms (Savarese et al., 2016).

Sequencing the coding regions of DNA or exons is referred to as Exome Sequencing (ES), previously Whole exome sequencing (WES). Several different capture-based methods are available to enrich, amplify and sequence fragmented DNA.

These methods utilize massive parallel sequencing (MPS), a type of high throughput sequencing (HTS). However, analyzing ES data requires heavy computational infrastructure and knowledge of sequencing data analysis. In a targeted gene panel sequencing, known causative genes and candidate genes

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with similar biochemical properties are included. Several diagnostic laboratories have custom-targeted gene panels that aid in the rapid molecular diagnosis of rare disease patients.

Examples of these are the MYOcap (Evila et al., 2016) and the Motorplex (Savarese et al., 2014) gene panels. In addition to these, several commercial diagnostic panels are nowadays also available from Illumina, Agilent, and Roche, and other private diagnostic services.

Alternatively, MPS methods are also used to sequence the genomic regions of DNA, including the coding and the non- coding part, referred to as Genome Sequencing (GS), previously Whole Genome sequencing (WGS). In recent years ES and GS strategies are increasingly becoming standard diagnostic approaches for Mendelian diseases (Xue et al., 2015). However, some technical challenges remain. Non- uniform coverage issues of complex sequence regions, segmental duplications, and satellite regions or ‘holes’ have been identified in all capture-based methods (Meienberg et al., 2015; Barbitoff et al., 2020) suggesting that neither method yet sequence all the targeted regions, entire exome or genome with uniform and adequate depth.

2.2.3.2 RNA sequencing

Despite the success of DNA sequencing methods based on MPS, the current diagnostic rate in myopathies is low, and additional sequencing strategies like transcriptome sequencing can add more levels of information. High throughput sequencing of tissue-specific mRNA (RNA-seq) from skeletal muscle biopsies or RNA obtained from fibroblasts of the patients reflect the disease specificity and can aid in the molecular diagnosis of unsolved myopathies. Recent studies have shown an increase in the rate of detecting novel pathogenic mutations in neuromuscular disorders via RNA-seq (Cummings et al., 2017; Gonorazky et al., 2019). However, RNA-seq is not a standalone diagnostic tool, and the framework for guidelines and interpretations of variant

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findings from RNA-seq for diagnostic uses is still in progress.

RNA-seq can also improve the interpretation of variants identified on DNA and provide information about possible allele-specific changes or splicing aberrations (Wang et al., 2009; Byron et al., 2016). Sequencing data from RNA have traditionally been used for differential expression profiling.

However, with the advancements in systems and network interaction studies, RNA-seq can be used to understand intramolecular and intermolecular RNA-RNA interactions (RRIs) or interactions with protein, thus providing valuable information on different biological processes in the interactome (Stark et al., 2019).

2.2.3.3 Variant pathogenicity

A significant challenge in analyzing sequencing data is to determine the pathogenicity of the variants. The American College of Medical Genetics and Genomics (ACMG), along with the Association of Molecular Pathology (AMP), has issued a set of guidelines for interpretation of variants in Mendelian diseases (Richards et al., 2015). These guidelines recommend the use of specific terms -"pathogenic," "likely pathogenic,"

"uncertain significance," "likely benign," and "benign"-to describe variants identified from MPS data. These guidelines suggest classifying the variants into these five categories based on different levels of evidence, e.g., minor allele frequency (MAF) in population data, in silico predictions, functional data, and segregation data.

Estimation of a variant frequency threshold can be made based on the mode of inheritance in the family and population frequency of the variant. Additionally, the segregation of variants with the phenotype in the family increases the value of the information provided for determining the variant pathogenicity. Consequences of the genetic variant on protein level are estimated with in silico prediction tools like CADD, SIFT, PolyPhen, and MutationTaster, but should just be

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considered preliminary assumptions. Functional studies in cell or animal models can be highly informative in asserting the damaging role of the genetic variant. Strong evidence of pathogenicity of a previously not reported variant requires a very low MAF in control population or public databases along with segregation data (or a confirmed de novo) and well- established functional studies showing the deleterious effects of the variants. On the other hand, strong evidence of the benign effect of the variant is considered when the MAF is too high in the population, the variant fails to segregate with the phenotype, and functional studies show no harmful effect of the variant.

Approximately 40% of variants observed in massively parallel sequencing data can be categorized as variants of uncertain or unknown significance (VUS) (Federici & Soddu, 2020). The interpretation of VUS, in both clinical and research settings, remains challenging for all Mendelian and complex diseases. The use of additional ‘omics’ data like transcriptomics, proteomics or metabolomics, functional studies, and network-based gene association studies can significantly improve the molecular understanding of most of these variants. Additionally, the use of variant pathogenicity guidelines (Richards et al., 2015) is a must in determining the pathogenicity score of a particular variant concerning the phenotype and the families being studies.

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2.3 Inclusion body myositis (IBM)

Idiopathic inflammatory myopathies (IIMs), also known as myositis, are disorders with muscle weakness and chronic inflammation in the muscle tissue (Lundberg et al., 2018).

Based on symptoms, such as muscle weakness, skin rash, and histopathology, IIMs include dermatomyositis, polymyositis, antisynthetase syndrome myositis, overlap myositis with systemic autoimmune disease, IBM, and immune-mediated necrotizing myopathy (IMNM). However, this classification system is limited as IBM is clinically and pathologically distinct from the other IIMs. In IBM patients, the onset of muscle weakness is insidious and often thought to have earlier (Badrising et al., 2005) than the mean age of onset of 61 to 68 years (Benveniste et al., 2011; Dobloug et al., 2012; Suzuki et al., 2012; Tan et al., 2013; Suzuki et al., 2016).

Histopathologically, characteristic features are endomysial infiltrates and the invasion of non-necrotic muscle fibers primarily by CD8⁺ T-cells besides the frequent rimmed vacuolated fibers in the muscle biopsies of IBM patients (Greenberg, 2019). Additionally, the selective pattern of atrophy and weakness in quadriceps and forearm distal muscles, i.e., long finger flexors and no response to immune therapies, presents IBM as an enigmatic entity in the IIM group.

The underlying molecular pathomechanisms of IBM are poorly understood, and consequently, no curative therapeutic strategy exists to treat IBM patients yet.

2.3.1 Epidemiology

According to a meta-analysis study, the latest estimates suggest a prevalence of 24-46 per million (Callan et al., 2017).

However, in different relatively isolated populations in Europe, the prevalence of IBM is slightly higher. In Finland, the prevalence of IBM is about 70 per million (Udd, 2007). In Ireland, it is about 112 per million (Lefter et al., 2017). The ratio

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of male patients to female patients is highly variable between 0.5 (Wilson et al., 2008) to 6.5 (Amato et al., 1996).

Due to poor understanding of the disease, IBM was initially often misdiagnosed in about 40-53% of patients (Needham et al., 2008; Suzuki et al., 2016; Callan et al., 2017). However, in recent years, the clinical and histopathological awareness of the disease has led to a better diagnosis encouraging a better course of prevalence studies in different populations. On average, it takes 4.6 to 5.8 years from the onset of symptoms to receive a correct diagnosis of IBM (Needham et al., 2008;

Dobloug et al., 2015; Suzuki et al., 2016).

2.3.2 Phenotype

IBM patients show a unique and relatively homogenous pattern of often asymmetric muscle weakness and atrophy in long finger flexors accompanied by atrophy and weakness in quadriceps and, later, the ankle dorsiflexors. Knee extensor weakness and ankle dorsiflexion weakness result in difficulties walking, climbing stairs, and poor rising from the squat, while finger flexor weakness results in a weak grip. These initial symptoms are often presumed as symptoms of aging or arthritis and occasionally lead to a misdiagnosis of polymyositis or a motor neuron disease (Greenberg, 2019).

Interestingly this pattern of weakness in finger flexion, knee extension, and ankle dorsiflexion suggests, apart from the quadriceps weakness, shows a clear overlap with the distal myopathies.

MRI in IBM patients shows characteristic involvement of the corresponding muscles (Tasca et al., 2015) including the distinct atrophy and simultaneous inflammatory edema in the quadriceps. Dysphagia in IBM patients is more evident with the progression of the disease and can often result in poor nutrition, weight loss, and pneumonia, contributing mainly to

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IBM-related fatalities (Oh et al., 2008; Cox et al., 2011; Price et al., 2016).

Muscle biopsies of IBM patients show characteristic muscle fiber degeneration, demonstrated by rimmed vacuoles accompanied by cytotoxic CD8⁺ T cell infiltration in non- necrotic fibers and rare congophilic protein accumulations or cytoplasmic inclusions. In addition, mitochondrial pathology and upregulation of MHC-class I antigen are vital components for the diagnosis. However, the precedence of either of these molecular pathological events has remained unclear as patients usually undergo diagnostic evaluations many years after the onset of symptoms. An earlier hypothesis suggests that the aging of muscle fibers and the accumulation of different misfolded or ubiquitinated proteins in muscle fibers may play a primary role in the pathogenesis leading to the degeneration of muscle fibers and consequently inviting immune response (Askanas & Engel, 2001). Alternatively, a widely supported theory of immune-mediated muscle fiber degeneration suggests that IBM is in fact, an autoimmune disease (Koffman et al., 1998; Badrising et al., 2004; Benveniste et al., 2015).

The diagnostic criteria defined by ENMC include two characteristic histopathological diagnostic markers of IBM, endomysial lymphocyte infiltrates with invasion in myofibers and the presence of rimmed vacuoles (Rose & Group, 2013).

However, a revised data-driven diagnostic criterion includes either invasion of non-necrotic muscle fibers or rimmed vacuoles (Lloyd et al., 2014) allowing a more sensitive diagnosis of Polymyositis-Mito or mitochondrial myositis patients as IBM. This division is also referred to as inflammatory myopathy with vacuoles, aggregates, and mitochondrial pathology (IM-VAMP). In IBM, the mitochondrial pathology refers to the presence of COX- negative and SDH-positive myofibers and less frequent ragged red myofibers. Multiple mtDNA deletions are part of the mitochondrial pathology and one of the diagnostic features in IBM (Oldfors et al., 1993; Rygiel et al., 2015). These features

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are accompanied by nonspecific myopathic changes: increased fiber size variation and number of internal nuclei. Cytoplasmic inclusions are very rare on light microscopy of muscle biopsy sections of IBM patients. The presence of rimmed vacuoles, mitochondrial pathology, and cytoplasmic inclusions is the degenerative part of IBM pathology. Overexpression of Class I, and to a lesser degree Class II, MHC in all muscle fibers together with the inflammatory cell response make the immune-mediated part of the pathology (Rodriguez Cruz et al., 2014).

Several studies have reported the varying degree of protein aggregation in IBM (Askanas et al., 1991; Mendell et al., 1991;

Askanas & Alvarez, 1992; Askanas et al., 1992a; Askanas et al., 1992b; Askanas et al., 1992c; Askanas et al., 1993a; Askanas et al., 1993b). However, the number of proteins and specificity of protein aggregation in IBM is still challenging to understand (Greenberg, 2009, 2019). Recent studies have shown the presence of more reliable autophagy biomarkers like TARDBP/TDP-43, SQSTM1/p62, and LC3 in IBM muscles (Weihl et al., 2008; Salajegheh et al., 2009; Hiniker et al., 2013). Lately, serum anti-cN1A antibodies have also been reported and are used as a supporting diagnostic marker of IBM. However, the specificity and sensitivity of these antibodies are still under study (Ikenaga et al., 2021; Paul et al., 2021).

The invasion of muscle fibers by highly differentiated CD8⁺ cytotoxic T-cells together with endomysial infiltrates is considered a hallmark feature of IBM (Greenberg et al., 2016;

Greenberg et al., 2019). The highly differentiated CD4⁺ and CD8⁺ cells are seen in both muscles and blood of IBM patients (Salajegheh et al., 2007; Pandya et al., 2010; Allenbach et al., 2014). Additionally, an increase in levels of chemokines and cytokines such as CXCL9 and CCL5 has been reported (Greenberg et al., 2002) suggesting upregulation of IFNg production. Simultaneously, increased expression of genes participating in interferon signaling is seen in IBM muscles

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(Raju et al., 2003; Ivanidze et al., 2011; Pinal-Fernandez et al., 2019).

2.3.3 Nomenclature issues

Historically, similar nomenclature and abbreviations largely used in previous literature have contributed to diagnostic issues in IBM. The abbreviation IBM refers to inclusion body myositis and not to inclusion body myopathy or a group of rimmed vacuolar diseases with overlapping clinical features (Greenberg, 2019). The classification of rimmed vacuolar myopathies with predominant distal muscle weakness includes several inherited diseases that have been previously termed inclusion body myopathies. An example of this is the GNE myopathy (Nonaka myopathy), previously known as hereditary inclusion body myopathy (hIBM) or IBM2 due to its hereditary nature and rimmed vacuoles in muscle biopsies of patients. Similarly, distal myopathy phenotypes with rimmed vacuolar pathology caused by pathogenic mutations in DES and MYH2 have been termed IBM1 (OMIM: 601419) and IBM3 (OMIM: 605637), respectively. Multisystemic proteinopathies (table 1) caused by pathogenic mutations in VCP, HNRNPA1, HNRNPA2B1, and SQSTM1 (digenic with TIA1) have also been previously referred to as Inclusion body myopathy with Paget disease with or without frontotemporal dementia (IBMFPD).

The term sporadic IBM or sIBM/s-IBM might separate the myositis phenotype from hIBMs (Askanas & Engel, 1993) referring to the former’s sporadic nature and no family history of weakness. However, the use of sporadic and hereditary terms with the abbreviation IBM gives a wrong impression about the inheritance and phenotype of IBM (Greenberg, 2019). The rare occurrence of familial cases, i.e., multiple/several diagnoses of IBM in different members of the same family, does increase the complexity of the disease nomenclature. However, the use of sporadic IBM and familial IBM (fIBM/f-IBM) is still better than outdated and confusing terms for inherited non-inflammatory myopathies with

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rimmed vacuoles. The use of these terms is often confusing and clutter the clinical literature, possibly even hampering the proper diagnosis of patients.

2.4 Molecular genetics of IBM

As noted previously, IBM shares clinical and pathological similarities with other late-onset rimmed-vacuolar distal myopathies, e.g., distal myopathy caused by mutations in DNAJB6, GNE myopathy (GNE), MSP1 (VCP), MSP2 (HNRNPA2B1), MSP3 (HNRNPA1), and MSP4 (SQSTM1 + TIA1). However, the latter follow Mendelian inheritance (except MSP4) and often have a positive family history and material available for studies. In IBM, due to the late age of onset and slowly progressive symptoms, often the parents are not available to study the vertical transmission of possibly pathogenic genetic variants. A couple of studies also observed rare variants in VCP and SQSTM1 in a few IBM patients (Weihl et al., 2015; Gang et al., 2016). However, these findings have not been replicated, and variant pathogenicity has not been explored further.

A hypothesis of a possible multi-factorial influence on pathomechanisms in IBM (Machado et al., 2014) is postulated, supported by negative DNA sequencing results, further indicating the need to explore a possible non-Mendelian inheritance in IBM.

2.4.1 Interpretation of rare variants in IBM

Since a few studies have observed rare variants in candidate genes such as VCP and SQSTM1 (Weihl et al., 2015; Gang et al., 2016), patients diagnosed with IBM should undergo sequencing of already known genes and candidate genes, especially those known to cause the overlapping rimmed- vacuolar phenotypes. On the one hand, identifying a previously known pathogenic variant in these genes in a probable-IBM patient can help re-evaluate the diagnosis. On the other hand,

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observations of rare/unique variants in these genes can help understand their incidence and burden with a good sample size.

A recent study also suggested enrichment of rare variants in FYCO1 and its association with IBM pathology (Güttsches et al., 2017). However, rare variants in FYCO1 are commonly seen in the population (unpublished data) and are seen routinely in sequencing analyses of neuromuscular patients. Careful statistical re-analysis of genetic data and a gene burden test on related genes can help understand the burden of rare variants in IBM patients.

2.4.2 Association studies in IBM

Previous studies of SNP genotyping and HLA typing have shown strong genetic linkage to the 8.1 ancestral haplotype region on chromosome 6 (Garlepp et al., 1994; Koffman et al., 1998; Kok et al., 1999; Lampe et al., 2003; Badrising et al., 2004; Price et al., 2004; O'Hanlon et al., 2005; Mastaglia et al., 2006; Scott et al., 2006; Needham et al., 2008; Mastaglia et al., 2009; Rojana-udomsart et al., 2011; Rojana-udomsart et al., 2012b; Rojana-udomsart et al., 2013; Rothwell et al., 2017;

Rothwell et al., 2019). These studies primarily identified association with HLA-DRB1 alleles, namely, *03:01, *01:01, and *13:01. Additionally, polymorphisms within the MHC region in the genes BTNL2 and NOTCH4 have also been included in susceptibility associations (Scott et al., 2011; Scott et al., 2012). Contrastingly, due to the rarity of the disease, these studies have remained underpowered to find higher resolution associations or other significant non-HLA associations. Due to this reason, traditional genotyping-based association studies at exome-wide (EWAS) or genome-wide (GWAS) level have not yet been performed in IBM.

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2.4.3 Gene expression studies in IBM

Previous studies have shown distinct gene expression profiles in IIMs. Using microarray-based methods, Greenberg and colleagues showed the molecular profile of different inflammatory myopathies (Greenberg et al., 2002). They showed heavy overexpression of cytokines and immunoglobulins in IBM. They also noticed increased expression of genes related to actin cytoskeleton that could provide interesting new information regarding muscle pathogenesis in IIMs. However, the small study size may partly limit the interpretation of gene expression differences correctly. Greenberg and colleagues later improved their findings by studying microarray data from 411 muscle samples, including 40 IBM muscles (Greenberg et al., 2019). They showed an IBM-specific signature of highly differentiated CD8+ T-cell effector memory and terminally differentiated effector cells or TEMRA. The authors also found a correlation of KLRG1 expression with T cell cytotoxicity and suggested that KLRG1 could be a promising therapeutic target for IBM patients.

Using an RNA-seq based method on a small cohort, Amici and colleagues suggested that perturbations of calcium regulations could be significant in IBM muscles (Amici et al., 2017). The authors supplemented their findings by comparing protein to transcript ratio in IBM vs. normal muscles. Pinal- Fernandez and colleagues studied differential signature of interferons in IIMs using RNA-seq. They identified that genes associated with type 1 interferon (IFN1) pathway are expressed low in IBM compared to other IIMs. In contrast, genes expressed in the type 2 interferon (IFN2) pathway are high in IBM and DM (Pinal-Fernandez et al., 2019). Later, Pinal- Fernandez and colleagues differentiated between these patterns using a specialized machine learning approach to suggest a unique gene expression profile in IBM (Pinal- Fernandez et al., 2020).

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While the above studies looked for changes in coding genes, a few studies also analyzed the non-coding genes in IIMs.

Hamman and colleagues compared different IIMs and showed a differential expression profile of lncRNAs such as H19, lncMyoD, and MALAT1 (Hamann et al., 2017). However, due to the small cohort size, the interpretation of these results could be challenging. Eisenberg and colleagues studied the miRNA profile of different primary muscle disorders using microarray- based methods (Eisenberg et al., 2007). They showed that miRNAs associated with MAPK signaling, T cell receptor signaling, and actin cytoskeleton are differentially expressed in the IBM transcriptome.

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3 Aims of the Study

This thesis aimed to study the genetics of late-onset sporadic patients with or without inflammatory features.

The specific objectives were:

1. To understand the molecular genetic background of sporadic inclusion body myositis using DNA and RNA sequencing data analysis.

2. To clarify molecular genetics of a late-onset previously unreported distal myopathy phenotype observed in apparently sporadic patients.

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4 Subjects and Methods

4.1 Sample and data collection

Patient data and sample collection are described for each project. Local ethics committees approved the study protocols, and ethical approval for the study is under HUS:195/13/03/00/11. Informed consent from the patients was obtained at the time of sample collection.

4.1.1 Finnish and Italian IBM patients (I)

Thirty patients from Finland and twelve patients from Italy with a negative family history of muscle weakness and diagnosis of IBM based on ENMC criteria (Rose & Group, 2013) were included in this study. The median age of onset of symptoms (median ± SD) was 60 ± 10.3 years in Finnish IBM patients and 66.5 ± 9.4 years in Italian IBM patients. The ratio of male to female IBM patients in the Finnish cohort was 1.5, and 1.4 in the Italian cohort. Genetic data from 94 non-myositis controls diagnosed with inherited neuromuscular disorders and 99 healthy Finnish controls from the 1000 genomes project were used. Blood samples were obtained from patients, and genomic DNA was extracted according to the manufacturer’s instructions using the Qiagen DNA extraction kit (Qiagen, Hilden, Germany) and stored at -20 °C for further use.

4.1.2 Extended IBM cohort (U)

Eighty-one Finnish IBM patients (including the 30 patients mentioned above) and twenty-nine Italian IBM patients were included in the enlarged cohort. The age of onset of symptoms for Finnish IBM patients was 60 ± 9.5 years, comprising 38 male and 43 female patients (ratio = 0.8). For Italian IBM patients, the age of onset was 63 ± 11, comprising 16 male and 13 female patients (ratio = 1.2). Blood samples were obtained from patients, and genomic DNA was extracted according to

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the manufacturer’s instructions using the Qiagen DNA extraction kit (Qiagen) and stored at -20 °C for further use.

The control group consisted of 256 Finnish hematopoietic stem cell transplantation (HSCT) register donors, and the DNA samples were collected at Finnish Red Cross Blood Service, Finland.

4.1.3 IBM patients, TMD patients, and amputees (II)

Muscle biopsies from twenty-four Finnish IBM, six TMD patients, and nine individuals that underwent lower leg amputation were also included. The median age of onset for IBM patients was 60 ± 11 years, and the age at muscle biopsy was 70 ± 9 years. Similarly, for TMD patients, the age of onset was 49 ± 11 years, and the age at biopsy was 54 ± 14 years. These nine biopsies from amputees were negative for pathologically defined muscle degeneration and did not show any signs of inflammation. Age at biopsy for these amputees was 70 ± 11 years. All muscle biopsies were snap-frozen and stored at –80

°C and processed for RNA extraction as detailed below. All extracted RNA samples were stored at –80 °C.

4.1.4 Patients and family members (III)

Data was collected from ten distal myopathy patients and eight asymptomatic family members from nine different families. All patients underwent clinical neurological examination. Blood samples were obtained from individuals, and genomic DNA was extracted according to the manufacturer’s instructions using the Qiagen DNA extraction kit (Qiagen) and stored at -20 °C for further use.

4.1.5 RNA extraction (II)

RNA was extracted from skeletal muscle biopsies of IBM and TMD patients and amputees. SpeedMill PLUS (Analytik Jena AG, Germany) was used to homogenize the muscle tissue.

Molecular Genetics of Inclusion Body Myositis and Late-Onset Rimmed-Vacuolar Distal Myopathy

Contents

List of Original Publications

Author Contributions

Abbreviations

Abstract

Referat

1 Introduction

2 Review of the Literature

3 Aims of the Study

4 Subjects and Methods