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Increasing Role of Titin Mutations in Neuromuscular Disorders

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Increasing Role of Titin Mutations in Neuromuscular Disorders Journal of Neuromuscular Diseases 3 (2016) 293–308 293 DOI 10.3233/JND-160158 IOS Press Review Increasing Role of Titin Mutations in Neuromuscular Disorders Marco Savaresea, Jaakko Sarparantaa,b, Anna Viholaa, Bjarne Udda,c,d and Peter Hackmana,∗ aFolkh¨alsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland b View metadata,Albert citation Einstein and similar College papers of Medicine, at core.ac.uk Departments of Medicine–Endocrinology and Molecular Pharmacology,brought to you by CORE Bronx, NY, USA provided by Helsingin yliopiston digitaalinen arkisto cNeuromuscular Research Center, University of Tampere and Tampere University Hospital, Tampere, Finland dDepartment of Neurology, Vaasa Central Hospital, Vaasa, Finland Abstract. The TTN gene with 363 coding exons encodes titin, a giant muscle protein spanning from the Z-disk to the M-band within the sarcomere. Mutations in the TTN gene have been associated with different genetic disorders, including hypertrophic and dilated cardiomyopathy and several skeletal muscle diseases. Before the introduction of next generation sequencing (NGS) methods, the molecular analysis of TTN has been laborious, expensive and not widely used, resulting in a limited number of mutations identified. Recent studies however, based on the use of NGS strategies, give evidence of an increasing number of rare and unique TTN variants. The interpretation of these rare variants of uncertain significance (VOUS) represents a challenge for clinicians and researchers. The main aim of this review is to describe the wide spectrum of muscle diseases caused by TTN mutations so far determined, summarizing the molecular findings as well as the clinical data, and to highlight the importance of joint efforts to respond to the challenges arising from the use of NGS. An international collaboration through a clinical and research consortium and the development of a single accessible database listing variants in the TTN gene, identified by high throughput approaches, may be the key to a better assessment of titinopathies and to systematic genotype–phenotype correlation studies. Keywords: TTN, titin, neuromuscular disorders, Limb-girdle muscular dystrophy (LGMD), Hereditary myopathy with early respiratory failure (HMERF), Late-onset autosomal dominant tibial muscular dystrophy (TMD), Congenital centronuclear myopathy (CNM), Early-onset myopathy with fatal cardiomyopathy (EOMFC), Multi-minicore disease with heart disease (MmDHD), Childhood-juvenile onset Emery-Dreifuss-like phenotype without cardiomyopathy INTRODUCTION Titin acts as a scaffold protein aiding in myofibril- lar assembly during myogenesis [2], as a molecular With its 363 coding exons and a full-length spring determining the passive elasticity of the mus- transcript of more than 100 kb [1] TTN gene encodes cle [3, 4], and as a mechanosensor serving various titin, the by far longest known polypeptide in nature. signaling functions [5, 6]. The longest human theoretical isoform of TTN would TTN mutations have to date been reported to cause produce a protein of 3,960 kDa containing 35,991 various cardiomyopathies [7, 8] and a range of skele- amino acids, although this isoform has not been tal muscle diseases and phenotypes listed below: observed [1]. ∗ – Late-onset autosomal dominant tibial muscular Correspondence to: Dr Peter Hackman, Folkhalsan Institute dystrophy (TMD) (MIM #600334); of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland. E-mail: – Young or early adult onset recessive distal peter.hackman@helsinki.fi. titinopathy; ISSN 2214-3599/16/$35.00 © 2016 – IOS Press and the authors. All rights reserved This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License (CC BY-NC 4.0). 294 M. Savarese et al. / Increasing Role of Titin Mutations in Neuromuscular Disorders – Limb-girdle muscular dystrophy type 2J in this review. Exon numbering will be according to (LGMD2J; MIM #608807); the HGVS recommendations [10] and to the current – Congenital centronuclear myopathy (CNM; Leiden database (LOVD) numbering (modified on MIM #255200); 11th October 2013, changing exon 47b to exon 48 – Early-onset myopathy with fatal cardiomyopa- and adding +1 to all subsequent exon numbers) [11]. thy, EOMFC (MIM #611705); The titin protein spans from the Z-disk to the M- – Multi-minicore disease with heart disease band [12]. Its modular structure is composed of four (MmDHD) including clinical variations; main parts (Fig. 1): the amino-terminal Z-disc region, – Childhood-juvenile onset Emery-Dreifuss-like the I-band and A-band regions, and the carboxyl- phenotype without cardiomyopathy; terminal part spanning the M-band. Titin is composed – Hereditary myopathy with early respiratory fail- of repeated immunoglobulin-like (Ig) and fibronectin ure (HMERF; MIM #603689); type 3-like (FN3) domains, interspersed by unique – Adult onset recessive proximal muscular dys- sequence regions [1]. It also contains the repetitive trophy. PEVK region, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues, in the I band, and a Mutations in titin will probably prove to be the serine/threonine kinase (TK) domain in the M-band. cause of many additional phenotypes of muscular More than 1 million splice variants could be gen- disorders in the coming years. erated theoretically by the TTN gene [13]. Indeed, Due to its huge size, it has not been possi- extensive alternative splicing results in a remarkable ble to sequence the entire TTN gene routinely in diversity of titin isoforms that can be divided into research and diagnostic laboratories until recently. three main classes based on the presence of the N2A Thus, before implementation of the next generation and N2B elements in the I-band region [1, 14, 15] sequencing (NGS) methods, only a limited amount (Fig. 1). Skeletal muscles express so-called N2A iso- of TTN mutations were identified. NGS sequencing forms, characterized by the inclusion of the N2A has enabled the rapid and thorough investigation of element and exclusion of the cardiac-specific N2B genetic material [9] and has resulted in an explosion element. In the heart, N2BA isoforms include both in the identification of new TTN variants. However, the N2B and N2A elements, while N2B isoforms use their clinical interpretation is a challenge. the N2B element only. The aforementioned isoforms Here, we focus on the current understanding of also differ in the lengths of the proximal tandem-Ig the titin gene and protein from a human disease and PEVK regions, which are longest in the N2A iso- perspective. In particular, we provide an overview forms and shortest in the N2B isoforms. Within each of the different neuromuscular disorders caused by isoform class, the tandem-Ig and PEVK regions also mutations in the TTN gene, reviewing the molecu- show variable expression in different muscles, and lar findings as well as the clinical data. Finally, we across developmental and physiological states. More- highlight the difficulties related to the interpretation over, the second last TTN exon 363 (Mex5), coding of the clinical significance of TTN variations and the for the is7 domain located in the M-band, is dif- need for further functional studies and bioinformatics ferentially spliced, producing is7– and is7+ isoforms tools. [16] The major isoform classes are represented in the THE TITIN GENE, ISOFORMS NCBI RefSeq database by the entries NM 133378 AND PROTEIN (N2A; NP 596869:3,680 kDa and 33,423 aa), NM 001256850.1 (N2BA; NP 001243779:3,780 The titin gene (MIM #188840), is located on the kDa and 34,350 aa), and NM 003319 (N2B; short arm of chromosome 2 (chromosomal band NP 003310:2,960 kDa and 26,926 aa) [1, 14, 15]. q31.2). It contains 363 coding exons and an additional The Novex-1 (NM 133432; NP 597676) and first non-coding exon [1]. The longest theoretical Novex-2 (NM 133437; NP 597681) isoforms are transcript (variant IC, NM 001267550.2), virtually similar to N2B, but they also include further 125 and obtained by the transcription of all the coding exons 192 amino acids encoded by the Novex-1 and Novex- (excluding the alternative C-terminal Novex-3 exon) 2 exons in the I-band. Finally, the much smaller and called “meta isoform”, has been adopted as the Novex-3 isoform (NM 133379; NP 596870:616 kDa gold standard for describing TTN variants, and will be and 5604 aa) only contains the N-terminal part of the used as reference for cDNA and protein numbering protein. This isoform, expressed on a low level in all M. Savarese et al. / Increasing Role of Titin Mutations in Neuromuscular Disorders 295 titin filament ZMZthin filament thick filament Ig domain FN3 domain unique sequence PEVK kinase (TK) Z-disc I-band A-band M-band proximal tandem-Ig distal tandem-Ig N2A / ex102–111 N2A PEVK / ex112–226 N2B / ex49 Z1 (Ig 1) / ex2 Novex-2 / ex46 M10 (Ig 152) / ex364 I106 (FN3 1) / ex253 / ex344 A150 (FN3 119) M1 (Ig 143) / ex359 Z9 (Ig 9) / ex28 / ex270 (FN3 11) I118 kinase (TK) / ex359 M-is7 / ex363 Z repeats / ex 8–14 I1 (Ig 10) / ex28 Novex-1 / ex45 I84 (Ig 80) / ex226 A1 (Ig 97) / ex271 A170 (FN3 132) / ex359 N C telethonin TPM PKA CAPN3 TPM CRYAB MyHC CaM FHL2 α-actinin actin PKG MARPs actin MyBPC NBR1 CAPN3 sAnk1 CAPN1 CRYAB CAPN1 p62 CMYA5 filamin C FHL1 MURF1 α-synemin nebulin FHL2 MURF2 OBSCN OBSL1 MYOM1 N2A (NP_596869) N2BA (NP_001243779) N2B (NP_003310) Novex-1 (NP_597676) Novex-2 (NP_597681) Novex-3 (NP_596870) Novex-3 Fig. 1. Top: A schematic view of the sarcomere, with titin filaments shown in red. One titin molecule, extending from the Z-disc to M- band, is highlighted. Middle: The modular structure of the titin protein (theoretical meta isoform). Titin is mostly comprised of repeated immunoglobulin-like (Ig; red) and fibronectin type 3-like (FN3; white) domains. Selected domains are labeled above the diagram with the classical titin nomenclature (sarcomere region Z/I/A/M+domain number; Bang et al.
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