The complexity of titin splicing pattern in human adult skeletal muscles

RESEARCH Open Access The complexity of titin splicing pattern in human adult skeletal muscles Marco Savarese 1,5*† , Per Harald Jonson 1† , Sanna Huovinen 2 , Lars Paulin 3 , Petri Auvinen 3 , Bjarne Udd 1,2,4 and Peter Hackman 1 Abstract Background: Mutations in the titin gene (TTN) cause a large spectrum of diseases affecting skeletal and/or cardiac muscle. TTN includes 363 coding exons, a repeated region with a high degree of complexity, isoform-specific elements, and metatranscript-only exons thought to be expressed only during fetal development. Although three main classes of isoforms have been described so far, alternative splicing events (ASEs) in different tissues or in different developmental and physiological states have been reported. Methods: To achieve a comprehensive view of titin ASEs in adult human skeletal muscles, we performed a RNASequencing experiment on 42 human biopsies collected from 12 anatomically different skeletal muscles of 11 individuals without any skeletal-muscle disorders. Results: We confirmed that the skeletal muscle N2A isoforms are highly prevalent, but we found an elevated number of alternative splicing events, some at a very high level. These include previously unknown exon skipping events and alternative 5′and 3′splice sites. Our data suggests the partial inclusion in the TTN transcript of some metatranscript-only exons and the partial exclusion of canonical N2A exons. Conclusions: This study provides an extensive picture of the complex TTN splicing pattern in human adult skeletal muscle, which is crucial for a proper clinical interpretation of TTN variants. Keywords: Titin, Titinopathies, RNA-sequencing, Exon usage, Alternative splicing events, Splicing pattern Background The TTN gene encodes titin, a muscle protein spanning from the Z-disk to the M-band within the sarcomere. The genomic structure of TTN is quite remarkable. It contains 364 exons (363 coding exons plus the first non-coding exon) and can theoretically generate more than one million splice variants [1,2]. It also has a large repeated region withahighdegreeofcomplexity[1]. Titin isoforms have traditionally been classified in three main categories based on the presence of the N2A and N2B elements within the I-band region [3–5]. N2A isoforms (mainly expressed in the skeletal muscles) contain the N2A element, but not the cardiac-specific N2B element. On the contrary, N2B isoforms only include the cardiac-specific N2B element. N2BA isoforms, expressed in the heart, include both the N2B and N2A elements. N2A and N2BA isoforms also include additional exons, resulting in a higher number of Ig and PEVK domains in the I-band region. Two further isoforms, named Novex-1 and Novex-2, are very similar to N2B but each of them includes an isoformspecific exon (exon 45 and exon 46, respectively). Finally, the Novex-3 isoform only contains the N-terminal part of titin due to an alternative stop codon in the Novex-3specific exon 48. Interestingly, specific exons included in the inferred complete metatranscript (NM_001267550.1) and referred to as metatranscript-only or meta-only exons are thought to be expressed only during embryonic development. Thereafter, they are not included in the canonical soleusderived N2A skeletal muscle isoform, or in any of the five cardiac isoforms. * Correspondence: [email protected] † Equal contributors 1 Folkhälsan Research Center, University of Helsinki, Helsinki, Finland 5 Folkhälsan Institute of Genetics, Department of Medical Genetics, University of Helsinki, Biomedicum, Haartmaninkatu 8, Pb 63, 00014 Helsinki, Finland Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Savarese et al. Skeletal Muscle (2018) 8:11 https://doi.org/10.1186/s13395-018-0156-z An extensive use of alternative splicing (AS) in different tissues or in different developmental and physiological states has been reported, resulting in a longer or smaller protein [2]. This reflects the global massive use of tissue-specific AS events (ASEs) which have been described in the skeletal muscle [6,7]. Although the presence of multiple different transcripts originating from TTN gene as consequence of ASEs has been partly suggested by experimental evidence [1,2,8], we still lack a clear picture of the global exon usage and of the subsequent splicing profile of TTN muscular transcripts. The introduction of RNA sequencing (RNA-Seq) methods has enabled a comprehensive study of the transcriptome [9]. Although early work focused on gene-expression analyses, RNA-Seq is a powerful tool for the identification and the studyofalternativeexonandsplicesiteusageandofnovel isoforms. It also allows an accurate quantification of relative transcript abundances [6,10]. In this study, we analyzed RNA sequencing data of human adult skeletal muscle tissues to obtain a comprehensive view of titin ASEs. This is crucial for a proper clinical interpretation of TTN variants that have been associated with a wide spectrum of human diseases and for an improved genotype-phenotype correlation [11–16]. Methods Skeletal muscle samples and RNA extraction Data was generated using 42 human skeletal muscle samples dissected from 12 anatomically different skeletal muscles (tibialis anterior,flexor hallucis longus, soleus,extensor digitorum longus,gracilis,semitendinosus,semimembranosus,vastus medialis,vastus lateralis,sartorius,biceps femoris,adductor magnus) collected from 11 adult individuals (7 males and 4 females) who had undergone above or below-the-knee amputation surgery for medical reasons other than neuromuscular disorders (Additional file 1:Table S1). A written informed consent was signed by all the patients and the Tampere University Hospital (Tampere, Finland) Ethics Committee approved the study. The samples (5 × 5 mm of size) were processed immediately after their removal to avoid tissue degradation as previously described [17]. Total RNA was extracted from the selected samples by the TRIzol reagent method, according to the manufacturer’sinstructions (Invitrogen, Life Technologies, Canada). RNA quality was checked with BioAnalyzer equipment using the RNA 6000 Nano Assay kit (Agilent Technologies, CA, USA). Library preparation, sequencing, and bioinformatics Indexed sequencing libraries were generated from 1 μg of total RNA, using the TruSeq Stranded Total RNA kit according to the manufacturer’sinstructions(Illumina, CA, USA). Single-end sequencing (86 bp reads) of multiplex libraries was performed on NextSeq500 instrument. Raw reads were mapped against the hg19 human reference genome using TopHat2 [18]. TopHat was also used for detecting and counting exon junctions. Alternative splice sites were evaluated using Human Splicing Finder (HSF) program [19]. For each exon, the inclusion rate was calculated as [(I/2)/[(I/2) + E], where Iis the number of reads supporting the exon inclusion (all junctions going into and exiting the exon) and E is the number of reads supporting its exclusion. Experimental validation of alternative splicing events For experimental validation of RNA-Seq results, cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Thermo Scientific, USA). RT-PCRs were performed using 1 μlofcDNAandaDreamTaq™ DNA Polymerase (Thermo Scientific). Primers were designed with Primer3 software (sequences available upon request). Amplified products were separated on 2% agarose gels and specific electrophoresis bands, corresponding to differently spliced products, were extracted using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Germany) and analyzed by Sanger sequencing. Publicly available data We also evaluated the presence of the ASEs in publicly available total mRNA sequencing data of adult gastrocnemius medialis from the ENCODE project (https://www.encodeproject.org; accession numbers ENCFF219LYV, ENCFF308RYZ, ENCFF569TCU, ENCFF408QZN, and ENCFF064NBB). Junctions were extracted from the available bam-files using regtools (https://github.com/griffithlab/regtools). Similarly, we analyzed RNA-Seq data from fetal skeletal muscles (accession numbers ENCFF009MKH, ENCFF084FDS, ENCFF121PKV, and ENCFF405BHX), fetal heart (accession numbers ENCFF111DKK, ENCFF686KAP, ENCFF167WVS, and ENCFF174EGJ), and adult heart (accession numbers ENCFF735RZM, ENCFF834OIQ, ENCFF608FZD, and ENCFF621SXE) from ENCODE. Results Before focusing on alternative splice events, we analyzed the canonical junctions, which are present in the previously reported isoforms, to evaluate their relative expression in human adult skeletal muscles. As expected, the junction 47–50, uniquely present in the previously identified skeletal long isoform N2A [1], is detected at very high level in all our samples (Fig. 1a). This confirms that most of the skeletal muscle transcripts belong to this class of isoforms. Savarese et al. Skeletal Muscle (2018) 8:11 Page 2 of 9 We then calculated the number of reads supporting each of the N2A canonical splicing events (Additional file 2: Table S2). Most of the canonical N2A junctions were identified. Interestingly, we noticed a very low number of reads supporting the inclusion of exon 11 (junctions linking exon 10–11 and exon 11–12). Similarly, we did not observe reads connecting exons 183 and 203. Then, we proceeded to the analysis of the alternative splice events. We identified 4039 unique splicing events, most of them in one or a few samples and supported by a very low number of reads. In order to eliminate the background sequencing noise and/or very weakly expressed transcripts, we applied a stringent quality control (QC)-filtering process, prioritizing only splicing events (n=498) supported by at least 1000 reads and identified in at least 14 samples. To reduce possible artefacts due to technical issues and obtain a less biased splicing pattern, we analyzed publicly available total mRNA sequencing data of adult gastrocnemius medialis from the ENCODE project. In a very conservative approach, we only focused on splicing events identified in our experimental samples as well as in the publicly available data (> 10 reads in ENCODE data). After that, we proceeded with a multistep analysis, based on two different categories of splicing events: (1) ASEs involving canonical splice sites (out of the repeated region and within this area) and (2) ASEs involving alternative splicing sites. 1) We identified 46 unreported exon junctions, involving canonical splice sites out of the repeated region (Table 1). All these 46 ASEs are predicted to maintain the frame. For 23 ASEs, we performed RTPCR and all confirmed the RNA-Seq results. We identified three ASEs (10–12; 10–13; 10–14), suggesting the skipping of exon 11. In line with the RNA-Seq results, RT-PCR confirms that exon 11 was poorly expressed in human adult skeletal muscle (Fig. 1b). Interestingly, 24 unreported junctions span metaonly exons, suggesting their partial inclusion in TTN human adult skeletal muscle transcripts (Table 1 and Fig. 1c). We identified a high number of reads involving the canonical splice sites of exons included in the a b c d e Fig. 1 Isoform identification and titin alternative splicing events in human skeletal muscle. aThe previously reported classes of isoforms differ from each other by the inclusion/exclusion of exons 48 (included only in Novex3 isoform) and 49 (included in all the other isoforms except the long skeletal muscle N2A-isoform). Our data suggests that N2A isoform is 20 times more expressed than Novex3. All the other isoforms have a very low expression. bWe identified a low number of reads connecting exon 11 to its flanking exons. On the contrary, a high number of reads connect exon 10 to exon 12 and 13, thereby skipping exon 11. In line with the RNA-Seq results, a standard RT-PCR (forward primer on exon 9 and reverse primer on exon 13, red arrows) and agarose gel electrophoresis show a very low abundance of the transcript including exon 11. M1 = 100 bp ladder. cSeveral RT-PCRs and agarose gel electrophoresis show a variable expression of metatranscript-only exons, confirming the RNASeq results. In particular, no expression of exons 163 and 165 is detected; on the contrary, all the other RT-PCRs result in a detectable band corresponding to the expected size. M1 = 100 bp ladder; M2 = 1 kb ladder; d = PCR from a control DNA; c = RT-PCR from a control cDNA (obtained by a retrotranscription of RNA extracted from gracilis muscle). dTitin repeated region is composed of nine exons/blocks (here represented by different colors and named B1-B9) repeated three times. Within the repeated region, linear expression of consecutive exons has been detected. Moreover, a number of alternative splicing events has been identified. eWe detected alternative splicing acceptors or donors leading to subtle changes in the produced protein. The splice-site strength for canonical splice sites (5′ss and 3′ss) as well as for alternative sites (alt 5′ss, alt 3′ss) has been calculated by Human Splice Finder (HSF) Savarese et al. Skeletal Muscle (2018) 8:11 Page 3 of 9 repeated area (Additional file 3: Supplementary Material 1, Additional file 4: Table S3 and Additional file 5: Table S4). Well-known bias due to such repetitive regions hampers a comprehensive and accurate study of this region. However, our data suggests the linear expression of consecutive exons within this area. We also identified a number of ASEs linking non-consecutive exons within the repeated elements (Fig. 1d). 2) We observed the usage of alternative splice sites (acceptors or donors) located next to the canonical sites. Most of these alternative splice sites (16/19) would produce an in-frame insertion or deletion of a few amino acids. The Human Splicing Finder (HSF) program displayed high splice site scores for most of these alternative splice sites, further suggesting their real use in TTN transcripts (Table 2and Fig. 1e). Based on the aforementioned splicing events passing our stringent QC filters, we calculated for each of the coding exons showing an alternative splicing, and not included in the repeated region, the number of reads supporting their inclusion or exclusion in TTN transcripts and a subsequent inclusion value (Table 3). It is noteworthy that 13 meta-only exons are expressed but only 7 have an inclusion value higher than 10%. On the other side, most of the canonical N2A exons, reported to be expressed in adult skeletal muscle, have a high inclusion value. Exon 11 as well as exons 155, 156, and 157 have an inclusion value lower than 50%, indicating that they are mostly spliced out. To evaluate the spatial and temporal expression of exon 11 and of meta-only exons, we examined a subset of publicly available RNA-Seq data from fetal skeletal muscles and fetal and adult hearts (Fig. 2). Interestingly, exon 11 is mostly expressed in fetal and adult hearts. Its expression is very low in adult and fetal skeletal muscles. Exon 148 has a similar expression in fetal and adult muscles, and it is mostly skipped in fetal and adult hearts. On the contrary, meta-only exons 213–217 are almost constitutively expressed in fetal muscles and their expression is halved in adult muscles. Table 1 Previously unreported junctions involving canonical splice sites out of the repeated region Donor exon Acceptor exon #TotReads #TotReads encode 10 12 63,681 31,420 10 13 37,864 22,425 10 14 25,860 19,371 36 38 2848 46 51 54 2319 52 85 88 8333 113 112 114 10,267 45 116 119 8651 1859 132 134 16,926 2843 137 143 1865 700 144 146 8300 297 146 151 10,262 805 146 152 3145 778 147 148 32,429 26,174 148 149 26,771 12,941 149 150 1304 951 150 151 1857 2836 153 158 12,300 116 154 158 35,902 4086 158 159 28,020 13,261 158 167 1129 45 158 168 3759 56 158 171 2965 279 158 172 7567 991 159 167 3117 2355 159 168 3192 1137 159 171 3722 3283 159 172 3627 1620 167 168 1830 2517 168 169 8111 7411 169 170 5910 5129 170 171 3237 8145 171 172 1439 8414 202 203 1358 15,787 203 209 1463 14 208 210 1741 27 212 213 28,311 11,949 213 214 8695 17,192 214 215 13,360 12,752 215 216 14,850 18,391 215 217 1647 1208 216 217 11,704 7775 Table 1 Previously unreported junctions involving canonical splice sites out of the repeated region (Continued) Donor exon Acceptor exon #TotReads #TotReads encode 217 218 10,983 15,909 219 222 1146 333 224 226 3126 2401 362 364 5458 2555 Metatranscript-only exons in italics Savarese et al. Skeletal Muscle (2018) 8:11 Page 4 of 9 A list of all detected ASEs that did not reach the minimum filtering criteria (i.e., a minimum of 14 out of 42 samples analyzed and at least 1000 supporting reads in total) or were not identified in the publicly available ENCODE data is included in Additional file 6: Table S5. Discussion Recent mRNA-Seq transcriptomic analyses show that most of multi-exonic genes are alternatively spliced [7, 10,20]. In particular, a vast majority of ASEs are tissue specific [10], and skeletal muscle seems to be among the tissues showing the highest numbers of tissue-specific ASEs [6,7,20]. Considering its 363 coding-exons and its genetic organization, a large number of ASEs were expected and partly reported in TTN transcripts. However, previous data, obtained by using different heterogeneous strategies in a pre-NGS era, did not provide a comprehensive view of the TTN splicing pattern and neither any unbiased repertoire of TTN ASEs in human adult skeletal muscles [1,2,8]. In our study, by performing RNA-Seq analysis using 42 adult human skeletal muscle samples, we identified in a reliable way a large number of ASEs, some of them at a very high level. We detected previously undescribed exon-exon junctions, suggesting novel, unreported skipping events. Exon 11, included in the canonical adult skeletal muscle isoform N2A, is mostly skipped in adult skeletal muscles. On the contrary, most of the so-called metatranscript-only exons are expressed in adult skeletal muscle at a variable level. Moreover, we identified alternative acceptors and donors leading to subtle changes in the produced protein. Although these events need to be experimentally validated, similar ASEs have already been described in other human genes and their functional relevance has been hypothesized [21–23]. With the exception of exon 11, the N-terminal exons, coding for the Z-disk part of titin, are mostly constitutively expressed. Exons 8 to 14 encode for seven copies of a specific domain, named Z-repeat (Zr) [24]. In particular, exon 11 encodes for Z-repeat 4 [24], and its differential splicing has been previously reported [25]. Sorimachi and colleagues reported that Z-repeats 1, 2, 3, and 7 are expressed in all striated rabbit muscles, whereas the expression of Zr4, 5, 6 (corresponding to exons 11–12 and 13) is dependent on developmental stage and tissue-type [25]. The differential splicing of the titin Z-disk seems to be part of a larger and more complex process able to modulate Z-disk interactions via Table 2 List of events involving alternative splice sites Donor Acceptor #Samples #Reads Frame HSF consensus value novel donor splice site (value for wt) HSF consensus value novel acceptor splice site (value for wt) #Reads encode c.669 (ex5) c.673 (ex6-alt acc) 31 1195 Yes –78.86 (85.41) 287 c.1398 (ex8) c.1399–3 (int8-alt acc) 42 6992 Yes –73.1 (80.21) 4456 c.9471 (ex40) c.9508 (ex41-alt acc) 19 10,437 Yes –76.95 (90.97) 101 c.22528 (ex78) c.22871 (ex80-alt acc) 27 1287 Yes –Unpredicted (77.00) 198 c.29124 (ex102-alt don) c.29228 (ex103-alt acc) 31 3807 No Unpredicted (88.47) 72.03 (79.27) 20 c.30754 (ex113) c.30757 (ex114-alt acc) 16 1537 No –72.87 (85.71) 15 c.31426 (ex118) c.31433 (ex119-alt acc) 31 4960 Yes –79.99 (81.96) 35 c.31762 (ex122) c.31769 (ex123-alt acc) 19 3455 Yes –86.28 (67.88) 33 c.32197 (ex127) c.32207 (ex128-alt acc) 25 1018 Yes –82.28 (78.21) 44 c.32392 (ex129) c.32399 (ex130-alt acc) 41 5055 Yes –82.29 (75.58) 121 c.33910 (ex145) c.33917 (ex146-alt acc) 18 1733 Yes –80.36 (77.55) 25 c.33994 (ex146) c.34301 (ex148-alt acc) 19 12,820 Yes –72.21 (73.01) 159 c.38058 (ex191-alt don) c.39484 (ex208-alt acc) 37 1038 Yes Unpredicted (76.37) 74.68 (80.08) 32 c.38058 (ex191-alt don) c.38980 (ex202-alt acc) 37 1427 Yes Unpredicted (76.37) 75.98 (77.27) 26 c.39063 (ex203-alt don) c.39484 (ex208-alt acc) 35 2300 Yes Unpredicted (77.92) 74.68 (80.08) 29 c.39147 (ex204-alt don) c.39484 (ex208-alt acc) 41 4744 Yes Unpredicted (76.37) 74.68 (80.08) 105 c.40786 (ex223) c.40790 (ex224 - alt acc) 30 1518 Yes –75.32 (94.42) 257 c.40876 (ex224) c.40880 (ex225 - alt acc) 24 1635 Yes –77.2 (91.6) 151 c.44646 (ex243-alt don) c.44914 (ex245) 20 10,706 No 83.39 (82.15) –15 alt don alternative donor, alt acc alternative acceptor Savarese et al. Skeletal Muscle (2018) 8:11 Page 5 of 9 Table 3 Exon usage Exon(s) Inclusion rate #Inclusion reads #Exclusion reads Skipping event ex1-10 Constitutively expressed ex11 Constitutively spliced out ex12 54% 147,944 63,724 10–13;10–14 ex13 79% 194,924 25,860 10–14 ex14– 36 Constitutively expressed ex37 98% 335,403 2848 36–38 ex38– 44 Constitutively expressed ex45– 46 Constitutively spliced out ex47 Constitutively expressed ex48 2% 5526 132,355 47–50 ex49 Constitutively spliced out ex50– 51 Constitutively expressed ex52 98% 222,051 2319 51–54 ex53 98% 252,059 ex54ex78 Constitutively expressed ex79 99% 262,245 1287 c.22,528–22,871 ex80– 85 Constitutively expressed ex86 91% 174,217 8333 85–88 ex87 93% 216,577 ex88– 112 Constitutively expressed ex113 90% 187,870 10,267 112–114 ex114116 Constitutively expressed ex117 92% 200,426 8651 116–119 ex118 92% 209,844 ex119132 Constitutively expressed ex133 66% 66,307 16,926 132–134 ex134– 137 Constitutively expressed ex138 97% 143,120 1865 137–143 ex139 96% 86,065 ex140 95% 77,132 ex141 96% 97,228 ex142 96% 100,706 ex143– 144 Constitutively expressed ex145 83% 82,433 8300 144–146 ex146 Constitutively expressed ex147 62% 86,205 26,227 c.33994–34,301;146– 151;146–152 Table 3 Exon usage (Continued) Exon(s) Inclusion rate #Inclusion reads #Exclusion reads Skipping event ex148 68% 72,020 16,913 146–151;146–152;147–149 ex149 67% 53,414 13,407 146–151;146–152 ex150 4% 3161 35,240 146–151;146–152;149–151 ex151 94% 94,107 3145 146–152 ex152– 153 Constitutively expressed ex154 82% 115,379 12,300 153–158 ex155 35% 52,812 48,202 153–158;154–158 ex156 34% 49,216 ex157 40% 64,075 ex158 Constitutively expressed ex 159 20% 52,844 103,121 158–167;158–168;158– 171;158–172;158–173; 158–175;158–182;158– 184;158–191;158–193; 158–204 ex160ex166 Constitutively spliced out ex 167 2% 6076 123,699 158–168;158–171;158– 172;158–173;158–175;158– 182;158–184;158–191;158– 193;158–204;159–168;159– 171;159–172;159–173;159– 175;159–184;159–193 ex 168 7% 16,892 116,748 158–171;158–172;158– 173;158–175;158–182;158– 184;158–191;158–193;158– 204;159–168;159–171;159– 172;159–173;159–175;159– 184;159–193 ex 169 6% 14,021 ex 170 4% 9147 ex 171 5% 11,363 110,061 158–172;158–173;158– 175;158–182;158–184;158– 191;158–193;158–204;159– 172;159–173;159–175;159– 184;159–193 ex172205 Repeated region ex206 81% 184,735 21,799 175–209;184–209;c.38058c.39484;193–209;c.39063c.39484;203–209;c.39147c.39484 ex207 68% 91,407 ex208 76% 86,726 13,717 175–209;184–209;193– 209;203–209 ex209 97% 99,982 1741 208–210 ex210– 212 Constitutively expressed ex213 26% 37,006 53,547 212–218 ex214 17% 22,055 ex215 22% 29,857 ex216 19% 26,554 55,194 212–218;215–217 ex217 19% 24,334 53,547 212–218 ex218ex219 Constitutively expressed Savarese et al. Skeletal Muscle (2018) 8:11 Page 6 of 9 splicing regulation. The N-terminal Z-disk region of titin binds a number of proteins, including alpha-actinin, nebulin, and filamin C that undergo a similar process of differential splicing [26–28]. As expected, most of the ASEs occur in the I-band region of titin, where a large number of exons are alternatively spliced [3,4]. It is noteworthy that exon 148, thought to be a meta-only exon, has an inclusion rate comparable to that of its neighboring exons in both adult and fetal skeletal muscles. Moreover, our experimental data as well as publicly available data suggests a significant expression of the meta-only exons 213, 214, 215, 216, and 217 in adult skeletal muscle, although their inclusion is higher in fetal muscles. In the M-band, we identified the previously reported splicing event (skipping of exon 363), producing the so called is7–and is7+ isoforms [29,30]. In line with previous data, exon 363 is skipped in about 10% of TTN transcripts in human adult skeletal muscle. As already discussed for the Z-disk splicing events, the regulation of alternative splicing events probably corresponds to modulation of interaction networks. For example, it is well known that the alternatively spliced is7 region, encoded by exon 363, binds the calcium-dependent protease calpain 3 (CAPN3) [31]. On the other hand, the role of the titin, and also nebulin, filament length (as a result of splicing events) on the sarcomere length and its passive elastic properties is still under debate [32–34]. Mutations in the TTN gene cause several different and heterogeneous skeletal muscle disorders with or without cardiac involvement, characterized by a variability in the age of onset, muscle involvement, and disease-course [11,12,35]. In addition, truncating mutations (TTNtv) have been associated with dilated cardiomyopathy (DCM) [13,14]. A genotype–phenotype correlation has been observed to some extent [11,15]. Mutations in metatranscript-only exons have recently been associated with a congenital titinopathy, characterized by arthrogryposis multiplex congenita and severe axial hypotonia as a form of congenital amyoplasia without cardiac involvement [36]. The hypothesis is that metatranscript-only mutations (mostly truncating mutations) specifically and selectively affect developmental isoforms, leading to a Fig. 2 Comparison of alternative splicing events among different tissues at different developmental stages. The analysis of publicly available total mRNA sequencing data from the ENCODE project shows that exon 11 is expressed only in cardiac muscles, whereas the expression of exon 148 is limited to skeletal muscles. Exons 213 and 217 show an increased expression in fetal skeletal (and, at least in part, cardiac) muscle compared to the adult expression. The reported values correspond to the inclusion values, based on the number of reads supporting each exon inclusion or exclusion in TTN transcripts Table 3 Exon usage (Continued) Exon(s) Inclusion rate #Inclusion reads #Exclusion reads Skipping event ex220 99% 168,189 1146 219–222 ex221 99% 194,654 ex222224 Constitutively expressed ex225 95% 124,798 3126 224–226 ex226243 Constitutively expressed ex244 93% 285,487 10,706 c.44,646–44,914 ex245362 Constitutively expressed ex363 91% 115,672 5458 362–364 ex364 Constitutively expressed Metatranscript-only exons in italics Savarese et al. Skeletal Muscle (2018) 8:11 Page 7 of 9 prenatal or congenital phenotype with a stable postnatal disease-course or weakness amelioration. On the contrary, proximal truncating mutations in canonical exons expressed on both alleles in adult isoforms lead to a premature truncated protein with nonsense mediated decay and would probably cause fetal death. The pathogenesis of TTNtv-related cardiomyopathies is probably more unclear; their penetrance is markedly reduced and they show a positional effect [14]. In particular, only TTNtv occurring in constitutive exons are significantly associated with DCM [14]. Deciphering the effective expression pattern of each TTN-exon, including meta-only exons, is crucial for a better understanding of TTN-related disorders. Our data clearly shows a variable expression for most of the metaonly exons (148, 150, 159, 167–171, 213–217), confirming, however, that some of them (160–166) are not expressed at all in human adult skeletal muscles. Our findings suggest the need for a more careful interpretation of the variants identified in a clinical setting. Here, we provided an accurate inventory of ASEs in human adult skeletal muscles, which suggest the presence of a high number of undescribed isoforms. Moreover, taking into account all the alternative splicing events occurring in TTN, we calculated a reliable inclusion value for titin exons. Further work remains to be done in order to refine our results. Long-read sequencing technologies, for example, will allow the identification of multiple splicing events along the same molecule, thereby elucidating how the individual splice events here described are connected, and thus confirming the presence of unreported isoforms. Similarly, a larger number of samples from each skeletal muscle type has to be analyzed in order to identify muscle-type specific ASEs or splicing patterns, considering that the current experimental setting has not identified any clear splicing difference among the muscles analyzed (Additional file 7: Table S6). The exonic usage and the subsequent isoform expression seem to be finely regulated among different developmental and physiological and/or pathological states [2,17,37]. A further refinement of TTN expression profiling in different tissues and/or different physiological and pathological states (including regenerating or injured muscles) would be of a great clinical relevance, deepening, for example, our understanding of the role of TTN variants in complex human diseases. Conclusions We have identified and partly characterized a large number of alternative splicing events in titin, providing the first RNA-Seq-based, accurate and comprehensive picture of TTN splicing pattern in adult human skeletal muscle. This same approach will probably unveil similar complex splicing patterns for other muscle transcripts. Additional files Additional file 1: Table S1. List of samples analyzed. (XLSX 11 kb) Additional file 2: Table S2. N2A splicing junctions. (XLSX 44 kb) Additional file 3: Supplementary Material 1: Titin repeated region. (DOCX 112 kb) Additional file 4: Table S3. Previously reported junctions in the repeated region. (XLSX 11 kb) Additional file 5: Table S4. Unreported junctions involving exons in the repeated region. (XLSX 12 kb) Additional file 6: Table S5. List of alternative splicing events not reaching the minimum filtering criteria or not identified in the publicly available ENCODE data. (XLSX 155 kb) Additional file 7: Table S6. Previously unreported junctions clustered accordingly to specific skeletal muscle types. (XLSX 17 kb) Abbreviations AS: Alternative splicing; ASEs: Alternative splicing events; HSF: Human Splicing Finder; NGS: Next-generation sequencing; PCR: Polymerase chain reaction; QC: Quality control; RNA-Seq: RNA sequencing; RT-PCR: Reverse transcriptase-polymerase chain reaction; TTNtv: Titin truncating variants Acknowledgements The authors would like to thank Meharji Arumilli for his advice in the analysis of RNA-Seq data. Funding This study was supported by Finnish Academy, Juselius Research Foundation, Association Française contre les Myopathies and Orion Research Foundation. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Raw data is available from the corresponding author on reasonable request. Authors’contributions All authors participated in designing all the studies. SH collected skeletal muscles. PHJ, SH, LP, PA, and PH conducted the RNA-sequencing. MS and PHJ analyzed the RNA-sequencing results and performed the experimental validation. MS, PHJ, BU, and PH wrote the manuscript. All authors have been involved with reviewing the manuscript and have approved the final version. Ethics approval and consent to participate A written informed consent was signed by all the patients and the Tampere University Hospital (Tampere, Finland) Ethics Committee approved the study. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 Folkhälsan Research Center, University of Helsinki, Helsinki, Finland. 2 Department of Pathology, Fimlab Laboratories, Tampere University Hospital, Tampere, Finland. 3 Institute of Biotechnology, University of Helsinki, Helsinki, Finland. 4 Vaasa Central Hospital, Vaasa, Finland. 5 Folkhälsan Institute of Genetics, Department of Medical Genetics, University of Helsinki, Biomedicum, Haartmaninkatu 8, Pb 63, 00014 Helsinki, Finland. Savarese et al. 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