RESEARCH ARTICLE

Inferring disease course from differential exon usage in the
wide titinopathy spectrum

Maria Francesca Di Feo<fnref n="1" />,<fnref n="2" /> ID, Ali Oghabian<fnref n="2" />, Ella Nippala<fnref n="2" />, Mathias Gautel<fnref n="3" />, Heinz Jungbluth<fnref n="3" />,<fnref n="4" />, Francesca Forzano<fnref n="5" />, Edoardo Malfatti<fnref n="6" /> ID, Claudia Castiglioni<fnref n="7" />, Ilona Krey<fnref n="8" />, David Gomez Andres<fnref n="9" />, Angela F. Brady<fnref n="10" />, Maria Iascone<fnref n="11" />, Anna Cereda<fnref n="12" />, Lidia Pezzani<fnref n="12" />, Daniel Natera De Benito<fnref n="13" /> ID, Andres Nascimento Osorio<fnref n="13" />, Berta Estévez Arias<fnref n="14" />, Sergei A. Kurbatov<fnref n="15" />,<fnref n="16" />, Tania Attie-Bitach<fnref n="17" />, Sheela Nampoothiri<fnref n="18" />, Erin Ryan<fnref n="19" />, Michelle Morrow<fnref n="19" />, Svetlana Gorokhova<fnref n="20" /> ID, Brigitte Chabrol<fnref n="21" />, Juha Sinisalo<fnref n="22" />, Heli Tolppanen<fnref n="22" />, Johanna Tolva<fnref n="23" />, Francina Munell<fnref n="24" />, Jessica Camacho Soriano<fnref n="25" />, Maria Angeles Sanchez Duran<fnref n="26" />, Mridul Johari<fnref n="2" />,<fnref n="27" /> ID, Homa Tajsharghi<fnref n="28" /> ID, Peter Hackman<fnref n="2" />, Bjarne Udd<fnref n="2" />,<fnref n="29" />,# & Marco Savarese<fnref n="2" />,# ID

Correspondence

Maria Francesca Di Feo, Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, and Maternal and Child Health (DINOGMI), University of Genoa, Genoa, Italy. Tel: +39 01056362801; Fax: +39 0102099227. E-mail: mariafrancescadifeo@gaslini.org

Abstract

**Objective:** Biallelic titin truncating variants (TTNtv) have been associated with a wide phenotypic spectrum, ranging from complex prenatal muscle diseases with dysmorphic features to adult-onset limb-girdle muscular dystrophy, with or without cardiac involvement. Given the size and complexity of TTN, reaching an unequivocal molecular diagnosis and precise disease prognosis remains challenging. **Methods:** In this case series, 12 unpublished cases and one already published case with biallelic TTNtv were collected from multiple international

<footnote n="1">¹Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, and Maternal and Child Health (DINOGMI), University of Genoa, Genoa, Italy</footnote>

<footnote n="2">²Folkhälsan Research Center, Helsinki, Uusimaa, Finland</footnote>

<footnote n="3">³Randall Division of Cell and Molecular Biophysics and Cardiovascular Division, King's College London BHF Centre of Research Excellence, London, UK</footnote>

<footnote n="4">⁴Paediatric Neurology, Neuromuscular Service, Evelina's Children Hospital, Guy's and St Thomas' Hospitals NHS Trust, London, UK</footnote>

<footnote n="5">⁵Clinical Genetics Department, Guy's and St Thomas NHS Foundation Trust, London, SE1 9RT, UK</footnote>

<footnote n="6">⁶Université Paris Est Créteil, INSERM, U955, IMRB, and Reference Center for Neuromuscular Disorders, APHP Henri Mondor University Hospital, Créteil, France</footnote>

<footnote n="7">⁷Clinica MEDS, Santiago de Chile, Chile</footnote>

<footnote n="8">⁸Institute of Human Genetics, University of Leipzig Hospitals and Clinics, Leipzig, 4275, Germany</footnote>

<footnote n="9">⁹Child Neurology Unit. Hospital Universitari Vall d'Hebron, Vall d'Hebron Research Institute (VHIR), Barcelona, Spain</footnote>

<footnote n="10">¹⁰North West Thames Regional Service, Northwick Park and St. Mark's Hospitals, Harrow, London, UK</footnote>

<footnote n="11">¹¹Medical Genetics Laboratory, ASST Papa Giovanni XXIII, Bergamo, Italy</footnote>

<footnote n="12">¹²Clinical Genetics Service, Pediatria 1—ASST Papa Giovanni XXIII, Bergamo, Italy</footnote>

<footnote n="13">¹³Neuropaediatrics Department, Hospital Sant Joan De Déu, Institut De Recerca Sant Joan De Déu, Barcelona, 08950, Spain</footnote>

<footnote n="14">¹⁴Neuromuscular Unit, Department of Neurology, Hospital Sant Joan De Déu, Barcelona, Spain</footnote>

<footnote n="15">¹⁵Voronezh NN Burdenko State Medical University, Voronezh, 394036, Russia</footnote>

<footnote n="16">¹⁶Saratov State Medical University, Saratov, 410012, Russia</footnote>

<footnote n="17">¹⁷Unité D'embryofoetopathologie, Service D'histologie-Embryologie-Cytogénétique, Hôpital Necker-Enfants Malades, Paris, France</footnote>

<footnote n="18">¹⁸Department of Pediatric Genetics, Amrita Institute of Medical Sciences & Research Centre, Kochi, Kerala, India</footnote>

<footnote n="19">¹⁹GeneDx, Gaithersburg, Maryland, USA</footnote>

<footnote n="20">²⁰Marseille Medical Genetics, Aix Marseille Université, Faculté Des Sciences Médicales Et Paramédicales, Marseille, France</footnote>

<footnote n="21">²¹Reference Center for Inherited Metabolic Diseases, Marseille University Hospital, Marseille, France</footnote>

<footnote n="22">²²Helsinki University Central Hospital, Helsinki, Finland</footnote>

<footnote n="23">²³Transplantation Laboratory, Department of Pathology, University of Helsinki, Helsinki, Finland</footnote>

<footnote n="24">²⁴Unitat De Malalties Neuromusculars Pediàtriques, Hospital Universitari Vall D'Hebron, Barcelona, Spain</footnote>

<footnote n="25">²⁵Histology Department, Vall D'Hebron University Hospital, Barcelona, Spain</footnote>

<footnote n="26">²⁶Maternal Fetal Medicine Unit, Department of Obstetrics, Universitat Autònoma de Barcelona, Hospital Vall D'Hebron, Barcelona, Spain</footnote>

<footnote n="27">²⁷Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia, Nedlands, Western Australia, Australia</footnote>

<footnote n="28">²⁸Division of Biomedicine, School of Health Sciences, University of Skovde, Skovde, Sweden</footnote>

<footnote n="29">²⁹Department of Musculoskeletal Diseases, Tampere University Hospital, Tampere, Pirkanmaa, Finland</footnote>
Received: 23 July 2024; Accepted: 7 August 2024

Annals of Clinical and Translational
Neurology 2024; 11(10): 2745-2755

doi: 10.1002/acn3.52189

medical centers between November 2022 and September 2023. TTN mutations were detected through exome or genome sequencing. Information about familial and personal clinical history was collected in a standardized form. RNA-sequencing and analysis of TTN exon usage were performed on an internal sample cohort including postnatal skeletal muscles, fetal skeletal muscles, postnatal heart muscles, and fetal heart muscles. In addition, publicly available RNA-sequencing data was retrieved from ENCODE. **Results:** We generated new RNA-seq data on TTN exons and identified genotype-phenotype correlations with prognostic implications for each titinopathy patient (whether worsening or improving in prenatal and postnatal life) using percentage spliced in (PSI) data for the involved exons. Interestingly, thanks to exon usage, we were also able to rule out a titinopathy diagnosis in one prenatal case. **Interpretation:** This study demonstrates that exon usage provides valuable insights for a more exhaustive clinical interpretation of TTNtv; additionally, it may serve as a model for implementing personalized medicine in many other genetic diseases, since most genes undergo alternative splicing.

Introduction

TTN gene encodes for the giant protein titin, which has a
crucial role in sarcomere development, structure, signal-
ing, and myofibrillar stability during muscle contraction-
relaxation. ¹,² It contains 363 coding exons plus the first
noncoding exon, which are all contained in the inferred
theoretical complete “metatranscript” isoform, identified
by the conventional TTN MANE Select reference tran-
script (NM_001267550.2).³

As expected, titin has a very complex splicing pattern,
with more than 1 million splice variants potentially
generated. ⁴,⁵ Overall, the diversity of titin isoforms pro-
duced by alternative splicing is thought to contribute to
the complexity and adaptability of muscle function, and
to the variability of disease involvement of anatomically
distinct muscles. ⁶,⁷

Considering this variability, the position of *TTN* vari-
ants is crucial to correlate the molecular findings with
the clinical phenotypes. For example, heterozygous trun-
cating variants in cardiac isoforms (with the principle
long cardiac isoform being N2BA) have been associated
with dilated cardiomyopathy (DCM), while truncating
variants in other regions do not necessarily imply car-
diac risk.⁸⁻¹⁰ Similarly, variants in the last canonical
exon (364) are associated with dominant tibial muscular
dystrophy, whereas biallelic TTNtv in the two last exons
with juvenile-early adult-onset recessive distal
titinopathy.¹¹⁻¹³ Variants in exon 344 cause usually
dominant hereditary myopathy with early respiratory
failure (HMERF).¹⁴ However, to date, our understanding
of the role of *TTN* exons and their differential expres-
sion throughout developmental stages and tissues has
been partial, so much as it appears we are barely
scratching the surface.

Biallelic truncating variants in metatranscript-only
exons have been associated with prenatal and congenital
severe myopathies.¹⁵⁻¹⁷ Remarkably, for some patients
with severe respiratory and feeding issues at birth, the dif-
ferential diagnosis includes a wide range of congenital
disorders.¹⁸ Also, the most severe congenital titinopathies
resemble syndromic phenotypes, affecting not only mus-
cles but also bone, heart, and other organs, with a quite
high rate of dysmorphisms (up to 36%).¹⁹ Notably, brain
abnormalities presenting both in prenatal and postnatal
life have been described in a few published cases,
although the presence of pathogenic variants in other
genes cannot be excluded.¹⁸,²⁰,²¹

Traditionally, metatranscript-only exons are not sup-
posed to be expressed in postnatal muscles, while, on the
other side, canonical exons are supposed to have a high
expression both in fetal and postnatal skeletal muscles.
However, clinical reports published in recent years suggest
that this may not always be the case.¹⁷,²²,²³

In our previous significant effort, we developed specific
recommendations for *TTN* variants interpretation.²⁴
Considering our increased knowledge of the phenotypic
spectrum and the crucial insights provided using
RNA-sequencing techniques, we propose a new compre-
hensive workflow for the clinical interpretation of genetic
findings in *TTN*, which relies on the current evidence on
exon usage.

**Material and Methods**

Clinical features analysis

We collected biallelic titinopathy cases that have been
brought to our attention by direct request for counseling
and one previously published case. In particular, the

<footnote>#These authors contributed equally to
this work.</footnote>
unpublished cases have been selected by geneticists from different international centers for carrying biallelic TTNtv. In order to avoid possible biases, we limited the study to cases with biallelic variants causing a premature stop codon (nonsense and indels causing a frameshift), excluding patients with variants predicted to alter the splicing.

All the cases have been clinically assessed either by gynecologists experienced in prenatal diagnostics for prenatal cases, by child neurologists for children, and by neuromuscular-expert neurologists for adults. We collected all the clinical data, including prenatal and family history information, in a standardized form (Table S1). Regarding the already published case, we collected extra details from the manuscript's authors.

## Molecular genetic analysis

Proband DNA was analyzed using short-read exome (ES) or genome (GS) sequencing in each referring institution. Sequencing data were analyzed using standard bioinformatic pipelines aiming at the identification of single nucleotide variants, small insertions or deletion (indels). All the recruited patients carried biallelic TTNtv. Segregation analysis was performed to confirm the phase of the variants.

## RNA-sequencing on samples cohort

We performed RNA-sequencing on a large samples cohort to determine the ratio at which each titin exon is included (i.e., not spliced out) among transcripts from four different tissue types (postnatal skeletal, postnatal cardiac, fetal skeletal, and fetal cardiac). For postnatal skeletal muscle analysis, we collected muscle samples dissected from both myopathic and non-myopathic individuals (41 individuals aging 0–89 years from different international centers) (Supplemental Material and Methods, Figure S1, Table S2).

Also, seven heart muscles samples (left ventricle) dissected from adult individuals that have undergone transplantation for ischemic heart disease were included (collected at the Department of Pathology, University of Helsinki, Finland). Fetal expression analysis was performed using publicly available database (ENCODE). Fetal skeletal muscles (n = 20) and fetal heart muscles (n = 2) from two different fetuses, without muscle pathology, were obtained from voluntary TOPs (collected at the Hospital Universitari Vall D'Hebron, Barcelona, Spain).

For long-read sequencing, data were generated from five different skeletal muscles and one fetal heart muscle, belonging to the same fetus.

The paired-end RNAseq reads were aligned using the splice-aware STAR alignment software (version 2.7.7a).²⁵

The Percentage Spliced In (PSI) values of TTN exons were measured using the Intron Exon Retention Estimator (IntEREst) R/Bioconductor package (V1.24.0).²⁶ For long reads, transcriptome analysis was performed with the SQANTI software with default parameters.²⁷ Only TTN transcripts (Ensembl ID ENSG00000155657) were included in the analysis and duplicates were filtered out. The extracted sequences were aligned and identified using the BLAST-like Alignment tool (BLAT).

## Results

We report 13 patients with biallelic truncating variants in the TTN gene; five out of 13 carry biallelic TTNtv in exons considered as “canonical” by the current classifications. To explain the clinical phenotype, we attempted to correlate signs and symptoms with exon usage data obtained by RNA-seq analysis.

The clinical details of each titinopathy patient are summarized in Tables 1 and 2 and described in greater detail in Table S1.

## Patients deceased before birth or in the perinatal period

Three cases (P1–P3) were unborn fetuses: P1 and P2 underwent termination of pregnancy because of severe prenatal findings such as intrauterine growth restriction (IUGR), fetal akinesia with hypo/amyoplasia and hydrops. P3 died in the first hour after birth. They all presented with arthrogryposis, fetal akinesia, and hypotonia and P3 also had unspecific facial dysmorphisms.

P1–P3 are compound heterozygous for TTNtv. They all carry one TTNtv located in an A-band or I-band exon highly expressed in skeletal muscles in all developmental stages (PSI 85%–95%), and another TTNtv on the other allele, located in a more variably expressed exon.

Interestingly, two of them P1 and P2 carry the second variant in canonical exons 156 and 149, which have a PSI of 26% and 25% in postnatal skeletal muscle, respectively, but 70% and 55%, respectively, in fetal muscles. Patient 3 carries a second TTNtv in exon 167, a metatranscript only exon with a high PSI in fetal muscles (63%) and a barely detectable expression in postnatal muscles (PSI = 4%).

In conclusion, all these patients carried biallelic TTNtv located in exons with a high PSI in fetal muscles, resulting in genotypes that appear severe and/or not to be compatible with life.

## Congenital cases improving with age

This group, including P4–9, is quite homogeneous in terms of onset (congenital) and clinical features, with all
<table><caption>**Table 1.** Clinical summary of the patients with biallelic TTNtv in compound heterozygosity and exon usage associated to each variant.</caption><thead><tr><th rowspan="2">ID</th><th rowspan="2">Clinical category</th><th rowspan="2">Prenatal signs and symptoms</th><th rowspan="2">Postnatal signs and symptoms</th><th rowspan="2">Improved after birth</th><th colspan="3">Variant 1</th><th colspan="3">Variant 2</th></tr><tr><th>Annotation (exon)</th><th>Exon usage in fetal SM (%)</th><th>Exon usage in adult SM (%)</th><th>Annotation (exon)</th><th>Exon usage in fetal SM (%)</th><th>Exon usage in adult SM (%)</th></tr></thead><tbody><tr><td>P1</td><td>Deceased before birth (TOP)</td><td>IUGR, amyoplasia, hydrops, arthrogryposis</td><td>NA</td><td>NA</td><td>c.14183dup (50)</td><td>99</td><td>97</td><td>c.35182_35188del (156)</td><td>70</td><td>26</td></tr><tr><td>P2</td><td>Deceased before birth (fetal death)</td><td>Amyoplasia, arthrogryposis</td><td>NA</td><td>NA</td><td>c.23386C&gt;T (82)</td><td>95</td><td>86</td><td>c.34408del (149)</td><td>55</td><td>25</td></tr><tr><td>P3</td><td>Deceased after birth</td><td>Arthrogryposis, amyoplasia, dysmorphisms</td><td>Respiratory insufficiency</td><td>NA</td><td>c.85348A&gt;T (327)</td><td>97</td><td>93</td><td>c.36100_36101del (167)</td><td>63</td><td>4</td></tr><tr><td>P5</td><td>Improving with age (infant)</td><td>Fetal akinesia, arthrogryposis</td><td>Hypotonia, respiratory insufficiency, dysphagia, congenital fractures</td><td>Yes</td><td>c.96464del (348)</td><td>94</td><td>90</td><td>c.36040A&gt;T (166)</td><td>39</td><td>1</td></tr><tr><td>P6</td><td>Improving with age (child)</td><td>Arthrogryposis</td><td>Hypotonia, respiratory insufficiency, dysphagia, congenital fractures</td><td>Yes</td><td>c.33055del (136)</td><td>77</td><td>51</td><td>c.38737G&gt;T (199)</td><td>15</td><td>2</td></tr><tr><td>P7</td><td>Improving with age (adult)</td><td>Arthrogryposis</td><td>Hypotonia, proximal and distal weakness, reduced vital capacity</td><td>Yes, independent walking acquired</td><td>c.67495C&gt;T (320)</td><td>92</td><td>84</td><td>c.38737G&gt;T (199)</td><td>15</td><td>2</td></tr><tr><td>P8</td><td>Improving with age (adult)</td><td>Arthrogryposis</td><td>Hypotonia, proximal and distal weakness</td><td>Yes, walking with aids</td><td>c.103531A&gt;T (359)</td><td>94</td><td>94</td><td>c.38661_38665del (198)</td><td>11</td><td>1</td></tr><tr><td>P10</td><td>Worsening with age</td><td>Not reported</td><td>Hypotonia, sluggish reflex</td><td>No, worsened, respiratory insufficiency at 18 months, motor delay, concentric hypertrophic cardiomyopathy</td><td>c.70978C&gt;T (327)</td><td>97</td><td>93</td><td>c.2047C&gt;T (13)</td><td>31</td><td>68</td></tr><tr><td>P11</td><td>Worsening with age (death at 6 months)</td><td>Arthrogryposis, threatened miscarriage</td><td>Hypotonia, respiratory insufficiency, dysphagia</td><td>No, worsened, death at 6 months of age</td><td>c.65163T&gt;A (312)</td><td>92</td><td>87</td><td>c.32680del (133)</td><td>21</td><td>30</td></tr><tr><td>P13</td><td>Unlikely case of titinopathy, TOP</td><td>Macrocephaly &gt;90 + p, vertebral fusion D7-D8-D9, multicystic lymphangioma of the mesentery</td><td>NA</td><td>NA</td><td>c.55939G&gt;T (289)</td><td>96</td><td>93</td><td>c.10439dupA (45)</td><td>4</td><td>0</td></tr></tbody></table>

IUGR, intrauterine growth restriction; SM, skeletal muscles; TOP, termination of pregnancy.
<table><caption>**Table 2.** Clinical summary of the patients with biallelic TTNtv in homozygosity and exon usage associated to each variant.</caption><thead><tr><th rowspan="3">ID</th><th rowspan="3">Clinical category</th><th rowspan="3">Prenatal signs and symptoms</th><th rowspan="3">Postnatal signs and symptoms</th><th colspan="3">Variant 1</th><th colspan="3">Variant 2</th></tr><tr><th rowspan="2">Improved after birth</th><th rowspan="2">Annotation (exon)</th><th rowspan="2">Exon usage in fetal SM (%)</th><th rowspan="2">Exon usage in adult SM (%)</th><th rowspan="2">Annotation (exon)</th><th rowspan="2">Exon usage in fetal SM (%)</th><th rowspan="2">Exon usage in adult SM (%)</th></tr><tr></tr></thead><tbody><tr><td>P4</td><td>Improving with age (infant)</td><td>IUGR, amyoplasia, arthrogryposis</td><td>Hypotonia, respiratory insufficiency, dysphagia, congenital fractures</td><td>Yes</td><td>c.40267G>T (217)</td><td>68</td><td>27</td><td>c.40267G>T (217)</td><td>68</td><td>27</td></tr><tr><td>P9</td><td>Improving with age (adult)</td><td>No</td><td>Hypotonia, distal arthrogryposis</td><td>Yes</td><td>c.38661_38665del (198)</td><td>11</td><td>1</td><td>c.38661_38665del (198)</td><td>11</td><td>1</td></tr><tr><td>P12</td><td>Worsening with age (adolescent)</td><td>No</td><td>HyperCKemia at 14 years of age</td><td>No</td><td>c.32656C>T (133)</td><td>21</td><td>30</td><td>c.32656C>T (133)</td><td>21</td><td>30</td></tr></tbody><tfoot><tr><td colspan="11">SM, skeletal muscles.</td></tr></tfoot></table>

patients having hypotonia and multiple contractures at birth, but variable respiratory involvement. Importantly, the conditions of all these five patients significantly improved after birth.

Patient 4, the most severe case within this group, is an 8-month-old child who presented prenatally with fetal akinesia, oligohydramnios, and intrauterine growth retardation (IUGR). He required intensive care with invasive respiratory support and nasogastric feeding. He also had a syndromic-like face with facial asymmetry with small eyes, an arched upper lip, severe micro retrognathia, and hyperconvoluted ear pinnae. He carries a homozygous TTNtv in the PEVK-encoding exon 217, which has a PSI of 70% in fetal muscles, 20% in postnatal muscles, and is not expressed in the heart. At the last neurologic evaluation (6 months of age), he showed spontaneous breathing with stable vital parameters and improving interactive skills, even though motor skills were still severely impaired.

Patient 5 is a 4-week-old infant born with arthrogryposis. He carries a TTNtv in the constitutively expressed “canonical” exon 348, located in the distal A-band, in compound heterozygosity with a TTNtv in exon 166, a metatranscript-only exon, with a PSI of 60% in fetal muscles and 0% in postnatal skeletal and cardiac muscles. His condition was reported stable at the last examination.

Patients 6, 7, 8, and 9 are myopathic patients aged 8–33 years. They were all born with congenital hypotonia and arthrogryposis; P6 and P7 required ventilation support, and P6 additional nasogastric tube feeding. Their conditions have significantly improved with age, and now they have a milder limb-girdle phenotype. None of them has cardiac involvement. Patients 6, 7, and 8 carry a TTNtv in an exon with high PSI in both fetal and postnatal skeletal muscles in compound heterozygosity with a TTNtv in the triplicated region of TTN (spanning from exon 173 to exon 199, encoding skeletal muscle PEVK sequences); patient 9 carries a homozygous TTNtv in the triplicated region. These exons have a low PSI in fetal skeletal muscles (15% approximately) and 0% in postnatal muscles.

To summarize, all six patients in this group have at least one variant in an exon with PSI decreasing from fetal to postnatal muscle stage.

## Congenital cases worsening with age

Patients 10, 11, and 12 showed less severe clinical phenotypes in the prenatal and perinatal period, but their signs and symptoms progressively worsened postnatally.

Patient 10 showed no contractures but diffuse hypotonia and respiratory difficulties at birth. Notably, he displayed concentric LV hypertrophy. He acquired
independent walking at 20 months. At the last examination (23 months old), he was on BiPAP for respiratory failure. He carries a TTNtv in the large and constitutively expressed exon 327, in compound heterozygosity with a TTNtv in exon 13, which has a PSI of 30% in fetal muscles and of 70% in postnatal muscles. Both exons have high PSI (90% approximately) in cardiac muscles. Exon 13 is in the Z-disk, which anchors antiparallel actin filaments from opposite sarcomere halves and forms the sarcomere boundary.²⁸ This exon is included in slow and cardiac muscles but not in fast muscle. By long-read isoform sequencing, we discovered seven novel fetal isoforms in the Z-disk region of TTN (Fig. S2). Differential usage of exons 11–13 explains a significant part of the variation between these previously unreported fetal isoforms. In our Iso-Seq data, exon 11 is infrequently used in fetal skeletal muscle; in all the data, it is included in only one out of 86 transcripts spanning the region. Instead, exon 13, is included in 45 out of 86 transcripts spanning this region.

Patient 11 presented with congenital arthrogryposis, hypotonia, and chest deformities; his condition deteriorated, and he died at 6 months of age of respiratory failure (see Table S1 for further details). He carried a TTNtv in the constitutively expressed exon 312, in compound heterozygosity with a TTNtv in the PEVK-encoding exon 133 which is an I-band differentially expressed exon with some fetal expression (PSI of 20% in fetal skeletal muscles) and a PSI of 30% approximately in postnatal muscles.

Patient 12 is an asymptomatic 14-year-old patient with recent onset of constant hyperCKemia (600–850) as the only sign of muscular disorder. His clinical history, including the perinatal period, is unremarkable. At ES, the only pathogenic finding was a homozygous TTNtv in exon 133.

In conclusion, all these patients have at least one TTNtv located in an exon with a very low PSI in fetal muscles but a higher PSI in postnatal muscles.

## An unlikely case of titinopathy

Patient 13 is an arthrogrypotic fetus with some unspecific syndromic-like signs (macrocephaly >90 + p, vertebral fusion). No heart or muscles anomalies were found. Termination of pregnancy was carried out at 35 weeks. ES analysis showed biallelic TTN variants: a TTNtv in the canonical exon 289, in compound heterozygosity with a TTNtv in exon 45 encoding Ig-domain 25, with an undetectable expression in both fetal and postnatal skeletal muscles in our RNAseq study. Exon 45 is a Novex-1-only exon, apparently expressed in the Novex1 isoform of cardiac muscle. According to Bang et al, the Novex-1

transcript is expressed at low levels in postnatal skeletal muscles.¹ We conclude that it seems unlikely that the fetus' phenotype might be caused by the identified biallelic TTNtv; however, excluding it with certainty would require detailed analysis of exon 45 usage in several fetal skeletal muscles.

## Discussion

To date, we do not yet have a proper understanding of the full range of titin isoforms. Nevertheless, in previous studies, many alternative splicing events (ASE) have been found, some of them at a very high level, suggesting the presence of a larger number of isoforms that are yet uncharacterized.⁴ In this study, we provided the first insight into the use of *TTN* exon usage in human fetal and postnatal skeletal muscles, both by short-read and long-read RNA-sequencing, and we used the data to assess the clinical meaning of truncated variants in newly identified patients.²⁸

PCA analysis on the samples cohort used for the RNA-sequencing analysis is reported in Figures S3 and S4. As expected, we found a clear distinction of the studied sample groups (i.e., fetal skeletal muscle, postnatal skeletal muscle, fetal cardiac muscle, and postnatal cardiac muscle) based on titin exon usage, but no association between the genetic status of the sample (myopathic or healthy controls) and titin expression profile.

With our analysis, we demonstrated that few exons are actually “canonical,” and most show a variable pattern (Figs. 1 and 2, Table S1). We speculate that biallelic truncating variants in canonical exons would most likely not be compatible with life, as they have never to date been reported in living individuals; nevertheless, we present five individuals with biallelic TTNtv in exons previously regarded as canonical: P1–P2 with TTNtv in exons 156 and 149, respectively, P10 carrying a TTNtv in exon 13, and P11–P12 carrying a TTNtv in exon 133 (Fig. 1). Remarkably, P10 and P12 are alive at 23 months and 15 years, respectively, and P12 with only hyperCKemia as referred symptom of muscle disease.

On the other hand, we show that some exons defined as “metatranscript-only” are also expressed in postnatal skeletal muscles, for example, exon 217 (P4). In addition, a case of a 33-year-old male patient with a homozygous truncating variant in the so-called metatranscript exon 170 was published recently.²³ Our data suggest that exon 170 has a PSI of 57% in fetal muscles and 10% in postnatal muscles, emphasizing that a new classification of *TTN* exons is needed. Moreover, we suggest that the terms canonical exons and metatranscript only exons should thereby be avoided, and each exon should be defined by its PSI in fetal or postnatal muscles.
<figure><img src="image_1.png" /><figcaption>**Figure 1.** Exon usage of "false" canonical exons. Although exons 13,133, 149, and 156 are traditionally described as canonical exons included in postnatally expressed skeletal muscle isoforms, our analysis shows that all of them have a variable PSI score. AH, postnatal heart; AM, postnatal muscles; FH, fetal heart; FM, fetal muscles.</figcaption></figure>

Our study, combining short-read RNA-sequencing and PacBio Iso-Seq, represents a first step toward a deeper understanding of titin isoforms.

By long-read sequencing, we found seven different novel fetal isoforms spanning the Z-disk (Fig. S2), which is a particularly interesting titin region, anchoring titin to the actin cytoskeleton at the sarcomere boundary. This region of titin includes the first four immunoglobulin-type domains (Z1–Z4) and a series of 45-residue repeats, called Z-repeats. As expected, differential usage of exons 11, 12, and 13 (which are part of the Z-repeats) explains a significant part of the variation between these isoforms, and that, in our data, exon 11 is very rarely used in fetal skeletal muscles. It is probable that multiple isoforms with differential exon usage of Z-repeats create minor but significant protein-level differences affecting titin anchoring. This is consistent with finding of different thickness and protein composition of Z-disks across species and muscle types.²⁹ Interestingly, exon 13, which hosts a TTNtv in the case of P10 (who presented with hypotonia and cardiomyopathy since birth), showed a higher inclusion level.

Regarding the complex I-band region of titin, we know that it is composed of two principal stretches composed by tandemly arranged Ig domains, intercalated by the PEVK sequence that has no defined structure and acts as an entropic elastic spring.² These exons are differentially expressed in tissue-specific transcripts, which results in the characteristic size of the main postnatal titin isoforms.⁴ However, in our fetal samples analyzed by long-read sequencing, we did not find any I-band pattern of so-called combined alternative splicing, where a certain

splicing event always takes place in combination with another splicing event. This observation seems to further confirm that, at prenatal age, I-band exons, especially from the PEVK exons, whose splice donor and acceptor sites are highly compatible, are not expressed "in blocks," as there are still no defined postnatal isoforms. These findings are also consistent with our short-read sequencing data indicating that some I-band exons are more expressed in fetal than in postnatal skeletal muscles. Indeed, some of these exons are mutated in cases of patients who improve after birth, for example, P6, P7, and P8 with variants in the triplicated region, which is part of the PEVK domain.

Clearly, our study has several limitations: first of all, a thorough characterization of titin splicing pattern would benefit from a larger number of samples.

Secondly, within the triplicated region (exons 173–199), there may be technical difficulties in mapping the single exon accurately. Furthermore, the applicability of our model may not always be straightforward for all titin variants. For example, splicing variants, which were not included in this study, pose additional challenges.³⁰ Further analyses will be needed to deepen our knowledge on differential exon usage: for example, according to the skeletal muscle type (fast-twitch, slow-twitch), body localization, stage of development (week of pregnancy), and other factors. Considering that titin is the largest and probably one of the most complex proteins in animals, we are far from being able to fully explain all the different signs and symptoms within the titinopathy spectrum. However, we show here that exon usage represents a valuable guidance to predict the prognostic trajectory of
<figure><img src="image_2.png" /><figcaption>**Figure 2.** TTN exon usage. (A) Heat map showing the PSI index score of TTN exons in skeletal and cardiac muscles in prenatal and postnatal samples. (B) PSI graphical representation in prenatal (red) and postnatal (green) skeletal muscles. (C) PSI scores of the mutated exon in postnatal (x-axis) and fetal (y-axis) skeletal muscles correlate with the clinical phenotype and the disease course, showing the three clusters: pre- or perinatal death (P1, P2, and P3 in blue); improving disease course (P5-P8 in pink); and worsening disease course (P9-P11 in dark gray).</figcaption></figure>

most of the biallelic titinopathy cases, with major impli-
cations for physicians and patients. As a proof, we
applied retrospectively this model to other published
cases of biallelic titinopathies, which were outlined in our
recent publication (Di Feo et al, 2023), and found that
most of the cases with clinical information on the prena-
tal phenotype and/or on the first years of life have a

disease course fitting the exon usage trajectory
(Table S4).¹⁵,¹⁸,²⁰,³¹,³²

Interestingly, our exon usage study not only provides a
better understanding of biallelic titinopathies but also
allows us to avoid misdiagnosis in prenatal cases. For
example, by combining clinical and molecular knowledge,
it will be possible to rule out that a TTNtv in an exon
with a very low PSI in fetal skeletal muscles could be the
cause of a severe prenatal phenotype.

Obviously, clinical predictions based on exon usage
data do not consider many factors, for example, the pres-
ence of modifying variants in other genes (e.g., SRPK3 or
RBM20) or the coexistence of other potentially damaging
TTN missense variants.33–35 In fact, in the presence of a
TTNtv in an exon with a PSI below 100%, the remaining
transcripts with the exon spliced out may, on the other
hand, include other potentially damaging variants. Future
investigations could shed new light on the importance of
in-cis variants in biallelic titinopathies and, perhaps even
change a genetic paradigm, in particular that the presence
of a variant in cis with a pathogenic variant in any inheri-
tance pattern is considered a criterion of benignity (BP2
according to ACMG guidelines.36

Also, there may be several variables in the clinical
course that we are unable to predict with exon usage data
alone (including response to treatments and infections).
In P2, we cannot exclude the possibility that if the fetus
had not gone through TOP, this case might have
improved, as the PSI of exon 149 in fetal muscles is 55%
while in the postnatal period it drops by about 30%.

Conclusions

With the advent of the genomic era, it has become crucial
to understand the clinical effects of variants in large and
complex genes. Not considering alternative transcripts
and differential tissue expression can lead to missed or
incorrect diagnoses, and this is still a major issue in many
diagnostic settings.37 TTN is one of the genes that face
frequent challenges in variant clinical interpretation, as
TTNtv are found in approximately 1 in 100 individuals
and very rare missense TTN variants in approximately 20
in 100.38,39 Our study provides a framework, based on
exon usage, that can be applied to other genetic diseases,
as recent studies have shown that 70% of the human
genes have at least 15 transcript isoforms.40

Acknowledgements

We thank all patients and families participating in this
study. We thank Lauri Snellman, Laboratory Technician,
for laboratory assistance.

Funding Information

M.S. received support from the Academy of Finland
(grant 339437), Association Française contre les Myopa-
thies (grant 23281), Sydäntutkimussäätiö, and Samfundet
Folkhälsan i Svenska, Finland. A.O. received supported by
Magnus Ehrnrooth Foundation. B.U. received support

from the European Joint Program on Rare Diseases (pro-
ject IDOLS-G), Academy of Finland, Juselius Foundation,
and Samfundet Folkhälsan i Svenska Finland. F.M.
received support from the European Joint Program on
Rare Diseases (project IDOLS-G) and Instituto de Salud
Carlos III, Spain (project number AC19/00048). P.H.
received support from the Jane and Aatos Erkko
foundation.

Conflict of Interest

The authors have no conflicts of interests to declare.

Author Contributions

M.F.D.F., M.S., B.U., and A.O. contributed to the concep-
tion and design of the study; M.F.D.F., M.G., H.J., F.F.,
E.M., C.C., I.K., D.G.A., A.F.B., M.I., A.C., L.P.,
D.N.D.B., A.N.O., S.A.K., T.A.B, S.N., E.R., M.M., S.G.,
B.C., J.S., J.T., F.M., J.C.S., M.A.S.D., and H.T. contrib-
uted to acquisition of data; M.F.D.F., A.O., and E.N.,
contributed to acquisition of data and data analysis;
M.F.D.F., M.S., B.U., M.J., E.N., and A.O. contributed to
drafting the text or preparing the figures.

Data Availability Statement

The data that support the findings of this study are avail-
able from the corresponding author upon reasonable
request.

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**Supporting Information**

Additional supporting information may be found online
in the Supporting Information section at the end of the
article.

Data S1.