Atypical phenotypes in titinopathies explained by second titin mutations.

Research Article Atypical phenotypes in titinopathies explained by second titin mutations Anni Evilä, MSc1, Anna Vihola, PhD1, Jaakko Sarparanta, MSc1, Olayinka Raheem, PhD2, Johanna Palmio, MD, PhD2, Satu Sandell, MD2,3, Bruno Eymard, MD, PhD4, Isabel Illa, MD, PhD5, Ricard Rojas-Garcia, MD, PhD5, Karolina Hankiewicz, MD5, Luis Negrão, MD, PhD6, Tuija Löppönen, MD, PhD7, Pekka Nokelainen, MD, PhD7, Mikko Kärppä, MD, PhD8, Sini Penttilä, MSc2, Mark Screen, MSc1, Tiina Suominen, PhD2, Isabelle Richard, PhD9, Peter Hackman, PhD1, and Bjarne Udd, MD, PhD1,2,10 From the 1Folkhälsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland; 2Neuromuscular Research Center, University of Tampere and Tampere University Hospital, Tampere, Finland; 3Seinäjoki Central Hospital, Department of Neurology, Seinäjoki, Finland; 4Institute of Myology, National Reference Center for neuromuscular disorders, University Hospital of Salpêtrière, Paris, France; 5 Neuromuscular Diseases Unit, Neurology Department, Hospital de la Santa Creu i Sant Pau, Universitat Autónoma de Barcelona, Barcelona, Spain; 6Neuromuscular Unit, Neurology Department, Coimbra University Hospital, Coimbra, Portugal; 7Department of Child Neurology, Kuopio University Hospital, Kuopio, Finland; 8Department of Clinical Medicine, Neurology, University of Oulu and Clinical Research Center, Oulu University Hospital, Oulu, Finland; 9 Généthon, Evry, France; 10Neurology Department, Vaasa Central Hospital, Vaasa, Finland

Corresponding author: Peter Hackman Corresponding author’s address: Folkhälsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland Corresponding author’s phone and fax: +358-9-19125069, +358-9-19125073 Corresponding author’s e-mail address: [email protected]

Running head: TMD complex cases

Number of words in abstract: 252 Number of words in main text: 3758 Number of figures: 4 (of which 1 in color) Number of tables: 2

Acknowledgement statement (including conflict of interest and funding sources):

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ana.24102

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Funding This study was supported by the Folkhälsan Research Foundation, the Association Française contreles Myopathies, the Academy of Finland, the Sigrid Jusélius Foundation, the Liv och Hälsa Foundation, and Tampere University Hospital Research Funds (B.U.).

Disclosures Dr. Nokelainen reports personal fees from Glaxo-Smith-Kline Finland, non-financial support from Novartis, outside the submitted work; Dr. Rojas-Garcia reports grants from FIS, EC08/00077, grants from FIS, BAE10/00047, personal fees from Pfizer, outside the submitted work; All other authors report no conflicts of interest.

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ABSTRACT

Objective: Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in eight patients of seven European families.

Methods: Clinical, histopathological and muscle imaging data of patients and family members was reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by RT-PCR and Sanger sequencing.

Results: Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of six patients, three with an LGMD2J phenotype. Novel frameshift mutations were identified in five patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in one patient, and one Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation.

Interpretation: The unequal expression levels of TTN transcripts in five probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism explaining phenotype variability.

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INTRODUCTION

Tibial muscular dystrophy (TMD, OMIM #600334) is an autosomal dominant distal myopathy caused by mutations in the titin gene (TTN). The disease is characterized by weakness and atrophy especially in the anterior compartment muscles of the lower leg, tibialis anterior, extensor hallucis longus and extensor digitorum longus1. Clinical symptoms typically begin at age 35 – 55 years and the disease is slowly progressive1. TMD was first described in Finnish patients; its prevalence in Finland is estimated to be >1/10,0002 which makes it the most common muscle disease in Finland. The Finnish founder mutation (FINmaj) is an 11-bp insertion-deletion mutation exchanging four amino acids in the 363rd and last exon of TTN (Mex6) which encodes the C-terminal Ig domain M10 of M-line titin3. In addition to the FINmaj mutation, we have reported several other mutations causing TMD in different European populations. These are missense or truncating mutations located in the last two TTN exons (Mex5 and Mex6)3-6 (Fig 1).

Homozygosity of the FINmaj mutation is rare but causes a completely different limb-girdle muscular dystrophy phenotype, LGMD2J (OMIM #608807)7, 8. LGMD2J is a severe childhood onset disease causing proximal muscle weakness in the first or second decade and progresses over the next twenty years to wheelchair confinement7, 8. One patient homozygous for the truncating French TMD mutation c.107890C>T (p.Q35964*) (NM_001267550.1) has also been reported with a proximal phenotype somewhat different to the homozygous Finnish LGMD2J patients. In the French patient the disease onset was at 25 years and the first muscle weakness occurred in the proximal upper limbs. Weakness and wasting progressed to all four limbs and the patient lost ambulation at the age of 56 years9.

TMD FINmaj-mutations in the last M10 domain of titin are predicted to cause cleavage of a larger part of the titin Cterminus since immunofluorescence studies of homozygote LGMD2J muscles showed an absence of titin M8/M9 domain epitopes3. Also Western blot analysis showed severe reduction of the C-terminal titin fragments in LGMD2J patient muscle5. Loss of protein interactions of C-terminal titin is thus a likely consequence of the TMD/LGMD2J mutations. Obscurin and obscurin-like 1 are known to interact with titin M10 domain and in LGMD2J muscles the FINmaj mutation leads to mislocalization of obscurin10. Next to the M10 domain is the is7 region, encoded by the second last exon 362 (Mex5), which contains an M-line binding site for the muscle-specific protease calpain 311. Calpain 3 is

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severely reduced in LGMD2J and it has been postulated that the LGMD2J-phenotype is caused by a loss-of-function mechanism including secondary calpain 3 abnormality12, 13, in contrast to the dominant late onset effect of the FINmaj mutation in TMD heterozygous patients.

Several families and patients with previously reported TMD-causing TTN mutations, have more complex, severe or unusual phenotypes5, 8. This study was initiated to clarify the molecular cause of the variant phenotypes observed in four Finnish and three Southern European families. Five patients proved to be compound heterozygotes with novel titin frameshift mutations combined with the previously reported TMD mutations. One Portuguese patient was homozygote for the previously reported Iberian TMD mutation, and one patient had the FINmaj mutation combined with a missense mutation in the A-band titin causing a new phenotype completely different from previously described titinopathies.

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SUBJECTS AND METHODS

Patients and biopsies DNA samples from eight patients of seven families were obtained from clinicians in different countries and screened for mutations in TTN. Four families were from Finland and one each from France, Spain and Portugal. Muscle biopsies were available for protein and mRNA studies in all probands. The study was approved by the local ethics committees and samples were obtained according to the Helsinki declaration.

Production and testing of monoclonal antibodies against the M10 domain The M10 domain of human titin was cloned into pGEX-6P-2 (GE Healthcare, Little Chalfont, UK). The GST-M10 was expressed in E. coli BL21 and affinity purified at Biomolecular Tools Finland Oy (Turku, Finland). The GST moiety was removed by cleavage with PreScission protease (GE Healthcare) and bound to glutathione sepharose resin. The M10 domain was purified by gel filtration and concentrated with Amicon Ultra cartridge (Merck Millipore, Billerica, MA, USA).

Monoclonal antibody development against the M10 domain conjugated to diphtheria toxin was carried out at BioGenes GmbH (Berlin, Germany). The hybridoma clones were screened by ELISA against native and denatured immunogen and further tested by Western blotting of wild-type and mutant titin is6-M10 constructs14 expressed in COS-1 cells, as well as muscle extracts. For the three clones selected for production, epitope mapping was performed on a SPOT array containing human and mouse M10 sequences as 15-mer peptides with a tiling of 3 residues (Peptide synthesis unit, Haartman Institute, University of Helsinki).

C-terminal M10 domain monoclonal titin antibodies Development of M10 monoclonal antibodies resulted in three clones (designated 7-4-4, 11-4-3, and 14-2-7) that efficiently stained C-terminal titin constructs in Western blotting, and recognized linear epitopes of human titin on peptide array. 11-4-3 recognized the sequence QGRFHI in the M10 domain of titin. The epitope is identical between human and mouse titin, and the antibody bound sequences from both species equally well on the peptide array. The

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specificity of the antibodies was further tested by running WB using skeletal muscle lysates from healthy control, FINmaj heterozygous TMD and FINmaj homozygous LGMD2J. All three antibodies detected several bands of various sizes, but only 11-4-3 showed specificity to titin C-terminus in skeletal muscle lysates as demonstrated by loss of most bands in the LGMD2J sample. Only two bands (approximate molecular weights 30 kDa and 100 kDa) remained unchanged, suggesting they represent unspecific cross reactions.

Western blotting Muscle biopsies were homogenized in reducing sample buffer containing 4% SDS and 10% β-mercaptoethanol, and heated 5 min at 100°C. SDS-PAGE and Western blotting were performed according to standard methods. Two different primary antibodies raised against titin M10, rabbit polyclonal antibody M10-15 and mouse monoclonal antibody 11-4-3 were used. In addition, mouse monoclonal calpain 3 antibody clones 2C4 and 12A2 (Leica Biosystems, Newcastle Upon Tyne, UK) were used, followed by HRP-conjugated secondary antibodies (Dako, Glostrup, Denmark) and ECL detection using the Pierce SuperSignal West Femto substrate (Thermo Fisher).

RNA extraction and reverse-transcription PCR (RT-PCR) Total RNA was extracted with RNeasy Fibrous Tissue Mini kit (Qiagen GmbH, Hilden, Germany) according to manufacturer’s instructions. cDNA was synthesized with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using random hexanucleotides. Primers spanning exon-exon junctions were designed for cDNA with Primer3 software. Fragments were amplified with DreamTaq™ DNA Polymerase (Fermentas), separated in agarose gel and isolated from gel for sequencing.

Sequencing Sanger sequencing of genomic DNA and cDNA was performed with standard techniques. Primer sequences are listed in Supplementary Table 1. PCR was performed using DreamTaq™ DNA Polymerase (Fermentas) according to standard protocol. PCR products were sequenced on an ABI3730xl DNA Analyzer (Applied Biosystems), using the Big-Dye Terminator v3.1 kit and analyzed with Sequencher 5.0 software (Gene Codes Corporation, Ann Arbor, MI, USA).

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RESULTS

Clinical characteristics of the patients Patient 1 A 16-year-old Finnish female was known to be heterozygous for the FINmaj mutation, inherited from her mother. The mother was diagnosed at age 50 with rather mild TMD, the father is healthy. The first symptoms occurred in early infancy with slightly delayed walking and no stage of crawling. At the age of four she was referred because of a peculiar walking and shortness of breath after walking 100 m. At the age of six she was unwilling to walk and had difficulties in climbing stairs. She had large calves, external rotation of the lower limbs while walking, and increased lordosis. Muscle biopsy showed early dystrophic findings with rimmed vacuolar pathology, while the immunohistochemistry of sarcolemmal membrane proteins was normal. Muscle MRI at age 12 showed no definite fatty replacement, but proximal muscle volume was decreased and there was minor oedema on STIR sequences in the right rectus femoris (Fig 2A). At age 16 she needs help in carrying her bag and getting up from the floor. She has mild contractures in the ankles, knees and elbows.

Patients 2 and 3 The patients 2 and 3 have been reported earlier in reference 8. Patient 2 was characterized with ‘infantile-onset generalized weakness’ and patient 3 as ‘childhood-onset LGMD’. Muscle imaging of patient 3 is shown in Figure 2B.

Patient 4 A 51-year-old Finnish male is heterozygous for the FINmaj mutation but with a phenotype differing completely from TMD, LGMD2J or any other described titinopathy. The patient’s father is asymptomatic and the mother has TMD with the FINmaj mutation. The first symptoms occurred at the age of 30 with muscle atrophy in the right calf. Ten years later muscle atrophy was observed also in the right thigh progressing later to the left thigh. The upper and left distal lower limbs remained normal. The patient started to use a cane after age 40. Muscle biopsy from the right vastus lateralis showed end-stage level non-specific dystrophic changes, whereas a second biopsy from the left vastus lateralis showed pronounced rimmed-vacuolar pathology without myofibrillar myopathy pathology. MRI findings were unique with fatty

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degenerative and atrophic changes in soleus and quadriceps on both sides and total tissue replacement by fat and connective tissue in the right gastrocnemius medialis and lateralis muscles (Fig 2C). In addition, similar less severe changes were observed in gluteus medius, adductor longus and semitendinosus on the right side.

Patients 5A and 5B Patients 5A and 5B (French family C), have been reported earlier in reference 5. Patient 5A is the mother of patient 5B. Muscle imaging of patient 5B is shown in Figure 2D.

Patient 6 The patient is a 36-year-old Spanish female heterozygous for the previously reported Iberian TMD mutation c.107889delA (p.K35963Nfs*9)5. She has an unusual early onset of the disease and a relatively severe phenotype, especially compared to her heterozygous mother who is asymptomatic still at the age of 58. Motor milestones were slightly delayed with walking after the age of 18 months, and general motor clumsiness during infancy. Walking difficulties and weakness slightly progressed and the first neurological examination at the age of 25 showed distal weakness in ankle dorsiflexors and mild weakness of hip flexors and knee flexors in the lower limbs. Muscle MRI, at the age of 26, displayed bilateral atrophy of soleus and tibialis anterior with more fatty replacement on the left side (Fig 2E). The muscles in the posterior compartment of the thighs showed atrophy on both sides and fatty degeneration more on the right side.

Patient 7 The patient, a 27-year-old Portuguese female, has a more severe distal TMD phenotype with early adult onset at 22 years of age and slowly progressive difficulty in running and frequent tripping. Neurological examination showed bilateral atrophy of the antero-lateral compartment of the legs and severe weakness of ankle dorsiflexion. Achilles tendon reflexes were abolished with mild Achilles contractures. Lower limb muscle MRI showed marked atrophy and fat infiltration of the anterolateral compartment and soleus muscles of the lower legs (Fig 2F). At the thigh level, hamstring muscles were severely replaced as were gluteus medius and minimus of the pelvic region. The patient proved to be homozygous for the previously reported Iberian TMD mutation c.107889delA (p.K35963Nfs*9)5. The parents, first degree cousins, are heterozygous for the same mutation, without symptoms or clinical signs of muscle disease at the age of 55-60 years,

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although subclinical myopathy with smaller lesions of fatty degeneration in gastrocnemius and soleus muscles were present on MRI of the father.

Patients' clinical characteristics are summarized in Table 1.

Molecular biological findings Titin and calpain 3 proteins were studied from muscle samples of patients by Western blotting. Reduction of C-terminal titin protein fragments of T (p.P30723S) in TTN exon 339. All these mutations were novel. Patient 7 showed the previously reported Iberian TMD mutation c.107889delA (p.K35963Nfs*9) homozygously. All found mutations and their consequences on protein level are summarized in Table 2. All 363 TTN

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exons from patient 3 were also sequenced but no additional mutation was identified with the primers used. Sequencing the DNA from both parents of patients 1, 2, 4 and 6, and from the mother of 5B confirmed that the new mutations were located on different alleles than the previously found and reported TMD mutations.

Subsequently RT-PCR and sequencing of cDNA from muscle biopsy was performed with primers designed for the TTN regions containing the new mutations found in patients 1, 2, 5A, 5B and 6. The sequencing chromatograms indicated a lower signal from the frameshift mutated alleles compared to the TMD alleles thus confirming earlier results of RT-PCR of exons Mex4-Mex6.

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DISCUSSION

Five of the studied patients with complex TMD phenotypes turned out to be compound heterozygotes, each with one previously known TMD mutation and five different novel frameshift mutations. Based on sequencing chromatograms of RT-PCR products from patients 1, 2, 5A, 5B and 6, there was in each case an indication of a much lower expression of the TTN allele with the frameshift mutation compared to the other allele with previously known TMD mutation. All the novel frameshift mutations cause a premature stop codon in the sequence more than 50-55 nucleotides upstream of the last exon-exon junction, which is likely to trigger nonsense-mediated decay (NMD)15. When the frameshift mutated mRNAs are degraded, the other TTN alleles with the known TMD mutations will be predominantly expressed. This may lead to compound heterozygous patients having a more severe phenotype or phenotypes similar to those homozygous for the corresponding TMD mutation. Since NMD is usually not complete, small amounts of truncated protein may still be expressed. As expected, the frameshift mutated alleles are recessive and alone do not cause disease: the fathers of patients 1 and 6 and the mother of patient 2, carriers of the frameshift alleles, were healthy.

A second mutation has not been found from patient 3 even though all TTN exons have been sequenced. It is, however, likely that also this patient has a frameshift mutation in the other TTN allele as there is loss of C-terminal epitopes on Western blotting and the RT-PCR results resemble those of patients 1, 2, 5A, 5B, and 6. A possible explanation for this putative second mutation escaping detection could be that the second mutation is a bigger frameshift-causing deletion or an inversion not detectable with Sanger sequencing, or that the primers used just do not identify the mutation despite showing good normal sequence. Other possibilities would include a deeper intronic mutation causing aberrant splicing, a mutation in the promoter or enhancer region of TTN, or an epigenetic change decreasing expression of the allele.

Loss of titin C-terminus in Western blotting in samples of patients 1, 2, 5A, 5B, 6, and 7 was indistinguishable from that seen in LGMD2J. This is in line with the predominant expression of the TMD alleles due to NMD of the other allele, leading to loss of functional C-terminal M10 domain at the protein level. Patient 3 showed residual staining of a high molecular weight titin band with the 11-4-3 mAb, although the smaller C-terminal titin fragments were missing. The case of patient 4 confirms the conclusion above, as the combination of the FINmaj and the novel A-band titin missense

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mutation shows less severe loss of titin C-terminus as compared to the FINmaj combined with a frameshift mutation (Fig 3).

Surprisingly, calpain 3 protein appeared normal in patient 5A with the Mex5 TMD mutation, in contrast to patients 1, 3, 6, and 7 who showed variable but clear calpain 3 reduction. Patient 4 showed a slightly reduced 94-kDa calpain 3 band, but proteolytic fragments were more pronounced, suggesting mild sample degradation that together with slightly lower total protein loading indicates calpain 3 is within normal range. Previously all FINmaj LGMD2J patients have shown severe reduction of calpain 3, whereas in heterozygous FINmaj TMD patients the amount of calpain 3 has been variably normal.

The Iberian TMD mutation c.107889delA (p.K35963Nfs*9) appears to have a reduced penetrance or recessive properties, since the parents of patient 7 and the mother of patient 6 are heterozygous for the mutation but seem to be subjectively healthy at age 55–60. It is also possible that they have a very late disease onset as indicated by minor changes on muscle MRI in the father of patient 7.

Patient 7, homozygous for the Iberian TMD mutation, and the compound heterozygous patients 5A, 5B and 6 have a more severe TMD phenotype rather than LGMD2J phenotype seen in FINmaj-homozygous patients and in patients 1 and 2 with FINmaj combined with a frameshift mutation. The reason why some homozygous TMD mutations or compound heterozygous TMD with frameshift mutation cause a more severe TMD while others cause LGMD2J is currently not known. The Iberian TMD mutation is located C-terminally from the FINmaj mutation, very close to titin C-terminus and could thus have a milder effect on the domain structure. However, one patient was recently reported to have the Iberian TMD mutation combined with a nonsense mutation and causing an LGMD2J phenotype16. Also a patient homozygous for the French Mex6 TMD mutation c.107890C>T (p.Q35964*), located next to the Iberian mutation and thus expected to have a comparable effect, showed a phenotype that resembled LGMD2J but also included involvement of distal muscles9.

The TMD phenotypes in patients 5A and 5B are interesting. The patients carry, in addition to the French Mex5 mutation, two different second mutations severely decreasing the expression level of the other allele on the mRNA level. The clear

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reduction of C-terminal titin, confirmed by Western blotting, is consistent with functional homozygosity for the Mex5 mutation; yet the patients show a severe TMD phenotype rather than LGMD2J. One factor contributing to the phenotype in both patients of this family could be the alternative splicing of Mex5: As the Mex5– (is7–) isoforms skip the mutation, C-terminal titin could actually retain some of its function, explaining why the patients do not develop the LGMD2J phenotype. However, the titin M10 immunoreactivity in the Western blot was very weak, and did not reveal significant amounts of normal Mex5– titin in the analyzed biopsies.

Mex5 encodes the is7 region containing a binding site for calpain 3. Surprisingly, Western blotting did not indicate a reduction of calpain 3 in patient 5A with the Mex5 TMD mutation, in contrast to patients with Mex6 TMD mutations. We have previously postulated that in the case of FINmaj mutation, the loss of calpain 3 binding could be due to proteolytic cleavage of titin upstream of the domains M8/M9 since LGMD2J (FINmaj homozygote) muscles are missing immunoreactivity to the M8/M9 domain antibody3 and they also have severely reduced calpain 312. As the French Mex5 TMD mutation truncates the is7 region C-terminally, close to the is7/M10 boundary, it is possible that the mutation does not directly interfere with the binding site of calpain 3. Moreover, since the mutation removes the entire M10 without causing domain unfolding, the mutant protein might be less prone to proteolytic cleavage, thus providing adequate sites for calpain 3 binding.

Patient 4 had the FINmaj mutation combined with a missense mutation c.92167C>T (p.P30723S) that changes a proline to a serine in the A140 domain of A-band titin. The patient has inherited the A-band mutation from his healthy father proving its recessive properties. Thus far reported pathogenic mutations in the A-band titin are missense mutations in the A150 domain causing hereditary myopathy with early respiratory failure (HMERF)17-21, missense mutations in different A-band regions causing arrhythmogenic right ventricular cardiomyopathy (ARVC)22 and truncating mutations in different A-band regions causing dilated cardiomyopathy23, 24. The phenotype of the patient however differs completely from previously described titinopathies. Although the patient has the FINmaj mutation, the symptoms are more severe and not similar to either TMD or LGMD2J. He has marked dystrophic changes in quadriceps femoris and soleus but tibialis anterior has a normal appearance on MRI. So far we do not have a plausible explanation for how this compound heterozygosity gives rise to the observed new titinopathy phenotype.

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Frameshift and nonsense mutations in different A-band regions of titin have previously been reported to cause dilated cardiomyopathy23,

24

. The mutations may cause expression of truncated titin proteins which are integrated into the

sarcomere and cause disease by means of dominant negative mechanism23. Truncated titin proteins are also expressed in patients with early-onset myopathy with fatal cardiomyopathy25 and in patients with core myopathy26 who have homozygous frameshift mutations in the M-line titin. None of the patients in our study showed cardiomyopathy. Instead the identified frameshift mutations in the compound heterozygous patients of our study very likely cause nonsensemediated decay with no or only small amounts of protein product expressed from the frameshift mutated allele.

Our results considerably expand the range knowledge of muscleular disorders caused by TTN mutations and suggest that the coexistence of second mutations may be a more common general mechanism causing phenotypic variability. In mMany muscular dystrophyies phenotype variations are known to exist in between patients with identical causative mutations, and our study may provide new insight for exploring the into one underlying mechanisms of those variations.

Acknowledgements We thank Merja Soininen, Helena Luque, Jaana Leppikangas and Satu Luhtasela for technical assistance. This study was supported by the Folkhälsan Research Foundation, the Association Française contreles Myopathies, the Academy of Finland, the Sigrid Jusélius Foundation, the Liv och Hälsa Foundation, and Tampere University Hospital Research Funds (B.U.).

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REFERENCES

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3. Hackman P, Vihola A, Haravuori H et al. Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin. Am J Hum Genet 2002;71(3):492-500.

4. Van den Bergh PY, Bouquiaux O, Verellen C et al. Tibial muscular dystrophy in a belgian family. Ann Neurol 2003;54(2):248-251.

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6. Pollazzon M, Suominen T, Penttila S et al. The first italian family with tibial muscular dystrophy caused by a novel titin mutation. J Neurol 2010;257(4):575-579.

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10. Fukuzawa A, Lange S, Holt M et al. Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band: Implications for hereditary myopathies. J Cell Sci 2008;121(Pt 11):1841-1851.

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11. Sorimachi H, Kinbara K, Kimura S et al. Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence. J Biol Chem 1995;270(52):3115831162.

12. Haravuori H, Vihola A, Straub V et al. Secondary calpain3 deficiency in 2q-linked muscular dystrophy: Titin is the candidate gene. Neurology 2001;56(7):869-877.

13. Charton K, Daniele N, Vihola A et al. Removal of the calpain 3 protease reverses the myopathology in a mouse model for titinopathies. Hum Mol Genet 2010;19(23):4608-4624.

14. Sarparanta J, Blandin G, Charton K et al. Interactions with M-band titin and calpain 3 link myospryn (CMYA5) to tibial and limb-girdle muscular dystrophies. J Biol Chem 2010;285(39):30304-30315.

15. Nagy E, Maquat LE. A rule for termination-codon position within intron-containing genes: When nonsense affects RNA abundance. Trends Biochem Sci 1998;23(6):198-199.

16. Ceyhan-Birsoy O, Agrawal PB, Hidalgo C et al. Recessive truncating titin gene, TTN, mutations presenting as centronuclear myopathy. Neurology 2013;81(14):1205-1214.

17. Ohlsson M, Hedberg C, Bradvik B et al. Hereditary myopathy with early respiratory failure associated with a mutation in A-band titin. Brain 2012;135(Pt 6):1682-1694.

18. Pfeffer G, Elliott HR, Griffin H et al. Titin mutation segregates with hereditary myopathy with early respiratory failure. Brain 2012;135(Pt 6):1695-1713.

19. Izumi R, Niihori T, Aoki Y et al. Exome sequencing identifies a novel TTN mutation in a family with hereditary myopathy with early respiratory failure. J Hum Genet 2013.

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21. Palmio J, Evila A, Chapon F et al. Hereditary myopathy with early respiratory failure: Occurrence in various populations. J Neurol Neurosurg Psychiatry 2013.

22. Taylor M, Graw S, Sinagra G et al. Genetic variation in titin in arrhythmogenic right ventricular cardiomyopathyoverlap syndromes. Circulation 2011;124(8):876-885.

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26. Chauveau C, Bonnemann CG, Julien C et al. Recessive TTN truncating mutations define novel forms of core myopathy with heart disease. Hum Mol Genet 2013.

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FIGURE LEGENDS

Figure 1. Titin M-line TMD mutations are located in Mex5 and Mex6 exons of TTN encoding the is7 region and the most C-terminal domain M10 in the sarcomere M-line. Calpain 3 has binding site in is7 and obscurin, obscurin-like 1 and myospryn bind to M10.

Figure 2. Muscle imaging of the patients A) Patient 1 at the age of 12. The musculature is still relatively well preserved. There is oedema in the right rectus femoris muscle when compared to the left side (Upper), but without dystrophic degenerative change. The left medial head of gastrocnemius shows oedema and its volume is reduced compared to the right side.

B) Patient 3 at the age of 30. All muscles are severely affected. All hamstring and adductor muscles show variable degrees of severe fatty degeneration and atrophy.

C) Patient 4 at the age of 51. The quadriceps and soleus muscles show advanced dystrophic changes. At the right side also the semitendinosus, gastrocnemius medialis and lateralis show severe fatty degeneration and marked atrophy. Less severe changes are seen in the adductor longus muscle on the right. The tibialis anterior muscles are relatively spared.

D) Patient 5B at the age of 40. Severe dystrophic fatty degeneration is seen in the antero-lateral compartments as well as soleus muscles of the lower legs.

E) Patient 6 at the age of 26 years: The posterior thigh muscles, particularly on the right side, show dystrophic changes. Most severely affected are the long head of biceps femoris, semitendinosus and semimembranosus muscles. On the lower legs the left tibialis anterior is already severely involved. The left gastrocnemius medialis is more severely affected than the right, and soleus more on the right side.

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F) Patient 7 at the age of 25. At the thigh level, biceps femoris (long head), semimembranosus and semitendinosus show moderate fatty degeneration, and quadriceps shows early changes on the left side. Gracilis is hypertrophic and biceps femoris (short head) is spared. The tibialis anterior, extensor hallucis longus, soleus and peroneal muscles are severely dystrophic and extensor digitorum longus replaced more on the left side. The gastrocnemius medialis and lateralis muscles are hypertrophic.

Figure 3. Western blotting A) Western blotting of control skeletal muscle lysate with the titin C-terminal antibodies M10-1 and 11-4-3 reveals a distinctive pattern of bands, representing C-terminal protein fragments of different lengths. The specificity of the bands is demonstrated in FINmaj TMD sample, which shows reduction of bands, and in FINmaj LGMD2J, where the immunoreactivity is nearly completely lost. M10-1 bands appear specific, whereas with 11-4-3, the bands of approximately 30 kDa and 90 kDa probably represent unspecific cross-reactions with unknown proteins. Especially the samples from patients 1, 5A, 6 and 7 show a fragment pattern similar to LGMD2J, with dramatic loss of titin C-terminal epitope recognition.

B) Western blotting of calpain 3 shows clear reduction of the full-size 90 kDa calpain 3 and its proteolytic fragments (30 kDa and 60 kDa with 2C4 and 12A2, respectively) in patients 1, 3, 6 and 7. Calpain 3 levels in patient 5A appear normal, whereas patient 4 shows slight reduction possibly caused by degradation as seen by enhanced protelytic fragment bands, and lower total protein loading. In addition, the lower sensitivity of 12A2 Ab may account for reduced 90 kDa band.

Figure 4. cDNA sequencing cDNA sequencing chromatograms of the TTN Mex6 FINmaj TMD region of a wildtype, FINmaj TMD patient and patient 1. The chromatograms indicate unequal expression of the two TTN alleles in patient 1 with lower expression of the other allele compared to the FINmaj TMD allele.

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Table 1. Clinical characteristics of TMD/LGMD2J complex patients.

Patient 1

Sex/Age/

Age at

Country

onset

F/16/

0

Finland

Patient 2

M/37/

18 mo

Symptoms at onset

Visible muscle

Disease

atrophies

progression

Delayed early motor

Hip region,

Progressive,

development and

lower back and

ambulant

frequent falls

lower limbs

Generalized weakness

NA

Muscle weakness

CK

Muscle

level

biopsy

Hand grip, pelvic, lower

17x

Dystrophy

limbs, unable to

UNL

RV

EMG

Myopathy

elevate/lift heavy objects

Finland

Slowly

Proximal and distal

2x

Myopathy

progressive,

lower limbs, upper limbs

UNL

RV

Progressive,

Proximal lower limbs,

3x

Myopathy

cane at age

loss of left ankle

UNL

RV

30

dorsiflexion

Myopathy

ambulant Patient 3

F/32/

6

Finland

Patient 4

M/51/

Waddling gait, mild

No

elbow contractures

30

Finland

NA

Atrophy of the right

Thighs, right

Slowly

Marked quadriceps and

2.5x

Dystrophy

Myopathy in

calf

calf

progressive,

right gastrocnemius

UNL

RV

the thighs

cane at age

weakness

Myopathy

40 Patient 5A

F/76/ France

30

Difficulties in standing on heels

Legs

Slowly

Pelvic, proximal and

1.5x

Dystrophy

progressive,

distal lower limbs, loss

UNL

RV

cane at age

of ankle dorsiflexion

56

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Patient 5B

M/49/

20

France

Difficulties in standing

Thighs, legs,

Progressive,

Pelvic, proximal and

2x

on heels

hands

cane at age

distal lower limbs, loss

UNL

45

of ankle dorsiflexion,

NA

Myopathy

Myopathy

dysphagia Patient 6

Patient 7

F/36/

Child-

Spain

hood

F/27/ Portugal

22

Walking difficulties

Posterior

Slowly

Ankle dorsiflexion, hip

2x

Mild

compartment

progressive,

flexion, knee flexion

UNL

myopathy

legs

ambulant

Walking difficulties,

Antero-lateral

Slowly

Loss of ankle

1.3x

Mild

occasional falls

compartment

progressive,

dorsiflexion

UNL

myopathy

legs

ambulant

RV NA

CK, creatine kinase; EMG, electromyography; UNL, upper normal limit; RV, rimmed vacuoles; NA, not available

Table 2. TTN mutations found in the DNA of the patients (transcript variant IC, NM_001267550.1 and transcript variant N2-A, NM_133378.4). Previously reported

New TTN mutation (IC)

TMD mutation (IC) Patient 1

Previously reported

New TTN mutation (N2-A)

Phenotype

LGMD2J

TMD mutation (N2-A)

Exon 363, FINmaj3

Exon 358 (M-line)

Exon 312, FINmaj3

Exon 307 (M-line)

c.107780-107790

c.101113delT

c.100076-100086

c.93409delT

delAAGTAACATGG

p.S33705Lfs*4

delAAGTAACATGG

p.S31137Lfs*4

insTGAAAGAAAAA

(paternal)

insTGAAAGAAAAA

(paternal)

p.3592735930delinsVKEK

p.3335933362delinsVKEK

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(maternal) Patient 2

Patient 3

(maternal)

Exon 363, FINmaj3

Exon 207 (PEVK region)

Exon 312, FINmaj3

Exon 161 (PEVK region)

(paternal)

c.39492dupT

(paternal)

c.32190dupT

Exon 363, FINmaj3

p.E13165fs*1

p.E10731fs*1

(maternal)

(maternal) Exon 312, FINmaj3

not found

(maternal) Patient 4

Patient 5A

Patient 5B

not found

LGMD2J

novel titinopathy

(maternal)

Exon 363, FINmaj3

Exon 339 (A-band)

Exon 312, FINmaj3

Exon 288 (A-band)

(maternal)

c.92167C>T

(maternal)

c.84463C>T

p.P30723S

p.P28155S

(paternal)

(paternal)

Exon 362, French5

Exon 352 (A-band)

Exon 311, French5

Exon 301 (A-band)

c.107647delT

c.98105delC

c.99943delT

c.90401delC

p.S35883Qfs*10

p.P32702Lfs*15

p.S33315Qfs*10

p.P30134Lfs*15

Exon 362, French5

Exon 357 (A-band)

Exon 311, French5

Exon 306 (A-band)

(maternal)

c.100558-100561dupACTG

(maternal)

c.92854-92857dupACTG

p.G33521Dfs*25 Patient 6

LGMD2J

severe TMD

severe TMD

p.G30953Dfs*25

Exon 363, Iberian5

Exon 318 (A-band)

Exon 312, Iberian5

Exon 267 (A-band)

c.107889delA

c.67089delT

c.100185delA

c.59385delT

p.K35963Nfs*9

p.K22364Rfs*24

p.K33395Nfs*9

p.K19796Rfs*24

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severe TMD

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Patient 7

(maternal)

(paternal)

(maternal)

(paternal)

Exon 363, Iberian5

-

Exon 312, Iberian5

-

(homozygote,

(homozygote,

maternal and paternal)

maternal and paternal)

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severe TMD

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Contributions Anni Evilä

acquisition of data, analysis and interpretation of data, drafting/revising manuscript

Anna Vihola

acquisition of data, analysis and interpretation of data, drafting/revising manuscript

Jaakko Sarparanta

analysis and interpretation of data, drafting/revising manuscript

Olayinka Raheem

acquisition of data, analysis and interpretation of data, revising manuscript

Johanna Palmio

analysis and interpretation of data, drafting/revising manuscript

Satu Sandell


Bruno Eymard


Isabel Illa


Ricardo Rojas-Garcia


Karolina Hankiewicz


Luis Negrao


Tuija Löppönen


Pekka Nokelainen


Mikko Kärppä


Sini Penttilä


Mark Screen


Tiina Suominen


Isabelle Richard


Peter Hackman

acquisition of data, analysis and interpretation of data, drafting/revising manuscript, study supervision

Bjarne Udd

study conception and design, acquisition of data, analysis and interpretation of data drafting/revising manuscript, study supervision

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Figure 1. Titin M-line TMD mutations are located in Mex5 and Mex6 exons of TTN encoding the is7 region and the most C-terminal domain M10 in the sarcomere M-line. Calpain 3 has binding site in is7 and obscurin, obscurin-like 1 and myospryn bind to M10. 170x51mm (300 x 300 DPI)

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Figure 2. Muscle imaging of the patients A) Patient 1 at the age of 12. The musculature is still relatively well preserved. There is oedema in the right rectus femoris muscle when compared to the left side (Upper), but without dystrophic degenerative change. The left medial head of gastrocnemius shows oedema and its volume is reduced compared to the right side. B) Patient 3 at the age of 30. All muscles are severely affected. All hamstring and adductor muscles show variable degrees of severe fatty degeneration and atrophy.

C) Patient 4 at the age of 51. The quadriceps and soleus muscles show advanced dystrophic changes. At the right side also the semitendinosus, gastrocnemius medialis and lateralis show severe fatty degeneration and marked atrophy. Less severe changes are seen in the adductor longus muscle on the right. The tibialis anterior muscles are relatively spared. D) Patient 5B at the age of 40. Severe dystrophic fatty degeneration is seen in the antero-lateral compartments as well as soleus muscles of the lower legs.

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E) Patient 6 at the age of 26 years: The posterior thigh muscles, particularly on the right side, show dystrophic changes. Most severely affected are the long head of biceps femoris, semitendinosus and semimembranosus muscles. On the lower legs the left tibialis anterior is already severely involved. The left gastrocnemius medialis is more severely affected than the right, and soleus more on the right side. F) Patient 7 at the age of 25. At the thigh level, biceps femoris (long head), semimembranosus and semitendinosus show moderate fatty degeneration, and quadriceps shows early changes on the left side. Gracilis is hypertrophic and biceps femoris (short head) is spared. The tibialis anterior, extensor hallucis longus, soleus and peroneal muscles are severely dystrophic and extensor digitorum longus replaced more on the left side. The gastrocnemius medialis and lateralis muscles are hypertrophic. 170x191mm (300 x 300 DPI)

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Figure 3. Western blotting A) Western blotting of control skeletal muscle lysate with the titin C-terminal antibodies M10-1 and 11-4-3 reveals a distinctive pattern of bands, representing C-terminal protein fragments of different lengths. The specificity of the bands is demonstrated in FINmaj TMD sample, which shows reduction of bands, and in FINmaj LGMD2J, where the immunoreactivity is nearly completely lost. M10-1 bands appear specific, whereas with 11-4-3, the bands of approximately 30 kDa and 90 kDa probably represent unspecific crossreactions with unknown proteins. Especially the samples from patients 1, 5A, 6 and 7 show a fragment pattern similar to LGMD2J, with dramatic loss of titin C-terminal epitope recognition. B) Western blotting of calpain 3 shows clear reduction of the full-size 90 kDa calpain 3 and its proteolytic fragments (30 kDa and 60 kDa with 2C4 and 12A2, respectively) in patients 1, 3, 6 and 7. Calpain 3 levels in patient 5A appear normal, whereas patient 4 shows slight reduction possibly caused by degradation as seen by enhanced protelytic fragment bands, and lower total protein loading. In addition, the lower

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sensitivity of 12A2 Ab may account for reduced 90 kDa band. 170x203mm (300 x 300 DPI)

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Figure 4. cDNA sequencing cDNA sequencing chromatograms of the TTN Mex6 FINmaj TMD region of a wildtype, FINmaj TMD patient and patient 1. The chromatograms indicate unequal expression of the two TTN alleles in patient 1 with lower expression of the other allele compared to the FINmaj TMD allele. 170x80mm (300 x 300 DPI)

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Supplementary Table 1. Primers used for sequencing TTN. gDNA primers (5'→3')

cDNA primers (5'→3')

TTN-ex1-F

TAGGTCCTCTCCCATCATGC

TTN-cDNA-ex205-209-F

AGTTGTGGAAGAGCCAGAGC

TTN-ex1-R

GGTCAGCACTGGCAAGAAAT

TTN-cDNA-ex205-209-R

GCACTTCTGGCACTTTTGCT

TTN-ex2-F

GGAGCAATCCATTTGGAGAA


GAGGTTACTGGCCTGATGGA

TTN-ex2-R

TGCTGGATTCTAACGGCAAT


AGGATTTTCCCTCCTCCAAA

TTN-ex3-F

TCCCTTCCTGCCTTCCTAAT


ACCACTCCAACCAAGATTCG

TTN-ex3-R

CTGCCCATAGCTTTTTCTGG


GCCAGAATCACCCCTGTCT

TTN-ex4-F

GCGAGTCATCACCTGGTTTT


TGACCCTCTAGAAGTGTCCTCA

TTN-ex4-R

ACCAGCTCTCTCCCCTTCTC


TCCCATGCCTTCAAGAGTTT

TTN-ex5-6-F

TCATCTTTCCCTTCCCACAG


GGCTACCACCAGCTCATCAT

TTN-ex5-6-R

GGACACTGAAGAAGCGAACC


GGTTGGTTCTGAAGGCTCTG

TTN-ex7-F

CCCTCTGAATGGGTTATGGA


TGTCAGCATAAGCCGCTCCAGA

TTN-ex7-R

GGCAGAAATCCAGTTGGAGA


TCAGGGTTGTCAGGTCATCTGTGT

TTN-ex8-F

TCTAGGTGGCTCAGTTTTCCA


GCCTCCTTCAGCAGTTTCAG

TTN-ex8-R

ACATCTCCGACCACCTTTTG


CAGTGGCAGAGTCAGATCCA

TTN-ex9-F

AGCTGGGATTACAGGTGTGC

TTN-ex9-R

GGGAGCATTCAGGGACACTA

TTN-ex10-F

TTCATTGGGCATCAGGAAAT

TTN-ex10-R

AGATGTGCCCTAAACTGTTCTG

TTN-ex11-F

CAATTTGCTAAGGCGCATGT

TTN-ex11-R

TGCAAATGAAATGGTGCAAG

TTN-ex12-F

TTGCCAGTGAAACAGTATGGA

TTN-ex12-R

GATGGATGAAAATCAAATGTGC

TTN-ex13-F

TGTGTTTGAAGCCCTGTTCA

TTN-ex13-R

GGTGAAGAGATAAGTGGAGAAAGG

TTN-ex14-15-F

GTCCTGATGCCCAGGAGATA

TTN-ex14-15-R

CCCCAGAGATGCTCTGTTTC

TTN-ex16-F

ACAGGCATGACAAGGAGCTT

TTN-ex16-R

AGGCAAGACTCCACTGGCTA

TTN-ex17-F

CCAGTGGAGTCTTGCCTTTC

TTN-ex17-R

CCCTTCTTCCTTGCCAAATA

TTN-ex18-F

CCATTTTGTGGCAGTTGAAA

TTN-ex18-R

GTGATGGCATGTGCATTAGG

TTN-ex19-20-F

TGTCCCTTTTTGGAACTTGG

TTN-ex19-20-R

TCGGTGGGGTGAGTAAATTC

TTN-ex21-F

TGCATTTTTCTCAATTTTCAGC

TTN-ex21-R

CAAAGACAATGGGGCAAAGT

TTN-ex22-F

TGGCTGGTTGGATTTGTTAG

TTN-ex22-R

TAACCCCGAGCTCATCACTT

TTN-ex23-F

GATGCCACAACATTCACTGG

TTN-ex23-R

TACTGTGGCAAGGAGCTGTG

TTN-ex24-F

CACAGCTCCTTGCCACAGTA

TTN-ex24-R

GCCATTTTAGCCCTCGATTT

TTN-ex25-F

AGTTGTGGGGGCAACAATAA

TTN-ex25-R

TGCTGGCCTGTGAAAATATG

TTN-ex26-27-F

CATATTTTCACAGGCCAGCA

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TTN-ex26-27-R

AGGCAGAATCTTGGCTGACA

TTN-ex28.1-F

ACCCCAACCCTGACATTGTA

TTN-ex28.1-R

TCAGGCAATTGGGATTCTTC

TTN-ex28.2-F

TGGAACAGATCACACATCTGC

TTN-ex28.2-R

TTCGAGACTTGAGCTCCACA

TTN-ex28.3-F

GAACCAAGGCCTGAGTTTCA

TTN-ex28.3-R

AACCTGAGGAATGGGGAAAG

TTN-ex29-F

AAACCTCCAGTCACGCATTC

TTN-ex29-R

CCACATGCTAAGGGTGACTTT

TTN-ex30-F

CCCATCAGCGGGTAGATATT

TTN-ex30-R

CAGGAATGGGAGGACTTACG

TTN-ex31-F

GCCTGCAGTGAGCATCATTA

TTN-ex31-R

TCACATCAGGGACTGACACC

TTN-ex32-F

CAGTGGGCGTGTCTCTGTC

TTN-ex32-R

TCCACAGGACATCAATTCCA

TTN-ex33-F

CGAACTCATGCTTCAGACGA

TTN-ex33-R

AAGGAATTTTGGGGGAAATG

TTN-ex34-F

GGGATTTCTTTGGTACCTTCTCA

TTN-ex34-R

TGTGTCTCAGGAAGGTTTAGAGG

TTN-ex35-F

TGCAGGGTGAGTAAGCAATG

TTN-ex35-R

AACAGTGGCATTTTCCAAGG

TTN-ex36-37-F

AGCAGTCAGCCCATGGTTAC

TTN-ex36-37-R

CCCACCCAATCAAGGATTTT

TTN-ex38-F

AATCTCCTCAACAGACGCACT

TTN-ex38-R

TGGTCTCTGGCTTCTTTCACT

TTN-ex39-40-F

GCTACACAATGATGAATACATGGAA

TTN-ex39-40-R

AAAGGATGGTGGTTAGAAAATG

TTN-ex41-F

TCACATGCCATTCATTGACC

TTN-ex41-R

GCTGGAACCACATGAATACCA

TTN-ex42-43-F

TTTTTCATGGGCTATTTGTCA

TTN-ex42-43-R

TGTGATGGAGGAGAAGCTGA

TTN-ex44-F

GGCATGCCCAGAGAAAAGTA

TTN-ex44-R

GGTAAAGGAGAGGCGAGACC

TTN-ex45-F

GGACAAGGCAGTAGGAGTGG

TTN-ex45-R

TGGACTGACCACAGTATGCAA

TTN-ex46-F

ATATTGCATCTGGCATGCTG

TTN-ex46-R

CATGGCAGAAAAGCTGCATA

TTN-ex47-F

TTTGGCATGGACTTTTTGGT

TTN-ex47-R

TCTAAACCAAGCATGCGACA

TTN-ex48.1-F

AAGCAGAAGAAGGCCATCAA

TTN-ex48.1-R

TGTAATAAACACTGGTGCAAAGC

TTN-ex48.2-F

AAAACCAATTCGCTGTGCTC

TTN-ex48.2-R

TGTGGATAATTCCCTTCAGGTT

TTN-ex48.3-F

AATGCCTGAAGAGCCTGAGA

TTN-ex48.3-R

GGGCTCTTGGGTGATGTTTA

TTN-ex48.4-F

TCCAGAAGAGCAGAGATTAAACC

TTN-ex48.4-R

GCAAGCTAAATCATTACAATCCA

TTN-ex49-F

TGCTGTAATCCATCTCACCAA

TTN-ex49-R

CTTGGTTGTTGGTCTCTCCA

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TTN-ex50-F

CCATTTTAAATTATGGCTCATGTC

TTN-ex50-R

AAAGTGTACTGACTGAATTGTTTGC

TTN-ex51-52-F

TTTGCGGAGTTTATGTTTCAGA

TTN-ex51-52-R

CTTCCATGGGGTAAGAAAGC

TTN-ex53-54-F

GGTGTTGAAAGTGTTTGGGAAT

TTN-ex53-54-R

TGGAGCAATGTCTCGATCTG

TTN-ex55-56-F

GGCCTAAAGTCAATCACTGAGC

TTN-ex55-56-R

GCAGGTTCTGTGGAAGGAAG

TTN-ex57-58-F

GGTGGAATGCAGTGCAAACT

TTN-ex57-58-R

CAGAAAAGCCAGTCCCTCAC

TTN-ex58-F

CCTTAATGCAAATCATTCCTTTTT

TTN-ex58-R

TCAGAGTGGCCTGGAAGG

TTN-ex59-60-F

GCCACTTTTGCTATAGGGATAATA

TTN-ex59-60-R

GAAATGCCCAATCCTCTCTG

TTN-ex61-62-F

CAAAAACAAATTAGTAGATTGACCTTT

TTN-ex61-62-R

CACCCCAGGCAAGAAAATCT

TTN-ex63-64-F

TTGCCCAGTGACCTGTTTTT

TTN-ex63-64-R

TGAATGATGGTGGTTCTGTGA

TTN-ex65-66-F

TGGGTAGGAGTTACAATATGTTTCTT

TTN-ex65-66-R

TCCAATGCAAACAGCAAAAC

TTN-ex67-F

TTCGGAAGCAGTAGCTGTGA

TTN-ex67-R

CTCTTGGAAAGAGGCAATGG

TTN-ex68-69-F

ATTCAGCCATTGCCTCTTTC

TTN-ex68-69-R

GGGGAGAAGGTGGTTTTCAT

TTN-ex70-71-F

TGCTGGACTCTACTTACCCATT

TTN-ex70-71-R

CACAGAAACAGAAAAGAGTGTTTGA

TTN-ex72-73-F

CCATCTCACTGGGAGAAACAA

TTN-ex72-73-R

CAGATGGAGTTGCAATTTTGA

TTN-ex74-75-F

AATTGCAACTCCATCTGAAGC

TTN-ex74-75-R

AAGGCGGTTCTAAGGAAGAAA

TTN-ex76-77-F

TGTCTCTTAAATCATTTGCATTCTTC

TTN-ex76-77-R

GCCACAGTTTGCAAAGAAAA

TTN-ex78-F

CATTGCCTTTCTTCTCATCCT

TTN-ex78-R

TTTGATTAATGTTGCCTCCAA

TTN-ex79-F

TCTTCAGCTACAAAAACAAGCTC

TTN-ex79-R

TGTTGAAGAATTGCAACCAAA

TTN-ex80-81-F

TTTTTCCTTTATAGTAAGCAAGTGTTC

TTN-ex80-81-R

TTTGAGGAAAATGATCATGGA

TTN-ex82-83-F

TGATCCATGATCATTTTCCTCA

TTN-ex82-83-R

CACCACGTCCACATGAAAGA

TTN-ex84-85-F

GCAAAAGCACTTCTCACTTGG

TTN-ex84-85-R

CGGTGGTTCTATGGTACAAAGG

TTN-ex86-87-F

GCTCTCTTTCTTTTCAAACATCC

TTN-ex86-87-R

AAATGGCACCAGCCTTTTG

TTN-ex88-F

GGCTGGTGCCATTTAGTTTC

TTN-ex88-R

AAGCACACCCACCCTCCTAT

TTN-ex89-F

TGCATAAATTTGTTTTCCAGAGC

TTN-ex89-R

TCGTTGAAGAGTCAAACAGGAA

TTN-ex90-F

TCTGAGGGAGTGGAAGATTGA

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TTN-ex90-R

TGGAGAGGAAACAAAGGGAA

TTN-ex91-92-F

CATCCACCAAAAATGCCTCT

TTN-ex91-92-R

TGGTATTGCCTCTGAATTTGC

TTN-ex93-94-F

CCGTCACAAAAGAGCCAAAT

TTN-ex93-94-R

CTGGGGCTAAAGTGACCAAA

TTN-ex95-96-F

TGCTGAACTGGAGTTGTTCG

TTN-ex95-96-R

AAAAATTAGGCCTCCCCAGA

TTN-ex97-98-F

TGCATTAGGGTTGGTCTCAG

TTN-ex97-98-R

AAAAACGACACCTTCAGGCT

TTN-ex99-F

GTTGGTTAGCCTTGATTTTTAACT

TTN-ex99-R

CCTTCGGGAAGCCACATAAT

TTN-ex100-F

GAGAGGAATAAACCAGGCACA

TTN-ex100-R

TGCAGATGAGCAACAACTGA

TTN-ex101-102-F

ACTGCCAACCTCCTAAGGCT

TTN-ex101-102-R

ATCTTGAGATGGATGGCCC

TTN-ex103-F

CCATTTTGCATCAGGGACTT

TTN-ex103-R

CTTGGCTTTGCTTGAGCAGT

TTN-ex104-105-F

GGAGCTGGAGAAGAAGAGGAA

TTN-ex104-105-R

GCATGTCATGGTGCAAGTCT

TTN-ex106-F

CACATCGCTAGCGCTAAACT

TTN-ex106-R

ACGCACCTTCGATTCTGAG

TTN-ex106-F

GAGCACATCTGCGTGTGATA

TTN-ex106-R

TCTACAGGTAGGGTTGCAGG

TTN-ex107-F

ATTTCTTCTCATTTTGGATTTTTGA

TTN-ex107-R

GGCTTAAATTATACATTCACTGGAAA

TTN-ex108-109-F

CAAAAGTTGGATGATTTTTCACC

TTN-ex108-109-R

TTCTGTGATTTACAAAATTATGCC

TTN-ex110-F

CTTACCTGCGTGGTTGCATA

TTN-ex110-R

AATGAAAAGTCTCATGCATTTCT

TTN-ex111-F

CTACTTTTATTTGTTCCGACTG

TTN-ex111-R

TTTTGTTTGATGATTTTCAGAG

TTN-ex112-F

AGCATTTTAGTTACTTACACCA

TTN-ex112-R

GAAAGTGAGTGAAGGGAGAG

TTN-ex113-F

CTCCTACAAACAACTAACAATG

TTN-ex113-R

CCCTCACTTCTTCTTTCTCT

TTN-ex114-F

GAAGCAGTTGGATGGATAGA

TTN-ex114-R

ACTGAGCAAAAATGAAGAAAAG

TTN-ex115-F

TCCCTTCTTATTCCTGTGTT

TTN-ex115-R

CTGATTTATACATTGCTGCC

TTN-ex116-117-F

GAGAGAGAGAATTTTAATAGGA

TTN-ex116-117-R

TTCAGATTTGTGAGTTACTTAG

TTN-ex118-119-F

ATGTGTTTGATCTGATGTCTTG

TTN-ex118-119-R

GGCTATAGGTATGCACACTG

TTN-ex120-F

GGTTTCTGAGGAACTGACAC

TTN-ex120-R

GATACAAGATGGATGCTAAGA

TTN-ex121-F

GTGAAGATTTATGCTTTAATGT

TTN-ex121-R

AACAGTAAAAACACAAGAAGAA

TTN-ex122-123-F

TTGTGCATGTGCCTATGTTCTT

TTN-ex122-123-R

TGAGCAACATAGAAGTGGCAAT

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Annals of Neurology

TTN-ex124-125-F

CTTCTATGTTGCTCATTTATTT

TTN-ex124-125-R

AGCACAGACCAGATGGAAAA

TTN-ex126-F

TGTACTGTGCAACTCTTCCC

TTN-ex126-R

TGTGGAAGAAGAAGAGACTT

TTN-ex127-F

GTTATCCATCTGTAGTGACC

TTN-ex127-R

GTCTCTTTTCATTGGTCTGT

TTN-ex128-129-F

TGCTATTTTGATTACTCTTTGA

TTN-ex128-129-R

TTATGCAAATGTGAAGGTATTA

TTN-ex130-131-F

CCCACAACTGTGTCACTTCA

TTN-ex130-131-R

ACAGCAGCAGAGAGAAAGAA

TTN-ex132-F

ACCTCCCCAGCAGAAAAACT

TTN-ex132-R

ACCACAAAAAGGCAGCCATA

TTN-ex132-133-F

TCTGGCTAGTATATTTTTTACG

TTN-ex132-133-R

CCTATTCCACTAGATTAGTTTT

TTN-ex134-F

TCAGGCACTGTTATGGTTGTCT

TTN-ex134-R

TTGCGTTTGTTTCATGCACT

TTN-ex135-F

ATTGTTACTATCAGTCATCC

TTN-ex135-R

TGTTTTATCTTGTTTGAGTG

TTN-ex136-F

CTTTTAAAGAACCAGAGAAG

TTN-ex136-R

AGGACAGACATGGAGGAAAC

TTN-ex137-138-F

ATTGATATTTTGAAGTGCTG

TTN-ex137-138-R

CCAACATTCTGCTGACAACT

TTN-ex139-140-F

ATTTCACATTTGGGCTCTGC

TTN-ex139-140-R

TCATTGGTGCTGCCAATAAC

TTN-ex141-142-F

CTACAGGTGCTTTTCACATTTT

TTN-ex141-142-R

CTGCTATGGCTCACCAAGTTA

TTN-ex143-144-F

GGGTAAAAAATGTGACACTAA

TTN-ex143-144-R

GTAATGGGGAAATTTGTATG

TTN-ex145-F

TTCTTTTTCATATCTATTGCTC

TTN-ex145-R

CAAAGCATCAATTATTATCAAC

TTN-ex146-F

TTGTACCCTTGAATAAATATCT

TTN-ex146-R

GATGAACAAAAGGATGGGAAA

TTN-ex147-F

CCTTTACCCTCTGTGATTGACC

TTN-ex147-R

CAGTGTTCCTGAACATCGTTAGA

TTN-ex148-F

CTTGGTTTCTCTTTTTTTGTC

TTN-ex148-R

AAGGAAGGAAATGGCATAGT

TTN-ex149-F

GGTCATTCCCAAGAAAGAGGA

TTN-ex149-R

ACAGTTCAACATCGGTGCAG

TTN-ex150-F

AGAGGCTCCAGCAGTAAAAAG

TTN-ex150-R

TAATTTGCCTGCCCAATTTC

TTN-ex151-F

AGTGAGTTTACACAAGCGTTT

TTN-ex151-R

GCATACTGGTGAATCTTAGAT

TTN-ex152-F

TTGGTATATGGGTGGTGTTCT

TTN-ex152-R

GTCCTTAATCAGTTCACTATC

TTN-ex153-154-F

GTAATGTTGGCGTTGTCTCT

TTN-ex153-154-R

CAACAATACACGAAAATCCAG

TTN-ex155-F

CTGGATTTTCGTGTATTGTTG

TTN-ex155-R

CAAAGGAAAGTTAGAGCAAGA

TTN-ex156-F

GACAAGCCATAGCACAAATAA John Wiley & Sons

TTN-ex156-R

AGACATCAAGTGGAATGCAAA

Annals of Neurology

TTN-ex157-F

TTATATCTCTCATGCTCTGCT

TTN-ex157-R

ACTTTGGGCTTATGTCTTTAC

TTN-ex158-159-F

TCGACAAAGCAATTCAGAGAAA

TTN-ex158-159-R

CTTTTCTGAAATCATACACTCTGATAA

TTN-ex160-F

GCCAATGCATTCTAGCCACT

TTN-ex160-R

GGTGGCTCAGGCACTTAAAA

TTN-ex161-F

CAATGATCCACTCTGAAGAAA

TTN-ex161-R

AAAAATGCCTCTGGTTGTATC

TTN-ex162-163-F

GCCACCCAAGAAAGTTGTTC

TTN-ex162-163-R

TGTAGCCACTTTGGCTTAAAAGA

TTN-ex164-F

TTCCCATCTGGAATGTTCTG

TTN-ex164-R

GGTTAAAAGGATCAGTGGAGACA

TTN-ex165-167-F

GCCATAAATATCACTTTGGTTCA

TTN-ex165-167-R

CAAGAATCAACACAATCAGGAAA

TTN-ex167-168-F

TCACACTAAACATAAAACTACT

TTN-ex167-168-R

ACCCACTATCCCACCATAAAA

TTN-ex169-F

AATCATGTCTGTGTTGACTCT

TTN-ex169-R

TGTTATGAAGACCGCTAGAAA

TTN-ex170-171-F

TTTCTAGCGGTCTTCATAACA

TTN-ex170-171-R

CACTTTCTTTTCAGGAACAAC

TTN-ex172-174-F

ATGTATCTCTTCTGCTCTGG

TTN-ex172-174-R

TAAAGGGGGAATATCGACTC

TTN-ex175-176-F

GGCCCTGGGGAAAGACTG

TTN-ex175-176-R

AGCCACGGGAATTTCTTTTT

TTN-ex176-178-F

TCGTCTGTACCCCTCACAATC

TTN-ex176-178-R

GCAGAAAAAGGACAGGGGTAA

TTN-ex178-180-F

ATCTCCTCTCCTACTGTAAA

TTN-ex178-180-R

TGTAAATGACGAGTTAGTGG

TTN-ex181-183-F

CTTTTTGATGTGCTGCTGGA

TTN-ex181-183-R

TACAACTTGTGAGATCGGCG

TTN-ex184-186-F

CAAAAAGCCAGAAGCTCCAC

TTN-ex184-186-R

GTGGGAGGAGCCTTAGGAAC

TTN-ex187-189-F

AAAACCAGAAGCTCCGATTG

TTN-ex187-189-R

TGACGAGTTAGTGGGTGCAG

TTN-ex190-191-F

TGGCTGTGGGTTTGTCATAA

TTN-ex190-191-R

GTGGAGCTTCTGGCTTTTTG

TTN-ex197-198-F

GTTCCTAAGGCTCCTCCCAC

TTN-ex197-198-R

CCAAGGGCATTTTCTTTTCA

TTN-ex199-201-F

GCAGTTGAAGACATTTTGTTCC

TTN-ex199-201-R

TGACGCTTAAACTCAATGACAA

TTN-ex200-202-F

AAAGTGCCAGTGACTCCTCC

TTN-ex200-202-R

AGCCTCAGGCACTTGAAAGA

TTN-ex203-205-F

CCTGAAAAGAAAGTGCCAGC

TTN-ex203-205-R

GACAAAAGCTCAGAGCACCC

TTN-ex206-208-F

GCTGAAGTTGTGGAAGAGCC

TTN-ex206-208-R

ATGTACCTTTTGCTGGTGGG

TTN-ex207-209-F

TTTTATAGTGCCCGAGGTGC

TTN-ex207-209-R

TAGAAATAGGCGCAAGTGGC

TTN-ex209-F

TGTCCCTGGGTTATTTGAGC

TTN-ex209-R

GCAACAGGAACTGGCTTTTC

TTN-ex210-211-F

TCGTGTTTAGTGTTTGTCCATCTT

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TTN-ex210-211-R

GTTGGGACCTTCTTCACTGG

Annals of Neurology

TTN-ex212-213-F

CCGCCTCCTAAAGGTACTCA

TTN-ex212-213-R

TGCAATATGGTTTTAACATAAATTCAC

TTN-ex214-215-F

GCCATGTCTCAGAAGCCATT

TTN-ex214-215-R

CAAAAATAAAGCAGTCAAACATGC

TTN-ex216-F

AATTGCCTTGGATCCTCAAA

TTN-ex216-R

CAGCAAAGGCACCAATAACA

TTN-ex217-218-F

GTGGCTTGTCTGGATGCTTT

TTN-ex217-218-R

ATGACAAGCAACCCAGGAAC

TTN-ex218-F

CAAACCACAGTCTTCCAGCA

TTN-ex218-R

ACAGAACATTCGATGGAGGC

TTN-ex219-F

CCCAGCATCAAAGTCACAAA

TTN-ex219-R

CGCTGACAGAATGGTTGAAA

TTN-ex220-F

TGATTTTTAGGCTACATTTCTTCA

TTN-ex220-R

TGTTTGCAAGATAAACCTTTTG

TTN-ex221-F

TCCATTCATGTATTTCCCTGC

TTN-ex221-R

GCCAATTGCATAGGAGAGTGA

TTN-ex222-223-F

TTCCAAGATACGTGGGCTTA

TTN-ex222-223-R

CAGCATAAACAGGGCTTGAA

TTN-ex224-F

GAGCATGACTCACCCTCCAT

TTN-ex224-R

TGAAATACCAGAAAACATGCTAGT

TTN-ex225-F

GCAGGCAGCCAAGTATGAAC

TTN-ex225-R

AAAACTTGGAAATAAGAGGTTTTG

TTN-ex226-F

AAACCTTAGTAGCAACTTGCATTG

TTN-ex226-R

AATACTGGGAAACGAGGTCC

TTN-ex227-228-F

TGTGAAATTTTTCCTATTCCTGAAA

TTN-ex227-228-R

AAAGCAACCCGAATCTAGGA

TTN-ex229-230-F

AGGGAATTCTCTGCTTACTTTTATT

TTN-ex229-230-R

GCTTTTAGAACTTGGCGTCCT

TTN-ex231-F

AAGTGCCATGTGAAATGGGT

TTN-ex231-R

TACTCCCCACTCCCATGATT

TTN-ex232-F

TCATGGGAGTGGGGAGTAAT

TTN-ex232-R

GAAGCTGTCAAAGGCTGTCC

TTN-ex232-233-F

TCATGGGAGTGGGGAGTAAT

TTN-ex232-233-R

TCCTGTTTGGAGTGAGGGTTA

TTN-ex234-F

TGCTTGGTTGTGTAGGGTGA

TTN-ex234-R

CAGTTTGCCACTTGTGTGCT

TTN-ex235-F

TCTGACTTCATCCACTGTCCA

TTN-ex235-R

CAGCACTTTCCTTCTCTTTGG

TTN-ex236-F

TCAAATAGTCCACCCCAAAATC

TTN-ex236-R

TAAGGAGTTGGGCTGCTTTC

TTN-ex237-238-F

TCCAAGAGATTGTCATTTCCC

TTN-ex237-238-R

TGTCCTGCATCCACTCTGAC

TTN-ex239-F

CACCATTTATTTGTTGCATTCC

TTN-ex239-R

TTTCACTCACATTCATTTTCTGA

TTN-ex240-F

GCTACCAGCTCTCCCCTTCT

TTN-ex240-R

ATCCTTCCTGCCTTCTGGTT

TTN-ex241-F

TCTAAATGAATTAAATGCACCCA

TTN-ex241-R

TCAGACTTTTGATTATTTGGTTGA

TTN-ex242-F

ACAGAACAAAGCCCTGGAGAJohn Wiley & Sons

TTN-ex242-R

AGTTAGGCCAAGAAGGGCAT

Annals of Neurology

TTN-ex243-F

TTTCCAGAAACCTCATTTTGAA

TTN-ex243-R

TGGTGACCAGAGAAGTTGTGA

TTN-ex244-245-F

CCTGTGTGAAGGAACAACAAGA

TTN-ex244-245-R

GCCAGTTCTGGGAAGAAAAA

TTN-ex246-247-F

TTTCTAACAATTCACACTTGCT

TTN-ex246-247-R

CTTGCACCAGAATGTGACAGA

TTN-ex248-249-F

TCAACATATTTTTCTCCTTTACAAC

TTN-ex248-249-R

GGAATTTCTGGAAAGAAAATGTG

TTN-ex250-F

TCGTGCTAGACTTTTTGTGGAA

TTN-ex250-R

AAATGAAAGACCCTCACAAGGA

TTN-ex251-F

TGATGTAGCTTGGCTACGTTT

TTN-ex251-R

GCTTTTAAATTGTGCTTTGCC

TTN-ex252-F

GGTAAGCCCTTGGGTCTCTC

TTN-ex252-R

TGGGGTAACGATGATGAAGG

TTN-ex253-F

TTGCAGTCTTTGCCTTCATC

TTN-ex253-R

TCATGCAGTAATTATTTCCCTTTTT

TTN-ex254-255-F

CGTAAATGGTATGCTCCAACTC

TTN-ex254-255-R

TGCACTTAAATCCATTGTTGG

TTN-ex255-256-F

TCAATGTGGATCATGGACATAAC

TTN-ex255-256-R

AATTCTTTGGGTGCACTTGG

TTN-ex257-F

GCCGAACTTGTCATTTCTCC

TTN-ex257-R

GAGGTAAAGGTAAGAAGTTGTAGCA

TTN-ex258-259-F

ATCTTTTGGGTTGGCGAAAG

TTN-ex258-259-R

AGATACACCGGCAGCATAGG

TTN-ex260-261-F

CCAGGGGTTTGAATTTTTGA

TTN-ex260-261-R

CCTTCCTTACCTAAGACGTTCA

TTN-ex262-263-F

AAAAGGGGATAAACTCTTTGCAG

TTN-ex262-263-R

TCATCTGTAATCCCAAGGAA

TTN-ex264-265-F

GGCTTCTTCTGCTTCTTACCA

TTN-ex264-265-R

TGAAAGTGCAGGCAGTCATT

TTN-ex266-267-F

AATGACTGCCTGCACTTTCA

TTN-ex266-267-R

TTCACCCAACGGGTACCTAA

TTN-ex268-269-F

TGGCTGCAAATTCTTAACTGTG

TTN-ex268-269-R

TGCAATGCATGAATAAAGGGTA

TTN-ex270-F

GCATTGCATTTTGACATACTCTG

TTN-ex270-R

GCCAGTTCATGAAGCCAATTA

TTN-ex271-F

TTTTTGCTTGGCACACAGAC

TTN-ex271-R

CCATAGTGCATCCATGTCCA

TTN-ex272-F

TGCTCAAGATCCAAAGCGTA

TTN-ex272-R

TGGCAACTCCATGAAACAAA

TTN-ex273-F

GTTGCCACTTTCCCATTTTG

TTN-ex273-R

AATGACTCACCTAACACACTGACA

TTN-ex274-F

ATAGCAACCCTTCCCACACA

TTN-ex274-R

CCCAGTATGACATAAAAATGCAA

TTN-ex275-F

GCATTTTTATGTCATACTGGGAAA

TTN-ex275-R

CAAATCAAACTCCCTGATGG

TTN-ex276-F

CCAAAACAGCACATGATCCA

TTN-ex276-R

AACTTCTACCCTGGCTGCTG

TTN-ex277-F

GCCTGGAGAAACACAACCTG

TTN-ex277-R

AACTGGACCAGGGACATCTG

TTN-ex278-F

TTCACAGGATTTTCCCAAGTG

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TTN-ex278-R

GGGAGTTTGAAGCCATAAAGC

Annals of Neurology

TTN-ex279-280-F

GGTTATGCCAAACAGAAGGC

TTN-ex279-280-R

CACTGGATGCCTTTTTGATGT

TTN-ex281-F

GGCTCAGTGTTCCGAAATGT

TTN-ex281-R

AGGAGCCTTATGTGTGTGGG

TTN-ex282-F

TCTTACAGTCATCCCTCCCAA

TTN-ex282-R

TTAGTCCCCAGAAACGGAAG

TTN-ex283-F

CCAGCACTTCATCAGCTTCA

TTN-ex283-R

AACAGGATTTAGCTCATATTTGTCA

TTN-ex283-285-F

CCAGCACTTCATCAGCTTCA

TTN-ex283-285-R

GAGTTTTCAGCAGTCTCCAGC

TTN-ex286-287-F

GACATACCCGAAGATGCACA

TTN-ex286-287-R

TGTGATGAGGCCAATCTTGA

TTN-ex287-288-F

GCAGGACAAAAGACTGCAAA

TTN-ex287-288-R

AGGCAGCTGTAAGGAGGACA

TTN-ex288-F

CACTGCCAGAGATCCGATATG

TTN-ex288-R

TCTGTGGAAATTGAAGGAATGA

TTN-ex289-290-F

TCCCTCAATGCTTTTTGCTC

TTN-ex289-290-R

TGGAGTTACGAGTCACGCTG

TTN-ex291-292-F

GGCATTGGAGAACCTCTTGA

TTN-ex291-292-R

GACAGAAGTTAATGGGATTGAGAA

TTN-ex293-294-F

ACCAGCAACAATTTTCCCAG

TTN-ex293-294-R

TGTTTGTTACACAGCATACAGCA

TTN-ex295-F

TTGAAGGCAAACAGCTTGAA

TTN-ex295-R

TCAAGTGAATGAAATGTACGGC

TTN-ex296-297-F

TTGCTATTTATGGGAGCAGAGA

TTN-ex296-297-R

GGCACAAAATGTTATTGCCA

TTN-ex298-299-F

GGCAATAACATTTTGTGCCA

TTN-ex298-299-R

GGCTGCCAAGTTAGATCGAC

TTN-ex300-301-F

TATTGGAGATCCAAGCCCAC

TTN-ex300-301-R

TCAAAATTTATTTTTAATGGCCTAA

TTN-ex302-303-F

GGTAAGAATTTTACTACTTAGGCCATT

TTN-ex302-303-R

ACATCTAGCTCCACGGATGG

TTN-ex304.1-F

GGTCTTGGTCTCCCTGACAC

TTN-ex304.1-R

TGGGGATGGTTTGCTTAGTC

TTN-ex304.2-F

AAATCTGATGGTGGCAGTCC

TTN-ex304.2-R

GCAACAATTGACCAGCCTTT

TTN-ex304.3-F

GACTAAGCAAACCATCCCCA

TTN-ex304.3-R

GGCTCACCTGGTCTGTCAAT

TTN-ex304.4-F

AACGTCCTTGACAGACCTGG

TTN-ex304.4-R

TCACAGCCACCATCATCTTC

TTN-ex304.5-F

CTGGCAGTTTTGTGGCCTAT

TTN-ex304.5-R

TCTGTGCTTGAGATTAAGAGTTGC

TTN-ex304.6-305-F

CCCTGTCAACAAGAGTGCAA

TTN-ex304.6-305-R

AGGGCATCCTGCTCTCACTA

TTN-ex306-F

CCTGCAGAGCTAAAGGAAGC

TTN-ex306-R

TTTTGAAGTGGGAGGGAGAA

TTN-ex307-F

AAATGCTTCTCCCTCCCACT

TTN-ex307-R

TCTTTCAGAAGTGGATTAAAATGG

TTN-ex308-F

TGTACATTGGAGCAAATCCA

TTN-ex308-R

GAGCTAGATTCCTCAGGGAGAA

John Wiley & Sons

TTN-ex309-310-F

Annals of Neurology

TTTGCACCAGTATAGCTCTCCA

TTN-ex309-310-R

TTGACTTTGGTGACTCGTGC

TTN-ex311-F

CTGAAAACCGATTTGGCATT

TTN-ex311-R

TTCTTAAATGGGAGTTGTAATCTTCA

TTN-ex312-F

GCAGCAGGGATGATAATGTG

TTN-ex312-R

GACCATCATGAATGCTGTTACAT

TTN-ex313-F

TCCATTCTTGAAAGAAGCAGC

TTN-ex313-R

TGAATGTCTTCTCCCACATTATTC

TTN-ex314-315-F

TAGAAAGACTGCAGCCCAGG

TTN-ex314-315-R

CCTGGGTGTTTAATGCTGCT

TTN-ex316-F

AGCAGCATTAAACACCCAGG

TTN-ex316-R

AGTACCCACCCAGCTCTCCT

TTN-ex317-318-F

AGGCCAGTCCCTGGAAATAC

TTN-ex317-318-R

CAGCCAATGCTACAGGATGA

TTN-ex319-F

TTCCCGTAAGCAAATGAACC

TTN-ex319-R

TGAGTGGGACACAAGAGTGC

TTN-ex320-321-F

AAAAGTTAATCGCCTGGCCT

TTN-ex320-321-R

CATGTTCAACTGTTCTCAGGGA

TTN-ex321-322-F

TGCGAGACATCCATTTGGTA

TTN-ex321-322-R

CATGCACCTGGGTTTTTCTT

TTN-ex322-323-F

AAAATGGCACTATGCTTGGG

TTN-ex322-323-R

GGAAGGAGGCTTAATTTGCTTT

TTN-ex324-F

CAGGTGCTATCAGTGCTCCA

TTN-ex324-R

GCTGACAGTGGCAGTGTTCT

TTN-ex325-326.1-F

TGTGAGGGAATTGACATGGA

TTN-ex325-326.1-R

CGAAGCTCGGCATCTAGTTC

TTN-ex326.2-F

TAGGAGACGAGGCCTGGATA

TTN-ex326.2-R

TTCACTGCCATCACTTGGAA

TTN-ex326.3-F

CAGTGGACCCACATCACAAC

TTN-ex326.3-R

ACTTTTGCCTGCCATGTTTT

TTN-ex326.4-F

GGCATGAGCCACTTTCTGAT

TTN-ex326.4-R

AGGCTACATTTGTCCATGCC

TTN-ex326.5-F

ACAGCGACTAATCCTGGTGG

TTN-ex326.5-R

CAGGTCAGCATCCAGTTCAA

TTN-ex326.6-F

GCAGGACTCAAGGCAACTTC

TTN-ex326.6-R

GCCCTTTTCATTCTGAGCTG

TTN-ex326.7-F

AAACCAGAGCATGATGGAGG

TTN-ex326.7-R

TCACTGATTGGCTCATTCCA

TTN-ex326.8-F

ACAACCACCTGGCAAATTGT

TTN-ex326.8-R

CCACTGTCGACACGTACTGC

TTN-ex326.9-F

GACACAATCGTGGTTCATGC

TTN-ex326.9-R

TTGGTTTGCTCCAAGAAAGG

TTN-ex326.10-F

GCGCCTGAGAGTAACTGGAC

TTN-ex326.10-R

AATTGGATCAGCAGTCTGGG

TTN-ex326.11-F

GGGAACCTCCTTTGTTGGAT

TTN-ex326.11-R

TCCCATACTGTGGTGGTTGT

TTN-ex326.12-F

TGATCCTCCAAAAGGACCTG

TTN-ex326.12-R

TCCTTGGCAGTTATTGGTCC

TTN-ex326.13-F

AACGTGATTTGCCTGATGGT

TTN-ex326.13-R

AAATCTGTAATGCGGCGTTT

TTN-ex326.14-F

AATCCATTTGTGCTTCCTGG

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TTN-ex326.14-R

ATTCTGTGGTGGTCCTGGAG

TTN-ex326.15-F

TTGGTCTCGAGAGGAAGGTG

TTN-ex326.15-R

TTTGTTGCCGTTACGGTGTA

TTN-ex326.16-F

CCAGTTATAGGCCGACCAAG

TTN-ex326.16-R

CGGCCATCAGGTAGATCTTT

TTN-ex326.17-F

GAATGCTTTGTTGCTCGTGA

TTN-ex326.17-R

TAACAGGCCCAGATTCCAAG

TTN-ex326.18-F

TTCAACTGAGGTACAGGCCC

Annals of Neurology

TTN-ex326.18-R

TGAGAATGCCTTCTGCCTTT

TTN-ex326.19-F

AGTGGTTGCTCAGGAGAGGA

TTN-ex326.19-R

GGTCTTCCTTTGAATGGCAC

TTN-ex326.20-F

GCTGCAGTTAACGAAAAGGG

TTN-ex326.20-R

TGCATTTACCAATTCCAGCA

TTN-ex326.21-F

TGCTTGTAACCTGGCAAGTG

TTN-ex326.21-R

GGTGGCTTGTTTCACGTTTT

TTN-ex326.22-F

GTGGCCGTTAATTGCAAAGT

TTN-ex326.22-R

GGTCAGAGCTATCACTGGGG

TTN-ex326.23-F

CAAAATTGTGGACTCAGGCA

TTN-ex326.23-R

GAAGGGTGTGAACCCAAAAG

TTN-ex327-F

AGCCTTGGTTGGTTCAGAAG

TTN-ex327-R

CAGGTGGTTCTGAAAAATGAG

TTN-ex328-F

TGAGTGACCCGAGAGAGCTT

TTN-ex328-R

TGGTAAAAGAAAACCTGGATCTT

TTN-ex329-330-F

CCAACTTTTCAAAGATCCAGG

TTN-ex329-330-R

CAGGAAAAGGTATGCGGAAA

TTN-ex331-332-F

AATGGCCAATGACCTTACTTT

TTN-ex331-332-R

AGCATATGCACAGGTTAGCG

TTN-ex333-F

TGGAAAGAATGCGAACATCA

TTN-ex333-R

CTTAGCAGGTGGACCTGGAG

TTN-ex333-334-F

TTGTACCTTGTTTTCCCTTCC

TTN-ex333-334-R

CAGGTCAATCTCTGGTGCCT

TTN-ex335.1-F

GTGCTGGACAAGGAGAACCT

TTN-ex335.1-R

ACTCCAGGACTGACTGCTCC

TTN-ex335.2-F

ATGGTGGTAGCGTCATCACA

TTN-ex335.2-R

AACTTTTTGCCAGAGGATGC

TTN-ex335.3-F

ATACGGAGTCAGCCAACCAC

TTN-ex335.3-R

TCTCCCAAACTCATATTGGTCA

TTN-ex336-F

CCATGGATGATTAAGACCTGG

TTN-ex336-R

TCCACTTAGCAACCTTGGGT

TTN-ex337-338-F

TTCCTGGCCTTGTGATATGG

TTN-ex337-338-R

GATGCATTTGCTTGGAAGGT

TTN-ex339.1-F

GCAGGCCACTTGATTCTGAT

TTN-ex339.1-R

TCCCTGCATCATTTCTGTTG

TTN-ex339.2-F

CCTACAGCTGTGTGGAGCAA

TTN-ex339.2-R

TTTCCATGTTACTTTGGGGG

TTN-ex339.3-F

TGTGAAGGCCAAGAATGATG

TTN-ex339.3-R

TAGACATGGCATTGGCAGAC

TTN-ex340-341-F

TTGCTGAACATCCATTTGGT

TTN-ex340-341-R

GTGTAATCGGGCATCCAGTT

TTN-ex342-343-F

CTGTCACTTGCCGAGATGAA

TTN-ex342-343-R

AAGACTGCCCTTCTCCCTTC

John Wiley & Sons

Annals of Neurology

TTN-ex344-F

GAAGGGAGAAGGGCAGTCTT

TTN-ex344-R

GCATGTGCTTTTCTAGACCATTC

TTN-ex345-346-F

GGTGAGTGTGGACAAGAGCA

TTN-ex345-346-R

GGTGATCAATCATAGGTCAAGC

TTN-ex347-F

AAAAGCTATCAGGCAAATAGGAAA

TTN-ex347-R

TGGGAAAGAAATCAGGCATT

TTN-ex348-349-F

TGAAAACATTCAGAATTCGTAACA

TTN-ex348-349-R

CCACAGGGTTAATTTTGTGACC

TTN-ex350-F

CCAACCAAAACCTAAGGAAAAC

TTN-ex350-R

TCAAAAAGGTGAATTTTCCCA

TTN-ex351-F

GGTGGCACAGCTAATTTTCAA

TTN-ex351-R

TTTGGGGTAGGGGGACTAAG

TTN-ex352-F

CTTAGTCCCCCTACCCCAAA

TTN-ex352-R

TGGAGGTTCTGGAGGATCTG

TTN-ex353-354-F

TGCCCTTTCAACCACAAAAT

TTN-ex353-354-R

GGGGGAATTAGCCTTAACTTG

TTN-ex355-F

CAAGGACAAAGGATTACAGATTGA

TTN-ex355-R

TGGTTTATCTGTTGGGGGAA

TTN-ex356-F

GGAACAGTTGATGCCATCCT

TTN-ex356-R

AAAGTGAAAGAAAAGGCACTTG

TTN-ex357-F

ATTTTGGACTCATATAACATTCTTTTT

TTN-ex357-R

CCTCCACCCCCAAGTTAATA

TTN-ex358.1-F

GGCTACCACCAGCTCATCAT

TTN-ex358.1-R

GGTTGGTTCTGAAGGCTCTG

TTN-ex358.2-F

TTATTAACTTGGGGGTGGAGG

TTN-ex358.2-R

TGCTTTCAAATGATTCATGGAG

TTN-ex358.3-F

TGGAATTGTCCATCGTTGTG

TTN-ex358.3-R

CCTCCTTCTTCACCAACTGC

TTN-ex358.4-F

GCAATTCGATCTCAGAAGGG

TTN-ex358.4-R

GGACAGTGGCTGACCATCTT

TTN-ex358.5-F

TACTGGCAAATGCAGAATGC

TTN-ex358.5-R

CTCTTCTTCAAGACGCAGCC

TTN-ex358.6-F

GAGATAGTGAGACCAGCCCG

TTN-ex358.6-R

TGAAAGGCTGCTGACTCAAA

TTN-ex358.7-F

ACTCCAGAGAGAACTCGCCC

TTN-ex358.7-R

ACGCTGTAATTGCCCTCATC

TTN-ex358.8-F

TAAGTACTTCTGCCCGCCAC

TTN-ex358.8-R

TGGCCTGTAGAATGCAAATG

TTN-ex359-F

TCTTCTAAATTCAGCTTCCCAAA

TTN-ex359-R

TGTGTGTTTCTGCTTTGGTGT

TTN-ex360-F

AAAAGGTGGGGGTCTCTTTC

TTN-ex360-R

TCTTCAGATGTGGAAGACATGG

TTN-ex361-F

ATCCCCTGAAATCGAATGGT

TTN-ex361-R

ACATCAGTTGGCTGTCCCTC

TTN-ex362-363-F

GGGTTATGCTGCTGTGTGTG

TTN-ex362-363-R

TCAGAAAGATTAGTCCGTGTGAAA

TTN-ex363.2-F

AGGGCCTGTGCCCTTATACT

TTN-ex363.2-R

CCAGGTTTTTCAGGTGCAAT

TTN-ex363.3-F

GGAGTGCCTGAATAGCTTGG

TTN-ex363.3-R

GCATGGGCTGTTTTGAACTT

John Wiley & Sons

Page 43 of 43

Similar phenotypes caused by mutations in OTOG and OTOGL.

Hereditary myopathy with early respiratory failure is caused by mutations in the titin FN3 119 domain.

Beneficial metabolic phenotypes caused by loss-of-function APOC3 mutations.

Pure and syndromic optic atrophy explained by deep intronic OPA1 mutations and an intralocus modifier.

Reply: Hereditary myopathy with early respiratory failure is caused by mutations in the titin FN3 119 domain.

Association of PRPS1 Mutations with Disease Phenotypes.

Clinical phenotypes of MAGEL2 mutations and deletions.

Reply: Hereditary myopathy with early respiratory failure is caused by mutations in the titin FN3 119 domain.

Reply: Hereditary myopathy with early respiratory failure is caused by mutations in the titin FN3 119 domain.

Variability in dentofacial phenotypes in four families with WNT10A mutations.

Hidden Genetic Variation in LCA9-Associated Congenital Blindness Explained by 5'UTR Mutations and Copy-Number Variations of NMNAT1.

Altered phenotypes associated with ampD mutations in Enterobacter cloacae.

C gene and associated phenotypes.

Human disease phenotypes associated with mutations in TREX1.

Suppression of defective-sporulation phenotypes by mutations in transcription factor genes of Bacillus subtilis.

Recurrent ETNK1 mutations in atypical chronic myeloid leukemia.

Atypical phenotype in two patients with LAMA2 mutations.

Visual consciousness explained by its impairments.

The pathological and molecular but not clinical phenotypes are maintained after second passage of experimental atypical bovine spongiform encephalopathy in cattle.

Expanding the clinical phenotypes of MT-ATP6 mutations.

Motility defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations.

[Paternal GNAS mutations: Which phenotypes? What genetic counseling?].

TOR1A mutations.

Cardiac sodium channel mutations: why so many phenotypes?