RESEARCH PAPER

Hereditary myopathy with early respiratory failure:occurrence in various populationsJohanna Palmio,1 Anni Evilä,2 Françoise Chapon,3 Giorgio Tasca,4 Fengqing Xiang,5

Björn Brådvik,6 Bruno Eymard,7 Andoni Echaniz-Laguna,8 Jocelyn Laporte,9

Mikko Kärppä,10 Ibrahim Mahjneh,11 Rosaline Quinlivan,12 Pascal Laforêt,7

Maxwell Damian,13 Andres Berardo,14 Ana Lia Taratuto,15 Jose Antonio Bueri,14

Johanna Tommiska,16 Taneli Raivio,16 Matthias Tuerk,17 Philipp Gölitz,18

Frederic Chevessier,19 Caroline Sewry,20 Fiona Norwood,21 Carola Hedberg,22

Rolf Schröder,19 Lars Edström,23 Anders Oldfors,22 Peter Hackman,2 Bjarne Udd1,2,24

▸ Additional material ispublished online only. To viewplease visit the journal online(http://dx.doi.org/10.1136/jnnp-2013-304965).

For numbered affiliations seeend of article.

Correspondence toDr Johanna Palmio,Department of Neurology,Neuromuscular Research Unit,Tampere University Hospitaland University of Tampere,Tampere FIN-33014, Finland;johanna.palmio@uta.fi

JP and AE contributed equally.

Received 14 January 2013Revised 24 March 2013Accepted 27 March 2013

To cite: Palmio J, Evilä A,Chapon F, et al. J NeurolNeurosurg PsychiatryPublished Online First:[please include Day MonthYear] doi:10.1136/jnnp-2013-304965

ABSTRACTObjective Several families with characteristic featuresof hereditary myopathy with early respiratory failure(HMERF) have remained without genetic cause. Thisinternational study was initiated to clarify epidemiologyand the genetic underlying cause in these families, andto characterise the phenotype in our large cohort.Methods DNA samples of all currently known familieswith HMERF without molecular genetic cause wereobtained from 12 families in seven different countries.Clinical, histopathological and muscle imaging data werecollected and five biopsy samples made available forfurther immunohistochemical studies. Genotyping, exomesequencing and Sanger sequencing were used to identifyand confirm sequence variations.Results All patients with clinical diagnosis of HMERFwere genetically solved by five different titin mutationsidentified. One mutation has been reported while fourare novel, all located exclusively in the FN3 119 domain(A150) of A-band titin. One of the new mutationsshowed semirecessive inheritance pattern with subclinicalmyopathy in the heterozygous parents. Typical clinicalfeatures were respiratory failure at mid-adulthood in anambulant patient with very variable degree of muscleweakness. Cytoplasmic bodies were retrospectivelyobserved in all muscle biopsy samples and these werereactive for myofibrillar proteins but not for titin.Conclusions We report an extensive collection offamilies with HMERF with five different mutations inexon 343 of TTN, which establishes this exon as theprimary target for molecular diagnosis of HMERF. Ourrelatively large number of new families and mutationsdirectly implies that HMERF is not extremely rare, notrestricted to Northern Europe and should be consideredin undetermined myogenic respiratory failure.

INTRODUCTIONHereditary myopathy with early respiratory failure(HMERF, OMIM #603689) was described as anautosomal dominant disease characterised by adultonset proximal and/or distal myopathy withrespiratory failure typically in ambulant patients.HMERF disease has been associated with two dif-ferent titin mutations.1–4 Muscle histopathologicalfeatures include a combination of cytoplasmic

bodies (CBs) and rimmed vacuoles.3 A diagnosticpattern of fatty degenerative changes in lower limbmuscles on MRI has been identified, showingmarked involvement of semitendinosus, obturator,sartorius, gracilis and iliopsoas muscles.3–6 Severalfamilies and patients with clinical, morphologicaland imaging features compatible with HMERFhave remained without molecular genetic cause,including two of the originally reported families1

and two previously described separate families.6 7

In the current large international study we identi-fied the recently reported titin A-band mutation3 4

in six families from various ethnic backgroundsand, moreover, four novel titin mutations in thesame A-band domain in six other families indicat-ing that the disease is not extremely rare andmaybe underdiagnosed.Mutations in TTN gene, encoding the giant

muscle protein titin, are known to cause several dif-ferent skeletal and/or cardiac myopathies.8–11 Adominant mutation in the kinase domain of M-linetitin leading to HMERF was first described in threefamilies from Sweden.2 Two recent reports revealeda novel titin A-band mutation in three Swedish andthree British families with HMERF.3 4 Based onour large international collection of families andnew mutations, the range of epidemiological, clin-ical and mutational spectrum of HMERF diseaseexpands considerably and includes unexpectedsemirecessive inheritance with one of themutations.

METHODSStudy protocol and patientsPatients belonged to 12 unrelated families: oneFrench (A), one Finnish (B), two Swedish (C, D),two British (E, I), two Italian (F, J), oneArgentinian (G), one German (H) and two Frenchwith Portuguese ancestry (K, L) (figure 1). FamiliesA–F and H–J correspond to the original Caucasianpopulations in these countries, whereas theArgentinian family has a Caucasian, probablyItalian background. The collection contains all fam-ilies with HMERF known to the authors withoutestablished molecular genetic cause. Among the 31affected patients, 16 were male and 15 female with

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a mean age at examination of 49 years (range 29–72 years). Allpatients had been clinically examined by neurologists includingmuscle strength evaluation (the Medical Research Council Scale,MRC) and medical and family histories. Pulmonary function

tests and echocardiography were performed, as well as electro-physiological examinations consisting of nerve conductionstudies and needle electromyogram (EMG), creatine kinasemeasurement and muscle imaging by CT or MRI. The diagnosis

Figure 1 Pedigrees of the families. DNA was collected from individuals marked with an asterisk *. Filled symbols are affected and open symbolsunaffected family members. Grey symbols represent members with subclinical manifestations.

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of HMERF was based on clinical symptoms of respiratory insuf-ficiency with muscle weakness and/or presence of CBs in musclebiopsy and a typical pattern of muscle involvement on MRI asdescribed previously.3 4 6 7 DNA samples of 31 affectedpatients, as well as 26 healthy family members were provided bythe clinicians in the different countries.

Muscle biopsies were obtained from proximal muscle (deltoidor thigh muscle, 20 samples). They were snap frozen and 8–10 mm sections were cut and examined using standard histo-chemical stainings including H&E, Gomori trichrome, reducednicotinamide adenine dinucleotide-tetrazolium reductase andATPase at pH 10.4, pH 4.6, and combined succinate dehydro-genase and cytochrome oxidase. Four samples were stained foractin with rhodamine conjugated phalloidin. Sections were alsoimmunostained for different myogenic antigens includingmyosin heavy chain (MyHC) isoforms (fetal, neonatal, slowand fast MyHC, MHC (major histocompatibility complex)class I). Five samples were available for additionalimmunohistochemistry.

Linkage studiesFluorescently labelled polymorphic microsatellite markers span-ning a region of 7 Mb in the TTN locus were used for genotyp-ing the families. Markers used were D2S2314, D2S1244,D2S138, D2S148, D2S2173, D2S300, D2S385, D2S324,D2S2978, D2S2261, D2S384, D2S364, and D2S350.Genotyping was performed using ABI3730xl DNA Analyzerand GeneMapper V.4.0 software (Applied Biosystems).

Exome sequencingTwo affected (II-3 and III-1) members and one healthy (II-1)member of French family A (figure 1) were exome sequencedat Axeq/Macrogen laboratory in South Korea. The capturemethod used was Illumina TruSeq Exome Enrichment.Captured DNA fragments were sequenced on an IlluminaHiSeq2000 platform using 100 bp paired-end reads. Sequencereads were aligned to the human reference genome (UCSChg19) using the Burrows-Wheeler Aligner.12 Variant calling wasmade with GATK.13 Variant quality/control data filtering wasperformed using the analysis and visualisation programmeRikuRator (unpublished), created by Riku Katainen from LauriAaltonen’s group at the University of Helsinki. To call avariant, the coverage was required to be at least two reads andthe mutated allele to be present in at least 20% of the reads.Only those variants that were shared by both the affected wereincluded and filtered against dbSNP132 and one healthymember of the family.

Sanger sequencingMutations were confirmed by Sanger sequencing. Sequencingprimers were obtained from Genethon and are available onrequest. PCR was performed with DreamTaq DNA Polymeraseaccording to standard protocol (Fermentas). PCR products weresequenced on an ABI3730xl DNA Analyzer (AppliedBiosystems), using the Big-Dye Terminator V.3.1 kit and analysedwith Sequencher V.5.0 software (Gene Codes Corporation).

RESULTSClinical characteristics of the patientsClinical data of 31 patients with HMERF are presented in theonline supplementary table and pedigrees of the 12 families infigure 1. The presenting symptom was either lower limb weak-ness (14/22) or respiratory failure (8/22) with a mean age ofonset of 36.6 years (range 16–53 years). In family D four

patients reportedly already had muscle weakness in childhoodbut no results of examinations performed in childhood wereavailable for confirmation. Muscle weakness was progressiveand usually symmetrical despite occasional asymmetry onimaging (table 1). Disease duration at the time of the latestexamination was on average 13 years (range 1–32 years) and themost typical symptoms consisted of distal and proximal lowerlimb weakness and respiratory insufficiency that needed invasiveor non-invasive ventilation. Neck flexor, abdominal and ankledorsiflexion weakness was marked. There was no upper limbweakness at onset. In the later course of the disease proximaland distal weakness in the upper extremities was observed in 19patients. The severity of muscle weakness and its rate of pro-gression varied from mild (no limb weakness in four patients) toloss of ambulation at age 36 years. One member of family A didnot have any weakness in limbs or respiratory muscles at age 38but did show mild pathognomonic findings in muscle biopsyand definite findings on muscle MRI. Patients in the youngergeneration of family B and two members of families C (C:IV-4)and D (D:IV-8) did not have respiratory symptoms at ages32–67 years but had evident findings on muscle biopsy andMRI. Cardiomyopathy was not manifest in any of the patients,based on clinical, electrocardiography, chest x-ray examinationsor echocardiography (nine patients). Creatine kinase activity wasnormal or slightly elevated. EMG was myopathic with normalnerve conduction studies in 17 patients. EMG was reported asneurogenic in one patient (B:III-3) and with mixed findings intwo (B:III-10, G:II-2).

There were 10 siblings in the second generation (figure 1, B:II) of family B, of whom six deceased patients had beenaffected. All four neurologically examined patients (B:II-3, -7,-9, -12) had documented muscle weakness and muscle biopsyobtained in two of them showed rimmed vacuolar myopathyand dystrophic findings. Three of the siblings (B:II-2, -4, -7) hadrespiratory failure, needed mechanical ventilation and died onaverage aged 65 years.

Clinical geneticsIn Italian family J the proband with homozygous mutation hadrelatively early adult onset of respiratory failure and the hetero-zygous parents were reported healthy at ages 52 years and55 years, which is compatible with a recessive mode of inherit-ance.7 After the same mutation in heterozygous state was identi-fied in two siblings in family L of Portuguese origin causing thesame disease but with considerably later onset, additionalstudies were performed by MRI in the parents of Italian family J(figure 2). Although subjectively healthy and with no muscleweakness on clinical testing, muscle MRI revealed clear path-ology compatible with the known pattern of muscle involve-ment in HMERF. In French-Portuguese family K, the parents ofthe homozygous proband were first cousins with no signs ofdisease on clinical examination at the age of 57 years and 61years, respectively. Muscle MRI was not performed. The parentsof two siblings in family L were reported to be healthy butmuscle MRI was not available.

Muscle imagingThe distribution and degree of fatty degenerative changes inmuscles of 18 patients were evaluated (table 1). Semitendinosusmuscle was moderately to severely affected in all patients andobturator, sartorius and gracilis muscles were similarly involvedin most. Other frequently affected muscles were gluteusminimus and iliopsoas. Changes in other pelvic and thighmuscles were more variable and quadriceps and biceps femoris

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Table 1 Muscle imaging findings

Patient Glma Glme Glmi Ilps Obt RF VL VI VM Sa Gr SM ST AM AL BF TA EH ED Pr Gm Gl S TP FP

A:II-3 3 3 3 3 3 3A:III-1 0 0 1 1 2 1B:III-3 0 0/2* 3 1 3 1 0 0 0 1 1/2 1 3 1 0 1 3 3 3 2/3 0 0/1 0/1 3 3B:III-5 1 3 3 2 3 1 0 1 0 3 3 2 3 1 1 1 3 3 3 3 1 2 1 2/3 1/2B:III-10 NA NA NA 2 3 0/2 0 0 0/1 3 3 1 3 1/2 2/3 1 1/3 3 0/1 2/3 0 1 1/2 2/3 2/3B:III-11 2 2 3 3 3 0 0 1 1 3 3 1 3 1/2 0/2 1 BFs 3 1/3 2/3 1 3 0 1 2 3 3C:IV-1 0 0 0 0 3 0 0 0 0 0 0 1 3 0 0 BFs 1 0 0 0 1 0 0 0 1/2 1C:IV-2 0 0 0 0 3 0 0 0 0 1 1 0 3 0 0 0 NA NA NA NA NA NA NA NA NAE:II-1 1 2 2 3 3 3 2 3 3 3 3 1 3 3 1 0 2 2 3 1 0 0 0 1 2F:II-1 2 2 3 3 3 2 1 1 1 3 3 0 3 0 0 0 2 2 2 3 0 0 0 3 †

H:III-2 1/2 1 2/3 1 2 2 1 3 2 1 3 1 3 0 0 BFs 3 3 3 3 3 2/3 2/3 1 2/3 2I:II-1 NA NA NA NA NA 0 0 0 0 1 3 1 3 0 0 0 1/0 1/0 1/0 1 0 1/0 1/0 1/2 0J:II-1 2 2 2 3 3 1 1 1 1 1 1 1 2 2 1 1 1 2 2 2 0 0 1 2 ‡

J:I-1 1 0 1 0 2 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0J:I-2 0 0 1 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 1 0 0 0 0 0K:II-1 3 3 3 3 3 1 1 1 1 3 3 2 3 3 3 3 3 3 3 3 1 1 3 3 3L:II-2 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0

*Right/left when there are asymmetrical findings.†FP: flexor hallucis longus=2; flexor digitorum longus=3.‡FP flexor hallucis longus=2; flexor digitorum longus=1.0, normal; 1, mild; 2, moderate; 3, severe; AL, adductor longus; AM, adductor magnus; BF, biceps femoris; BFs, biceps femoris short head; ED, extensor digitorum longus; EH, extensorhallucis longus; FP, flexor hallucis and digitorum longus; Gl, gastrocnemius lateralis; Glma, gluteus maximus; Glme, gluteus medius; Glmi, gluteus minimus; Gm, gastrocnemius medialis;Gr, gracilis; Ilps, iliopsoas; NA, not assessed; Obt, obturator; Pr, peroneus longus; RF, rectus femoris; S, soleus; Sa, sartorius; SM, semimembranosus; ST, semitendinosus; TA, tibialisanterior; TP, tibialis posterior; VI, vastus intermedius; VL, vastus lateralis; VM, vastus medialis.

Figure 2 Muscle imaging findings of proband and his parents in family J. Muscle imaging of the homozygous proband ( J:II-1) shows fattyreplacement of iliopsoas, abdominal and obturator muscles and all gluteal muscles are moderately involved (A). At the thigh level semitendinosusand adductor magnus are the most involved as are extensor hallucis and digitorum longus, tibialis posterior and peroneus longus muscles on thelower legs (B). MRI in his heterozygous mother shows particular involvement of obturator muscle (C) and selective involvement of semitendinosusmuscle in his father (D).

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were relatively spared. In the lower legs gastrocnemius medialisand lateralis, as well as soleus muscles were relatively preserved,while in all other muscles the changes were moderate to severe.The homozygous proband of family J ( J:II-1) had the typicalphenotype with pathognomonic findings on imaging. His het-erozygous parents without clinical symptoms had mild to mod-erate fatty degenerative changes on MRI; the father particularlyin semitendinosus muscles and the mother in obturator muscles(figure 2).

Muscle histopathology and immunohistochemistryTypical pathological findings were fibre-size variation andincrease of internal nuclei (table 2). CBs were observed in allsamples, although in some samples only in a few fibres. Rimmedvacuolar pathology was another constant feature but rimmedvacuoles did not appear in the same fibres with CBs. CBs werepresent in the sections of four out of the five samples availablefor additional immunohistochemical evaluation, ranging fromone or a few fibres to 10–15% fibres harbouring CBs. Theywere generally found in subsarcolemmal position, often formingsubsarcolemmal rings, and were different in size, but all dis-played similar immunohistochemical features (figure 3). In par-ticular, CBs were positive with antimyotilin, anti-αB-crystallinantibodies, and also contained actin and dystrophin. Desminwas absent from the core of the bodies but sometimes positive

in a thin surrounding halo and detectable in areas of myofibril-lar disarray. These myofibrillar disruption areas were alsopresent in other fibres in the central position, and were positivewith antimyotilin and anti-αB-crystallin antibodies. However,CBs were not reactive with antititin antibodies, and TDP-43(TAR DNA binding protein-43) and p62/SQSTM1 present inrimmed vacuoles were absent from them as well. p62/SQSTM1also showed a dotted appearance in some hypotrophic fibres,and was positive in the areas of myofibrillar disarray betweenCBs. CBs did not display affinity for ubiquitin, whereas ubiqui-tin positivity was sometimes detected at the periphery of thebodies and in abnormal fibres as a diffuse cytoplasmic increase.The autophagosome marker LC3 labelled rimmed vacuoles butwas largely absent from CBs.

Linkage studiesHaplotype segregation using markers at the TTN locus 2q31was identified in all familial materials available. In the Frenchfamily (Family A) all the affected patients shared the samehaplotype that was not present in any of the healthy membersof the family. The Finnish family (B) showed segregation of adifferent haplotype in the patients, which was also partiallypresent in the Swedish (C and D), one British (E), one Italian(F) and one Argentinian (G) families. All patients in these fam-ilies with the C30071R mutation shared a haplotype including

Table 2 Muscle biopsy findings

Patient MuscleAgeat Bx

Fibre sizevariation

Incr. ofint.nuclei

Fattychange Fibrosis Necrosis

Rimmedvacuoles

Cytoplasmicbodies

Other proteinaggregation Other

A:I-I VL 72 y y n n n y y nA:II-3 VL 44 y y n n n y y n

A:III-1 VL 22 y n n n n n y nB:III-3 VL 55 y n n n n y y yB:III-5 VL 56 y y n n n n y nB:III-10 RF 53 y y y y n y y nB:III-11 VL 57 n n n n n n y (one fibre) y Irregular NADH stainingC:IV-1 VL 40 y y n n n y y nD:III-2 VL 50 y y y y n y y n Irregular NADH stainingD:III-10 VL 47 y y y n n y y Dystrophin Irregular NADH stainingE:II-1 NR 56 y y y y y y y (one fibre) y Congo red material,

grouped atrophy*F:II-1 Q 52 n n n n n n y (one fiber) nG:II-2 D 48 y n y y n y y n Autophagic vacuoles,

irregular NADH staininggrouped atrophy†

H:II-2 NR 24 y y NA NA NA y y n Ring fibres, target fibresH-III-2 D 20 y y n y y y y Desmin,

phalloidin, dysRing fibres, autophagicvacuoles, irregular NADHand COX staining

I:II-1 NR 57 y y n n y y y nJ:II-1 D 32 y y n n y y y n Lack of SDH and COX

stainingK:II-1 D 27 y y n n y y y n Sectorial distribution of

morphological lesionsL:II-1 D 52 y y n n n y y nL:II-2 D 56 y y n n n y y EM‡ Sectorial distribution of

morphological lesions

*In addition: COX-negative fibres, cores phalloidin +, ↑ MHC-1, ↑utrophin.†In addition: Z-line widening and streaming.‡EM findings: Several vacuoles that are empty or containing myeloid figures.D, deltoid; COX, cytochrome oxidase; EM, electron microscopy; n, no; NA, not available; NADH, nicotinamide adenine dinucleotide; NR, not retrieved; Q, quadriceps; RF, rectus femoris;SDH, succinate dehydrogenase; VL, vastus lateralis; y, yes.

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markers D2S300 and D2S385. The shared haplotype was lessthan 1.3 Mb in size (less than 1.1 cM). Patients in one Italianfamily ( J) and in two French families with Portuguese ancestry(K and L) showed segregation of yet another haplotype whichincluded markers D2S300 and D2S385 and was less than1.3 Mb in size (less than 1.1 cM). In Italian family J and French(Portuguese) family K both probands showed this identical shorthaplotype on both chromosomes with extended haplotypemarker allele sharing in family K consistent with the parentsbeing first cousins.

Exome sequencingExome sequencing was performed on two affected and onehealthy member of French family A. When variants shared byboth affected patients were filtered against dbSNP132 and thehealthy member of the family, only one variant was foundwithin the linked haplotype in the TTN gene. The variantg.274367C>G was located in TTN exon 343 and it caused oneamino acid change, p.P30068R.

Sanger sequencingSince the new mutation in the French family and the previouslyreported HMERF A-band mutation3 4 were both in TTN exon343, all other families were screened for exon 343 and fourmore mutations were identified (table 3). The causative muta-tion was identified in every patient with HMERF in our series.One of these mutations g.274375T>C (p.C30071R) previouslyreported in a few Swedish and UK families3 4 was now identi-fied in one Finnish, one UK, one Italian, one Argentinian and inthe two Swedish families. One of the new mutationsg.274436C>T (p.P30091L) has been observed in an exomesequencing project in one single patient, but without furtherconfirmation of its possible pathogenicity.14 This mutation wasnow identified in heterozygosity in two sibs in one Frenchfamily, and in homozygosity in the proband of the secondFrench family with Portuguese ancestry, as well as in homozy-gosity in the proband of Italian family J. The German mutation

g.274426T>C (p.W30088R) and the new British mutationg.274428G>C (p.W30088C) were identified by direct sequen-cing of the candidate region and could be directly associatedbecause of the identical phenotype and segregation in theaffected patients only. Mutations were not present indbSNP132, 1000 Genomes or NHLBI (the National Heart,Lung, and Blood Institute, Maryland, USA) Exome SequencingProject databases. TTN exon 343 was sequenced from 102Finnish and 96 Italian healthy controls and none of them hadany of these mutations.

All five mutations are missense mutations changing one aminoacid in the protein. The p.P30068R mutation changes a hydro-phobic amino acid to a positively charged amino acid, the p.C30071R mutation changes a small neutral amino acid to alarge positively charged amino acid, the p.W30088C mutationchanges a large amino acid to a small amino acid which mayaffect cysteine-cysteine bindings, the p.W30088R mutationchanges a hydrophobic amino acid to a positively chargedamino acid, and the p.P30091L mutation changes a rigid aminoacid to a flexible amino acid. All of the mutations are located inthe same FN3 119 domain (A150) in A-band titin (figure 4).

DISCUSSIONHMERF disease was first described in single families more than20 years ago.6 15 Two of these families showed linkage to thechromosome 2q31 locus16 and the first titin mutation associated

Table 3 Mutations identified in the families

Family gDNA Protein

A g.274367C>G p.P30068RB, C, D, E, F, G g.274375T>C p.C30071R

I g.274426T>C p.W30088RH g.274428G>C p.W30088CJ, K, L g.274436C>T p.P30091L

Figure 3 Immunohistochemical findings. Immunohistochemical evaluation shows cytoplasmic bodies (CBs) on Gomori trichrome (A) and H&E (B),frequently in subsarcolemmal position. CBs are reactive for αB-crystallin (C), myotilin (D) and actin (E) but not for titin (F). p62/SQSTM1 (G) anddesmin (H) are mostly absent from the core of the CBs but may show increased expression in the surrounding cytoplasm and in other areas ofmyofibrillar disarray. Antibodies against the following proteins were applied: ubiquitin (DakoCytomation), dystrophin (Novocastra NCL-DYS-2),desmin (Biogenex, USA), myotilin (Novocastra, UK), αB-crystallin (Novocastra, UK), actin (Invitrogen, California, USA), titin (Novocastra, UK), p62/SQSTM1 (Santa Cruz Biotechnology, Inc) and data in the text for TDP-43 (Proteintech), LC3 (Novus Biologicals). Immunohistochemical stainings wereperformed on the BenchMark (Roche Tissue Diagnostics/Ventana Medical Systems Inc) immunostainer using the official protocol of the BenchMarkimmunostainer, visualised with a peroxidase based detection kit. (B–H) are serial sections.

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with HMERF was identified in the M-band kinase domain.2 Thegenetic cause in the remaining families was elusive until newstudies using exome sequencing eventually identified a new dom-inant titin mutation in three Swedish families and in three UKfamilies in the distal part of A-band titin.3 4 Since both titin muta-tions showed an identical pattern of muscle involvement onMRI3 4 and histopathology, these parameters were used toreassess two previously reported families,6 7 two of the originalSwedish families without established genetic diagnosis, and otherunreported HMERF families regarding possible titin mutations.

In our larger collection of patients with HMERF in 12 unre-lated families from different Caucasian populations one previ-ously reported and four novel mutations were identified. Ourfindings emphasise the geographically wide occurrence, theimportance of titin as a causative gene of HMERF disease andparticularly the role of exon 343 as a mutational hotspotregion. Furthermore, this study considerably expands our under-standing of the clinical presentation and provides new insightinto molecular pathology.

Previous linkage studies in French family A suggested that thetitin gene locus was excluded.17 New muscle MRI studies leadto reclassification of some of the individuals in the family.Because of the identical pattern of muscle involvement with theother families with HMERF, A-band titin Sanger sequencingwas also performed, besides exome sequencing, resulting in thenovel p.P30068R mutation to be identified. As expected, afterreclassification, new linkage studies showed that the disease wasindeed linked to the titin locus.

The p.C30071R mutation, now identified in six families fromfive different populations, and previously reported in threeSwedish and three British families,3 4 is the most frequent muta-tion in HMERF so far. The mutation mediates a dominant effectwith full penetrance. The age of onset with this mutation variesfrom 16 years to 53 years being usually after age 30–35 years. Allthese families share a genomic region less than 1.3 Mb in size

suggesting an ancestral founder. The p.P30068R, p.W30088Cand p.W30088R mutations are novel and occur in single familieswith dominant inheritance and full penetrance. The age of onsetand disease severity in patients with these new mutations iswithin the range of the phenotypes with the p.C30071R muta-tion. However, the fifth mutation identified, p.P30091L, in fam-ilies J, K and L, shows a different penetrance and is neithercompletely dominant nor completely recessive. The parents ofthe probands in families J and K showed no signs of muscledisease and therefore the probands appeared to be sporadicpatients. However, because the same mutation occurred in het-erozygous state in family L, although with a milder phenotype,additional studies were needed. In families J and K the homozy-gous p.P30091L probands ( J:II-1 and K:II-1) have a more severedisease with earlier onset and more rapid progression than theheterozygous patients of family L. Furthermore, in family J theheterozygous parents had definite signs of subclinical muscledisease on MRI with fatty degenerative change in HMERFtypical muscles. In family L, the heterozygous patients hadrespiratory insufficiency although at much later age and withoutclear limb muscle weakness. Their muscle MRI findings were alsomilder. Since this novel p.P30091L mutation may or may notcause clinically manifest disease in heterozygous state, and itcauses a clearly more severe phenotype in homozygosity, weprefer to call this mutation semirecessive or semidominant. Thenew mutations reported are currently undergoing further func-tional studies, although, segregation with the phenotype inreported families and the fact that after the first submission threeother new mutations in the same exon were reported,18 19 makeall these mutations very likely pathogenic.

The most typical feature of HMERF is respiratory failure atmid-adulthood in an ambulant patient at the first visit. In thecase of absent or undiagnosed respiratory symptoms, more spe-cific clues to enable correct diagnosis can be obtained frommuscle imaging and histopathology. Our results on muscle

Figure 4 Titin mutations. Sanger sequencing first confirmed the new French mutation (A). Four other mutations (B–E) were found when allhereditary myopathy with early respiratory failure families were sequenced. All found mutations are located in the same FN3 119 domain of A-bandtitin (F and G). All substituted amino acids are conserved (H, UCSC genome browser). Titin references: GenBank: AJ277892, UniProt: Q8WZ42.

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imaging so far confirm the characteristics and pathognomonicpattern of muscle involvement in this disease.3–5 7

Muscle pathology is another key to diagnosis. CBs, a hallmarkof the disease, were observed in all samples. However, in somebiopsies they were present in one or a few fibres only and could beeasily overlooked in the first reading. Rimmed vacuolar pathologywas another consistent finding but these focal degenerativechanges did not occur in the same fibres that contained CBs.Myofibrillar changes, Z-disk alterations and CBs that bind phal-loidin (a marker for F-actin), and also contain desmin, myotilin,αB-crystallin, VCP (valocin-containing protein), and dystrophin,have been demonstrated in previous HMERF studies.1 3 4 7 17 20

Moreover, p62/SQSTM1 containing cytoplasmic inclusions hasbeen observed.2 In our new series of immunohistochemistrystudies including three different mutations (two dominant and thesemirecessive) we were able to detail the different types of accumu-lations and aggregations in three different cytoplasmic abnormal-ities: (1) CBs contained dystrophin, myotilin, actin andαB-crystallin; (2) rimmed vacuoles contained ubiquitin, TDP-43,p62/SQSTM1 and LC3 positive components; and (3) in regionswith myofibrillar disarray between CBs or centrally in some fibresthe expression of desmin, ubiquitin and p62/SQSTM1 wasincreased. Surprisingly, the most compelling finding was that CBswere not reactive for titin. Some features in patients with HMERFoverlap with myofibrillar myopathies (MFM). However, the darkand hyaline changes in the cytoplasm of affected fibres on tri-chrome staining characteristics for MFM are largely lacking inHMERF. Instead the routine stainings are showing CBs which arenot a hallmark of MFM.21

The spectrum of different human titinopathies is growing butnone of the other described forms8–10 22 show the unique path-ology of fibres with multiple CBs apart from rimmed vacuolatedfibres, which are both caused by the mutant titin protein.Immunohistochemistry findings indicate that the mutant proteinitself is not aggregating and not seeding the CBs but seems totrigger the aggregation of other sarcomeric proteins. The firsttitin mutation causing HMERF in the kinase domain of M-linetitin leads to disruption of the kinase associated proteincomplex with Nbr1, p62/SQSTM1 and MuRF2.2 This wasshown to lead to the mislocalisation of the multiproteincomplex, which is involved in ubiquitin-mediated regulation oftranscription, protein turnover via the ubiquitin proteasomesystem and autophagy-mediated protein turnover via the inter-action with ubiquitinated proteins and LC3 of p62/SQSTM1and nbr1.23 However, this kinase mutation has later been shownto occur as a variant (rs140319117) with a frequency of 0.0018among European Americans that warrants comprehensive newassessment of the mechanism. The A-band region of titin has acentral role in controlling myosin thick filament positioning andfunction,24 25 but the specific links of this particular FN3 119domain (A150) to dysregulated protein recycling and autophagyturnover remain to be clarified.

Five different mutations causing HMERF disease in variouspopulations identified in exon 343 of TTN makes this a first-step target for molecular diagnosis and the range of mutationsindicates that HMERF disease is not extremely rare and is notrestricted to the Scandinavian population.

Author affiliations1Department of Neurology, Neuromuscular Research Unit, Tampere University andUniversity Hospital, Tampere, Finland2Department of Medical Genetics, Folkhälsan Institute of Genetics and HaartmanInstitute, University of Helsinki, Helsinki, Finland3CHU de Caen, Neuromuscular Disorders Competence Centre, and UniversityHospital of Caen, Caen, France

4Don Carlo Gnocchi Onlus Foundation, Milan, Italy5Department of Womeńs and Childreńs Health, Center for Molecular Medicine,Karolinska Institute, Stockholm, Sweden6Department of Neurology, Institution of Clinical Sciences, Lund University, Lund,Sweden7Institute of Myology, National Reference Center for neuromuscular disorders,University Hospital of Salpêtrière, UPMC, Paris, France8Department of Neurology, Hôpitaux Universitaires, Strasbourg, France9Department of Translational Medecine and Neurogenetics, IGBMC, UniversityStrasbourg, Illkirch, France10Department of Clinical Medicine, Neurology, University of Oulu and ClinicalResearch Center, Oulu University Hospital, Oulu, Finland11Department of Neurology, University of Oulu and Pietarsaari Hospital, Oulu,Finland12MRC Centre for Neuromuscular Disease, Institute of Neurology, National Hospital,London, UK13Cambridge University Hospitals, Cambridge, UK14Neuromuscular Section, Hospital Universitario Austral (HUA), Buenos Aires,Argentina15Department of Neuropathology, Institute for Neurological Diseases, FLENI, BuenosAires, Argentina16Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland17Department of Neurology, University of Erlangen-Nuremberg, Erlangen, Germany18Department of Neuroradiology, University of Erlangen-Nuremberg, Erlangen,Germany19Departments of Neuropathology, University of Erlangen-Nuremberg, Erlangen,Germany20Wolfson Centre for Inherited Neuromuscular Diseases, RJAH Orthopaedic Hospital,Oswestry, UK21Department of Neurology, King’s College Hospital NHS Foundation Trust, London,UK22Department of Pathology, University of Gothenburg, Sahlgrenska UniversityHospital, Gothenburg, Sweden23Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden24Department of Neurology, Vaasa Central Hospital, Vaasa, Finland

Acknowledgements Dr R Carlier and Dr N Romero, Myology Institutecollected data and provided general advice. Pr D Hénin, Claude-Bernard BichatHospital, Department of Pathology, Genethon and AFM collected data and providedgeneral advice. Dr E Bertini, Department of Neurosciences, Unit of MolecularMedicine, Bambino Gesu Children’s Hospital collected data and provided generaladvice. Dr E Ricci Institute of Neurology, Catholic University School of Medicine,Rome, Italy collected data and provided general advice. Dr Maria Saccoliti MD,Department of Neuropathology, Institute for Neurological Diseases, FLENI, BuenosAires, Argentina performed electron microscopy. MSc Maritta Kariniemi,Neuromuscular Research Unit, Tampere Finland assisted with the preparation offigure 3.

Contributors Study conception and design: AO and BU. Acquisition of data: JP,AE, FC, GT, BB, BE, AE-L, JL, MK, IM, RQ, MD, AB, ALT, JAB, MT, PG, FC, FN, RS,LE, AO, PH and BU. Analysis and interpretation of data: JP, AE, FC, GT, FX, BE, PL,JT, TR, CS, CH, RS, LE, AO, PH and BU. Drafting of the manuscript: JP, AE, PH, BU.Critical revision of the manuscript for important intellectual content: FC, GT, FX, BB,BE, AE-L, JL, MK, IM, RQ, PL, MD, AB, ALT, JAB, MT, PG, FC, CS, FN, CH, RS, LE,AO, PH and BU. Obtained funding: BU. Administrative, technical and materialsupport: PH and BU. Study supervision: PH and BU.

Funding This study was supported by the Folkhälsan Research Foundation, theAcademy of Finland, the Sigrid Jusélius Foundation, the Liv o Hälsa Foundation andTampere University Hospital Research Funds (BU).

Competing interests RQ has received consultancy fee from Global, lecture feefrom Genzyme, and Muscular Dystrophy Campaign grant. PL has received grants andhonorarium from Genzyme Company. He is a member of the Pompe advisory Boardfor Genzyme. JT and TR have received funding from Academy of Finland, and FX hasreceived funding from ALF-grant (medical training and clinical research). JL’sinstitution has received grants from ANR and AFM. He is employed by INSERM. Allother authors report no conflicts of interest.

Patient consent Obtained.

Ethics approval Systemic collection of clinical data and all genetic studiesin Finland were approved by the Ethics committee of Tampere University Hospital,Finland.

Provenance and peer review Not commissioned; externally peer reviewed.

REFERENCES1 Edström L, Thornell LE, Albo J, et al. Myopathy with respiratory failure and typical

myofibrillar lesions. J Neurol Sci 1990;96:211–28.

8 Palmio J, et al. J Neurol Neurosurg Psychiatry 2013;0:1–9. doi:10.1136/jnnp-2013-304965

Neuromuscular

group.bmj.com on April 25, 2013 - Published by jnnp.bmj.comDownloaded from

2 Lange S, Xiang F, Yakovenko A, et al. The kinase domain of titin controls musclegene expression and protein turnover. Science 2005;308:1599–603.

3 Pfeffer G, Elliott HR, Griffin H, et al. Titin mutation segregates with hereditarymyopathy with early respiratory failure. Brain 2012;135:1695–713.

4 Ohlsson M, Hedberg C, Brådvik B, et al. Hereditary myopathy with earlyrespiratory failure associated with a mutation in A-band titin. Brain2012;135:1682–94.

5 Birchall D, von der Hagen M, Bates D, et al. Subclinical semitendinosus andobturator externus involvement defines an autosomal dominant myopathy with earlyrespiratory failure. Neuromuscul Disord 2005;15:595–600.

6 Tasca G, Mirabella M, Broccolini A, et al. An Italian case of hereditary myopathywith early respiratory failure (HMERF) not associated with the titin kinase domainR279W mutation. Neuromuscul Disord 2010;20:730–4.

7 Chapon F, Viader F, Fardeau M, et al. Familial myopathy with "cytoplasmic body"(or "spheroid") type inclusions, disclosed by respiratory insufficiency (in French). RevNeurol (Paris) 1989;145:460–5.

8 Gerull B, Gramlich M, Atherton J, et al. Mutations of TTN, encoding the giantmuscle filament titin, cause familial dilated cardiomyopathy. Nat Genet2002;30:201–4.

9 Hackman P, Vihola A, Haravuori H, et al. Tibial muscular dystrophy is a titinopathycaused by mutations in TTN, the gene encoding the giant skeletal-muscle proteintitin. Am J Hum Genet 2002;71:492–500.

10 Carmignac V, Salih MA, Quijano-Roy S, et al. C-terminal titin deletions cause anovel early-onset myopathy with fatal cardiomyopathy. Ann Neurol2007;61:340–51.

11 Herman DS, Lam L, Taylor MR, et al. Truncations of titin causing dilatedcardiomyopathy. N Engl J Med 2012;366:619–28.

12 Li H, Durbin R. Fast and accurate short read alignment with Burrows-WheelerTransform. Bioinformatics 2009;25:1754–60.

13 McKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: a MapReduceframework for analyzing next-generation DNA sequencing data. Genome Res2010;20:1297–303.

14 Vasli N, Bohm J, Le Gras S, et al. Next generation sequencing for moleculardiagnosis of neuromuscular diseases. Acta Neuropathol 2012;124:273–83.

15 Edström L, Thornell LE, Eriksson A. A new type of hereditary distal myopathy withcharacteristic sarcoplasmic bodies and intermediate (skeletin) filaments. J Neurol Sci1980;47:171–90.

16 Nicolao P, Xiang F, Gunnarsson LG, et al. Autosomal dominant myopathy withproximal weakness and early respiratory muscle involvement maps to chromosome2q. Am J Hum Genet 1999;64:788–92.

17 Xiang F, Nicolao P, Chapon F, et al. A second locus for autosomal dominantmyopathy with proximal muscle weakness and early respiratory muscle involvement:a likely chromosomal locus on 2q21. Neuromuscul Disord 1999;9:308–12.

18 Pfeffer G, Barresi R, Wilson IJ, et al. Titin founder mutation is a common cause ofmyofibrillar myopathy with early respiratory failure. J Neurol Neurosurg Psychiatry2013 Mar 13. [Epub ahead of print]

19 Izumi R, Niihori T, Aoki Y, et al. Exome sequencing identifies a novel TTN mutationin a family with hereditary myopathy with early respiratory failure. J Hum Genet2013 Feb 28. [Epub ahead of print]

20 Chinnery PF, Johnson MA, Walls TJ, et al. A novel autosomal dominant distalmyopathy with early respiratory failure: clinico-pathologic characteristics andexclusion of linkage to candidate genetic loci. Ann Neurol 2001;49:443–52.

21 Selcen D, Ohno K, Engel AG. Myofibrillar myopathy: clinical, morphological andgenetic studies in 63 patients. Brain 2004;127:439–51.

22 Udd B, Vihola A, Sarparanta J, et al. Titinopathies and extension of the M-linemutation phenotype beyond distal myopathy and LGMD2J. Neurology2005;64:636–42.

23 Waters S, Marchbank K, Solomon E, et al. Interactions with LC3 and polyubiquitinchains link nbr1 to autophagic protein turnover. FEBS Lett 2009;583:1846–52.

24 Bucher RM, Svergun DI, Muhle-Goll C, et al. The structure of the FnIII TandemA77-A78 points to a periodically conserved architecture in the myosin-bindingregion of titin. J Mol Biol 2010;401:843–53.

25 Labeit S, Gautel M, Lakey A, et al. Towards a molecular understanding of titin.EMBO J 1992;11:1711–16.

Palmio J, et al. J Neurol Neurosurg Psychiatry 2013;0:1–9. doi:10.1136/jnnp-2013-304965 9

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doi: 10.1136/jnnp-2013-304965 published online April 19, 2013J Neurol Neurosurg Psychiatry

 Johanna Palmio, Anni Evilä, Françoise Chapon, et al. failure: occurrence in various populationsHereditary myopathy with early respiratory

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