Clinical and genetic diversity of nemaline myopathy from a single neuromuscular center in Korea

Accepted Manuscript Clinical and genetic diversity of nemaline myopathy from a single neuromuscular center in Korea

Jong-Mok Lee, Jeong Geun Lim, Jin-Hong Shin, Young-Eun Park, Dae-Seong Kim PII: DOI: Reference:

S0022-510X(17)34385-X doi:10.1016/j.jns.2017.10.020 JNS 15615

To appear in:

Journal of the Neurological Sciences

Received date: Revised date: Accepted date:

5 July 2017 19 September 2017 9 October 2017

Please cite this article as: Jong-Mok Lee, Jeong Geun Lim, Jin-Hong Shin, Young-Eun Park, Dae-Seong Kim , Clinical and genetic diversity of nemaline myopathy from a single neuromuscular center in Korea. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jns(2017), doi:10.1016/ j.jns.2017.10.020

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Manuscript title Clinical and genetic diversity of nemaline myopathy from a single neuromuscular center in Korea

T

Authors

SC RI P

Jong-Mok Leea, Jeong Geun Limb, Jin-Hong Shina, Young-Eun Parkc, Dae-Seong Kima,c*

a

NU

Affiliations

Department of Neurology, Research Institute for Convergence of Biomedical Science

MA

and Technology, Pusan National University Yangsan Hospital Department of Neurology, Keimyung University School of Medicine

c

Department of Neurology, Pusan National University School of Medicine

ED

b

CE

PT

Running title; Clinical and genetic diversity of nemaline myopathy

Corresponding author

AC

Dae-Seong Kim, MD, PhD Department of Neurology, Pusan National University Yangsan Hospital 20 Geumo-ro, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 50612, Republic of Korea. Tel: +82-55-360-2450 Fax: +82-55-360-2521 E-mail: [email protected]

ACCEPTED MANUSCRIPT

Acknowledgements We deeply appreciate to Kirsi Kiiski, Vilma-Lotta Lehtokari and other staffs of the Folkhälsan Institute of Genetics and The Department of Medical and Clinical Genetics at University of Helsinki for their contribution of identifying copy number variations in

SC RI P

T

triplicate region of NEB using array CGH.

Funding

This research was supported by Basic Science Research Program through the National

MA

and Technology (2013R1A1A2005521)

NU

Research Foundation of Korea (NRF) funded by the Ministry of Education, Science,

Conflict of interest

AC

CE

PT

ED

The authors state that there is no conflict of interest.

ACCEPTED MANUSCRIPT

Abstract Nemaline myopathy (NM), the most common of the congenital myopathies, is caused by various genetic mutations. In this study, we attempted to identify the causative mutations of NM and to reveal any specific genotype–phenotype relationship in Korean

SC RI P

T

patients with this disease. We investigated the clinical features and genotypes in 15 pathologically diagnosed NM patients, using whole exome sequencing (WES) combined with targeted sequencing and array-based comparative genomic hybridization. This strategy revealed pathogenic causative mutations in seven patients (46.7%), among

NU

whom mutations in the nebulin gene (NEB) were the most frequent (5 patients, 33.3%). Copy number variation (CNV) abnormality in NEB was not observed in any of our

MA

patients. In those with NEB-associated NM, the clinical spectrum was highly variable regardless of the mutation type. However, the majority of patients showing anterior

ED

lower leg weakness were associated with mutations located between NEB exons 166

PT

and 177. We concluded that the combination of WES and targeted Sanger sequencing is an effective strategy for analyzing genotypes in patients with NM, and that CNV in

CE

NEB may not be a frequent cause of this disease among Koreans.

AC

Keywords; nemaline myopathy, NEB, whole exome sequencing, comparative genomic hybridization, copy number variation

ACCEPTED MANUSCRIPT

1. Introduction Nemaline myopathy (NM), the most common of the congenital myopathies, is pathologically characterized by the presence of nemaline rods in skeletal muscle fibers. The nemaline rods are accumulations of degraded Z-disc components as well as broken

SC RI P

T

thin filaments [1].

NM is a genetically heterogeneous disease that is caused by pathological variants in the genes encoding components of the thin filaments and associated proteins. The thin filament of skeletal muscle consists of nebulin (NEB), tropomyosin-β and -γ (TPM2 and

NU

TPM3), actin (ACTA1), and troponin T (TNNT1) [2-6]. The actin and tropomyosin proteins are double-helical structures encircling the nebulin protein, and are bound to

MA

the Z-disc at the C-terminal and to tropomodulin at the N-terminal. The troponin T adjacent to tropomyosin regulates contraction in accordance with calcium ions. The

ED

stabilization of nebulin and tropomodulin is controlled by a muscle-specific protein,

PT

Kelch-like family member 40 (KLHL40) (Fig. 1) [7]. The dysregulation of these genes breaks the thin filament components directly or indirectly [8].

CE

The clinical spectrum of NM is known to be diverse [9-11]. Generally, NM can be classified into six clinical forms on the basis of age of onset and severity of motor and

AC

respiratory involvement [12]. In the severe neonatal form of NM, patients typically present with severe muscular hypotonia and respiratory failure at birth. Dilated cardiomyopathy or multiple joint contractures can be complicated. Amish NM is a clinically distinct autosomal recessive disease with neonatal onset and early childhood lethality. It is associated with TNNT1 mutations and has been described only in a single Amish family [6] and a single Dutch case [13]. Patients with intermediate congenital NM fail to achieve motor developmental milestones, or they become wheelchair-

ACCEPTED MANUSCRIPT

dependent and/or develop respiratory failure by the age of 11 years, although they initially show anti-gravity movement or independent respiration at birth. In the typical congenital form of the disease, patients show spontaneous anti-gravity limb movement, and respiratory involvement is not obvious. Patients may show delayed gross motor

T

development with proximal limb and bulbar weakness, which is static or progresses

SC RI P

very slowly. However, most of the patients with typical congenital NM are able to lead independent lives [10]. Childhood-onset NM is frequently inherited as an autosomal dominant trait, and patients usually develop their first symptom in their late first or

NU

second decade. Although a minority of the patients may become wheelchair-bound at adult age, most of them can lead independent ambulation. In adult-onset NM, patients

MA

develop generalized muscle weakness between the ages of 20 and 50 years and most of them can lead independent lives. In childhood- and adult-onset NM, cardiac

ED

involvement or respiratory failure is rare [14, 15]. Although NM is associated with many genetic causes, the genotype–phenotype

PT

correlation is not so clear in many of the cases. Furthermore, the large size of the genes

CE

associated with NM makes the genetic diagnosis a challenging task. For example, NEB, which is being considered as the most common causative gene of NM, has an enormous

AC

size with more than 180 exons. In this regard, the recent introduction and development of next-generation sequencing technology could be useful for exploring the genetic etiologies of NM. This notion led us to investigate and identify NM-causative gene mutations in affected patients. First, we attempted to identify causative mutations in all NM-related genes, using currently available technologies; namely, targeted Sanger sequencing of small-sized NM-related genes, whole exome sequencing (WES), and array-based comparative

ACCEPTED MANUSCRIPT

genomic hybridization (array-CGH) for the analysis of copy number variation (CNV) of NEB. Second, once the genetic diagnosis was established, we attempted to reveal the specific relationship between genotypes and clinical phenotypes, such as clinical

AC

CE

PT

ED

MA

NU

SC RI P

T

features or muscle biopsy findings.

ACCEPTED MANUSCRIPT

2. Methods 2.1. Patients We retrospectively recruited 15 patients from 14 unrelated families, who were diagnosed with NM at the neuromuscular clinic of Pusan National University Hospital

SC RI P

T

and Pusan National University Yangsan Hospital since 1998. The diagnosis of NM in all patients was based on the presence of nemaline rods in their muscle biopsy. Clinical information was collected from the patient’s medical records; gender, presence of affected family members, age at onset, initial symptom, distribution of muscle weakness,

NU

respiratory muscle involvement, and associated dysmorphic features. Laboratory findings, including serum creatine kinase (CK) levels and electrocardiogram and

MA

electromyography readings, were also obtained and analyzed. Written informed consent was obtained from all the patients examined for genomic DNA sequencing, and this

ED

study was approved by the ethical review board of Pusan National University Yangsan

PT

Hospital (IRB No. 05-2014-063). 2.2. Pathological analysis

CE

Open muscle biopsy was performed under local or general anesthesia on the clinically affected limb muscles. The biopsied muscles were flash frozen and stored at –80 °C

AC

until used. The serial frozen sections were stained with the following histochemical stains; hematoxylin and eosin (H&E), modified Gomori trichrome, nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), and adenosine triphosphatase, at varying pH values. Muscle specimens were also prepared for electron microscopy observation. In brief, the specimens were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer. After shaking with a mixture of 4% osmium tetroxide, 1.5% lanthanium nitrate, and 0.2 M s-collidine

ACCEPTED MANUSCRIPT

for 2–3 hours, the samples were embedded in epoxy resin. Semi-thin sections of 1-μm thickness were stained with toluidine blue, whereas ultrathin sections of 50-nm thickness were stained with uranyl acetate and lead citrate. 2.3. DNA analysis

T

Genomic DNA extracted from each patient’s blood or skeletal muscles was used. First,

SC RI P

all coding exons and exon-intron boundaries of ACTA1 and TPM3 were amplified by PCR and analyzed by Sanger sequencing [5]. If no pathogenic mutation was identified in these two genes, other causative genes of congenital myopathies, including NM-

NU

related genes (NEB, CFL2, TPM2, TNNT1, KBTBD13, KLHL40, KLHL41, LMOD3, MYO18B, and MYPN), were evaluated by WES [2, 3, 6, 7, 15-20]. Exome capture was

MA

performed using the Illumina TruSeq Exome Enrichment Guide. Sequence reads were mapped to the human reference genome assembly (GRch37/hg19) using Integrative

ED

Genomics Viewer (IGV; Broad Institute, Cambridge, MA, USA). Variants potentially affecting protein structure or function, including nonsynonymous variants, frameshifts,

PT

or variants affecting splicing, were investigated. The variants showed a depth of more

CE

than 30 and a minor allele frequency of less than 0.01. We evaluated the impact of mutations using the SIFT/PROVEAN and Polyphen2 prediction system in the case of

AC

point mutations. The splice sites were evaluated with Human Splicing Finder [21]. Sanger sequencing was performed to confirm the mutations found by WES. Because WES has clear limitations in reading the triplicate region of NEB, Sanger fill- in was performed for patients in whom a single heterozygous variant or no variant was identified. In addition, all patient samples were sent to the Folkhälsan Institute of Genetics at the University of Helsinki for CNV analysis by targeted array-CGH, as

ACCEPTED MANUSCRIPT

previously described [22]. Information of the primer sequences used is available upon

AC

CE

PT

ED

MA

NU

SC RI P

T

request.

ACCEPTED MANUSCRIPT

3. Results Overviews of the clinical features and genetic analysis of the 15 patients with NM are summarized in Table 1 and Figure 2, respectively. In terms of age of onset and clinical features, we could classify our patients into three

SC RI P

T

groups: (1) the intermediate congenital form, (2) the typical congenital form, and (3) mild childhood-onset NM. None of our patients were classified as severe neonatal type, Amish NM, or mild adult-onset NM. Two patients (patients 1 and 2), who developed their first symptom at birth, were classified as intermediate congenital NM. Both of

NU

them showed generalized muscular hypotonia, and they developed respiratory failure requiring mechanical ventilation during the neonatal period. One patient showed

MA

marked limb muscle weakness with an inability to move the limbs against gravity. Five patients (patients 3, 4, 5, 6, and 7) had the typical congenital form of NM. In this group,

ED

two patients manifested subclinical weakness of the respiratory musculature (patients 4

PT

and 7), and one (patient 6) showed predominant calf weakness. Eight patients (patients 8, 9, 10, 11, 12, 13, 14, and 15) were classified into benign childhood-onset NM. Motor

CE

developmental delay with gait disturbance was the most common clinical manifestation, and facial paresis was evident in one patient (patient 10). Pes cavus or equinovarus

AC

deformity was observed in seven patients (patients 8, 9, 10, 11, 12, 13, and 14). This group could be further subdivided into six patients with predominant distal leg weakness, and one patient with evenly distributed weakness (patient 9). In one patient (patient 11), clinical information regarding muscle weakness distribution was not available. Among the six patients with predominant distal leg weakness, four (patients 8, 10, 13, and 14) showed predominant peroneal muscle weakness with foot drop, whereas

ACCEPTED MANUSCRIPT

the peroneal and calf muscles were equally affected in the other two individuals (patients 12 and 15). Six patients, all of whom were classified as having mild childhood-onset NM (patients 8, 9, 10, 13, 14, and 15), underwent muscle CT or MRI. Five of them manifested distal leg

T

dominant muscle weakness, whereas the sixth patient (patient 9) showed mild

SC RI P

generalized muscle weakness that was not confined to a specific muscle group. In patients 13 and 14, muscle CT showed preferential involvement of the bilateral tibialis anterior muscle. Patient 15 revealed a high signal change in the tibialis anterior, soleus,

NU

and gastrocnemius muscles in T2-weighted images of muscle MRI (Fig. 3A). Although patient 8 presented with predominant bilateral foot drop, his muscle CT showed a

MA

predominant affection of the right gastrocnemius muscle (Fig. 3B). Slight involvement of the gastrocnemius muscle with sparing of the anterior compartments of the lower legs

ED

and thigh muscles was observed in patient 10 (Fig. 3C). Patient 9 showed a patch-like distribution of abnormal intensities in the muscle CT (Fig. 3D). Serum CK levels were

PT

generally normal, except for one patient with an ACTA1 mutation (patient 7) who

CE

showed a markedly elevated serum CK level (5,370 IU/L; normal 7–151 IU/L) and associated hypertrophic cardiomyopathy.

AC

Modified Gomori trichrome staining of the biopsied muscle tissues showed characteristic nemaline bodies in all patients, except for one individual who was classified with an intermediate congenital form associated with TPM3 mutation (patient 2). In this patient, all muscle fibers were extremely atrophic, and the nemaline body was not readily identifiable under the light microscope, but was evident under the electron microscope. The locations of nemaline bodies in the muscle fibers were variable, being either in the cytoplasm or the subsarcolemmal area, or both, and did not show any

ACCEPTED MANUSCRIPT

significant correlation with the clinical phenotype or genes affected (Fig. 4). In most of the patients, the nemaline bodies were present in type 1 fibers, except for patient 5, in whom nemaline bodies were exclusively observed in atrophic type 2 fibers. Interestingly, two patients also showed core lesions in the NADH-TR stain at the same

T

time (central cores in patient 10 and minicores in patient 15). The clinical, pathological,

SC RI P

and genetic features of both patients had been previously reported elsewhere, and both had predominant distal leg weakness and mutations in NEB [23]. Other frequently observed pathological abnormalities included increased fiber size variability (13/15),

NU

type 1 fiber predominance (12/15), endomysial fibrosis (8/15), type 1 fiber atrophy (2/15), and increased number of muscle fibers with internal nuclei (2/15). However,

MA

none of these findings showed clear correlation with the patient’s clinical phenotype or genotype. Under electron microscopy, all showed the presence of characteristic

ED

filamentous aggregations, which were caused from disruption of the Z-band (Fig. 5). Dominant pathogenic mutations affecting TPM3 (patient 2) and ACTA1 (patient 7) were

PT

identified by Sanger sequencing [24]. Subsequent investigation revealed 16 additional

CE

mutations affecting NEB in 12 patients (Figure 2); namely, four nonsense mutations, three missense mutations, six small del/ins, and three splice site mutations. Eight

AC

mutations were novel (i.e., not reported elsewhere; bolded in Table 1), and their effects, evaluated using the prediction system SIFT/PROVEAN, Polyphen2, or Human Splicing Finder, were all found to be deleterious. Two known mutations (Ser8228Ser and Arg7970Serfs*48) were shared by two patients, respectively [23, 25]. In all patients with NEB-associated NM, at least one nonsense or frameshift mutation was identified, except in one patient in whom only a single heterozygous deletion of a single amino acid was identified (patient 5).

ACCEPTED MANUSCRIPT

None of our patients with NM possessed abnormal CNV of the triplicate region of NEB. Finally, we were able to confirm a genetic diagnosis in seven of the 15 patients with NM. Of the other eight patients, only a single heterozygous mutation in NEB was identified in seven individuals, and no mutation was identified in any NM-related gene

AC

CE

PT

ED

MA

NU

SC RI P

T

in the eighth individual (patient 4).

ACCEPTED MANUSCRIPT

4. Discussion In this study, we aimed to identify genetic defects in 15 patients with pathologically confirmed NM using WES combined with targeted sequencing, and were able to identify causative mutations in seven patients. Among the genes investigated, NEB was

SC RI P

T

the most common causative gene of NM in our series. Two other pathogenic variants detected by targeted Sanger sequencing consisted of a mutation affecting TPM3 and ACTA1 individually. Although the exact proportion is uncertain, mutations in NEB have been attributed to 50% of NM cases [26]. Our study showed that autosomal recessive

NU

pathogenic variants in NEB accounted for 38% of our pool, which is compatible with previous reports and shows that NEB is also the major causative gene of NM in Korea

MA

[27, 28].

WES has already shown a strong impact on the investigation of pathogenic variants in

ED

congenital myopathy, which has many candidate genes [29]. In our study, we identified

PT

pathogenic compound heterozygous variants of NEB in two patients using WES only. Among 11 patients in whom WES had identified either a single heterozygous mutation

CE

only or no significant mutation, three patients were revealed by Sanger sequencing to have additional pathogenic heterozygous variants at repetitive regions of NEB, and the

AC

genetic diagnosis was confirmed. The reason why WES had shown a relatively low detection rate in NEB is most likely due to the short read length of this technique [25, 30]. Because NEB has a highly repetitive region between exons 82 and 105, it is most likely that mapping errors cannot be avoided using current WES technology with its limited read length. Given that we had seven patients harboring only a single causative heterozygous mutation in NEB and one genetically undiagnosed patient, it is strongly suggested that cryptic pathogenic variants or large deletions in the highly repetitive

ACCEPTED MANUSCRIPT

region of NEB may not be detectable using the current standard technology of WES or Sanger sequencing [22]. In this regard, a recently developed single- molecule, real-time, new-generation sequencing technology that offers longer read lengths may be useful for overcoming this problem [31].

T

Recently, abnormal CNV in the triplicate region of NEB was implicated as a frequent

SC RI P

cause of NM [32]. However, no CNV abnormality was identified in our patients. Although the number of samples studied was small, this finding suggesting that CNV abnormality is not a common cause of NEB-related NM among Koreans may reflect an

NU

ethnic difference in the genetic background of the disease [32].

The clinical spectrum of NM was wider than previously recognized [25, 33]. Patients

MA

with TPM3- or ACTA1-associated NM typically showed onset at birth or in infancy. In ACTA1-associated NM, intrafamilial phenotypic variability has also been reported [27].

ED

The results of our study are compatible with the previous study, because our patients with TPM3 (patient 2) or ACTA1 (patient 7) mutations presented their symptoms at

PT

birth or in infancy [34]. In patients with NEB-associated NM, the age of onset and

CE

clinical severity were highly variable. Whereas both patients with the intermediate congenital form showed profound limb weakness associated with respiratory or

AC

swallowing difficulties, those with mild childhood-onset NM manifested mild limb weakness and many of them were still ambulatory. One of the most remarkable findings in our series is that only two cases were associated with respiratory failure, which is much fewer than that seen in previous studies [33, 35]. This is most likely because we definitely had a smaller number of patients with severe phenotype than the other study series (none with severe neonatal form and only two with intermediate congenital form). Another point is that distal leg weakness was frequent in NEB-associated NM, occurring

ACCEPTED MANUSCRIPT

in one patient with typical congenital NM (patient 6) and in six with mild childhoodonset NM (patients 8, 10, 12, 13, 14, and 15). Among them, four developed predominant weakness in the ankle extensors (patients 8, 10, 13, and 14) and one manifested predominant ankle flexor weakness (patient 6). In the other two patients,

T

ankle extensors and flexors were equally affected (patients 12 and 15).

SC RI P

In the previous studies using muscle MRI, the rectus femoris, vastus intermedius, and tibialis anterior muscles had been considered as the most frequently affected leg muscles in patients with NEB-related NM, and it had been suggested that predominant

NU

gastrocnemius muscle involvement may exclude the diagnosis of NEB-related NM [36]. However, another study showed definite involvement of the hamstring muscles in

MA

addition to the rectus femoris muscle and pronounced involvement of the gastrocnemius muscle in a patient with NEB mutations [37]. In our study, patient 14 with mild

ED

childhood-onset NM and distal dominant phenotype exhibited similar findings. His muscle MRI showed a selective involvement of muscles of the posterior group at the

PT

thigh level. Moreover, among his lower leg muscles, the tibialis anterior, gastrocnemius

CE

medialis, and soleus muscles were selectively affected (Fig. 3A). Thus, our study clearly showed that affection of the gastrocnemius muscle is not unusual in patients

AC

with NEB-associated NM, which had been reported to be rare in the literature [38]. The correlation between muscle pathology and clinical features in NM is still controversial. In a previous study, it was suggested that the number of rods observed in muscle pathology and the residual amount of nebulin protein in western blot analysis are correlated with clinical severity [39]. Another study suggested that the features of sarcomeric disturbance in electron microscopy findings are also correlated with muscle weakness in NM [40]. However, another study using a large number of pathology

ACCEPTED MANUSCRIPT

specimens showed that the numbers and localization of nemaline rods correlated poorly with clinical severity in NM [41]. We were not able to identify any significant correlations between the numbers or localization of rods with the clinical findings, such as age of onset and clinical severity of muscle weakness. Given the relatively small

T

patient number in our study, further study using a larger sample size will be needed to

SC RI P

elucidate this issue.

In terms of the genotype–phenotype correlation with regard to reported mutations in NEB, the clinical severity of our cohort differed from that of reported cases. Although

NU

the splice site mutation c.24579G>A (p.Ser8228Ser) was identified in two patients with mild childhood-onset NM in our series, the same mutation has been reported in the

MA

typical congenital form of the disease [25]. A small deletion leading to frame shifting (c.23908_23911delAGAG [p.Arg7970Serfs*48]), identified in two of our patients with

ED

mild childhood-onset NM manifesting as predominant distal leg weakness (patients 13 and 14), was associated with typical congenital NM in a previous report [33]. The

PT

clinical phenotype of two nonsense mutations identified from patients with relatively

CE

severe phenotypes in our cohort (c.8425C>T [p.Arg2809X] and c.23245C>T [p.Arg7749X]) was not available in the previous reports [25, 42]. Only patient 9,

AC

harboring the c.1674+1G>T mutation, showed similar clinical presentation to that of a reported case of mild childhood-onset NM [25]. This discrepancy could be explained by the unshared second mutation, which may have a potential role in muscle function. In addition, we were not able to identify any clear relationship between the mutation type and the patient’s clinical phenotype. For example, patient 12, who had compound heterozygous nonsense and frameshift mutations, was classified as mild childhood-

ACCEPTED MANUSCRIPT

onset NM, whereas another patient with compound heterozygous nonsense and missense mutations (patient 1) presented with severe phenotype (Table 1). Interestingly, the proportion of patients with distal manifestations in our cohort (47%) was significantly larger than that from a previous study employing a larger number of

T

families (showing only 10 out of 159 families (6%)) [25, 43, 44]. Although this

SC RI P

discrepancy could be due to the bias from the small cohort size, the higher frequency of distal manifestation in our cohort seems to be meaningful. Moreover, the majority of patients showing anterior lower leg weakness in our cohort had mutations located

NU

between NEB exons 166 and 177. Donner et al. had reported that the alternatively spliced exons 166–177 express 20 different transcripts in adult human tibialis anterior

MA

muscle, where exons 167 and 175 expressed the majority of these transcripts, which strongly suggests that mutations at this location could lead to clinical involvement of

ED

the anterior compartment of the lower legs [30]. A recent research study has suggested that the severity of the clinical features is

PT

determined by the degree of alteration of the binding affinity among nebulin,

CE

tropomyosin, and actin by the location and type of mutations in NEB [45]. Further study is needed in order to clarify this issue.

AC

In summary, our study showed that NEB is the most common NM-causative gene in Korean patients with this disease. Although combined WES and Sanger sequencing is an effective strategy for the detection of pathogenic variants in NM, the highly repetitive region in NEB is the major obstacle to a definite genetic analysis. Furthermore, contrary to the study results in western countries, abnormal CNV in the triplicate region of NEB may not be a frequent cause of NM among Koreans. Further study is required in order to establish the relationship between the genotypes and clinical features of NM.

ACCEPTED MANUSCRIPT

References [1] A.S. McElhinny, S.T. Kazmierski, S. Labeit, C.C. Gregorio, Nebulin: the nebulous, multifunctional giant of striated muscle., Trends Cardiovasc Med 13(5) (2003) 195-201. [2] K. Pelin, P. Hilpela, K. Donner, C. Sewry, P.A. Akkari, S.D. Wilton, D.

SC RI P

T

Wattanasirichaigoon, M.L. Bang, T. Centner, F. Hanefeld, S. Odent, M. Fardeau, J.A. Urtizberea, F. Muntoni, V. Dubowitz, A.H. Beggs, N.G. Laing, S. Labeit, A. de la Chapelle, C. Wallgren-Pettersson, Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy., Proc. Natl. Acad. Sci. U.S.A. 96(5) (1999)

NU

2305-2310.

[3] K. Donner, M. Ollikainen, M. Ridanpää, H.-J. Christen, H.H. Goebel, M. de Visser,

MA

K. Pelin, C. Wallgren-Pettersson, Mutations in the beta-tropomyosin (TPM2) gene--a rare cause of nemaline myopathy., Neuromuscul Disord 12(2) (2002) 151-158.

ED

[4] N.G. Laing, S.D. Wilton, P.A. Akkari, S. Dorosz, K. Boundy, C. Kneebone, P.

PT

Blumbergs, S. White, H. Watkins, D.R. Love, A mutation in the alpha tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy., Nat Genet 9(1)

CE

(1995) 75-79.

[5] K.J. Nowak, D. Wattanasirichaigoon, H.H. Goebel, M. Wilce, K. Pelin, K. Donner,

AC

R.L. Jacob, C. Hübner, K. Oexle, J.R. Anderson, C.M. Verity, K.N. North, S.T. Iannaccone, C.R. Müller, P. Nurnberg, F. Muntoni, C. Sewry, I. Hughes, R. Sutphen, A.G. Lacson, K.J. Swoboda, J. Vigneron, C. Wallgren-Pettersson, A.H. Beggs, N.G. Laing, Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy., Nat Genet 23(2) (1999) 208-212.

ACCEPTED MANUSCRIPT

[6] J.J. Johnston, R.I. Kelley, T.O. Crawford, D.H. Morton, R. Agarwala, T. Koch, A.A. Schäffer, C.A. Francomano, L.G. Biesecker, A novel nemaline myopathy in the Amish caused by a mutation in troponin T1., Am J Hum Genet 67(4) (2000) 814-821. [7] G. Ravenscroft, S. Miyatake, V.-L. Lehtokari, E.J. Todd, P. Vornanen, K.S. Yau,

T

Y.K. Hayashi, N. Miyake, Y. Tsurusaki, H. Doi, H. Saitsu, H. Osaka, S. Yamashita, T.

SC RI P

Ohya, Y. Sakamoto, E. Koshimizu, S. Imamura, M. Yamashita, K. Ogata, M. Shiina, R.J. Bryson-Richardson, R. Vaz, O. Ceyhan, C.A. Brownstein, L.C. Swanson, S. Monnot, N.B. Romero, H. Amthor, N. Kresoje, P. Sivadorai, C. Kiraly-Borri, G.

NU

Haliloglu, B. Talim, D. Orhan, G. Kale, A.K. Charles, V.A. Fabian, M.R. Davis, M. Lammens, C.A. Sewry, A. Manzur, F. Muntoni, N.F. Clarke, K.N. North, E. Bertini, Y.

MA

Nevo, E. Willichowski, I.E. Silberg, H. Topaloğlu, A.H. Beggs, R.J.N. Allcock, I. Nishino, C. Wallgren-Pettersson, N. Matsumoto, N.G. Laing, Mutations in KLHL40 are

ED

a frequent cause of severe autosomal-recessive nemaline myopathy., Am J Hum Genet 93(1) (2013) 6-18.

PT

[8] C.A.C. Ottenheijm, H. Granzier, New insights into the structural roles of nebulin in

CE

skeletal muscle., J Biomed Biotechnol 2010 (2010) 968139. [9] D. Sanoudou, A.H. Beggs, Clinical and genetic heterogeneity in nemaline myopathy

AC

– a disease of skeletal muscle thin filaments., Trends Mol Med 7(8) (2001) 362-368. [10] C. Wallgren-Pettersson, C.A. Sewry, K.J. Nowak, N.G. Laing, Nemaline myopathies., Semin Pediatr Neurol 18(4) (2011) 230-238. [11] L. Levesque, M.R. Del Bigio, S. Krawitz, A.A. Mhanni, A de novo dominant mutation in ACTA1 causing congenital nemaline myopathy associated with a milder phenotype: expanding the spectrum of dominant ACTA1 mutations., Neuromuscul Disord 23(3) (2013) 239-242.

ACCEPTED MANUSCRIPT

[12] C. Wallgren-Pettersson, A.H. Beggs, N.G. Laing, 51st ENMC International Workshop: Nemaline Myopathy. 13-15 June 1997, Naarden, The Netherlands., Neuromuscul. Disord., 1998, pp. 53-56. [13] W.L. van der Pol, J.F. Leijenaar, W.G.M. Spliet, S.W. Lavrijsen, N.J.G. Jansen,

T

K.P.J. Braun, M. Mulder, B. Timmers-Raaijmakers, K. Ratsma, D. Dooijes, M.M. van

SC RI P

Haelst, Nemaline myopathy caused byTNNT1 mutations in a Dutch pedigree., Mol Genet Genomic Med 2(2) (2014) 134-137.

[14] C. Lomen-Hoerth, M.L. Simmons, S.J. Dearmond, R.B. Layzer, Adult-onset

NU

nemaline myopathy: Another cause of dropped head., Muscle Nerve 22(8) (1999) 11461150.

MA

[15] M. Yuen, S.A. Sandaradura, J.J. Dowling, A.S. Kostyukova, N. Moroz, K.G. Quinlan, V.-L. Lehtokari, G. Ravenscroft, E.J. Todd, O. Ceyhan-Birsoy, D.S. Gokhin, J.

ED

Maluenda, M. Lek, F. Nolent, C.T. Pappas, S.M. Novak, A. D'Amico, E. Malfatti, B.P. Thomas, S.B. Gabriel, N. Gupta, M.J. Daly, B. Ilkovsk i, P.J. Houweling, A.E. Davidson,

PT

L.C. Swanson, C.A. Brownstein, V.A. Gupta, L. Medne, P. Shannon, N. Martin, D.P.

CE

Bick, A. Flisberg, E. Holmberg, P. Van den Bergh, P. Lapunzina, L.B. Waddell, D.D. Sloboda, E. Bertini, D. Chitayat, W.R. Telfer, A. Laquerrière, C.C. Gregorio, C.A.C.

AC

Ottenheijm, C.G. Bonnemann, K. Pelin, A.H. Beggs, Y.K. Hayashi, N.B. Romero, N.G. Laing, I. Nishino, C. Wallgren-Pettersson, J. Melki, V.M. Fowler, D.G. MacArthur, K.N. North, N.F. Clarke, Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy., J Clin Invest 124(11) (2014) 4693-4708. [16] C.W. Ockeloen, H.J. Gilhuis, R. Pfundt, E.J. Kamsteeg, P.B. Agrawal, A.H. Beggs, A. Dara Hama-Amin, A. Diekstra, N.V.A.M. Knoers, M. Lammens, N. van Alfen,

ACCEPTED MANUSCRIPT

Congenital myopathy caused by a novel missense mutation in the CFL2 gene., Neuromuscul Disord 22(7) (2012) 632-639. [17] N. Sambuughin, K.S. Yau, M. Olivé, R.M. Duff, M. Bayarsaikhan, S. Lu, L. Gonzalez-Mera, P. Sivadorai, K.J. Nowak, G. Ravenscroft, F.L. Mastaglia, K.N. North,

T

B. Ilkovski, H. Kremer, M. Lammens, B.G.M. van Engelen, V. Fabian, P. Lamont, M.R.

SC RI P

Davis, N.G. Laing, L.G. Goldfarb, Dominant mutations in KBTBD13, a member of the BTB/Kelch family, cause nemaline myopathy with cores., Am J Hum Genet 87(6) (2010) 842-847.

NU

[18] V.A. Gupta, G. Ravenscroft, R. Shaheen, E.J. Todd, L.C. Swanson, M. Shiina, K. Ogata, C. Hsu, N.F. Clarke, B.T. Darras, M.A. Farrar, A. Hashem, N.D. Manton, F.

MA

Muntoni, K.N. North, S.A. Sandaradura, I. Nishino, Y.K. Hayashi, C.A. Sewry, E.M. Thompson, K.S. Yau, C.A. Brownstein, T.W. Yu, R.J.N. Allcock, M.R. Davis, C.

ED

Wallgren-Pettersson, N. Matsumoto, F.S. Alkuraya, N.G. Laing, A.H. Beggs, Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as

PT

an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy., Am J Hum

CE

Genet 93(6) (2013) 1108-1117.

[19] E. Malfatti, J. Böhm, E. Lacène, N. Romero, J. Laporte, A premature stop codon in

AC

MYO18B is associated with severe nemaline myopathy with cardiomyopathy, Neuromuscul Disord 25 S186. [20] S. Miyatake, S. Mitsuhashi, Y.K. Hayashi, E. Purevjav, A. Nishikawa, E. Koshimizu, M. Suzuki, K. Yatabe, Y. Tanaka, K. Ogata, S. Kuru, M. Shiina, Y. Tsurusaki, M. Nakashima, T. Mizuguchi, N. Miyake, H. Saitsu, K. Ogata, M. Kawai, J. Towbin, I. Nonaka, I. Nishino, N. Matsumoto, Biallelic Mutations in MYPN, Encoding

ACCEPTED MANUSCRIPT

Myopalladin, Are Associated with Childhood-Onset, Slowly Progressive Nemaline Myopathy., Am J Hum Genet 100(1) (2017) 169-178. [21] F.-O. Desmet, D. Hamroun, M. Lalande, G. Collod-Béroud, M. Claustres, C. Béroud, Human Splicing Finder: an online bioinformatics tool to predict splicing

T

signals., Nucleic Acids Res 37(9) (2009) e67.

SC RI P

[22] K. Kiiski, L. Laari, V.-L. Lehtokari, M. Lunkka-Hytönen, C. Angelini, R. Petty, P. Hackman, C. Wallgren-Pettersson, K. Pelin, Targeted array comparative genomic hybridization--a new diagnostic tool for the detection of large copy number variations in

NU

nemaline myopathy-causing genes., Neuromuscul Disord 23(1) (2013) 56-65. [23] Y.-E. Park, J.-H. Shin, B. Kang, C.-H. Lee, D.S. Kim, NEB-related core-rod

MA

myopathy with distinct clinical and pathological features., Muscle Nerve 53(3) (2016) 479-484.

ED

[24] S.Y. Kim, Y.-E. Park, H.-S. Kim, C.-H. Lee, D.H. Yang, D.S. Kim, Nemaline myopathy and non-fatal hypertrophic cardiomyopathy caused by a novel ACTA1

PT

E239K mutation., J. Neurol. Sci. 307(1-2) (2011) 171-173.

CE

[25] V.-L. Lehtokari, K. Kiiski, S.A. Sandaradura, J. Laporte, P. Repo, J.A. Frey, K. Donner, M. Marttila, C. Saunders, P.G. Barth, J.T. den Dunnen, A.H. Beggs, N.F.

AC

Clarke, K.N. North, N.G. Laing, N.B. Romero, T.L. Winder, K. Pelin, C. WallgrenPettersson, Mutation update: the spectra of nebulin variants and associated myopathies., Hum Mutat 35(12) (2014) 1418-1426. [26] N.B. Romero, S.A. Sandaradura, N.F. Clarke, Recent advances in nemaline myopathy., Curr Opin Neurol 26(5) (2013) 519-526.

ACCEPTED MANUSCRIPT

[27] M.C. Sharma, D. Jain, C. Sarkar, H.H. Goebel, Congenital myopathies – a comprehensive update of recent advancements., Acta Neurol Scand 119(5) (2009) 281292. [28] M.W. Lawlor, C.A. Ottenheijm, V.-L. Lehtokari, K. Cho, K. Pelin, C. Wallgren-

T

Pettersson, H. Granzier, A.H. Beggs, Novel mutations in NEB cause abnormal nebulin

SC RI P

expression and markedly impaired muscle force generation in severe nemaline myopathy., Skelet Muscle 1(1) (2011) 23.

[29] M. Scoto, T. Cullup, S. Cirak, S. Yau, A.Y. Manzur, L. Feng, T.S. Jacques, G.

NU

Anderson, S. Abbs, C. Sewry, H. Jungbluth, F. Muntoni, Nebulin (NEB) mutations in a childhood onset distal myopathy with rods and cores uncovered by next generation

MA

sequencing., Eur J Hum Genet 21(11) (2013) 1249-1252. [30] K. Donner, M. Sandbacka, V.-L. Lehtokari, C. Wallgren-Pettersson, K. Pelin,

ED

Complete genomic structure of the human nebulin gene and identification of alternatively spliced transcripts., Eur J Hum Genet 12(9) (2004) 744-751.

PT

[31] A. Rhoads, K.F. Au, PacBio Sequencing and Its Applications., Genomics

CE

Proteomics Bioinformatics 13(5) (2015) 278-289. [32] K. Kiiski, V.-L. Lehtokari, A. Löytynoja, L. Ahlstén, J. Laitila, C. Wallgren-

AC

Pettersson, K. Pelin, A recurrent copy number variation of the NEB triplicate region: only revealed by the targeted nemaline myopathy CGH array., Eur J Hum Genet 24(4) (2016) 574-580. [33] D. Piga, F. Magri, D. Ronchi, S. Corti, D. Cassandrini, E. Mercuri, G. Tasca, E. Bertini, F. Fattori, A. Toscano, S. Messina, I. Moroni, M. Mora, M. Moggio, I. Colombo, T. Giugliano, M. Pane, C. Fiorillo, A. D'Amico, C. Bruno, V. Nigro, N. Bresolin, G.P. Comi, New Mutations in NEB Gene Discovered by Targeted Next-

ACCEPTED MANUSCRIPT

Generation Sequencing in Nemaline Myopathy Italian Patients., J Mol Neurosci 59(3) (2016) 351-359. [34] B. Ilkovski, N. Mokbel, R.A. Lewis, K. Walker, K.J. Nowak, A. Domazetovska, N.G. Laing, V.M. Fowler, K.N. North, S.T. Cooper, Disease severity and thin filament

T

regulation in M9R TPM3 nemaline myopathy., J. Neuropathol. Exp. Neurol. 67(9)

SC RI P

(2008) 867-877.

[35] C. Wallgren-Pettersson, K. Pelin, K.J. Nowak, F. Muntoni, N.B. Romero, H.H. Goebel, K.N. North, A.H. Beggs, N.G. Laing, E.I.C.O.N. Myopathy, Genotype-

NU

phenotype correlations in nemaline myopathy caused by mutations in the genes for nebulin and skeletal muscle alpha-actin., Neuromuscul Disord 14(8-9) (2004) 461-470.

MA

[36] V. Straub, P.G. Carlier, E. Mercuri, TREAT-NMD workshop: pattern recognition in genetic muscle diseases using muscle MRI: 25-26 February 2011, Rome, Italy.,

ED

Neuromuscul Disord 22 Suppl 2 (2012) S42-53. [37] H. Jungbluth, C.A. Sewry, S. Counsell, J. Allsop, A. Chattopadhyay, E. Mercuri, K.

PT

North, N. Laing, G. Bydder, K. Pelin, C. Wallgren-Pettersson, F. Muntoni, Magnetic

CE

resonance imaging of muscle in nemaline myopathy., Neuromuscul Disord 14(12) (2004) 779-784.

AC

[38] M.M.R. Kathryn N North, Nemaline Myopathy. https://www.ncbi.nlm.nih.gov/books/NBK1288/, 2012 (accessed 11 June 2015). [39] E. Malfatti, V.-L. Lehtokari, J. Böhm, J.M. De Winter, U. Schäffer, B. Estournet, S. Quijano-Roy, S. Monges, F. Lubieniecki, R. Bellance, M.T. Viou, A. Madelaine, B. Wu, A.L. Taratuto, B. Eymard, K. Pelin, M. Fardeau, C.A.C. Ottenheijm, C. WallgrenPettersson, J. Laporte, N.B. Romero, Muscle histopathology in nebulin-related nemaline

ACCEPTED MANUSCRIPT

myopathy: ultrastrastructural findings correlated to disease severity and genotype., Acta Neuropathol Commun 2 (2014) 44. [40] M.M. Ryan, B. Ilkovski, C.D. Strickland, C. Schnell, D. Sanoudou, C. Midgett, R. Houston, D. Muirhead, X. Dennett, L.K. Shield, U. De Girolami, S.T. Iannaccone, N.G.

T

Laing, K.N. North, A.H. Beggs, Clinical course correlates poorly with muscle

SC RI P

pathology in nemaline myopathy., Neurology 60(4) (2003) 665-673.

[41] J.-M. Hong, S.-M. Kim, I.-N. Sunwoo, S.-H. Kim, T.-S. Kim, D.-S. Shim, Y.-C. Choi, Clinical heterogeneity in Korean patients with nemaline myopathy., Yonsei Med J

NU

51(2) (2010) 225-230.

[42] V.-L. Lehtokari, K. Pelin, M. Sandbacka, S. Ranta, K. Donner, F. Muntoni, C.

MA

Sewry, C. Angelini, K. Bushby, P. Van den Bergh, S. Iannaccone, N.G. Laing, C. Wallgren-Pettersson, Identification of 45 novel mutations in the nebulin gene associated

ED

with autosomal recessive nemaline myopathy., Hum. Mutat. 27(9) (2006) 946-956. [43] C. Wallgren-Pettersson, V.-L. Lehtokari, H. Kalimo, A. Paetau, E. Nuutinen, P.

PT

Hackman, C. Sewry, K. Pelin, B. Udd, Distal myopathy caused by homozygous

CE

missense mutations in the nebulin gene., Brain 130(6) (2007) 1465-1476. [44] V.-L. Lehtokari, K. Pelin, A. Herczegfalvi, V. Karcagi, J. Pouget, J. Franques, J.F.

AC

Pellissier, D. Figarella-Branger, M. von der Hagen, A. Huebner, B. Schoser, H. Lochmüller, C. Wallgren-Pettersson, Nemaline myopathy caused by mutations in the nebulin gene may present as a distal myopathy., Neuromuscul Disord 21(8) (2011) 556562. [45] M. Marttila, M. Hanif, E. Lemola, K.J. Nowak, J. Laitila, M. Grönholm, C. Wallgren-Pettersson, K. Pelin, Nebulin interactions with actin and tropomyosin are altered by disease-causing mutations., Skelet Muscle 4 (2014) 15.

ACCEPTED MANUSCRIPT

Figure 1. Schematic diagram depicting the structural organization of the protein players in nemaline myopathy. A, A-band; I, I-band; M, M-line; Z, Z-disc; KLHL, Kelch-like protein. Figure 2. Flow diagram of the sequence in genetic investigation.

T

Figure 3. Magnetic resonance imaging (A) and computed tomography (B, C, and D)

SC RI P

findings of distal legs. In patient 15, the bilateral tibialis anterior, gastrocnemius medialis, and left soleus muscles show high T2 signals (A, arrows). In computed tomography, low-density lesions are observed in the right soleus and gastrocnemius

NU

muscles in patient 8 (B, arrows), as well as in the bilateral gastrocnemius and soleus muscles in patient 10 (C, arrows). In patient 9, a patch-like distribution of abnormal

MA

intensities is evident (D). (E) Normal computed tomography of lower legs. Figure 4. Distribution of nemaline rods in muscle fiber observed under a light

ED

microscope. In patients 3 (A), 7 (B), 13 (C), and 14 (D), the nemaline rods are scattered across the muscle fiber. On the other hand, in patients 5 (E), 6 (F), 9(G), and 12 (H), the

PT

nemaline rods are located mainly at the subsarcolemmal areas. All slides were stained

CE

with modified Gomori trichrome stain. Bar: 50 µm (A–H). Figure 5. Electron microscopy findings of nemaline myopathies. In patient 8, electron-

AC

dense nemaline rods (arrow) are clearly observed along with the Z-line disruptions (arrowhead) (A). In patient 12, nemaline rods are accumulated mainly in the subsarcolemmal areas (B). In patient 2, numerous nemaline rods are scattered in atrophic muscle fibers, which were not clearly observed under the light microscope (C). In patient 7, only Z-line disruptions are observed. (D). Bars indicate 5 µm in (A) and (B), 2 µm in (C), and 1 µm in (D). All sections were stained with uranyl acetate and lead citrate.

MA

NU

SC RI P

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Fig. 1

MA

NU

SC RI P

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Fig. 2

AC

Fig. 3

CE

PT

ED

MA

NU

SC RI P

T

ACCEPTED MANUSCRIPT

SC RI P

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

Fig. 4

NU

SC RI P

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

Fig. 5

ACCEPTED MANUSCRIPT

Table 1. Clinical and laboratory data of fifteen patients with nemaline myopathy Pat ient nu 1 2 3 4 5 6 7 8 9 10 11 12 mb er

Upp er extre mity , Prox imal Low er extre mity , Prox imal Ankl e, Dors iflex ion Ankl e, Plan tar flexi on Resp irato

Typi cal

Typ ical

Typ ical

Chi ldh ood ons et

Chil dhoo d onset

Childho od onset

Chil dho od onse t

3 Mo /F

1 Yr /F

5 Yr / M

3 Mo /F

6 Mo /M

6 Yr / F

20 Yr / M

16 Yr /M

15 Yr / M

9 Yr / M

9 Yr /F

At birth

At bir th

At birt h

At birt h

At birth

1 Yr

10 Mo

5 Yr

3 Yr

5 Yr

Poor sucki ng

Hy po to nia

Poo r suc kin g

Poo r suc kin g

Wea k cryin g

Mot or dev elo pm ent dela y

Mo tor dev elo pm ent del ay

Ac hill es ten don con trac ture

Slow runn er after 3 years old

None

M ot he r

No ne

No ne

None

N/ A

No ne

No ne

2

3+

5

N/ A

3

2

4

4

Chil dhoo d onset

14

15

Child hood onset

Child hood onset

Chi ldh ood ons et

T

Ty pic al

24 Yr /M

20 Yr /M

43 Yr / M

6 Yr

9 Yr

9 Yr

9Yr

10 Yr

Distal lower extremit y weaknes s

Toein gait

Dista l lowe r extre mity weak ness

Unsta ble gait and slow runner

Slow runner

Foo t dro p for 4 yea rs

Non e

N/A

N/A

None

Broth er

Broth er

Bro ther

NU

SC RI P

25 Yr / M

MA

ED

Fam ily histo ry Mus cle wea knes s

Typ ical

4

2

N/ A

4

4

N/A

5

4

4+

5

PT

Clini cal featu re at onse t

Int er me dia te

CE

Age / Gen der Sym pto m onse t age

Inter medi ate

AC

Sub grou p

13

N/ A

2

4

4

5

4

4

N/A

5

5

5

3

2

3+

4

N/ A

2

5

4+

1-2

4

2

N/A

4+

2

1

2

2

3+

4

N/ A

2

3

4+

5

4

4+

N/A

4+

5

4

2

Posit ive

M ec ha

Ne gati ve

Pne um oni

N/A

Neg ativ e

Ort hop nea

Ne gati ve

Nega tive

Negativ e

Neg ativ e

Nega tive

Negati ve

Negati ve

Neg ativ e

ACCEPTED MANUSCRIPT

ry invo lvem ent

a

at 15 Yr

15

25

7

119

N/ A

5,3 70

151

24

N/A

N SR

N/ A

N/ A

NSR

NS R

N/ A

N/ A

NSR

N/ A

N/A

My opa thic

N/A

scat tere d

AC TA 1

142

No rm al

N/ A

N/ A

N/A

N/ A

Elec trom yogr aphy

N/A

M yo pat hic

N/ A

My opa thic

Nor mal

My opa thic

My opa thic

Loca tion of rods in LM

scatt ered

No vis ibl e in L M

scat tere d

sub sar col em ma

subsa rcole mma

Sca tter ed, sub sarc ole mm a

Gen e

NEB

TP M 3

NE B

No ne

NEB

NE B

†‡

c.3 2T >A †

‡

ex16 1;c.2 3245 C>T *

AC

ex84/9 2/100; c.1269 8A>T† p.R28 09X p.L44 23W p.D42 33V

p. M1 1K

ED

PT

CE

ex61;c .8425 C>T* ex87/9 5/103; c.1326 8T>G

p.R7 749 X

ex29;c .2875_ 2877d elTG T*

p.C95 9del

ex13 9;c.2 0928 delG

c.71 5G> A†

*

p.L6 977 Nf s* 5

SC RI P

N/A

Pred icted cons eque nce

N/A

118

118

103

NSR

NS R

NSR

N/A

N/A

N/ A

Trivial TR

N/A

N/A

N/A

N/A

N/ A

N/A

N/A

Myo pathi c

Myop athic

Myop athic

My opa thic

scat tere d

Scatt ered, subs arcol emm a

Scattere d, subsarco lemma

N/A

subs arcol emm a

scatter ed

scatter ed

Sca tter ed

NE B

NEB

NEB

NEB

NEB

NEB

NEB

NE B

ex80 ;c.11 930 G> A *‡

ex82/ 90/98; c.124 78A> T†‡ int18; c.167 4+1G >T*

ex39;c.46 17_4629d elTTATA AAGCAG AT insAAA* ex175;c.2 4684G>A*

ex167;c .23908_ 23911d elAGA G*

ex167;c .23908_ 23911d elAGA G*

MA

Card iac ultra sono grap hy

Hy pert rop hic car dio my opa thy

Nucl eotid e chan ge

47

T

36

NU

Seru m CK, U/L (nor mal; 0145) Elec troca rdio grap hy

nic al ve nti lat io n

p.E2 39K

p.W 3977 X

p.I416 0F

p.H1539Q fs*12 p.S8228S

int14 4;c.2 1522 +3A> G*

ex82/ 90/98; c.1239 7delA ‡

ex88/ 96/10 4;c.13 389G >A ‡

p.M4 133X p.W4 463X

ex33 ;c.33 87de lC* ex17 5;c.2 4684 G>A *

p.R797 0Sfs*48

p.R797 0Sfs*48

p.Y1 130 Mfs *53 p.S8 228 S

Abbreviations; Intermediate, intermediate congenital form; Typical, typical congenital form; Childhood, mild nemaline myopathy with childhood onset; CK, creatine kinase; N/A, not applicable; LM, light microscope; Mo, months; NSR, normal sinus rhythm; TR, tricuspid valve regurgitation; Yr, years Novel mutations are bold. Variant was identified by whole exome sequencing* or Sanger sequencing†. T he exon 82-89, 90-97, and 98-105 are identical. Therefore, this variant may be in any of these locations‡ . NEB, NG_009382, NM_001271208; TPM3, NG_008621, NM_152263; ACTA1, NG_006672, NM_001100

ACCEPTED MANUSCRIPT

Frequency

Increased fiber size variation

13/15 (87%)

Type 1 fiber predominance

12/15 (80%)

Endomysial fibrosis

8/15 (53%)

Type 1 fiber atrophy

2/15 (13%)

Increased fibers with internal nuclei

2/15 (13%)

Core lesion

2/15 (13%)

AC

CE

PT

ED

MA

NU

SC RI P

Abnormal pathologic findings

T

Table 2. Summary of muscle pathology findings

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI P

T

Highlights  Whole exome sequencing is useful for genetic diagnosis of nemaline myopathy.  NEB is the most common causative gene among Korean patients with nemaline myopathy.  Distal leg weakness is frequent in NEB associated childhood onset nemaline myopathy.